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recent years were associated with substantial development of enteral nutrition , and particularly , home enteral nutrition ( hen ) . for the last ten to 20 years , hen has been fully reimbursed in many european countries which has contributed to increased popularity of this treatment modality [ 8 , 13 ] . also , in poland , hen has been developed and is reimbursed by the national health fund ( nhf ) since 2007 . according to the regulations of nhf , however , enteral feeding is reimbursed only if there is no possibility of oral nutrition , which excludes oral supplementation or sip feeding included in the espen definition of enteral feeding . enteral nutrition is required in all patients who are unable to cover their energetic and nutritional requirements by means of normal ingestion despite , at least partially , retained function of the gastrointestinal tract . potential advantages of hen in comparison to traditional hospital - based enteral nutrition include shorter hospitalization , lower direct costs of therapy and lower risk of secondary malnutrition - associated complications . according to sparse literature data , children constitute a minority among patients receiving hen . published epidemiologic data on the administration rates of enteral / parenteral home nutrition is very limited and available only in a few countries [ 2 , 3 , 9 , 15 ] . in turn , reliable data on the administration rates of hen is required to assess potential benefits associated with this treatment modality along with types and rates of associated complications . additionally , it can be helpful in confronting requirements for this type of procedure with available resources . finally , actual data is necessary for planning prospective studies of therapeutic outcomes of enteral nutrition . constantly updated registries constitute the most optimal source of reliable data on hen service availability and utilization rates . despite their obvious advantages , only a few such national registries exist , however [ 2 , 3 , 7 , 15 ] . since it was reimbursed and thus available , the dynamic increase in the number of patients who were offered hen services by polish medical centers was observed during recent years . however , still there are some regions with no specialized centers offering hen services for children . therefore , the aim of this first nationwide study was to assess the availability of pediatric hen services in poland in order to identify potential areas that should be improved . this study was based on a retrospective 1-year analysis of hen services that were offered to polish children in 2010 . all public healthcare services in poland are covered by the regional branches of the same funder , national health fund . therefore , all 16 polish provinces have the same hen reimbursement criteria and procedures . according to these regulations , each healthcare provider is responsible for qualifying patients to nutritional treatment , training patients ( or their parents in the case of underage subjects ) in hen procedures , providing diets and all related devices , follow - up visits and all other necessary medical services . in january 2010 , the questionnaire , developed by the polish society for clinical nutrition of children and previously tested for reliability and validity , was sent to all regional centers providing pediatric hen services in poland ( n = 14 ) . in each center , the results of the survey were analyzed with an aid of statistica 8 ( statsoft ) package . the analysis included the number of pediatric patients who received hen on january 1st 2010 and december 31st 2010 , their demographic characteristics and geographical distribution . the prevalence rate of hen was expressed as the number of children receiving this type of service per one 1 million of the inhabitants in the analyzed region . furthermore , the distributions of indications and methods of enteral nutrition administration were analyzed , along with the potential reasons of withdrawal from the hen program . the number of children receiving hen in 2010 increased by 21% ( from 433 to 525 patients ) compared to january 1st 2010 . within the study period , 73 patients were withdrawn from the hen program due to various reasons ( table 1 ) , and 165 new subjects were qualified to the enteral feeding ( 31.43%).table 1exclusion criteria from polish pediatric hen program in 2010criterionnumberlack of satisfactory outcome1complications associated with enteral nutrition2lack of patient s acceptance7lack of caregivers acceptance7satisfactory weight gain20patient s death17other19total73 exclusion criteria from polish pediatric hen program in 2010 the fraction of children receiving hen increased substantially in 2010 , from 11.34 per 1 million inhabitants on january 1st to 13.75 per 1 million on december 31st . marked differences were observed in terms of geographical distribution of this parameter , from zero ( in two provinces ) to up to 30 pediatric patients per 1 million inhabitants ( fig . 1geographic distribution of polish children receiving hen in 2010 geographic distribution of polish children receiving hen in 2010 most patients receiving hen ( n = 419 , 79.8% ) were supervised by specialized pediatric centers , while the remainder ( n = 106 , 20.2% ) were handled by medical centers serving both children and adults . median age of patients receiving hen in 2010 was 6 years ( range : 9 months18 years ) . in most cases , hen was prescribed due to neurological disorders ( n = 337 , 64.2% ) . other indications for hen are summarized in table 2 . in most cases ( n = 450 , 85.71% ) , enteral nutrition was administered by means of gastrostomy ( table 3).table 2indications for enteral nutrition in polish children receiving hen in 2010indicationnumberpercentagecerebral palsy16431.2encephalopathy529.9spinal muscular atrophy ( sma)448.4muscular dystrophy61.1other neurological disorders7113.5genetic syndromes9317.7short bowel syndrome10.2inflammatory bowel disease30.6other gastrointestinal diseases265.0cystic fibrosis203.8chronic renal failure152.9chronic liver failure10.2oncological diseases51.0malformation syndrome112.1congenital heart disease40.8metabolic disorders91.7total525100.0table 3methods of enteral nutrition administration in polish children receiving hen in 2010methodnumberpercentagenasogastric tube5911.2gastrostomy45085.7nasojejunal tube20.4jejunostomy40.8gastrojejunal tube101.90total525100.0 indications for enteral nutrition in polish children receiving hen in 2010 methods of enteral nutrition administration in polish children receiving hen in 2010 this first polish nationwide survey on hen prevalence rates among pediatric patients included data from all centers offering this type of specialized services . throughout 2010 nonetheless , the fraction of polish pediatric patients receiving hen ( 13.75 per 1 million inhabitants ) was still markedly lower compared to published data from other countries : 48.23 cases per 1 million in some regions of italy and 95.6 per 1 million in the united kingdom . it should be remembered , however , that 2010 corresponded to already the fourth year of pediatric hen reimbursement / availability in poland . one can assume this as one potential reason for both relatively low prevalence of hen services and dynamic increase in prescription rates of enteral feeding . home enteral nutrition was not prescribed to children living in two out of 16 provinces , probably due to the lack of centers offering hen services in these administrative regions of poland . in another five provinces , however , there were several provinces where markedly higher hen administration rates ( above 15 per 1 million inhabitants ) were reported . similar regional differences in hen distribution in children were previously reported from spain and italy [ 3 , 15 ] . one should confer this highly variable hen administration rates in poland to the prevalence rates of pediatric disorders that usually require enteral nutrition in respective regions . any discrepancies between these two measures would suggest potential regional deficiency of hen services and eventually substantiate funding new pediatric centers . our survey documented that neurologic disorders constituted principle indication for enteral nutrition in polish children ( 64.2% ) . also , in other european countries , chronic neurologic disorders predominate among indications for hen , but the fractions of pediatric patients receiving this treatment modality due to neurological reasons are lower : 28.23% in spain , 35% in france , and 50% in italy . . revealed substantial change in the distribution of indications for hen administered in french children . in 2000 , chronic neurologic disorders constituted the most frequent indication for hen , in contrast to 1989 when enteral nutrition was usually prescribed due to primary alimentary disorders . compared to other european countries , our study revealed markedly lower percentage of pediatric patients who received hen due to respiratory and circulatory tract disorders , and chronic kidney or liver failure . perhaps this underrepresentation of indications other than neurological and alimentary disorders resulted from recent educational activities of the polish society for clinical nutrition of children . during our certified courses , we focused on prescribing hen in chronic neurological and alimentary conditions . the results of this study suggest , however , that during future courses provided by the society more attention should be paid to other disorders that may potentially require enteral nutrition . recent evidence suggests that cystic fibrosis is a condition which definitely can benefit from enteral nutrition [ 6 , 10 , 12 , 16 , 17 ] . in one study , cystic fibrosis patients corresponded to 23% of pediatric patients receiving enteral nutrition in france . in contrast , our survey revealed only 20 ( 3.6% ) cystic fibrosis cases among children who were offered hen in 2010 . taking into account rough estimates of cystic fibrosis prevalence in poland ( n = 1,500 ) , enteral nutrition was prescribed to only 1.3% of this group , hence to six - fold lower fraction compared to tube - feed cystic fibrosis cases managed in the united states .

published epidemiologic data on the administration rates of enteral / parenteral home nutrition is very limited . the aim of this first nationwide study was to assess the availability of pediatric home enteral nutrition ( hen ) services in poland . the questionnaire was sent to all regional centers providing pediatric hen services in poland ( n = 14 ) . the analysis included the number of pediatric patients who received hen in 2010 , their demographic characteristics and geographical distribution . furthermore , the distributions of indications and methods of enteral nutrition administration were analyzed , along with the reasons of withdrawal from the hen program . the number and fraction of children receiving hen increased in 2010 , from 433 ( 11.34 per 1 million inhabitants ) on january 1st to 525 ( 13.75 ) on december 31st . marked differences were observed in geographical distribution of this parameter , from zero to up to 30 pediatric patients per 1 million inhabitants . median age of patients was 6 years ( range : 9 months18 years ) . in most cases , hen was prescribed due to neurological disorders ( n = 337 , 64.2% ) , and administered by means of gastrostomy ( n = 450 , 85.71% ) . this study revealed the dynamic development of pediatric hen services in poland but also documented their potential regional shortages .

Introduction Materials and methods Results Discussion

wild - type and rpa12 cells were grown to stationary phase in a synthetic complete medium . we used microarrays to study the effect of rpa12 deletion on the cellular metabolism of yeast and identified that distinct class of genes were up - regulated in rpa12 strain . yeast wild - type and deletion strains : euroscarftrizol : invitrogen , cat . no . 10 - 296 - 028synthetic complete mediayeast nitrogenous base : difcoyeast drop - out : sigma - aldrich yeast wild - type and deletion strains : euroscarf trizol : invitrogen , cat . no . 10 - 296 - 028 synthetic complete media yeast nitrogenous base : difco yeast drop - out : sigma - aldrich wild - type and rpa12 cells were first inoculated in yeast extract , peptone and 2% dextrose ( ypd ) medium . stationary phase cells were subcultured in a synthetic complete ( sc ) medium containing 2% dextrose as a carbon source along with kanamycin ( 50 g / ml ) at 30 c . after 24 h , the cells were pelleted and washed with phosphate - buffered saline to remove the remaining medium . these samples were hybridized to the affymetrix yeast genome 2.0 array according to the manufacturer 's instructions . all the original microarray data or raw data ( cel file ) were first normalized using the robust multiarray average ( rma ) method that consisted of three steps : a background adjustment , quantile normalization and finally summarization . all above procedures were done by rma algorithm in gene springgx11.5 software from agilent technologies . the genes of low intensity information content in each data set were filtered as follows : first , the probes of intensities < 20.0 percentile in the raw data were excluded and then the probes whose intensities ' coefficient of variation ( cv ) < 50.0% at least 1 out of 4 types remained . in differential gene expression ( dge ) analyses , we identified many genes involved in cellular metabolism are differentially expressed in rpa12 strain . normalized data were filtered for probe sets between 20 and 100 percentile . fold change ( fc ) analysis was performed in gene spring 11.0 using the threshold fc 1.0 and fc 2.0 . fold change 2.0 was selected because the number of gene lists were large in fc 1.0 . wild - type and rpa12 cells were grown to stationary phase in a synthetic complete medium . we used microarrays to study the effect of rpa12 deletion on the cellular metabolism of yeast and identified that distinct class of genes were up - regulated in rpa12 strain . no . 10 - 296 - 028synthetic complete mediayeast nitrogenous base : difcoyeast drop - out : sigma - aldrich yeast wild - type and deletion strains : euroscarf trizol : invitrogen , cat . no . 10 - 296 - 028 synthetic complete media yeast nitrogenous base : difco yeast drop - out : sigma - aldrich wild - type and rpa12 cells were first inoculated in yeast extract , peptone and 2% dextrose ( ypd ) medium . stationary phase cells were subcultured in a synthetic complete ( sc ) medium containing 2% dextrose as a carbon source along with kanamycin ( 50 g / ml ) at 30 c . after 24 h , the cells were pelleted and washed with phosphate - buffered saline to remove the remaining medium . these samples were hybridized to the affymetrix yeast genome 2.0 array according to the manufacturer 's instructions . all the original microarray data or raw data ( cel file ) were first normalized using the robust multiarray average ( rma ) method that consisted of three steps : a background adjustment , quantile normalization and finally summarization . all above procedures were done by rma algorithm in gene springgx11.5 software from agilent technologies . the genes of low intensity information content in each data set were filtered as follows : first , the probes of intensities < 20.0 percentile in the raw data were excluded and then the probes whose intensities ' coefficient of variation ( cv ) < 50.0% at least 1 out of 4 types remained . in differential gene expression ( dge ) analyses , we identified many genes involved in cellular metabolism are differentially expressed in rpa12 strain . normalized data were filtered for probe sets between 20 and 100 percentile . fold change ( fc ) analysis was performed in gene spring 11.0 using the threshold fc 1.0 and fc 2.0 . fold change 2.0 was selected because the number of gene lists were large in fc 1.0 . to identify the rpa12-regulated genes , the expression profiles of rpa12 strain with wild - type ( by4741 ) were compared . on the basis of microarray data analyses , we observed that there are significant changes in the gene expression profile in rpa12 as compared to wild - type . the upregulation of all metabolic genes in rpa12 strain suggested that rpa12 could be a master regulator of whole cellular metabolism .

the ribosomal rna ( rrna ) biosynthesis is the most energy consuming process in all living cells and the majority of total transcription activity is dedicated for synthesizing rrna . the cells may adjust the synthesis of rrna with the availability of resources . rrna is mainly synthesized by rna polymerase i that is composed of 14 subunits . deletion of rpa12 , 14 , 39 and 49 are viable . rpa12 is a very small protein ( 13.6 kda ) , and the amount of protein in the cells is very high ( 12,000 molecules per cell ) , but the role of this protein is unknown in other cellular metabolic processes ( kulak et al . , 2014 [ 1 ] ) . rpa12 consists of two zinc - binding domains and it is required for the termination of rrna synthesis ( mullem et al . , 2002 [ 2 ] ) . deletions of rpa12 in saccharomyces cerevisiae and schizosaccharomyces pombe cause a conditional growth defect ( nogi et al . , 1993 [ 3 ] ) . in s. pombe , c - terminal deletion behaves like wild - type ( imazawa et al . , 2001 [ 4 ] ) . this prompted us to investigate in detail the physiological role of rpa12 in s. cerevisiae , we performed the microarray of rpa12 strain and deposited into gene expression omnibus under gse68731 . the analysis of microarray data revealed that the expression of major cellular metabolism genes is high . the amino acid biosynthesis , nonpolar lipid biosynthesis and glucose metabolic genes are highly expressed . the analyses also revealed that the rpa12 cells have an uncontrolled synthesis of cell metabolites , so rpa12 could be a master regulator for whole cellular metabolism .

Direct link to deposited data Experimental design, materials and methods Experimental design Materials Sample preparation Statistical analysis of the microarray data Results

muscle strength exercise is an important therapeutic technique in physical therapy , which is often performed for recovery from disuse atrophy . although the precise mechanism of satellite cell activation induced by hepatocyte growth factor ( hgf ) is unclear , it is one of the key events in this recovery process1,2,3,4,5 . in fact , previous studies using experimental animal models have demonstrated that satellite cell activation and hgf production are induced by activities that cause continuous muscle contraction , such as high - intensity running for 30 min , synergist muscle ablation , contralateral continuous overloading by ablation of a sciatic nerve , and reloading6,7,8,9,10,11,12,13,14 . however , in clinical physical therapy , it is difficult to perform high - intensity exercise for 30 min or longer . therefore , in this study , we examined the effects of short muscle strength exercise on hgf expression and satellite cell activation for clinical application . to examine the changes during postnatal growth , 20 male sprague - dawley rats ( 212 weeks old ; charles river japan , shizuoka , japan ) were used . the rats were housed in a temperature - controlled room ( 2024 c ) with a 12-h light / dark cycle and ad libitum access to laboratory chow and water . under anesthesia with an intraperitoneal injection of pentobarbital sodium ( 5 mg/100 g body weight ) , 2- , 4- , 8- , and 12-week - old rats were perfused with ice - cold phosphate buffered saline ( pbs , ph 7.2 ) , and the right plantaris muscle was extracted and trimmed of excess fat . for real - time polymerase chain reaction ( pcr ) , the center of the muscle was stored at 80 c until use . to investigate the behavior of satellite cells , 61 male sprague - dawley rats ( 8 weeks old ; initial body weight 293361 g ; charles river japan ) were used . in all rats , the right ankle was fixed to a metal plate by wrapping the foot with a strap . then , the right sciatic nerve was exposed , and a silver electrode was placed on the nerve . the rats were randomly allocated to either the stimulation ( stim ) group or the non - stim group . in the stim group , isometric contractions of the right plantaris muscle were induced by stimulating the sciatic nerve with 150-ms supramaximal single square pulses ( 0.05 ms , 100 hz , 5 v ) once every second for 5 min , using an electronic stimulator ( nihon kohoden , tokyo , japan ) . the rats were perfused with ice - cold pbs under anesthesia on days 1 , 3 , and 7 , and the plantaris muscle of the right hindlimb was removed and trimmed of excess fat . for real - time pcr , the center of the muscle was stored at 80 c until use . additionally , for immunofluorescence analysis , the center of the muscle was placed in tissue - tek o.c.t . compound ( sakura finetek japan , tokyo , japan ) , snap - frozen in liquid nitrogen - cooled isopentane , and stored at 80 c until use . all animal care and treatment procedures were performed in accordance with the guidelines for the care and use of laboratory animals at kanazawa university , and all protocols were approved by the committee on animal experimentation of kanazawa university . total rna was isolated from muscle samples using the rneasy fibrous tissue mini kit ( qiagen , tokyo , japan ) . genomic dna was degraded with dnase i. the purity of the extracted rna was determined by measuring optical density ( taitec , tokyo , japan ) at 260 and 280 nm , where a 260/280 nm ratio of 1.82.0 was the optimal result . rna concentration was measured at 260 nm , and the muscle total rna concentration was calculated on the basis of total rna . first - strand cdna was reverse - transcribed for each muscle sample using the primescript first strand cdna synthesis kit ( takara , tokyo , japan ) according to the manufacturer s protocol . one microgram of total rna , random primers , and a dntp mixture in a 10-l total reaction volume were incubated at 65 c for 5 min , followed by quick - cooling on ice . then , 5 primescript buffer , rnase inhibitor , and primescript rtase were added to a 20-l total reaction volume , which was incubated at 30 c for 10 min , followed by 42 c for 60 min . finally , the reverse transcript reaction mixture was heated to 95 c for 5 min to stop the reaction . real - time pcr was performed with a lightcycler st300 ( roche diagnostics , tokyo , japan ) using the sybr green intercalator method . amplification was performed in a 20-l total reaction volume using 2 l of each rt reaction mixture with the following primers : hgf ( sense : 5-cttaaacatttcccagctagtc-3 ; antisense : 5-ctcgtaataaaccatctgcgt-3 ) ; hgf receptor ( c - met ) ( sense : 5-taggattcggtcttcaagtag-3 ; antisense : 5-aaatcagcaaccttgacagt-3 ) ; myogenic differentiation 1 ( myod ) ( sense : 5-actacagcggcgactcagac-3 ; antisense : 5-actgtagtaggcggcgtcgt-3 ) ; myogenin ( sense : 5-tgaatgcaactcccacagc-3 ; antisense : 5-cagacatatcctccaccgtg-3 ) ; and glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) ( sense : 5-aacgggaaacccatcacca-3 ; antisense : 5-cggagatgatgacccttttg-3 ) . the real - time pcr samples were initially denatured for 10 s at 95 c . pcr was then carried out for 40 cycles with a denaturation step of 5 s at 95 c , and an annealing and extension step of 20 s at 60 c , followed by melting curve analysis . the reaction conditions were optimized to improve the efficiency of the standard curve analysis . for each primer set , pcr specificity was judged based on the presence of a single product at the end of the 40 cycles , as determined by melting curve analysis that showed a single peak at the melting temperature of the product . the mrna expressions of hgf , c - met , myod , and myogenin were normalized against that of gapdh . the relative content of the respective mrna in the 2-week - old rats was compared with that in 4- , 8- , and 12-week - old rats . additionally , the relative content of the respective mrna in the stimulated muscle was compared with that in the non - stimulated muscle . for immunofluorescence analysis , 9 muscle samples from day 1 , 3 , 7 on stim group were cut into 10-m - thick transverse sections using a cryostat . the transverse sections were then fixed in 70% ethanol for 30 min at 4 c . non - specific binding sites were blocked with 5% normal goat serum in pbs for 30 min at room temperature . the sections were incubated with 1:20 rabbit polyclonal anti - hgf alpha antibody ( sc-7949 ; santa cruz biotechnology , santa cruz , ca , usa ) or 1:100 mouse monoclonal anti - phospho - tyrosine antibody ( # 9411 ; cell signaling , tokyo , japan ) in blocking buffer overnight at 4 c . the sections were then incubated with a 1:600 dilution of anti - rabbit alexa fluor 488 ( life technologies japan , tokyo , japan ) and alexa flour 546 ( life technologies japan ) in blocking buffer for 20 min at room temperature , and mounted with prolong gold antifade reagent with 46-diaminino-2-phenylindole ( dapi ; life technologies japan ) . fluorescent signals were captured under fluorescence microscopy ( biozero bz-8100 ; keyence , osaka , japan ) . differences of variance between the 2-week - old rats and the 4- , 8- , and 12-week - old rat in the postnatal growth experiment were analyzed by using dunnett s multiple comparison test , and differences of variance between the stim and non - stim groups were examined by using the student s t - test or welch s test , followed by the f - test . in the growth phase , the plantaris muscle wet weight to body weight significantly increased between 2 and 8 weeks of age compared to that at 2 weeks of age , and then plateaued thereafter ( table 1table 1.morphological and molecular biological changes during postnatal growthpostnatal weeks2 wk4 wk8 wk12 wkn = 5n = 5n = 5n = 5body weight ( g)32.8 1.188.4 2.3278.4 6.2419.0 7.6plantaris muscle wet weight ( mg)16.2 0.671.7 7.3298.6 17.7464.5 15.9muscle wet weight / body weight ( mg / g)0.99 0.031.62 0.14 * 2.14 0.10 * 2.21 0.09*hgf mrna1.00 0.330.57 0.13 * 0.44 0.25 * 0.35 0.09*myod mrna1.00 0.140.86 0.060.59 0.19 * 0.73 0.20data are expressed as mean standard deviation . * p < 0.01 , compared with 2 wk . p < 0.01 , compared with 4 wk ) . the mrna expression of hgf was significantly high at 2 weeks of age , and then decreased thereafter ( table 1 ) . the mrna expression of myod was significantly lower at week 8 than at week 2 ( table 1 ) . < 0.01 , compared with 4 wk the mrna expressions of myod , myogenin and c - met significantly increased in the stimulated muscles compared to the expressions in the non - stimulated muscles at day 1 , and the high expression levels continued up to day 7 ( table 2table 2.mrna expressions in the plantaris muscle contracted with a 5-min electrical stimulationdays after stimulation137non - stim groupstim groupnon - stim groupstim groupnon - stim groupstim groupn = 8n = 8n = 9n = 9n = 9n = 9hgf1.00 0.441.02 0.391.00 0.601.73 0.901.00 0.391.65 0.69*c - met1.00 0.301.30 0.11 * 1.00 0.392.42 0.97**1.00 0.332.39 1.18**myod1.00 0.312.06 0.64**1.00 0.274.94 2.31**1.00 0.123.01 1.26**myogenin1.00 0.162.12 1.31 * 1.00 0.2920.51 12.5**1.00 0.292.66 1.65*data are expressed as mean standard deviation . * p < 0.05 , compared with the non - stim group for the same number of days . * * p < 0.01 , compared with the non - stim group for the same number of days . ) . the mrna expression of hgf was significantly high only at day 7 ( table 2 ) . furthermore , immunofluorescence analysis showed positive signals of hgf and phospho - tyrosine in the same locations from day 1 to day 7 ( fig . 1fig . 1.immunofluorescence images of the localization of hepatocyte growth factor and phospho - tyrosine in the plantaris muscle contracted with a 5-min electrical stimulation . muscle sections at 1 , 3 , and 7 days after 5-min electrical stimulation are stained for hepatocyte growth factor ( hgf ; green ) and phospho - tyrosine ( red ) . hgf and phospho - tyrosine - immunopositive cells are indicated by arrows . ) . * p < 0.05 , compared with the non - stim group for the same number of days . * * p < 0.01 , compared with the non - stim group for the same number of days . immunofluorescence images of the localization of hepatocyte growth factor and phospho - tyrosine in the plantaris muscle contracted with a 5-min electrical stimulation . muscle sections at 1 , 3 , and 7 days after 5-min electrical stimulation are stained for hepatocyte growth factor ( hgf ; green ) and phospho - tyrosine ( red ) . in the present study , the plantaris muscle wet weight to body weight , and the mrna expressions of hgf and myod , plateaued at 8 weeks of age during postnatal growth . these results are consistent with those of previous studies using hgf enzyme - linked immunosorbent assay and immunohistochemistry11 , 15 , 16 . therefore , 8-week - old rats in which the effect of growth was small were used in the subsequent study . myod and myogenin belong to a class of myogenic differentiation factors , which are expressed in myogenic cells during proliferation and differentiation , and in the present study , expression of these genes indicated satellite cell activation11 , 17,18,19 . the mrna expressions of myod , myogenin , and c - met immediately increased after the 5-min electrical stimulation . in contrast , the mrna expression of hgf increased on day 7 , and not immediately after stimulation . however , strong signals of hgf and phospho - tyrosine were observed in the same locations immediately after electrical stimulation . proteolytic activation of hgf protein by the hgf - specific serine protease hgf activator , is required for binding to c - met20 , 21 . the active form of hgf has been reported to increase after muscle injury3 . additionally , the active form of hgf has been reported to be released in the extracellular matrix where it undergoes enzymolysis by matrix metalloproteinases induced by mechanical stimulation such as stretch stimulation and/or muscle injury22,23,24 . hgf - bound c - met is tyrosine - phosphorylated , and mediates mitogenesis and morphogenesis25 . in the present study , the hgf- antibody recognized both the active and inactive forms of hgf , and the anti - phospho - tyrosine antibody did not recognize phospho - c - met . therefore , in this study , the occurrence of these activation processes is unclear . hgf expression has been reported to be induced by several growth factors and cytokines including hgf itself26 , 27 . the increase in the expression of hgf mrna on day 7 may have occurred through an indirect mechanism . the results indicate that activation of satellite cells related to hgf does not only occur during continuous muscle activities , such as down - hill running for 30 min , synergist ablation , and reloading6,7,8,9,10,11,12,13,14 , but also occurs after activities that induce short muscle contraction , such as a 5-min electrical stimulation . however , in this study , electrical stimulation induced tetanic contraction of the plantaris muscle , which does not easily occur in daily life activities . additionally , muscle hypertrophy occurs upon repeated muscle strength exercise over approximately 3 weeks . therefore , skeletal muscle hypertrophy can be achieved with repeated short muscle contractions , as shown in this study . in physical therapy , achieving a curative effect with exercise in a limited amount of time is very important . in the present study , we demonstrated that the biological signal of muscle hypertrophy triggered by hgf was induced with a 5-min electrical stimulation that caused muscle contractions . this study provided evidence for the use of short muscle strength exercise in physical therapy .

[ purpose ] the present study aimed to determine the effects of short muscle strength exercise on hepatocyte growth factor expression and satellite cell activation . [ subjects ] the study included 72 212-week - old male sprague - dawley rats . [ methods ] the rat plantaris muscle was contracted with a 5-min electrical stimulation of the sciatic nerve , and then , the mrna expressions of hepatocyte growth factor and myogenic regulatory factors in the plantaris muscle were determined , and the phosphorylation of the hepatocyte growth factor receptor ( c - met ) was examined . [ results ] the mrna expressions of hepatocyte growth factor and myogenic regulatory factors increased after a short muscle contraction compared to that un - contraction . immunofluorescence analysis showed the expression of hepatocyte growth factor protein and the possibility that downstream biological changes occurred in the hepatocyte growth factor - bound c - met . [ conclusion ] our results demonstrated that activation of satellite cells induced hepatocyte growth factor expression during muscle contraction with a short 5-min electrical stimulation , which simulates short muscle strength exercise in physical therapy . the present study provides evidence for the use of short muscle strength exercise in physical therapy .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

from october 1996 to november 2002 , 30 consecutive patients with hepatocellular carcinomas underwent transarterial chemoembolization followed by hepatic resection at our institution . of these patients , we included the patients who met the two following criteria : ( a ) two or more four - phase ( i.e. , hepatic arterial , portal venous , and delayed phase , and unenhanced image ) helical cts were taken after the first transarterial chemoembolization , and ( b ) hepatic resection was done within one month of the last follow - up ct . eighteen patients had one tumor , four patients had two tumors , one patient had three tumors and one patient had six tumors . they underwent lobectomy ( n = 22 ) , segmentectomy ( n = 1 ) or liver transplantation ( n = 1 ) after transarterial chemoembolization . of these , 20 patients could undergo hepatic resection because they had recovered enough hepatic function to endure hepatic surgery , as compared to the time when they had undergone the initial transarterial chemoembolization . there were 21 men and three women , and their ages ranged from 38 to 73 years ( mean age ; 54 years ) . all the patents in the study had liver cirrhosis as a result of either hepatitis b ( n = 23 ) , or hepatitis c ( n = 1 ) . twenty - six hepatocellular carcinomas were diagnosed by the pathologic results for those patients that demonstrated viable tumor in the resected specimens . of the nine totally necrotic tumors , the diagnosis of three hepatocellular carcinomas was established based on the results of percutaneous needle biopsy . the remaining six hepatocellular carcinomas showed the characteristic laboratory findings ( e.g. , elevated -fetoprotein levels and viral markers ) in combination with the characteristic radiological findings on the follow - up ct images . the mean diameter of the hepatocellular carcinomas was 3.7 cm ( range ; 0.5 - 12 cm ) . seventeen patients underwent multiple treatments of transarterial chemoembolization ( range ; 2 - 5 treatments , mean ; 2.9 treatments ) . the remaining seven patients underwent it once . the mean interval between the transarterial chemoembolization and the follow - up ct was 20 days ( range ; 3 - 23 days ) , and the mean interval between the last ct and surgery was 14 days ( range ; 1 - 30 days ) . the ct scans were performed with a helical scanner ( hispeed advantage ; ge medical systems , milwaukee , wi , usa ) . the scanning parameters were 120 kvp , 180 mas , 7 mm section collimation and a 7 mm / sec table speed during a single breath - hold helical acquisition of 25 - 30 seconds , depending upon the liver size . the images were obtained in a craniocaudal direction and they were reconstructed every 7 mm to provide contiguous or overlapping sections for the unenhanced and enhanced images . after the acquisition of the unenhanced images , the hepatic arterial phase , the portal venous phase and the delayed phase images were obtained with delays of 30 , 60 and 180 seconds , respectively , after the start of injecting 120 ml of nonionic iodinated contrast material ( iopamiro 300 , bracco , milano , italy and ultravist 300 , schering , berlin , germany ) through the antecubital vein at a rate of 3 ml / sec . transarterial chemoembolization was performed with a mixture of iodized oil ( lipiodol ; guerbet , aulnay - sous - bois , france ) and doxorubicin hydrochloride ( adriamycin ; kyowa hakko kogyo , tokyo , japan ) in all the patients . we used 3 mg of doxorubicin hydrochloride and 1 cc of iodized oil per 1-cm diameter of the tumor . for particle embolization , we also used gelfoam powder ( upjohn , kalamazoo , mi , usa ) if a patient had neither child class c cirrhosis nor thrombosis in the major portal vein . the pathologic specimens were sectioned at 5-mm intervals and the tumor necrosis was estimated as a percentage . the pathologic findings that were used as the standards of reference revealed that nine of the 35 hepatocellular carcinomas were totally necrotic . one observer had 24 years experience at abdominal imaging and other two observers had five years experience each . the observers were informed of the patients ' histories in as much as they knew that all the patients had undergone transarterial chemoembolization for hepatocellular carcinoma , but the observers were blinded to the pathologic findings concerning the presence of viable tumor in each patient . first , the observers reviewed the last four - phase ct taken before liver resection . second , the observers repeated the review two weeks after the first interpretation with using the last ct combined with the previous serial ct images . the observers were obliged to review the second - to - the - last ct . further review of the other previous ct examinations was determined by the respective decision of the observers for each case . all images were evaluated using a 2,000 2,000 picture archiving and communication systems ( pacs ; ge medical systems integrated imaging solutions , mt prospect , il , usa ) monitor . the images were initially evaluated using two window settings ( window level ; 60 hu , window width ; 350 hu , and window level ; 110 hu , window width ; 200 hu ) , and then the window settings were adjusted as needed . images were interpreted for the presence , number , size and site of the viable tumors . each observer also recorded his or her degree of confidence as to whether a lesion seen on an image represented a viable tumor . the attenuation of each lesion in relation to that of the liver ( i.e. , hypoattenuation , hyperattenuation , isoattenuation or mixed attenuation ) was subjectively assessed . the diagnostic confidence for each lesion was scored using a five - point scale ( 1 , not viable tumor ; 2 , probably not viable tumor ; 3 , possibly viable tumor ; 4 , probably viable tumor ; 5 , definitely viable tumor ) at each interpretation session . for the objectivity and reproducibility of the image analysis performed in this study , we regarded the following lesions as viable areas of tumors on the last ct images : ( a ) a hyperattenuating or isoattenuating lesion seen during the hepatic arterial phase and as a hypoattenuating lesion seen during the portal venous phase or the delayed phase , ( b ) an area of mixed attenuation seen on the hepatic arterial phase images that showed a hypoattenuating portion either on the portal venous phase or the delayed phase , and ( c ) a nodule that was seen as being isoattenuation on the hepatic arterial phase , the portal venous phase or the delayed phase images , but it was seen as hypoattenuation on the unenhanced images ( 4 ) . a lesion that showed wedge - shaped hyperattenuation during the hepatic arterial phase and that appeared as isoattenuation on the portal venous phase , on the delayed phase images and on the unenhanced images was considered to be a noncancerous lesion . when the last ct along with the previous serial ct images was reviewed , a lesion that showed a newly presenting defect within an iodized oil - containing nodule was regarded as a viable portion of tumor . a small peritumoral area with suspicious enhancement on the hepatic arterial phase of the last ct , which showed an iodized oil - containing liver parenchyma on the previous serial ct images , was considered as an arterioportal shunt and not as a viable tumor . also , a long - standing hypoattenuating area ( longer than one year ) that did not change after contrast enhancement within a nodule of hepatocellular carcinoma was not considered viable tumor . a radiologist ( the study coordinator ) and they evaluated the location ( upper or lower , right or left , and anterior or posterior ) and the size of the viable portions within the tumors on both the ct and the pathologic examinations . a binominal receiver operating characteristic ( roc ) curve was calculated for each observer 's confidence rating data with using maximum - likelihood estimation . we evaluated the diagnostic accuracy of the last ct alone and the last ct combined with the previous serial ct images by calculating the area under each observer - specific binomial roc curve ( denoted as the az index ) ( 5 ) . the lesions that were assigned a score of three to five were considered as diagnosed viable tumors . the sensitivity and specificity were calculated for each observer and for each different review of the cts , and the statistical analysis of their differences was assessed using the mcnemar test ( spss , version 10.0 ; spss , chicago , il , usa ) . the false negative rates for the detection of viable tumors were also calculated for the review of the last ct alone and for the review of the last ct along with the previous serial ct images . statistics were used to assess the interobserver agreement for the presence of a viable portion of tumor with the review of the last ct alone and with the review of the last ct combined with the previous serial ct images . the degree of agreement was categorized as follows : values of 0.00 - 0.20 were considered to indicate poor agreement , values of 0.21 - 0.40 were considered to indicate fair agreement , values of 0.41 - 0.60 were considered to indicate moderate agreement , values of 0.61 - 0.80 were considered to indicate good agreement and values of 0.81 - 1.00 were considered to indicate excellent agreement ( 5 ) . from october 1996 to november 2002 , 30 consecutive patients with hepatocellular carcinomas underwent transarterial chemoembolization followed by hepatic resection at our institution . of these patients , we included the patients who met the two following criteria : ( a ) two or more four - phase ( i.e. , hepatic arterial , portal venous , and delayed phase , and unenhanced image ) helical cts were taken after the first transarterial chemoembolization , and ( b ) hepatic resection was done within one month of the last follow - up ct . eighteen patients had one tumor , four patients had two tumors , one patient had three tumors and one patient had six tumors . they underwent lobectomy ( n = 22 ) , segmentectomy ( n = 1 ) or liver transplantation ( n = 1 ) after transarterial chemoembolization . of these , 20 patients could undergo hepatic resection because they had recovered enough hepatic function to endure hepatic surgery , as compared to the time when they had undergone the initial transarterial chemoembolization . there were 21 men and three women , and their ages ranged from 38 to 73 years ( mean age ; 54 years ) . all the patents in the study had liver cirrhosis as a result of either hepatitis b ( n = 23 ) , or hepatitis c ( n = 1 ) . twenty - six hepatocellular carcinomas were diagnosed by the pathologic results for those patients that demonstrated viable tumor in the resected specimens . of the nine totally necrotic tumors , the diagnosis of three hepatocellular carcinomas was established based on the results of percutaneous needle biopsy . the remaining six hepatocellular carcinomas showed the characteristic laboratory findings ( e.g. , elevated -fetoprotein levels and viral markers ) in combination with the characteristic radiological findings on the follow - up ct images . the mean diameter of the hepatocellular carcinomas was 3.7 cm ( range ; 0.5 - 12 cm ) . seventeen patients underwent multiple treatments of transarterial chemoembolization ( range ; 2 - 5 treatments , mean ; 2.9 treatments ) . the remaining seven patients underwent it once . the mean interval between the transarterial chemoembolization and the follow - up ct was 20 days ( range ; 3 - 23 days ) , and the mean interval between the last ct and surgery was 14 days ( range ; 1 - 30 days ) . the ct scans were performed with a helical scanner ( hispeed advantage ; ge medical systems , milwaukee , wi , usa ) . the scanning parameters were 120 kvp , 180 mas , 7 mm section collimation and a 7 mm / sec table speed during a single breath - hold helical acquisition of 25 - 30 seconds , depending upon the liver size . the images were obtained in a craniocaudal direction and they were reconstructed every 7 mm to provide contiguous or overlapping sections for the unenhanced and enhanced images . after the acquisition of the unenhanced images , the hepatic arterial phase , the portal venous phase and the delayed phase images were obtained with delays of 30 , 60 and 180 seconds , respectively , after the start of injecting 120 ml of nonionic iodinated contrast material ( iopamiro 300 , bracco , milano , italy and ultravist 300 , schering , berlin , germany ) through the antecubital vein at a rate of 3 ml / sec . transarterial chemoembolization was performed with a mixture of iodized oil ( lipiodol ; guerbet , aulnay - sous - bois , france ) and doxorubicin hydrochloride ( adriamycin ; kyowa hakko kogyo , tokyo , japan ) in all the patients . we used 3 mg of doxorubicin hydrochloride and 1 cc of iodized oil per 1-cm diameter of the tumor . for particle embolization , we also used gelfoam powder ( upjohn , kalamazoo , mi , usa ) if a patient had neither child class c cirrhosis nor thrombosis in the major portal vein . the pathologic specimens were sectioned at 5-mm intervals and the tumor necrosis was estimated as a percentage . the pathologic findings that were used as the standards of reference revealed that nine of the 35 hepatocellular carcinomas were totally necrotic . one observer had 24 years experience at abdominal imaging and other two observers had five years experience each . the observers were informed of the patients ' histories in as much as they knew that all the patients had undergone transarterial chemoembolization for hepatocellular carcinoma , but the observers were blinded to the pathologic findings concerning the presence of viable tumor in each patient . first , the observers reviewed the last four - phase ct taken before liver resection . second , the observers repeated the review two weeks after the first interpretation with using the last ct combined with the previous serial ct images . the observers were obliged to review the second - to - the - last ct . further review of the other previous ct examinations was determined by the respective decision of the observers for each case . all images were evaluated using a 2,000 2,000 picture archiving and communication systems ( pacs ; ge medical systems integrated imaging solutions , mt prospect , il , usa ) monitor . the images were initially evaluated using two window settings ( window level ; 60 hu , window width ; 350 hu , and window level ; 110 hu , window width ; 200 hu ) , and then the window settings were adjusted as needed . images were interpreted for the presence , number , size and site of the viable tumors . each observer also recorded his or her degree of confidence as to whether a lesion seen on an image represented a viable tumor . the attenuation of each lesion in relation to that of the liver ( i.e. , hypoattenuation , hyperattenuation , isoattenuation or mixed attenuation ) was subjectively assessed . the diagnostic confidence for each lesion was scored using a five - point scale ( 1 , not viable tumor ; 2 , probably not viable tumor ; 3 , possibly viable tumor ; 4 , probably viable tumor ; 5 , definitely viable tumor ) at each interpretation session . for the objectivity and reproducibility of the image analysis performed in this study , the important criteria for a viable tumor and for the noncancerous lesions we regarded the following lesions as viable areas of tumors on the last ct images : ( a ) a hyperattenuating or isoattenuating lesion seen during the hepatic arterial phase and as a hypoattenuating lesion seen during the portal venous phase or the delayed phase , ( b ) an area of mixed attenuation seen on the hepatic arterial phase images that showed a hypoattenuating portion either on the portal venous phase or the delayed phase , and ( c ) a nodule that was seen as being isoattenuation on the hepatic arterial phase , the portal venous phase or the delayed phase images , but it was seen as hypoattenuation on the unenhanced images ( 4 ) . a lesion that showed wedge - shaped hyperattenuation during the hepatic arterial phase and that appeared as isoattenuation on the portal venous phase , on the delayed phase images and on the unenhanced images was considered to be a noncancerous lesion . when the last ct along with the previous serial ct images was reviewed , a lesion that showed a newly presenting defect within an iodized oil - containing nodule was regarded as a viable portion of tumor . a small peritumoral area with suspicious enhancement on the hepatic arterial phase of the last ct , which showed an iodized oil - containing liver parenchyma on the previous serial ct images , was considered as an arterioportal shunt and not as a viable tumor . also , a long - standing hypoattenuating area ( longer than one year ) that did not change after contrast enhancement within a nodule of hepatocellular carcinoma was not considered viable tumor . a radiologist ( the study coordinator ) and they evaluated the location ( upper or lower , right or left , and anterior or posterior ) and the size of the viable portions within the tumors on both the ct and the pathologic examinations . a binominal receiver operating characteristic ( roc ) curve was calculated for each observer 's confidence rating data with using maximum - likelihood estimation . we evaluated the diagnostic accuracy of the last ct alone and the last ct combined with the previous serial ct images by calculating the area under each observer - specific binomial roc curve ( denoted as the az index ) ( 5 ) . the lesions that were assigned a score of three to five were considered as diagnosed viable tumors . the sensitivity and specificity were calculated for each observer and for each different review of the cts , and the statistical analysis of their differences was assessed using the mcnemar test ( spss , version 10.0 ; spss , chicago , il , usa ) . the false negative rates for the detection of viable tumors were also calculated for the review of the last ct alone and for the review of the last ct along with the previous serial ct images . statistics were used to assess the interobserver agreement for the presence of a viable portion of tumor with the review of the last ct alone and with the review of the last ct combined with the previous serial ct images . the degree of agreement was categorized as follows : values of 0.00 - 0.20 were considered to indicate poor agreement , values of 0.21 - 0.40 were considered to indicate fair agreement , values of 0.41 - 0.60 were considered to indicate moderate agreement , values of 0.61 - 0.80 were considered to indicate good agreement and values of 0.81 - 1.00 were considered to indicate excellent agreement ( 5 ) . during the two analyses of the ct images , each observer detected all 35 hepatocellular carcinomas that were pathologically demonstrated in the resection specimens . the mean diagnostic accuracies ( az values ) for the depiction of viable tumor with the last ct alone and with the review of previous serial ct images for all the observers were 0.885 and 0.901 , respectively ( p > 0.05 ) ( table 1 ) . the sensitivity , specificity and diagnostic accuracy of the last ct alone were 72% , 91% and 78% , respectively , and the corresponding values for the review of the last ct combined with the previous serial ct images were 78% , 97% and 84% , respectively ( table 2 ) , and the differences were not statistically significant . although these differences in the diagnostic accuracies were not statistically significant , the review of the previous images enabled the observers to render a correct diagnosis for three ( 9% ) tumors . two of these three tumors showed as being slightly hyperattenuating focal areas adjacent to the iodized oil - containing tumors on the hepatic arterial phase images of the last ct , and these two tumors appeared as isoattenuating areas on the unenhanced images . the second to the last ct showed that these areas appeared as parenchymal uptake around the iodized oil - containing tumors ( fig . 1 ) , and the two observers changed their confidence levels to probably not viable tumor . the one remaining tumor showed as an interval developed defect within the iodized oil - containing nodule that was not significantly enhanced on the last ct . on the basis of this feature , the two observers changed their confidence level to probably viable tumor . the pathology revealed a 70% necrotic hepatocellular carcinoma . after reviewing of the last ct and the previous serial ct images , all three observers assessed the 10 compact iodized oil - containing tumors as being non - viable . of these thus , the compact iodized oil - containing tumors showed a mean necrosis rate of 98.5% . all of the 16 false - negative lesions that were diagnosed by each observer after reviewing the last ct combined with the previous serial ct images showed 90% or greater necrosis on the pathologic examination ( table 3 ) . particularly , four tumors of these lesions were diagnosed as negative lesions simultaneously by all the observers with both the review of the last ct alone and the review of the last ct with the previous serial ct images . 2 ) . the remaining two showed no definite enhancement in the iodized oil defect portion on the hepatic arterial phase ct . the interobserver agreement ( statistic ) for the presence of a viable tumor was 0.828 to 0.943 for the review of the last ct alone and 0.826 to 0.885 for the additional review of the previous serial ct images ( table 4 ) . the interobserver agreement for the review of the last ct with or without the previous serial ct images was considered excellent . the criteria suggested by the world health organization and that is used for evaluating the effect of chemotherapy on cancer can not be applied to the evaluation of transarterial chemoembolization therapy because any tumor reduction can seldom be recognized before one month after the treatment ( 6 ) . thus , other criteria have been evaluated to determine the efficacy of transarterial chemoembolization treatment for hepatocellular carcinoma by using iodized oil retention in a tumor and the enhancement on ct ( 7 , 8) . ( 9 ) suggested that the overall cumulative recurrence rate of hepatocellular carcinoma was 23% after one year , 55% after two years and 67% after three years , and 45% of the recurrences occurred adjacent to a primary site that was considered controlled by the transarterial chemoembolization . hence , periodic ct follow - up or angiography might be recommended even though there may be the appearance of complete remission for hepatocellular carcinoma . with the advent of helical ct and with the other rapid technical advances , multiphasic helical ct has recently become a more useful imaging modality for the detection and characterization of liver lesions , for the follow - up after local treatment or surgical excision , and for the assessment of the hemodynamic changes in the liver . however , to the best of our knowledge , there has been no previous study that has compared the preoperative ct and the review of the previous serial ct images with the resected specimens for the evaluation of the viable portion of hepatocellular carcinoma treated with transarterial chemoembolization . in the past , some investigators have reported on the diagnostic efficacy of the hepatic arterial phase ct and the portal venous phase ct for the detection of hypervascular tumors , and especially hepatocellular carcinoma ( 10 , 11 ) . ( 1 ) have suggested that the delayed phase is important for the detection of small hepatocellular carcinomas that are less than 2 cm , for confirming or increasing the confidence level for the detection of equivocal nodules on the arterial or portal venous phase images ( because those nodules are usually more conspicuous on the delayed phase than on the portal venous phase imaging ) , and for the differentiation of arterioportal shunting from the true hepatocellular carcinoma . in our study , this concept was used to depict a viable tumor among the arterioportal shunt areas . for the hepatocellular carcinoma treated with transarterial chemoembolization , takayasu et al . ( 8) have suggested that the iodized oil deposition in a tumor could be considered as necrosis . choi et al . ( 7 ) have also proposed that the complete retention of iodized oil in a tumor and the surrounding liver demonstrated the best therapeutic effects . kim et al . ( 4 ) have recently pointed out that unenhanced images could be of additional diagnostic value to supplement the enhanced biphasic helical ct images for the patients treated with transarterial chemoembolization for hepatocellular carcinoma by facilitating the differentiation of the true lesions from the hyperattenuating pseudolesions that simulate viable tumor . further , they stated that these unenhanced images can also aid in the detection of any isoattenuation lesion on the hepatic arterial phase and the portal venous phase because of the hypoattenuation on the unenhanced images . we also evaluated the unenhanced images , and we used the criteria suggested by kim et al . in addition to the criteria in the previous studies ( 1 , 4 , 7 , 10 , 11 ) , we considered the long - standing hypoattenuating areas within an iodized nodule on the enhanced images of the serial ct images as necrotic lesion despites a lack of iodized oil . on the other hand , a lesion that showed an interval developed defect within an iodized nodule was considered as a viable portion of tumor . in our study , although no significant statistical difference was found between the review of the last ct alone and the review of the last ct along with the previous serial ct images , the additional review of the previous serial ct images allowed the observers to render a correct diagnosis for three ( 9% ) lesions . we believe that many abdominal radiologists actually do review some of the previous ct images when they find an equivocal viable lesion on the liver ct following transarterial chemoembolization . according to our results , after the review of the last ct combined with the previous serial ct images , all the false - negative lesions showed 90% or greater necrosis on the pathologic examination . thus , if a substantial ( larger than 10% ) viable tumor is present in an iodized nodule , we can depict it on ct with a thorough review of the previous serial ct images . first , as this is a retrospective study , all the patients of the study group did not undergo regular follow - up ct and transarterial chemoembolization because of suspected complications or because of personal preference . second , we included a relatively small number of hepatocellular carcinomas , and so this might show no significant statistical difference between the review of the last ct alone and the review of the last ct along with the previous serial ct images . finally , although we thoroughly reviewed the pathologic specimens alongside the ct images , any perfect area - by - area histopathologic correlation was actually impossible . by referring to the location and size of viable tumors within the tumors , however , we tried to match the ct and histopathologic findings as closely as possible . in our results , two of the three tumors that had a correct diagnosis rendered after the review of previous images appeared as parenchymal uptake around the iodized oil - containing tumors on the second to the last ct the pathology demonstrated totally necrotic tumors , and this revealed that the suspicious hyperattenuating focal areas adjacent to the iodized oil - containing tumors on the hepatic arterial phase images of the last ct were pseudolesion . for the third hepatocellular carcinoma with 70% necrosis , we could not perfectly match the interval developed defect within the iodized oil - containing nodule and the 30% viable tumor portion on the pathologic specimen . also , we do not know the reason why the two compact iodized oil - containing tumors seen on ct had viable tumor portions on the pathology examination . in conclusion , for depicting the viable tumor in hepatocellular carcinoma patients treated with transarterial chemoembolization , although the difference in the diagnostic accuracies was not found to be statistically significant in our study , a review of the multiphasic helical ct combined with the previous serial ct images can help render a correct diagnosis for those lesions incorrectly diagnosed with the review of only the last ct .

objectivethe purpose of our study was to assess whether a review of multiphasic helical ct combined with the previous serial ct images could be helpful to depict a viable tumor in hepatocellular carcinoma treated with transarterial chemoembolization.materials and methodstwenty - four consecutive patients with 35 hepatocellular carcinomas underwent transarterial chemoembolization followed by hepatic resection . first , three radiologists independently analyzed the last ct images taken before resection for the presence of viable tumor . a second analysis was then performed using the last ct combined with the previous serial ct images . the ct analyses were then compared with the pathologic results . the added value of the review of the previous serial ct images was evaluated by performing a receiver operating characteristic analysis . the sensitivity , specificity and diagnostic accuracy for the depiction of viable tumor were also assessed , and the characteristics of the false - negative lesions were pathologically evaluated.resultsthe mean diagnostic accuracies ( az values ) for the depiction of viable tumor with using the last ct alone and with the review of the previous serial ct images for all observers were 0.885 and 0.901 , respectively , which were not significantly difference ( p > 0.05 ) . however , the additional review of the previous serial ct images allowed the observers to render a correct diagnosis for three lesions that had been incorrectly diagnosed with the review of last ct alone . the sensitivity , specificity and diagnostic accuracy of the last ct along with the review of the previous serial ct images were 78% , 97% and 84% , respectively . all of the 16 false - negative lesions diagnosed by each observer showed 90% or greater necrosis on the pathologic examination.conclusionfor the depiction of viable tumor in hepatocellular carcinoma treated with transarterial chemoembolization , although the difference in the diagnostic accuracies was not statistically significant , a review of the multiphasic helical ct combined with the previous serial ct images could help reach a correct diagnosis for those lesions incorrectly diagnosed with the review of the last ct alone .

MATERIALS AND METHODS Patient Selection Multiphasic Helical CT Transarterial Chemoembolization Techniques Pathologic Analysis Image Analysis Statistical Analysis RESULTS DISCUSSION

this narrowing can lead to entrapment of the nerve roots and to neurogenic claudication , i.e. pain in the lower extremities while walking or standing . degenerative lumbar spinal stenosis is the most common reason for lumbar surgery in patients beyond the age of 65 years . the standard surgical management for degenerative lumbar spinal stenosis is decompression of the spinal canal and the entrapped nerves [ 35 ] . the long - term outcome of this surgery depends on the maintenance of sufficient intervertebral stability . a too large resection may cause instability of the vertebral column [ 611 ] , sometimes resulting in pars interarticularis fractures and spondylolisthesis [ 1214 ] . on the other hand , removing too little bone does not resolve the stenotic or radicular symptoms . the effect of laminectomy and facetectomy on the stability of the lumbar spine has been studied in vitro [ 8 , 10 ] as well as in finite element studies [ 6 , 1518 ] . these studies show that after laminectomy and facetectomy the stability is impaired , which would increase the risk of spondylolisthesis . in addition , in vitro experiments have shown that anterior shear loading can lead to bony failure of the posterior elements of the spine , with the pars interarticularis being most frequently affected [ 1922 ] . similarly it has been shown in in vitro experiments that after removal of the posterior elements compression and bending stiffness are reduced [ 23 , 24 ] and the strength in shear loading is smaller [ 22 , 25 ] . none of these studies measured the shear strength of the vertebral column after laminectomy and a partial facetectomy . the aim of this study was to investigate the shear strength , in terms of ultimate shear force and displacement to failure and shear stiffness , of the porcine lumbar spine after laminectomy and partial facetectomy , comparable with surgery for spinal stenosis . we hypothesized that laminectomy and partial facetectomy would cause a major decrease of the shear strength . eight porcine lumbar spines of domestic pigs ( mean weight 78 kg , mean age 7 months ) were obtained from the slaughterhouse . each lumbar spine was sectioned into two motion segments for testing : l2l3 and l4l5 . one of the motion segments was assigned to the control group and one to the treatment group , while counterbalancing l2l3 and l4l5 segments over the treatment group and the control group so that both groups consisted of eight l2l3 and eight l4l5 segments . in the treatment group , a laminectomy and a partial facetectomy were applied : first , a laminectomy was made with a mechanical 10-mm oscillating saw at level l2 or l4 by removing the spinous process and part of the lamina ( fig . 1 ) . before performing the partial facetectomy the width of the pars interarticularis was measured with a calliper rule ( accuracy one tenth of a millimetre ) , both at the left and right sides . at half of the width of the pars the facet joint was left intact . from here , on both sides a partial facetectomy to the midline was performed using a mechanical 4-mm oscillating saw ( fig . 2 ) . subsequently , the vertebral bodies were embedded in neutral position in cups with a low melting point ( 48c ) bismuth alloy , with the articulating parts remaining outside the bismuth . prior to embedding screws were drilled into the body of both vertebrae up to a depth of 7 mm to avoid any movement in the bismuth . for both the treatment and the control groups , the transverse processes were partially removed and for the control group , removal of these bony ends was applied to promote solid embedding and did not affect the arch or the articulating parts of the vertebra.fig . the black lines indicate where the laminectomy was performed dorsal view of a segment before testing . the double black arrows show the facet joint using a dead weight connected through a pulley system to the plateau , segments were loaded with a compression force of 1,600 n. this load was selected to allow for comparison with previous work and was a compromise between applying compression forces that are sufficiently large to simulate spine loads that occur in vivo when large shear forces are present [ 2628 ] , but low enough to avoid damage due to compression forces alone . an anterior shear force was applied on the cranial vertebra using a hydraulic materials testing machine ( model 8872 ; instron & ist , canada ) . the caudal vertebra was fixed on a plateau that could move in axial and horizontal direction , without allowing any movement in the shear direction ( fig . the only structures resisting shear displacement of the cranial vertebra were the articulations with the caudal vertebra . force and displacement were recorded and digitized at 100 hz ( instron fast track 2 ) . the test was stopped after hearing a clear crack or after a large force reduction was seen.fig . the segments were loaded with 1,600 n compression force by a dead weight ( a ) along the x - axis , and an anterior shear force ( b ) along the y - axis . the caudal vertebra ( c ) could freely move in x - direction and axially rotate around the x - axis ; the cranial vertebra ( d ) could only move along the y - axis when shear was loaded a schematic picture of the experimental setup . the segments were loaded with 1,600 n compression force by a dead weight ( a ) along the x - axis , and an anterior shear force ( b ) along the y - axis . the caudal vertebra ( c ) could freely move in x - direction and axially rotate around the x - axis ; the cranial vertebra ( d ) could only move along the y - axis when shear was loaded for each of the 16 motion segments tested , the ultimate shear force to failure , i.e. the peak force in the load displacement curve , and the displacement at the instant of ultimate shear force were determined . the average shear stiffness of the segments was calculated from the load displacement curve . the deformation was linear , with an r > 0.997 for each individual test , after an initial phase ( i.e. after a load of 400 n ) and up to the first visible sign of failure ( i.e. up to over 900 n in all specimens ) , and therefore the average stiffness between loads of 400 and 900 n was calculated . a wilcoxon signed ranks test was applied to the ultimate shear force at failure , to the displacement at failure and to the shear stiffness between 400 and 900 n. the test was applied both for the factors treatment group ( treatment and control ) and vertebral level ( l2l3 and l4l5 ) . furthermore , the pars width prior to treatment , averaged over the left and right side , was tested for a difference between l2l3 and l4l5 , again using a wilcoxon signed ranks test . the statistical analyses were performed using spss for windows version 17.0 ( spss inc . , after testing , the mode of failure was established for each specimen by inspection prior to and after dissection . eight porcine lumbar spines of domestic pigs ( mean weight 78 kg , mean age 7 months ) were obtained from the slaughterhouse . each lumbar spine was sectioned into two motion segments for testing : l2l3 and l4l5 . one of the motion segments was assigned to the control group and one to the treatment group , while counterbalancing l2l3 and l4l5 segments over the treatment group and the control group so that both groups consisted of eight l2l3 and eight l4l5 segments . in the treatment group , a laminectomy and a partial facetectomy were applied : first , a laminectomy was made with a mechanical 10-mm oscillating saw at level l2 or l4 by removing the spinous process and part of the lamina ( fig . 1 ) . before performing the partial facetectomy the width of the pars interarticularis was measured with a calliper rule ( accuracy one tenth of a millimetre ) , both at the left and right sides . at half of the width of the pars the facet joint was left intact . from here , on both sides a partial facetectomy to the midline was performed using a mechanical 4-mm oscillating saw ( fig . 2 ) . subsequently , the vertebral bodies were embedded in neutral position in cups with a low melting point ( 48c ) bismuth alloy , with the articulating parts remaining outside the bismuth . prior to embedding screws were drilled into the body of both vertebrae up to a depth of 7 mm to avoid any movement in the bismuth . for both the treatment and the control groups , the transverse processes were partially removed and for the control group , removal of these bony ends was applied to promote solid embedding and did not affect the arch or the articulating parts of the vertebra.fig . the black lines indicate where the laminectomy was performed dorsal view of a segment before testing . using a dead weight connected through a pulley system to the plateau , segments were loaded with a compression force of 1,600 n. this load was selected to allow for comparison with previous work and was a compromise between applying compression forces that are sufficiently large to simulate spine loads that occur in vivo when large shear forces are present [ 2628 ] , but low enough to avoid damage due to compression forces alone . an anterior shear force was applied on the cranial vertebra using a hydraulic materials testing machine ( model 8872 ; instron & ist , canada ) . the caudal vertebra was fixed on a plateau that could move in axial and horizontal direction , without allowing any movement in the shear direction ( fig . the only structures resisting shear displacement of the cranial vertebra were the articulations with the caudal vertebra . force and displacement were recorded and digitized at 100 hz ( instron fast track 2 ) . the test was stopped after hearing a clear crack or after a large force reduction was seen.fig . the segments were loaded with 1,600 n compression force by a dead weight ( a ) along the x - axis , and an anterior shear force ( b ) along the y - axis . the caudal vertebra ( c ) could freely move in x - direction and axially rotate around the x - axis ; the cranial vertebra ( d ) could only move along the y - axis when shear was loaded a schematic picture of the experimental setup . the segments were loaded with 1,600 n compression force by a dead weight ( a ) along the x - axis , and an anterior shear force ( b ) along the y - axis . the caudal vertebra ( c ) could freely move in x - direction and axially rotate around the x - axis ; the cranial vertebra ( d ) could only move along the y - axis when shear was loaded for each of the 16 motion segments tested , the ultimate shear force to failure , i.e. the peak force in the load displacement curve , and the displacement at the instant of ultimate shear force were determined . the average shear stiffness of the segments was calculated from the load displacement curve . the deformation was linear , with an r > 0.997 for each individual test , after an initial phase ( i.e. after a load of 400 n ) and up to the first visible sign of failure ( i.e. up to over 900 n in all specimens ) , and therefore the average stiffness between loads of 400 and 900 n was calculated . a wilcoxon signed ranks test was applied to the ultimate shear force at failure , to the displacement at failure and to the shear stiffness between 400 and 900 n. the test was applied both for the factors treatment group ( treatment and control ) and vertebral level ( l2l3 and l4l5 ) . furthermore , the pars width prior to treatment , averaged over the left and right side , was tested for a difference between l2l3 and l4l5 , again using a wilcoxon signed ranks test . the statistical analyses were performed using spss for windows version 17.0 ( spss inc . , the mode of failure was established for each specimen by inspection prior to and after dissection . no bone abnormalities were found in the tested segments before the shear load was applied . the main site of failure during shear loading ( table 1 ) was the pars interarticularis for the laminectomy / facetectomy group and the arch for the control group . the median ultimate shear force to failure with laminectomy and partial facetectomy ( table 1 ) was 1,645 n ( range 1,0661,985 ) and this was significantly ( p = 0.012 ) smaller than the median ultimate shear force to failure of the control segments ( 2,113 n , range 1,3382,659 ) . the median displacement at the instant of ultimate shear force with laminectomy and partial facetectomy was 9.3 mm ( range 7.511.0 ) and this was not significantly different ( p = 0.069 ) from the control segments ( median 11.7 mm , range 6.113.1).table 1ultimate shear force to failure , stiffness , displacement and failure site for each of the specimens tested in the present studytestl2l3l4l5ultimate shear force to failure ( n)place of occurrence of fracturepars width right ( mm)pars width left ( mm)average stiffness between 400 and 900 n load ( n / mm)displacement ( mm)treatment group1x1,116pars right + left12.312.5170.87.52x1,537pars right11.311.9186.38.23x1,066pars right10.010.1119.29.04x1,476pars right + left12.511.9171.49.25x1,985pars right + left11.411.1208.511.06x1,754pars right + end plate11.912.4210.59.47x1,794pars right + left12.712.5215.89.48x1,753pars left + end plate10.810.8216.79.5median1,645197.49.3range1,0661,985119.2216.77.511.0control group1x1,712arch right10.610.7231.78.32x2,136arch left11.711.5188.113.13x1,338arch left12.713.9222.76.14x1,995arch left9.710.2200.811.05x2,386arch left12.012.7210.212.66x2,517pars left + end plate10.710.9250.212.27x2,659arch left11.411.3232.812.68x2,091arch left12.612.9208.811.1median2,113188.1250.211.7range1,3382,659216.56.113.1 ultimate shear force to failure , stiffness , displacement and failure site for each of the specimens tested in the present study the median shear stiffness between 400 and 900 n was 197.4 n / mm ( range 119.2216.7 ) with laminectomy and partial facetectomy and this was significantly ( p = 0.036 ) smaller than the median stiffness of the control segments ( 216.5 n / mm , 188.1250.2 ) . in percentage the median ultimate shear force to failure with laminectomy and partial facetectomy was 22% smaller than in the control group . the median width of the pars interarticularis ( table 1 ) was significantly ( p = 0.018 ) smaller for the l2l3 segments ( 10.8 mm , range 10.011.4 ) than for the l4l5 segments ( 12.4 mm , range 11.613.3 ) . however , despite this difference in width between the l2l3 and l4l5 segments , those segments did not differ in ultimate shear force to failure ( p = 0.327 ) or in stiffness ( p = 0.575 ) . inspection of load - displacements curves showed , in some cases , sudden small changes in the stiffness of the segments ( fig . 5 ) . we assume that these small changes in stiffness during a test were caused by micro fractures or by a small rotation of one of the segments.fig . 4the tweezers show a pars interarticularis fracture after testing a laminectomy and partial facetectomy segmentfig . the grey arrow shows the ultimate shear force to failure the tweezers show a pars interarticularis fracture after testing a laminectomy and partial facetectomy segment load - displacement curves for anterior shear loading . the present study showed that the ultimate shear force to failure of the lumbar porcine spine after laminectomy and partial facetectomy was significantly smaller than the ultimate shear force to failure of the control segments . the shear stiffness of the segments on which the laminectomy and the partial facetectomy were performed , was also significantly smaller . this confirms our hypothesis and is also in line with model studies [ 1518 ] and in vitro studies [ 10 , 24 ] showing reduced stability of the spine after laminectomy and partial facetectomy . thus , the pars interarticularis contributes substantially to the resistance to shear forces at the lumbar spine and , as such , provides important protection against shear injury . removal of 50% of the pars interarticularis with a laminectomy resulted , in the present study , in a median reduction of the ultimate shear force to failure of 22% . clinically , these results can explain the pars interarticularis fractures and spondylolisthesis that are sometimes seen after decompression of the spinal canal [ 1214 , 19 , 30 ] . our results suggest that decompression surgery reduces the injury threshold in shear loading of the lumbar spine . patients should be informed about their potentially reduced shear injury threshold , with the possibility of causing pars interarticularis fractures and spondylolisthesis . prevention programmes , aimed at reducing shear loading , may be useful in reducing the incidence of post - surgical shear load induced injury . our results underline the importance of preserving as much of the pars interarticularis as possible during surgery , but the percentage reduction of ultimate shear force to failure is not proportional with the percentage bone removed . the percentage reduction of ultimate shear force to failure is only about half of the percentage bone that is removed . partially , this can be explained by the fact that the pars interarticularis is not the only structure resisting shear forces . in fact , the intervertebral disc has been shown to be one of the primary shear - resisting structures when applying forward shear forces [ 19 , 22 , 25 , 31 ] and it has been shown to be responsible for 62.574% of the ultimate shear strength [ 19 , 22 , 31 ] . another explanation for the finding that removal of 50% of the bone of the pars interarticularis did not result in a 50% decrease of ultimate shear force to failure , is that the pars interarticularis is not the weakest link prior to surgery . as can be seen in table 1 , the location of failure in the control specimens was seven times the arch of the vertebra and only one time the pars interarticularis . in the present study , the median ultimate shear force to failure in the control group was 2,113 n , which is comparable with other studies [ 22 , 25 , 31 ] . the shear stiffness reduction in the present study ( 9% ) was even smaller than the reduction in ultimate shear force to failure ( 22% ) . comparably , van dieen et al . found only 32% increased amplitude in shear displacement after complete removal of the posterior elements . those relatively low percentages may be related to the 1,600 n compression load used in the present study . it has been shown that the shear stiffness of spinal motion segments increases with compressive loading [ 32 , 33 ] . indeed , without compressive loading , lu et al . found a reduction of stiffness of 78% after removing the complete posterior elements . the difference between the stiffness reduction ( 9% ) and the ultimate shear force reduction ( 22% ) in the present study might be speculated to be due to an increased bony contribution to the stiffness when approaching the ultimate shear load . it can be noted from table 1 that fractures were not symmetrically distributed over the left and right sides . all of the specimens were exactly levelled in the middle of the bismuth during testing and the anterior shear load was symmetrically applied on the vertebras in the bismuth . we found no explanation for the occurrence of the asymmetrical arch fractures , and we assume that it is a coincidence . a limitation of the present study is that we used porcine spines with a mean age of 7 months and not adult human spines . we chose pigs for their specimen homogeneity related to a comparable age , weight , diet , activity level and genetic background between animals . the main difference is the smaller size , while morphological characteristics are similar to human lumbar spines . multiple studies have shown that mechanical properties like compressive and shear strength in porcine specimens are similar to human lumbar spines [ 22 , 25 , 35 ] . another limitation of the present study is that we used only a single level of compression force . as indicated earlier , compression is known to increase the shear stiffness of the intervertebral disc , so that the percentages found in the present study would probably be higher when the compression force would have been lower . we selected the present load level because it is a physiologically reasonable level of compression with the applied shear loads and this load was selected to allow for comparison with previous work . it should be noted that surgery for spinal stenosis is mainly applied in patients exceeding the age of 65 years . osteoporosis likely reduces both the stiffness and the ultimate shear strength of the pars interarticularis . initially , this results in a loss of ( shear ) stiffness of the disc , but severely degenerated discs may show enhanced stiffness [ 19 , 30 , 31 , 3440 ] . therefore , the overall effect of aging on the ultimate shear strength is hard to predict . another limitation of the present study was that we did not , as common in this kind of surgery , use a kerrison rongeur to remove the bone . the reason was that pilot work suggested that we could more accurately remove 50% with a mechanical saw . nevertheless , the removed bone may not have been exactly 50% . furthermore , that the strength and stiffness of the removed bone was different from the strength and stiffness of the remaining bone can not be excluded . bone density as well as precise quantification of the amount of bone removed might be obtained from 3d ct images , but this was not applied in the present study . in this study we did not investigate the effects of partial facetectomy and laminectomy on shear strength separately . this choice was made because of the destructive nature of the tests in combination with the goal to simulate the surgical procedure used in patients with spinal stenosis . cyclic loading might , through visco - elastic behaviour of the intervertebral disc , shift load to the posterior elements , thereby enhancing the effect of partial facetectomy and laminectomy on shear strength . in conclusion , the present study showed that in porcine segments , laminectomy and partial ( 50% ) facetectomy resulted in 22% reduction in ultimate shear force to failure and 9% reduction in shear stiffness . although relatively small , these effects may explain the high incidence of pars interarticularis fractures and spondylolisthesis in patients after spinal decompression surgery .

degenerative lumbar spinal stenosis is the most common reason for lumbar surgery in patients in the age of 65 years and older . the standard surgical management is decompression of the spinal canal by laminectomy and partial facetectomy . the effect of this procedure on the shear strength of the spine has not yet been investigated in vitro . in the present study we determined the ultimate shear force to failure , the displacement and the shear stiffness after performing a laminectomy and a partial facetectomy . eight lumbar spines of domestic pigs ( 7 months old ) were sectioned to obtain eight l2l3 and eight l4l5 motion segments . all segments were loaded with a compression force of 1,600 n. in half of the 16 motion segments a laminectomy and a 50% partial facetectomy were applied . the median ultimate shear force to failure with laminectomy and partial facetectomy was 1,645 n ( range 1,0661,985 ) which was significantly smaller ( p = 0.012 ) than the ultimate shear force to failure of the control segments ( median 2,113 , range 1,3382,659 ) . the median shear stiffness was 197.4 n / mm ( range 119.2216.7 ) with laminectomy and partial facetectomy which was significantly ( p = 0.036 ) smaller than the stiffness of the control specimens ( median 216.5 , 188.1250.2 ) . it was concluded that laminectomy and partial facetectomy resulted in 22% reduction in ultimate shear force to failure and 9% reduction in shear stiffness . although relatively small , these effects may explain why patients have an increased risk of sustaining shear force related vertebral fractures after spinal decompression surgery .

Introduction Methods Specimens and specimen preparation Biomechanical testing procedure Analysis Results Discussion

spinal injuries in small children are rare with a reported incidence of 0.2 to 0.5% of all fractures or dislocations and 1.5 to 3% of all lesions in the spine.1 it has been shown that the younger the child at the time of injury , the more likely the upper cervical spine is affected , with over 50% of spinal injuries in small children affecting this region . this relates to the child 's greater head - to - body ratio , where more forces are centered around the junction between the large head and the smaller body.2 a subset of cervical spine fractures is the odontoid process fracture . in small children , this fracture typically involves the cartilaginous plate that separates the odontoid process from the body of the axis . the most common mechanism of such an injury is a head - on motor vehicle accident with a toddler restrained in a backseat by a four - point children 's seat harness . previous biomechanical investigations using simulation have shown vehicle speed greater than 40 km / h is sufficient to create the shearing forces required to cause such injuries in children under the age of 3.3 however , despite the increasing numbers of motor vehicle accidents occurring annually , resulting in increasing numbers of pediatric fractures , the optimal management of odontoid process fractures in small children remains undetermined . the current literature reports these fractures can be successfully treated with nonoperative management including cervical spine immobilization as well as operative interventions ranging from closed reduction and external fixation to posterior fixation or fusion of c1/c2.4 we present a case of a conservatively managed type 2 displaced odontoid process fracture in a 2-year - old girl . a 2-year - old girl who was previously healthy re - presented to the emergency department following a motor vehicle accident 6 days earlier . the parents describe a head - on motor vehicle accident at 90 km / h , with the toddler restrained in the backseat by a four - point child seat harness . the fracture was missed on initial presentation to the emergency department , which included a cervical spine x - ray ; however , she remained reluctant to move her head and became distressed when not lying flat . on repeat presentation , she was comfortable at rest but lacked head control . her neurologic examination was unremarkable , but a cervical spine computed tomography ( ct ) revealed a 45-degree flexed type 2 odontoid process fracture . she remained neurologically intact throughout and by 12 weeks had regained head control and a full range of movement without pain . repeat ct scans were performed at 6 weeks , 10 weeks , and 6 months postinjury . these revealed callus formation at 6 weeks , followed by remodeling toward normal alignment by 6 months ( fig . 1 ) . serial sagittal computed tomography images of a conservatively managed odontoid process fracture in a 2-year - old : ( a ) 45 degrees at initial presentation ; ( b ) 45 degrees at 6 weeks postinjury with callus formation ; ( c ) 40 degrees at 10 weeks postinjury ; ( d ) 25 degrees at 6 months postinjury . this case represents the common mechanism of injury for this fracture in the pediatric population . furthermore , it illustrates the difficulties in diagnosis , where patients may present without neck pain but rather a feeling of instability . in the pediatric population , reports of these symptoms are often very difficult to attain from the patient and therefore clinicians should maintain a high index of suspicion in a child who becomes distressed with movement or the requirement of head control and who has a significant mechanism of injury . if plain film x - rays are difficult to discern on initial presentation , further imaging is required . we utilized a ct scan to further delineate the bony injury , and due to the difficulty in interpreting the plain film x - rays in this child , we used limited upper cervical ct scans to monitor fracture healing . however , such scans should be used with caution in children due to the concerns of the ionizing radiation dose delivered.5 6 although limited scans deliver a low dose , they should nonetheless be avoided where possible.5 6 within the adult population , displaced type 2 odontoid process fractures can be treated operatively or nonoperatively , depending on the patient age , comorbidities , fracture pattern , and displacement.7 8 however , the management of such fractures in the pediatric population remains unclear . to our knowledge , there has only been one other case describing the management of a displaced type 2 odontoid process fracture in a small child . in this publication , bhagat and coauthors described operative intervention of a displaced type 2 odontoid process fracture in a 2-year - old girl.9 although they did not describe when union occurred , they noted marked remodeling of the fracture over a 30-month period . with such paucity of literature on this topic , it is unknown whether operative intervention aids fracture union and functional outcome in the small child . however , our case illustrates that odontoid process fractures in small children can be successfully managed without operative intervention despite significant angulation . in this case , developing union , with callus formation , was noted at 6 weeks postinjury , and progressive remodeling was seen to occur by the 6-month follow - up . we utilized a semirigid aspen collar rather than a rigid cervical spine orthosis , such as a halo vest traction , because of the higher rate of complications , such as pin site infections , pin loosening , brain abscesses , skin breakdown , and stiffness , associated with halo traction.10 11 furthermore , the bulk and size of halo traction in small children limits their mobilization and , due to their poor head control and crawling rather than upright gait , risks progressive loss of spinal alignment if the brace is not tightly fitted . the concern , however , with nonrigid immobilization is that of poor compliance , with the brace able to be removed by the patient or parents . in addition , within the adult population , higher union rates are reported with rigid halo vest immobilization compared with nonrigid immobilization , although this is debated and has not been analyzed in the pediatric population.12 13 in conclusion , this case illustrates that displaced type 2 odontoid process fractures in small children can be successfully treated conservatively in a semirigid cervical orthosis .

study design case report . objective odontoid process fractures represent an uncommon injury in small children , with their optimal management remaining unclear . we present a case of conservatively managed displaced type 2 odontoid process fracture in a small child . methods we analyzed clinical and radiographic outcomes of a restrained 2-year - old girl involved in a motor vehicle accident who sustained a displaced type 2 odontoid process fracture and was treated conservatively in a semirigid aspen collar . results the fracture progressively healed with callus formation evident at 6 weeks . by 12 weeks , the patient was asymptomatic and had regained head control and a full range of movement . radiographic remodeling of the fracture was seen to improve over the 6-month follow - up . conclusion this case illustrates that displaced type 2 odontoid process fractures can be successfully managed conservatively in small children .

Introduction Case Report Discussion

chronic graft versus host disease ( cgvhd ) belongs to the most serious and frequent ( 3070% ) complications in patients undergoing hemopoietic stem cells transplantation ( hsct ) for hematological malignancies . musculoskeletal and skin cgvhd , by inducing fibrotic changes in tissues , may result in reduction of joints range of motion ( rom ) , loss of muscular strength and , finally , in functional impairment , compromising the activities of daily living ( adl ) . supportive cares , such as physical rehabilitation and occupational therapy , although recommended as ancillary therapy , have been rarely experienced in such a condition . , recently presented , dealt with a single case of motor rehabilitation in a patient with cgvhd - related contractures . at the best of our knowledge , it represents the first reported case of extensive cgvhd with skin and musculoskeletal involvement treated with physical rehabilitation in an adult patient . thereby , we described an additional case of a patient with similar cgvhd involvement recently treated with physical rehabilitation within our homecare program . a 39-year - old man affected by chronic myeloid leukemia ( cml ) in third relapse after allogeneic hsct and not eligible for further active therapy was referred to our homecare service as advanced / terminal patient in june 2008 . at diagnosis , in 1992 , he had been submitted to hla identical sibling allogeneic hsct , obtaining complete remission ( cr ) ; in 1998 , a disease relapse , as accelerate phase of cml , occurred . therefore , a second hla identical sibling allogeneic hsct , from a different donor , was performed and both hematological and cytogenetic remission were achieved . in 2004 , the patient developed a second hematological relapse ( myeloid blast crisis ) and he was given donor lymphocyte infusions ( dli ) and imatinib ; after three dlis at escalating doses , a molecular cr was obtained . however , 8 months later , he developed extensive cgvhd ( skin , mouth , eye ) and , therefore , immunosuppressive therapy ( steroids and extracorporeal photoferesis ) was started , without improvement , such that cgvhd progressed with the addition of lung involvement . therefore , the patient received multiple lines of immunosuppressive drugs ( rituximab , cyclosporine , plaquenil and mycophenolate ) with only a poor response . meanwhile , in june 2007 , he developed a third relapse ( extramedullary lung and bone involvement ) and was treated with imatinib and dasatinib without response and therefore he was considered not eligible for further causal therapy . at admission in the homecare service , the patient presented with an extensive skin and musculoskeletal cgvhd ; barthel index ( bi ) , as adl measure , was 40 ( moderate severe reduction ; normal = 100 ) , as a result of diffuse contractures with severe reduction of joints rom [ table 1 ] ; secondary , legs muscles hypotrophy was increased due to spinal cord disease - related compression . the patient was assisted with a fully homecare program , with medical and nursing periodic examination , transfusions support and motor rehabilitation . both physical and occupational therapy were promptly started , with particular attention to stretching exercises for joints ; planned intensity was 3 sessions per week . after 4 weeks of treatment , planned intensity was respected and no rehabilitation - related complications were noted . although bi did not improve , the rom of treated joints increased and the mean rom improvement ( expressed as percentage of baseline value ) was 52.5% [ table 1 ] ; both motor skills , psychological aspects and patient quality of life had significant amelioration . after 5 weeks , rehabilitation was discontinued because of infectious pneumonia and , 1 month later , an attempt to restart failed due to rapid deterioration . in november 2008 , range of motion in major joints : normal value , baseline value ( t0 ) , after 1 month of treatment ( t1 ) and improvement expressed as percentage of baseline value ( ) musculoskeletal involvement , in the course of cgvhd , is a rare phenomenon ; larger reported series describe cgvhd - related fasciitis or myositis in less than 1% of the patients undergoing allogeneic sct . musculoskeletal cgvhd , expression of an immunological response toward recipient antigens , is considered an organ involvement in the course of a widespread disease , and systemic immunosuppressive therapy is the standard approach of treatment . however , local treatment should be used in order to improve response and to reduce toxicity , allowing a prompt and rapid escalation of systemic treatment . although data derived from experiences in different immunologically mediated musculoskeletal contractures suggest an important role of physical rehabilitation , such a therapy remains a poorly explored issue in patients with cgvhd , with few data reported in adult patients . although limited , confirm the possibility of almost temporary results , also in long - lasting cgvhd - related contractures . monitoring for musculoskeletal involvement in patients at risk for or with initial features of cgvhd is required , both to prospectively evaluate cgvhd - related muscoloskeletal involvement and to enroll patients at an initial disease stage . clinical trials have to be developed to adequately assess feasibility , safety and efficacy of motor rehabilitation in response to the need to prevent disease progression toward rom limitation and consequent disability .

chronic graft versus host disease ( cgvhd ) is a frequent complication of allogeneic stem cell transplantation . extensive musculoskeletal and skin involvement may induce severe functional impairment , disability and quality of life deterioration . physical rehabilitation is recommended as ancillary therapy in these forms , but experiences are sparse . a 39-year - old man affected by musculoskeletal and skin chronic graft versus host disease ( cgvhd ) was treated with a homecare - based motor rehabilitation program during palliation for disease progression . significant functional improvement was obtained . motor rehabilitation should be strongly considered for patients with musculoskeletal cgvhd , both in the palliative and in the curative phase of disease .

INTRODUCTION CASE REPORT DISCUSSION

complex regional pain syndrome ( crps ) type ii , known as causalgia , develops after a nerve injury , and tends to show the more painful and more severe symptoms of crps . the ordinary strategy in crps treatment is often multi - disciplinary , with the use of various kinds of medications and physical therapies combined with sympathetic nerve block . even with several treatments , it is very difficult to manage the acute stage of crps type ii . and neurostimulation ( spinal cord stimulator ) could be surgically implanted to control the pain by directly stimulating the spinal cord for the treatment of intractable crps . a systematic review concluded that spinal cord stimulation appears to be an effective therapy in the management of patients with crps type i ( level a evidence ) than type ii ( level d evidence)14 ) . moreover , there is evidence to reveal that scs is a cost - effective treatment for crps type i. we report a case of acute stage crps type ii treated with new anti - inflammatory agent [ polydeoxyribonucleotide ( pdrn ) solution ] . as far as we know , this is 1st case report showing effectiveness of pdrn for the treatment of crps type ii . a 32-year - old female patient came to the hospital complaining of left leg numbness . she had experienced a traumatic event , a fall into a waterway , one month ago . after which , she had severe low back pain and was evacuated to the nearby general hospital . computed tomography ( ct ) showed an l5 left transverse process fracture and an s2 body fracture , and absolute bed rest ( abr ) for one month was recommended ( fig . her left leg numbness was progressively aggravated . to evaluate whether symptom aggravation during the abr period was related to the patient 's history of a left - side l5s1 discectomy 2 years previously the mri did not show recurrent disc material , but it did show an annular tear at l45 and postoperative scar tissue at l5s1 , and the previous doctor recommended continued conservative management . after the period of abr was complete , the patient began ambulation and was transferred to our hospital in her hometown . she complained of left leg numbness initially upon arrival , and the more she walked , the more the pain was aggravated , showing hyperalgesia and allodynia . even as the symptoms seemed to subside somewhat , skin flushing still appeared on the left lower leg when the patient was in a standing position ( fig . 2 ) . the persistent skin discoloration seemed to worsen as time went on , despite frequent sympathetic nerve blocks using steroid . to rule out the possibility of arterial injury , we performed lower extremity ct angiography , and there was no sign of arterial injury around the fracture site . we concluded that her symptoms resulted from lumbosacral plexus injury due to the transverse process fracture . at that time , it was 2 months after the initial trauma . because this was the acute phase , we wanted to prevent the disease from progressing to the chronic phase . our clinics have been performing prolotherapy using pdrn solution , and we are familiar with the effects of pdrn . we injected pdrn solution at the ventral surface of the left l5 transverse process , superior and inferior , one ampoule ( 3 cc ) each by using needles used for medial branch block ( fig . allodynia and hyperalgesia were improved one day after the procedure and the skin flushing was diminished ( fig . the patient was discharged 3 days after the pdrn injection and at the 1-month follow - up , her symptoms were very much improved . autonomic disturbance can lead to significant pain in patients with peripheral nerve injuries and is characteristic of crps . severe burning pain , allodynia , hyperalgesia , careful attempts to protect the involved extremity from movement or contact , and evidence of autonomic hyper- or hypoactivity are features of crps . vascular changes such as skin flushing and discoloration and temperature fluctuations are hallmarks of this deranged autonomic activity . crps type i usually occurs after a minor injury to the extremity ( e.g. , ankle sprain ) , whereas crps type ii occurs after significant injury to a major mixed nerve ( e.g. , brachial plexus injury ) , such as in our case . crps may change within a patient over time , particularly in the transition from " warm crps " ( acute ) to " cold crps " ( chronic)5 ) . several pathophysiological concepts have been proposed to explain the complex symptoms of crps : 1 ) facilitated neurogenic inflammation ; 2 ) pathological sympatho - afferent coupling ; and 3 ) neuroplastic changes within the cns11 ) . management of crps remains challenging , with many patients having only partial or minimal relief with treatment . pharmacotherapy is based on individual symptoms and includes steroids , free radical scavengers , antineuralgic agents , and finally agents interfering with bone metabolism ( calcitonin , bisphosphonates ) . nonsteroidal anti - inflammatory drugs ( nsaids ) , corticosteroids , cyclooxygenase ( cox)-2 inhibitors , and free radical scavengers are used in crps with intent to treat for pain as well as for inflammation . however , crps - related inflammation may be largely neurogenic , initiated by inflammatory mediators from the terminals of afferent nociceptors , and no drugs have been studied for this type of inflammation8 ) . sympathetic nerve block is traditionally recognized as an important procedure , both in the diagnosis and treatment of crps . the pain relief following sympathetic nerve block generally outlasts the effect of the local anesthetic13 ) . if after numerous therapies , a patient 's pain is intractable , sympathectomy or spinal cord stimulation ( scs ) may be recommended as surgical treatment alternatives . recently , scs seems to be the general trend , and it is reported that scs is effective in reducing the chronic neuropathic pain of crps type i914 ) . specific treatment of crps type ii at the chronic stage is also reported , but to our knowledge , no specific interventional or invasive treatment for control of acute stage crps type ii has been reported6 ) . pdrn ( placentex ) is a low molecular weight dna complex extracted from trout sperm . the adenosine a2a receptor alters the cytokine system by decreasing secretion of inflammatory cytokines such as tumor necrosis factor- , macrophage inflammatory protein 1 , and interleukin ( il)-6 . the adenosine a2a receptor is also associated with increased circulation of il-10 , anti - inflammatory cytokine4 ) . therefore , pdrn has an anti - inflammatory effect and could be a potential pharmacological treatment for inflammatory diseases such as rheumatoid arthritis315 ) . this also explains how pdrn can help to control the acute phase of crps , such as in our case . however , this does n't seem to be enough to explain the dramatic result of our case . the adenosine a2a receptor also has a tissue regeneration effect , as indicated by its stimulation of endothelial cell migration and proliferation . activation of this receptor is also associated with increased levels of vascular endothelial growth factor , which enhances tissue function712 ) . promoting cell proliferation by acting as a mitogen for fibroblasts and , endothelial cells could result in extracellular matrix production and remodeling , and can be useful for burn treatment and prolotherapy210 ) . we suppose that this regeneration mechanism has an additional effect on our case . because we have been performing prolotherapy in our clinics , we have observed these mechanisms of pdrn , not only the regeneration effect , but also the anti - inflammatory effect . pdrn has been found to be a safe modality , and no harmful effect has been reported1 ) . patients with acute stage crps type ii need appropriate treatment to prevent progression to the chronic phase . treatment is based on a multidisciplinary approach , and pdrn could be an option for the treatment of the acute inflammatory phase of crps type ii .

complex regional pain syndrome ( crps ) type ii is a syndrome that develops after nerve injury . symptoms may be severe , and vary depending on the degree of sympathetic nerve involvement . as yet , there is no satisfactory treatment . we report the case of a female patient who had an l5 left transverse process fracture and an s2 body fracture , who developed symptoms of crps type ii in her left lower leg that were aggravated during ambulation in spite of absolute bed rest for one month after the trauma . several treatments , including bed rest , medication , and numerous nerve blocks were attempted , but the pain persisted . we finally tried injection of polydeoxyribonucleotide ( pdrn ) solution at the left l5 transverse process fracture site because we knew of the anti - inflammatory effect of pdrn . one day after this treatment , her symptoms had almost disappeared and three days later , she was discharged . we will also further discuss the possibility of using pdrn solution for the treatment of crps .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

the prevalence of childhood overweight and obesity gradually increases , even in preschool - age children . globally , more than 20 million children who are below the age of 5 are overweight . the finding that overweight in preschool children is an indicator of five times more overweight in adolescence and four times more overweight in adulthood is compared with normal - weight counterparts . since chronic conditions such as diabetes , cardiovascular disorders , hypertension , stroke , asthma , and certain neoplastic disorders may occur in late adulthood , prevention and early interventions for overweight or obesity are a real concern . although the body mass index ( bmi ) is used as a marker of obesity , it may be a less sensitive indicator in early childhood . a few studies on preschool children showed a weak correlation between bmi and metabolic risk factors [ 19 , 36 ] . thus , it may be considered that bmi is an unreliable marker in this age group since central adiposity which is associated with metabolic risk factors is not adequately reflected by bmi . therefore , early identification of children s central adiposity is important [ 40 , 41 ] . the waist circumference ( wc ) , one of the anthropometric techniques , is strongly correlated with metabolic risk factors and now becomes widely used . in addition , wc is an essential diagnostic criterion for metabolic syndrome according to the national cholesterol education program ( ncep ) adult treatment panel iii ( atp iii ) and the international diabetes federation ( idf ) [ 40 , 41 ] . there are recent reports indicating that the onset of risk factors for metabolic syndrome under the age of 10 years can be considered . the ncep and idf criteria for metabolic syndrome may be modified to explain metabolic risks under the age of 10 years [ 14 , 30 , 45 ] . thus , wc cutoffs and reference values in preschool children can be useful for identifying children with metabolic risk and might be helpful for early intervention . the preschool age is a difficult group for achievement and anthropometric measure while school - age children are easily accessible . therefore , although many countries have their own reference values for wc [ 1 , 1721 , 27 , 35 , 39 ] , a few studies cover preschool - age children [ 11 , 12 , 21 , 37 ] . the aim of this study was to establish reference values of waist circumference in turkish preschool children . furthermore , 50th and 90th percentiles of wc were compared with the ones in a few other countries [ 11 , 37 ] . we analyzed the data of anthropometry of the turkish children aged 06 years ( atca-06 ) . the atca-06 study was conducted from september 2009 to may 2010 in one of the five great cities of turkey with about 1,200,000 residents . the sampling design of the study was a two - stage probability sampling for preschool children living in kayseri . the primary sampling unit was the family health centers ( aile sagligi merkezi , asm ) located in the city center and suburbs . at the first stage , children were selected from 21 asms in kayseri by stratifying according to the socio - economic levels of their parents . children aged 06 years old were randomly selected among the list of district midwives and they were invited to asms with their parents . those infants and children , whose parents did not accepted , were invited again to participate in the study by midwives . a total of 2,947 children ( 1,471 boys and 1,476 girls ) whose parents gave consent were included in the study . after data collection , we removed 230 ( 119 boys and 110 girls , 7.9 % of sample size ) subjects from the study because of reasons such as missing data in the interviewing forms , children with growth disorders , or using any kind of medication which could interfere with growth . then , to obtain normally distributed data , the higher and lower limits ( 3rd97th percentiles ) for each gender and quarter age were removed ( n = 115 ) . chronological age was calculated by subtracting the date of birth from the date of observation . each quarter year elapsed from their birthday was noted . the who group recommended that early growth patterns should be documented in intervals shorter than 3 months [ 10 , 13 ] . so , we calculated by age and gender in 06-year - old children in quarter - year intervals except for the 028-day newborn period . parents written consent was obtained prior to the study and the procedures were in accordance with those outlined by the declaration of helsinki . waist circumference was measured with a non - stretchable tape at the midpoint of the lowest rib cage and the iliac crest , to the nearest 0.1 cm , at the end of a gentle expiration . construction of the centile curves was performed with the lms chart maker pro version 2.3 software program ( the institute of child health , london ) , which fits smooth centile curves to reference data . this method summarizes percentiles at each age based on the power of age - specific box the final curves of percentiles are produced by three smooth curves representing l ( lambda , skewness ) , m ( mu , median ) , and s ( sigma , coefficient of variation ) ( lms ) . descriptive statistics for each quarter year ( e.g. , 38 m , etc . ) within sex were calculated by spss version 15.0 ( chicago , il , usa ) . we analyzed the data of anthropometry of the turkish children aged 06 years ( atca-06 ) . the atca-06 study was conducted from september 2009 to may 2010 in one of the five great cities of turkey with about 1,200,000 residents . the sampling design of the study was a two - stage probability sampling for preschool children living in kayseri . the primary sampling unit was the family health centers ( aile sagligi merkezi , asm ) located in the city center and suburbs . at the first stage , children were selected from 21 asms in kayseri by stratifying according to the socio - economic levels of their parents . children aged 06 years old were randomly selected among the list of district midwives and they were invited to asms with their parents . those infants and children , whose parents did not accepted , were invited again to participate in the study by midwives . a total of 2,947 children ( 1,471 boys and 1,476 girls ) whose parents gave consent were included in the study . after data collection , we removed 230 ( 119 boys and 110 girls , 7.9 % of sample size ) subjects from the study because of reasons such as missing data in the interviewing forms , children with growth disorders , or using any kind of medication which could interfere with growth . then , to obtain normally distributed data , the higher and lower limits ( 3rd97th percentiles ) for each gender and quarter age were removed ( n = 115 ) . chronological age was calculated by subtracting the date of birth from the date of observation . each quarter year elapsed from their birthday was noted . the who group recommended that early growth patterns should be documented in intervals shorter than 3 months [ 10 , 13 ] . so , we calculated by age and gender in 06-year - old children in quarter - year intervals except for the 028-day newborn period . parents written consent was obtained prior to the study and the procedures were in accordance with those outlined by the declaration of helsinki . waist circumference was measured with a non - stretchable tape at the midpoint of the lowest rib cage and the iliac crest , to the nearest 0.1 cm , at the end of a gentle expiration . construction of the centile curves was performed with the lms chart maker pro version 2.3 software program ( the institute of child health , london ) , which fits smooth centile curves to reference data . this method summarizes percentiles at each age based on the power of age - specific box the final curves of percentiles are produced by three smooth curves representing l ( lambda , skewness ) , m ( mu , median ) , and s ( sigma , coefficient of variation ) ( lms ) . descriptive statistics for each quarter year ( e.g. , 38 m , etc . ) within sex were calculated by spss version 15.0 ( chicago , il , usa ) . there were 1,471 ( 50 % ) boy and 1,476 ( 50 % ) girl infants . the descriptive statistics for waist circumference for boys and girls by age are given in table 1 . in tables 2 and 3 , the selected waist circumference percentiles including 3rd , 10th 25th , 50th , 75th , 90th , and 97th percentiles are shown for each age and gender . the lms parameters for waist circumference are shown in table 4 . the mean and 1 , 2 , 3 standard deviations added and subtracted wc for each age group and gender are shown in tables 5 and 6.table 1descriptive statistics for waist circumference for boys and girls by ageageboysgirlsnmedian ( min max)nmedian ( min max)028 days10831.00 ( 28.0034.00)10831.00 ( 26.0040.00)28 days3 months4938.00 ( 30.5044.80)4437.50 ( 31.5045.00)3<6 m7041.00 ( 33.0048.00)6340.00 ( 34.5049.50)6<9 m7842.00 ( 35.0553.05)6441.13 ( 32.0049.00)9<12 m7144.00 ( 35.1052.00)7542.50 ( 34.6051.25)12<15 m5843.63 ( 39.5051.00)5643.50 ( 38.5050.00)15<18 m5744.00 ( 40.3050.50)6144.00 ( 39.0050.00)18<21 m5444.00 ( 39.5051.00)7343.80 ( 39.0050.00)21<24 m5745.00 ( 39.5051.00)6645.00 ( 38.1050.00)24<27 m3646.40 ( 41.0052.00)5246.00 ( 41.0053.00)27<30 m5648.00 ( 41.5054.00)4847.00 ( 41.0052.00)30<33 m5748.00 ( 41.0054.00)4846.70 ( 41.0051.00)33<36 m5648.00 ( 41.0054.00)4449.00 ( 43.0053.30)36<39 m5048.75 ( 45.0055.50)5149.00 ( 43.0057.00)39<42 m3750.00 ( 45.0056.75)5348.50 ( 43.0057.00)4245 m4150.00 ( 45.0055.20)4048.00 ( 42.5058.00)45<48 m5149.50 ( 45.5056.40)4450.75 ( 42.5058.00)48<51 m3850.90 ( 45.0057.00)4450.00 ( 45.0058.00)51<54 m4151.00 ( 45.0062.00)4551.00 ( 45.0061.30)54<57 m4651.70 ( 46.0060.60)6052.00 ( 45.0059.00)57<60 m4452.00 ( 48.0061.00)3851.50 ( 45.8061.50)60<63 m5452.75 ( 46.0062.00)2250.65 ( 45.0061.40)63<66 m4752.00 ( 46.0063.00)4652.75 ( 46.0061.00)66<69 m4652.00 ( 47.0068.00)5752.00 ( 45.062.00)69<72 m4752.00 ( 46.0066.00)5552.00 ( 45.0061.00)72<75 m4254.00 ( 48.0064.00)4151.60 ( 47.0064.60)75<78 m3051.75 ( 47.0060.00)3551.50 ( 47.0062.00)78<81 m3053.50 ( 47.0066.00)2453.00 ( 48.0066.00)81<84 m2055.00 ( 47.1066.00)1953.00 ( 47.0066.00)table 2smoothed age - specific waist circumference percentile values for boysage3p5p10p25p50p75p90p95p97p028 days26.627.328.229.731.232.533.634.334.728 days3 months32.232.833.835.437.239.040.541.442.03<634.935.536.538.240.042.043.744.845.56<936.537.138.139.841.743.745.546.747.49<1237.738.339.240.942.844.946.848.048.712<1538.639.240.241.843.745.847.748.949.715<1839.440.040.942.644.546.648.549.850.618<2140.140.741.643.345.247.349.350.551.421<2440.841.442.343.945.948.050.051.352.224<2741.442.042.944.646.548.650.752.052.927<3042.042.643.545.247.149.351.452.753.630<3342.643.244.145.747.749.952.053.454.433<3643.143.744.646.248.250.452.654.155.136<3943.644.245.146.748.751.053.254.755.739<4244.144.645.647.249.251.553.855.356.4424544.545.146.047.649.752.054.455.957.045<4844.945.546.448.050.152.554.956.557.648<5145.345.846.848.450.552.955.457.158.251<5445.646.247.148.850.953.355.957.658.854<5746.046.547.549.151.353.756.358.159.357<6046.346.947.849.551.654.156.858.659.960<6346.647.248.149.851.954.557.259.160.463<6646.947.448.450.152.354.857.659.560.966<6947.247.748.650.452.555.258.060.061.469<7247.448.048.950.652.855.558.460.461.9727547.748.249.250.953.155.858.760.862.3757847.948.549.451.153.456.159.161.262.8788148.148.749.651.453.656.459.461.663.2818448.448.949.951.653.956.659.762.063.7table 3smoothed age - specific waist circumference percentile values for girlsage3p5p10p25p50p75p90p95p97p028 days27.027.628.429.831.433.234.936.036.728 days3 months32.032.633.535.036.838.740.641.742.53<634.535.136.037.639.541.543.344.545.26<936.136.737.639.341.143.145.046.146.99<1237.337.938.840.442.344.346.247.348.112<1538.238.839.741.443.345.347.248.349.115<1839.039.640.542.244.146.148.049.250.018<2139.740.341.242.944.846.948.850.050.821<2440.340.941.943.545.547.649.650.851.624<2740.941.542.544.246.148.350.351.552.427<3041.542.143.144.746.748.951.052.353.230<3342.042.643.645.347.349.551.653.053.933<3642.543.144.145.847.850.152.253.654.636<3942.943.544.546.248.350.652.854.355.239<4243.343.944.946.748.851.153.454.955.9424543.744.345.347.149.251.653.955.556.545<4844.144.745.747.449.652.054.456.057.148<5144.445.046.047.850.052.454.956.557.651<5444.745.346.348.150.352.855.457.058.254<5745.045.646.648.450.753.255.857.558.757<6045.345.946.948.751.053.656.258.059.260<6345.546.247.249.051.353.956.658.459.763<6645.846.447.449.351.654.257.058.860.166<6946.046.747.749.551.954.557.359.260.669<7246.346.947.949.852.154.857.759.661.0727546.547.148.150.052.455.158.060.061.4757846.747.348.350.252.655.458.460.461.8788146.947.548.650.452.855.758.760.862.2818447.147.748.850.653.155.959.061.162.6table 4the power of a box cox transformation ( l ) , the median ( m ) , and the coefficient of variation ( s ) for waist circumference for both gendersageboysgirlslmslms028 days3.03031.1850.0670.19231.4490.08128 days3 months1.36037.2060.0700.14736.8130.0753<60.61140.0420.0700.09839.4880.0726<90.20141.6910.0700.07841.1340.0699<120.06142.8350.0680.09942.3200.06812<150.25843.7310.0670.14343.2660.06715<180.42444.5030.0660.20744.0790.06618<210.57845.2100.0660.28844.8140.06621<240.72945.8790.0650.38345.4980.06624<270.88146.5170.0650.48946.1410.06627<301.03447.1230.0650.60046.7420.06630<331.18847.6950.0640.71547.3060.06633<361.34248.2360.0640.83347.8330.06636<391.49348.7470.0640.94948.3240.06739<421.64249.2290.0651.06548.7830.06742451.78749.6840.0651.17849.2120.06845<481.92750.1140.0651.28949.6140.06848<512.06250.5190.0651.39649.9910.06951<542.19250.9030.0661.50050.3460.06954<572.31851.2660.0661.60050.6800.07057<602.43851.6110.0661.69750.9960.07060<632.55351.9380.0671.79051.2950.07063<662.66452.2500.0671.88051.5800.07166<692.77052.5480.0671.96751.8520.07169<722.87152.8330.0672.05152.1120.07272752.96853.1070.0682.13152.3620.07275783.06253.3710.0682.20952.6020.07278813.15253.6250.0682.28452.8340.07381843.23953.8700.0692.35653.0570.073table 5the mean and 1 , 2 , and 3 standard deviations added and subtracted wc for boysage3 sd2 sd1 sdmean+1 sd+2 sd+3 sd028 days36.627.129.030.932.834.736.628 days3 months47.531.534.737.941.144.347.53<6 m49.834.837.840.843.846.849.86<9 m51.435.438.641.845.048.251.49<12 m53.736.740.143.546.950.353.712<15 m52.638.141.043.946.849.752.615<18 m52.539.041.744.447.149.852.518<21 m53.038.541.444.347.250.153.021<24 m52.439.442.044.647.249.852.424<27 m55.440.943.846.749.652.555.427<30 m55.542.545.147.750.352.955.530<33 m55.941.944.747.550.353.155.933<36 m56.642.145.047.950.853.756.636<39 m56.943.946.549.151.754.356.939<42 m59.943.446.750.053.356.659.94245 m58.044.046.849.652.455.258.045<48 m58.045.047.650.252.855.458.048<51 m61.144.647.951.254.557.861.151<54 m62.144.147.751.354.958.562.154<57 m64.144.648.552.456.360.264.157<60 m61.646.649.652.655.658.661.660<63 m64.445.449.253.056.860.664.463<66 m64.145.148.952.756.560.364.166<69 m70.843.348.854.359.865.370.869<72 m64.844.348.452.556.660.764.872<75 m65.446.450.254.057.861.665.475<78 m63.645.148.852.556.259.963.678<81 m68.745.750.354.959.564.168.781<84 m69.645.650.455.260.064.869.6table 6the mean and 1 , 2 , and 3 standard deviations added and subtracted wc for girlsage3 sd2 sd1 sdmean+1 sd+2 sd+3 sd028 days23.526.128.731.333.936.544.328 days3 months28.331.434.537.640.743.853.13<6 m31.534.437.340.243.146.054.76<9 m31.935.038.141.244.347.456.79<12 m33.436.539.642.745.848.958.212<15 m34.937.840.743.646.549.458.115<18 m35.838.641.444.247.049.858.218<21 m36.539.041.544.046.549.056.521<24 m36.839.542.244.947.650.358.424<27 m37.440.443.446.449.452.461.427<30 m37.740.743.746.749.752.761.730<33 m39.141.644.146.649.151.659.133<36 m37.741.344.948.552.155.766.536<39 m40.743.546.349.151.954.763.139<42 m39.042.445.849.252.656.066.24245 m38.541.945.348.752.155.565.745<48 m38.842.746.650.554.458.370.048<51 m41.244.347.450.553.656.766.051<54 m39.943.747.551.355.158.970.354<57 m40.544.247.951.655.359.070.157<60 m39.043.447.852.256.661.074.260<63 m40.344.047.751.455.158.869.963<66 m42.045.649.252.856.460.070.866<69 m38.843.247.652.056.460.874.069<72 m39.543.747.952.156.360.573.172<75 m38.543.147.752.356.961.575.375<78 m42.345.749.152.555.959.369.578<81 m41.145.549.954.358.763.176.381<84 m39.744.248.753.257.762.275.7 descriptive statistics for waist circumference for boys and girls by age smoothed age - specific waist circumference percentile values for boys smoothed age - specific waist circumference percentile values for girls the power of a box cox transformation ( l ) , the median ( m ) , and the coefficient of variation ( s ) for waist circumference for both genders the mean and 1 , 2 , and 3 standard deviations added and subtracted wc for boys the mean and 1 , 2 , and 3 standard deviations added and subtracted wc for girls in comparison to 10th , 50th , and 90th percentiles between genders , boys wc were longer than girls after the infancy period ( fig . 1the comparison of 10th , 50th , and 90th percentile between genders the comparison of 10th , 50th , and 90th percentile between genders we also compared the frequency of wc higher than 90th percentile between each gender to reveal the fluctuations in abdominal obesity prevalence ( fig . 2 ) . the 10th , 50th , and 90th percentiles of our study were compared with other studies , the methodologies of which are similar to ours ( figs . 3 and 4).fig . 3the comparison of 10th , 50th , and 90th percentiles of our wc ( turkey ) with the preschool children of german ( ger ) and european american ( eu am ) for boysfig . 4the comparison of 10th , 50th , and 90th percentiles of our wc ( turkey ) with the preschool children of german ( ger ) and european american ( eu am ) for girls the frequency of wc higher than 90th percentile between each gender the comparison of 10th , 50th , and 90th percentiles of our wc ( turkey ) with the preschool children of german ( ger ) and european american ( eu am ) for boys the comparison of 10th , 50th , and 90th percentiles of our wc ( turkey ) with the preschool children of german ( ger ) and european american ( eu am ) for girls this is the first cross - sectional study for age- and gender - specific references of 0- to 6-year - old turkish children . in addition to finding a slightly low wc in girls compared with boys , in comparison with other studies in similar age groups , the wc of boys and girls were lower . the age- and gender - specific prevalence of abdominal obesity was also calculated . because of difficulty in sampling in infancy and preschool children and the high number of studies about obesity prevalence in school children and adolescence , we concentrated on infants and preschool children [ 22 , 42 ] . early - onset obesity ( < 6 years ) not only increases the risk of metabolic and cardiovascular disorders in adulthood but also indicates that the risk of adult obesity would be increased by at least 2550 % [ 14 , 25 , 31 ] . then , this study would at least provide the opportunity of determining the validity of the preceding statement by comparing with future similar cross - sectional studies in our population . there is a moderate correlation between obesity in early childhood and in adulthood . in a report by nader et al . , the authors stated that preschool children whose bmis were higher than 50p would have six - times - higher risk of overweight in later childhood . in addition to these findings , obesity at the age of 5 was proposed as the predictor of metabolic status at 9 years old . then , early interventions to treat obesity would not only have a short time but would also have a long period of consecutions . wc references which were determined in this study would be useful to determine metabolic risks in early childhood since a similar study was not done in our population . it is known that there is a decrease in bmi from infancy to 6 years old , the so - called adiposity rebounds , but because of individual variations during this period any two children who have the same bmi may be at different levels of adiposity . since the onset of adiposity rebound varies individually in this period , children whose adiposity onset is thus , in preschool children ( < 6 years old ) , we may conclude that the use of bmi to predict adiposity is not reliable . additionally , in this period , non - uniform segmental growth leads the need to use wc but not bmi since bmi reflects total body fat content but not body fat distribution [ 3 , 4 , 32 ] . bmi may define large central fat deposits as normal , while it has poor sensitivity to define central obesity . since wc in children has a good correlation with insulin resistance , it is considered as a useful tool to predict the risk of developing metabolic and cardiovascular complications . the correlation between wc and central adiposity is also confirmed by dual - energy x - ray absorptiometry . the rationale of this study depends on this hypothesis that segmental fat distribution is best reflected by wc compared with bmi . the primary contribution of this study is to provide the first wc references in turkish preschool children to our knowledge . our findings indicate that the increase in wc through early childhood is faster than 1.5 to 6 years . according to our findings , predominance in central adiposity for boys compared with girls may be an indicator of sexual dimorphism in early childhood in accordance with previous studies [ 15 , 38 ] . although there are different cutoffs to define central obesity , we used wc > 90th percentile since it was proposed as the prerequisite in the definition of metabolic syndrome . in addition to smoothed references ( 3rd , 10th 25th , 50th , 75th , 90th , and 97th percentiles ) , we presented all descriptive characteristics of our data ( mean , median , standard deviations ) to let other researchers make comments on our results . the cited methods to define cutoff for wc can be stated as : z score 1.3 by fredrick , z score 1.5 by taylor , at > 75th by moreno , and age- and sex - specific 90th by katzmarzyk [ 12 , 18 , 26 , 39 ] . increased wc > 90th percentile is proposed by ncep - atp iii and idf , but cutoff for wc to define metabolic syndrome is lacking before 10 years old [ 9 , 40 , 41 ] . however , in recent studies , to diagnose metabolic risk , it is suggested that modified idf and ncep criteria can be used in the pre - pubertal period ( 610 years ) [ 6 , 14 , 45 ] . in these modified criteria , wc > 90th percentile was accepted as cutoff value to define metabolic risk at older than 10 years . although cutoff for abdominal obesity is not determined under 6 years , wc > 90th percentile may be speculated at this level of knowledge [ 6 , 14 ] . according to this criterion , we found total abdominal obesity prevalence as 10.4 % ( boys 10.1 % , girls 10.7 % ) , where age - adjusted prevalence values were also given . we also compared the frequency of wc higher than 90th percentile between each gender and found significant fluctuations until the age of 2 years . after 2 years , wc showed a small decline until 3 years and it was followed by a gradual increase until 6 years in both genders , where abdominal obesity was higher in girls during this period ( fig . 2 ) . the observation that the increase in abdominal obesity was shifted towards the early years can be interpreted as early rebound . although bmi and wc do not completely reflect the same metabolic parameters , early onset of abdominal obesity may require the onset of rebound obesity for bmi . since wc reflects primarily the body fat distribution but not total body fat , inconsistent obesity rebounds in wc and bmi must be evaluated separately . there are few studies about wc percentiles for children under 6 years old . in a study conducted in sweden on children less than 5 year old , in another study conducted in 316-year - old indian children , there were calculated percentiles which can be compared with our references . we selected two previous studies providing wc references with a similar method in the same age of caucasians . the other one was a study conducted in 218-year - old european american children in usa [ 11 , 37 ] . in comparing our data with these two studies , we found that our wc references were significantly lower than those ( figs . 3 and 4 ) . these differences may be explained by dissimilarities in ethnic , geographical , and nutritional behaviors as well as lifestyle . even in the pre - pubertal period , these dissimilarities may be observed . we consider that behaviors related to sedentary activities such as watching television and/or playing with electronic games are established in the preschool period . our data can then be used to determine the risk of central obesity in early childhood . we can conclude that wc together with bmi is a useful clinical tool to detect preschool - age children who may be at a higher risk for cardiovascular diseases .

waist circumference , a proxy measure of abdominal obesity , is associated with cardio - metabolic risk factors in childhood and adolescence . although there are numerous studies about waist circumference percentiles in children , only a few studies cover preschool children . the aim of this study was to develop age- and gender - specific waist circumference smoothed reference curves in turkish preschool children to determine abdominal obesity prevalence and to compare them with reference curves obtained from different countries . the design of the study was cross - sectional . a total of 2,947 children ( 1,471 boys and 1,476 girls ) aged 06 years were included in the study . the subjects were divided according to their gender . waist circumference was measured by using a standardized procedure . the age- and gender - specific waist circumference reference curves were constructed and smoothed with lms method . the reference values of waist circumference , including 3rd , 10th 25th , 50th , 75th , 90th , and 97th percentiles , and standard deviations were given for preschool children . waist circumference values increased with age , and there were differences between genders . the prevalence of abdominal obesity was calculated as 10.1 % for boys and 10.7 % for girls . having compared our data with two other countries data , we found that our waist circumference data were significantly lower . this is the first cross - sectional study for age- and gender - specific references of 0- to 6-year - old turkish children . the gender- and age - specific waist circumference percentiles can be used to determine the risk of central obesity .

Introduction Materials and methods Subjects Measurements Statistical analysis Results Discussion

these cells normally line body cavities , specifically pleura , peritoneum , pericardium , and testis . they can be localized or diffuse . initially it occurred in miners and millworkers who were directly exposed to asbestos in their work environment . subsequently , mesothelioma was diagnosed in plumbers , carpenters , defence personnel , and insulation installers . furthermore people living in industrial areas were inadvertently exposed to asbestos fibres released in the atmosphere . elsewhere , for example , in countries where asbestos is still unregulated , the prospects are even worse . there is widespread interest in this disease by doctors and the general public , because millions of people have been exposed to asbestos fibres . in addition to its substantial personal and health care costs , malignant mesothelioma is associated with considerable compensation costs . the predicted total economic burden of malignant mesothelioma related to compensation for asbestos exposure in the next 40 years is up to $ 200 billion for the united states and $ 80 billion for europe . malignant pleural mesothelioma ( mpm ) has caused and is still causing violent debate between protagonists and antagonists of the three options in relation to treatment . surgery , radiotherapy , and chemotherapy are endlessly discussed , often with limited scientific evidence to support strongly held viewpoints . diagnosis and palliation will be part of the surgeon 's role but everyone 's big hope and question is is there a chance of surgical cure ? until recently it was thought that local radiotherapy directed to surgical sites prevents spreading of tumour and radiotherapy can provide relief of chest - wall pain . however , an up - to - date systematic review paper by price concluded that there is currently no evidence to support the routine role of radiotherapy in patients with mesothelioma . surgery became part of management options in the 1970s and large series have been published in relevant journals [ 14 , 15 ] . the two surgical procedures commonly used are pleurectomy with decortication ( p / d ) and extrapleural pneumonectomy ( epp ) . p / d is a more limited procedure and requires less cardiorespiratory reserve ; it has a morbidity of 25% and a mortality of 2% . data from the uk thoracic surgical register of the society for cardiothoracic surgery in great britain and ireland showed that a limited number of patients underwent surgery for mesothelioma in the past decade . single centre retrospective studies reported favourable results with epp . in some hospitals , this procedure , within a multimodal treatment strategy , became the standard of care . the median survival after epp within mars is consistent with 10 , 12 , 13 , and 14 months in larger observational studies . a best evidence topic paper addressing the question of whether p / d is superior to palliative care in the treatment of patients with malignant pleural mesothelioma came to the following conclusion : p / d is a morbid operation that is associated with significant peri - operative mortality and complication rates . the antagonists of surgery argue that , when a patient commences trimodality ( chemotherapy , radiotherapy , and surgery ) treatment , it will take six to nine months to complete it . this is a time during which the patient is in and out of hospital and sometimes travelling long distances from home . the primary objective of the current review is to determine if surgery improves all - cause mortality and/or quality of life in patients with mpm . we will discuss the different surgical options ( epp versus p / d ) and their relevant advantages and disadvantages including mortality and morbidity . this is a combination of chemotherapy either before ( neoadjuvant ) or after ( adjuvant ) surgery , an operation that aims to remove the tumour and radiotherapy . the evidence around the use of different chemotherapeutic regimes will be discussed as well as the evidence on radiotherapy approaches . the overall success rates of the trimodality therapy will be discussed in relation to survival , freedom from recurrence , and quality of life ( symptom relief from breathlessness and pain ) . electronic search was performed of pubmed , ovid , cochrane central register of controlled trials , cochrane database of systematic reviews , and web of science from january 1990 to january 2013 . in order to identify all potentially relevant studies we combined mesothelioma and pneumonectomy or mesothelioma and pleurectomy / decortication as keywords . we reviewed abstracts from the recent european association of cardiothoracic surgery meetings and i searched the british thoracic society website for relevant material . the reference lists of all retrieved studies were reviewed for further identification of relevant papers . the articles that were found were screened using the inclusion and exclusion criteria ( previously described in detail in the protocol of this review ) . eligible studies for the present review included those in which patients with histologically proven mpm were treated with surgery ( epp or p / d ) either as the only treatment or as part of a protocol involving adjuvant or neoadjuvant chemotherapy and/or radiotherapy . we did not exclude any forms of chemotherapy or radiotherapy and there was considerable variability between studies in this respect . patient selection may have varied between institutions and within an institution at different time periods . we attempted to identify all relevant randomised controlled trials ( rcts ) ; however , it became clear from the initial search that we would have to include retrospective series and nonrandomised phase i and ii trials as the number of rcts was small . we included studies with more than 20 participants , age greater than 18 ( adults ) , life expectancy at least 12 weeks , and at least moderate performance status either by who or karnofsky criteria . review articles were also excluded to avoid duplication of results ; however , their references were reviewed . we reviewed their references and included some of the studies that were relevant to my systematic review . we reviewed their references and we also used similar headlines ( to theirs ) for the summary tables that we constructed . we did not find any systematic review looking at the role of both epp and p / d in the management of mpp . recurrence - free survival , symptomatic status , and complications were not always reported or measured and we will discuss this below . the time period that the studies cover extends from 1980 to 2012 . over those two decades the diagnostic modalities have evolved ( earlier studies did not have modern scanners available , e.g. ) , the management has changed from single treatment approach to triple therapy ( surgery , chemotherapy , and radiotherapy ) , the chemotherapeutic agents and radiotherapy strategies have improved or changed , even surgical technique has become more consistent , and surgeons have acquired better skill with experience . furthermore the postoperative hospital care and support has improved . this evolution has made it possible , in our days , to have diagnosis of mesothelioma at earlier stage and protocol and multidisciplinary team guided management , with better drugs and more skilful surgeons on the background of modern health care institutions with specialist interest . all these factors influence operative mortality and survival , which are the hard endpoints of this review . the staging system for mesothelioma has changed throughout the years and the definitions of p / d and epp have only recently started to become more consistent . these factors make meaningful comparison of reported results an extremely difficult and sometimes impossible task . as the main focus of this review , from an intervention point of view , is surgery for mesothelioma we have classified relevant studies into p / d and epp . we will critically summarise first the studies that include p / d and epp results , then studies involving only p / d , and lastly studies which focus only on epp . the characteristics of each publication included as well as their main findings are depicted in the relevant tables . ( tables 1 and 2 for p / d publications and tables 3 and 4 for epp publications ) . the study by schipper et al . was a retrospective review of a single institution over an eighteen - year period . they classified p / d into total and subtotal depending on how extensive the surgical resection was . epp was associated with acceptable mortality and had a median survival significantly better than that for subtotal p / d . they documented their concern regarding the fact that improved survival for epp came at a cost of high morbidity . in the paper by luckraz et al . the surgeon who first described surgical management of pleural mesothelioma in the 1970s ( butchart ) is one of the authors . they did statistical analysis of their results and found that the use of adjuvant therapy ( radiotherapy , chemotherapy , or both ) was associated with an increased postoperative survival . on univariate analysis , p / d combined with chemotherapy and radiotherapy was the strongest predictor of prolonged survival . an important limitation is that , during the study period , the chemotherapy and radiotherapy regimens changed significantly so it was impossible to make meaningful comparisons . they conclude that despite the serious limitations in their study their overall results represent real life their recommendation was to undertake a randomised controlled trial to determine the relative role of each procedure . . attempted to perform a straightforward comparison of epp and p / d in their institution , by retrospectively reviewing their results . the selection of which patient will undergo epp and which will undergo p / d depended on stage of the mpm , the overall medical fitness of the patient , and requirements of several prospective clinical trials performed during that time period . in patients not participating in protocols that predefined either epp or p / d , the decision to perform an epp or p / d was based on surgeons ' intraoperative judgement on which procedure was appropriate in order to completely remove the tumour . also tumour stage and patients ' medical condition influenced the decision for epp or p / d . the patients had chemotherapy or radiotherapy according to which clinical trial they were participating in . univariate analysis showed that more aggressive tumour histology and higher stage were associated with significantly worse survival . a limitation of this study is that the investigators did not have comorbidity data . whether a patient received epp or p / d was very much based on how fit the patient was , so this introduced selection bias in the study . the authors were not able to support the use of one or the other surgical approach . their choice of whether to perform an epp or a p / d was based on the degree of the tumour invasion into the lung as well as fitness of the patient for each surgical procedure . they concluded that older age , nonepithelial histology , and pathologic stages iii - iv disease had a significantly negative impact on survival . their findings show that younger patients with a stages i - ii epithelial tumour were good candidates for radical surgery , either p / d or epp . aziz et al . reported a retrospective review of their 10-year experience in their centre . p / d was considered for locally extensive disease but only for relief of pain or shortness of breath . the decision to perform an epp as opposed to a p / d was based on how much the tumour had spread within the lung and whether patients were well enough to go through major surgery . during the more recent years they also decided not to operate on patients older than 60 . this was because they believed that younger patients would be able to tolerate the operation better and therefore have better operative mortality results . they did not find any significant difference in survival between patients undergoing p / d or having no surgery at all . systemic chemotherapy was not offered to patients who underwent p / d because this was essentially an attempt to reduce the tumour load in the lung , not to completely remove it , so chemotherapy seemed futile as an option . lang - lazdunski et al . in a ground - breaking and certainly controversial paper compared p / d with hyperthermic pleural lavage against epp . this is the first prospective but not randomised study which is hinting on the possibility of treating patients with mesothelioma with p / d as a surgical curative attempt ( complete resection ) . the authors did multivariate analysis and concluded that epithelioid histology , p / d , and completeness of resection were independent prognostic factors of completion of trimodality therapy . this means that patients who had p / d were well enough after the surgery to undergo chemotherapy and radiotherapy . rena and casadio published their single centre 11-year experience with epp and p / d . importantly they included a quality - of - life ( qol ) assessment in their comparison . it is the only study of its kind and their findings agree with lang - lazdunski et al . in supporting superiority of p / d over epp . qol parameters were similar at baseline for patients undergoing either procedure but p / d patients had a better qol at 6 and 12 months . all parameters were improved among p / d patients with the exception of postoperative pain , which was similar in the two groups . the authors thought that this was because both epp and p / d use a posterolateral thoracotomy incision to get to the lung , which is associated with a lot of pain after the surgery . patients with lower stage mpm ( i and ii ) who had p / d had a significantly better quality of life when compared with that of those submitted to epp . patients with histological diagnosis of epithelial mesothelioma had similar survival regardless of which surgical procedure they had . they also found that patients who underwent epp had worse long - term survival than those who underwent p / d . this finding needs further exploration as in theory epp achieves complete removal of mpm whereas p / d does not . therefore we would expect epp patients to live longer but the opposite was found in this study . maintained a prospectively updated database which they used to analyse patients that underwent p / d only . exclusion criteria that made them unfit for epp were clinical stage t4 or m1 ( cancer had extensive spread within the lung or had given metastases elsewhere in the body ) , mediastinoscopy proven stage n2 ( cancer had spread to lymph nodes of the opposite side ) , age > 70 years , poor lung function due to smoking or other lung problems ( predicted postoperative fev1 < 40% ) , and right or left ventricular systolic dysfunction ( poor myocardial contractility ) . p / d was classified into radical ( r ) and nonradical ( nr ) depending on how extensive the resection of the pleura and tumour were . they found that patients undergoing radical p / d had a survival advantage over those who underwent nonradical p / d . there was a difference though in the distribution of the histological subtypes of the disease : there were proportionately more patients with epithelioid cell type ( a less aggressive type ) in group r ( 40 epithelioid , 4 sarcomatoid , and 7 biphasic ) than in group nr ( 28 epithelioid , 11 sarcomatoid , and 12 biphasic ) . in contrast to the majority of the other studies included in this review , nakas et al . believe that with radical p / d they can achieve complete macroscopic clearance of tumour . most surgeons think that p / d results in high rates of local recurrence of mpm . an important limitation in their paper is that they did not have an established protocol regarding adjuvant treatment following p / d since these decisions were at the discretion of referring oncologists . martin - ucar et al . performed p / d for symptomatic control of patients diagnosed with mpm . indications included shortness of breath , chest pain , and empyema ( collection of pus in the chest cavity ) . radiotherapy and chemotherapy were not given to patients undergoing p / d unless there was clinical evidence of disease progression . they found that at 3 weeks and 6 months after p / d the patients felt less breathlessness and less chest pain . however , despite good symptom control in those who survived , mortality outweighed the benefits after 3 months . in multivariate analysis , an aggressive cell type and weight loss were predictors of poor symptom control . patients with epithelial cell type and no weight loss were significantly more likely to retain symptomatic control than those with more aggressive cell type and loss of weight . a recent paper by lang - lazdunski et al . looked at patients unsuitable to undergo epp or those that refused epp . they developed an alternative multimodality therapy plan for these patients based on p / d and hyperthermic pleural lavage ( hot water at 40c mixed with betadine was used to wash the pleural cavity ) followed by prophylactic radiotherapy and adjuvant chemotherapy . a large proportion of patients were referred to their hospitals for enrolment into the mesothelioma and radical surgery ( mars ) trial involving epp during the 20052008 period . those patients not wishing to undergo epp or to enrol on the mars trial were offered the alternative treatment described above . selection bias is therefore an issue in this study as well as small sample size and nonrandomisation . those patients that had complete macroscopical removal of the mpm tumour had better survival than those who underwent an incomplete resection . the authors concluded that treatment with p / d and hyperthermic pleural lavage was associated with low morbidity and mortality and therefore it could represent an alternative to the classical trimodality regimen . have published one of the largest series of patients treated with trimodality therapy for mesothelioma . this is one of the earliest publications on this topic and one of the most quoted papers that we encountered in our literature search . they found that multimodality therapy including epp is feasible in selected patients , microscopic resection margins affect long - term survival and patients with epithelial subtype ( less aggressive ) , margin - negative ( complete removal of mpm with surgery ) , extrapleural node - negative ( tumour not spread to lymph nodes elsewhere in the body ) resection had extended survival . they also proposed a revision to the staging system that was used at that time and it has since been revised . krug et al . conducted a multicentre phase ii study in the us to assess performance of a treatment regime which consisted of neo - adjuvant chemotherapy , followed by epp , followed by radiotherapy in patients with stages i to iii disease . a rigorous assessment of each patient 's fitness was done before enrolment to determine respiratory and cardiac function . among those who underwent epp , median survival was 21.9 months . among patients who completed radiotherapy , median survival was 29.1 months , 1-year survival was 90.0% , and 2-year survival was 61.2% . remarkably the parameters histological type of the tumour , gender , clinical stage of mpm , and lymph node spread did not influence survival . relapse - free rates among epp patients were 63.8% at 1 year and 38.9% at 2 years . one limitation of this study is that a mediastinoscopy to assess whether tumour had spread to lymph nodes outside the lung was not required for staging and data regarding how many patients underwent mediastinoscopy was not recorded . furthermore these patients were highly selected on the basis of stage of disease ( early stages ) , good performance status , and satisfactory heart and lung function . the authors concluded that the treatment algorithm with induction chemotherapy , epp , and then hemithoracic radiation is feasible and effective , but only a subgroup of patients experience long survival . did a retrospective review of 46 patients treated with trimodality therapy over a ten - year period in a single centre . median survival for stages 0 , ii , iii , and iv patients was 17 , 33 , 21 , and 24 months , respectively . if the mpm involved lymph nodes , that was a poor prognostic finding , with a median survival of 11 months , compared with 34 months if node - negative . the authors estimate that , for every patient deemed acceptable of trimodality therapy , about another 4 mesothelioma patients were turned down because of advanced disease or comorbidities . a limitation is that the choice of chemotherapy agent , dose , and schedule were at the discretion of the oncologist . during the last 4 years , also patients were referred to epp after they had already started on or completed chemotherapy at other institutions . de perrot et al . conducted a retrospective analysis of 60 patients that were selected to undergo trimodality therapy for mesothelioma in canada . disease - free survival was influenced by whether mpm had invaded lymph nodes or not ( pathologic nodal status ) and to a lesser extent by the histologic type of mpm . the median disease - free survival was 12 months for patients with n2 disease ( mpm spread to lymph nodes outside the affected lung ) , 44 months for patients with n1 disease ( mpm spread to lymph nodes of the affected lung ) , and not reached for patients with n0 disease ( no lymph node involvement ) . after multivariate analysis , the presence of n2 disease remained a significant prognostic marker of worse outcome despite completion of the trimodality therapy . histological type , extent of lung invasion ( t status ) , gender , and age were not significant . weder et al . did a multicentre trial of patients undergoing trimodality therapy for mesothelioma and they assessed quality - of - life parameters as well as survival . the levels of distress did not change over time ( from 0 to 6 months ) . physical symptoms and activity showed worsening after surgery , but there was a recovery back to normal after 36 months . the authors concluded that qol impairment was not a major issue ; however , they stressed the importance of a specialised experienced team . only 22 patients managed to complete the full intervention including chemotherapy , epp , and radiotherapy . they reduced the total radiation dose administered to patients and included additional cardiopulmonary and respiratory function tests which led to a more careful selection of patients . however this amendment made meaningful interpretation of results impossible as the baseline characteristics of the groups before and after the change in protocol were very different . van schil et al . published a study , which was original in that their primary endpoint was success of treatment . this was defined as a patient who received the full protocol treatment within the defined time frames and was still alive 90 days after the end of protocol treatment without progression or evidence of high toxicity related to the chemotherapy or the radiotherapy . only 24 ( 42.1% ) patients met the primary endpoint definition of success . median progression - free survival for all the 57 patients who were eligible and started treatment was 13.9 months and 1-year survival rate was 54.4% . trimodality treatment was completed in 37 ( 64.9% ) patients and median treatment duration was 184 days . embarked in a very ambitious project in 2005 to conduct a randomised controlled trial ( mars ) across 12 centres in the uk , which would definitively determine the role of epp in the management of mesothelioma in the context of trimodality treatment . 12-month recurrence - free survival in the epp group was 34.8% and median recurrence - free survival was 7.6 months . median quality - of - life scores seemed to be lower for the epp group than the no epp group , with the lowest median score shortly after surgery ; however , there were no statistically significant differences between treatment groups . at the time mars was being planned , pemetrexed was not yet the standard of care in the uk . unfortunately during recruitment , the chemotherapy standard of care for mesothelioma changed , and patients recruited later were more likely to receive cisplatin and pemetrexed than those recruited earlier in the study . at the start of the trial the authors calculated that 670 patients would be needed to identify any statistically significant difference between epp and no epp with overall survival as the primary outcome . because of the anticipated difficulty in recruitment of such a high number of patients to a trial comparing epp with a nonsurgical approach , the mars researchers designed a feasibility trial with the objective of randomly assigning 50 patients within 1 year to assess the possibility of completing a larger trial to clarify the role of epp . the study was therefore not designed to test the benefit of epp for patient outcome and any conclusions were speculative . moreover it could be argued that this feasibility study was partly unsuccessful , because it took 3 years to compile 50 patients . . reviewed retrospectively 56 patients across three centres in italy who had undergone epp followed by different modalities of radiotherapy . recurrent tumour ( considered as the first site of relapse ) was local in two cases ( within the lung cavity ) , nodal in three cases ( into regional or distant lymph nodes ) , and distant ( metastasis in other organs ) in 13 cases . baseline characteristics , patient selection , and follow - up periods varied between institutions . tables 2 and 4 summarise survival and peri - operative outcomes for p / d and epp , respectively . for p / d median survival ranged from 8.1 to 32 months . mortality ranged from 0% to 9.8% and morbidity ranged from 5.9% to 55% . for epp median survival ranged from 6.9 to 46.9 months . this large variability makes it difficult to reach safe conclusions in relation to the efficacy and safety of each procedure and echoes the issues that i have discussed at the beginning of section 3 . the principles of surgical management for cancer are similar for all types of solid tumours . surgery for mpm aims at removing all the tumour that can be seen during the operation ( macroscopic disease ) whereas chemotherapy and radiotherapy aim to kill any remaining cancer cells locally ( in the lung cavity ) or in other organs ( microscopic disease ) . two surgical techniques exist.epp involves resection of the ipsilateral ( same side as the tumour ) lung , visceral and parietal pleura ( mesothelial linings of lung and chest wall ) , ipsilateral hemidiaphragm , and pericardium with reconstruction of the latter two structures to prevent cardiac and visceral herniation ( movement of the heart or abdominal organs into the chest cavity after the lung has been removed ) . the description of the epp surgical technique is common knowledge in the thoracic surgical community without much variation . because of this uniformity in the definition of epp , results from different hospitals can be compared , differences in outcomes can be studied , and meaningful conclusions can be reached.p / d is an operation that has not been standardised yet . the reasons behind this are probably multifactorial . from the current review it has become clear that p / d was considered a sort of compromise procedure reserved for patients who were not fit enough from cardiovascular or respiratory point of view to undergo the definitive epp procedure . however in recent years some surgeons tend to favour it over epp mainly due to the fact that most studies have shown no superiority of epp in terms of survival and also much more complications related to epp . epp involves resection of the ipsilateral ( same side as the tumour ) lung , visceral and parietal pleura ( mesothelial linings of lung and chest wall ) , ipsilateral hemidiaphragm , and pericardium with reconstruction of the latter two structures to prevent cardiac and visceral herniation ( movement of the heart or abdominal organs into the chest cavity after the lung has been removed ) . the description of the epp surgical technique is common knowledge in the thoracic surgical community without much variation . because of this uniformity in the definition of epp , results from different hospitals can be compared , differences in outcomes can be studied , and meaningful conclusions can be reached . it has become clear that p / d was considered a sort of compromise procedure reserved for patients who were not fit enough from cardiovascular or respiratory point of view to undergo the definitive epp procedure . however in recent years some surgeons tend to favour it over epp mainly due to the fact that most studies have shown no superiority of epp in terms of survival and also much more complications related to epp . the international association for the study of lung cancer staging committee ( iaslc ) recommended that the following terminology should be used in the mesothelioma staging project.extrapleural pneumonectomy ( epp ) : en bloc resection of the parietal and visceral pleura with the ipsilateral lung , pericardium , and diaphragm.extended pleurectomy / decortication ( epd ) : parietal and visceral pleurectomy to remove all gross tumour with resection of the diaphragm and/or pericardium.pleurectomy/decortication ( p / d ) : parietal and visceral pleurectomy to remove all gross tumour without diaphragm or pericardial resection.partial pleurectomy : partial removal of parietal and/or visceral pleura for diagnostic or palliative purposes but leaving gross tumour behind . extrapleural pneumonectomy ( epp ) : en bloc resection of the parietal and visceral pleura with the ipsilateral lung , pericardium , and diaphragm . extended pleurectomy / decortication ( epd ) : parietal and visceral pleurectomy to remove all gross tumour with resection of the diaphragm and/or pericardium . pleurectomy / decortication ( p / d ) : parietal and visceral pleurectomy to remove all gross tumour without diaphragm or pericardial resection . partial pleurectomy : partial removal of parietal and/or visceral pleura for diagnostic or palliative purposes but leaving gross tumour behind . in addition , this will also provide uniform descriptors to be used in future research and improve pathologic staging and efforts to provide accurate prognosis to patients . current evidence and the iaslc report led several international mesothelioma interest group members ( latest meeting in boston 2012 ) to conclude that both surgical options ( p / d or epp ) are valid as long as they aim at complete removal of the cancer . furthermore they should be performed as part of a multimodality treatment for mpm . the choice of which one to use for each patient depends on the spread of mpm within the lung cavity , the preference and experience of the surgeon , and also which one is favoured at different hospitals . furthermore , it was collectively agreed that multimodality treatment should be performed in centers of high expertise and by surgeons who have achieved morbidity and mortality rates similar to what is reported in the current literature . for the time being , there is no evidence - based answer as to which procedure p / d or epp is the more appropriate technique to achieve long - term survival in patients with mpm . epp has been reported to have better survival but comes at a higher cost of peri - operative mortality and morbidity . p / d is associated with not only less peri - operative risk , but also less long - term success in controlling the spread or recurrence of mpm . the largest report comparing both procedures in a retrospective multicentre study on 663 patients , combining the experience of three large centers in the united states , concluded that the study emphasises the similarities in outcome after epp or p / d . the studies that were reviewed in this paper are highlighting the fact that nowadays the challenge is how to carefully select the patients that would benefit from each procedure ( epp or p / d ) . the timing of the operation and whether chemotherapy should come before or after surgery also vary between centres . the controversy around this issue remains and the debate in the scientific community is ongoing . there is one statement however that scientists seem to agree on : patients with histologically proven mpm and resectable tumour who could tolerate the three treatment modalities should be considered for a multimodal approach and be included in a trial if possible . despite more than two decades of intensive research into possible treatments for mpm , a landmark randomised controlled trial with the acronym emphacis demonstrated the superiority of cisplatin combined with pemetrexed in the management of patients with mpm . survival increased from 9.3 to 12.1 months and this was also associated with quality - of - life benefit . since publication of this study the combination of cisplatin and pemetrexed has replaced all previous chemotherapeutic agents as the main therapy for patients who are having chemotherapy as part of multimodality therapy and for those who have too high risk to undergo a surgical procedure . previously , worldwide research into drug development used to rely on empirical testing of new agents in clinical trials . in vitro or in vivo molecular targets were used extensively . in the current century genomic medicine was based on our efforts to identify targets which may have some clinical significance . some of the cell changes are called passenger mutations because although they are present they do not give a growth advantage to the cancer cell . unfortunately , no mutations of clinical value ( that can be targeted by drugs or other interventions ) have yet been identified in mesothelioma , and certainly none have been transferred into practical clinical application . traditionally , adjuvant rt has been given through fields from in front of as well as behind the patient in order to include the hemithorax where the mpm is located in its entirety . the heart , liver , kidneys , and stomach are protected by using radiotherapy shields over them . however in this way the dose that the chest receives at the edges of the shields is uncertain . therefore sometimes the radiologists end up giving too much or too little radiation to the chest . intensity - modulated radiation therapy ( imrt ) is a complex technique that was designed to overcome the problems associated with the traditional technique . with imrt normal organs are protected against receiving any radiation , and higher doses are delivered to the chest . therefore this is safer for the patient and more precise way of delivering rt to the mpm . current research focuses on arc therapy and helical tomotherapy which are rotational radiotherapy techniques that deliver radiation from more angles than imrt . they have been shown to achieve good results in preventing spread of the mpm ; however this comes with a cost : pneumonitis ( reactive inflammation of the lung which has been radiated ) is a common complication . research is ongoing in current trials to determine if theoretical advantages of new radiotherapy techniques can be translated into clinical benefit for mesothelioma patients . this systematic review has shown that epp and p / d for patients with mpm can be performed with an acceptable perioperative mortality rate in specialised centres . however , the evidence for long - term survival in patients operated - on in the context of tmt in the current literature is inconsistent . these encouraging results demonstrate the potential benefit that surgery can offer for patients treated by a multidisciplinary approach in specialist high volume centres .

introduction . malignant pleural mesothelioma ( mpm ) is an aggressive cancer arising from pleural mesothelium . surgery aims to either cure the disease or control the symptoms . two surgical procedures exist : extrapleural pneumonectomy ( epp ) and pleurectomy / decortication ( p / d ) . in this systematic review we assess current evidence on safety and efficacy of surgery . methods . five electronic databases were reviewed from january 1990 to january 2013 . studies were selected according to a predefined protocol . primary endpoint was overall survival . secondary endpoints included quality of life , disease - free survival , disease recurrence , morbidity , and length of hospital stay . results . sixteen studies were included . median survival ranged from 8.1 to 32 months for p / d and from 6.9 to 46.9 months for epp . perioperative mortality was 0%9.8% and 3.2%12.5% , respectively . perioperative morbidity was 5.9%55% for p / d and 10%82.6% for epp . average length of stay was 7 days for p / d and 9 days for epp . conclusion . current evidence can not definitively answer which procedure ( epp or p / d ) is more beneficial in terms of survival and operative risks . this systematic review suggests that surgery in the context of trimodality therapy offers acceptable perioperative outcomes and long - term survival . centres specialising in mpm management have better results .

1. Introduction 2. Methods 3. Results 4. Discussion: Current and Future Research 5. Conclusion

an intracranial arachnoid cyst ( iac ) is a developmental brain disorder in which fluid accumulates within a split of the arachnoid membrane . these structures do not fit the anatomical definition of a cyst and should be referred to as pseudocysts or diverticula . however , we have adopted the widely used term arachnoid cyst in this article . the cavity does not usually communicate with the subarachnoid space ( non - communicating cyst ) . however , iacs communicating with the subarachnoid space have been reported in humans . in humans , iacs are often incidental findings , but their clinical incidence seems to be more frequent than previously thought in humans , as well as in dogs , although this is hard to confirm . iacs represent 1% of all intracranial masses in humans , and they have been sporadically reported in dogs , where they occur most commonly in the quadrigeminal cistern . only three cases have been reported in cats ; all were located in the caudal fossa at the level of the quadrigeminal cistern . this case describes an iac with an unusual location treated conventionally by cystoperitoneal shunt ( cps ) placement . a 1-year - old male neutered european cat was presented to the small animal neurology department at vetagro sup , lyon , after a 24 h history of focal seizures and one generalised seizure . the cat had about 12 complex partial seizures characterised by orofacial motor signs ( chewing , licking , and eye and facial twitching ) , hypersalivation , obtundation , tremors of the thoracic limbs and urination . routine biochemistry ( glucose , urea , creatinine , alkaline phosphatase , alanine aminotransferase , total proteins , total cholesterol and triglyceride ) and bile acid stimulation were normal . magnetic resonance imaging ( mri ) of the brain was performed using a 0.2-tesla mri system ( e - scan xq ; esaote ) . a large , well - demarcated , extra - axial cystic lesion was present in the left temporal area , mainly beneath the piriform lobe . it measured 1.6 cm in width , 1.7 cm in height and 2.1 cm in length at its largest points . the signal intensity of the content was identical to cerebrospinal fluid ( csf ; hypointense on t1-weighted and fluid - attenuated inversion recovery images , hyperintense on t2-weighted images ) ( figures 1 and 2 ) . this lesion did not enhance after administration of gadolinium ( dotarem ; guerbet ) . t1-weighted transverse magnetic resonance image ( left ) demonstrating a hypointense extra - axial lesion in the left temporal lobe ( open arrowhead ) . the lesion is hyperintense in t2-weighted images ( right ) t1-weighted parasagittal magnetic resonance image demonstrating a hypointense lesion in the left temporal lobe ( open arrowhead ) the degree of parenchymal compression of the forebrain was calculated with a method previously described by matiasek et al for iac in dogs . the mri features were consistent with the diagnosis of a supratentorial left arachnoid cyst with left hemisphere ( and particularly temporal lobe ) compression . the seizures were controlled with anticonvulsant treatment started after admission ( phenobarbital 2.5 mg / kg q12h ) . the catheter can be observed as a hypointense signal on the right ( filled arrowhead ) a dorsolateral approach to the skull was chosen , lateral to the sagittal crest . a peritoneal catheter was tunneled to the left hypochondrium and introduced into the peritoneal cavity by mini - laparotomy . it was anchored to the temporal bone and fixed to a low - pressure valve in the neck , under the muscular fascia . however , persistent modifications of the brain parenchyma were observed in the temporal lobe , including decreased thickness of the temporal lobe compared with the contralateral side and slight signal abnormalities in t2-weighted images ( figure 3 ) . four months after treatment discontinuation , epilepsy recurred and the cat presented two focal and one generalised seizures over 24 h. mri was performed again and showed the same lesions as described 3 weeks after surgery ( figure 3 ) . phenobarbital treatment was restarted at the initial dose ( 2.5 mg / kg q12h ) ( figure 4 ) . ( a , c ) t2-weighted image signal abnormalities ( diffuse hyperintensity ) in the former cyst location ( filled arrowhead ) at ( a ) 3 weeks and ( c ) 21 months after surgery . ( b , d ) t1-weighted image showing parenchymal atrophy in the former cyst location ( open arrowhead ) compared with the contralateral side at ( b ) 3 weeks and ( d ) 21 months differential diagnoses for intracranial cystic lesions are extensive but histological analysis of the lesions can give an accurate diagnosis . in our case , the lesions considered in the differential diagnoses list in cats , according to veterinary literature , are parasite cysts , cystic neoplasms , abscesses , cysts with a non - neural epithelial lining or ex vacuo - type cysts . the lack of contrast enhancement , extra - axial position and homogenous csf - like aspect of this cyst makes all other possibilities than an iac less likely . to our knowledge , this is the first case of a supratentorial arachnoid cyst reported in a cat . the cats were three persian - breed males ranging in age from 10 weeks to 4 years . the cysts were diagnosed with mri in two cases or during necropsy in one case . the cyst was in the caudal fossa at the level of the quadrigeminal cistern in all cases , and in one case it extended rostrally to the cerebral hemisphere . canine iac tends to occur mainly in the quadrigeminal cistern , with other infratentorial locations sporadically reported ( eg , cerebellomedullary angle ) . in humans , iac are mainly supratentorial , with 5060% of them occurring in the middle cranial fossa . they occur less often in the caudal fossa ( 10% ) , wherein they occur most frequently in the cerebellopontine angle and not the quadrigeminal cistern . in our case , the cystic lesion was mainly in the forebrain region , spreading slightly to the brain stem , and it looked more similar to human than canine iac . iac is often an incidental finding in humans , and it has recently been suggested that this may also be true for dogs ; however , numerous canine symptomatic cases have been reported . matiasek et al evaluated brain compression in 28 dogs with quadrigeminal cysts and concluded that occipital lobe compression of > 14% was always associated with clinical signs ( focal seizures , generalised seizures or both ) . no such statistics exist in cats because only three cases of quadrigeminal cysts have been reported , and their clinical significance is difficult to assess . the first case was a 1-year - old cat with a history of uncontrollable seizures . anticonvulsant therapy ( diazepam and phenobarbital ) stopped the seizures for 5 months , but the cat relapsed and was euthanased . the second reported case was a 4-year - old cat with a 12 month history of episodic collapse and altered mentation that was presented after a severe episode of non - ambulatory tetraparesis and cranial nerve abnormalities . the third case was a 10-week - old kitten that died after a 24-h period of lethargy with decreasing levels of consciousness . in our case , the clinical significance of the cyst , located in the left temporal area , is difficult to assess . the type of seizures ( complex partial seizures with predominant orofacial motor signs ) is similar to those observed with hippocampal necrosis . this value is given on an indicative basis and can not be compared to the percentages found in the study by matiasek et al ( 12 23% ) because the location of the cyst was not the same . recurrence of seizures after resolution of the cyst can be explained by the persistence of lesions in the temporal lobe ( atrophy and diffuse hyperintensity in the hippocampal structures in t2-weighted images ) ( figure 3 ) . management of iac in dogs is usually achieved by either cyst fenestration or , more commonly , by cps placement . in humans , it is reported that cps reduces the cyst permanently , while fenestration may require secondary intervention . arachnoid cysts do not have an epithelial lining and should be described as pseudocysts or diverticula , but the inner membrane is believed to have secretory activity . in veterinary medicine , both fenestration and shunting are described as treatments , but no study reports the therapeutic superiority of any of them . in our case , cps placement resulted in no complications during surgery and lead to total resolution of the cyst 21 months after surgery ( mri assessment ) . shunting was chosen because it is considered as an efficient and low - morbidity method , and is reported in the literature more frequently . although this technique does not allow histological analysis of the cyst , to avoid catheter leaking , it is considered a less invasive method than fenestration . to our knowledge , this is the first reported case of a supratentorial arachnoid cyst in a cat , as all other iacs mentioned in the literature were quadrigeminal cysts . the location of the cyst in the temporal lobe is unusual in cats and dogs but is consistent with the predominant location in humans . the semiology of the seizures suggests that the cyst had clinical significance , even if this can not be definitively proven . shunting could be considered a reference method in iac surgical therapy but too few cases are reported in veterinary medicine to assess the therapeutic superiority of either cps or fenestration .

arachnoid cysts are defined as an accumulation of fluid within the arachnoid membrane . feline intracranial arachnoid cysts are seldom reported , with only three cases in the veterinary literature . a 1-year - old male neutered european cat with a 24 h history of seizures was presented to the small animal neurology department at vetagro sup , lyon . magnetic resonance imaging ( mri ) revealed a large intracranial arachnoid cyst ventral to the brain in the left temporal area . cystoperitoneal shunt placement resulted in complete resolution of the cyst without recurrence ( follow - up mris 3 weeks and 21 months after surgery ) . anticonvulsant treatment ( phenobarbital 2.5 mg / kg q12h ) was initiated at presentation and gradually stopped after 17 months . seizures recurred 4 months after ending treatment , and seizure therapy was therefore restarted at the initial dose . we report a case of an intracranial arachnoid cyst in an unusual location not previously described . a cystoperitoneal shunt resolved the cyst without complications . maintenance anticonvulsant treatment was required to control symptomatic epilepsy .

Introduction Case description Discussion Conclusions

intraperitoneal lymphangioma ( il ) is a type of benign cyst with an incidence of approximately 1% . lesions often take place in mesentery , retroperitoneum . most previously reported ils are asymptomatic and are often incidentally found through imaging investigation or during surgery for other unrelated causes . current advancements in radiographic techniques and a deeper realization of il have increased the possibility of imaging and clinical characterization of the abdominal cystic lesion . however , a range of other abdominal lesions including cystic teratomas , enteric cysts , pancreatic psuedocyst , and alimentary tract duplication may masquerade as lymphangioma . in this study , we investigated the evidence of clinic characteristics and imaging features that can contribute to the clinic impression . in addition , we detailed the reasons for misdiagnosis and the therapeutic method of cystic lymphangioma originating in the abdomen treated in our institution , in addition to investigating postoperative recurrences and processing modes . the medical records of 21 patients who were admitted for intra - abdominal lymphangioma between april , 2003 and july , 2013 in our institution were retrospectively analyzed . in our series , the relationship between age , sex , symptoms , physical signs , and location of lymphangioma of each patient were summarized . all patients had an ultrasound ( us ) , abdominal computed tomography ( ct ) scan and/or magnetic resonance imaging ( mri ) . for these three examination methods , the number , location , size of cysts , and whether the cysts were unilocular or multilocular , as well as radiological manifestations of il , were collected . follow - up information was obtained through clinical interviews , and recurrences were discussed in detail . we stated that the protocol for the research project had been approved by a suitably constituted ethics committee of the institution and that it conforms to the provisions of the declaration of helsinki . statistical package for the social sciences ( international business machines corporation , new york , ny , usa ) version 18.0 was used for descriptive analysis . statistical package for the social sciences ( international business machines corporation , new york , ny , usa ) version 18.0 was used for descriptive analysis . twenty - one cases consisted of retroperitoneal , mesenteric , pancreatic and adrenal cysts , and a cyst in the hepatorenal fossa was also reported [ table 1 ] . the number of males and females was 10 and 11 , respectively , with a sex proportion of 1.1 . there were five children , with an age range of 415 years , and 16 adults ( 2780 years ) in our series [ table 2 ] . the incidence of each age group was shown in table 3 . the analysis based on sex revealed that there was no gender predilection . summary of the 21 patients with intraperitoneal lymphangioma clinical characteristics of the 21 intraperitoneal lymphangioma patients * the number of male and female was 10 and 11 , respectively , with a sex proportion of 1.1 ; mean age was 40 ( range 480 ) years ; the size of cysts range varied from 3.67 cm to 18.33 cm with an average size of 7.30 3.28 cm . us : abdominal ultrasound ; ct : abdominal computed tomography scan ; mri : magnetic resonance imaging ; il : intraperitoneal lymphangioma . represents that these patients do not have any symptoms . distribution of age and location of the 21 patients with intraperitoneal lymphangioma clinically , symptoms happened in 14 of the 21 ( 67% , 14/21 ) cases in our series . the relationship between locations and symptoms of the 21 patients with intraperitoneal lymphangioma was summarized in table 4 . the main symptom was intermittent dull abdominal pain , which could be aggravated by the erect position . fatigue was also found in eight patients ( 38% , 8/21 ) ( the location of the mass of these eight patients included mesentery [ n = 3 ] , retroperitoneal [ n = 3 ] , hepatorenal fossa [ n = 1 ] , and pancreas [ n = 1 ] ) . three patients ( 14% , 3/21 ) complained of persistent abdominal pain , sometimes accompanied by nausea and vomiting when the pain become serious ( the locations were , respectively , mesentery [ n = 1 ] and retroperitoneal [ n = 2 ] ) . seven patients ( 33% , 7/21 ) showed no discomfort , and in four of these cases , either the patient or a family member could touch the lump without assistance ( the lumps of those seven patients were located in mesentery [ n = 2 ] , retroperitoneal [ n = 4 ] , or adrenal gland [ n = 1 ] ) . the participants had histories of pain varying in duration from 4 days to 3 years . intestinal obstruction symptoms ( abdominal pain and distension , without exhaust ) indicated the existence of an abdominal lump in one patient ( 5% , 1/21 ) whose diagnosis was mesenteric lymphangioma . a man with acute appendicitis showed right lower quadrant pain in the abdomen with nausea and vomiting , and we found a pancreatic mass in the process of the preoperative imaging examination . the physical examination revealed an abdominal mass in 16 patients ( 76% , 16/21 ) , and of these , eight patients ( 50% , 8/16 ) could feel pain when the location of the il was pressed . although a significant percentage of patients had clinical manifestation , there were no positive findings in the physical examination . symptoms and location of the 21 patients with intraperitoneal lymphangioma three of the 5 ( 415 years ) children ( 60% , 3/5 ) complained of the sensation of abdominal pain of varying degrees , and a palpable mass could be touched in the other two kids , who were completely asymptomatic . in these five children , four were mesenteric , and one was retroperitoneal . in the 16 adults ( 2780 years ) , 10 cases ( 63% , 10/16 ) described a feeling of discomfort , which included two cases of mesenteric lymphangioma , six cases of retroperitoneal lymphangioma , one case of pancreatic lymphangioma , and one case located in the hepatorenal fossa . the lumps of four patients were found upon routine examination , and no symptoms were present ; the lumps were found in the adrenals ( n = 1 ) and retroperitoneum ( n = 3 ) , respectively . in addition , a man was admitted upon consequence discovering a palpable mass himself , resulting in a diagnosis of renal lymphangioma . the abdominal us and ct scan were carried out in the 21 intraabdominal cystic lesions that could be the culprit of symptoms and/or signs [ figure 1 ] . only one person was checked by mri , which better clarified the nature of the mass [ figure 2 ] . radiological studies revealed a unilocular cyst in 16 patients , and five multilocular masses were defined by the septae dividing the cystic spaces . the locations of the cysts are summarized in table 5 . abdominal computed tomography scan ( a ) and computed tomography contrast enhancement ( b ) show an intra - abdominal cystic mass . computed tomography clearly showed a cystic mass with homogeneous internal density and no contrast enhancement after intravenous injection . coronal images ( a and b ) , sagittal image ( c ) . magnetic resonance imaging showed the mass as hyperintense on t2-weighted sequences , suggesting fluid content , with regular margins , thin walls , and internal septa . summary of cyst types and location of the 21 patients with intraperitoneal lymphangioma , n ( % ) in our cases , even in a patient with remote hemorrhage , calcifications were not described . nineteen cases ( 90% , 19/21 ) were accurately diagnosed as lymphangioma prior to surgery based on clinic features and imaging examinations . for example , a woman was diagnosed as having cystadenoma of the pancreas due to evidence of an enhanced echo on the back wall of the cyst under us and mild enhancement of the wall of the cyst , as finding on abdominal ct . in addition , abundant signaling of blood flow was noted on the abdominal us and a ct revealed that the cyst had an uneven density giving the clinic impression of sole angioma in one boy . the size of the cysts varied from 3.67 to 18.33 cm , with an average size of 7.30 3.28 cm . the most common site of these cysts was the retroperitoneum ( n = 10 , 48% , 10/21 ) with a medial scale of 6.6 cm , followed by the mesentery ( n = 7 , 33% , 7/21 ) with a mean size of 9.1 cm . the pancreas ( n = 2 , 9% , 2/21 , 6 cm ) , adrenals ( n = 1 , 5% , 1/21 , 7 cm ) , and hepatorenal fossa ( n = 1 , 5% , 1/21 , 8 cm ) were less frequent locations . histopathological findings were similar in all specimens : dilated lymphatic vessels , lymph , lymphocytes , and flattened epithelial cells of the cyst inside wall [ table 6 ] . surgical treatment was performed on 20 patients , while one a 57-year - old man refused the surgery . the primary surgical approach was total excision of the masses by laparotomy performed in 16 patients . in the remaining three patients with mesenteric lymphangioma , the cyst and a small portion of the intestine were removed to excise the mass completely . it is worth mentioning that one cyst , including its base , was treated laparoscopically [ figure 3 ] . two patients ( 10% , 2/21 ) were in an emergent condition that needed to immediate intervention , a young woman suffered from small bowel obstruction secondary to a large mesenteric mass , and the other presented with acute appendicitis that was complicated by lymphangioma . there were two cysts that had evidence of remote hemorrhage , since the hydatid fluid manifested as brown fluid . it is worth mentioning that we extracted milky liquid from a cyst of an elderly woman . in the rest of the 17 patients , in all , 17 ( 85% , 17/20 ) of the 20 patients were followed up with an average of 5.8 years ( 210 years ) . recurrence of il at the original site occurred in two patients ( 10% , 2/20 ) whose , we separated the mass from the surrounding tissues as completely as possible without damaging the normal structures . therapy after the recurrence included excision of the mass together with the accretive tissues in the two individuals , as shown in table 7 . management and recurrence of the 20 * il patients who received surgery the mean years of follow - up is 5.75 2.83 . some scholars treat this as a hematoma with many dilated lymphatic channels demonstrating multiple cystic spaces which is widely regarded as a developmental abnormality . the most popular sites of il include the mesentery and retroperitoneum areas for children , but il may also be acquired later in life after an exogenous insult such as radiation or surgery . some existing research states that mesenteric lymphangioma occurs more frequently than retroperitoneal , although , in our study , retroperitoneal lymphangioma ( 48% ) had a higher prevalence than did mesenteric lymphangioma ( 33% ) . other possible abdominal sites for the tumor include the liver , spleen , kidney , ligamentum hepatoduodenale , gall bladder , the falciform ligament , and the omentum . the true incidence of il is obscure , and there appears to be no true on sex predilection . also reported that il has a female preponderance in adult because of endogenous estrogens , while some reports demonstrated a male predominance . of the three histological types of the lymphangioma described , all masses were determined to be cysts in our research , although intraperitoneal cavernous masses have been reported in the literatures . in the ordinary course of events , symptoms are stimulated by the primary mass ( 52% ) , while in a few circumstances , they are triggered by complications ( 5% ) . for instance , in our study , acute abdominal pain and ceasing exhaust air with nausea and vomiting was the result of intestinal obstruction secondary to mesenteric lymphangioma . it is of interest that two patients with intracystic remote hemorrhage did not complain of discomfort . previous literatures , however , have reported that acute hemorrhage invariably produces acute abdominal diseases . the mean diameter of the mesenteric cysts ( 8.57 4.54 cm ) was lager compared to the retroperitoneal masses ( 5.83 2.05 cm ) , and consequently , the rate of symptoms in mesentery cysts was 71% and the rate was 42% in retroperitoneum masses . furthermore , complications such as intestinal obstruction , torsion , and inflammation , are more likely to occur in large lymphangioma . the research also states that indisposition induced by masses has something to do with the size which was confirmed in our research . our study further considered that the location , compression of adjacent tissues , and distribution of vessels and nerves of the cysts might be the primary cause of symptoms , which were also linked to one 's pain threshold . the mechanism of pain might relate to nerve traction on the cyst wall and compression of the adjacent nerve , vessel or organ of the gradually increased mass , which again leads to nonspecific gastrointestinal reactions . goh et al . found that younger patients , including children , were more likely to have obvious and more acute symptoms , and our study substantiates this observation [ table 2 ] . symptoms were absent in 33% cases , which was higher than what was noted in the previous research . in a typical us image we may observe fluid in the sonolucent area with the use of enhancement effects , and the boundary of the cysts and internal separate structures are well visible . ct clearly showed the relationship between cystic and adjacent tissues , while typically , contrast administration fails to enhance the imaging of the cyst walls , as illustrated in figure 1 . unfortunately , occasional enhanced capsule walls or abundant flow signals might cause confusion in the diagnosis of lymphangioma . awareness of this unusual occurrence can lead to a correct diagnosis . in the case of adequate realization of lymphangioma in the abdomen , in our study , the accuracy rate of preoperative diagnosis was 90% , which contradicts the literature , which states that preoperative diagnosis is difficult . in addition , ct is more valuable in the diagnostic process than the other available diagnostic tools . mri is infrequently used for the diagnosis of lymphangioma in the literatures , but it enables better preoperative evaluation of intra - abdominal cysts . pathologically , these lesions may be unilocular or multilocular and contain fluid , which may range from a clear , straw - colored liquid to brown liquid , possibly indicating remote hemorrhage . generally , the occurrence of these lesions is single , multiple lesions are reported to be a rare event . surgical resection should be administered immediately after the establishment of a diagnosis of lymphangioma , since the masses gradually increase in size . as the masses increase , there is a growing incidence of complications that will appear over time , such as infection , hemorrhage , intestinal obstruction ( our case ) , and tumor growth which may prevent complete removal of the cysts , leading to the increased possibility of recurrence and/or loss of adjacent structures . as described above , three patients lost part of their intestine , and two cases experienced recurrence due to incomplete excision . some surgeons worry about the infiltrative nature of some lesions and the difficulty in achieving complete resection , however , observation of an unambiguous wall is practical , both in radiological studies and during the surgery , and it was feasible to perform complete excision in 90% of the patients with a recurrence rate of 10% and hardly any morbidity . hence , it is necessary to excise segmental normal adjacent tissues that appeared to be continuous with the cyst , a recommendation that does contradict the recommendation in some previous research . placement of a drainage tube after surgery is essential for preventing chylous ascites . in recent years , laparoscopic excision was given more attention as a potential therapy for intra - abdominal lymphangioma with increasing use and popularity of laparoscopy . from our own perspective some scholars treat this as a hematoma with many dilated lymphatic channels demonstrating multiple cystic spaces which is widely regarded as a developmental abnormality . the most popular sites of il include the mesentery and retroperitoneum areas for children , but il may also be acquired later in life after an exogenous insult such as radiation or surgery . some existing research states that mesenteric lymphangioma occurs more frequently than retroperitoneal , although , in our study , retroperitoneal lymphangioma ( 48% ) had a higher prevalence than did mesenteric lymphangioma ( 33% ) . other possible abdominal sites for the tumor include the liver , spleen , kidney , ligamentum hepatoduodenale , gall bladder , the falciform ligament , and the omentum . the true incidence of il is obscure , and there appears to be no true on sex predilection . also reported that il has a female preponderance in adult because of endogenous estrogens , while some reports demonstrated a male predominance . of the three histological types of the lymphangioma described , all masses were determined to be cysts in our research , although intraperitoneal cavernous masses have been reported in the literatures . in the ordinary course of events , symptoms are stimulated by the primary mass ( 52% ) , while in a few circumstances , they are triggered by complications ( 5% ) . for instance , in our study , acute abdominal pain and ceasing exhaust air with nausea and vomiting was the result of intestinal obstruction secondary to mesenteric lymphangioma . it is of interest that two patients with intracystic remote hemorrhage did not complain of discomfort . previous literatures , however , have reported that acute hemorrhage invariably produces acute abdominal diseases . the mean diameter of the mesenteric cysts ( 8.57 4.54 cm ) was lager compared to the retroperitoneal masses ( 5.83 2.05 cm ) , and consequently , the rate of symptoms in mesentery cysts was 71% and the rate was 42% in retroperitoneum masses . furthermore , complications such as intestinal obstruction , torsion , and inflammation , are more likely to occur in large lymphangioma . the research also states that indisposition induced by masses has something to do with the size which was confirmed in our research . our study further considered that the location , compression of adjacent tissues , and distribution of vessels and nerves of the cysts might be the primary cause of symptoms , which were also linked to one 's pain threshold . the mechanism of pain might relate to nerve traction on the cyst wall and compression of the adjacent nerve , vessel or organ of the gradually increased mass , which again leads to nonspecific gastrointestinal reactions . found that younger patients , including children , were more likely to have obvious and more acute symptoms , and our study substantiates this observation [ table 2 ] . symptoms were absent in 33% cases , which was higher than what was noted in the previous research . in a typical us image we may observe fluid in the sonolucent area with the use of enhancement effects , and the boundary of the cysts and internal separate structures are well visible . ct clearly showed the relationship between cystic and adjacent tissues , while typically , contrast administration fails to enhance the imaging of the cyst walls , as illustrated in figure 1 . unfortunately , occasional enhanced capsule walls or abundant flow signals might cause confusion in the diagnosis of lymphangioma . awareness of this unusual occurrence can lead to a correct diagnosis . in the case of adequate realization of lymphangioma in the abdomen , in our study , the accuracy rate of preoperative diagnosis was 90% , which contradicts the literature , which states that preoperative diagnosis is difficult . in addition , ct is more valuable in the diagnostic process than the other available diagnostic tools . mri is infrequently used for the diagnosis of lymphangioma in the literatures , but it enables better preoperative evaluation of intra - abdominal cysts . clinical , pathologic diagnosis is the gold standard . pathologically , these lesions may be unilocular or multilocular and contain fluid , which may range from a clear , straw - colored liquid to brown liquid , possibly indicating remote hemorrhage . generally , the occurrence of these lesions is single , multiple lesions are reported to be a rare event . surgical resection should be administered immediately after the establishment of a diagnosis of lymphangioma , since the masses gradually increase in size . as the masses increase , there is a growing incidence of complications that will appear over time , such as infection , hemorrhage , intestinal obstruction ( our case ) , and tumor growth which may prevent complete removal of the cysts , leading to the increased possibility of recurrence and/or loss of adjacent structures . as described above , three patients lost part of their intestine , and two cases experienced recurrence due to incomplete excision . it should be noted that cystic lymphangioma may give rise to hodgkin lymphoma . some surgeons worry about the infiltrative nature of some lesions and the difficulty in achieving complete resection , however , observation of an unambiguous wall is practical , both in radiological studies and during the surgery , and it was feasible to perform complete excision in 90% of the patients with a recurrence rate of 10% and hardly any morbidity . hence , it is necessary to excise segmental normal adjacent tissues that appeared to be continuous with the cyst , a recommendation that does contradict the recommendation in some previous research . placement of a drainage tube after surgery is essential for preventing chylous ascites . in recent years , laparoscopic excision was given more attention as a potential therapy for intra - abdominal lymphangioma with increasing use and popularity of laparoscopy . from our own perspective

background : intraperitoneal lymphangioma ( il ) used to be thought of as a benign lymphatic malformation with a low rate of preoperative diagnosis . this retrospective study aimed to explore the connection between the cysts and clinical manifestation and imaging characteristics , and to study diagnostic confusion , therapeutic principles and potential recurrent reasons , to further enhance the comprehension of this rare disease.methods:here , we retrospectively reviewed 21 patients diagnosed with il . age , sex , complaints , physical findings , and imaging features of each patient were documented . the therapies , postoperative complications and treatments were discussed.results:symptomatology included eight patients ( 38% ) with intermittent dull pain in the abdomen , and three patients ( 14% ) complained of abdominal persistent pain . the physical examination revealed an abdominal mass in 16 patients ( 76% ) , and eight ( 38% ) were reported no discomfort . il was correctly established preoperatively in 19 patients ( 90% ) . patients were treated using laparotomy , except one who was treated with laparoscopy . two recurrences were noted during follow-up.conclusions:il should be suspected in any patient with a mobile abdominal mass and surgery is required immediately after discovery of the tumor .

I M Statistical analysis R D Location and histological type Symptoms Imaging examination and diagnosis Therapy and recurrence Financial support and sponsorship Conflicts of interest

the database , danbio , is a nationwide , danish register for research use in rheumatologic diseases such as rheumatoid arthritis ( ra ) , axial spondyloarthritis ( ax spa ) , and psoriatic arthritis ( psa ) . it also serves as a clinical database that monitors clinical quality of treatment by the use of selected quality indicators for patients with ra in denmark . up to the year 2000 , no routine - based nationwide reporting of patients with ra existed in denmark , and patient files rarely comprised quantitative patient - reported outcome data . the introduction of new biological disease - modifying antirheumatic drugs ( bdmards ) triggered the formation of a nationwide voluntary register , which aimed to survey indications for treatment , efficacy , and adverse events in rheumatologic patients who received biological therapies in routine care . in the beginning , data were collected on paper forms , which were subsequently scanned into the register at the danbio office.1 in 2006 , an online version of danbio was introduced , and the aims were extended to include patients regardless of treatment and also to collect patient - reported outcomes regarding , eg disability , pain , lifestyle , and quality of life by the use of dedicated touch screens in the waiting room ( figure 1 ) . this is routinely done before consultation with the doctor , and the information from the touch screen is available during the consultation . danbio has been approved by the national board of health as a clinical quality register , and since 2006 , reporting to the register has been mandatory . for clinical quality registers , the usual requirement for obtaining an informed patient consent before registration is not needed . only rarely do patients object to be included in the register , and in these cases , it is up to the physician to decide whether to register the patient or not . ra , ax spa , and psa are chronic diseases that require lifelong monitoring and treatment . a patient is therefore reported to the database at the time of diagnosis , referral to specialized treatment , including biological therapy at the hospital , or in a private rheumatologic clinic . at the first registration , the diagnosis , date of diagnosis , age , sex , and previous medical treatment are registered . at later visits , and at least once yearly , information regarding patient s disease activity ( including pain and functional status ) is collected via touch screens , and objective measures ( eg , swollen and tender joint counts , c - reactive protein ) are entered by the physician . the patient groups with ax spa and psa are not included at the moment in the national clinical quality program , and therefore reporting is only mandatory for patients with ra and for the other patient groups only when treated with bdmards . in the beginning of 2015 , the danbio cohort comprised 26,000 patients with ra , 3,200 patients with ax spa , and 6,200 patients with psa treated with conventional synthetic dmards and/or bdmards . some variables are collected via touch screens ( patient - reported outcome measures [ proms ] ) , and others are entered by the physician . the use of touch screens for data collection has been validated to perform equally well as paper forms ( figure 1).2 the variables are collected in a standardized format , often using tick boxes , which have been shown to have a high validity.1 figure 2 shows the scoreboard for entering and reviewing individual patient data . in connection with ie , queries from danbio and research projects , the departments fill in missing data by checking against the hospital patient records the completeness of data is generally high ( ie , 96% , 93% , 83% , 90% , and 93% for information on the name of the treating doctor , diagnosis , year of diagnosis , das28 , and haq , respectively ) . national coverage of danbio is assessed annually in two ways : for patients in biological treatment by comparing to those registered in the patient record systems of each rheumatologic department ( 94% in 2013 ) and by calculation of all incident patients with ra in danbio by comparison to the danish national patient registry ( npr ; 85% in 2013 ) . since npr is an administrative register3 in which ra diagnosis may be registered for patient contacts before the final diagnosis of the patient has been established , it can not serve as a golden standard ; however , it is the most valid nationwide register available for such comparison . further , local medical record audits have shown that the diagnosis in danbio is valid . therefore , it is assumed that the true coverage of danbio is higher than this estimate.4 at least once - a - year follow - up information is collected in danbio , including proms such as the level of function and level of pain . disease activity , type of treatment , treatment effectiveness , and side effects of treatment ( if any ) are registered by the physician.5 systematic monitoring of patients with ra with real - time feedback to the physician is feasible . it is documented that regular registration of disease activity for patients with ra results in improvement in disease activity , although the goal of treat to target is not achieved in a substantial proportion of patients in routine care.6 the current quality indicators for ra are shown in table 2 . ( ie , remission or low disease activity ) by follow - up of patients with measurement and registration of disease activity level , functional status , pain , quality of life , and x - ray status . a standard for each indicator is set by the danbio steering committee and by consensus among the members of the danish society of rheumatology setting goals for how large a fraction of patients who should meet the indicator criteria . the indicator results are published at country , regional , and hospital department levels in the annual clinical quality report.4 an example of supplementary results from the quality report is presented in figure 3 that gives the number of adult rheumatologic patients initiating bdmards in denmark over time and the differences in prescription pattern between geographical regions.4 ( ie , remission or low disease activity ) by follow - up of patients with measurement and registration of disease activity level , functional status , pain , quality of life , and x - ray status . a standard for each indicator is set by the danbio steering committee and by consensus among the members of the danish society of rheumatology setting goals for how large a fraction of patients who should meet the indicator criteria . the indicator results are published at country , regional , and hospital department levels in the annual clinical quality report.4 an example of supplementary results from the quality report is presented in figure 3 that gives the number of adult rheumatologic patients initiating bdmards in denmark over time and the differences in prescription pattern between geographical regions.4 danbio is used in routine care for systematic monitoring of the patients , but it is also an extensive research database . danbio data have been used for research with focus on both short- and long - term evaluation of treatment responses , remission rates , and drug adherence.710 furthermore , research has been performed on cancer incidence and precancerous development following treatments with biological treatment regimes,11,12 as well as on the influence of tobacco smoking on treatment response.13 more than 60 peer - reviewed articles have been published to date with the use of danbio data and > 170 abstracts have been presented at international conferences and congresses . danbio is easily linked to other data sources by the use of the unique personal identification number , assigned to all danish citizens . with regulatory approval for specific research purposes , danbio data can be merged with ie , the npr , the danish cancer registry , or administrative registers holding socioeconomic information in statistic denmark . a nationwide biobank ( ie , blood and synovial fluid ) associated with danbio has been established since 2015 through public funding . danbio has an independent steering committee with representatives from the danish society of rheumatology , drfo ( danske reumatologer og fysiurgers organisation ) , junior rheumatologists ( yngre reumatologer ) , and the hospital owners . the daily administration is handled in the danbio general office at rigshospitalet , glostrup , and is staffed by the head of secretariat as well as a secretary . danbio has been approved as a clinical quality register by the danish authorities the national board of health and the danish data protection agency and is sponsored by the public hospital owners ( danish regions ) under the organization of the danish clinical registries a national improvement program . the pharmaceutical companies that provide biological treatments in rheumatology contribute to the development of the information technology platform through unrestricted grants , which have been approved by the public hospital owners . the sponsors have no influence on the register setup , data collection , data analysis , or publication of results . danbio serves as a powerful register for research in rheumatologic diseases and additionally as a clinical quality register with the aim of monitoring quality by the use of selected clinical indicators for patients with ra in denmark . the register has existed since 2000 , and patients diagnosed at the hospital or at private rheumatologic clinic are registered . since 2006 , the registration , including proms , has been electronically registered directly into the database with the use of touch screens . danbio has high nationwide coverage of patients with rheumatologic diseases and high completeness on key data variables . data have been used for a large number of research projects and each year a national clinical quality report is published .

introductiondanbio is a research register and a data source for rheumatologic diseases ( rheumatoid arthritis [ ra ] , axial spondyloarthritis , and psoriatic arthritis ) for monitoring clinical quality at the national , regional , and hospital levels.study populationthe register includes patients with rheumatologic diseases who are treated at a hospital or a private rheumatologic clinic . registration is mandatory for all patients with ra regardless of treatment and also for patients with other diagnoses if treated with biological disease - modifying antirheumatic drugs . since 2006 , the registration has been done electronically , including patient - reported outcome measures registered electronically by the patients with the use of touch screens.main variablescore variables such as diagnosis , year of diagnosis , age , and sex are registered at the beginning . data entered at later visits included the following : patient - reported outcomes for disease activity , pain , fatigue , functional status , and physician - reported objective measures of disease activity , treatment , c - reactive protein , and , when indicated , imaging . for subgroups of patients , the variables such as quality of life , sociodemographic factors , lifestyle , and comorbidity are also registered.descriptive datathe danbio cohort comprised 26,000 patients with ra , 3,200 patients with axial spondyloarthritis , and 6,200 patients with psoriatic arthritis in 2015 . danbio has high nationwide coverage and completeness on key data variables . more than 60 original papers as well as annual reports of clinical quality ( since 2005 ) have been published.conclusiondanbio is a powerful register for research in rheumatologic diseases and furthermore serves as a clinical quality register with the aim of monitoring treatment quality in patients with ra in denmark .

Introduction Study population and data collection Main variables Follow-up Quality indicators Examples of research Administrative issues and funding Conclusion

stereotactic body radiotherapy ( sbrt ) for non - surgical treatment of early - stage primary lung cancer has recently been investigated in clinical practice , during which several problems regarding lung cancer treatment planning have been identified [ 15 ] . the development of 4d - computed tomography ( ct ) has made it possible to visualize breathing - induced tumor motion , shape and volume during a respiratory cycle . respiration - induced tumor motion may result in considerable differences between the planned and actually delivered dose . second , inhomogeneous corrections are reported to lead to larger dose differences for lung cancer patients . aarup et al . reported that dose differences to the target obtained using the monte carlo ( mc ) calculation and treatment planning systems using pencil beam calculation were influenced by lung density ( 0.4 to 0.1 g cm ) . the international commission on radiation units and measurements ( icru ) recommended the use of dose volume instead of point dose at the reference point . dose calculation to the planning target volume ( ptv ) is complicated , however , because it can be affected by low lung density . if the dose prescription is defined at the ptv , monitor units should be adjusted to allow sufficient ptv coverage . on this basis , we defined that 99% of the gross tumor volume ( gtv ) should be covered by 100% of the prescribed dose ( d99 = 100% ) using the mc calculation . however , the dose should be calculated with confirmation that the dose prescription is defined to the gtv with tumor motion . the purpose of this study was to validate the dose prescription defined to the gross tumor volume ( gtv ) 3d and 4d dose distributions . figure 1 shows the basic virtual phantom , which has a simulated gtv of 10 mm diameter with an equivalent water density of 1.0 g cm . the density of the equivalent lung surrounding the gtv was defined as 0.25 g cm . the selected gtv sizes were intended to represent the average target dimensions in lung cancer sbrt . the influence of gtv motion was investigated with motion amplitudes of 5 , 10 and 20 mm . the ptv was generated by adding a uniform margin of 8 mm to the itv to account for setup uncertainties and mechanical inaccuracy . a leaf margin of 2 mm was added to the ptv , and the isocenter was positioned in the center of the ptv . the plan was calculated using the mc calculation in an iplan rt dose , ver 4.1.2 ( brainlab , germany ) treatment planning system . beam energy for all plans were 6-mv photon beams , and gantry angles were 0 , 72 , 144 , 216 and 288 , which is consistent with simple sbrt treatment planning . 1.schematic overview of the foundation model with center of the mid - expiration gtv ( mid ) , 10 mm relative to the end - expiration gtv ( ex ) , and end - inspiration gtv ( in ) surrounded by lung - equivalent material ( grey ) and a 20 mm wall of water - equivalent material ( black ) . margin to the ptv ( black solid line ) was 8.0 mm in all directions . schematic overview of the foundation model with center of the mid - expiration gtv ( mid ) , 10 mm relative to the end - expiration gtv ( ex ) , and end - inspiration gtv ( in ) surrounded by lung - equivalent material ( grey ) and a 20 mm wall of water - equivalent material ( black ) . margin to the ptv ( black solid line ) was 8.0 mm in all directions . dose prescription was defined as d99 = 100% of the gtv at mid - expiration phase ( mid - gtv ) . delivered dose to the gtv with movement was validated using the end - expiration ( ex - gtv ) and end - inspiration ( in - gtv ) phases . the reference images were changed to evaluate the effect of movement of the gtv on dose distribution . all planning parameters ( beam arrangement , leaf positions , isocenter position and monitor unit ) remained unchanged . the mc calculation was performed using the full mlc geometry simulation accuracy optimized model with a spatial resolution of 2 mm and variance of 2% . target coverage was evaluated in each breathing phase as dose to 99% of the volume of the gtv , determined as a dose volume histogram ( dvh ) . delivered dose was defined as a given dose to the gtv , which was assigned a density of 1.0 g cm in the breathing phase . a total of 94 patients treated with sbrt between october 2010 and october 2011 were included in this analysis . patient characteristics are shown in table 1 . as a routine procedure for the planning of stereotactic radiotherapy , 3d - ct ( general electric company , 4-slice brightspeed qx / i scanner ) was used to acquire a whole lung image series under free breathing using a system developed in - house to suppress tumor motion , the air - bag system . the air - bag system consists of a non - elastic air bag connected to a second smaller elastic air bag . the first air - bag is placed between the patient 's body surface and a hipfix device ( civco , usa ) and secured by a pressure adjustment via the elastic air - bag . 4d - ct was performed to more accurately determine tumor shape , volume and position at different phases of the breathing cycle . the ct images had a slice thickness of 2.5 mm with a gantry rotation time of 1.0 s. each image was tagged with the corresponding phase of the respiratory cycle and then sent to the advantage workstation ( general electric company , waukesha , wi ) using advantage 4d - ct software . the 4d datasets were categorized into four phases of the respiratory cycle ( 0% , 25% , 50% and 75% ) , with 0% representing maximum inspiration . image quality of the 4d - ct was sufficient for tumor evaluation in all patients . the visible tumor was delineated as the gtv in the ct pulmonary window of the 4d - ct images . the 3d tumor motion vector , which was individually measured in each direction , was calculated as follows : where x was the lateral , y was the anterior - posterior and z was the cranio - caudal direction . dose prescription was conformed to 99% of the gtv , as in the phantom study . the number of beams was from five to eight with non - coplanar arrangements . table 1.patient characteristicspatients ( n)94gender male ( n)53 ( 56% ) female ( n)41 ( 44%)age ( years)72 ( 3489)gross tumor volume ( cm)8.9 ( 0.377.5)planning target volume ( cm)52.0 ( 9.7232.3)target location ( lobe ) upper and middle ( n)46 ( 48% ) lower ( n)48 ( 52% ) patient characteristics all planning parameters on the reference plan of the 3d - ct images were copied to the four breathing phase 4d - ct images to recalculate the dose , which was influenced by breathing . d99 of the delivered dose to the gtv in the 4d plan in this study was defined as the average of each d99 for the four breathing phases . the 3d and 4d calculations were compared using the student paired t - test , and tumor motion in the upper and lower locations were compared using the student unpaired t - test . the analyzed data were displayed as mean standard deviation with ranges in parentheses among 94 clinical plans . figure 2 shows the dose distribution and dose - volume histogram ( dvh ) of the delivered dose to the ex - gtv , mid - gtv and in - gtv , calculated at the amplitude of 10 mm . the average d99 of delivered dose to the gtv with motion amplitudes of 5 , 10 and 20 mm was 99.2% , 99.0% and 98.5% , respectively . 2.dose distribution and dose volume histogram were calculated at the amplitude of 10 mm , showing the delivered doses to ex - gtv ( black dashed line ) , mid - gtv ( black solid line ) and in - gtv ( grey dotted line ) . dose distribution and dose volume histogram were calculated at the amplitude of 10 mm , showing the delivered doses to ex - gtv ( black dashed line ) , mid - gtv ( black solid line ) and in - gtv ( grey dotted line ) . tumor motion in patients with upper / middle lobe and lower lobe tumors , for which movement was greatest in the cranio - caudal direction , was 2.2 1.9 mm ( range 0.18.2 mm ) and 6.2 5.0 mm ( range 0.121.4 mm ) , respectively ( p < 0.05 ) ; that in the lateral direction was 0.9 0.7 mm ( range 0.13.6 mm ) and 0.9 0.6 mm ( range 0.12.9 mm ) , respectively ( p = 0.74 ) ; and that in the anterior - posterior direction was 1.4 1.0 mm ( range 0.15.0 mm ) and 1.4 1.2 mm ( range 0.14.7 mm ) , respectively ( p = 0.99 ) . tumor motion vector for patients with upper / middle and lower lobe tumors was 2.8 1.9 mm ( range 0.19.9 mm ) and 6.3 5.0 mm ( range 0.122.0 mm ) , respectively , with this difference in tumor motion being significant ( p < 0.05 ) . all patients with upper / middle lesions exhibited between 0 and 10 mm of tumor motion . tumor movement of more than 20 mm was seen in one patient , with a lower lobe tumor . 3.histograms of the tumor motion vector of upper / middle lung tumors ( a ) and lower lung tumors ( b ) using an abdominal compression system developed in - house . histograms of the tumor motion vector of upper / middle lung tumors ( a ) and lower lung tumors ( b ) using an abdominal compression system developed in - house . figure 4 shows the dose distribution and dvh of the delivered dose to the gtv for the four breathing phases for representative patients . the lower isodose lines hardly differ between breathing phases , while the higher isodose lines are synchronized with the tumor motion . d99 to the gtv in the 4d calculation was 99.7% , 99.9% , 100.3% and 100.0% at 0% , 25% , 50% and 75% , respectively . dose to the gtv in the inspiration phase was slightly lower than that in the expiration phase . the dose difference between maximum and minimum d99 of the gtv in 4d calculations was 0.6 1.0% ( range 0.24.6% ) . average d99 of the gtv in 4d calculations for most of the patients was almost 100% ( 99.8 1.0% ) . no significant difference in dose to the gtv was seen between 3d and 4d dose calculations ( p = 0.67 ) . 4.(a ) dose volume histograms and ( b ) dose distributions of gtv for each breathing phase in one patient ( tumor motion : 11 mm ) . ( a ) dose volume histograms and ( b ) dose distributions of gtv for each breathing phase in one patient ( tumor motion : 11 mm ) . the japan clinical oncology group ( jcog ) and the radiation therapy oncology group ( rtog ) have planned multi - institutional trials of sbrt for non - small - cell lung cancer [ 45 ] . in the jcog 0403 protocol , dose prescription is defined as the point dose at the isocenter of the ptv with inhomogeneous correction , such as the pencil beam convolution with batho power law and clarkson with effective path length correction , but this prescription is not accurate for dose calculations of lung cancer . in contrast , the rtog 0236 protocol defines dose prescription as the volume dose at the periphery of the ptv without inhomogeneous correction . these clinical trials reported around 90% of local control rates have been report [ 13 ] . because these reports do not cover the test results of inhomogeneous correction , the actual dose delivered to the tumor can not be accurately determined . inhomogeneous corrections were necessary to prevent large discrepancies between planned and actual delivered doses to individual patients . takahashi et al . reported that collapsed cone convolution and superposition calculation plans were close to the mc calculation plan and the actual dose distributions obtained in lung sbrt . the external border of the ptv is covered by a lower isodose surface than that usually used in conventional radiotherapy planning , typically around 80% [ 1112 ] . therefore , prescription dose was defined to the ptv , which was differently covered at 65% and 80% , with normalization to 100% at the isocenter in inhomogeneous correction . this increases the amount of normal tissue in the field , however , which could lead to increased toxicity . the ptv is a tool designed to ensure that the tumor receives an adequate absorbed dose . this study revealed that tumor motion influenced the gtv on inhomogeneous dose distribution using the mc calculation . our present phantom study suggests that the actually delivered dose to the gtv was more stable than that expected from the planned dose , owing to the generation of secondary electrons from the tumor . additionally , the decreased dose to the gtv resulted from larger tumor motion , indicating the need for particular attention to the larger tumor motion induced by breathing . nevertheless , no dose difference to the gtv between 3d and 4d calculations was observed for our present lung cancer patients because tumor motion was small . measured 3d motion in 20 patients using a 2 mm gold marker implanted in or near the tumor during real - time imaging . they reported that the greatest average amplitude of tumor motion in the cranial - caudal direction was 12 2 mm for lower lobe tumors , and 2 1 mm in both the lateral and anterior - posterior directions for upper and lower lobe tumors , respectively . the motion range of tumors for many patients can be suppressed to less than 5 mm using the air - bag system . abdominal compression has been widely used to minimize respiratory - associated tumor movement during sbrt to lung and liver tumors . abdominal compression can be used to suppress respiratory diaphragm motion , which leads to suppressed tumor motion . the reduction in tumor motion may lead to a decrease in dose to the normal lung structure . an effective approach to resolving motion without increasing margins is to gate and synchronize the radiation to specific phases of the respiratory cycle . most lung cancer patients are elderly , and these techniques require a longer time for beam delivery , making it more difficult for patients as they must maintain normal breathing over the delivery time . we therefore selected the free - breathing technique using abdominal compression . a dose difference between maximum and minimum at the d99 of gtv for each phase was > 3% in eight patients . the tumor motion vector for these patients was 3.1 1.9 mm ( range 1.56.4 mm ) . figure 5 shows the dose distribution at the worst case for end - inspiration and end - expiration phases , in which the tumor was located near the diaphragm . the delivered dose to the gtv at the end - inspiration phase was lower than that at the end - expiration phase . dose distributions were different in each breathing phase , owing to secondary scatter generated from the diaphragm . reported that the ctv was still covered by almost 100% of the dose under conditions of tumor movement , but the peripheral dose was reduced by the low - density material . if the microscopic extension margin is added and dose prescription is defined to the ctv , the ctv includes the low - density material , leading to a similar dose result to that for the ptv . 5.dose distributions for ( a ) end - inspiration ( b ) end - expiration in one patient ( tumor motion : 5.1 mm ) . dose distributions for ( a ) end - inspiration ( b ) end - expiration in one patient ( tumor motion : 5.1 mm ) . for a regular free - breathing ct simulation , images could be acquired at a random phase . selection of the reference image for 3d calculation at the end - breathing phase , however , may result in a larger dose difference between the 3d and 4d dose calculations . in the case of 4d , we renormalized calculation to achieve an average delivered gtv dose of more than 100% . 4d treatment planning provides more accurate dose calculations to the tumor with regard to relative changes in shape , volume , position and density during respiration . our approach to gtv for respiration - induced tumor motion achieved adequate tumor coverage and spared normal tissue . we investigated the effect of respiratory motion on the delivered dose to the gtv in 4d dose distribution . no significant differences were found between 3d and 4d dose calculations in treatment planning for lung cancer patients using the mc calculation . particular attention should be paid to not only tumor motion but also the structure around the tumor . this study supports the clinical acceptability of treatment planning based on the dose prescription defined to the gtv .

the purpose of this study was to validate the dose prescription defined to the gross tumor volume ( gtv ) 3d and 4d dose distributions of stereotactic radiotherapy for lung cancer . treatment plans for 94 patients were generated based on computed tomography ( ct ) under free breathing . a uniform margin of 8 mm was added to the internal target volume ( itv ) to generate the planning target volume ( ptv ) . a leaf margin of 2 mm was added to the ptv . the prescription dose was defined such that 99% of the gtv should receive 100% of the dose using the monte carlo calculation ( iplan rt dosetm ) for 6-mv photon beams . the 3d dose distribution was determined using ct under free breathing . the 4d dose distribution plan was recalculated to investigate the effect of tumor motion using the same monitor units as those used for the 3d dose distribution plan . d99 ( 99% of the gtv ) in the 4d plan was defined as the average d99 in each of the four breathing phases ( 0% , 25% , 50% and 75% ) . the dose difference between maximum and minimum at d99 of the gtv in 4d calculations was 0.6 1.0% ( range 0.24.6% ) . the average d99 of the gtv from 4d calculations in most patients was almost 100% ( 99.8 1.0% ) . no significant difference was found in dose to the gtv between 3d and 4d dose calculations ( p = 0.67 ) . this study supports the clinical acceptability of treatment planning based on the dose prescription defined to the gtv .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

new - onset atrial fibrillation ( noaf ) in the context of acute myocardial infarction ( ami ) is common and usually induces severe hemodynamic dysfunction and has been shown to be an independent predictor of mortality among ami patients . in addition , among the patients complicated with noaf , the incidence of stroke , bleeding , and reinfarction are all higher than those without noaf . the secondary decrease in cardiac output aroused by impairment of atrial contraction , atrioventricular synchrony , and irregular rr interval may be partly responsible for the poor prognosis . however , the potential mechanisms may be complex and have not been well - characterized . whether higher killips class is an independent predictor of noaf remains controversial , so we performed this comprehensive meta - analysis to explore the potential relationship between higher killips class and noaf . we conducted this analysis according to the guidelines of the meta - analysis of observational studies in epidemiology group . we included prospective or retrospective observational studies with a primary objective to analyze the association between high killips class ( > i ) and noaf after ami . titles and abstracts of all articles were evaluated and rejected following inclusion criteria : ( 1 ) human subjects with ami ; ( 2 ) killips class at admission evaluated ; ( 3 ) retrospective / prospective cohort studies ; ( 4 ) baseline data available ; ( 5 ) enough follow - up period for detect noaf ; ( 6 ) noaf following ami for patients with different killips class mentioned ; ( 7 ) ami type mentioned ; ( 8) enough sample size ( n > 100 ) of patients from individual study . we carefully searched on - line database of pubmed , web of science , china national knowledge infrastructure , and the cochrane controlled trials register databases until february 2015 to identify relevant studies . we used the following keywords : atrial fibrillation ( af ) , new - onset and killips , heart failure and myocardial infarction . titles and abstracts , as well as the reference lists of all the identified reports , were examined independently by two reviewers ( zhang ey and cui l ) in order to include potentially relevant studies . the two reviewers agreed on the inclusion / exclusion status in 90% of the reviewed studies . disagreements were resolved by discussion or consensus of a third reviewer ( li zy ) . in addition , a manual search was conducted using review articles on this topic , bibliographies of original papers , and abstracts of the scientific sessions of the , the american heart association , the european society of cardiology , heart rhythm society , and american college of cardiology during the past 2 years . to limit heterogeneity secondary to differences between study designs , the quality of each study was evaluated according to the guidelines developed by the evidence - based medicine working group and the united states preventive task force . we applied the point score system assessed by the following characteristics : ( 1 ) clear description of inclusion and exclusion criteria ; ( 2 ) study sample representative for mentioned population ; ( 3 ) clear description of sample selection ; ( 4 ) full specification of clinical and demographic variables ; ( 5 ) follow - up during hospitalization ; ( 6 ) no loss of follow - up ; ( 7 ) clear definition of killips class ; ( 8) clear definition of outcomes and outcome assessment ; ( 9 ) temporality ( assessment of killips class once at presentation ) ; ( 10 ) adjustment of possible confounders in multivariate analysis . if one of these key points was not mentioned clearly in a study , we considered it not performed data extraction was performed by two blind investigators ( zhang ey and cui l ) independently with a standard data extraction form to determine eligibility for inclusion . the following information collected was tabulated : ( 1 ) publication details : first author 's last name , publication year ; ( 2 ) characteristics of included studies : study population , definition of noaf , detection method , quality score , risk estimate and nation ; ( 3 ) baseline data of the studied population : sample size , age , gender , killips > i ( % ) , noaf ( % ) , current smoker ( % ) , left atrium diameter , left ventricle ejection fraction ( lvef ) , st - elevation myocardial infarction ( % ) , anterior myocardial infarction ( % ) , diabetes mellitus ( % ) , hypertension ( % ) , previous infarction ( % ) , coronary revascularization ( % ) . cardiac function on admission was scored according to the killips classification scheme class i iv , i as absence of heart failure , ii as presence of rales and/or jugular venous distention , iii as presence of pulmonary edema and iv as cardiogenic shock . killips class i at presentation was defined as severe cardiac dysfunction in our meta - analysis . the magnitude of association between high killips and noaf following ami was measured by adjusted odds ratio ( or ) or or with 95% confidence intervals ( cis ) . finally , we examined the heterogeneity with the standard chi - square test of heterogeneity . a pooled effect was calculated with a random - effects model when the chi - square test for heterogeneity was found significant , through which we could take within - study and between - study variance into account , while not significant , a fixed - effects model was still used . sensitivity analysis was done by dropping studies and checking the consistency of the overall effect estimate . all the jobs above were performed with review manager version 5.2 ( revman ; the cochrane collaboration , oxford , uk ) . we included prospective or retrospective observational studies with a primary objective to analyze the association between high killips class ( > i ) and noaf after ami . titles and abstracts of all articles were evaluated and rejected following inclusion criteria : ( 1 ) human subjects with ami ; ( 2 ) killips class at admission evaluated ; ( 3 ) retrospective / prospective cohort studies ; ( 4 ) baseline data available ; ( 5 ) enough follow - up period for detect noaf ; ( 6 ) noaf following ami for patients with different killips class mentioned ; ( 7 ) ami type mentioned ; ( 8) enough sample size ( n > 100 ) of patients from individual study . we carefully searched on - line database of pubmed , web of science , china national knowledge infrastructure , and the cochrane controlled trials register databases until february 2015 to identify relevant studies . we used the following keywords : atrial fibrillation ( af ) , new - onset and killips , heart failure and myocardial infarction . titles and abstracts , as well as the reference lists of all the identified reports , were examined independently by two reviewers ( zhang ey and cui l ) in order to include potentially relevant studies . the two reviewers agreed on the inclusion / exclusion status in 90% of the reviewed studies . disagreements were resolved by discussion or consensus of a third reviewer ( li zy ) . in addition , a manual search was conducted using review articles on this topic , bibliographies of original papers , and abstracts of the scientific sessions of the , the american heart association , the european society of cardiology , heart rhythm society , and american college of cardiology during the past 2 years . to limit heterogeneity secondary to differences between study designs , the quality of each study was evaluated according to the guidelines developed by the evidence - based medicine working group and the united states preventive task force . we applied the point score system assessed by the following characteristics : ( 1 ) clear description of inclusion and exclusion criteria ; ( 2 ) study sample representative for mentioned population ; ( 3 ) clear description of sample selection ; ( 4 ) full specification of clinical and demographic variables ; ( 5 ) follow - up during hospitalization ; ( 6 ) no loss of follow - up ; ( 7 ) clear definition of killips class ; ( 8) clear definition of outcomes and outcome assessment ; ( 9 ) temporality ( assessment of killips class once at presentation ) ; ( 10 ) adjustment of possible confounders in multivariate analysis . if one of these key points was not mentioned clearly in a study , we considered it not performed data extraction was performed by two blind investigators ( zhang ey and cui l ) independently with a standard data extraction form to determine eligibility for inclusion . the following information collected was tabulated : ( 1 ) publication details : first author 's last name , publication year ; ( 2 ) characteristics of included studies : study population , definition of noaf , detection method , quality score , risk estimate and nation ; ( 3 ) baseline data of the studied population : sample size , age , gender , killips > i ( % ) , noaf ( % ) , current smoker ( % ) , left atrium diameter , left ventricle ejection fraction ( lvef ) , st - elevation myocardial infarction ( % ) , anterior myocardial infarction ( % ) , diabetes mellitus ( % ) , hypertension ( % ) , previous infarction ( % ) , coronary revascularization ( % ) . cardiac function on admission was scored according to the killips classification scheme class i iv , i as absence of heart failure , ii as presence of rales and/or jugular venous distention , iii as presence of pulmonary edema and iv as cardiogenic shock . killips class i at presentation was defined as severe cardiac dysfunction in our meta - analysis . the magnitude of association between high killips and noaf following ami was measured by adjusted odds ratio ( or ) or or with 95% confidence intervals ( cis ) . finally , we examined the heterogeneity with the standard chi - square test of heterogeneity . a pooled effect was calculated with a random - effects model when the chi - square test for heterogeneity was found significant , through which we could take within - study and between - study variance into account , while not significant , a fixed - effects model was still used . sensitivity analysis was done by dropping studies and checking the consistency of the overall effect estimate . all the jobs above were performed with review manager version 5.2 ( revman ; the cochrane collaboration , oxford , uk ) . we found a total of 3732 records via the primary articles search . among them , 1512 duplicates were discarded . however , after screening the titles and abstracts , 2194 studies were excluded because they were either review articles , laboratory studies , irrelevant to the current analysis , impertinent type of statistics , noncohort studies or retrospective studies . after the detailed evaluation , 5 prospective observational studies finally met the inclusion criteria [ figure 1 ] . the proportion of higher killips classification ranged from 11.7% to 25.0% , while incidence of noaf from 5.2% to 13.6% . among the 10,053 patients included in our meta - analysis the characteristics of each included studies and baseline data of the patients from corresponding study are depicted in tables 1 and 2 , respectively . the meta - analysis of these studies demonstrated that killips score > i was associated with higher incidence rate of noaf after ami [ or = 2.29 , 95% ci 1.962.67 , p < 0.00001 , figure 2 ] , no significant differences were found between individual trials ( p = 0.14 and i = 43% ) . characteristics of studies included in meta - analysis af : atrial fibrillation ; noaf : new - onset af ; ecg : electrocardiogram ; aor : adjusted odd ratio ; or : odd ratio . baseline clinical characteristics of included studies af : atrial fibrillation ; lad : left atrium diameter ; lvef : left ventricle ejection fraction ; stemi : st - elevated myocardial infarction ; dm : diabetes mellitus ; htn : hypertension ; n / a : not applicable . the risk of noaf following ami by higher killips class . our results indicated the strong predictive value of higher killips score for noaf after ami . late recent research showed only noaf perform independent predictive value for in - hospital mortality since different clinical characteristic and therapeutic implications , we should differ noaf from existing af . data from the randomized assessment of pexelizumab in ami trial showed the patients who developed noaf ( 6.3% ) even exceeded those with af at baseline ( 4.8% ) . the risk of noaf in the setting of ami was 8.2% in our meta - analysis . patients with noaf following ami were found to develop complications more easily , which includes cardiogenic shock , reinfarction , ventricular arrhythmias , and with worse laboratory or echocardiographic features . with the increase of the killips class , maximum killips class iii and iv appeared an independent predictor of overall mortality , new - onset af during acute coronary syndromes also carried a higher risk of death . greater incidence of killips class on admission usually indicates larger infarction size and correlates with a higher possibility of developing noaf in our result , which in turn aggravates the cardiac dysfunction . abundant evidence has suggested that noaf indicates higher rate of heart failure potentially , while in turn more serious heart failure could prompt greater likelihood of developing noaf , some studies have pointed out that noaf may be considered a symbol of the current cardiac insufficiency . prior studies have reported consistently that noaf is mainly associated with the increase in left ventricular end - diastolic volume and pressure , which indirectly increase left atrial wall tension and induces af occurrence . noaf among ami patients had been proved associated with the presence of left ventricular function impairment and atrial ischemia . rapid elevation of intra - atrial pressure with passive stretching of the left atrium was proved to facilitate af via activation of some ion channels in animal experiment , complicated with increased af vulnerability and shortening of the atrial effective refractory period . in a study paying attention of gender and weight , guenancia et al . found lvef impairment and higher killips class after ami usually reflect an acute increase in cardiac filling pressure and trigger noaf . therefore , severe left ventricular dysfunction and increased left atrial pressure may trigger noaf in ami patients . moreover , dorje et al . have indicated the predictive value of bnp for noaf in ami patients treated with primary pci , which to some extent supported the relationship between killips class and noaf . olgin et al . have reported for long time that the alterations of autonomic tone may also contribute to the noaf . jons et al . have also certified the key role of sympathetic cardiac autonomic activity dysfunction in noaf during ami . all above supported the potential relationship between noaf following ami and cardiac function reflected by killips score . considering our result , the killips class at admission could predict noaf within very period after ami , and as an easily obtainable parameter , may help to stratify risk for noaf in the setting of ami . although the total number of observational patients was large , first , the method for defining noaf was not accordant between various trials in our study . second , the type of noaf was not precisely classified , which may bring bias while confounding paroxysmal noaf into persistent or even permanent noaf . moreover , some included studies in our meta - analysis did not provide adjusted or of higher killips class for predicting noaf due to nonsignificant p value in univariate regression analysis . future investigations are warranted to clarify the mechanisms of noaf further . in conclusion , patients with noaf in the context of ami could be independently predicted by killips class > although the total number of observational patients was large , there were still several limitations in our meta - analysis . first , the method for defining noaf was not accordant between various trials in our study . second , the type of noaf was not precisely classified , which may bring bias while confounding paroxysmal noaf into persistent or even permanent noaf . moreover , some included studies in our meta - analysis did not provide adjusted or of higher killips class for predicting noaf due to nonsignificant p value in univariate regression analysis . future investigations are warranted to clarify the mechanisms of noaf further . in conclusion , patients with noaf in the context of ami could be independently predicted by killips class >

background : recent observational studies have shown that patients with higher killips score ( > i ) have higher risk of new - onset atrial fibrillation ( noaf ) following acute myocardial infarction ( ami ) , while others drew a neutral conclusion . the ultimate predictive value of high killips class on noaf remained obscure.methods:pubmed , web of science , china national knowledge infrastructure , and the cochrane controlled trials register databases were searched until february 2015 . of the 3732 initially identified studies , 5 observational studies with 10,053 patients were analyzed.results:the meta - analysis of these studies showed that higher killips score on admission was associated with higher incidence of noaf following ami ( odds ratio = 2.29 , 95% confidence interval 1.962.67 , p < 0.00001 ) , while no significant differences exist among individual trials ( p = 0.14 and i2 = 43%).conclusions : killips class > i was associated with the higher opportunity of developing noaf following ami .

I M Inclusion criteria Search strategies Quality assessment Data extraction Definition of important parameters Statistical analysis R D Limitations

nearly all of the photoactive iridium(iii ) complexes that are used as emitters in electroluminescent devices , as dyes in solar cells , in nonlinear optics ( nlo ) , as photoredox catalysts , as solar fuels , and in bioimaging contain conjugated five - membered chelated ligands , such as the commonly used 2-phenylpyridine ( ppyh ) , 2,2-bipyridine ( bpy ) , acetylacetonate ( acac ) , and picolinate ( pic ) . photoactive iridium complexes containing a six - membered cyclometalating chelate are very rare , and the few reported examples can be categorized into two families of complexes : those containing conjugated or nonconjugated bidentate cyclometalating ligands . for instance , in 2008 , song et al . obtained a phosphorescent ir(iii ) complex [ ir(dfb - pz)2(fptz ) ] ( where ( dfb - pz)h = 2,4-difluorobenzyl - n - pyrazole and fptz = 3-trifluoromethyl-5-(2-pyridyl)triazole ) containing a nonconjugated n - benzylpyrazole ligand to form a six - membered chelated framework . this complex is a blue emitter in dichloromethane with em = 437 and 460 nm ( pl = 10% and e = 0.10 s ) . the methylene spacer of the cyclometalated ligand effectively interrupts the -conjugation to produce a significant blue shift , compared to [ ir(dfppy)2(fptz ) ] ( where dfppyh = 2-(2,4-difluorophenyl)pyridine , which uses a five - membered ring chelate c^n ligand and the same ancillary ligand ( em = 460 and 489 nm in dichloromethane ) . complex [ ir(bis[2-(n - carbazolyl)pyridinato - n , c)picolinate ] ( [ ir(cpy)2(pic ) ] ) containing a six - membered chelating framework where the ligand is fully conjugated , leading to yellow luminescence in the recrystallized solid state with em = 538 nm ( pl = 5% ) . in an ongoing effort in our group to develop charged blue - emitting phosphors for solution - processed light - emitting electrochemical cells ( leecs ) and organic light emitting diodes ( oleds ) , we investigated the coordination of 2-benzhydrylpyridine ( bnpyh2 ) derivatives with ir(iii ) in order to access six - membered chelate complexes t1t3 ( figure 1a ) . surprisingly , given the prior art , upon sequential treatment of ircl36h2o with bnpyh2 and then 4,4-di - tert - butyl-2,2-bipyridine ( dtbubpy ) in a one - pot reaction , t1 was not obtained . instead the formation of 1 arises from a highly unusual double c h bond activation of the bnpyh2 ligand , which binds to the iridium in a tripodal fashion . following a similar synthetic protocol , analogues 2 and 3 , functionalized with either electron - donating tert - butyl or electron - withdrawing trifluoromethyl groups meta to the ir this is the first report of an iridium complex of the form [ ir(c^n^c)(n^n)cl ] and one in which the tridentate bis - cyclometalated ligand is a tripod featuring two six - membered metallacycles . indeed , the complex with the closest binding motif is [ ir(bppy)(bpy)cl ] , where bppy is 2-([1,1-biphenyl]-3-yl)pyridine . this previous complex possesses a related [ ir(c^c^n)(n^n)cl ] structure , but the bppy ligand coordinates to the iridium in the more commonly observed planar five - membered chelate fashion . it is a very poorly luminescent near - ir emitter in ch2cl2 ( em = 725 nm , pl = 8.4 10% ) . ir(iii ) complexes bearing monocyclometalating planar tridentate bis(five - membered ) chelate ligands ( e.g. , n^n^c or n^c^n ) and a cyclometalating bidentate ligand , c^n , have on the other hand been more widely explored , while kozhevnikov has reported dinuclear ir(iii ) complexes with a bridging ligand featuring two n^c^n motifs . ( a ) scheme for the proposed synthesis of the initial targets ( t1t3 ) and the synthesis of complexes 13 . ( a , i ) ircl36h2o , 2-ethoxyethanol / h2o ( 3:1 ) , reflux , 19 h ; ( ii ) dtbubpy , reflux , 6 h. ( b ) aq . nh4pf6 . compounds l1 , l2 , and l3 ( scheme 1 ) were synthesized in two steps via a grignard reaction followed by a reduction step and obtained as solids in good yields . a mixture of the corresponding proligand and ircl36h2o in 2-ethoxyethanol / h2o ( 3:1 ) was refluxed . after 19 h , dtbubpy 4,4-di - tert - butyl-2,2-bipyridyl was added at once , and heating was continued for 6 h to give the neutral complexes 13 as solids in a one - pot synthesis in 45% , 51% , and 55% yield , respectively ( figure 1a ) . complexes 13 were characterized by h , c , and , for 3 , f nmr spectroscopy ; esi - hr mass spectra ; elemental analysis ; and melting point determination ( see figures s21s30 in the supporting information ( si ) for nmr and esi - hr mass spectra ) . ( a , 1 ) mg , 1,2-dibromoethane , thf , n2 , reflux , 4 h. ( 2 ) methyl picolinate , thf , 0 c to r.t . , 90 min ; ( b , 1 ) hoac , 57% hi . ( 2 ) naohaq . , 0 c to r.t . ; ( c , 1 ) pbr3 , reflux , 2 h. ( 2 ) zn , hoac , ( 3 ) naohaq . , 0 c to r.t . the structures of 13 were determined by single - crystal x - ray diffraction ( figure 1b , table s1 ) . all three complexes possess a distorted octahedral geometry with the tridentate tripod ligand coordinated to the iridium to form two six - membered chelated rings . both cyclometalating carbon atoms are trans to the pyridine rings of the dtbubpy , and the pyridyl unit of the bnpy - type ligands is trans to the chloride . cl bond [ 2.375(3 ) for 1 , 2.3612(8 ) for 2 , and 2.369(2 ) for 3 ] is in the same range as that found for [ ir(tpy)(dmbpy)cl ] ( 2.357 , where tpy = 2,2:6,2-terpyridine and dmbpy = 4,4-dimethyl-2,2-bipyridine ) but is significantly shorter ( by ca . 0.1 ) than the ir cl bond in other cyclometalated tridentate ir(iii ) complexes . cc^n^c bonds [ 2.048(13 ) and 2.064(6 ) for 1 , 2.028(4 ) and 2.031(3 ) for 2 , and 2.017(7 ) and 2.027 for 3 ] , this leads also to a correspondingly shorter ir nc^n^c bond [ 2.055(11 ) for 1 , 2.044(3 ) for 2 , and 2.032(7 ) for 3 ] compared to the ir ndtbubpy bonds [ 2.158(10 ) and 2.159(11 ) for 1 , 2.127(3 ) and 2.140(3 ) for 2 , and 2.122(6 ) and 2.133(5 ) for 3 ] . the bite angle of the n^n ligand is unremarkable at 75.60(4) for 1 , 75.85(12) for 2 , and 76.1(2) for 3 and in line with cationic ir(iii ) complexes of the form [ ir(c^n)2(n^n ) ] . owing to the presence of the six - membered chelates , the c ir c bond angle is significantly larger ( 85.60(5) for 1 , 85.65(15) for 2 , and 84.(3 ) for 3 ) than the ndtbubpy ir the electrochemical behavior for 13 was evaluated by cyclic voltammetry ( cv ) and differential pulse voltammetry ( dpv ) in deaerated ch2cl2 solution at 298 k at a scan rate of 50 mv s using fc / fc as the internal reference and referenced with respect to sce . the electrochemistry data can be found in table 1 , and the voltammograms are shown in figure 2 . all complexes exhibit a quasi - reversible single electron oxidation peak , which is attributed to the ir(iii)/ir(iv ) redox couple with contributions from the bnpy - type ligand . complex 2 displays a lower oxidation potential ( 0.80 v ) than 1 ( 0.87 v ) , both of which are notably lower than [ ir(mesppy)2(dtbubpy)]pf6 ( e1/2;ox . = 1.17 v in deaerated ch2cl2 , where mesppy is 2-phenyl-4-mesityl - pyridinato ) . conversely , 3 shows a significantly anodically shifted oxidation potential at 1.14 v. the cvs of 13 show irreversible reduction waves that are monoelectronic as inferred from the respective dpvs . dft calculations ( figure 3a ) indicate that both the homo and homo1 , which are close in energy ( see figure s34 in the si ) , involve the iridium and chloride atoms and the two phenyl rings of the bnpy ligand . the lumo is almost exclusively localized on the dtbubpy ligand , while the lumo+1 is primarily on the pyridyl ring of the bnpy ligand . the reduction potentials of 1 and 2 are each found at 1.82 v , while the reduction wave of 3 at 1.62 v is anodically shifted by 200 mv compared to 1 and 2 . all three complexes are significantly more difficult to reduce than [ ir(mesppy)2(dtbubpy)]pf6 ( e1/2;red . cyclic voltammograms ( in solid lines ) and differential pulse voltammetry ( in dotted lines ) carried out in degassed ch2cl2 at a scan rate of 50 mv s , with fc / fc as the internal reference , referenced to sce ( 0.46 v vs sce ) . ( a ) representation of the four frontier mos of 1 . ( b ) side and top views of the spin density distribution for the lowest triplet state ( t1 ) of 1 . [ ru(bpy)3]pf6 in mecn as reference ( pl = 1.8% in aerated mecn at 298 k ) . measurements were carried out in degassed ch2cl2 at a scan rate of 50 mv s with fc / fc used as the internal reference , and referenced with respect to sce ( fc / fc = 0.46 v in ch2cl2 ) . the normalized uv vis absorption spectra of 13 recorded in ch2cl2 at 298 k are depicted in figure 4 and the data summarized in table s2 in the si . the invariance of the intense high - energy ( on the order of ( 11.5 ) 10 m cm ) absorption bands below 300 nm are ascribed to * ligand - centered ( lc ) transitions localized on the dtbubpy ligand . two moderately intense bands ( on the order of ( 35 ) 10 m cm ) in the region of 340360 nm and 390405 nm are assigned to mixed charge - transfer transitions with the former consisting of more metal - to - ligand / ligand - to - ligand charge - transfer ( mlct / llct ) character while the latter , according to td - dft calculations , implicate an intraligand ct ( ilct ) from the phenyl rings to the pyridyl heterocycle of the bnpy - type ligand ( see figure s34 and table s3 in the si ) . weak bands ( on the order of 10 m cm ) with onsets between 470 and 510 nm and tailing to 580 nm are attributed to a mixture of mlct / llct and spin - forbidden mlct / llct transitions involving the dtbubpy ligand . introduction of the tert - butyl groups in 2 results in a small red - shift of the ct bands below 340 nm , whereas the trifluoromethyl groups in 3 produce a significant blue - shift of these bands , trends that are corroborated by td - dft calculations ( figure s35 in the si ) . normalized uv vis absorption and photoluminescence spectra of 13 in ch2cl2 at 298 k. the normalized photoluminescence ( pl ) spectra of 13 in degassed ch2cl2 are shown in figure 4 , and the data are summarized in table 1 . upon photoexcitation at 420 nm , all complexes show a broad and unstructured profile , indicative of an emission with mixed ct character . in line with the trends observed in the absorption spectra and the oxidation potentials in the cvs , the emission maxima are 581 , 619 , and 630 nm for 3 , 1 , and 2 , respectively . these emission maxima match very closely to the vertical phosphorescence energies calculated by spin - unrestricted dft , which predicts emissions at 573 , 613 , and 622 nm , respectively . the calculations reveal that the emissive triplet state is localized on the iridium , chlorine , and dtbubpy but does not include significant contributions from the bnpy ligand ( figures 3b and s36 in the si ) . the photoluminescence quantum yield ( pl ) of 1 is 8% , which is lower than that of the yellow - emitting [ ir(ppy)2(dtbubpy)]pf6 ( pl = 64% in ch2cl2 , em = 570 nm , where ppyh is 2-phenylpyridine ) . the radiative rate constants , kr , for 1 and 2 are similar ( 2.52 vs 2.51 10 s , respectively ) . however , 2 exhibits a significantly larger nonradiative rate constant , knr ( 39.33 10 s ) , compared to 1 ( 28.93 10 s ) . complex 3 possesses both the largest kr ( 3.62 10 s ) and the smallest knr values ( 10.31 10 s ) in accordance with the energy gap law . in conclusion , a new family of luminescent iridium(iii ) complexes bearing an unprecedented tripodal bis(six - membered ) chelate tridentate ligand has been prepared through a highly unusual double cyclometalation reaction . current efforts are focused on further modulating the electronics through a combination of modifications of the ancillary di - imine and the monodentate chloride ligands . this unprecedented tripodal ligand opens new perspectives for the design of tridentate ir luminophores . an oven - dried flask was charged under a nitrogen atmosphere with magnesium turnings ( 0.911 g , 37.50 mmol , 5 equiv ) and thf ( 80 ml ) followed by 2 ml of 1,2-dibromoethane . after the observation of gas evolution , the corresponding bromo derivative ( 22.50 mmol , 3 equiv ) in thf ( 40 ml ) was added dropwise . the reaction mixture was heated under stirring and kept at reflux for 4 h , resulting in a color change of the solution to gray . the reaction mixture was then cooled in an ice bath , and a solution of methyl picolinate ( 7.50 mmol , 1.00 equiv ) in thf ( 40 ml ) was added carefully . the solution was allowed to warm to room temperature and was stirred for 90 min . the combined organic layers were dried over mgso4 , and the solvent was evaporated , leaving a residue , which was purified over silica ( 10% etoac in petroleum ether as the solvent ) . the desired fractions were combined , and the solvent was evaporated , leaving the title compound . compound a1 was prepared according to the general procedure and was obtained as a colorless solid ( 1.686 g , 6.45 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.60 ( d , j = 4.7 hz , 1h ) , 7.64 ( td , j = 7.8 , 1.8 hz , 1h ) , 7.407.21 ( m , 11h ) , 7.12 ( d , j = 7.9 hz , 1h ) , 6.28 ( s , 1h ) . c nmr ( 101 mhz , cdcl3 ) : 163.2 , 147.7 , 146.1 , 136.4 , 128.1 , 127.9 , 127.3 , 122.9 , 122.3 , 80.8 . hr - ms ( ftms ) [ m + h ] calculated ( c18h15noh ) : 262.1226 . found : 262.1226 . chn calcd for c18h15no : c , 82.73 ; h , 5.79 ; n , 5.36 . found : c , 82.68 ; h , 5.73 ; n , 5.41 . compound a2 was prepared according to the general procedure and was obtained as a colorless solid ( 1.994 g , 5.34 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.58 ( d , j = 3.2 hz , 1h ) , 7.677.60 ( m , 1h ) , 7.337.28 ( m , 4h ) , 7.247.12 ( m , 6h ) , 6.19 ( s , 1h ) , 1.30 ( s , 18h ) . c nmr ( 101 mhz , cdcl3 ) : 163.8 , 150.1 , 147.7 , 143.3 , 136.4 , 127.9 , 124.9 , 123.1 , 122.3 , 80.6 , 34.6 , 31.5 . hr - ms ( ftms ) [ m + h ] calculated ( c26h31noh ) : 374.2478 . found : 374.2476 . chn calcd for c26h31no : c , 83.60 ; h , 8.37 ; n , 3.75 . found : c , 83.45 ; h , 8.51 ; n , 3.88 . compound a3 was prepared according to the general procedure and was obtained as a beige solid ( 1.893 g , 4.76 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.64 ( d , j = 7.4 hz , 1h ) , 7.757.69 ( m , 1h ) , 7.60 ( s , 4h ) , 7.44 ( s , 4h ) , 7.31 ( d , j = 8.5 hz , 1h ) , 7.13 ( d , j = 9.7 hz , 1h ) , 6.50 ( s , 1h ) . c nmr ( 101 mhz , cdcl3 ) : 161.5 , 149.4 , 148.2 , 136.9 , 130.4 , 130.1 , 129.7 , 129.4 , 128.5 , 125.2 , 125.1 , 125.1 , 125.1 , 123.1 , 122.7 , 80.4 , 77.4 , 77.1 , 76.7 . hr - ms ( ftms ) [ m + h ] calculated ( c20h13f6noh ) : 398.0974 . found : 398.0965 . chn calcd for c20h13f6no : c , 60.46 ; h , 3.30 ; n , 3.53 . found : c , 60.51 ; h , 3.36 ; a mixture of a1 ( 0.837 g , 3.21 mmol ) , aqueous 57% hi ( 2.5 ml ) , and hoac ( 13 ml ) was heated to 100 c for 4 h. the resulting mixture was then cooled to 0 c and basified to ph 9 with an aqueous naoh solution ( 2 m ) . ethyl acetate ( 100 ml ) was added , and the mixture was washed successively with an aqueous nahso3 solution and brine . the combined organic layers were dried over mgso4 , and the solvent was evaporated . the residue was purified over silica ( 10% etoac in petroleum ether as the solvent ) . the desired fractions were combined and the solvent evaporated leaving a beige solid ( 0.788 g , 3.21 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.64 ( d , j = 4.1 hz , 1h ) , 7.667.59 ( m , 1h ) , 7.33 ( t , j = 7.3 hz , 4h ) , 7.24 ( dd , j = 21.8 , 7.2 hz , 6h ) , 7.187.10 ( m , 2h ) , 5.76 ( s , 1h ) . c nmr ( 101 mhz , cdcl3 ) : 163.2 , 149.5 , 142.7 , 136.4 , 129.4 , 128.4 , 126.5 , 123.8 , 121.4 , 59.4 . hr - ms ( ftms ) [ m + h ] calculated ( c18h15nh ) : 246.1277 . found : 246.1277 . a mixture of the a2 ( 0.900 g , 2.41 mmol ) , aqueous 57% hi ( 2.70 ml ) , and hoac ( 13.20 ml ) was heated to 100 c for 4 h. the resulting mixture was then cooled to 0 c and basified to ph 9 with an aqueous naoh solution ( 2 m ) . ethyl acetate ( 100 ml ) was added , and the mixture was washed successively with an aqueous nahso3 solution and brine . the combined organic layers were dried over mgso4 , and the solvent was evaporated . the residue was purified over silica ( 10% etoac in petroleum ether as the solvent ) . the desired fractions were combined and the solvent evaporated , yielding the title compound as oil ( 0.655 g , 1.83 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.628.56 ( m , 1h ) , 7.627.56 ( m , 1h ) , 7.31 ( s , 4h ) , 7.10 ( s , 6h ) , 5.62 ( s , 1h ) , 1.29 ( s , 18h ) . c nmr ( 126 mhz , cdcl3 ) : 163.8 , 149.6 , 149.2 , 139.9 , 136.5 , 129.0 , 125.4 , 123.9 , 121.4 , 58.7 , 34.5 , 31.5 . hr - ms ( asap ) [ m + h ] calculated ( c26h31nh ) : 358.2535 . found : 358.2534 . a mixture of a3 ( 0.500 g , 1.26 mmol , 1 equiv ) and pbr3 ( 25 ml ) was vigorously stirred and heated and kept at 110 c for 2 h. the mixture was then cooled to r.t . and was carefully poured onto ice , and aqueous naoh ( 2 m ) was added until the ph was neutral . the organic layer was dried over mgso4 , and the solvent was evaporated , leaving a residue which was dissolved in acetic acid ( 50 ml ) . then , zinc dust ( 0.799 g , 12.60 mmol , 10 equiv ) was added . after 1 h , 20 ml of water was carefully added , and aqueous naoh ( 2 m ) was added until the ph was neutral . the organic layer was dried over mgso4 , and the solvent was evaporated , leaving a residue which was purified over silica ( 10% etoac in petroleum ether as the solvent ) . the desired fractions were combined , and the solvent was evaporated , leaving colorless oil ( 0.117 mg , 0.31 mmol ) . yield : 24% . h nmr ( 400 mhz , cdcl3 ) : 8.63 ( ddd , j = 4.9 , 1.9 , 0.9 hz , 1h ) , 7.66 ( td , j = 7.7 , 1.9 hz , 1h ) , 7.57 ( d , j = 8.2 hz , 4h ) , 7.30 ( d , j = 8.1 hz , 4h ) , 7.20 ( ddd , j = 7.6 , 4.8 , 1.1 hz , 1h ) , 7.10 ( dt , j = 7.9 , 1.1 hz , 1h ) , 5.75 ( s , 1h ) . c nmr ( 126 mhz , cdcl3 ) : 161.4 , 150.1 , 146.0 , 137.0 , 129.8 , 129.3 , 125.7 , 125.3 , 124.0 , 122.2 , 58.9 hr - ms ( ftms ) [ m + h ] calculated ( c20h13f6nh ) : 382.1030 . found : 382.1023 . a mixture of the corresponding ligand ( 1.2 equiv ) and ircl36h2o ( 1 equiv ) in 2-ethoxyethanol ( 15 ml ) and h2o ( 5 ml ) was heated under stirring to 125 c . after 19 h , 4,4-di - tert - butyl-2,2-bipyridine ( 1.5 equiv ) was added , and heating was continued . after 6 h , the solvent was evaporated , leaving a solid , which was filtered over silica ( 1% meoh in ch2cl2 ) . the desired fractions were combined , and the solvent was evaporated , leaving a solid which was washed with diethyl ether . the general one - pot protocol using 0.114 g ( 0.38 mmol ) of ircl36h2o was followed , and 1 was obtained as a red solid ( 0.127 g , 0.0.13 mmol ) . h nmr ( 400 mhz , cd2cl2 ) : 8.75 ( d , j = 5.8 hz , 2h ) , 8.42 ( d , j = 1.7 hz , 2h ) , 7.70 ( dd , j = 7.3 , 1.4 hz , 2h ) , 7.53 ( d , j = 7.5 hz , 1h ) , 7.49 ( dd , j = 5.9 , 1.9 hz , 3h ) , 7.24 ( dd , j = 7.1 , 1.6 hz , 2h ) , 7.19 ( d , j = 5.9 hz , 1h ) , 6.90 ( dtd , j = 26.4 , 7.3 , 1.5 hz , 4h ) , 6.456.39 ( m , 1h ) , 5.28 ( s , 1h ) , 1.50 ( s , 18h ) . c nmr ( 126 mhz , cd2cl2 ) : 165.4 , 162.6 , 157.4 , 151.8 , 151.3 , 143.9 , 141.4 , 138.7 , 137.0 , 125.8 , 124.0 , 123.8 , 123.4 , 122.7 , 122.1 , 120.8 , 69.7 , 35.9 , 30.8 . hr - ms ( asap ) [ m cl ] calculated ( c36h37irn3 ) : 704.2618 . found : 704.2618 . chn calcd for c36h37clirn33/2 h2o : c , 56.42 ; h , 5.26 ; n , 5.48 . found : c , 56.45 ; h , 5.24 ; n , 5.28 . the general one - pot protocol using 0.088 g ( 0.30 mmol ) of ircl36h2o was followed , and 2 was obtained as a red solid ( 0.129 g , 0.15 mmol ) . h nmr ( 400 mhz , cd2cl2 ) : 8.79 ( d , j = 5.9 hz , 2h ) , 8.42 ( d , j = 2.0 hz , 2h ) , 7.80 ( d , j = 2.1 hz , 2h ) , 7.497.42 ( m , 4h ) , 7.13 ( d , j = 7.7 hz , 3h ) , 6.89 ( dd , j = 7.7 , 2.1 hz , 2h ) , 6.38 ( ddd , j = 7.0 , 5.9 , 2.0 hz , 1h ) , 5.22 ( s , 1h ) , 1.51 ( s , 18h ) , 1.32 ( s , 18h ) . c nmr ( 126 mhz , cd2cl2 ) : 166.0 , 162.6 , 157.5 , 151.7 , 151.3 , 147.8 , 141.1 , 140.4 , 136.8 , 135.9 , 123.7 , 123.6 , 122.7 , 122.4 , 120.8 , 118.8 , 68.6 , 35.9 , 34.9 , 32.1 , 30.9 . [ m cl ] calculated ( c44h53irn3 ) 816.3869 . found : 816.3867 . chn calcd for c44h53clirn3 : c , 62.06 ; h , 6.27 ; n , 4.93 . found : c , 61.96 ; h , 6.31 ; n , 5.02 . the general one - pot protocol using 0.060 g ( 0.20 mmol ) of ircl36h2o was followed , and 3 was obtained as a yellow solid ( 0.096 g , 0.11 mmol ) . h nmr ( 400 mhz , cd2cl2 ) : 8.59 ( d , j = 5.8 hz , 2h ) , 8.44 ( d , j = 2.0 hz , 2h ) , 7.98 ( d , j = 2.0 hz , 2h ) , 7.627.48 ( m , 4h ) , 7.37 ( d , j = 7.7 hz , 2h ) , 7.207.15 ( m , 3h ) , 6.50 ( ddd , j = 7.5 , 5.9 , 1.7 hz , 1h ) , 5.46 ( s , 1h ) , 1.51 ( s , 18h ) . c nmr ( 126 mhz , cd2cl2 ) : 163.5 , 163.3 , 157.3 , 151.9 , 151.1 , 147.4 , 142.2 , 137.6 , 134.7 , 127.6 , 126.7 , 124.5 , 124.3 , 123.5 , 123.4 , 121.2 , 119.4 , 69.2 , 36.0 , 30.8 . f nmr ( 376 mhz , cd2cl2 ) : 61.58 . [ m cl ] calculated ( c38h35f6irn3 ) : 840.2364 . found : 840.2379 . chn calcd for c38h35f6irn3 : c , 52.14 ; h , 4.03 ; n , 4.80 . found : c , 52.10 ; h , 4.16 ; n , 4.74 .

a new family consisting of three luminescent neutral ir(iii ) complexes with the unprecedented [ ir(c^n^c)(n^n)cl ] architecture , where c^n^c is a bis(six - membered ) chelating tridentate tripod ligand derived from 2-benzhydrylpyridine ( bnpy ) and n^n is 4,4-di - tert - butyl-2,2-bipyridine ( dtbubpy ) , is reported . x - ray crystallography reveals an unexpected and unusual double c h bond activation of the two distal nonconjugated phenyl rings of the bnpy coupled with a very short ir cl bond trans to the pyridine of the bnpy ligand . depending on the substitution on the bnpy ligand , phosphorescence , ranging from yellow to red , is observed in dichloromethane solution . a combined study using density functional theory ( dft ) and time - dependent dft ( td - dft ) corroborates the mixed charge - transfer nature of the related excited states .

Introduction Results and Discussion Conclusions Experimental Section

pain is highly prevalent in older people and is associated with depression , falls , poor quality of life , sleep disturbance , mobility limitation and decline in physical function [ 1 , 2 ] . the prevalence of pain doubles from 2 years to 1 month before death . up to two - thirds of people with advanced cancer report pain . it has been recognized that optimizing pain management in people with cancer requires an individualized approach which seeks to maximize pain relief but minimize the risk of adverse drug events ( ades ) [ 5 , 6 ] . frailty is a geriatric syndrome characterized by a decreased homeostatic reserve resulting in an increased susceptibility to ades . however , analgesic prescribing for frail older people is challenging due to increased heterogeneity in drug disposition and response , multi - morbidity and changes in body composition . analgesic selection is complicated by the risk drug drug and drug disease interactions . additionally , since frailty is associated with comorbidity and older age , many frail older people are excluded from participation in clinical trials of analgesics . cross - sectional studies have identified an association between pain and frailty [ 2 , 10 ] . to our knowledge , only one previous study has specifically investigated analgesic use and frailty . in this study , more than 65 % of frail older people used analgesics but nearly half wanted their physicians to pay greater attention to pain management . no previous studies have investigated the association between analgesic use , pain and frailty in older people with cancer . this is important because both frailty and analgesic - related ades may be exacerbated by cancer and chemotherapy . a patient s first presentation to a geriatric oncology outpatient clinic represents an opportunity to optimize pain management . the objective of this study was to investigate and characterize the relationship between analgesic use and pain by frailty status in older outpatients with cancer . we expected a higher prevalence of pain among outpatients who were frail , but a less pronounced relationship between analgesic use and pain level due to clinicians being reluctant to prescribe analgesics to people perceived to be at high risk of adverse drug events . this knowledge is important for developing safe and effective strategies for analgesic use in these outpatients . the study participants and data collection have been described previously . all patients aged 70 years and over who presented at the medical oncology outpatient clinic at the royal adelaide hospital between january 2009 and july 2010 completed a structured data collection instrument . the instrument captured each participant s age , sex , diagnoses , medications , general pain ( 10-point visual analogue scale , vas ) , instrumental activities of daily living ( iadls ) , karnofsky performance scale ( kps ) physical function ( sf-36 ) , self - reported weight loss over the past 6 months , exhaustion [ 15 , 16 ] and distress ( via a 10-point vas ) . the instrument was completed by the outpatient with or without involvement from a family member , and any sections that were incomplete were completed with a nurse at their first visit to the clinic . self - reported medication use was verified at the first visit by a nurse with access to each outpatient s medical records , and any medications that were not self - reported were recorded . data about prescription , non - prescription and complementary and alternative medications ( cams ) were collected separately to ensure a full history was obtained . the validity of the medication lists was estimated by comparing a sample of 30 medication lists to those obtained by clinical pharmacist interviews . there was a greater than 70 % concordance overall , with approximately 80 % concordance for prescription medications . analgesics were defined as opioids ( buprenorphine , codeine + paracetamol , dextropropoxyphene , fentanyl , methadone , morphine , oxycodone and tramadol ) , paracetamol and non - steroidal anti - inflammatory drugs ( nsaids ; celecoxib , diclofenac , ibuprofen , ibuprofen + codeine , indomethacin , meloxicam and naproxen ) . frailty was defined using a modified version of fried s frailty phenotype [ 15 , 21 , 22 ] which considered five criteria : dependence in at least one iadl , weight loss of > 5 % over the past 6 months , an exhaustion score of at least three , kps < 70 % and dependence in at least one sf-36 physical function domain . outpatients were classed as robust if they had none of the criteria , pre - frail if they had one or two of the criteria and frail if they had 3 of the criteria . demographic and clinical characteristics were summarized numerically and graphically by frailty status ( robust , pre - frail or frail ) . logistic regression was used to compute unadjusted and adjusted odds ratios ( ors ) and 95 % confidence intervals ( 95 % cis ) for the relationship between analgesic use and pain score for each frailty group separately . the analyses were adjusted for age , charlson s comorbidity index ( cci ) and sex . the study was approved by the royal adelaide hospital human research ethics committee , the university of south australia human research ethics committee and the monash university human research ethics committee . the study participants and data collection have been described previously . all patients aged 70 years and over who presented at the medical oncology outpatient clinic at the royal adelaide hospital between january 2009 and july 2010 completed a structured data collection instrument . the instrument captured each participant s age , sex , diagnoses , medications , general pain ( 10-point visual analogue scale , vas ) , instrumental activities of daily living ( iadls ) , karnofsky performance scale ( kps ) physical function ( sf-36 ) , self - reported weight loss over the past 6 months , exhaustion [ 15 , 16 ] and distress ( via a 10-point vas ) . the instrument was completed by the outpatient with or without involvement from a family member , and any sections that were incomplete were completed with a nurse at their first visit to the clinic . self - reported medication use was verified at the first visit by a nurse with access to each outpatient s medical records , and any medications that were not self - reported were recorded . data about prescription , non - prescription and complementary and alternative medications ( cams ) were collected separately to ensure a full history was obtained . the validity of the medication lists was estimated by comparing a sample of 30 medication lists to those obtained by clinical pharmacist interviews . there was a greater than 70 % concordance overall , with approximately 80 % concordance for prescription medications . analgesics were defined as opioids ( buprenorphine , codeine + paracetamol , dextropropoxyphene , fentanyl , methadone , morphine , oxycodone and tramadol ) , paracetamol and non - steroidal anti - inflammatory drugs ( nsaids ; celecoxib , diclofenac , ibuprofen , ibuprofen + codeine , indomethacin , meloxicam and naproxen ) . frailty was defined using a modified version of fried s frailty phenotype [ 15 , 21 , 22 ] which considered five criteria : dependence in at least one iadl , weight loss of > 5 % over the past 6 months , an exhaustion score of at least three , kps < 70 % and dependence in at least one sf-36 physical function domain . outpatients were classed as robust if they had none of the criteria , pre - frail if they had one or two of the criteria and frail if they had 3 of the criteria . demographic and clinical characteristics were summarized numerically and graphically by frailty status ( robust , pre - frail or frail ) . logistic regression was used to compute unadjusted and adjusted odds ratios ( ors ) and 95 % confidence intervals ( 95 % cis ) for the relationship between analgesic use and pain score for each frailty group separately . the analyses were adjusted for age , charlson s comorbidity index ( cci ) and sex . the study was approved by the royal adelaide hospital human research ethics committee , the university of south australia human research ethics committee and the monash university human research ethics committee . robust , pre - frail and frail outpatients had a median age of 77 , 75 and 78 years , respectively ; 65 , 54 and 62 % being male . robust , pre - frail and frail outpatients used a median of four , five and seven medications , respectively . outpatients who were frail had higher pain scores ( fig . 1 ; medians of 1 , 2 and 5 for robust , pre - frail and frail outpatients , respectively ) and had a higher prevalence of analgesic use ( corresponding percentages of 21 , 34 and 53 % ; fig . 1 ) . analgesic users who were frail used more medications ( medians of six , eight and nine for robust , pre - frail and frail outpatients who used analgesics , respectively ) . of the analgesic users in the robust group ( n = 21 ) , five ( 24 % ) used an opioid , 14 ( 67 % ) used paracetamol , five ( 24 % ) used an nsaid and three ( 14 % ) used more than one analgesic . of the analgesic users in the pre - frail group ( n = 65 ) , 32 ( 49 % ) used an opioid ( most commonly oxycodone ; n = 15 ) , 44 ( 68 % ) used paracetamol , 18 ( 28 % ) used an nsaid ( most commonly celecoxib ; n = 15 ) and 30 ( 46 % ) used more than one analgesic . of the analgesic users in the frail group ( n = 50 ) , 29 ( 58 % ) used an opioid ( most commonly oxycodone ; n = 19 ) , 31 ( 62 % ) used paracetamol , four ( 8 % ) used an nsaid and 24 ( 48 % ) used more than one analgesic.fig . 1distributions of pain score and analgesic use by frailty status distributions of pain score and analgesic use by frailty status figure 2 displays the unadjusted relationship between analgesic use and pain by frailty status . for robust outpatients , there was weak evidence for a 30 % relative increase in the odds of analgesic use between outpatients who differed by one unit of pain score after adjusting for age , cci and sex ( 95 % ci for the or 0.9951.71 , p = 0.0532 ) . for pre - frail outpatients , there was evidence for a negative quadratic ( i.e. concave ) relationship between the log odds of analgesic use and pain ( adjusted or for the quadratic coefficient : 0.952 , 95 % ci 0.9100.993 , p = 0.0244 ) . for frail outpatients , there was an 8 % relative increase in the adjusted odds of analgesic use between outpatients who differed by one unit of pain score , but no statistical evidence for association ( 95 % ci 0.9341.26 , p = 0.298).fig . 2unadjusted associations between analgesic use and pain score by frailty status unadjusted associations between analgesic use and pain score by frailty status this was the first study to investigate the association between analgesic use and pain in older outpatients with cancer . the main findings were that analgesic use increased linearly with pain in robust and frail outpatients , but there was a concave relationship between analgesic use and pain in pre - frail outpatients . there was a graded association between pain , analgesic and overall medication according to frailty status . robust outpatients had a lower prevalence of pain , analgesic and overall medication use than frail outpatients . the magnitude of the positive linear relationship between analgesic use and pain was most pronounced in the robust group , which may indicate that clinicians feel that the benefits of analgesics in robust older people with cancer outweigh potential risks . for the frail group , this positive linear relationship was less pronounced , with lesser prescribing of analgesics per unit difference of pain . this may reflect clinicians caution with prescribing analgesics in this vulnerable group of this cohort . the higher prevalence of pain in pre - frail and frail outpatients suggests possible underuse of pharmacological and non - pharmacological treatment approaches . this is consistent with previous research demonstrating that age - related physiological changes , polypharmacy and multi - morbidity are potential barriers to effective pain management . interestingly , the relationship between analgesic use and pain was concave in the pre - frail group . this may represent hesitation in prescribing analgesics to pre - frail older people with cancer experiencing higher levels of pain . these people may have been susceptible to ades , drug drug interactions and/or progression to frailty . recognizing and treating pain in pre - frail outpatients is important because pain is associated with depression and can negatively impact quality of life and function [ 26 , 27 ] . our study also demonstrated differences in the types of analgesics prescribed according to frailty status . people who were robust were more likely to receive nsaids ( n = 5 , 24 % ) and less likely to receive opiates ( n = 5 , 24 % ) when compared to people who were frail ( nsaids n = 4 , 8 % and opiates n = 29 , 58 % ) . this may reflect prescribers desire to avoid ades and drug drug interactions in frail people . additionally , people who were pre - frail had high levels of both nsaids ( n = 18 , 28 % ) and opiates ( n = 32 , 49 % ) , reflecting the complexity of treating pain in this group . demographic , clinical and medication data were collected using a structured data collection instrument and verified at the initial consultation . the measure of pain used in this study is well established and has been shown to be valid , reliable and appropriate for overall pain in a clinical setting . however , since it was a measure of general pain , we could not assess the prevalence of particular types of pain , such as neuropathic or musculoskeletal . nevertheless , this non - discriminatory measure of pain was consistent with our non - discriminatory definition of analgesic use it is possible that outpatients with more advanced cancer may be more likely to be frail , and may experience more pain , regardless of the analgesic used . we did not distinguish between regular and as - needed use of analgesics , or investigate the use of adjuvant therapies or non - pharmacological treatments . the study was conducted in a single outpatient oncology clinic , hence the results may not be generalizable to other geriatric oncology cohorts . as this study was cross - sectional , it was not possible to describe individual outpatient trajectories of analgesic use over time , and how these trajectories related to pain and frailty transitions . thus longitudinal studies are warranted . demographic , clinical and medication data were collected using a structured data collection instrument and verified at the initial consultation . the measure of pain used in this study is well established and has been shown to be valid , reliable and appropriate for overall pain in a clinical setting . however , since it was a measure of general pain , we could not assess the prevalence of particular types of pain , such as neuropathic or musculoskeletal . nevertheless , this non - discriminatory measure of pain was consistent with our non - discriminatory definition of analgesic use it is possible that outpatients with more advanced cancer may be more likely to be frail , and may experience more pain , regardless of the analgesic used . we did not distinguish between regular and as - needed use of analgesics , or investigate the use of adjuvant therapies or non - pharmacological treatments . the study was conducted in a single outpatient oncology clinic , hence the results may not be generalizable to other geriatric oncology cohorts . as this study was cross - sectional , it was not possible to describe individual outpatient trajectories of analgesic use over time , and how these trajectories related to pain and frailty transitions . thus longitudinal studies are warranted . in this cohort of older people recently diagnosed with cancer , analgesic use increased linearly with pain in robust and frail outpatients , and there was evidence for a concave relationship between analgesic use and pain in pre - frail outpatients . our findings suggest additional strategies are needed to optimize analgesic use in older outpatients with cancer , particularly in pre - frail outpatients experiencing high levels of pain . future research should investigate the role of analgesics as part of a comprehensive and high quality approach to pain management in these outpatients .

backgroundpain management can be challenging in frail older people with cancer due to drug drug interactions and heightened susceptibility to adverse drug events.objectiveto investigate the relationship between analgesic use and pain by frailty status in older outpatients with cancer.methodsa total of 385 consecutive patients aged 70 years and over who presented to an outpatient oncology clinic between january 2009 and july 2010 completed structured assessments of analgesic use ( opioids , paracetamol or non - steroidal anti - inflammatory drugs ) , pain ( 10-point visual analogue scale ) and clinical factors . frailty was derived using modified fried s frailty phenotype . logistic regression was used to compute adjusted odds ratios ( ors ) and 95 % confidence intervals ( cis ) for the relationship between analgesic use and pain for each frailty group ( robust , pre - frail or frail).resultsfor robust outpatients ( n = 101 ) , there was weak evidence for a 30 % relative increase in the adjusted odds of analgesic use between outpatients who differed by one unit of pain score ( 95 % ci 0.9951.71 , p = 0.0532 ) . for pre - frail outpatients ( n = 190 ) , there was evidence for a negative quadratic relationship ( adjusted or for the quadratic coefficient : 0.952 , 95 % ci 0.9100.993 , p = 0.0244 ) . for frail outpatients ( n = 94 ) , there was an 8 % relative increase in the adjusted odds of analgesic use between outpatients who differed by one unit of pain score , but no statistical evidence for association ( 95 % ci 0.9341.26 ; p = 0.298).conclusionsthese findings can be considered for the ongoing development of safe , effective strategies for analgesic use in older outpatients with cancer .

Introduction Methods Study Population and Data Collection Measures and Definitions Statistical Analysis Ethical Considerations Results Discussion Strengths and Limitations Conclusions

in addition to being the first cause of mortality for persons under the age of 45 , injury represents the disease with the highest financial burden . furthermore , the demographic shift along with the incessant development of new and costly technologies have led to an explosion in the costs of trauma care.[13 ] in the light of evidence suggesting that acute clinical trauma care is suboptimal and that patient outcomes vary across trauma centers , health care authorities worldwide are expressing the urgent need for information about health care performance . performance in health care is widely measured using donabedian 's structure - process - outcome model . structure refers to the physical environment of the health care facility including human resources , process refers to clinical interventions in individual patients , and outcome refers to the status of the patient at the end of an episode of care . according to this model , improvements in structure influence process and ultimately performance evaluation in trauma began in 1976 , when the american college of surgeons committee on trauma ( acs - cot ) introduced a series of 12 audit filters , updated to 22 in 1993 . however , these audit filters are based uniquely on clinical processes and patient outcome . in organised trauma systems , structure is traditionally evaluated in a qualitative fashion with on - site accreditation visits by a committee of external experts who verify adherence to a series of criteria recommended by acs - cot . no quantitative indicator of structural performance has been proposed for trauma center evaluation despite the widespread use of accreditation procedures . the objective of this study was to develop and validate a structural performance indicator that can be ascertained from accreditation visit data and used to drive performance improvement efforts . the study was based on the inclusive trauma system of the province of quebec , canada . the quebec trauma system was instated in 1993 and involves regionalized care from urban level i trauma centers through to rural community hospitals . at the end of the study period , the system included 6 level i ( including 2 pediatric ) , 4 level ii , 21 level iii , and 28 level iv centers . standardized prehospital protocols ensure that major trauma cases are taken to these hospitals and standing agreements regulate interhospital transfers within the system . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . the quebec accreditation process was performed by an independent trauma medical counsel comprising 30 medical experts from provincial trauma centers . the medical counsel evaluated each center using a checklist of items based on acs - cot criteria . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . we first developed a standardized evaluation grid to extract qualitative information from the accreditation reports ( appendix ) . this grid included 73 items grouped under three themes commitment , trauma program , and procedural protocols that were based on the acs - cot checklist . a preliminary series of 10 randomly selected reports were transposed independently onto the grid by five experts from the trauma medical counsel to standardize the transposition process . after this run - in period , a single evaluator transposed all reports onto the evaluation grid . a series of 12 randomly selected reports were duplicated and inserted under a fictitious hospital among the reports to verify intraobserver reliability . to generate a quantitative score from the evaluation grid , each item was scored either on a four - point likert scale ( 0-very negative to 4-very positive ) or on a binary scale ( 0-absent/4-present ) . weights were then assigned to each item of the grid by group of 10 experts from the trauma medical counsel according to perceived importance . the structural performance score was calculated for each trauma center as the weighted sum of the 73 items , standardized to range between 0 and 100 ( see appendix ) . a higher score thus represents better structural performance ; a score of 0 would be given to a trauma center with all items absent or very negative whereas a score of 100 would be given to a trauma center with all items present or very positive . trauma center accreditation grid : items and their individual weights ( sum = 100 points ) since the accreditation reports were written in short form , elements of the evaluation grid were not all thoroughly documented in each report . in addition , some items were not scored because they were not applicable to a particular level of trauma center ( see appendix ) . sensitivity analyses to verify the robustness of results to this treatment of missing data are presented in the results section . structural performance scores are presented using modified rank plots whereby trauma centers are ordered by designation level and volume . because data are based on only one accreditation visit , confidence intervals ( ci ) we , therefore , identified outliers by plotting 2 standard deviation around the global mean . the structural performance indicator was evaluated in terms of intra - observer reliability , discrimination between trauma centers , construct validity , and forecasting according to recommendations for evaluating composite indicators proposed by the agency for healthcare research and quality . intraobserver reliability was evaluated by calculating quadratic weighted kappa statistics on each item for a sample of 12 randomly selected reports that were coded twice by the same evaluator . discrimination was defined as the ability of the indicator to differentiate performance between trauma centers and was evaluated with the coefficient of variation , calculated as the ratio of the standard deviation of the structural performance scores to their mean multiplied by 100 . the ci of the coefficient of variation was generated using boostrap resampling ( n = 500 ) . coefficients of variation are expressed as a percentage where higher values indicate higher between - center variation . a coefficient of variation with a lower 95% ci above 10% was considered to reflect a distribution with high variation , synonym of good between - center discrimination . construct validity was defined as the degree of association between the structural performance indicator and other measures of quality and was assessed by evaluating the correlation of the structure scores with the following : i ) designation levels , ii ) trauma patient volume , iii ) accreditation recommendations . forecasting was defined as the ability of the structural performance indicator to predict structural performance over time . for this analysis we calculated structural performance scores using accreditation reports completed between 1994 and 1999 during the run - in period of the quebec trauma system ( data not used in any other analyses ) . we then evaluated the correlation between structural performance scores generated from the two waves of accreditation visits ( 1994 - 1999 and 1998 - 2005 ) . since the first wave of visits was only available for 57/59 trauma centers , the correlation coefficient was based on n = 57 . sensitivity analyses were performed to assess the robustness of the structural performance scores to the treatment of missing data items and the weighting scheme . for missing data items , we evaluated the correlation in trauma center structural performance scores when missing data values were coded 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) instead of 2 ( neutral ) . for the weighting scheme , we evaluated the correlation in trauma center ranks when equal weights were used over consensus - based weights . the study was based on the inclusive trauma system of the province of quebec , canada . the quebec trauma system was instated in 1993 and involves regionalized care from urban level i trauma centers through to rural community hospitals . at the end of the study period , the system included 6 level i ( including 2 pediatric ) , 4 level ii , 21 level iii , and 28 level iv centers . standardized prehospital protocols ensure that major trauma cases are taken to these hospitals and standing agreements regulate interhospital transfers within the system . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . the quebec accreditation process was performed by an independent trauma medical counsel comprising 30 medical experts from provincial trauma centers . the medical counsel evaluated each center using a checklist of items based on acs - cot criteria . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . the quebec accreditation process was performed by an independent trauma medical counsel comprising 30 medical experts from provincial trauma centers . the medical counsel evaluated each center using a checklist of items based on acs - cot criteria . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . we first developed a standardized evaluation grid to extract qualitative information from the accreditation reports ( appendix ) . this grid included 73 items grouped under three themes commitment , trauma program , and procedural protocols that were based on the acs - cot checklist . a preliminary series of 10 randomly selected reports were transposed independently onto the grid by five experts from the trauma medical counsel to standardize the transposition process . after this run - in period , a single evaluator transposed all reports onto the evaluation grid . a series of 12 randomly selected reports were duplicated and inserted under a fictitious hospital among the reports to verify intraobserver reliability . to generate a quantitative score from the evaluation grid , each item was scored either on a four - point likert scale ( 0-very negative to 4-very positive ) or on a binary scale ( 0-absent/4-present ) . weights were then assigned to each item of the grid by group of 10 experts from the trauma medical counsel according to perceived importance . the structural performance score was calculated for each trauma center as the weighted sum of the 73 items , standardized to range between 0 and 100 ( see appendix ) . a higher score thus represents better structural performance ; a score of 0 would be given to a trauma center with all items absent or very negative whereas a score of 100 would be given to a trauma center with all items present or very positive . trauma center accreditation grid : items and their individual weights ( sum = 100 points ) since the accreditation reports were written in short form , elements of the evaluation grid were not all thoroughly documented in each report . in addition , some items were not scored because they were not applicable to a particular level of trauma center ( see appendix ) . sensitivity analyses to verify the robustness of results to this treatment of missing data are presented in the results section . structural performance scores are presented using modified rank plots whereby trauma centers are ordered by designation level and volume . because data are based on only one accreditation visit , confidence intervals ( ci ) we , therefore , identified outliers by plotting 2 standard deviation around the global mean . the structural performance indicator was evaluated in terms of intra - observer reliability , discrimination between trauma centers , construct validity , and forecasting according to recommendations for evaluating composite indicators proposed by the agency for healthcare research and quality . intraobserver reliability was evaluated by calculating quadratic weighted kappa statistics on each item for a sample of 12 randomly selected reports that were coded twice by the same evaluator . discrimination was defined as the ability of the indicator to differentiate performance between trauma centers and was evaluated with the coefficient of variation , calculated as the ratio of the standard deviation of the structural performance scores to their mean multiplied by 100 . the ci of the coefficient of variation was generated using boostrap resampling ( n = 500 ) . coefficients of variation are expressed as a percentage where higher values indicate higher between - center variation . a coefficient of variation with a lower 95% ci above 10% was considered to reflect a distribution with high variation , synonym of good between - center discrimination . construct validity was defined as the degree of association between the structural performance indicator and other measures of quality and was assessed by evaluating the correlation of the structure scores with the following : i ) designation levels , ii ) trauma patient volume , iii ) accreditation recommendations . forecasting was defined as the ability of the structural performance indicator to predict structural performance over time . for this analysis we calculated structural performance scores using accreditation reports completed between 1994 and 1999 during the run - in period of the quebec trauma system ( data not used in any other analyses ) . we then evaluated the correlation between structural performance scores generated from the two waves of accreditation visits ( 1994 - 1999 and 1998 - 2005 ) . since the first wave of visits was only available for 57/59 trauma centers , the correlation coefficient was based on n = 57 . sensitivity analyses were performed to assess the robustness of the structural performance scores to the treatment of missing data items and the weighting scheme . for missing data items , we evaluated the correlation in trauma center structural performance scores when missing data values were coded 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) instead of 2 ( neutral ) . for the weighting scheme , we evaluated the correlation in trauma center ranks when equal weights were used over consensus - based weights . level i trauma centers admitted on average 900 patients per year that met trauma registry criteria whereas mean volume was below 40 for rural level iv centers [ table 1 ] . level i / ii centers admitted more seriously injured patients who more frequently presented with a head injury than lower level centers . characteristics of trauma centers in the quebec trauma system ( 1999 - 2006 ) the mean structural performance score among the 59 trauma centers was 47.4/100 ( 95% ci : 43.6 - 51.1 ) . most points were lost on trauma program ( mean of 12.8/33.4 ) , followed by procedural protocols ( 20.3/44.4 ) , and lastly commitment ( 15.6/22.2 ) . there were two institutional outliers - one level iii center was a high performer whereas one level iv center was a low performer [ figure 1 ] . structural performance scores for the 59 trauma centers in the quebec trauma system ( 1998 - 2005 ) intraobserver agreement was almost perfect ( kappa > 0.8 ) for 66 of the 73 items ( 90% ) in the evaluation grid . between - center discrimination of structural performance was good . the coefficient of variation of the structural performance scores was 31.2% with a bootstrap 95% ci of 25.5 - 37.6% , indicating that the distribution of structural performance scores had high variation . of the 59 trauma centers in the system , 20 ( 33.9% ) maintained their designation status as is , 6 ( 10.2% ) were asked to make specific changes with no on - site control visit , 15 ( 25.4% ) were asked to make changes with a control visit and 18 ( 30.5% ) had their designation level revoked . in terms of construct validity , accreditation decisions had a strong correlation with structural performance scores [ table 2 ] ; median scores were 52/100 for those that conserved their status as is , 47/100 for centers with changes to make but no control visit , 43/100 for centers with changes and a control visit , and 35/100 for centers with revoked status ( p = 0.0008 ) . the indicator of structural performance also correlated with trauma center designation level [ table 2 ] . median structural performance scores for level i through iv were 63/100 , 52/100 , 54/100 , and 40/100 , respectively , showing decreasing scores with decreasing level of accreditation ( p = 0.002 ) . we observed a weaker but statistically significant correlation with patient volume whereby higher volumes were associated with higher structural performance scores ( p = 0.05 ) . no correlation was observed between structural performance scores in the first wave of accreditation visits , performed just after implementation of the trauma system in 1993 , and those in the second wave performed between 1998 and 2005 [ figure 2 ] . association of structural performance scores with the accreditation decision , designation level , and trauma center volume ( 1998 - 2005 ) correlation between structural performance scores derived from accreditation visits performed in the first wave ( 1994 - 1999 ) and the second wave ( 1998 - 2005 ) structural performance scores calculated by attributing a value of 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) to all missing data items had a high correlation with scores calculated by attributing a value of 2 [ table 3 ] . between - center variation was unchanged , as expected . correlations with accreditation decision , designation level and volume were similar as was the correlation between the two waves of visits . the correlation between structural performance scores based on consensus weights and equal weights for each item of the grid was almost perfect : r = 0.99 ( 95% ci 0.98 - 0.99 ) . sensitivity analyses : comparison of study results when missing data values were coded 2 ( neutral ) with study results when missing data values were coded 0 ( very negative / absent ) or 4 ( very positive / present ) structural performance scores calculated by attributing a value of 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) to all missing data items had a high correlation with scores calculated by attributing a value of 2 [ table 3 ] correlations with accreditation decision , designation level and volume were similar as was the correlation between the two waves of visits . the correlation between structural performance scores based on consensus weights and equal weights for each item of the grid was almost perfect : r = 0.99 ( 95% ci 0.98 - 0.99 ) . sensitivity analyses : comparison of study results when missing data values were coded 2 ( neutral ) with study results when missing data values were coded 0 ( very negative / absent ) or 4 ( very positive / present ) in this study , we have demonstrated the feasibility of deriving a quantitative composite indicator of structural performance using trauma system accreditation reports based on asc - cot criteria . the proposed indicator shows good reliability , discriminates between centers , and correlates well with designation level , volume , and accreditation status . lack of correlation over time may suggest that accreditation reports need to be filled out in a more standardized manner but may also be an indication that structural performance fluctuates over time . the proposed indicator can be used to describe structural performance and identify trauma center outliers to drive quality improvement efforts . indicators of structural performance are an essential part of trauma center performance evaluation . in addition , they are easier to assess than process or outcome indicators as they are collected at the hospital rather than the patient level and they may also be more easily actionable . data used to calculate the proposed indicator are already routinely collected in most trauma systems on periodic accreditation visits . furthermore , many items could be collected at a distance rather than at on - site visits . the observed association of the proposed structural performance indicator with designation level and volume is an indication of construct validity and suggests that the indicator is likely to have good predictive criterion validity as both designation level and volume have been shown to have a negative correlation with risk - adjusted rates of mortality , severe disability at discharge , and hospital length of stay . in addition , the near perfect correlation between structural performance scores based on consensus and equal weights suggests that the latter could be used with no impact on results . this would simplify calculation of structural performance scores as while weights allow administrators to account for the perceived importance of structural aspects , they require periodic updating according to changes in best practise guidelines and may not be exportable across trauma systems . the observed inability of the score to forecast performance at a later time could be due to the lack of standardization of the accreditation process between the first visit during the run - in period and the second visit , performed when the system had had a chance to mature . this and the presence of missing data suggest that the accreditation process would benefit from better standardization . indeed , in our system , the upcoming round of visits will be based on mandatory electronic completion of all items in the evaluation grid . however , the observed lack of temporal correlation may also be explained by important changes in structural performance over time . of interest , among the themes that are part of the structural score , we observed better forecasting for procedural protocols ( r = 0.19 ; 95% ci -0.08;0.43 ) than for the trauma program ( r = 0.009 ; 95% ci -0.25;0.27 ) or commitment ( r = -0.05 ; 95% ci -0.30;0.22 ) . however , a recent scoping review and systematic review on trauma quality indicators did not identify any studies that implemented or validated a composite indicator of structural performance for trauma care . in other health care sectors , few studies have looked at quantifying hospital structural performance . daley et al . used on - site visits to measure structural performance for veteran 's affairs medical centers and developed a quantitative score using consensus - based ratings . they demonstrated a relationship between this indicator of structural performance and risk - adjusted mortality / morbidity rates . the index of hospital quality includes a composite indicator of structure for 12 medical specialities derived by a one - factor solution from principal components analysis that is used by the us news and world report ( national opinion research center ) to generate us hospital rankings . although trauma care is not evaluated per se in us hospital rankings , the structural indicator includes an indicator of the presence and level of trauma care . this study was based on accreditation data from a mature integrated trauma system that follows acs - cot designation criteria . in addition , we were able to evaluate intra - rater reliability and partly address the internal validity of the score . however , limitations include the lack of standardized data collection , problems related to composite scores , and generalizability beyond the quebec trauma system . the accreditation visit reports used in the present study lacked standardization because they were not performed in the context of quantitative performance evaluation . we addressed this problem by retrospectively extracting qualitative information from reports and transposing it onto a standardized evaluation grid . however , missing data items were frequent , which may have influenced the final structural performance scores . a value of 2 ( neutral ) was attributed to missing data items of the evaluation grid . this would have led to an underestimation of between - center variation and is likely to have led to an underestimation of associations of structural performance scores with designation level , volume , and accreditation status . however , the results of sensitivity analyses suggest that the strategy used to address missing data items was adequate . to address this problem , future accreditation visits will be based on mandatory prospective electronic completion of all items in the evaluation grid . the proposed indicator of structural performance is a composite measure and while it is a useful indicator of global performance , it is associated with the limitations inherent to composite scores . it may mask item - specific differences across centers and may make action difficult as it is hard to identify the specific causes of high / low performance . for this reason , composite scores should always be accompanied by item - specific data . there are several different methods for deriving composite scores and there is no clear consensus on which method is optimal . we used a simple weighted average ( consensus or equal - weights ) over more complex methods such as principal components analysis or latent variable models partly because these methods would have required at least 10 observations ( trauma centers ) per grid item ( i.e. , 10 84 trauma centers ) to generate accurate results . finally , the evaluation grid used in our trauma system is based on acs - cot criteria but evaluation items are likely to vary across systems . therefore , while this study provides evidence of the feasibility of generating a quantitative structural performance indicator from report data , methods will have to be adapted to different reporting formats . in addition , the validity and reliability of the proposed composite indicator of trauma center structural performance will need to be assessed in other trauma systems . this study was based on accreditation data from a mature integrated trauma system that follows acs - cot designation criteria . in addition , we were able to evaluate intra - rater reliability and partly address the internal validity of the score . however , limitations include the lack of standardized data collection , problems related to composite scores , and generalizability beyond the quebec trauma system . the accreditation visit reports used in the present study lacked standardization because they were not performed in the context of quantitative performance evaluation . we addressed this problem by retrospectively extracting qualitative information from reports and transposing it onto a standardized evaluation grid . however , missing data items were frequent , which may have influenced the final structural performance scores . a value of 2 ( neutral ) was attributed to missing data items of the evaluation grid . this would have led to an underestimation of between - center variation and is likely to have led to an underestimation of associations of structural performance scores with designation level , volume , and accreditation status . however , the results of sensitivity analyses suggest that the strategy used to address missing data items was adequate . to address this problem , future accreditation visits will be based on mandatory prospective electronic completion of all items in the evaluation grid . the proposed indicator of structural performance is a composite measure and while it is a useful indicator of global performance , it is associated with the limitations inherent to composite scores . it may mask item - specific differences across centers and may make action difficult as it is hard to identify the specific causes of high / low performance . for this reason , composite scores should always be accompanied by item - specific data . there are several different methods for deriving composite scores and there is no clear consensus on which method is optimal . we used a simple weighted average ( consensus or equal - weights ) over more complex methods such as principal components analysis or latent variable models partly because these methods would have required at least 10 observations ( trauma centers ) per grid item ( i.e. , 10 84 trauma centers ) to generate accurate results . finally , the evaluation grid used in our trauma system is based on acs - cot criteria but evaluation items are likely to vary across systems . therefore , while this study provides evidence of the feasibility of generating a quantitative structural performance indicator from report data , methods will have to be adapted to different reporting formats . in addition , the validity and reliability of the proposed composite indicator of trauma center structural performance will need to be assessed in other trauma systems . in summary , we have proposed a structural performance indicator that can be derived from trauma center accreditation visit reports and we have provided evidence of its reliability and internal validity . the indicator can be used to describe performance , identify institutional outliers , and inform accreditation decisions with the goal of driving performance improvement efforts . further research needs to evaluate the influence of structural performance measured by the proposed indicator on clinical process performance and ultimately , on patient outcome . the observed variability in structural performance across centers and the change in structural scores over time underline the importance of evaluating structural performance in trauma systems at regular intervals to drive quality improvement efforts .

background : indicators of structure , process , and outcome are required to evaluate the performance of trauma centers to improve the quality and efficiency of care . while periodic external accreditation visits are part of most trauma systems , a quantitative indicator of structural performance has yet to be proposed . the objective of this study was to develop and validate a trauma center structural performance indicator using accreditation report data.materials and methods : analyses were based on accreditation reports completed during on - site visits in the quebec trauma system ( 1994 - 2005 ) . qualitative report data was retrospectively transposed onto an evaluation grid and the weighted average of grid items was used to quantify performance . the indicator of structural performance was evaluated in terms of test - retest reliability ( kappa statistic ) , discrimination between centers ( coefficient of variation ) , content validity ( correlation with accreditation decision , designation level , and patient volume ) and forecasting ( correlation between visits performed in 1994 - 1999 and 1998 - 2005).results : kappa statistics were > 0.8 for 66 of the 73 ( 90% ) grid items . mean structural performance score over 59 trauma centers was 47.4 ( 95% ci : 43.6 - 51.1 ) . two centers were flagged as outliers and the coefficient of variation was 31.2% ( 95% ci : 25.5% to 37.6% ) , showing good discrimination . correlation coefficients of associations with accreditation decision , designation level , and volume were all statistically significant ( r = 0.61 , -0.40 , and 0.24 , respectively ) . no correlation was observed over time ( r = 0.03).conclusion : this study demonstrates the feasibility of quantifying trauma center structural performance using accreditation reports . the proposed performance indicator shows good test - retest reliability , between - center discrimination , and construct validity . the observed variability in structural performance across centers and over - time underlines the importance of evaluating structural performance in trauma systems at regular intervals to drive quality improvement efforts .

INTRODUCTION MATERIALS AND METHODS Study population Data Accreditation data Patient-level data Development of the structural performance indicator Validation of the structural performance indicator Sensitivity analyses RESULTS Sensitivity analyses DISCUSSION Strengths and limitations CONCLUSION

angiosarcomas are poorly differentiated neoplasms composed of stands of endothelial cells and have a very narrow lumen filled with blood cells . angiosarcomas frequently originate from the vascular wall , usually from medium and large sized veins . they are malignant but rarely result in recurrences or metastases to lungs and lymph nodes . angiosarcomas are commonly detected during the initial six months of life , since they usually involve the soft tissues of head and skin . they are the most frequently found benign neoplasms of the liver in children . in adults angiosarcomas are very rare findings , being mostly described in the liver , lungs , brain and bones . a 68-year - old patient was admitted due to loose bowel movements with traces of blood observed for a period of two weeks . the symptoms were initially recorded by the patient 6 months prior to admission and took the form of blood in the stool . sigmoidoscopy revealed an elevated ulcerative lesion 20 mm in diameter , located 12 cm from the anal verge along with two small polyps ( 4 mm and 8 mm ) situated 25 cm from the anal verge . histopathology of the ulceration showed a tubular adenoma with chronic mucosa inflammation . above the splenic flexure of the transverse colon , the colonic mucosa was pink and glossy with a mesh of dilated vessels suggestive of chronic inflammation . remaining segments of the large intestine were normal on sigmoidoscopic examination . in spite of the benign pathologic result the patient was qualified for en bloc surgical resection on these aforementioned findings . a hard , exophytic tumor ( 5 cm 5 cm ) was found intra - operatively on the mesorectal side of the rectum . the patient underwent resection of the upper rectum and sigmoid colon ( with formation of end - to - end anastomosis with mechanical stitches ) with regional lymphadenectomy . no complications were noted in the postoperative period and the patient was discharged eight days after surgery . histopathology of the tumor revealed a g3 malignant angiosarcoma with chronic inflammation of the colonic mucosa ( figs . the surgical resection margin was negative and no metastases were found in regional lymph nodes . fragment of intestinal wall with neoplastic infiltration of muscular layer . on the left visible intestinal mucosa with lymphoid tissue . he , magnification 100 atypical , epithelioid cells without tendency to form blood vessel structures . . only a few well - documented cases of angiosarcoma in this location have been reported in the literature [ 313 ] . the rare occurrence and high aggressiveness of this tumor preclude designing an optimal therapeutic approach . surgical treatment seems to be the only effective treatment modality in cases of this malignancy with a colorectal localization . the reported cases of angiosarcoma have documented early spread of this malignancy after surgical treatment and a short overall survival . among these cases was a 77-year - old patient reporting a history of constipation and bleeding from the lower alimentary tract , who was operated on for colorectal angiosarcoma . numerous liver metastases were detected following the resection of the primary tumor and the patient died six months after surgery . another reported case involved a 72-year - old patient with pulmonary metastases and colorectal angiosarcoma as the underlying primary malignancy . he was re - operated on several months later due to massive rectal bleeding , and intraperitoneal spread was detected . additionally , the case of a 60-year - old female patient was presented , who was also operated on due to colorectal angiosarcoma . little is known on the long - term outcomes of adjuvant treatment ( radiotherapy and chemotherapy ) following the surgical resection of angiosarcomas . komorowski presented a case involving a 19-year - old patient , who was diagnosed with this malignancy . the patient was operated on and received four subsequent courses of chemotherapy and radiotherapy . at follow - up after 18 months , the patient was still alive with no signs of recurrence . up to now , the longest postoperative survival in a colorectal angiosarcoma patient was reported by smith et al . , where a 16-year - old female patient survived 36 months after surgery . however , in most of the above - described cases , patients diagnosed with colorectal malignancies died within one year after resection due to neoplastic spread or postsurgical complications . in contrast , our patient survived four years after angiosarcoma resection and died due to myocardial infarction . therefore , this is the first reported case of such long - term survival ( 48 months ) after resection of colorectal angiosarcoma . the surgical approach implemented in our patient proved efficient , and seems the only effective modality in cases of angiosarcoma of such unusual locations as the large intestine . further research is needed in order to verify the role of adjuvant treatment after surgical resection .

hemangioendotheliomas are poorly differentiated neoplasms built from strands of endothelial cells and have a very narrow lumen filled with blood cells . hemangioendotheliomas are commonly detected during the initial six months of life , concerning mainly soft tissues and skin . it is the most common bening liver cancer in children . in adults , hemangioendotheliomas are very rare findings , being mostly described in the liver , lungs , brain and bones . hemangioendotheliomas of the large intestine are extremely rare in adults . cancer developing there is of high degree of malignancy . the authors present the case of 68 year old patient in whom preoperative benign tumor with ulcers were diagnosed . intraoperative macroscopic picture suggested malignant process . the patient underwent anterior rectal resection with regional lymphadenectomy . the postoperative histopathological evaluation with immunohistochemical studies angiosarcoma was diagnosed . a four year survival free of cancer was obtained , and the patient died due to myocardial infarct . literature describes the short periods of survival of patients with vascular sarcoma of the colon . the most common reason for decease are metastases with massive haemorrhages . bleeding tendency results from growing kasabach - merritt syndrome . it is characterized by profound thrombocytopenia and coagulopathy . the presented patient is a unique example of angiosarcoma with long period of survival .

Introduction Case report Discussion None

large - scale clinical trials have created a robust evidence base to inform much of what is now standard acute - stroke practice.13 the classical clinical trial is designed to test efficacy of a particular intervention over a comparator , for example , placebo or usual care . to facilitate comparison between the groups requires a standard measure of outcome that is relevant and suited to the clinical question , valid for the population studied , and meaningful to the research team . in those trials that describe interventions designed to impact on quantifiable physiological variables , such as glycemia or blood pressure , choice of end - point assessment choice of assessment strategy is more challenging for a chronic , nonprogressive , or variably progressive disorder with potential multisystem effects such as cerebrovascular disease . hard clinical end points such as stroke mortality or stroke recurrence are useful , but do not fully capture the potential devastating effect of a disabling but survivable stroke . as stroke represents the leading global cause of adult disability,4 an important consideration for any study of stroke interventions this is recognized by regulatory authorities , who now recommend a measure of functional recovery / disability as primary or coprimary end point for stroke intervention trials . although the focus of this review will be functional assessment tools for stroke trials , these instruments also have utility in clinical practice . as functional assessment scales give a numerical value to abstract concepts such as disability , they can be used to objectively quantify deficits and track change over time . in clinical practice , an appreciation of how to describe stroke recovery in terms of common stroke scales allows for development of a common language between professionals caring for stroke survivors that facilitates comparisons of patients and services . within a single review , it would be impossible to review all stroke - specific and generic scales that may be needed in a stroke survivor s journey ( figure 1 ) . for the interested reader , we recommend a number of reference works.57 we recognize there is also extensive literature on assessment strategies for cognitive function poststroke . we will not review cognitive testing , suffice to say that there are a multitude of tools available with little consistency in choice of assessment.8 a large number of stroke - assessment scales are described , with novel scales frequently appearing ( and often subsequently disappearing ) in the literature . for those who are new to functional assessment , the large and varied nature of available scales and tools may seem daunting . the world health organization s international classification of functioning , disability and health ( who - icf)9 gives a conceptual framework that can aid classification of the scales and help decide on the appropriate measure for a particular purpose . who - icf describes levels of pathology ( in this case , the stroke lesion ) , impairments ( the direct loss of function ) , activity limitation ( formerly called disability ) , and societal participation ( formerly called handicap ) . the who - icf grades do not exist in isolation ; they interact and often create feedback loops . for example , an ischemic stroke ( pathology ) may cause a hemianopia ( impairment ) ; this may lead to poor mobility ( activity limitation ) and may restrict the stroke survivor from driving ( societal participation limitation ) . these problems may result in a fall with soft - tissue injury ( impairment ) , and fear of falling may cause the stroke survivor to forgo usual hobbies and activities ( societal participation limitation ) ( figure 2 ) . measures of pathology ( for example , size of infarct on imaging ) or impairment ( for example the medical research council motor assessment scale ) are straightforward to perform and interpret , but give little useful information on how stroke affects the individual . for this reason , impairment scales are often used in early phase trials . for phase iii studies , activity measures or measures of participation although not part of the who - icf , a further concept of quality of life ( qol ) is also described , and tools exist for its measurement . measures of qol give a far more detailed assessment , but as a result can be more burdensome to the patient and are often more difficult to interpret ( figure 3 ) . clinimetrics is the study of properties of clinical assessment tools;10 the term is derived from the theory of psychometrics.11 classical test theory describes important properties such as validity and reliability.12 other important factors for clinical scales are acceptability , both to patient and to assessor , and responsiveness to change . although in psychometrics , classical test measures are increasingly being superseded by contemporary theories of item response , the measures of validity , reliability , and responsiveness remain important for understanding clinical scales , and we will discuss them in turn . the clinimetric property of validity seeks to assess whether a scale measures the concept it purports to measure . adequate validity is essential for a stroke scale to have clinical utility , as a functional assessment tool that does not measure function is meaningless . validity can be assessed in various complementary ways.57 there is no gold standard for poststroke function , so assessment of criterion validity , where a scale is compared to a reference standard , is not possible . however , concurrent validity can be applied to a stroke scale by comparing it with another measure that purports to measure a similar construct ; for example , comparing a novel impairment scale with an established scale . face validity is an assessment of whether a priori the scale should measure the concept of interest , usually assessed by experts in the field . content validity asks whether the various items of a scale can adequately describe the concept of interest . prognostic or predictive validity for a stroke scale may be examined by , for example , studying if an impairment scale is associated with longer - term stroke outcomes . important reliability measures include the reproducibility of repeat scoring by the same observer ( intraobserver reliability or test retest reliability ) and between scorers ( interobserver variability ) . whether all items within a scale measure the same construct is a further measure of reliability , usually termed internal consistency . in contemporary stroke trials , where many thousands of stroke survivors may be assessed by hundreds of international research teams , reliability of assessment is clearly paramount . whereas validity of a scale is inherent , reliability of assessment may be modified . various methods to improve consistency of assessment are employed in large - scale trials , including training in use of scales , certification exams , and use of standardized protocols . while validity is relative , reliability can be objectively described . there is no consensus on the optimal method to measure reliability , although kappa statistics are frequently used in the biomedical literature to assess agreement . a kappa of 0 would imply no agreement other than that expected by chance , and perfect agreement is scored as 1.0 . traditionally , a kappa greater than 0.6 is taken as sufficient agreement to justify use of a scale . various forms of statistical weighting of kappa values can be used to give a measure of the degree of difference between raters.13,14 increasingly , more sophisticated analyses , such as that of bland altman , are being used to assess reliability.15 responsiveness can be thought of as the ability to detect meaningful change over time . meaningful change is clearly a subjective term , and will vary with the context in which the scale is used . the issue of responsiveness and the ability to detect small but meaningful change is especially important for a condition with high incidence and prevalence , such as stroke . if a scale does not pick up change in function , treatment effects that are modest for the individual but potentially important at a population level could be missed . the ideal scale would be easy and quick to administer , acceptable to patients and researchers , valid for its chosen purpose , reliable , and responsive to meaningful clinical change . there is no ideal stroke measure that fulfills all these criteria ( nor is there ever likely to be ) . although some guidance on stroke assessment for trials is emerging , debate continues as to the relative strengths and limitations of differing assessment strategies , and there is no consensus as to the optimal outcome measure(s ) for use . the stroke literature describes a variety of instruments , generic and specific to stroke , for functional assessment of recovery . a recent analysis of tools used in stroke trials suggests substantial heterogeneity in choice of assessment measure and in method of application.16 use of bespoke , nonvalidated assessments is still seen , although less commonly than previously . certain assessments are used more frequently than others and are increasingly recommended by specialist societies.17 for the non - stroke specialist , a basic knowledge of the more prevalent stroke scales will allow for improved understanding and critical analysis of stroke studies . we will describe three common stroke assessments : the national institutes of health stroke scale ( nihss ) , the modified rankin scale ( mrs ) and the barthel index ( bi ) . for each scale , we will discuss history , development , and application , and use the scales to further discuss the importance of clinimetric properties . the scientific study of assessment scales , particularly stroke assessment , is rapidly expanding and it would be impossible to comprehensively cover all areas . in this review , we will not describe the optimal analysis of functional scales for stroke trials . debate continues as to the relative merits of various statistical techniques , including dichotomization and use of the complete range of a scale , with the differing approaches having vocal proponents.18,19 equally , we will not consider the literature on outcome assessment in animal models of stroke.20,21 the nihss is a 15-item scale that standardizes and quantifies the basic neurological examination , paying particular attention to those aspects most pertinent to stroke . the nihss provides an ordinal , nonlinear measure of acute stroke - related impairments by assigning numerical values to various aspects of neurological function.22 the scale incorporates assessment of language , motor function , sensory loss , consciousness , visual fields , extraocular movements , coordination , neglect , and speech.22 it is scored from 0 ( no impairment ) to a maximum of 42 . a standardized approach to assessment , starting with fundamental assessments such as level of consciousness , is recommended , and guidance is given on how to score where the stroke survivor is not able to respond to commands . the nihss was developed in the early 1980s as a research tool to allow consistent reporting of neurological deficits in acute - stroke studies , particularly the early trials of thrombolysis and putative neuroprotectants.22 the nihss was developed through a robust consensus approach , taking the most informative measures from existent stroke - examination scales ( toronto stroke scale , oxbury initial severity scale , and cincinnati stroke scale ) and creating a composite scale that was further reviewed by a panel of stroke researchers and amended ( further items were added to ensure the assessment was as comprehensive as possible ) . the resulting scale was piloted and refined in a controlled trial of naloxone in acute stroke . it has since been used as primary or coprimary end point in landmark trials of thrombolytic agents and is commonly used in clinical acute - stroke practice . using a factor - analysis process , utility of individual components of the nihss has been assessed.23 this work has formed the basis for development of the modified nihss or mnihss , which removed components deemed unreliable.24 the resulting 11-point mnihss has been prospectively assessed , and improved reliability is described.25 as the standard nihss is already fairly quick to perform and reliable , it is debatable whether a shorter scale is needed , and at present the mnihss is not frequently used in trials or practice . further amendments to the nihss have been described to facilitate use of the scale in prehospital settings.26 a pediatric nihss ( pednihss ) is also described.27 the nihss has many advantages as a stroke outcome - assessment tool . it is relatively straightforward and takes around 6 minutes to perform , with no need for additional equipment . in the acute - stroke environment , it has been suggested that a change in the nihss of more than 2 points represents clinically relevant early improvement or deterioration.28 nihss scores are reliable across observers , and this has been demonstrated both in cohorts of neurology - trained and non - neurologist raters . the availability of a reliable method for neurological exam that is suitable for nonspecialists is a particular strength of the nihss . reliability and validity has also been demonstrated for remote nihss assessment via telemedicine.29 the interobserver reliability of the nihss is further improved by the various training materials available . training resources now exist , such as dvds and online educational aids , as well as pocket - sized nihss summary scales . practitioners can undergo certification to demonstrate their proficiency in assessment and interpretation of the nihss.30 content validity of the nihss has been demonstrated , although high internal consistency suggests that certain items of the nihss may be redundant . the nihss has predictive validity , as initial score is a robust predictor of in - hospital complication and outcome at 3 months.31,32 correlations with objective measures of stroke severity , such as size of infarct on imaging , provide further evidence of nihss validity.23,33,34 compared with bi and mrs , the nihss is the more sensitive outcome score , requiring potentially smaller sample sizes to detect relevant therapeutic effects.23,35 the nihss is responsive to change and can measure impairment throughout the expected range of stroke severity.36 a criticism of the nihss relates to its validity in certain nondominant - hemisphere stroke syndromes . it is well recognized that an individual can score 0 on the nihss , despite having evidence of ischemic stroke , particularly in the posterior circulation territory.37 examination of the component subscales of the nihss reveals a focus on limb and speech impairments and relatively little attention to , for example , cranial nerve lesions . similarly , when the nihss is used to predict dependent living , lower scores are seen in posterior circulation events compared to anterior circulation.38 there are radiological correlates , when quantifying extent of cerebral damage for a specified nihss score , the median volume of right - hemisphere strokes is larger than the volume of left - hemisphere strokes , suggesting nondominant strokes are required to be more severe to reach the same grading on the nihss.39 as an impairment scale , the nihss can give only limited information on how stroke has affected the individual stroke survivor . excellent outcome from stroke ; a hemianopia that precludes driving and may necessitate loss of employment would score nihss 1 , but for the individual this may not seem an excellent result . adapted from the maryland disability index , the bi authors florence i mahoney and dorothea w barthel intended their scale for use as a simple index of independence , useful in scoring improvement in rehabilitation.40 first described in the 1950s and published in 1965 , the bi was developed to assist in discharge planning from long - term care wards . with time , the bi has been adopted by other disciplines and is a recommended assessment in older adult care.41 the bi is the most commonly used functional measure in stroke - rehabilitation settings and the second most commonly used functional outcome measure across stroke trials.16,42 many scales have been described that take the name some authors have sought to modify or adapt the bi from the original ; these include reducing the number of items,43 extending it with the addition of cognitive and social domains,44 and attempts to further subdivide the outcomes to include different degrees of assistance.45 however , each of these requires independent validation , as it is known that even comparatively minor adaptations alter the validity of a tool and accuracy of responses.46 for consistency , it is recommended that a single bi measure is used ; the scale as described by wade and collin47 has been used in many trials . scores range from 0 and 100 , with a higher score indicating greater independence ( table 1 ) . while this can be useful for statistical analysis , it is more informative in practice to present the scores for the individual domains . an unresolved issue for trials is how to define a good bi outcome , with significant heterogeneity within the published literature48 and attempts to subcategorize , based on total score.49,50 a popular interpretation of bi scores is that subjects with bi > 80 are generally independent and should be able to return home ; while subjects with bi < 40 are very dependent.51 other interpretations of favorable and unfavorable bi outcomes have been described : statistical modeling looking at differing bi scores as a trial end point suggested a score of 95/100 was the optimal descriptor of an excellent outcome and that 75/100 was the best cut point for defining a poor outcome.52 validity of bi is well described . the scale is recognized as a valid prognostic tool following stroke , in particular as predictor of recovery , level of care required,53 and duration of rehabilitation required following stroke.54 bi scores correlate with other stroke - assessment scales,55 including other more detailed adl scales.56 interobserver reliability is usually quoted as a strength of the bi , and reliability has been demonstrated in nonstroke populations.57 systematic review of reliability of bi in stroke also suggests reasonable reliability , although few multicenter reliability studies are available.58 the original bi is not without its limitations . as a scale of primarily physical function , it does not reflect the burden on the individual of communication and cognitive deficits that can result from a stroke event.59 for clinical trials , the bi lacks a result to represent stroke mortality , and this can complicate analysis of results . however , the major limitation of the bi for clinical trial use is its responsiveness to change . although in certain stroke - care settings , the bi is as sensitive to change as other scales,60 a score must be able to represent changes throughout the entire spectrum of potential functional outcomes . it is in this regard that the foor and ceiling effects of the bi become apparent.50 floor and ceiling describes the phenomenon by which the score does not change from minimum or maximum despite clinical change.61 for example , a stroke patient in a neurointensive care setting can make significant gains but still score a total 0 on the bi ; conversely , a patient who is discharged from hospital and independent may still have substantial functional problems but will score 100 on the bi . given this limitation , the bi may be best suited to stroke survivors requiring inpatient rehabilitation , while other scales may be needed to assess functional change in those with more major or minor stroke symptoms.62 the bi can be considered as a measure of basic adl ( self - care and mobility ) . extended activities of daily living ( e - adl ) measures.63 the term instrumental adl was first used in lawton and brody s work , and a lawton i - adl scale is described.64 a validated measure that has been used with stroke survivors is the nottingham extended adl scale , which asks participants to reflect their actual activities over the preceding weeks , rather than simply what they have the capability to do.65,66 the nottingham extended adl scale compares favorably to the bi , and is less susceptible to the ceiling effects described.67 the mrs is a 6-point , ordinal hierarchical scale that describes global disability with a focus on mobility ( table 2 ) . the original rankin scale was developed by the scottish physician john rankin to describe the positive outcomes he was achieving in his prototypic stroke unit.68 although not originally intended as an assessment for clinical trials , a slightly modified version of rankin s eponymous scale was used as end point in the first multicenter stroke trial ( the uk tia study).69 since this time , the mrs has grown in popularity and is now the most commonly used functional measure in stroke trials , and has been the primary or coprimary outcome in most recent large - scale stroke trials.16 a further variation of the mrs , the oxford handicap scale , has been described but is not commonly used by trialists . in contemporary stroke studies , the mrs is often used both as a measure of premorbid ability to assist in selection of patients and as final outcome measure . the mrs has many potential strengths , and it is acceptable to patient and assessor , with nonstandardized interviews taking around 5 minutes to complete.70 concurrent validity is demonstrated by strong correlation with measures of stroke pathology ( for example , infarct volumes ) and agreement with other stroke scales.71,72 the six potential scores on the mrs ( 05 ) describe a full range of stroke outcomes , with a score of 6 usually added to denote death . with a limited number of scores , the mrs may be less responsive to change than some other scales ; however , a single - point change on the mrs will always be clinically relevant . the principle limitation of the mrs is its reliability , with the potential for substantial interobserver variability . a study describing the interobserver variability of the mrs is available ; indeed , in the first clinical studies that used the mrs as end point , the trialists described interobserver variability for a third of subjects interviewed by paired assessors.73 a systematic review and meta - analysis of studies describing interobserver variability of the mrs reports pooled reliability across ten published studies ( n = 587 patients ) of kappa = 0.46.74 those studies that assessed mrs reliability with multiple raters and centers ( ie , similar to a contemporary clinical trial ) revealed a worryingly low agreement of kappa = 0.21.75 this level of inconsistency will impact on the validity of the trial results and conclusions . the statistical noise created by the interobserver variability will increase the possibility of a type ii error , ie , a beneficial treatment effect is missed . it has been postulated that problems with mrs reliability may have partly explained a series of unexpected neutral results in large - scale neuroprotectant studies . there are published examples of nonstroke studies whose results were fundamentally altered when statistical analysis accounted for observer variation.76 recognizing the problems of reliability in standard mrs assessments , trialists have explored various interventions to improve consistency in scoring . usual mrs interviews are unstructured , and researchers vary considerably in their length of interview and number of questions asked . more structured approaches to assessment have been described , from a comprehensive scripted interview75 to use of anchoring questions that require a yes / no answer.77 the groups that developed these assessments describe substantial improvements in reliability . however , improvements have not been seen when the structured interviews have been tested by independent centers.78 training in use of the mrs can also offer potential to improve consistency . as with the nihss and bi , an online training resource is available with an accompanying certification exam.79 a further trial modification that may improve reliability is to record mrs interviews and have a remote consensus grading by experienced stroke trialists . two other modifications to mrs assessment are commonly used and deserve some discussion : using proxies to substitute for stroke survivors in the mrs interview and calculating a prestroke mrs . stroke survivors often have physical , language , or cognitive impairments that may complicate a standard face - to - face interview . in this situation , an informant who knows the patient often supplements the interview or substitutes it completely . while this approach makes intuitive sense , we should not assume validity , and clinimetric analysis is still required . a recent systematic review of proxy stroke scales ( the mrs was not included ) suggested that the properties of certain proxy - based assessments may differ from equivalent standard assessments.80 a study of proxy mrs described suboptimal reliability and validity , and recommended that direct mrs interview with the patient should be the preferred assessment if possible.81 in stroke trials , traditional statistical analyses assess numbers achieving a good functional outcome . to improve trial power , prestroke mrs has been used in many landmark stroke trials , where prestroke mrs > 2 is used as the exclusion criterion during participant selection . the wording of the mrs grades is not suited to such prestroke assessment , and it is perhaps unsurprising that when formally assessed , prestroke mrs had only moderate reliability and validity.82 in view of improving longer - term survival and functional outcomes following stroke,83 it could be argued that assessments against participation or qol will become increasingly important.84 certainly evaluations of health - related qol in stroke survivors can provide a rich description of the multifaceted effects of a stroke , providing insights above those recorded with traditional impairment and activity measures.85 measuring health - related qol in stroke presents particular challenges . important predictors and components of qol following stroke will vary at different periods following the event.86 thus , we must balance having a suitably comprehensive assessment that is sensitive to the nuances of qol against the time and burden required for this assessment . carer / family - based assessments of the patient s qol are often biased , with the proxies reporting poorer outcomes than the subject.87 various qol scales have been proposed , some generic and some specific to stroke / brain injury . qol scales should be subject to the same rigor of clinimetric assessment as any other scale . it is evident from the published literature that for qol there is a propensity to generate new scales rather than validating existing ones.88,89 it has been argued that qol can be assessed by asking just two questions assessing dependency and problems.90 an alternative approach is to apply existing health - related qol scales or to use disease - specific scores . the short form 36 ( sf-36 ) is a generic scale intended for patient completion that assesses eight domains of health - related qol derived from the medical outcomes study ( table 3).91 although the sf-36 is validated for stroke patients,92 noncompletion bias and marked floor and ceiling effects may limit its utility.93,94 the generic qol scale , euro - qol , was developed based on the findings of an international postal survey.95 the self - completion questionnaire requires assessment across five domains complemented by a visual analog scale ( table 3).96 euroqol has been validated in stroke populations.97 however , noncompletion bias is recognized : in one study , only 61% of stroke survivors could complete the scale without external assistance.97 the stroke - specific qol scale was developed based on interviews with stroke survivors.98 it is based on twelve domains ( table 3).93 the scale is validated in stroke populations , and values for minimal detectable change and clinically important difference99 are established . many assessment tools , spanning various functional domains , are available to clinicians and researchers working with stroke survivors . we have given a favor of the marked heterogeneity in use of assessment scales . lack of consistency in outcome assessment has hindered comparative research and meta - analysis , and so we would recommend that future researchers use a common set of outcome assessments . no perfect stroke - assessment scale exists , and in this review we have deliberately avoided suggestions that one scale is better than an other . we have focused on the three most commonly used stroke scales ( mrs , bi , nihss ) as exemplars . these scales have been validated , are familiar to many , and have proven utility , with each suited to differing assessment scenarios . assessment , we would recommend continuing use of the three core assessment scales : the mrs as an outcome if the study is describing global disability , the nihss for studies looking at neurological impairment , and the bi for studies looking at basic adl . trialists and clinicians can supplement these core assessments with specific tools suited to the clinical scenario / research question . increasing awareness of the importance of clinimetric properties has highlighted deficiencies and potential limitations with stroke functional assessment . clinicians and researchers should always select their assessment tool(s ) based on the question of interest and the evidence base around clinimetric properties . where , as is often the case , the research around clinimetric properties of a scale is sparse , we would encourage researchers to design and conduct their own clinimetric studies .

as stroke care has developed , there has been a need to robustly assess the efficacy of interventions both at the level of the individual stroke survivor and in the context of clinical trials . to describe stroke - survivor recovery meaningfully , more sophisticated measures are required than simple dichotomous end points , such as mortality or stroke recurrence . as stroke is an exemplar disabling long - term condition , measures of function are well suited as outcome assessment . in this review , we will describe functional assessment scales in stroke , concentrating on three of the more commonly used tools : the national institutes of health stroke scale , the modified rankin scale , and the barthel index . we will discuss the strengths , limitations , and application of these scales and use the scales to highlight important properties that are relevant to all assessment tools . we will frame much of this discussion in the context of clinimetric analysis . as they are increasingly used to inform stroke - survivor assessments , we will also discuss some of the commonly used quality - of - life measures . a recurring theme when considering functional assessment is that no tool suits all situations . clinicians and researchers should chose their assessment tool based on the question of interest and the evidence base around clinimetric properties .

Why measure functional outcomes in stroke trials? Which functional measure to use Clinimetric properties of scales National Institutes of Health Stroke Scale Barthel Index Modified Rankin Scale Quality of life Conclusion

allogeneic hematopoietic stem cell transplantation ( allo - hsct ) is an established treatment for hematologic malignancy , and more than 15,000 procedures are performed worldwide each year . after hsct , chronic graft - versus - host disease ( cgvhd ) is the most common cause of morbidity and mortality . indeed , the incidence of cgvhd is reported to be 6080% during long - term follow - up , and the incidence has recently been increasing because of the extensive use of unrelated donor transplants , older donor age , increased use of donor leukocyte infusion , and peripheral blood stem cell transplantation ( pbsct ) . symptoms of cgvhd can vary depending on the site of involvement , which may include the skin , eyes , oropharynx , or respiratory and gastrointestinal tracts . however , renal involvement associated with gvhd , particularly glomerulopathy , is very rare . in general , renal injury after hsct occurs due to hemodynamic compromise , medications , radiation , or thrombotic microangiopathy , which manifests as tubulointerstitial nephropathy . cases of nephrotic or nephritic syndrome after hsct have recently been reported , and these glomerulopathies are presumably related to cgvhd . herein , we report a case of membranous nephropathy ( mn ) in a patient who underwent hsct 21 months before this unusual nephrotic syndrome ( ns ) developed . a 39-year - old female patient was admitted to our hospital due to generalized edema and fatigue . the patient was diagnosed with aplastic anemia 3 years previously , and had no history of diabetes or hypertension . most importantly , she had undergone allogeneic pbsct from her human leukocyte antigen - identical brother 21 months before admission following myeloablative conditioning chemotherapy with cyclophosphamide and anti - thymoglobulin . grade iv acute gastrointestinal gvhd accompanied by diarrhea developed 12 days after transplantation despite gvhd prophylaxis with cyclosporine , methotrexate , and steroids , for which a continuous maintenance regimen of cyclosporine and prednisolone resulted in resolution . the patient also suffered from cytomegalovirus colitis 4 months after transplantation , and recovered after a 2-week administration of gancyclovir while cyclosporine was discontinued and prednisolone was tapered to 5 mg / day . eighteen months after cytomegalovirus infection , the patient suddenly developed generalized edema and gained 5 kg of body weight over a 2-week period . at this time initial laboratory tests showed the following values : hemoglobin , 10.5 g / dl ; platelets , 32010/l ; serum albumin , 2.2 g / dl ; total cholesterol , 402 mg / dl ; low - density lipoprotein cholesterol , 248 mg / dl ; serum creatinine , 0.77 mg / dl ; random urine protein - to - creatinine ratio ( upcr ) , 7.85 g / g ; and 24-hour urinary protein excretion , 5.03 g / day . hepatitis b surface antigen , hepatitis c antibody and anti - nuclear antibody titers were undetectable , and serum concentrations of c3 , c4 , and immunoglobulins g , a , and m were within reference range . 1a ) , eight nonsclerotic glomeruli were normocellular without mesangial expansion . the glomerular basement membrane was not thickened and double contours or subepithelial spikes were not noted . the interstitium was moderately infiltrated by mononuclear inflammatory cells , which immunohistochemical staining confirmed as cd3 + t cells ( fig . electron microscopy demonstrated numerous nodular electron - dense deposits that were mainly located in the subepithelial space along with diffusely effaced epithelial foot processes ( fig . as such , oral prednisolone at a dose of 1 mg / kg was immediately started , and an 8-week treatment resulted in partial remission with a upcr of 1.9 g / g . because the patient was intolerant to the side effects of corticosteroid treatment , prednisolone was tapered . two weeks later , however , the random upcr increased to 7.58 g / g ; thus , cyclosporine at a dose of 5 mg / kg was added . complete remission was achieved as demonstrated by a upcr of 0.23 g / g 4 months after the combined treatment with cyclosporine and 10 mg / day of low - dose prednisolone . acute kidney injury ( aki ) is a common complication in patients with allo - hsct . in general , aki can be attributed to preexisting renal disease , underlying malignancy , previous chemotherapies , irradiation , and various nephrotoxic agents such as antimicrobials , antifungals , and antivirals . accordingly , acute tubular necrosis , hemolytic uremic syndrome , thrombotic microangiopathy , and radiation nephritis are the most common forms of aki after hsct . however , glomerulopathy such as ns or nephritis rarely occurs as a manifestation of cgvhd . the notion that graft - versus - host reaction is directly related to glomerular injury has recently been gaining acceptance . one of these reports showed that nine of 889 patients who underwent allo - hsct from 1994 to 2002 developed ns , with an overall incidence of 1% . when only sibling transplant patients with or without cgvhd were taken into account , the incidence of ns was 1.8% and 0.6% , respectively , suggesting that it is not a common complication . glomerular diseases tend to occur in allogeneic pbsct patients from unrelated donors who received nonmyeloablative therapy shortly after immunosuppression was decreased or stopped , , and the median time elapsed to the onset of ns from cessation of immunosuppression was 1.5 months . regarding pathologic features , mn ( 61% ) was the most common type of ns , followed by minimal change disease ( 22% ) . approximately 70% of patients had concomitant cgvhd at the onset of ns and , in particular , 7593% of patients with posthsct mn had recent or current cgvhd , . in addition , development of ns appears to be a late event after hsct because ns was diagnosed 15.524 months after hsct . in line with these findings , our patient developed ns 21 months after allogeneic pbsct , whereby mn was confirmed by renal biopsy . unlike previous observations , the patient did not show other features of cgvhd before the onset of ns , although she did experience acute gvhd 12 days after hsct . however , a prior episode of acute gvhd is unlikely to be related to the development of glomerular disease considering its late onset after hsct . despite the high incidence of cgvhd , only a small portion of patients with cgvhd developed glomerular disease , and this low incidence is no different than that of the whole hsct population , . furthermore , cgvhd was not evident in approximately 30% of patients who presented with ns , suggesting that ns can develop irrespective of gvhd . in this regard , it is not clear whether preexisting cgvhd can indeed contribute to the development of glomerular disease . nevertheless , in this case , immunohistochemical studies clearly identified t cells in the interstitium . because complete donor chimerism was confirmed , these t cells originated from the donor and were presumed to be associated with kidney injury . this finding may be nonspecific because kidney injury could be caused by other conditions such as drugs or infections . however , considering the fact that the patient had no evidence of kidney injury associated with such conditions before the onset of ns , this pathologic finding partly explains the presence of gvhd in the kidney . previous observations showed that ns usually occurred shortly after immunosuppressive medications were stopped or their dose was reduced . however , it is uncertain whether cessation or reduction of immunosuppression may play a role in the development of ns . in fact , 40% of patients that have ns may develop glomerular disease while on immunosuppressive agents . although controversy remains over the role of preexisting cgvhd in posthsct ns , it has been suggested that immune complex , which is formed as a result of gvhd , can deposit within the glomeruli and cause damage . in particular , b - cell dysregulation with a high prevalence of autoantibodies to cellular antigens and minor histocompatibility antigens has been demonstrated in patients with cgvhd , , , . besides b - cell dysregulation in fact , cgvhd is considered an immune reaction between donor cd4+/th2 cells and the recipient 's minor histocompatibility complex antigens . furthermore , prehsct conditioning chemotherapy or irradiation can cause renal damage , which renders hiding autoantigen exposed or altered , ultimately leading to selective activation of alloreactive donor cd4 + cells and the production of immune complex . the pathogenesis of hsct - associated ns can not be explained by a single factor , and is more likely to be attributed to the combination of b cell dysregulation , alloreactive t cells , preconditioning chemotherapy , etc . treatment of hsct - associated ns has not yet been defined , but , similar to idiopathic ns , corticosteroids and cyclosporine have been most commonly used . other immunosuppressive agents such as cyclophosphamide , mycofenolate mofetil , and rituximab have also been attempted to induce remission . overall responsiveness to these drugs appears favorable : according to a previous report , 27% and 62% of patients with mn attained complete and partial remission , respectively , and 90% of patients with minimal change disease achieved complete remission . likewise , in our case , cyclosporine in addition to corticosteroids resulted in complete resolution of proteinuria , and the patient has been in complete remission . in conclusion , we present a case of posthsct mn as a rare manifestation of gvhd . however , ns occurred without other features of cgvhd while the patient was on low - dose corticosteroids . our report contradicts previous assumptions that concomitant gvhd is responsible for the development of ns , suggesting that ns can develop as a new , independent manifestation of gvhd .

nephrotic syndrome ( ns ) rarely occurs after hematopoietic stem cell transplantation ( hsct ) as a late manifestation of graft - versus - host disease ( gvhd ) . herein , we report a case of hsct - associated membranous nephropathy in a female patient with aplastic anemia . the patient received an allogeneic hsct from her human leukocyte antigen - identical brother following myeloablative conditioning chemotherapy . ns occurred 21 months after hsct without any concurrent features of chronic gvhd . the patient was treated with prednisolone and cyclosporine after renal biopsy confirmed membranous nephropathy , and achieved complete remission . our report contradicts previous assumptions that concomitant chronic gvhd is responsible for the development of ns , suggesting that ns can develop as a new , independent manifestation of gvhd .

Introduction Case report Discussion Conflict of interest

re - expansion pulmonary oedema ( repe ) is a rare complication after re - inflation of a chronically collapsed lung , following pneumothorax or effusion . it has an incidence of about 1% , but is associated with high mortality ( 20% ) . re - expansion pulmonary oedema is usually ipsilateral and the risk is highest after rapid re - expansion of the lung , which has been collapsed for 3 or more days . other contributory factors are large pneumothorax , young patients ( between 20 and 39 years ) and the method of re - expansion . although most patients recover within 7 days , severe repe due to sequestration of large quantities of fluid in the lung may lead to hypoxaemia , hypotension , shock , and death . though conventional ventilatory therapy may be useful in mild cases of repe , no definitive treatment is known for severe forms . repe has been described following re - expansion of lung after pneumothorax , pleural effusion , resection of mediastinal tumours , thorascopies and has also been reported after diaphragmatic hernia repair . the aim of this case report is to emphasise the possibility of repe arising after diaphragmatic hernia reduction , and the significance of differential lung ventilation ( dlv ) as a successful ventilatory management option in severe cases of repe . a 22-year - old male presented to the hospital with a history of blunt trauma to the abdomen 3 months ago , along with respiratory distress of 3 days duration . radiological investigations revealed left sided diaphragmatic hernia with abdominal contents in the left hemi - thorax and mediastinal shift to the right . pre - operatively , patient was anxious , unable to lie down supine with minimal chest expansion on the left side . he was having respiratory rate ( rr ) of 48/min , pulse rate 126/min , and blood pressure ( bp ) of 90/54 mmhg . modified rapid sequence induction was performed in a propped up position using 60 mg intravenous ( iv ) ketamine , 2 mg iv midazolam and 75 mg iv succinylcholine and the patient was intubated with 8.0 mm internal diameter cuffed endotracheal tube ( ett ) . right radial artery was cannulated for the purpose of invasive arterial bp monitoring and left subclavian central venous catheter was secured and ionotropic support was initiated with iv dopamine 10 g / kg / min . surgery revealed a grossly distended stomach , transverse colon and spleen in the left hemi - thorax . once the abdominal contents from the left hemi - thorax were reduced , re - expansion of the left lung was achieved . the saturation improved from 94% to 98% transiently , after which it suddenly decreased to 93% along with the drop in bp . the ionotropic support was increased and copious amount of pink frothy fluid in the ett was noticed . there was incessant production of fluid , which was flooding the ett and the anaesthesia circuit . since it developed dramatically after left lung re - expansion , the possibility of repe was considered . in order to avoid contamination of the right lung , we secured the airway with 39 french left sided double lumen tube ( dlt ) , confirmed the correct position of the dlt with fibreoptic bronchoscope and ventilated both lungs intraoperatively . there was continuous drainage of the pink fluid from the bronchial lumen of the dlt , which was intermittently suctioned . the arterial blood gas ( abg ) investigation revealed ph 7.18 , partial pressure of carbon dioxide ( pco2 ) 59 mmhg , partial pressure of oxygen ( po2 ) 117 mmhg ; bicarbonate 22 meq / l on fractional of inspired oxygen concentration ( fio2 ) of 0.8 . rest of the intraoperative period was uneventful and the patient was shifted to intensive care unit ( icu ) for further management , with dlt in situ . patient was sedated with continuous infusion of iv morphine and midazolam . in icu , conventional ventilation with dlt was continued for first 6 h with fio2 of 0.8 using synchronized intermittent mandatory ventilation ( simv ) with tidal volume ( tv ) of 500 ml , rr of 14/min , and positive end expiratory pressure ( peep ) of 8 mmhg . abg revealed ph 7.34 with pco2 35 mmhg , po2 58 mmhg and bicarbonate 19.3 meq / l . pulse oximetry showed 89 - 90% saturation and chest radiograph revealed left repe [ figure 1 ] . chest radiograph showing left sided re - expansion pulmonary oedema as there was deterioration in oxygenation on conventional ventilation , dlv was initiated using two ventilators of the same company ( ge datex ohmeda engstrom carestation ) [ figure 2 ] . differential lung ventilation being administered using two ventilators lung salvage ventilatory strategy was adopted for ventilating the left affected lung , with fio2 of 0.8 using simv with pressure control ( pc ) 17 cm h2o , rr 14/min , peep of 14 mmhg . the ventilatory setting for the right lung was fio2 of 0.4 using simv , ps of 14 cmh2o , rr 14/min , and peep of 5 mmhg . patient tolerated the dlv with morphine and midazolam infusion , without the requirement for paralysis . the saturation and haemodynamics gradually improved , and abg after 24 h on same ventilatory settings revealed ph 7.42 with pco2 36 mmhg , po2 127 mmhg and bicarbonate 24.2 meq / l . chest radiograph showing the resolution of left sided re - expansion pulmonary oedema after differential lung ventilation compliance of the left lung gradually improved , and dlv was continued for 72 h until both lungs were ventilated with identical ventilatory settings of fio2 0.4 using simv , ps of 14 cmh2o , rr 14/min , and peep of 5 mmhg . there was further improvement in the clinical condition of the patient , and trachea was extubated . after 2 days of intermittent non - invasive ventilation , patient was put on 40% venturi and was shifted to step down icu . re - expansion pulmonary oedema is a rare but fatal complication , which occurs after rapid re - expansion of a chronically collapsed lung . multiple possible aetiologies play a role in development of repe , but the exact cause is not yet understood . contributory factors include increased pulmonary vasculature permeability due to anoxic damage to capillary endothelium and neovascularisation , reperfusion related free radical injury and mechanical damage to blood vessels due to overstretching during re - expansion . the patient may present with dramatic onset of respiratory failure and circulatory collapse , along with copious pink frothy secretions . utmost vigilance is required for early recognition and optimal management of repe , as symptoms worsen rapidly for first 24 - 48 h. if mortality is prevented in the first 48 h , recovery is usually complete . in repe , the affected lung has poor compliance , but is overperfused , whereas the unaffected lung has better compliance , yet hypoperfused . though continuous positive airway pressure , asynchronous dlv , high frequency jet ventilation and extracorporeal membrane oxygenation have proven beneficial in the treatment of repe , a definitive treatment modality has not yet been described . as conventional mode of ventilation a salvage strategy of ventilation was used for the affected lung with a high peep and low tv goals , whereas a lung protective strategy was adopted for the right lung with minimisation of fio2 , peep , peak inspiratory pressure and tv . this helped in improvements in compliance and ventilation - perfusion in the affected lung , and hence we were able to gradually wean off the ventilatory support and extubate successfully . dlt prevents the unaffected lung from being flooded with the oedema fluid from the contralateral lung , and facilitates dlv , which not only protects the affected lung with poor compliance , but also prevents the volutrauma and barotrauma to normal compliant lung . prevention of development of repe post thoracocentesis include the use of low negative pressure ( < 20 cmh2o ) for suction during tube thoracostomy and restricting the drainage to about 1.5 l of pleural fluid . though repe has a high mortality rate , if fatality is avoided at an early stage , patient recovery is usually complete . dlv may be the initial ventilatory treatment strategy , and we should not wait for deterioration to occur by starting conventional lung ventilation as the pathophysiology of repe necessitates dlv .

re - expansion pulmonary oedema ( repe ) is a rare complication following re - inflation of a chronically collapsed lung , which is often fatal . we present a case of a 22-year - old male who presented to the hospital with severe respiratory distress and a history of blunt abdominal trauma 3 months back . he was diagnosed to have left sided diaphragmatic hernia with a mediastinal shift to the right , and was posted for emergency repair of the same . after surgical decompression of the left hemi - thorax and reduction of the abdominal contents , re - expansion of the left lung was achieved , following which patient developed repe . a left sided double lumen tube was then inserted to prevent flooding and cross contamination of the right lung and ventilation of both lungs was maintained intraoperatively . post - operatively , repe was successfully managed by differential lung ventilation with a lung salvage strategy to the left lung and a lung protective strategy to the right lung .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

early childhood caries is a significant public health problem in both developing and industrialized countries which continues to affect babies and preschool children worldwide . early childhood caries ( ecc ) is not life - threatening but it may contribute to suboptimal health and failure to thrive . ecc is a chronic , transmissible , infectious disease with a complex and multifactorial etiology . the short - term consequences of untreated decay in children 's teeth include pain , with up to 12% of 5-year olds reported to have experienced tooth ache , systemic infection and abscesses . untreated caries may lead to early loss of the primary dentition and affect the growth and maturation of the secondary , adult dentition . in fact , decay in the primary dentition is the best predictor for decay in the permanent dentition ; poor dental health and disease often persist to adulthood , affecting speech articulation , growth , and dietary practices . thus , poor dental health has a significant impact on the growth and cognitive development of child by interfering with nutrition , concentration and subsequently school participation.[68 ] ecc prevalence varies from population to population ; however , children of disadvantaged subpopulations , regardless of race , ethnicity or culture , have been found to be most vulnerable . in england the prevalence reported ranges from 6.8 - 12% and in usa prevalence varies from 11.0 - 53.1% . in asia , in the far east region , which seems to have one of the highest prevalence and severity for the disease , the prevalence in three - year - olds ranges from 36 to 85%,[914 ] while in india a prevalence of 44% has been reported for caries in 8- to 48-month - olds . another study reported a caries prevalence of 54.1% in the preschool children of hubli and dharwad city . there has been a considerable amount of information about prevalence of early childhood caries in pre - schoolers in other parts of the world , whereas in india , not much baseline data is available . hence the aim of this study was to find out the prevalence of early childhood caries among 3 - 5 year old pre - schoolers in kindergartens of schools of marathahalli , bangalore . a cross sectional study was conducted among 3 - 5 years old pre - schoolers in schools of marathahalli , bangalore . a list of schools having kindergartens was obtained from deputy director of public instructions ( ddpi ) office , bangalore south . all the schools with kindergartens in marathahalli were included in the study . out of the eight schools , six schools permitted to conduct the study . before conducting the study , ethical clearance was obtained from the institutional review board . also , prior permission to conduct the study was obtained from the authorities of respective schools . voluntary written informed consent was obtained from parents of the children participating in the study before the clinical examination . all the children aged 3 - 5 years attending the schools of marathahalli , bangalore whose parents gave consent to participate in the study and who co - operated during the oral examination were included in the study . children with acute infections of oral cavity which interfered with oral examination were excluded from the study . a total of 717 pre - schoolers participated in the study . before the start of the survey , intra - examiner reliability was assessed on 10 subjects on different days and the examiner repeated her examinations on them . the kappa co - efficient value for intra - examiner reliability with respect to decayed , indicated for extraction and filled teeth index ( deft ) was 0.8 which reflected high degree of conformity in observations . a pilot study was carried out in one school chosen using simple random sampling , on all the 3 - 5 year old pre - schoolers present on the day of examination to check the feasibility and relevance of proforma , to have prior idea regarding the estimate of the time taken to examine each patient and the survey was planned accordingly . early childhood caries was diagnosed using early childhood caries diagnostic criteria , consistent with the nidcr ( national institute of dental and craniofacial research ) workshop statement . both d1 and d2 stages of caries were recorded . clinical examination was conducted at respective schools by making child sit on ordinary chair with back rest , with the examiner sitting in front of the chair and deft index was recorded using gruebell 's criteria . the d component included decayed deciduous teeth ( both initial and cavitated lesions ) , e component included deciduous teeth extracted due to caries or indicated for extraction , and f included filled teeth . descriptive statistics that included mean , standard deviation and percentages were calculated for each of the categories . student t - test , anova was used to find out significant differences in mean deft between groups . significance for all statistical tests was predetermined at a probability value of 0.05 or less . the study population consisted of 717 pre - schoolers in the age range of 3 - 5 years . out of 717 children , 317 ( 44.2% ) were females and 400 ( 55.8% ) were males . 201 ( 28% ) were 3 years old , 243 ( 33.9% ) were 4 years old and 273 ( 38.1% ) were 5 years old with a mean age of 4.10 ( 0.8 ) years . the prevalence of early childhood caries among the study population was 40% with a mean dmft of 1.89 3.3 while the sic index of the study population was 5.51 . out of 201 3 year old children , 90 ( 44.8% ) had early childhood caries and 111 ( 55.2% ) did not had caries . among 243 4 year old children , 85 ( 35% ) had early childhood caries and 158 ( 65% ) were caries free . out of 273 5 year old children , 112 ( 41% ) had early childhood caries and 161 ( 59% ) did not had caries . 126 ( 39.7% ) females and 161 ( 40.3% ) males had early childhood caries [ table 1 ] . age and gender wise prevalence of early childhood caries the mean dmft of 3 year old was 1.86 2.9 , of 4 year old was 2.0 3.8 and in 5 year old was 1.81 3.1 . the mean dmft in females was 1.71 2.9 while in males it was 2.03 3.6 . however , this difference was also not statistically significant ( p = 0.198 ) [ table 2 ] . mean dmft of study population according to age and gender the pattern of tooth wise distribution of early childhood caries among the study population showed that the most severely affected teeth were maxillary central incisors ( 19.5% and 20.2% for maxillary right and maxillary left central incisor respectively ) followed by maxillary lateral incisors ( 14.9% and 14.6% for maxillary right and maxillary left lateral incisor respectively ) and mandibular second molars ( 14.9% on right side of the arch and 14.4% on left side of the arch ) . the caries pattern was fairly symmetrical across the arches [ table 3 ] . pattern of tooth wise distribution of early childhood caries this present study was conducted on 717 pre - schoolers aged 3 - 5 years in kindergartens of schools of marathahalli , bangalore , karnataka , india . early childhood caries is a lifestyle disease that begins when the child 's teeth erupt in the oral cavity . the distinctive pattern of decay rapidly spreads from one tooth to another and involves the surfaces of teeth that are usually not at risk . the children were examined both for non cavitated ( including white spot lesions ) and cavitated lesions . no attempt was made to use a dental explorer to confirm cavitation of the lesions due to the young age of the children . this ensured their compliance with the examination without adversely affecting their cooperation and behavior in the dental environment in future . this study documented a 40% prevalence of early childhood caries which is in equivalence to karnataka state average ( 40.5% ) , our country average ( 40 - 60% , 52% ) and studies done by saravanan s ( 44.4% ) and simratvir , et al . caries prevalence ( 40% ) was higher in this population in comparison to studies conducted by wyne , askarizadeh , et al . who reported a prevalence of 27.3% and 17.2% respectively . on the other hand , the prevalence of the present study was less in comparison to studies done by mazhari and sadeghi . this can be attributed to the inclusion of early carious lesions and non cavitated lesions into consideration for diagnosis in the present study . the study population in the present study had a mean dmft of 1.89 3.3 . this mean dmft values are in conformity with the karnataka state average ( 1.7 ) , studies by simratvir , sudha p. , horowitz , et al . however , it was much lesser in comparison to study done by wyne who reported a mean dmft of 8.6 ( 3.4 ) and sadeghi who reported a mean dmft of ( 2.6 3.15 ) . since the caries was diagnosed entirely on visual examination in the present study , this certainly resulted in an underestimation of the actual caries status ; hence it is possible that the true carious lesion prevalence would be higher than reported in our present study . the other contributory factors could be varied diagnostic criteria used for diagnosis of early childhood caries , genetic predisposition , lack of parental education and varied socioeconomic status among the population studied , lack of dietary and oral hygiene discipline , in addition to very late first dental visit for routine checkup.[61820 ] the mean deft among the 3 year old children was 44.8% ( dmft 1.86 2.9 ) which was less in comparison to that reported by schroth , et al . similar difference was found for 4 year old and 5 year old children who reported a mean dmft of 2.0 3.8 in 4 years old and 1.81 3.1 in 5 years old in the present study while it was 13.9 2.8 in 4 years old and 13.7 2.9 in 5 year old according to scroth , et al . however the results of the present study were similar to those done in ludhiana , hubli - dharwad which shows that most of the lesions recorded were of d category ( decayed teeth , which required treatment ) . this clearly reflect that the availability of oral health services , oral health awareness , socioeconomic status and attitude of masses towards oral health greatly influence the distribution of def components . the mean dmft was slightly high among boys compared to the girls with a mean dmft of 2.03 3.6 and 1.71 2.9 respectively which was similar to the studies by simratvir , et al . however this difference was not statistically significant ( p = 0.081 ) which was consistent with the other studies.[6102223 ] the early childhood caries pattern among the present study group showed that maxillary right and left central incisors showed highest prevalence of caries ( 19.5% and 20.2% respectively ) followed by mandibular second molars ( 14.9% and 14.4% on left and right side respectively ) and the mandibular incisors showed the lowest prevalence of caries being 0.3% and 0.1% . it is noteworthy that majority of the children , who had decay , had anterior tooth involvement as well ; clearly indicative of the fact that besides oral hygiene , erratic nursing habit might have also contributed to caries . lack of dexterity in this age group and negligence on the part of caretakers might have accounted for poor oral health . also , besides oral hygiene the present study showed a sic index value of 5.51 for 3 - 5 year old pre - schoolers . this was slightly higher than the sic index of 4.11 in brazilian population reported by carvalho , et al . this aims at focusing the interest on the neglected and needy high risk group pre - schoolers and calls for a more specific and targeted actions . past caries experience is an important indicator for future caries risk and children with high caries levels will most likely be those adults requiring complex and expensive treatments in the future . effective early prevention measures will cut down treatment expenditure for this caries prone group in their adult life . thus , the aim should be population based prevention to reduce the overall caries burden of this underprivileged preschool age group . early childhood caries is a public health problem that warrants the attention and resources of the community . this study showed a 40% prevalence of early childhood caries among 3 - 5 year old pre - schoolers in marathahalli , bangalore . efforts to increase awareness of the public on the prevalence , severity and impact of ecc on general and oral health , growth and development of children should be undertaken . awareness on the diagnosis , prevention and treatment of ecc should be increased among dentists , physicians , paediatricians , nurses , midwives and other community health workers involved in care of preschool children . thus , the result of the present study calls for a need to focus on pre - schoolers oral health and parental education for prevention and early detection of early childhood caries .

background : dental caries among preschool children is still a major public health problem in many developing countries including india . hence the aim of the present study was to find out the prevalence of early childhood caries among 3 - 5 year old pre - schoolers in schools of marathahalli , bangalore.materials and methods : a cross sectional study was conducted on 717 pre - schoolers in 6 schools of marathahalli . clinical examination was performed and deft index was recorded using gruebell 's criteria . early childhood caries was diagnosed using early childhood caries diagnostic criteria , consistent with the nidcr workshop statement . data was analysed using spss 15.0 and descriptive statistics was applied . chi - square test was used to find out the significant differences . the level of significance was taken at p value < 0.05.results:prevalence of early childhood caries was 40% with a mean deft of 1.89 ( + 3.3 ) and significant caries index score was 5.51 . 44.8% of 3 year old had early childhood caries , 35% of 4 year old children and 41% of 5 year old had early childhood caries . almost , all of deft was due to untreated caries.conclusion:the results of the present study calls for a need to focus on pre - schoolers oral health and parental education for prevention and early detection of early childhood caries . a high significant caries index in this study population indicates a more targeted approach for high risk pre - schoolers .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

a 36-year - old woman with no history of any trauma exhibited a palpable mass on the volar ulnar aspect of her left middle finger at the level of the distal interphalangeal joint for a year . physical examination revealed a 11.5-cm hard , non - tender , non - movable mass . plain radiographs of the finger revealed a calcifying soft tissue mass associated with large cortical scalloping on the volar ulnar side of the distal phalanx ( fig . the distal interphalangeal joint was normal in appearance . on the high - resolution ultrasound ( us ) exam ( hdi5000 , philips medical system , bothell , wa ) with a high - frequency ( 15 - 7 mhz ) compact linear transducer , a heterogeneously hyper - echoic mass with surface lobulation and extrinsic cortical erosion by the mass was clearly observable ( fig . surgery revealed a flesh - colored lobulated mass was firmly attached to the periosteum of the distal phalanx and eroded it . microscopic sections revealed a spindle - cell neoplasm with scattered calcifications and chondroid differentiation ( fig . 3 ) . a pathologic diagnosis of calcifying aponeurotic fibroma was made . during the last nine months calcifying aponeurotic fibroma usually appears in the first decade of life and most of the lesions manifest within the first 2 decades of life . for this reason , the tumor has since been reported in the fingers , wrist , forearm , elbow , upper arm , neck , abdominal wall , lumbar paravertebral area , leg , ankle , and thigh ( 4 , 5 ) . fibromatoses can be divided into two subdivisions , based on anatomic location and on age of presentation . one is the " juvenile fibromatoses " including juvenile aponeurotic fibroma , congenital generalized fibromatosis , fibromatosis coli , fibrous hamartoma of infancy , recurring digital fibrous tumor of reye , juvenile hyaline fibromatosis , diffuse infantile fibromatosis , and hereditary gingival fibromatosis . the second subdivision includes the aggressive fibromatoses , plantar - palmar fibromatosis , and nodular fasciitis ( 1 ) . radiologically , calcifying aponeurotic fibroma may show a soft tissue mass with no associated osseous lesions and a fine stippling of focal calcification ( 4 ) . however , in extremely rare cases , occasional scalloping of the cortex ( 1 , 3 ) and thickening of the bone ( 2 ) have been reported in pediatric patients . to our knowledge , there has been no case with involvement of the distal digital phalanx . on mri , kwak et al . ( 7 ) reported that the signal intensity of the calcifying aponeurotic fibroma was lower than that of muscle on t1- and t2-weighted images . in our case , us also clearly demonstrated the extent and character of the soft tissue mass with internal calcific foci and cortical erosive changes of the adjacent bone . the recurrence rate after surgical excision of this tumor is high and generally reported at 50% ( 8) . this necessitates an accurate diagnosis and complete excision . therefore , evaluation of the extent of the mass and its relationship with surrounding soft tissue or bone is important . the differential diagnosis of calcifying aponeurotic fibroma differs depending on the patient 's age , location of the lesion , presence of the calcification , and osseous involvement . calcifying aponeurotic fibroma in adults should be differentiated from other fibrous tumors such as aggressive fibromatoses , plantar - palmar fibromatosis , and nodular fasciitis . however , in this case , considering distal phalangeal erosive soft tissue mass with calcification , the differential diagnoses were parosteal / soft tissue chondroma , synovial sarcoma , and the calcified epidermoid . in summary , calcifying aponeurotic fibroma is a rare soft tissue tumor that presents as a painless mass primarily on the volar surface of the hands and plantar aspects of the feet in juveniles , but this tumor should be also included in differential diagnoses of any mass with calcification and adjacent bone involvement in the distal phalanx of the finger . in addition , us could be useful for the preoperative evaluation of digital calcifying aponeurotic fibroma .

calcifying aponeurotic fibroma is a rare soft tissue tumor that occurs in the distal extremities of children and adolescents . we report ultrasound and x - ray findings of a calcifying aponeurotic fibroma in the finger of a 36-year - old woman , associated with distal phalangeal bone involvement .

CASE REPORT DISCUSSION

more than half of the benign tumors are myxomas , and the rest are lipomas , papillary fibroelastoma , and rhabdomyomas . sarcoma ( including angiosarcoma , undifferentiated sarcoma , malignant fibrous histiocytoma , leiomyosarcoma , and osteosarcoma ) is the most often reported neoplasm occupying 95% of malignant cardiac tumors . surgical resection is the most common and effective therapy for both benign and malignant tumors . surgical approaches have varied as science and technology has advanced , and cardiac neoplasms can be excised using different procedures . few articles have been published that summarize the surgical experience within a large population and the comprehensive surgical approaches used over two decades . in this study the cardiovascular surgery database at our center was searched for records of all patients who had cardiac tumor excision from january 1993 through to may 2016 . three operation approaches were included : 1 ) conventional procedure with median sternotomy ; 2 ) robotically assisted procedure with da vinci surgical system ( intuitive surgical , sunnyvale , ca , usa ) ; and 3 ) mini - thoracotomy procedure via right anterolateral approach . both robotically assisted procedure and mini - thoracotomy procedure were minimally invasive surgeries where cardiopulmonary bypass was established via right internal jugular venous cannulation , right femoral arterial , and venous cannulation . and tumors were excised through a small working port , which were especially small in robotically assisted procedures which were performed between the ribs . patient information , including demographics , cardiac comorbidities , symptoms , lesion characteristics , operation information , and in - hospital and six - month follow - up data were recorded . patient clinical characteristics and operation information were reviewed , and operation parameter , postoperative complications , and short - term prognosis among robotic , mini - thoracotomy , and conventional procedures were compared . categorical data were described as numbers and percentages and were compared using the chi - square analysis . normally distributed data were compared using analysis of variance ; data with non - conformity of normal distribution were compared by mann - whitney u test or kruskal - wallis test . all statistical analyses were performed with spss , version 17 ( spss , inc . , chicago , il , usa ) . the cardiovascular surgery database at our center was searched for records of all patients who had cardiac tumor excision from january 1993 through to may 2016 . three operation approaches were included : 1 ) conventional procedure with median sternotomy ; 2 ) robotically assisted procedure with da vinci surgical system ( intuitive surgical , sunnyvale , ca , usa ) ; and 3 ) mini - thoracotomy procedure via right anterolateral approach . both robotically assisted procedure and mini - thoracotomy procedure were minimally invasive surgeries where cardiopulmonary bypass was established via right internal jugular venous cannulation , right femoral arterial , and venous cannulation . and tumors were excised through a small working port , which were especially small in robotically assisted procedures which were performed between the ribs . patient information , including demographics , cardiac comorbidities , symptoms , lesion characteristics , operation information , and in - hospital and six - month follow - up data were recorded . patient clinical characteristics and operation information were reviewed , and operation parameter , postoperative complications , and short - term prognosis among robotic , mini - thoracotomy , and conventional procedures were compared . categorical data were described as numbers and percentages and were compared using the chi - square analysis . normally distributed data were compared using analysis of variance ; data with non - conformity of normal distribution were compared by mann - whitney u test or kruskal - wallis test . all statistical analyses were performed with spss , version 17 ( spss , inc . , chicago , il , usa ) . we identified a total of 225 patients with mean age of 50.713.3 years ( 61.3% female and 38.7% male ) ( table 1 ) and mean body mass index of 23.64.1 kg / m . among the 225 patients , 31 patients had hypertension ( 13.8% ) , 15 patients had diabetes ( 6.7% ) , 19 patients had coronary artery disease ( 8.4% ) , nine patients had valvular heart disease ( 4.0% ) , and 30 patients had cerebral vascular disease ( 13.3% ; 27 patients within three months , and three patients exceed three months ) . the cardiac function of most patients were fine according to new york heart association ( nyha ) classification grading of cardiac function : 61 patients were nyha i ( 27.1% ) , 129 patients were nyha ii ( 57.3% ) , 30 patients were nyha iii ( 13.3% ) , and five patients were nyha iv ( 2.0% ) . patients with heart neoplasm presented diverse symptom , and 42 patients ( 18.7% ) were asymptomatic . up to 164 patients ( 72.9% ) complained of cardiac symptoms including dyspnea and orthopnea . cerebral vascular symptoms occurred in 27 patients ( 12.0% ) including the patients combined with cardiac symptom . peripheral embolism happened in 18 patients ( 8.0% ) ; and two patients ( 1.3% ) had pulmonary embolism , both of whom had emergency operations . a total of 232 lesions were founded in 228 operations ( table 2 ) . up to 201 lesions occurred in the left atrium ( la ) , 23 lesions in the right atrium ( ra ) , six in the left ventricle ( lv ) , and two in the right ventricle ( rv ) . neoplasms that occurred in three chambers ( la , lv , and rv ) were seen in one patient , biatrial lesions ( la and ra ) occurred in two patients , and 225 patients had a tumor in a single chamber . the mean size of tumors was 4.83.4 cm according to echocardiogram measurements ( product of maximum diameter ) . among the tumors , 220 lesions ( 94.8% ) were myxoma . about 5.2% of the neoplasms were non - myxoma , including three fibromas ( 1.3% ) , two cardiac lipomas ( 0.9% ) , two thrombus tumors ( 0.9% ) , one atrial hemangioma ( 0.4% ) , one parasites tumor ( 0.4% ) , one papillary fibroelastoma ( 0.4% ) , one sarcoma ( 0.4% ) , and one inflammatory myofibroblastic tumor ( 0.4% ) . for the 225 patients , a total of 228 operations were performed in our center , 68.4% of which were conventional procedures via a median sternotomy approach . concomitant procedures for cardiac comorbidities were performed on 18 patients , including nine valve surgery , eight coronary artery bypass graft and one pericardiectomy . robotically assisted procedure was performed in 60 patients ( 26.3% ) without other concomitant procedures . mini - thoracotomy procedures using the right anterolateral approach were completed on 12 patients ( 5.3% ) . redo - operations for recurrent cardiac neoplasms were performed on five patients ( 2.2% ) through a conventional approach , four patients had the operation for a second time , and one patient for a third time . the patient who had three operations was found to have neoplasm re - occurrence in january 2016 and had not yet returned for an operation . conventional procedures were the preferred approach in patients who had complex cardiac comorbidities ( p=0.000 ) , poor heart function ( p=0.005 ) , emergency status , and re - do operation . operative complication rate after operation was 15.8% ( 36/228 ) ; 20 patients ( 8.8% ) had complications with arrhythmia ; 17 patients ( 7.5% ) had atrial fibrillation ( af ) , two patients ( 0.9% ) had supraventricular tachycardia , ( svt ) , one patient ( 0.4% ) had af combined with ventricular premature beat ( vpb ) , 11 patients ( 4.8% ) had delayed mechanical ventilation ( dmv ) with ventilation time > 24 hours , one patient ( 0.4% ) had complications with cerebral infarction , one patient ( 0.4% ) had pleural effusion ( pf ) , one patient ( 0.4% ) had complications with pneumothorax , one patient ( 0.4% ) suffered from subcutaneous emphysema , and one patient ( 0.4% ) presented with dmv and pf . patients with nyha iii / iv , recurrent neoplasm excision , comorbidity operation , and other chamber lesions were excluded . a total of 156 patients accepted the conventional procedure , and 84 patients received the left atrial tumor resection after excluding . robotic procedure and mini - thoracotomy procedure were performed on 60 and 12 patients respectively , 53 and 10 patients had left atrial neoplasm resection after screening respectively ( table 3 ) . cardiopulmonary bypass ( cpb ) time was not significantly different between the robotic group and the conventional group ( p=0.196 ) . however , cpb time in the mini - thoracotomy group was longer than the robotic group ( p=0.034 ) and the conventional group ( p=0.002 ) . tumors were more often removed via left atriotomy using the robotic procedure and more frequently resected via the right atrium when using the mini - thoracotomy and the conventional procedure group ( p=0.000 ) . differences were not noted in operative complications ( p=0.835 ) including arrhythmia ( p=0.212 ) , dmv ( p=0.614 ) , and thoracic complication ( p=0.645 ) among the three groups . in six - month follow - up , main adverse events ( maes ) , including arrhythmia , cardiac death , stroke , and non - cerebral systemic embolism were seen in 11 patients ( 13.1% ) in the conventional group , six patients ( 11.3% ) in the robotic group and three patients ( 30% ) in the mini - thoracotomy group , which was not statistically significantly different among the groups ( p=0.285 , with 4.7% lost to follow - up ) . we identified a total of 225 patients with mean age of 50.713.3 years ( 61.3% female and 38.7% male ) ( table 1 ) and mean body mass index of 23.64.1 kg / m . among the 225 patients , 31 patients had hypertension ( 13.8% ) , 15 patients had diabetes ( 6.7% ) , 19 patients had coronary artery disease ( 8.4% ) , nine patients had valvular heart disease ( 4.0% ) , and 30 patients had cerebral vascular disease ( 13.3% ; 27 patients within three months , and three patients exceed three months ) . the cardiac function of most patients were fine according to new york heart association ( nyha ) classification grading of cardiac function : 61 patients were nyha i ( 27.1% ) , 129 patients were nyha ii ( 57.3% ) , 30 patients were nyha iii ( 13.3% ) , and five patients were nyha iv ( 2.0% ) . patients with heart neoplasm presented diverse symptom , and 42 patients ( 18.7% ) were asymptomatic . up to 164 patients ( 72.9% ) complained of cardiac symptoms including dyspnea and orthopnea . cerebral vascular symptoms occurred in 27 patients ( 12.0% ) including the patients combined with cardiac symptom . peripheral embolism happened in 18 patients ( 8.0% ) ; and two patients ( 1.3% ) had pulmonary embolism , both of whom had emergency operations . a total of 232 lesions were founded in 228 operations ( table 2 ) . up to 201 lesions occurred in the left atrium ( la ) , 23 lesions in the right atrium ( ra ) , six in the left ventricle ( lv ) , and two in the right ventricle ( rv ) . neoplasms that occurred in three chambers ( la , lv , and rv ) were seen in one patient , biatrial lesions ( la and ra ) occurred in two patients , and 225 patients had a tumor in a single chamber . the mean size of tumors was 4.83.4 cm according to echocardiogram measurements ( product of maximum diameter ) . among the tumors , 220 lesions ( 94.8% ) were myxoma . about 5.2% of the neoplasms were non - myxoma , including three fibromas ( 1.3% ) , two cardiac lipomas ( 0.9% ) , two thrombus tumors ( 0.9% ) , one atrial hemangioma ( 0.4% ) , one parasites tumor ( 0.4% ) , one papillary fibroelastoma ( 0.4% ) , one sarcoma ( 0.4% ) , and one inflammatory myofibroblastic tumor ( 0.4% ) . for the 225 patients , a total of 228 operations were performed in our center , 68.4% of which were conventional procedures via a median sternotomy approach . concomitant procedures for cardiac comorbidities were performed on 18 patients , including nine valve surgery , eight coronary artery bypass graft and one pericardiectomy . robotically assisted procedure was performed in 60 patients ( 26.3% ) without other concomitant procedures . mini - thoracotomy procedures using the right anterolateral approach were completed on 12 patients ( 5.3% ) . redo - operations for recurrent cardiac neoplasms were performed on five patients ( 2.2% ) through a conventional approach , four patients had the operation for a second time , and one patient for a third time . the patient who had three operations was found to have neoplasm re - occurrence in january 2016 and had not yet returned for an operation . conventional procedures were the preferred approach in patients who had complex cardiac comorbidities ( p=0.000 ) , poor heart function ( p=0.005 ) , emergency status , and re - do operation . operative complication rate after operation was 15.8% ( 36/228 ) ; 20 patients ( 8.8% ) had complications with arrhythmia ; 17 patients ( 7.5% ) had atrial fibrillation ( af ) , two patients ( 0.9% ) had supraventricular tachycardia , ( svt ) , one patient ( 0.4% ) had af combined with ventricular premature beat ( vpb ) , 11 patients ( 4.8% ) had delayed mechanical ventilation ( dmv ) with ventilation time > 24 hours , one patient ( 0.4% ) had complications with cerebral infarction , one patient ( 0.4% ) had pleural effusion ( pf ) , one patient ( 0.4% ) had complications with pneumothorax , one patient ( 0.4% ) suffered from subcutaneous emphysema , and one patient ( 0.4% ) presented with dmv and pf . patients with nyha iii / iv , recurrent neoplasm excision , comorbidity operation , and other chamber lesions were excluded . a total of 156 patients accepted the conventional procedure , and 84 patients received the left atrial tumor resection after excluding . robotic procedure and mini - thoracotomy procedure were performed on 60 and 12 patients respectively , 53 and 10 patients had left atrial neoplasm resection after screening respectively ( table 3 ) . cardiopulmonary bypass ( cpb ) time was not significantly different between the robotic group and the conventional group ( p=0.196 ) . however , cpb time in the mini - thoracotomy group was longer than the robotic group ( p=0.034 ) and the conventional group ( p=0.002 ) . tumors were more often removed via left atriotomy using the robotic procedure and more frequently resected via the right atrium when using the mini - thoracotomy and the conventional procedure group ( p=0.000 ) . differences were not noted in operative complications ( p=0.835 ) including arrhythmia ( p=0.212 ) , dmv ( p=0.614 ) , and thoracic complication ( p=0.645 ) among the three groups . in six - month follow - up , main adverse events ( maes ) , including arrhythmia , cardiac death , stroke , and non - cerebral systemic embolism were seen in 11 patients ( 13.1% ) in the conventional group , six patients ( 11.3% ) in the robotic group and three patients ( 30% ) in the mini - thoracotomy group , which was not statistically significantly different among the groups ( p=0.285 , with 4.7% lost to follow - up ) . the kaplan - meier curve for maes is presented in figure 1 . primary cardiac neoplasm is a disease with a low prevalence and favorable prognosis after operation . tumors occur more often in the middle - aged and elderly population , and the morbidity is higher in females . cardiac myxoma rarely occurs in young patients , and only 0.4% of patients were under the age of 10 years in our center study . in a previous study , young patients with cardiac myxoma were often in syndromic context of so - called carney complex ( cnc ) , which was first described as the syndrome of myxomas , spotty pigmentation , and endocrine over activity by carney at the mayo clinic in 1985 and has been found to be associated with germline mutation prkar1a . the majority of patients usually present with dyspnea and orthopnea , and some patients have other symptom such as syncope , presyncope , angina , and hemoptysis . systemic embolism is frequently seen and appeared on 20% of patients in our center and cerebral embolism was seen more often than peripheral artery . pulmonary embolism was rare , and usually occurred in patients with right heart neoplasm , and regularly required emergency operation . echocardiography is an ideal imaging modality since it is simple , non - invasive , widely available , and of low cost . only one patient had tumors arising from three chambers and the tumors re - occurred seven months after the operation . myxoma is the most common histological type with a ratio of 94.8% in our institution , which was higher than that of 7889% in previous japanese studies and 4250% in american studies . benign tumors such as fibroma , lipoma , hemangioma , and papillary fibroelastoma always have favorable prognosis after surgery operation . sarcoma , which is a malignant neoplasm , is rarely seen and usually has a poor prognosis . in a previous study , only 31.9% of patients with sarcoma underwent effective surgical resection because of advanced - stage tumors , metastatic extracardiac malignancy , and high comorbidity burdens . since the first excision of a left atrial myxoma was performed by swedish cardiovascular surgeon clarence crafoord in 1954 , atrial arrhythmia , which occurred in 8.8% of patients in our center , was the most common complication . in one large series , transient atrial arrhythmias were seen in 26% of patients but only 2% of patients needed a permanent pacemaker . andrew et al . found the recurrence rate of myxoma after resection was 13% , but was much more common with familial myxomas than with sporadic tumors ( 22% versus 3% ) . local recurrence of myxoma is uncommon but may be related to inadequate resection , multi - centricity , origin in chamber other than the left atrium , familial tumors , or disease complexes such as cnc . minimally invasive surgery ( mis ) with a healing cosmetic incision has been applied to cardiac surgery more and more often due to advances in optics and instrumentations . the safety and efficacy of minimally invasive cardiac tumor resection has been confirmed in previous studies for select patients . in our study , robotic - assisted procedures and mini - thoracotomy procedures of the left atrial myxoma resection had no significant differences in operative parameters such as cross clamp time and operative duration compared with a conventional procedure . but the cpb time in the mini - thoracotomy group was longer than the other two groups , which may have been due to the steep learning curve of the surgery . the in - hospital mortality , para - operative complications , and six - month maes of the three groups had no differences according to our experience . in our previous study , patients who accepted robotic - assisted myxoma resection had a better quality of life after their operation and went back to work much earlier compared with patients who had a conventional surgical procedure . however , we believe that it is more sensible to perform traditional open chest surgery on patients who have complex cardiac comorbidities , poor heart function , emergency status , and re - do operations . minimally invasive surgery of cardiac tumor resection can be an alternative to conventional operation in select patients .

backgroundprimary cardiac tumors are rare but have favorable surgical prognosis . previous studies have been small series studies with limited surgical approaches . to date , few studies have examined the clinical features associated with different surgical procedures.material/methodsin a search of the cardiovascular surgery database of our institution , we retrospectively identified 225 patients who had cardiac tumor resection from january 1993 to may 2016 . the patients clinical characteristics and operation information were reviewed , and the operation parameters , postoperative complications , and short - term prognosis among robotic , mini - thoracotomy , and conventional procedures in our center were compared.resultsa total of 228 operations were performed , including 156 traditional open surgeries ( 68.4% ) , 60 robotically assisted neoplasm resections ( 26.3% ) , and 12 mini - thoracotomy procedures ( 5.3% ) . among 232 lesions , myxoma ( 94.8% ) was the most common neoplasm , and the remainders were fibroma ( 1.3% ) and lipoma ( 0.9% ) . operative complications occurred in 36 patients ( 15.8% ) . arrhythmia ( 8.8% ) was the first common complication , and delayed mechanical ventilation ( 4.8% ) ranked second . the overall risk of recurrence of myxoma was 2.7% . the cardiopulmonary bypass ( cpb ) time in the mini - thoracotomy group was longer than in the robotic group ( p=0.034 ) and the conventional group ( p=0.002 ) . there were no significant differences in cross clamp time ( p=0.266 ) or complications ( p=0.835 ) among the three groups . the in - hospital survival rate was 100% in all patients . there were no significant differences in main adverse events among the three groups at six - month follow - up ( p=0.285).conclusionsprognosis for cardiac neoplasm surgical resection is favorable for primary cardiac tumors . the minimally invasive surgery of cardiac tumor resection can be an alternative to conventional operations in selected patients .

Background Material and Methods Patient selection Data recording Data analysis Statistical analysis Results Patient characteristics Patient symptom Lesion characteristics Operation information Conventional versus minimally invasive procedure Discussion Conclusions

the gag reflex is a physiological response which safeguards the airway from foreign bodies . gagging can become a conditioned response that makes dental treatment difficult or impossible for both the individual and the dentist . clinicians successfully treat many patients with mild gagging problems using only minor procedural modifications or behaviour techniques . generally , the ultimate goal for patients is to make routine dental care possible by helping them unlearn the behaviour that leads to gagging . therefore , strategies to overcome the gag reflex in treatment have focused on behaviour modification using systemic desensitization and distraction methods . some patients suffered from such severe gagging that behaviour techniques did not sufficiently reduce gagging in dentistry . in these patients pharmacological management ( sedatives [ propofol , midazolam , nitrous oxide e.g. ] , antihistamine , or parasympathetic depressants ) was thought to be the last alternative to eliminate the reflex [ 4 - 6 ] . we present three cases which describe the rehabilitation of a female and two paediatric patients who have exaggerated gag reflex requiring prosthetic and orthodontic procedures with the help of intranasal midazolam . a 50 year old , 65 kg female patient was referred to the prosthetic department of seluk university for rehabilitation of their oral function and aesthetic . she had malformed anterior teeth in the maxilla and left first molar present in the mandible . the treatment was planned as providing partial removable dentures retained with fixed partial dentures . intranasal midazolam 2.5 mg ( demizolam , dem medikal , istanbul ) was employed to facilitate the taking of accurate dental impressions without gagging during the whole procedure by the anaesthesiologist . the patient was given verbal information about the procedure at an assessment visit prior to the impression appointment . the patient had no gag reflex and allowed the dentist to apply the impression tray accurately in the mouth . the patient was controlled after sedation for 45 minutes before she was discharged . in addition , planning for horse - shoe palatal connector reduced palatal coverage area , thereby , providing less interference for tongue and reduced gag reflex ( figure 1 and 2 ) . initial intraoral photograph of patient 1 . a 15 year old , 56 kg child patient was referred to the prosthetic department of seluk university . patient was suffering from chewing dysfunction resulting from maxillary tooth agenesis and deficiency . he did not allow the dentist to put the tray into his mouth and this encouraged us to administer midazolam intranasally for its inhibitory effect on gagging . intranasal midazolam 2.5 mg ( demizolam , dem medikal , istanbul ) were applied incrementally in both nostrils . patient was able to overcome his gagging reflex and completed the treatment by the help of midazolam . an 11 year old , 45 kg child patient was referred to the orthodontic department of seluk university . it was learnt from the medical history of the patient that he used regular medication for hyperactivity . in extraoral clinical examination , it was observed that the patient had a skeletal class iii malocclusion and concave profile . in the intraoral and radiographic examination , it was detected that the patient was in mixed dentition stage and angle class ii molar relationship on both sides . according to the facial midline , the maxillary midline was 1 mm on the left and the mandibular midline was 1 mm on the right . the orthodontic treatment plan included non - extraction treatment after using face mask . during obtaining initial records , taking impression procedure was elicited patient s hypersensitive gagging reflex . therefore , it was decided to administer intranasal midazolam in advantage to take precise dental impressions without gagging during the whole procedure . after patient s parents were given verbal information about the procedure , 2.5 mg intranasal midazolam ( demizolam , dem medikal , istanbul ) were applied incrementally in both nostrils . five minutes later , the patient felt himself relaxed and the level of sedation was sufficient to take dental impression . we experienced the benefit of intranasal midazolam to facilitate taking precise dental impressions in problematic gagging patients intolerable to dental therapy . all patients suffered moderate burning in the nostril after delivery of intranasal midazolam ( figure 3 and 4 ) . retentive denture prosthesis is a major factor in achieving a successful result when providing partial removable dentures . the best results are obtained when the prosthesis bases contain all anatomic landmarks in the upper and lower arches . this is really complicated when the patients anamnesis tells us the difficulty with the impression phase of treatment due to a hypersensitive gag reflex . intranasal midazolam may be safely used to facilitate the taking of dental impressions in these patients . in the aetiology of gagging , anatomical abnormalities or neural hypersensitivity in the oropharynx have been identified as somatogenic factors in the development of the gag reflex . in a previous study by sata et al . , it was reported that psychological factors were identified in all patients with strong anxiety indicated as the probable cause of the gag reflex . however same authors were stated that the gag reflex was caused by touching the oral mucosa in most of patients with some difference in degree of severity . in our patients , a pre - treatment oral examination was not performed to determine gagging severity but a hypersensitive gag reflex was developed during taking impression in all patients . however it may not be easy to distinguish between two aetiological factors because physical stimuli may still provoke gagging of psychogenic origin . a number of techniques for reduction of gagging have been suggested , including distraction of patient s attention from the dental procedure , relaxation , hypnosis , acupuncture , drugs and general anaesthesia ( ga ) . ga is also conducted in a patient with extremely problematic gagging who is intolerable to dental therapy under intravenous sedation . the advantage of ga is complete elimination of the reflex , but costs should be considered in comparison to the benefit of dental care . as another alternative , inhalation sedation using nitrous oxide is the most common method of delivery of conscious sedation in dentistry . nitrous oxide inhalation sedation was shown to be useful for control of the gagging reflex . nitrous oxide is a commonly used pharmacologic agent but has the main disadvantage in especially children of being a nasal inhalation agent . one of the popular medications available to the dentist for use in children is midazolam . midazolam is a short - acting benzodiazepine with a short half - life , in children , of two hours compared to 18 hours for diazepam . midazolam has been used for preoperative sedation by the intramuscular , rectal , oral and nasal routes . the injections are painful and injection procedure is the most common fear of the children . the anaesthetics used orally and rectally start to relieve symptoms in a longer time . this technique has advantages when compared with oral administration as the bioavailability of intranasal administered midazolam is approximately 55% , compared with 15% when administered orally . the rate of onset and recovery are more rapid , but intranasal burning is the main disadvantage of the nasal application of midazolam . the nasal anaesthesia is popular as a useful and safe way . the short absorption time and the fast relief process of anaesthetics transferred to the systemic circulatory system are important advantages of nasal anaesthesia . no serious adverse effects were observed and the patients remained cooperated in the impression phase as in the beginning of the treatment ; they had no gagging during the entire procedure which was completed to the full satisfaction of all involved . these three cases showed that intranasal midazolam could be very effective treatments in exaggerated gag reflex in paediatric and adult patients . for some patients , however , severe gagging can be elicited by the dentist s fingers or instruments contacting the oral mucosa or even by nontactile stimuli , for example , patients seeing the dentist or remembering a previous dental experience . some clinicians swab the sensitive mucosal areas with topical anaesthetics , whereas others suggest that impressions should be made under general anaesthesia or hypnosis . anaesthetic sprays are difficult to control and their use may result in increased risk of toxicity . intranasal midazolam has been found to be effective in doses ranging from 0.2 to 0.6 mg / kg ( maximum 15 mg ) when used for conscious sedation and as a premedicant for children . in our clinic ; 2.5 mg or 5 mg intranasal midazolam is being used according to the anaesthesiologists preference in adults and 0.5 mg / kg ( maximum 10 mg ) in child . in this clinical report the reason of this quantity of dose preference for the child patients is their weights ( 56 and 45 kg ) and no sedation effect is required for them . the beneficial effects of midazolam include sedation , anxiolysis , and reduction of postoperative vomiting . our patients were given written consent before orthodontic and prosthetic treatment and also verbal information about the midazolam procedure at an assessment visit prior to the impression appointment . premedication with oral midazolam has shown to be more effective than parental presence or placebo in reducing gagging anxiety and improving compliance at induction of anaesthesia . intranasal drug administration is relatively quick , simple , and may have benefits over transmucosal routes or rectal administration , which requires more patient cooperation . and , intravenous way is not needed . the use of conscious sedation with inhalational , oral , or intravenous agents may temporarily eliminate gagging during dental treatment . in dental procedures ; midazolam is accepted as one of the most effective agents to reduce gagging reflex due to its sedative effects . in a study by murphy et al . which has investigated the effect of intravenously administered midazolam on the sensitivity of upper airway reflexes in volunteers , the authors concluded that midazolam produced significant depression of upper airway reflex sensitivity . and this conclusion may also explain our clinical results . besides the sedative effect of midazolam , the intranasal midazolam was found to be very useful for taking impressions probably effecting on the depression of upper airway reflex sensitivity . further investigations by using quantitative methods are needed to be done to clearly prove this effect of midazolam in dentistry .

abstractbackgroundgagging can become a conditioned response that makes dental treatment difficult or impossible for both the individual and the dentist . the aim of this study is to report a 50 year old female and two children ( 15 and 11 years old ) with exaggerated gag reflex referred to faculty of dentistry of seluk university.methodsduring obtaining records , taking impression procedure was elicited the patients hypersensitive gagging reflex . therefore , it was decided to administer intranasal midazolam to facilitate the taking of accurate dental impressions . after given verbal information about the procedure , 2.5 mg intranasal midazolam ( demizolam , dem medikal , istanbul ) were applied incrementally in both nostrils.resultsfive minutes later ; patients had no gagging reflex and allowed the clinician to take satisfying impressions . no serious adverse effects were observed and the patients remained cooperative during the entire procedure which was completed to the full satisfaction of all involved . the short absorption time and the fast relief process of anaesthetics transferred to the systemic circulatory system are important advantages of nasal application.conclusionsintranasal midazolam may be very useful for the treatment of gag reflex probably effecting on the depression of upper airway reflex sensitivity .

INTRODUCTION CASE DESCRIPTION AND RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS

affective disorders are a leading cause of disabilities worldwide , and the etiology of these many affective disorders such as depression and posttraumatic stress disorder ( ptsd ) is due to hormone changes , which includes hypothalamus - pituitary - adrenal ( hpa ) axis in the peripheral nervous system and neuromodulators in the central nervous system . the hypothalamic - pituitary - adrenal ( hpa ) axis plays a pivotal role in stress induced physiological changes , so that the levels of hpa axis , such as cortisol , are a good biomarker for certain mental disorders . in the central nervous system , neuromodulators , such as norepinephrine ( ne ) , are regarded as a critical part of the central stress circuitry , which is consistent with pharmacological studies indicating that medical treatment acts by increasing the concentration of catecholamine . hpa axis and ne both can interact and interfere with each other ; for example , ne release in the central nervous system facilitates activation of the hypothalamic - pituitary - adrenal axis in response to acute stress . the major function of lc / ne system is to induce fight or flight hormonal hypothalamus - pituitary - adrenal ( hpa ) stress axis and the peripheral sympathoadrenal autonomic response system are possibly very good biomarkers for the physiological stresses , while the activities of the locus coeruleus ( lc ) or ne levels in the central nervous system are very good biomarkers for the mental stress . stress can be defined as any threat , either real or perceived , to the homeostasis and wellbeing of an organism . stress can be induced by two broad and qualitatively differentiated categories of stressors : physiological stress and psychological stressors . physiologic stress is a physical threat to the wellbeing , and the psychogenic stress is the cognitive processing or interpretation of the stimulus as stressful . the first category is a real and imminent physiological threat to health and wellbeing , and the second psychogenic stressor is cognitive interpretation of the stimulus as a stressful event . the locus coeruleus ( lc ) neurons can be activated by both stressful events , which means that information from both the external and the internal environment can activate lc / ne system . many electrophysiological and neurochemical studies have shown that the brain ne system is physically and robustly activated by a diverse array of acutely stressful stimuli . the locus coeruleus ( lc ) is regarded as a part of the central stress circuitry [ 6 , 7 ] , because robust activation of the lc was reported after stressful stimuli . the lc is the largest norepinephrine ( ne ) in the brain and projects axons to almost all brain regions . it is a pure ne nucleus in rodents , comprising approximately 1500 cells on each side of the brain stem in rats . the hpa axis is regulated by a number of neural and hormonal inputs to the hypothalamus , and noradrenergic system has been implicated as one of the important systems for the hpa stress axis , mainly through its action upon the corticotrophin - releasing factor neurons in the paraventricular nucleus ( pvn ) of the hypothalamus . the ne afferents to the pvn originate mainly from the medullary ne nuclei and reach the pvn via the ventral ne bundle . the crf neurons in the pvn play the most important role in handling neuroendocrine stress response . the pvn contain neurons producing vasopressin and oxytocin , also regarded as hormones to cope with stressors , as well as regulating the water and electrolytes balance and parturition , respectively . the functional significance of this pathway in mediating stress responses was demonstrated in many studies , such as lesion of na pathway . microinjection of ne directly into the pvn of conscious rats increases crf gene expression in the pvn in parallel with acth release from the pituitary . the ne influence upon the pvn crf neurons stress induced activation of ne neurotransmission facilitates behavioral responses evoked by acute stress [ 9 , 11 ] , whether these responses represented inhibition of ongoing behavior , consistent with the behavioral function of lc / ne to say no , or activation of behavior that would not otherwise occur in the absence of acute stress . an inability to appropriately initiate or regulate aspects of the stress response has been proposed as a potentially critical factor in the pathophysiology of various stress - related disorders . the lc has been implicated in a variety of physiological functions , such as fear and anxiety . multiple brain regions have been implicated in emotional processing including anxiety and fear , the amygdala , the lc , the septohippocampal system , and the raphe nuclei . consistently , the noradrenergic system in the brain is considered to play an important role in emotion , and central responses to stress , and has also been implicated in affective disorders . a considerable amount of evidence has suggested the relationship between the central ne and fear / anxiety . emotions are one possible means by which stress responses may be tailored to specific stress . ne is especially important to conditioned fear because fear learning occurs during times of threat and stress . dysregulation of lc / ne systems has been implicated in stress and its related psychiatric diseases such as depression , posttraumatic stress disorder ( ptsd ) , and other anxiety disorders . this notion is supported by the knowledge of the pharmacological actions of many psychotherapeutic drugs , including tricyclic antidepressants and noradrenaline reuptake inhibitors . the central noradrenergic system is also involved in panic disorders , especially posttraumatic stress disorders , because an alpha-2-adrenergic agonist , clonidine , is effective in alleviating the symptoms of patients with panic disorders . it was more than 30 years ago that the functional relationship of the lc with fear or anger was first suggested in monkeys by redmond et al . : electrical stimulation of the lc resulted in particular behaviors that were observed in fearful or threatening situations in the wild . ne is especially important to conditioned fear because fear learning occurs during times of threat and stress . thereafter , considerable effort was made to examine whether the ablation of the lc of rodents elicits the behaviors consistent with those in monkeys . however , the rats with 6-ohda induced lc ablation were more reluctant to leave a familiar place and took longer to consume the food pellets in an unfamiliar place , suggesting an increase in fear following the lesion . this is exactly opposite to the predictions of the fear hypothesis derived from monkey studies . similarly , increase in neophobia ( i.e. , fear in response to novelty ) was also observed in subsequent studies . the reason for this discrepancy between monkey and rat studies is not clear . it might be possible that ne has different controls over behaviors in different animals , for example , in prey and predators ( anger in prey , fear in predator ) . for example , when a deer meets a lion , ne is released in the brains of both animals , but the reaction of the deer is flight ( fear ) , while the reaction of a lion is fight ( anger ) . so fear and anger are twin emotions coming from the same neuromodulator ne , but with different reaction depending upon the inner state of the animals . ( master yoda ( george lucas ) ) fear is the path to the dark side . ( master yoda ( george lucas ) ) yoda was so wise and insightful that he said this . it is so true that you can test it in every situation when you are fearful ; you will see it quickly turns into anger . everything in the world , whether it is positive or negative , occurs in ways expected or unexpected by us . our ancestors negotiating rich environments were faced with a set of hugely complex inference and learning problems , involving many forms of variability . therefore , our ancestors evolved an adaptive mechanism to first have a safety check for everything in the world with available information , to see whether they are as expected or unanticipated . if something happens as anticipated , people feel calm ; instead if it happens surprisingly , the first reaction of people would be to get scared and angry . this is an early evolutionary adaptation to allow better coping with dangerous and unexpected situations . therefore , fear and anger are due to unexpected ways in which these things happen . let us take one example from izard 's paper ; when rafe was hit from the back by a wheelchair , the first reaction of him was to get scared and angry and he showed angry expression and clenched fist . but after he turned back to see rebecca , a person with hemiplegia whose wheelchair had gone out of control and caused her to crash into rafe , rafe 's understanding changed his anger to sadness and sympathy . so when something happens , people will first evaluate whether it is dangerous ( fear / anger ) or not ( calm ) and next evaluate whether it fits into their need ( happy / sad ) ( figure 1 ) . similar emotional flow happens all the time in our everyday life : life is normally calm ; everything is as expected ; then something unexpected happens ; people first feel unsafe and scared ( fear ) and then blame ( anger ) the reasons for the unexpectancy after fear is gone ; then people feel happy after successfully coping with the unsafe stimulation and/or feel sad if the individual failed to cope successfully . finally , the stressful events go away , and people calm down . this kind of emotional wave , big or small , long or short , constitutes our everyday stressful emotional flow . so fear - anger - happiness - sadness - calm constitutes the rainbow of emotions or emotional flow in our everyday life ( figure 1 ) . even though fear and anger are the most frequently experienced emotions we have , there is no clear definition about them , let alone the relationship reports about them . from above , we can find that fear and anger are twin emotions that always come together at stressful events , usually fear first and anger next in tandem . for example , when an aggressive driver cuts in front of you , your first reaction is being scared ( fear ) , and next you will feel angry after and only if fear is gone . similarly , lazarus proposed a relational concept for stimulus and emotion : emotions are due to a relationship with the environment that the person regards as significant for his wellbeing . lazarus borrowed the concept of appraisal from arnold and elaborated the concept as a key factor for emotions : emotional processes depend on the predictability of the stressful events . he distinguishes two basic forms of appraisal : primary and secondary appraisal , and he proposed that the primary appraisal and its induced emotions are faster activating , automatic processing , which is similar to the safety need . indeed , lazarus distinguished three types of stressful events : harm , threat , and challenge , which are related to primary appraisal . the secondary appraisal concerns coping options , which include blame or credit , coping potential , and future expectations . it seems that the primary appraisal is related to fear , and the secondary one is related to anger ( figure 1 ) . therefore , these two kinds of appraisals are related to safety needs : for personal safety , which are related to the unexpected ways of stimulus occurring . evolutionarily , fear is related to threats , or aversive unconditional stimuli , such as pain and height . due to our rational ability to think in the future and fantasize about what might happen , fear is getting broader in humans , such as being afraid of losing the needed things , in addition to getting the threat , because of the uncertainty / unexpectancy . actually , the major reason is the uncertainty , because no matter what the hedonic value is , it has two sides of uncertainty , get it and lose it , which make us worry . anger is a nature passion aroused by a real or fancied injury or insult and involves a desire for retaliation . anger always comes after fear at stressful events , and anger is due to fear , or more accurately , anger is due to the unexpected things , which induced fear . therefore , it seems that fear and anger are twin emotions that always come together , but fear and anger exclude each other in that they never occur at the same time in the same person . we give the equation below for the relationship between anger and fear , which fits in both duration and tension : ( 1)the total amount of stress = fear ( duration , tension)+anger ( duration , tension ) . therefore , for a certain amount of stress , if fear is stronger , anger would be relatively less . for example , there is no time left for anger while riding a rollercoaster . on the contrary , when the fear is less , anger should dominate , such as betrayal by a lover . at most of the time , fear and anger come in tandem with fear first and anger next . for example , after long time preparation for the test , nancy was told to have failed in the test . she was scared at first , and then she was angry after she found out that it was not her fault , and her fear was gone for it was the teacher 's mistake . fear and anger are basic emotions , which are built into us to allow us to react to dangerous situations , just like in other animals . yet the problems come because we have the rational ability to think in the future and past and fantasize about so we might be angry , sometimes unperceived , because fear , most of which is based on subjective thinking , can quickly overload causing us to react in anger . anger is the secondary reaction to fear ; if we are fearful , we will also have the tendency to be angry . therefore , we have to keep our fears checked in order to control our anger . anger plays a significant role in everyday life , but there was no clear scientific definition about it before . if there is any , it would be very confusing ; for example , anger is an internal , mental , subjective feeling state with associated cognitions and physiological arousal patterns . barries gave a simple definition : anger is the emotion humans experience when they do not get what they should or must get ; therefore , anger is due to unexpectancy . it would be very useful to break down anger types , because anger takes many forms , such as bitterness , malice , clamor , envy , resentment , intolerance , criticism , revenge , wrath , hatred , sedition , jealousy , attack , gossip , sarcasm , and unforgiveness . hughes divided anger into three types : the first form of anger , named hasty and sudden anger by joseph butler , is connected to the impulse for self - preservation . the second type of anger is named settled and deliberate anger and is a reaction to perceived deliberate harm of unfair treatment by others . the third type of anger is dispositional and is related more to the character traits than to instincts or cognition . irritability , sullenness , and churlishness postures are examples of the last form of anger . one of the most common forms of breaking down anger into anger traits is habitual anger responses such as those determined by the state - trait anger expression inventory . this test addresses four different categories : trait anger , anger - out , anger - in , and anger control . anger - in itself refers more to the affective experience and is not to be confused with hostility or aggression . anger - out implies that a person is more likely to deal with anger through outward expression such as verbal or physical behavior . the last factor , anger control , refers to the tendency to engage in behavior that is intended to reduce overt anger expression . anger is experienced more often than other emotions , and it is very intense and results in adrenaline rush . there are many people that have anger ( fear ) problems , and their problems are due to wrong beliefs . our definition clearly states that anger is due to unexpectancy , so expectancy plays a key role in anger . so ellis proposed that anger is not caused by the things that happen to us but is the result of wrong belief in our minds : we like to put the things we would like to have , wish to have , hope to have , and prefer to having into things we believe we must have , should have , ought to have , are outght to have , and demand to have [ sic ] . so in one sentence , anger / fear is due to expectancy , or anticipation , which depends on your belief . anger pushes others away for people are often fearful of being exposed to angry people . even the angry people themselves tend to isolate themselves from the world , because the angrier they get and the more the ne release , the more fearful they will be next time , and this results in social phobia . in the emotional flow , for the phobia patients , their problems might be that they can not successfully accomplish the emotional flow ( fear - anger - happiness - sadness - calm ) . the best way to remove fear is anger ; these patients have problems with expression of anger , so their emotions are checked at emotional flowing from fear to anger . depression . depression is characterized by unrelenting sadness accompanied by an inability to derive pleasure from positively hedonic situations . therefore , depression might be related to the primary appraisal , the worrying about safety , instead of hedonic satisfaction . indeed , excessive self - blame and feeling worthless are symptoms of major depression episodes across cultures , which is similar to lazarus 's secondary appraisal . so depressed patients have problems with anger or with coping appraisal , or their problem is due to inability to cope with the unsafe stressful situation , and they showed inward anger . recently , compelling evidence from genetic studies and pharmacological studies points to dysfunction of the central catecholaminergic network . genetic analysis suggests that adhd is caused by multiple genes , including dopamine receptors , the adrenergic receptors , and possibly also other monoamine transporters . reduced levels of ne and da have been demonstrated in rodent models of adhd and in adhd patients , consistent with pharmacological studies indicating that medical treatment acts by increasing the concentration of catecholamine . a growing number of studies suggest that the activation of brain ne neurotransmission by acute stress acts to facilitate an array of neuroendocrine , autonomic , behavioral components of integrated , organismic response to stress . given such a widespread modulatory effect on a number of adaptive responses to stress , it is possible that dysregulation of the brain ne system may represent a potential substrate for the stress - related psychopathology , such as depression , ptsd , or other anxiety disorders . therefore , the neuromodulator levels in the cerebral spinal fluid and blood flow might act as biomarkers for stress induced mental disorders .

affective disorders are a leading cause of disabilities worldwide , and the etiology of these many affective disorders such as depression and posttraumatic stress disorder is due to hormone changes , which includes hypothalamus - pituitary - adrenal axis in the peripheral nervous system and neuromodulators in the central nervous system . consistent with pharmacological studies indicating that medical treatment acts by increasing the concentration of catecholamine , the locus coeruleus ( lc)/norepinephrine ( ne ) system is regarded as a critical part of the central stress circuitry , whose major function is to induce fight or flight behavior and fear and anger emotion . despite the intensive studies , there is still controversy about ne with fear and anger . for example , the rats with lc ablation were more reluctant to leave a familiar place and took longer to consume the food pellets in an unfamiliar place ( neophobia , i.e. , fear in response to novelty ) . the reason for this discrepancy might be that ne is not only for flight ( fear ) , but also for fight ( anger ) . here , we try to review recent literatures about ne with stress induced emotions and their relations with mental disorders . we propose that stress induced ne release can induce both fear and anger . adrenaline rush or norepinephrine rush and fear and anger emotion might act as biomarkers for mental disorders .

1. Introduction 2. NE and Stress 3. NE and Emotions 4. Stress Induced Emotional Flows 5. Typical Mental Disorders 6. Conclusions

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

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

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

in a previous issue of critical care , nseir and colleagues presented an article regarding continuous control of endotracheal cuff pressure and tracheal wall damage . among the pathogenic mechanisms responsible for ventilator - associated pneumonia ( vap ) , oropharyngeal colonization by potentially pathogenic microorganisms and silent aspiration of subglottic secretions around the tracheal tube cuff several approaches have been proposed such as placing patients in the semirecumbent position , continuous aspiration of subglottic secretions ( cass ) above the tracheal tube cuff , oropharyngeal decontamination by antiseptics , and the application of antiseptic - impregnated endotracheal tubes . the key element of the proposed pathogenesis of vap appears to be aspiration of colonized oropharyngeal and subglottic secretions . appropriate control of the endotracheal tube cuff pressure ( pcuff ) may therefore serve as a major prevention target . intubated patients were recommended to be managed with pcuff values between 20 and 30 cmh2o to provide a sufficient seal without compromising mucosal perfusion . the routine management of cuff inflation consists of periodic manual checking of the pcuff , which does not ensure the appropriate maintenance of the pcuff during continuous tracheal intubation . moreover , the manual checking of the pcuff may cause either overinflation or deflation of the cuff and may cause aspiration of contaminated secretions to the lower airway during the manoeuvre . leaks and loss of pcuff are frequent in intubated patients , and a persistent pcuff below 20 cmh2o was an independent risk factor for vap in one study . consequently , appropriate maintenance of pressure of the tracheal tube cuff is recommended in recent guidelines . in a previous issue of the journal , nseir and coworkers describe a pneumatic device for the continuous control of the pcuff in an animal model . the aim of the study was to assess whether the continuous control of the pcuff results in reduced tracheal ischaemic lesions in mechanically ventilated piglets . for this purpose , the authors compared the pneumatic device with the manual control of pcuff in a randomized trial . the pneumatic device provided effective continuous control of the pcuff , with longer periods of pcuff within the target values than piglets managed with manual control . this device is therefore potentially useful for clinical practice in order to avoid both excessive inflation and deflation of the cuff . hyperaemia and haemorrhages in the trachea were observed at the cuff contact area in all animals , however , with no differences between animals with and without the pneumatic device . several devices that provide an automatic and continuous effective control of the pcuff have been described in the literature . most of these devices are not automatic , some devices need frequent control by the attending staff , and other devices operating in a more automatic and continuous way are complex , requiring the use of special and expensive equipment that may not be available routinely . it is probable that these issues concerning complexity and cost could explain the lack of continuous automatic control of cuff inflation in clinical practice . we have described a simple and cheap device that is very effective for the routine maintenance of adequate cuff inflation during mechanical ventilation that does not require any specific equipment . a recent randomized clinical trial in mechanically ventilated patients comparing this device with the routine manual control of cuff inflation , however , showed no benefits in the prevention of vap . these findings suggest that other factors than cuff inflation influence the microaspiration of secretions to the lower airways around the tracheal tube cuff . commercially available high - volume low - pressure tracheal tubes such as those used in the study often form folds around the cuff , hence allowing leakage of secretions pooled above the tube cuff in studies in vitro , even at pcuff levels similar to those used by nseir and colleagues in piglets . several devices have consequently been recently developed in order to overcome this problem . among those devices , the microcuff endotracheal high - volume low - pressure tube features an ultrathin ( 7 m ) poly - urethane cuff membrane around an inner conventional inflatable cuff . this tube is effective in preventing fluid leakage around the cuff in an in vitro setup . the combination of this device with cass is effective in preventing both early - onset and late - onset vap in a recent clinical study . one of the potential concerns of all these devices , particularly cass , is the potential damage of the tracheal wall . in an animal sheep model , berra and in the study by nseir and colleagues , the tracheal lesions found could be explained , at least in part , by the high inflation pressure they applied eight times daily via 50 ml during 30 min . this is not the current clinical practice in humans , and after this study it should be completely avoided . cass = continuous aspiration of subglottic secretions ; pcuff = endotracheal tube cuff pressure ; vap = ventilator - associated pneumonia . the present study was supported by ciberes ( isciii - cb06/06/0028 ) , fis 02 - 0744 , separ 2001 , and 2005 sgr 00822 .

continuous control of tracheal tube cuff inflation using a pneumatic device resulted in severe tracheal wall damage in ventilated piglets . this damage was similar in piglets managed with manual control of cuff inflation . the periodic hyperinflation of the tube cuff used in both groups of this study may explain these results . this manoeuvre should be avoided in clinical practice .

None Abbreviations Competing interests Acknowledgements

stroke is the leading cause of adult disability in the developed world.1 , 2 in ischemic stroke , a blood clot ( thrombus ) leads to an area of brain experiencing a significant drop in cerebral blood flow , which can ultimately result in death of this hypoperfused tissue . collateral blood flow gives rise to the ischemic penumbra , which is defined as an area of hypoperfused tissue that is at risk of death ( infarction ) if hypoperfusion persists , and is separate from tissue that has already died ( or programmed to die ) soon after stroke onset ( core).3 the concept of the ischemic penumbra led to the notion that opening an occluded blood vessel in order to re - establish blood flow to the ischemic area ( reperfusion ) would rescue the ischemic penumbra from death3,4 ( figure 1 ) . reperfusion can be achieved through the use of systemic thrombolytic drugs , although thrombolytic agents may theoretically be more effective if delivered directly into the clot ( intra - arterial delivery ) or if assisted with ultrasound insonation of the clot ( sono - thrombolysis ) . a thrombus can originate from several different causes , which may alter its susceptibility to thrombolytic drugs . the main causes of thrombus formation in ischemic stroke are atherosclerosis ( in - situ thrombus or thromboembolic ) or forming in the heart ( cardioembolic ) . it was originally hypothesized that the source of thrombi had a large determining influence on clot composition and therefore potentially upon susceptibility to thrombolytic treatment as thromboembolic clots tend to be platelet and fibrin rich and were formed in high flow areas ( often referred to white clots ) , whilst cardioembolic can contain tissue debris such as fat , air or bacteria interwoven inside the platelet and fibrin mesh which were formed in low flow areas ( often referred to as red clots).5,6 however recent histological studies analyzing the thrombi extracted from patients using intra - arterial clot retrieval showed thrombi composition was similar between cardiac and arterial origins.7 atherosclerotic plaque rupture or cardio - embolic thrombosis ( typically due to atrial fibrillation ) leads to activation of the coagulation cascade as well as platelet activation . in the cascade , zymogens ( free floating inactive coagulation factor precursors ) are converted to an activated coagulation factor by interaction with the atherosclerotic plaque / thrombus.8 each active zymogene is able to activate nearby zymogens , leading to a large localized reaction of coagulation factors . activated platelets then catalyze an interaction between activated coagulation factors ( zymogens ) to aid in the generation of thrombin by conversion of the soluble protein fibrinogen to insoluble fibrin , forming a blood clot . there are two blood coagulation cascades ( intrinsic and extrinsic pathways ) , that have separate initial pathways but converge on a common pathway9 ( figure 2).10 thrombolytic drugs dissolve ( lyse ) thrombi in the vascular bed by activating plasminogen to form plasmin . plasmin is a proteolytic enzyme that breaks the crosslinks between fibrin molecules to destabilise the structural integrity of blood clots . the main types of thrombolytic drugs used in ischemic stroke to activate plasminogen are urokinase / streptokinase and tissue plasminogen activators ( eg , alteplase ) . thrombolytic drug development has undergone at least three generations with the aim of enhancing fibrin specificity or reducing inhibition of thrombolysis by plasminogen activator inhibitor type 1 ( table 1 ) . tissue plasminogen activator ( tpa ) is a serine protease found on the endothelial cells lining blood vessels and is involved in the breakdown of blood clots ( thrombus ) . a thrombus is composed of fibrin monomers that are cross - linked through lysine side chains which tpa binds too . lysine binding of tpa results in a activation of plasminogen only around a thrombus , which minimises activation of circulating plasminogen . the lysine side chains have a high affinity for binding with plasminogen , making the thrombus plasminogen rich . the tpa enzyme binds to fibrin components of a thrombus and catalyses plasminogen conversion to plasmin by cleavage of the arginine - valine bond at positions 560 and 561 to break down a clot by degrading the fibrin matrix of a thrombus . plasmin then breaks the thrombus down into fibrin degradation products due to the plasmin 's protease action in dissolving the thrombus.11 however , on fibrin bound plasmin , the inhibitory effect of alpha 2-antiplasmin and type 1 plasminogen activeator inhibitor restricts lysine binding.12 streptokinase is a first generation thrombolytic agent . therefore streptokinase is not a protease but binds to plasminogen for the generation of plasmin and is not restricted at the site of thrombus formation . because of its non lysine specificity , streptokinase , produces more fibrin degradation products ( fibrinogensis ) as a result of widespread lytic action in the body . fibrinogensis occurs when there are high levels of fibrin in the blood and can cause thrombosis , haemorrhage or tissue oedema . although much less expensive , this makes streptokinase a less attractive agent for acute treatments in ischemic stroke than tpa products . studies of streptokinase in acute stroke were stopped due to an increase in mortality compared to placebo due to increased haemorrhage rates.13,14 the specificity of tpa drugs ( alteplase , retaplase , tenecteplase and desmoteplase ) for plasminogen bound fibrin means that conversion of plasminogen to plasma occurs in clots with minimal circulating plasma . the 1999 prolyse in acute cerebral thromboembolism ii trial ( proact ii ) tested the benefit of intra arterial delivery of 9 mg of recombinant prourokinasea ( r - prouk ) in 180 patients . despite an increased rate of symptomatic intracranial haemorrhage the study found a significant benefit in patients treated with r - prouk at 90 days.15 to date , the proact ii study is the only positive phase iii intra - arterial trial . some centres use r - prouk intravenously due to its low cost compared to other thrombolytic drugs . the second generation thrombolytic drug alteplase , is a recombinant form of human rtpa and has undergone the most study in the clinical stroke setting . recombinant techniques mean that molecular cloning is used to bring genetic material together from multiple sources that would not otherwise be found together , to create new dna sequences , which can be used to manufacture drugs . alteplase is a purified glycoprotein ( a protein with sugar chains covalently attached to polypeptide side chains ) of 527 amino acids that is synthesised from the complementary dna ( cdna ) of natural human tissue - type plasminogen activator found in human melanoma cells and is made up of five structural components : a protease , epidermal growth factor ( egf ) and two kringle domains ( figure 3 ) . the lysine binding sites of alteplase are on the kringle 2 domain and are why alteplase has a high binding affinity to thrombi . in terms of pharmacodynamics , rtpa has a short half life of around 5 min , and therefore requires an infusion after a bolus injection in the acute ischemic stroke setting . the majority of ischemic stroke clinical research has revolved around alteplase , as it is the only food and drug administration ( fda ) approved drug for ischemic stroke . fda approval for alteplase was licensed for the management of acute myocardial infarction in 1987 , acute massive pulmonary embolism in 1990 and acute ischemic stroke in 1996.16 the total dose of alteplase for acute ischemic stroke is divided into a 10% bolus and 90% as an infusion over 60 min . small dose - range studies for alteplase in human stroke found increasing neurological benefit up to a dose of 0.85 mg / kg , but an increased rate of haemorrhage with a dose of 0.95 mg / kg.17 the ground - breaking ninds - rtpa study was the first positive phase iii acute stroke trial . this trial showed that early treatment ( within 3 hours ) of an ischemic stroke with alteplase substantially improved clinical outcomes . an alteplase dose of 0.9 mg / kg was used in the ninds trial.18 and found no significant increase in the risk of haemorrhage.19 the ideal dose of alteplase is still under debate with the original alteplase dose - ranging studies being quite small . it is felt that asian people may be more prone to haemorrhage from tpa , although this is not based on level one evidence . to this point , the standard dose of alteplase in japan in 0.6 mg / kg based on a single japanese study.20 on the other hand , there is justifiable concern that a reduced dose of alteplase will not successfully lyse larger clots , typically in more proximal locations . it has been reliably demonstrated that long clots on non contrast computed tomography ( ncct ) show a reduced rate of reperfusion and subsequent clinical improvement.21 indeed , a recent study of 130 patients using non - contrast ct to measure occlusion length identified that the tpa dose was independently predicative of thrombus resolution , with complete resolution in only 8% of patients . the average occlusion reduced by 20% , while 15% of patients had an increase in clot length following tpa administration . therefore tpa dose was associated with thrombus resolution and improved clinical outcome.22 following the ninds study , which showed that early treatment ( within 3 hours ) was the key to improving ischemic stroke outcome , there have been several studies investigating extending this treatment time window . the european cooperative acute stroke study ( ecass ) i and ii looked at administering alteplase between 0 and 6 hours after ischemic stroke onset and did not find an overall benefit , with an increased rate of haemorrhage in ecass i. however ecass i and ii did not use advanced imaging and may have had a high rate of patients with very little penumbra to salvage , especially in the 3 - 6 hours time window . ecass iii subsequently demonstrated evidence of good outcome following treatment with 0.9 mg / kg alteplase between 3 and 4.5 hours in patients < 80 years after symptom onset using only ncct to exclude patients with haemorrhage or oedema . as a result of ecass iii , many countries have amended the license for stroke treatment with rtpa to 4.5 hours following ncct assessment . there has been much interest in using advanced imaging to visualise the volume of the penumbra and infarct core , particularly with a view to extending the treatment window for thrombolysis . the echoplanar imaging thrombolytic evaluation trial ( epithet ) randomised ischemic stroke patients 3 - 6 hours after symptom onset to alteplase or placebo and used acute magnetic resonance imaging ( mri ) to define the core and penumbra ( ' diffusion - perfusion mismatch ' ) . the primary hypothesis of this phase ii trial was that patients with perfusion - diffusion mismatch would have less infarct growth with alteplase than with placebo . although the primary outcome was negative ( mean geometric infarct growth ) all secondary outcomes were positive and confirmed that alteplase attenuated infarct growth in mismatch patients . other analyses from the epithet dataset have shown that the diffusion infarct core is the strongest predictor of response to thrombolysis , with a chance of good outcome dropping if the core > 25 ml and there being virtually no benefit > 70 ml.23,24 pooled analysis from epithet and another trial of alteplase 3 - 6 hours after ischemic stroke , the diffusion and perfusion imaging evaluation for understanding stroke evolution study ( defuse ) , showed that patients in the 3 - 6 hours time window with a favourable imaging profile , i.e. a large penumbra , and small infarct core.25 as stroke occurs predominantly in the aged population , with > 45% of patients receiving alteplase being over 70 years old,26 there is a concern about decreasing benefit from thrombolysis with age . the main concerns are over the possibility of aged related renal and hepatic impairments affecting the metabolising and clearance of alteplase as well as the possibility of an increased risk of haemorrhagic transformation of the infarct following alteplase administration . there is also concern that the elderly tend to have subtle pre - morbid cognitive dysfunction and also may have less capacity for brain plasticity and recovery after stroke . however , the international stroke trial 3 ( ist3 ) , recently found there was no direct association between alteplase administration and haemorrhage rate in elderly patients selected for alteplase treatment using ncct imaging.27 this was the largest stroke thrombolytic trial ever , and randomised 0 - 6 hours patients to 0.9 mg / kg alteplase or placebo ( without using advanced imaging selection ) . although the primary outcome was negative , ist3 confirmed that patients treated within 3 hours of symptom onset clearly benefited from alteplase , and the elderly had as much benefit as did younger patients . tenecteplase ( tnk - tpa ) is a third generation point mutation tissue plasminogen activator created by recombinant dna technology from a mammalian cell line . like alterphase tenecteplase has modifications at three sites of the protein structure on the complementary dna template that differentiate tnk from alterphase , such as substitution of threonine 103 with asparagine , and a substitution of asparagine 117 with glutamine , both within the kringle 1 domain , and a tetra - alanine substitution at amino acids 296 - 299 in the protease domain ( figure 4 ) . these structural and functional changes to the cdna mean that tenecteplase has a longer half life and greater binding affinity for fibrin and better resistance to inactivation by endogenous inhibitor ( pai-1 ) compared to alteplase . the amino acids that were replaced at the three positions are called t , n , and k according to the one letter code for amino acids , giving the drug the name tnk . tenecteplase is expressed with carbohydrate side chains linked to the glycosylation sites of the polypeptide . the carbohydrate side chains enlarge the molecule , reducing its elimination and prolonging its plasma half life . tenecteplase can be administered by iv bolus , without the need for follow - up infusion , with a half life of 17 minutes . tenecteplase has been approved for acute myocardial infarction , and was demonstrated to be superior to alteplase.28,29 in ischemic stroke , the tenecteplase dose used for myocardial infarction ( 0.5 mg / kg ) initially did not show benefit when using standard clinical criteria to select patients for treatment , with an increase in symptomatic haemorrhage.30 however when patients were selected for treatment based on acute computed tomography perfusion imaging to identify the volume of the infarct core and penumbra within 3 - 6 hours of symptom onset , treatment with tenecteplase at low dose ( 0.1 mg / kg ) appeared to be superior to alteplase in a non - randomised study.31 a subsequent randomised phase iib study using multimodal ct to select patients ( vessel occlusion , small core , large penumbra ) found that moderate ( 0.25 mg / kg ) and low ( 0.1 mg / kg ) doses did lead to significantly better patient outcomes than standard dose alteplase ( 0.9 mg / kg ) . tenecteplase was associated with significantly increased reperfusion , early neurological improvement and improved 3 month functional outcome32 with a strong dose - dependent relationship , with a 0.25 mg / kg dose achieving significant reperfusion and neurological improvement compared to 0.1 mg / kg . furthermore , despite the enhanced efficacy of tenecteplase for the larger proximal clots , there was not an increase in ich ( in fact a trend towards lower rates ) . it was noted in 1932 that vampire bat saliva interfered with the haemostatic mechanisms of the host.33 the dna sequencing of four plasminogen activators in vampire bat saliva were completed in 1991 , with activator alpha 1 ( rdspa1 , desmoteplase ) being the most active34 and shows a 72% homology to human t - pa.35 desmoteplase is structurally similar to alteplase , but is missing the plasmin sensitive cleavage site and lysine binding kringle 2 domain . this results in desmoteplase being more selective for fibrin ( 2increase in catalytic activity ) , and having no known effect on the blood brain barrier . desmoteplase has a half life of 4 hours ( compared to alteplase 5 minutes , reteplase 13 minutes , tenecteplase 17 minutes ) . a small phase three trial of desmoteplase in acute ischemic stroke ( dias 2 ) has been completed and but demonstrated no benefit in patients selected for treatment biased on acute advanced neuroimaging findings.17,36 the trial randomised patients to desmoteplase or placebo 3 - 9 hours after stroke onset using imaging selection with ct perfusion or mri , assessed visually for mismatch between core and penumbra volume of > 20% by the local investigator . this patient selection paradigm followed the promising phase 2 studies , dias and dedas , which demonstrated increased reperfusion and strong trends to improved outcome with desmoteplase compared to placebo . apart from a very small sample size for a phase iii study , it is thought the predominant reason for lack of benefit seen with desmoteplase was the lack of standardisation of the advanced imaging assessment . additionally , dias 2 did not have a minimum or maximum volume of infarct or penumbra volume as inclusion criteria , as such , many patients had small perfusion lesions with a favourable natural history that would have made detecting a difference with desmoteplase treatment challenging even with a larger sample size . subsequent analyses of pooled data from dias and dias-2 identified that patients with a proximal cerebral vessel occlusion or high grade stenosis had much greater mismatch tissue volumes and a positive response to desmoteplase compared to placebo.37 the rate of early ( effective ) recanalization using alteplase is 25% in patients with a proximal middle cerebral artery occlusion and 10% in patients with an internal carotid occlusion.38 additionally , the rate of re - occlusion , a situation where a thrombus moves down a vascular tree but does not dissolve and re - occludes a distal blood vessel , is as high as 30% with alteplase.39 therefore , methods to enhance recanalization are required . intra arterial ( ia ) thrombolysis and more recently clot retrieval have become increasingly popular acute stroke treatments , albeit without strong evidence . the proact ii trial was the first ia thrombolysis trial to show benefit ( compared to placebo ) . subsequent phase ii trials such as interventional management of stroke ( ims ) i and ii suggested benefit of a ' bridging ' iv / ia approach when compared to iv rtpa alone . with evolution of technology , there has been an increased interest in mechanically removing clots.40 however , three recent ia clot retrieval tials using first generation endovascular devices have recently been published including , ims iii , synthesis and mr rescue , all failed to show benefit compared to iv rtpa . this mostly likely relates to delays in revascularisation compared to iv treatment , and also to poor patient selection . for example , mr rescue not only had a very late time to endovascular treatment ( median > 6 hours ) , but the pre - treatment baseline infarct core volume was exceptionally large ( median 60 ml).41,42 these trials with the first generation clot retrieval devices ( merci and penumbra ) had quite poor recanalization rates , which may be another reason why they failed to show improved clinical outcomes compared to iv tpa alone . however , there is unbridled enthusiasm for the newer ' thrombectomy ' devices such as solitaire43 and trevo,44 with phase ii studies showing much better recanalization than with the first generation devices . however , it is unlikely any device will prove superiority to iv tpa unless clot retrieval can occur at a similar time point to iv treatment . however there is still a trend towards increased haemorrhage in patients undergoing ia treatments regardless of time to treatment . sono enhanced thrombolysis has been shown in vitro and in vivo to accelerate the enzymatic activity of thrombolysis.45 one proposed method of action , is that ultrasound pops the microbubbles in a targeted blood vessel to generate micro - streaming of blood to the occlusion as a delivery mechanism for tpa . additionally , ultrasound causes enlargement of the fibrin mesh to allow better binding and penetration of tpa into a thrombus.46 phase 2 clinical trials have shown that continuous transcranial ultrasound at 2 mhz significantly increased the rate of early recanalisation in patients treated with alteplase.47 however these studies did not use the gold standard of angiography to assess recanalisation , rather they relied on transcranial ultrasound changes , which are less well validated . there has also been interest in administering intravenous micro - bubbles as a contrast agent in order to increase the available volume of micro - bubbles for ultrasound to affect , however this approach may increase risk of ich.48 glycoprotein iib / iiia inhibitors may prevent platelet activation induced by thrombolysis and promote a more complete and rapid action of thrombus breakup.49 glycoprotein inhibitors are used to prevent platelet activation to stop new blood clots forming to avoid re - occlusion . platelet activation by adenosine diphosphate ( adp , stored in blood platelets and is released on platelet activation which can be blocked by clopidogrel ) leads to a conformational change in platelet gpiib / iiia receptors that induces binding to fibrinogen . however recent myocardial studies have shown that gpiib / iiia inhibitors lead to more effective reperfusion , but also increased the rate of intracranial haemorrhage.50 for ischemic stroke there is currently little clinical data , with only small case series indicating a low rate of symptomatic intracranial haemorrhage after duel alteplase and gpiib / iiia administration.51,52 nanotherapeutics aim to increase tpa delivery to the vessel occlusion . the current concept of thrombolysis is to inject a thrombus dissolving drug into the blood stream and hope that it reaches the occlusion site in a high enough concentration to totally dissolve the thrombus . the chosen doses of clot - lysing drugs that can be administered are a trade off between the potential risk of bleeding and the required dosage to have an effect . however , the more distal an occlusion is , the less likely there is to be effective drug delivery regardless of dose . one recent approach to guide tpa delivery is through the use of shear - activated nanotherapeutics ( sa - nts).53 however nanoparticles are only in phase one clinical trials . treatment of ischemic stroke with thrombolysis is a trade off between the risk of haemorrhage and the possible benefit . clinical trials have shown that treatment with tpa in selected patients results in highly clinically significant improvement . however the criteria on how we best identify these highly treatmentresponsive patients are still undergoing investigation . there is much interest in the use of neuroimaging to enhance our ability to identify these responders but this approach is yet to be proven in a phase iii clinical trial.54 while better thrombolytics and drug delivery mechanisms such as sono - thrombolysis may increase the breakdown of a thrombus , there is still the major risk of haemorrhage brought about by damaged to the blood brain barrier by the stroke . it is unknown if a resulting haemorrhage is caused by interactions of thrombolytic drugs and the blood brain barrier , or extensive brain damage brought on by an established infarct . therefore it is unknown if better drugs or mechanisms will reduce the risk of haemorrhage after stroke , or if appropriately selecting patients with a large penumbra and without a large core using imaging reduces the rate of post lysis haemorrhage . the recent failure of the endovascular trials to show benefit compared to iv alteplase demonstrate that we should focus on early treatment with lytics and concentrate on either enhancing their delivery , and/or on better agents such as tenecteplase .

the cornerstone of acute ischemic stroke treatment relies on rapid clearance of an offending thrombus in the cerebrovascular system . there are various drugs and different methods of assessment to select patients more likely to respond to treatment . current clinical guidelines recommend the administration of intravenous alteplase ( following a brain noncontract ct to exclude hemorrhage ) within 4.5 hours of stroke onset . because of the short therapeutic time window , the risk of hemorrhage , and relatively limited efficacy of alteplase for large clot burden , research is ongoing to find more effective and safer reperfusion therapy , as well as focussing on refinement of patient selection for acute reperfusion treatment . studies using advanced imaging ( incorporating perfusion ct or diffusion / perfusion mri ) may allow us to use thrombolytics , or possibly endovascular therapy , in an extended time window . recent clinical trials have suggested that tenecteplase , used in conjunction with advanced imaging selection , resulted in more effective reperfusion than alteplase , which translated into increased clinical benefit . studies using desmoteplase have suggested its potential benefit in a sub - group of patients with large artery occlusion and salveageable tissue , in an extended time window . other ways to improve acute reperfusion approaches are being actively explored , including endovascular therapy , and the enhancement of thrombolysis by ultrasound insonation of the clot ( sono - thrombolysis ) .

Introduction Thrombus formation and fibrinolysis Thrombolytic drugs Enhancing thrombolysis delivery

zr - labeled monoclonal antibodies ( mabs ) have recently been useful in clinical trials for positron emission tomography ( pet ) imaging of various cancers due to the favorable pairing of the radioisotope s half - life ( t1/2 = 3.3 days ) and the antibody s pharmacokinetic properties , complemented by the high sensitivity and quantitative properties of pet . pertuzumab ( perjeta ) is an fda - approved mab for the treatment of human epidermal growth factor receptor 2 ( her2)-positive metastatic breast cancer , in combination with trastuzumab ( herceptin ) , another her2specific monoclonal antibody . pertuzumab binds to domain ii of the human epidermal growth factor receptor-2 ( her2 ) epitope , distinct from the trastuzumab epitope located within domain iv . both antibodies elicit antibody - dependent cellular cytotoxicity ( adcc ) ; however , the predominant mechanisms responsible for the synergy in downregulating her2 expression are that pertuzumab inhibits her2 dimerization with other epidermal growth factor receptors and trastuzumab inhibits intracellular signaling . zr - labeled trastuzumab has been evaluated as a pet imaging agent for detecting her2 expression in trastuzumab - treated or in nave patients , with promising results toward the stratification of patients mostly likely to respond to trastuzumab therapy . however , investigations with pertuzumab as a pet imaging agent have not been explored , to our knowledge . pertuzumab has only been investigated in vivo as a spect imaging agent by radiolabeling with in and lu . however , the sensitivity of pet imaging may be advantageous for further developing pertuzumab as an imaging agent . a qualitative comparison of fluorescently labeled cy5-trastuzumab and cy5-pertuzumab in the presence and absence of either unconjugated trastuzumab or pertuzumab has been conducted in her2 + xenograft models , which suggests imaging with cy5-pertuzumab in the presence of unconjugated trastuzumab and imaging with cy5-trastuzumab in the presence of unconjugated pertuzumab do not alter the tumor accumulation of each cy5-labeled antibody . however , a quantitative comparison between the tumor accumulation of radiolabeled trastuzumab and pertuzumab as imaging agents during trastuzumab therapy has yet to be investigated . furthermore , in silico studies suggest the binding of trastuzumab induces a change of her2 conformation , permitting greater exposure of the pertuzumab binding site and enhancing pertuzumab affinity . toward this end herein , we demonstrate the specificity of zr - pertuzumab in her2 + tumors in vivo and quantitatively show enhanced tumor accumulation of zr - pertuzumab in the presence of trastuzumab . pertuzumab ( perjeta ) and trastuzumab ( herceptin ) were purchased from roche ( south san francisco , ca ) . desferrioxamine - p - benzyl - isothiocyanate ( dfo - bz - ncs ) was purchased from macrocyclics ( dallas , tx ) . the automated production of zr - oxalate was carried out in house , adapted from previously established methods and neutralized to ph 6.87.2 as previously developed for optimal radiolabeling . all other chemicals were purchased from sigma - aldrich ( st . louis , mo ) unless stated otherwise . her2expressing breast cancer cell lines bt-474 and skbr3 as well as her2nonexpressing mda - mb-231 were purchased from the american type culture collection ( atcc , manassas , va ) and cultured in iscove s modified dulbecco s medium ( imdm ) containing 10% fetal bovine serum ( fbs ) and 50 g / ml gentamycin ( complete media ) in a humidified incubator with 5% co2 at 37 c . reagents for cell culture were purchased from life technologies ( grand island , ny ) unless stated otherwise . conjugation of dfo - bz - ncs to pertuzumab and subsequent radiolabeling with zr - oxalate were performed following previous methods . briefly , pertuzumab ( 10 mg / ml ) was conjugated to 8-fold molar excess of dfo - bz - ncs dissolved in dimethyl sulfoxide in 0.1 m sodium carbonate buffer ( ph 9 ) at 37 c for 1 h. the resulting dfo - pertuzumab conjugate was purified and buffer exchanged into 1 m hepes buffer ( ph 7.1 ) using gel filtration spin columns ( mwco 40 kda , thermo fisher scientific , rockford , il ) . dfo - pertuzumab was radiolabeled with neutralized zr - oxalate using a ratio of 296:1 kbq:g at 37 c for 3060 min for a final ph between 6.87.2.zr-pertuzumab with radiochemical yields 95% as determined by instant thin - layer chromatography was used for in vitro and in vivo studies without further purification . dfo - bz - ncs - pertuzumab conjugate and unmodified pertuzumab were buffer exchanged in 0.25 m nh4oac buffer twice using zeba spin desalting columns ( thermo scientific , rockford , il ) following supplier s instructions . the final concentration of protein was quantified using a bicinchoninic acid ( bca ) assay ( thermo scientific , rockford , il ) . a 10 l aliquot was loaded into an offline electrospray capillary pulled in house using borosilicate tubing on a laser based puller ( both b100 - 58 - 10 tubing and p-2000 puller were from sutter instrument company , novato , ca ) to perform the native mass spectrometry measurement . the collision energy at the trap and transfer region was adjusted for desolvation of the ions . the pulled capillaries were coated with gold using a sputter coating machine in the electron microscopy core at washington university in st . the sample solution was infused to a hybrid ion - mobility quadrupole time - of - flight mass spectrometer ( q - im - tof , synapt g2 hdms , waters inc . , milford , ma ) . the instrument was operated under gentle esi conditions ( capillary voltage 1.51.8 kv , sampling cone 20 v , extraction cone 2 v , source temperature 30 c ) . the collision energy at the trap and transfer region was adjusted for dissociating the complex . the instrument was externally calibrated up to 8000 m / z with the clusters produced by esi of a nai solution . the peak picking and data processing was performed in masslynx ( v 4.1 ) and driftscope software ( water inc . , milford , ma ) . the number of dfo per pertuzumab was calculated by ( m / z of conjugate m / z of unconjugated)/(dfo molecular weight ) z , where m / z represents the observed mass to charge ratio and z represents the charge species . the immunoreactivity of zr - pertuzumab was determined using the lindmo et al . other cell uptake experiments were performed in her2 + bt-474 and skbr3 , and her2 mda - mb-231 cells , in which 250 l of 1 10 cells / ml were seeded in 24-well plates . cells were allowed to adhere overnight at 37 c in a 5% co2 atmosphere . the media was removed and replaced with 125 l of fresh complete media and 125 l of 50 ng / ml of zr - pertuzumab , saving aliquots in microcentrifuge tubes as a standard . the cells were washed three times with pbs , trypsinized , and transferred to microcentrifuge tubes . radioactivity associated with cells was counted in a beckman 8000 gamma counter ( beckman coulter inc . , the percentage of bound radioactivity was calculated as the ratio of bound to the total radioactivity added per well multiplied by 100 . equal number of cells per well were confirmed by counting the cells using a cellometer auto t-4 cell counter ( nexcelom , lawrence , ma ) . competitive binding assays were performed on skbr3 cells adhered to 24-well plates as described above . cells were incubated with 1.7 10 m zr - pertuzumab in the presence of different concentrations ( 1.7 101.7 10 m ) of unlabeled pertuzumab or trastuzumab competitor in triplicate wells at 4 c for 1.5 h. the cells were washed with pbs and dissociated from the wells as described above and assayed in a gamma counter . the percentage of zr - pertuzumab bound in the presence of competitor / no competitor ( b / b0 100 ) was plotted against the log concentration of added competitor and fitted with a sigmoidal dose skbr3 cells ( 1 10 cells in 400 l ) were seeded into 12-well plates and allowed to adhere overnight in the cell culture incubator . zr - pertuzumab or zr - trastuzumab positive control antibody was diluted to 50 ng / ml using complete media and 125 l of each radiolabeled antibody was added to their designated wells . the plates were incubated at 37 c in a 5% co2 atmosphere for 0.5 , 1 , 3 , 17 , 24 , 28 , 42 , and 48 h. as a control , some reactions were performed at 4 c . at each time point , the cells were washed three times with 400 l of cold pbs and the cells were dissociated from the well via incubation with 400 l of trypsin for 3 min at 37 c . the cells were washed again with 200 l of cold pbs , transferred to a microcentrifuge tube via pipet , and sedimented at 7000 g using a benchtop centrifuge . the supernatant was collected and the cells were treated with 100 l of 0.1 m sodium citrate ( ph 2 ) for 5 min to remove any surface bound zr - mab . the cells were sedimented at 7000 g , the supernatant was combined with the previously collected supernatant , and assayed in the gamma counter . the amount of zr - mab bound on the cell surface and internalized was calculated as previously described . all animal experiments were conducted according to the guidelines of the institutional animal care and use committee ( iacuc ) and approved by the washington university animal studies committee . in vivo pet imaging and biodistribution studies were conducted in severely combined immunodeficient female nog mice ( taconic , hudson , ny ) . mice were inoculated with 150 l of 13 10 cells / ml ( bt-474 , skbr3 , and mda - mb231 ) suspended in saline with no artificial stimuli . mice were injected via tail - vein with 100 l of 3.7 mbq of zr - pertuzumab ( specific activity 222296 mbq / mg ) in saline . studies in the presence of unlabeled antibodies were performed with a tail vein injection of 100 l of 10 mg / ml of either pertuzumab or trastuzumab in saline , between 560 min prior to the injection of zr - pertuzumab in the bt-474 xenograft . static pet images were acquired for 20 min using an inveon micropet / ct scanner ( siemens , knoxville , tn ) at 7 days p.i . the images were reconstructed using maximum a posteriori probability ( map ) algorithm and coregistered with ct images using image display software ( inveon research workplace workstation , siemens , schenectady , ny ) . regions of interest ( roi ) were drawn for tumor uptake and analyzed as standard uptake values ( suv ) using the formula suv = ( mbq / ml ) ( animal weight ( g))/injected dose ( mbq ) . mice were sacrificed and organs were harvested , weighed , and assayed in the gamma counter for biodistribution studies . radioactivity associated with each organ was expressed as percentage of injected dose per gram of organ ( % id / g ) . tumors and spleen were fixed with 4% paraformaldehyde at 4 c until radioactivity decayed for about 1 week . tumors were paraffin - embedded , sliced , mounted on slides and stained with hematoxylin and eosin ( h&e ) by the washington university pulmonary morphology core facility . immunohistochemical staining was performed by first blocking the slides with goat serum following instructions from a vectastain elite abc kit ( vector laboratories , burlingame , ca ) and incubated with 1:200 dilution of a polyclonal goat anti - her2 antibody ( santa cruz biotechnology , santa cruz , ca ) in pbs for 1 h at room temperature . secondary antibody incubation was performed following the kit s instructions and colorimetric development was achieved using a 3,3-diaminobenzidine ( dab ) substrate ( vector laboratories , burlingame , ca ) . digital images were collected on a nikon optiphot-2 microscope equipped with a zeiss axiocam digital camera . cox analysis indicated that the variances among the test groups were unequal and a logarithm transformation of the suv and biodistribution data was necessary for variance stabilization . a one - way anova was performed on the log transformation data , followed by group comparisons using the tukey method to control multiple comparisons . statistical analysis was performed using graphpad prism v6 ( graphpad software inc . , la jolla , ca ) and sas v9.3 ( sas institute , cary , nc ) . although statistical significance was performed on the transformed data , the results are presented in the original metric for ease of discussion . the conjugation of dfo - bz - ncs to pertuzumab was performed by combining 8 mol equiv of chelator at ph 9 for 1 h at 37 c , followed by immediate purification . esi - ms determined up to 4 chelates per antibody conjugate , calculated from the mass shift of the unconjugated pertuzumab ( figure 1 ) . subsequent radiolabeling with zr yielded specific activities of 222370 mbq / mg with 95% radiochemical yield as determined by instant thin layer chromatography . ( a ) unconjugated pertuzumab depicted in + 25 , + 24 , and + 23 charge states and ( b ) dfo - bz - ncs - pertuzumab conjugate in the same charge states as the unconjugated control . the immunoreactivity of zr - pertuzumab was determined using the lindmo assay in her2 + skbr3 cells . the immunoreactive fraction was calculated to be 72.2% , indicating retention of antigen - binding ( figure 2a ) . the specificity of zr - pertuzumab was investigated in vitro by comparing its binding in her2 + ( skbr3 and bt-474 ) and her2 ( mda - mb-231 ) cells . cell binding of zr - pertuzumab to skbr3 , bt-474 , and mda - mb-231 cells was 94.7 2.6 , 95.2 19.1 , and 3.8 0.1% of total radioactivity added , respectively ( figure 2b ) . significantly greater binding was observed in her2 + compared to her2 cells ( p < 0.0001 ) . skbr3 and bt-474 cells have been reported to have a similar number of her2 receptors , 647811 834 and 10 59513 917 fmol / mg of protein , respectively . cell binding studies with zr - pertuzumab in skbr3 and bt-474 were consistent with pegram et al . studies , as no statistical difference was observed between skbr3 and bt-474 cells ( p > 0.05 ) . competitive binding studies showed decreased uptake of the radiolabeled antibody with increasing concentrations of unlabeled pertuzumab , with a half maximal effective concentration ( ec50 ) of 2.4 0.11 nm ( figure 2c ) . the absence of competitive binding with unlabeled trastuzumab is consistent with an her2 epitope binding distinction between zr - pertuzumab to domain ii and trastuzumab to domain iv of her2 . furthermore , the binding of zr - pertuzumab is increased by 30% in the presence of high concentrations ( 0.022 m ) of unlabeled trastuzumab . this observation is also in agreement with the in silico studies by fuentes et al . conformational changes occur upon trastuzumab binding resulting in the increased affinity of pertuzumab for its her2 binding epitope . lastly , a comparative rate of cellular internalization study was conducted at 37 c with zr - pertuzumab and zr - trastuzumab . the internalization of both antibodies was similar ; with 50% of zr - pertuzumab internalizing at 23 h and zr - trastuzumab at 21 h ( figure 2d ) . the control reactions at 4 c when receptor internalization is reduced showed minimal internalization of both radiolabeled antibodies at 48 h and demonstrated the efficiency of the acid wash treatment to separate surface - bound from internalized antibody . these studies showed zr - pertuzumab was as effective as zr - trastuzumab in binding her2 in vitro , despite different binding sites . in vitro cell binding studies show zr - pertuzumab retains immunoreactivity and specifically binds to a different her2 binding site than trastuzumab . ( a ) immunoreactivity of zr - pertuzumab in skbr3 cells was determined with a y intercept of 1.39 0.04 and calculated an immunoreactive fraction of 72.2% . ( b ) cell uptake studies at 37 c in bt-474 ( her2 + ) , skbr3 ( her2 + ) , and mda - mb-231 ( her2 ) demonstrate significantly higher zr - pertuzumab uptake in the her2 + cell lines compared to the her2 control ( p < 0.001 ) . no statistical difference was observed between bt-474 and skbr3 ( p > 0.05 ) . ( c ) competitive binding studies of zr - pertuzumab with unlabeled pertuzumab in skbr3 cells at 4 c yields an ec50 of 2.4 0.11 nm , and no competition was observed with unlabeled trastuzumab . ( d ) cell internalization studies in skbr3 cells at 37 c up to 48 h demonstrate 50% of zr - pertuzumab internalizes at about 20 h , similar to zr - trastuzumab . control reactions when receptor internalization is reduced at 4 c show minimal internalization of radiolabeled antibodies at 48 h. subcutaneous tumor xenografts in severely combined immunodeficient mice have been reported to have excellent tumor take rates without the need for exogenous stimuli such as matrigel or estrogen pellets . we observed slow tumor growth for bt-474 and mda - mb-231 , which was achieved in 46 weeks with tumor take rates of 93% ( 13/14 ) and 100% ( 7/7 ) , respectively . the skbr3 tumor take rate , even at high cell concentration ( 1 10 cells / ml ) was 22% ( 3/14 ) and this low sampling hampered our investigation of zr - pertuzumab in this model . , in bt-474 xenografts and zr - pertuzumab accumulation in the bt-474 tumor was higher than in mda - mb-231 with max suv of 10.5 6.1 , and 2.2 0.8 , respectively ( p = 0.0006 , figure 3a , b ) . these data were analyzed as the logarithmic transformation to more precisely represent the means due to the high variances observed . max suv analysis showed increased tumor uptake of zr - pertuzumab in bt-474 in the presence of unlabeled trastuzumab ( 19.0 5.4 ) although not statistically significant ( p > 0.05 ) and may be attributed to the high variance in each test group . the accumulation of zr - pertuzumab was reduced in the presence of unlabeled pertuzumab with suv of 2.2 2.5 ( p = 0.0004 ) , a value equivalent to the tumor uptake in the mda - mb-231 model ( p > 0.05 ) . post - pet biodistribution studies significantly correlate with pet imaging quantification yielding pearson s coefficient of r = 0.98 , with a linear fit of r = 0.98 ( p = 0.01 , supporting information figure s1 ) . the tumor uptake was higher for bt-474 with 47.5 32.9% id / g ( n = 7 ) compared to the mda - mb-231 xenograft with 9.5 1.7% id / g ( n = 7 , p = 0.0009 ) . blocking with unlabeled pertuzumab had a significant reduction in tumor uptake , 8.5 6.6% id / g ( n = 4 , p = 0.0006 ) . the tumor accumulation of zr - pertuzumab showed a significant increase of 173 74.5% id / g in the presence of unlabeled trastuzumab ( n = 3 , p = 0.01 ) . the variance instability for the biodistribution studies is shown in supporting information figure s2 , showing the need to perform statistical analyses on the logarithmic transformations of the data . low accumulation was observed in most normal organs particularly the blood , muscle , and heart with 0.05 0.02 , 0.7 0.2 , and 1.0 0.1% id / g , respectively , for the bt-474 xenografts . in vivo studies show high and specific tumor uptake of zr - pertuzumab in her2 + tumors , enhanced in the presence of trastuzumab . ( a ) maximum intensity projection of pet / ct images of zr - pertuzumab in bt-474 ( her2 + ) and mda - mb-231 ( her2 ) breast cancer xenografts at 7 days p.i . ( b ) max suv quantification of zr - pertuzumab tumor uptake in bt-474 was 10.5 6.1 , increased in the presence of unlabeled trastuzumab ( 19.0 5.4 ) , decreased in the presence of a blocking concentration of unlabeled pertuzumab ( 2.2 2.5 ) , and decreased in mda - mb-231 xenografts ( 2.2 0.8 ) . ( c ) biodistribution studies correlate with pet imaging quantification with 47.5 32.9% id / g for zr - pertuzumab in bt-474 , 173 74.5% id / g in the presence of unlabeled trastuzumab ( p = 0.01 ) , 8.5 6.6% id / g in the presence of unlabeled pertuzumab ( p = 0.0006 ) , and 9.5 1.7% id / g in the her2 mda - mb-231 . biodistribution of zr - pertuzumab in her2 + bt-474 and her2 mda - mb-231 xenografts . a cohort of bt-474 xenografts were coinjected with either unlabeled trastuzumab ( checkered bars ) or unlabeled pertuzumab ( horizontal line bars ) . zr - pertuzumab alone with no other cotreatments added were studied in bt-474 ( black bars ) or mda - mb-231 ( unfilled bars ) . interestingly , the spleen showed a relatively high tracer accumulation , greater than 100% id / g , in these severely combined immunodeficient mice . this unexpected outcome led us to investigate whether this phenomenon was a consequence of these tumors ; thus , we performed biodistribution studies of zr - pertuzumab in nontumor bearing nogs and in the conventional athymic nu / nu mice as a control . similar spleen uptake of 119.7 17.9% id / g ( n = 2 ) was observed in nontumor bearing nog mice at 7 days p.i . however , only 2.7 1.7% id / g ( n = 2 ) was observed in nontumor bearing athymic nu / nu mice ( supporting information figure s3 ) . thus , the high spleen uptake is unique to the severely combined immunodeficient mice . we hypothesize that the absence of immunogenic response ( e.g. , the lack of t , b , and natural killer cell functions ) as well as the abnormal macrophage activity in the nog strain are involved in the catabolism of zr - pertuzumab to the spleen . in contrast , conventional strains of mice that still have some intact immunogenic response ( e.g. , with functional natural killer cells ) , catabolize antibodies primarily in the liver . further investigations are needed to determine the pharmacokinetic properties of humanized antibodies in different types of preclinical models . following biodistribution studies , tumor slices from the bt-474 and mda - mb-231 groups were stained for her2 expression ( brown ) using a commercially available anti - her2 antibody and counterstained with tartrazine ( yellow ) ( supporting information figure s4 ) . her2 expression in bt-474 tumors was pronounced , visibly outlining the her2 extracellular membrane , whereas the mda - mb-231 tissue stained negative for her2 expression . furthermore , we observed negative her2 staining in the spleen , confirming the absence of human her2 in this organ ( data not shown ) . h&e stains confirmed tumor viability . these results showed that the specific targeting of zr - pertuzumab to her2 in vivo were in agreement with the her2 expression levels ex vivo . our findings illustrate zr - pertuzumab specifically accumulates in her2 + tumors , enhanced by the presence of trastuzumab . this study is consistent with in silico studies conducted by fuentes et al . , where a conformational change in her2 occurs upon trastuzumab binding , likely inducing greater exposure of this binding site leading to the enhanced affinity of pertuzumab . our in vitro and in vivo studies show increased cellular and tumor uptake of zr - pertuzumab in the presence of excess unlabeled trastuzumab , respectively ( figure 2c , figure 3 ) . these different studies did not show a comparable degree of enhanced uptake of zr - pertuzumab , with only 30% increased cellular uptake in vitro at 4 c within 1.5 h of incubation and almost 400% increased tumor uptake in vivo within 7 days of bloodstream circulation . this apparent discrepancy may be due to differences in the kinetic properties of zr - pertuzumab in the more artificial environment in vitro compared to the complex environment in vivo over several days . the 7 day pet imaging after administration of zr - pertuzumab was performed to allow clearance of the radiolabeled antibody as exemplified by previously studied zr - labeled monoclonal antibodies , one of the advantages of imaging with zr over shorter - lived radioisotopes . an earlier imaging time point at 5 days p.i . was performed in a small cohort of mice , and no differences in tumor uptake were observed between 5 and 7 days p.i . ( data not shown ) , which confirms the in vivo stability of this radiopharmaceutical . additionally , the increased bloodstream residence of zr - pertuzumab may also contribute to its increased tumor uptake in the presence of trastuzumab . the absence of t , b , and nk cells part of the immune response and the abnormal macrophage activity in this nog host may contribute to the inefficiency of the clearance mechanism for antibodies , as evidenced by the high spleen uptake of zr - pertuzumab . in contrast , the less immunocompromised strains such as athymic mice which require estrogen pellet implantation for bt-474 tumor engraftment have some functional immunogenic response to mediate the hepatobiliary clearance of antibodies more efficiently . as such , higher liver and lower spleen uptake were observed for nontumor bearing athymic mice compared to the nog strain ( supporting information figure s3 ) . differences in clearance mechanisms for antibodies in different preclinical models may be an interesting future study ; however , this should not impede the translation of zr - pertuzumab to clinical trials . in a previous study , scheuer et al . investigated pertuzumab as an optical imaging agent labeled with a cy5 fluorescent dye , to assess the synergistic therapeutic effect of the combination of pertuzumab and trastuzumab in kpl-4 human breast cancer xenografts . this study found similar tumor uptake between cy5-pertuzumab alone and cy5-pertuzumab in combination with unlabeled trastuzumab . no enhancement of cy5-pertuzumab tumor uptake in the presence of trastuzumab was reported , possibly due to differences in experimental conditions such as the increased injected mass of the cy5-pertuzumab , in which its unlabeled fraction may block some her2 binding sites or the reduced sensitivity of optical imaging compared to pet . a limitation of our study is the high variance in zr - pertuzumab tumor uptake in the bt-474 xenografts . this observation may be a consequence of tumor growth variability among the nogs . despite the high variance in the means , zr - pertuzumab demonstrates high affinity and selectivity for her2 + tumors in vivo . although radiolabeled trastuzumab has had much success in imaging her2 expression in breast cancer patients who were trastuzumab nave or were undergoing trastuzumab therapy , radiolabeled pertuzumab may be a more favorable alternative as it binds to a distinct her2 epitope . imaging with pertuzumab while treating with trastuzumab or the trastuzumab antibody drug conjugate , t - dm1 , would allow a noncompetitive and cooperative binding interaction with her2 . currently , in - labeled pertuzumab is being investigated in breast cancer patients using single - photon emission computed tomography ; however , the sensitivity of pet and the resolution of zr will provide excellent images with reduced radioactivity exposure to the patient . thus , pet imaging using zr - pertuzumab may be advantageous to monitor patient response to her2-targeted therapy involving trastuzumab to more sensitively detect her2 expression . zr - pertuzumab as a pet imaging agent detects her2 expression selectively in breast cancer xenograft models with increased tumor uptake in the presence of trastuzumab . zr - pertuzumab is a promising radiopharmaceutical for noninvasive imaging of her2 + breast cancer undergoing trastuzumab therapy to enhance the sensitivity of her2 detection in vivo .

pertuzumab is a monoclonal antibody that binds to her2 and is used in combination with another her2specific monoclonal antibody , trastuzumab , for the treatment of her2 + metastatic breast cancer . pertuzumab binds to an her2 binding site distinct from that of trastuzumab , and its affinity is enhanced when trastuzumab is present . we aim to exploit this enhanced affinity of pertuzumab for its her2 binding epitope and adapt this antibody as a pet imaging agent by radiolabeling with 89zr to increase the sensitivity of her2 detection in vivo . here , we investigate the biodistribution of 89zr - pertuzumab in her2expressing bt-474 and her2nonexpressing mda - mb-231 xenografts to quantitatively assess her2 expression in vivo . in vitro cell binding studies were performed resulting in retained immunoreactivity and specificity for her2expressing cells . in vivo evaluation of 89zr - pertuzumab was conducted in severely combined immunodeficient mice , subcutaneously inoculated with bt-474 and mda - mb-231 cells . 89zr - pertuzumab was systemically administered and imaged at 7 days postinjection ( p.i . ) followed by terminal biodistribution studies . higher tumor uptake was observed in bt-474 compared to mda - mb-231 xenografts with 47.5 32.9 and 9.5 1.7% id / g , respectively at 7 days p.i ( p = 0.0009 ) and blocking studies with excess unlabeled pertuzumab showed a 5-fold decrease in bt-474 tumor uptake ( p = 0.0006 ) , confirming the in vivo specificity of this radiotracer . importantly , we observed that the tumor accumulation of 89zr - pertuzumab was increased in the presence of unlabeled trastuzumab , at 173 74.5% id / g ( p = 0.01 ) . biodistribution studies correlate with pet imaging quantification using max suv ( r = 0.98 , p = 0.01 ) . collectively , these results illustrate that 89zr - pertuzumab as a pet imaging agent may be beneficial for the quantitative and noninvasive assessment of her2 expression in vivo especially for patients undergoing trastuzumab therapy .

Introduction Materials and Methods Results Discussion Conclusions

data on case investigation timelines in 2002 were collected from records at state and local health departments and public health laboratories in each of 6 states for < 100 salmonella spp . participating states included 1 with a large population ( > 6 million ) , 3 with a medium - sized population , and 2 with a small ( < 2 million ) population from 5 different geographic regions . rules mandated reporting of diagnosed cases from physicians or clinical laboratories to local health departments ( 2 states ) , to the state health department ( 2 states ) , or to both ( 2 states ) . cases were selected by systematically choosing every nth record on the basis of the number of cases reported and the number sampled . for 1,319 cases , dates were collected for the following : onset of symptoms ( 873 [ 66% ] ) , stool specimen collection ( 1,088 [ 82% ] ) , culture result ( 633 [ 50% ] ) , report to state or local health department ( 553 [ 42% ] ) , submission of isolate to public health laboratory ( 882 [ 98% ] of 899 isolates that were submitted ) , case interview ( 648 [ 49% ] ) , and molecular subtyping by pulsed - field gel electrophoresis ( pfge ) ( 634 of 635 isolates that were subtyped ) . although stool culture result dates were recorded for 633 cases , most were for final culture results based on confirmation by the public health laboratory . for each case , intervals between milestones were calculated from the dates available . for 112 outbreaks of foodborne disease , dates were collected for the following : implicated meal or event ( 100 [ 89% ] ) , onset of symptoms of index case - patients ( 112 [ 100% ] ) , first stool collection ( 65 [ 79% ] of 82 outbreaks for which stool samples were collected ) , foodborne illness complaint or report of outbreak - related case to health department ( 99 [ 88% ] ) , initiation of outbreak investigation activities ( 90 [ 80% ] ) . for each outbreak , intervals were calculated from the dates available . the median intervals from onset of symptoms to surveillance milestone events for individual cases were as follows ( table 1 ) : collection of stool samples , 24 days ; initial stool culture results , 58 days ; case report to health department , 79 days ; isolate submission to public health laboratory , 810 days . for case - patients who were interviewed , the median interval from onset of symptoms to interview was 12 days for e. coli o157:h7 cases , 14 days for salmonella spp . and shigella spp . cases , and 18 days for campylobacter spp . cases . for isolates that were subtyped by pfge , the median intervals from onset of symptoms to subtyping were 15 days for e. coli o157:h7 , 18 days for salmonella spp . , and 21 days for shigella spp . * pfge , pulsed - field gel electrophoresis . a higher percentage of isolates were submitted to the public health laboratory in states where submission was required ( 98% for salmonella spp . isolates , 100% for e. coli o157:h7 ) compared to states where submission was not required ( 75% for salmonella spp . however , no difference was found between these states in length of time for isolates to be submitted . of 112 confirmed foodborne disease outbreaks , 83 ( 74% ) had an etiologic agent confirmed by laboratory testing ( table 2 ) ( 5 ) . of 29 outbreaks that were not confirmed , norovirus was the suspected cause in 17 ( 59% ) outbreaks , and toxigenic bacteria were suspected in 7 ( 24% ) outbreaks . median intervals from onset of symptoms to outbreak complaint or recognition were 1 day for bacterial toxins , 3 days for norovirus , 8 days for e. coli o157:h7 and campylobacter spp . , and 16 days for salmonella spp . overall , 83 ( 74% ) outbreaks were detected by a consumer complaint , 12 ( 11% ) were detected by a healthcare provider , 11 ( 10% ) were detected by pfge cluster evaluation , and 6 ( 5% ) were identified through an interview with an individual case - patient . intervals from onset of symptoms to consumer complaint ( median 3 days , range 021 days ) or to report by healthcare provider ( median 3 days , range 011 days ) were similar . outbreaks identified by case interview ( median 11 days , range 616 days ) or pfge cluster evaluation ( median 23 days , range 783 days ) followed case surveillance timelines described above . the median interval from detection of the outbreak to the initiation of the first outbreak investigation step was 0 days ( range 041 days ) for all outbreaks . the median duration of exposure for all outbreaks with a confirmed etiologic agent was 1 day ( range 121 days ) . however , 12 ( 29% ) of 41 norovirus , 2 ( 67% ) of 3 e. coli o157:h7 , and 9 ( 75% ) of 12 salmonella spp . the median duration of multiday outbreaks was 4 days for norovirus ( range 213 days ) , 5 days for e. coli o157/h7 outbreaks ( range 56 days ) , and 10 days for salmonella spp . the multiple steps between onset of a foodborne illness and its investigation by a public health agency result in delayed recognition of outbreaks caused by reportable enteric diseases . one important way to speed the detection of outbreaks is to encourage clinicians to immediately notify health departments when they suspect a patient is part of an outbreak . since many outbreaks caused by e. coli o157:h7 and salmonella spp . last multiple days , physician reporting concurrent with stool collection may provide opportunities for a public health intervention that could prevent outbreak - associated cases . the speed with which clinical laboratories receive , process specimens , and report results varies by setting , agent , and location . the lack of detail available about these steps is an important limitation of this study . however , health departments generally receive reports from clinicians a median of 2 days after the culture result , and isolates are submitted to public health laboratories within 23 days of the initial culture result . these data suggest that improving physician and laboratory reporting practices and logistics could shorten the reporting timeline by 1 or 2 days for most cases . timeline elements directly under control of public health agencies include the interval from case report to interview and from submission of the isolate to subtyping by pfge . our results demonstrate more variability for these intervals than for earlier steps in enteric disease surveillance . in particular , half of e. coli o157:h7 cases but less than one fourth of salmonella spp . cases were contacted by a local health department on the same day the report was received . in addition , outbreaks caused by e. coli o157:h7 were detected a median of 8 days sooner than outbreaks caused by salmonella spp . given the risk for hemolytic uremic syndrome after e. coli o157:h7 infections and the potential for person - to person transmission , such attention is warranted . even so , the intervals from onset of symptoms to pfge subtyping documented in the nationwide outbreak of e. coli o157:h7 infections associated with spinach demonstrated that little has changed across the public health system from 2002 to 2006 ( 6 ) . infection reinforce the need to increase the timeliness of case follow - up , molecular subtyping , and the linkage of results between them that can reduce delays in the investigation of foodborne outbreaks ( 7 ) .

we reviewed timeline information for a sample of salmonella spp . , shigella spp . , campylobacter spp . , and escherichia coli o157:h7 cases and all confirmed foodborne outbreaks reported in 6 states during 2002 . increasing the timeliness of case follow - up , molecular subtyping , and linkage of results are critical to reducing delays in the investigation of foodborne outbreaks .

The Study Conclusions

she presented with a 5-day history of behavioral change , in the form of incomprehensive talking visual and auditory hallucinations , and short attention span . she had sleep disturbance , labile mood , and decreased appetite with loss of sphincter control . one month prior to this episode , she had one attack of unprovoked generalized tonic - clonic brief seizure . her father had hypothyroidism that was , well controlled on treatment . on physical examination , she was agitated , hemodynamically stable , and afebrile . she had hallucinations and abnormal facial movements , but otherwise there was no neurological deficit . a full work - up including metabolic screening , toxicology screening , brain mri , cerebrospinal fluid ( csf ) analysis , and septic screening were all negative . her thyroxine ( t4 ) and thyroid stimulating hormone ( tsh ) were normal , but thyroid antibodies were elevated : thyroglobulin=383 ( normal range < 115 iu / ml ) , and thyroid peroxidase= 195 ( normal range < 34 iu / ml ) . the working diagnosis was hashimoto thyroiditis , and she was treated with intravenous immunoglobulin ( 400 mg / kg / day for 5 days ) . she showed a quick improvement in her condition , and returned to her baseline within 2 weeks . electroencephalography showing diffuse slow background activity without epileptiform discharges . a 9-year - old boy presented with a history of behavioral changes associated with aggressiveness and excessive crying for one week . he then started to develop a series of seizures and status epilepticus . on examination , he was encephalopathic , with a glasgow coma scale of 9/15 , hemodynamically stable , and afebrile . results of brain mri were normal , and csf showed 24 cells / mm normal range < 5 , mainly mononuclear . the anti - nmdar antibodies were high in the csf ( 1:30 ; normal range<1:1 ) and serum ( 1:160 ; normal range<1:10 ) . other work - up including septic work - up , toxicology , and metabolic screening , he was considered to have anti - nmdar encephalitis , and treated with intravenous immunoglobulin , steroids , rituximab , and anti - epileptics . the outcome was good , and he returned to normal within 9 months of treatment . a 7-year - old girl was referred because of acute psycho - behavioral changes , 2 weeks after febrile illness . her vital signs were within normal limits . a systemic examination including neurological examination was normal . she was diagnosed with lgi1 antibodies encephalitis and started on intravenous immunoglobulins and pulse methylprednisolone with a good outcome . she presented with a 5-day history of behavioral change , in the form of incomprehensive talking visual and auditory hallucinations , and short attention span . she had sleep disturbance , labile mood , and decreased appetite with loss of sphincter control . one month prior to this episode , she had one attack of unprovoked generalized tonic - clonic brief seizure . her father had hypothyroidism that was , well controlled on treatment . on physical examination , she was agitated , hemodynamically stable , and afebrile . she had hallucinations and abnormal facial movements , but otherwise there was no neurological deficit . a full work - up including metabolic screening , toxicology screening , brain mri , cerebrospinal fluid ( csf ) analysis , and septic screening were all negative . her thyroxine ( t4 ) and thyroid stimulating hormone ( tsh ) were normal , but thyroid antibodies were elevated : thyroglobulin=383 ( normal range < 115 iu / ml ) , and thyroid peroxidase= 195 ( normal range < 34 iu / ml ) . the working diagnosis was hashimoto thyroiditis , and she was treated with intravenous immunoglobulin ( 400 mg / kg / day for 5 days ) . she showed a quick improvement in her condition , and returned to her baseline within 2 weeks . a 9-year - old boy presented with a history of behavioral changes associated with aggressiveness and excessive crying for one week . he then started to develop a series of seizures and status epilepticus . on examination , he was encephalopathic , with a glasgow coma scale of 9/15 , hemodynamically stable , and afebrile . results of brain mri were normal , and csf showed 24 cells / mm normal range < 5 , mainly mononuclear . the anti - nmdar antibodies were high in the csf ( 1:30 ; normal range<1:1 ) and serum ( 1:160 ; normal range<1:10 ) . other work - up including septic work - up , toxicology , and metabolic screening , he was considered to have anti - nmdar encephalitis , and treated with intravenous immunoglobulin , steroids , rituximab , and anti - epileptics . the outcome was good , and he returned to normal within 9 months of treatment . a 7-year - old girl was referred because of acute psycho - behavioral changes , 2 weeks after febrile illness . peri - oral movements were also observed . there was no history of headache , vomiting or drug ingestion . her vital signs were within normal limits . a systemic examination including neurological examination was normal . she was diagnosed with lgi1 antibodies encephalitis and started on intravenous immunoglobulins and pulse methylprednisolone with a good outcome . we describe 3 patients with immune - mediated encephalopathy / encephalitis who presented in the initial phase with predominant psychiatric symptoms . the presenting symptoms included mainly psycho - behavior changes , aggressivity and hallucinations . immune - mediated encephalopathies / encephalitis are increasingly being recognized in children with antibodies , mainly to nmdar , but also to lgi1 , caspr2 ( previously attributed to voltage- gated potassium channel antibodies complex ) or other cns antigens.5,7 most of the patients present with a combination of recurrent seizures / status epilepticus , decreased level of consciousness , and motor neurological deficits . a recent large cohort - based study of patients with immune - mediated encephalitis revealed that 4% of patients presented with isolated psychiatric episodes.8 support for this paradigm comes from another study involving 43 children with a first episode of acute psychosis . antibodies to the dopamine d2 receptor were identified in 3 children , and to the nr1 subunit of the nmdar in 6 children , whereas no such antibodies were found in a group of 43 healthy control children.9 the mechanisms of psychiatric manifestations in antibodies mediated encephalitis are still not clear . the nmda - receptor blockade in vivo produces schizophrenia - like symptoms in healthy individuals . ion channel disturbance and hyponatremia have also been described as features of schizophrenia , previously attributed to medication - induced or psychogenic polydipsia.8,9 the findings from this study have several practical implications . there is growing evidence that a proportion of psychosis / psychiatric manifestations in children may be immune - mediated , and psychiatrists should consider this etiology in each presentation of first - episode psychosis . if positive , these patients should be considered for treatment with immunotherapy . on the other hand , several antipsychotic drugs , such as haloperidol , must be used with caution in these conditions , as they have been observed to exacerbate motor symptoms in patients with anti - nmdar encephalitis . we therefore recommend that all patients with a new - onset psychosis , or neuropsychiatric manifestations including seizures , autonomic dysfunction , movement disorders , or rapid deterioration of the level of consciousness should be evaluated for a possible antibody - associated encephalitis . this assessment should include , as a minimum , a neurological examination and early testing for antibodies against the nmdar , lgi1 , caspr2 , ampa , gad , gabab antibodies , and thyroid antibodies . the eeg can be useful : and encephalopathic pattern or extreme delta brushes pattern should prompt early referral . mri of the brain may be normal ( although the finding of temporal lobe or limbic system signal changes is very suggestive of an immune - mediated encephalopathy ) . the aim of these recommendations is the early identification and improved treatment for patients with this disorder . in conclusion , autoimmune mechanisms have been recently implicated in a number of neuropsychiatric disorders , classically linked to a pure psychiatric disease . delay in diagnosis and treatment with immunomodulatory drugs can lead to permanent sequelae . in cases of acute psychosis in children ,

new - onset psychosis in children represents a complex presenting symptom . psychosis can be attributable to a combination of factors and etiologies , and all possible causes must be systematically examined . there is growing evidence that a proportion of psychosis / psychiatric manifestations in children may be immune - mediated , and physicians should consider this etiology in each presentation of first - episode psychosis . immune - mediated encephalopathies / encephalitis are increasingly being recognized in children with antibodies to n - methyl - d - aspartate receptor , leucine - rich glioma - inactivated 1 or other central nervous system antigens such as contactin - associated protein - like 2 , glutamic acid decarboxylase , alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or gamma - aminobutyric acid b. in this study , we describe 3 cases of immune - mediated encephalopathy / encephalitis with prominent psychiatric symptoms at presentation , and suggest a practical diagnostic and treatment approach for children with acute psychosis of an immune - mediated cause .

Case Report Patient 1 Patient 2 Patient 3 Discussion

within and beyond the world of nanoscience , images abound . there are lots of colourful images of structures at the nanoscale , based on visualizations of digital data created by scanning probe microscopy or other imaging instruments ( as are made available to colleague nanoscientists ) . there are also artist s impressions , ranging from impressions of entities at the nanoscale to envisioning nanoachievements to be realized in the near or long - term future , including science - fiction type images of nanobots . one can distinguish imaging , that is , creating images based on data and aiming at resemblance , from imagining , where imagination is mobilized to create a vision of the nanoworld . actually , imaging and imagining are always entangled , already in so - called scientific visualizations where expectations about how the nanoscale could look like steer the choices involved in making images . a subsequent question then is what such entangled images stand for , not only representing in the sense of resemblance , but also in terms of what images do when representing the nanoscale . a clear example are images which demonstrate of technoscientific achievements . in practice , there is a continuum ranging from visualizations of the nanoscale adapted from original instrument - based data to so - called artist s impressions ( which need not be produced by artists ; the phrase is used to indicate some freedom in visualizing ) of how the nanoscale could look like , or how audiences may expect that it looks . this occurs already within science , for example when images indicate possible functions of complex molecular structures . to capture the continuum of imaging and imagining , we will write imag(in)ing.1 we will start by analyzing occurrences of imag(in)ing and the tensions involved . while imag(in)ing has to do with representation , which is often focused on making something present through resemblance , we emphasize representation as standing for and being able to political representation ( cf . ) . standing for and being able to act for , is somewhat independent of what is being stood for. thus , this notion of representation is broader than the epistemological puzzles , and debates , about reality ( out there and/or in contrast with fiction ) . in the final part of our paper right representation is , in terms of images that become iconic , and in that sense stand for , and are allowed to speak for , nanotechnology . the nanoscale , invisible to the naked eye , is visualized through the use of a variety of imaging instruments . at the outset , it is not clear what the right imaging tools would be , and this relates to the basic problem how one can know whether the visualizations obtained actually visualize the nanoscale harry collins highlighted the problem of choice between instruments that would be adequate to the task of studying unknown phenomena a problem which he introduced as an experimenter s regress because there is no independent check of the correctness of the choice . for the production of visualizations there is the additional question about the ways to produce images / pictures that lead to reliable representations of the invisible . just as the experimenter s regress can ( and will ) be stopped in practice when expectations about the unknown phenomenon become shared , expectations about how the invisible phenomena should look like , can provisionally stop the visualization regress (: 5 ) . a subsequent imaging challenge is to reach audiences which are not familiar with the production processes of the images . there will be ( .. ) a diffuse message about the nanoscale which has to be presented as clearly and convincingly as possible (: 9 ) to intended audiences . artist s impressions may do such a job better than data - based images . in visualization practices expectations about what can be seen at the nanoscale and how the nanoscale should be visualized are to some extent internalized as rules about how to do it . the data - based images are manipulated to highlight the information they should convey , which is an accepted practice in the domain of science , as long as the scientific content remains unchanged . artist s impressions of how the nanoscale could look like are similar , in the sense that imaging and imagining are also entangled . in other words visualizations of the nanoscale as well as artist s impressions are used in scientific journals to show research results.2 such practices are not uncontested . correctly , make it to the cover of scientific journals.3 when they do so , they come to stand for scientific achievements , and cover pictures are often presented on research groups websites next to their list of publications . 1artist s impression of an array of nanotubes fets overlaid with gold source and drain electrodes . this image was used for a cover of science , and was subsequently criticized by ottino , who argued that if the carbon atoms are visible , then the much larger gold atoms in the structure should also be on view courtesy of : c. dekker , tu delft / tremani artist s impression of an array of nanotubes fets overlaid with gold source and drain electrodes . this image was used for a cover of science , and was subsequently criticized by ottino , who argued that if the carbon atoms are visible , then the much larger gold atoms in the structure should also be on view as an emerging technology , nanotechnology comprises present achievements and visions of what it may become . while such combinations occur for all sorts of sciences and technologies ( see van lente on promising technologies ) this appears particularly striking in nanotechnology . mody argues that nanotechnology , in contrast to , for example , physics and chemistry , seems decidedly non - presentist and that nanotechnologists work as much in this future world as in the present (: 108 ) . then , there is the design orientation in nanotechnology , where a possible future is projected to be realized by a present design . with computer simulations playing an important role , much of nanotechnology s created reality thus has a virtual , yet - to - be - realized quality . kaiser adds another twist:they [ future visions ] have been invented and are presented with the strong probability claim that they will become reality in the future . from a modal logical point of view , they do nt represent possible worlds in the fictional universe ; on the contrary , they constitute future worlds in ours (: 667 ) . they [ future visions ] have been invented and are presented with the strong probability claim that they will become reality in the future . from a modal logical point of view , they do nt represent possible worlds in the fictional universe ; on the contrary , they constitute future worlds in ours (: 667 ) . if one wants to use categories like reality and fiction ( as actors often do ) , one can say that boundaries between reality and fiction are blurred in the nanorealm , and this will be reflected in images of nanotechnology . scientists produce images equivalent to artist s impressions that show what could be possible applications for nanotechnology . images of bucky balls , molecular machines , new uses of carbon nanotubes , are created to make a possible future present . artists offer their ( own or commissioned ) visions of nanotechnology s potential future applications . as soon as the future is incorporated , there is no sharp boundary between images that articulate directions of further research and more open - ended visions of new performances which then merge with futuristic visions . some actors will attempt to separate science ( reality ) from science fiction. this can be a tactic to have their project prevail over that of others ( this tactic has been deployed by critics of drexler s visions of molecular manufacturing , cf . ) . or it can be a defensive move to avoid being called to account for possible negative implications of investing in nanotechnology research ( that s only fiction ) . arguments for disentangling the future from the present also occur in relation to lay - audiences . such an argument would run like : it should be made clear which images are realistic and which are fictive , so as to avoid giving the false impression that ( say ) nanobots have already been built . ottino extended his crusade for correctness to the nanolouse image ( see fig . 2 ) : fig . 2the nanolouse image ; over - hyped or a possible application of nanotechnology in medicine in the future . source : ottino the image looks so real that it is easy to imagine a viewer being fooled into believing it has already been built . this is important in terms of public reaction , especially in the backdrop of scenarios such as in michael crichton s new novel prey , which portrays swarms of self - replicating nanomachines destroying all other life forms that they encounter (: 476 ) . the nanolouse image ; over - hyped or a possible application of nanotechnology in medicine in the future . source : ottino the image looks so real that it is easy to imagine a viewer being fooled into believing it has already been built . this is important in terms of public reaction , especially in the backdrop of scenarios such as in michael crichton s new novel prey , which portrays swarms of self - replicating nanomachines destroying all other life forms that they encounter (: 476 ) . ottino links his criticism with a concern about public acceptance of nanotechnology , if such images should come to stand for nanotechnology in the public eye . the public , however , may well be more discerning than he projects it to be . still , the image does contain an ambivalence , which is brought out in how it has been presented on the bbc website ( www.bbc.co.uk ) . on one occasion it was positioned as the 2002 winner of the visions of science award : the more over - hyped applications see tiny machines roaming the body to cure diseases. on another occasion , realistic possibilities were emphasized : the nanolouse image intends to show one of the possible applications of nanotechnology in medicine in the future microscopic machines roaming the body , injecting or taking samples for tests. continuing with this example , it is interesting that the nanolouse image has been often used in the world of science . in the period 20052007 , the image was used in presentations about drug delivery , nano - probes and ( n)mems , as an illustration and occasionally as a possible future application .4 in general terms , images of nanotechnology that import the future into the present are used to communicate what nanotechnology is as well as to indicate nanotechnology s potential . in doing so , within science and beyond , such images are used strategically to put audiences in desired positions and at the same time to strengthen one s own position . an interesting example is how the foresight nanotech institute used the nanogear image to position itself . on their website , they present the nanogear image ( fig . 5 ) ( and the image of the strained - shell sleeve bearing ) to indicate that 1 day , surely , productive nanosystems will be realized . respirocytes ( artificial blood cells able to carry 236 times more oxygen than human s red blood ) as designs , implying that they can ( and thus will ) be built in the future . what is new in nanotechnology is that these images are about aimed - for possibilities rather than an actual construction plan of such a future . the nanogear image is an extreme example , but these kind of images and their use are widespread . nanoscientists play with the entanglement of data - based imaging and impressionistic imagining , and with the entanglement resulting from projecting of the future into the present . they produce and use images to illustrate scientific breakthroughs and to create expectations about the potential realization of scientific achievements , or to highlight socio - political debates . in addition , the aesthetic appeal of the images that are produced as visualizations of the nanoscale , is actively pursued , for example through impressive colouring , as in ibm s image of the quantum corral ( fig . 7 ) . the fact that images are now given a name , and an evocative name at that , is an indication that they are seen as works of art , which have titles.5 this link with the world of art , at least with the world of being creative in making images , is not just an excursion of scientists . an interesting development is how artists make use of , or even get involved in , the production of images through imaging instruments . as chris orfescu noted , there appear to be two ways to do so : nanolandscapes in which given structures of matter at the nanoscale are depicted , and nanosculptures , which are created through the manipulation of matter at the nanoscale by nanoscientists , and then visualized.6 one sees how such images offer a view to a new world that is in the process of being explored . many of the nano- and microsculptures like the nano - guitar , nano - opera house , nano - toilet and the nano - bull ( see fig . interestingly , the methods which are used to create such nanosculptures are of scientific interest . fig . a team of japanese engineers has created the smallest sculpture of a bull , which could only be viewed with a scanning electron microscope . a team of japanese engineers has created the smallest sculpture of a bull , which could only be viewed with a scanning electron microscope . source : kawata et al . for example , kawata et al . created a model bull , measuring 10 by 7 m , which is about the size of a red blood cell . the nano - bull was used to show the potential of two - photon photopolymerization . but the fact that it was a bull was the immediate message , and kawata et al.s claim about their technoscientific achievement piggy - backed ( if we may say so ) on this message . another example comes from a nanoart project in which imaging tools like the scanning tunneling microscope ( stm ) and atomic force microscope ( afm ) are deliberately used for artistic purposes . scali and goode produced the smallest map ever of the continent africa , created through the use of a scanning electron microscope ( sem ) and visualized with an afm ( see fig . the actual size is 200 m 130 m , and it is meant to highlight the paradox of africa being geographically and anthropological central to our world , yet unrecognizable , unexplored , and invisible (: 408 ) . apart from the political message that requires text to be conveyed , a visualization is produced to create an aesthetic paradox : a piece of art that you can never see . yet that exists and carries a message.7 at the same time , it shows the research group s abilities to work at the nanoscale and to visualize it , and so illustrate / demonstrate the innovative character of nanotech developments . the artwork is a small sliver of silicon , visualized with an afm , and only visible by using the same instrument . the artwork is a small sliver of silicon , visualized with an afm , and only visible by using the same instrument . source : http://www.nanoarte.it in this way , nanoart also becomes a showcase for nanoscience . by crafting impressive nanosculptures , scientists capacities to manipulate matter at the nanoscale are highlighted , and expectations are created about the possible applications of the new methods . then there are artists ( often graphic artists ) who create impressions of how the nanoscale could look , or give their impressions of possible future applications for nanotechnology developments . they can be commissioned to do so by scientists to enhance the visual appeal of their achievements . the aesthetic appeal of images becomes more relevant when images are intended to circulate more widely and to reach broader audiences . this happens when imaging contests are organized where beautiful visualizations of the nanoscale are selected , and which then may become linked to more general issues and foreground nanotechnology developments and science in general , and , importantly , the beauty of the nanoscale . such images might then stand for nanotechnology . the image of the nanoflower , for example , is a nanosculpture , it was created in a laboratory , and its colouring was intentionally chosen to enhance its appeal to wider audiences.8 it was indeed used as a general illustration in texts explaining nanotechnology . how can images of nanotechnology become iconic , in the sense of standing for nanotechnology and its further development ? there are two parts to this question about the process and what is involved . the overall process is their reception and further circulation , where the message that is conveyed also plays a role . at some moment , the image has so much standing that it can stand for nanotechnology in its own right . secondly , there are struggles between actors with an interest in which image will eventually come to stand for nanotechnology . in a sense , whether an image becomes iconic or not is the outcome of such struggles . on the basis of these two processes 2 ) , the nanogear ( see fig . 5 ) and the ibm - nanologo ( see fig . 6).9fig . 5the nanogear image . the nanogear image features a complex molecular structure which is envisioned to be used to build products from the bottom - up . this structure is designed through molecular construction software developed by nanorex . it stands for the idea that 1 day atoms could be used as the ultimate building blocks . source : http://nanoengineer-1.com/content/index.php?option = com_content&task = view&id = 52&itemid = 62fig . the ibm logo was created at the nanoscale by moving 35 xenon atoms on a nickel surface . this image showed that atoms could not only be visualized , but also be moved through the use of an stm . the nanogear image features a complex molecular structure which is envisioned to be used to build products from the bottom - up . this structure is designed through molecular construction software developed by nanorex . it stands for the idea that 1 day atoms could be used as the ultimate building blocks . source : http://nanoengineer-1.com/content/index.php?option = com_content&task = view&id = 52&itemid = 62 ibm nano - logo . the ibm logo was created at the nanoscale by moving 35 xenon atoms on a nickel surface . this image showed that atoms could not only be visualized , but also be moved through the use of an stm . eigler and schweizer in the case of the nanolouse image , we have traced its circulation and reception ( see ) . after winning the science visions award in 2002 , the nanolouse image draws on , and reinforces , ideas about magic bullets redressing our sorrows . while contested within science that it might create wrong ideas about nanotechnology when presented to lay audiences ( cf . ) it was actually nanoscientists who continued to use the nanolouse image in their presentations , making it available on their websites . there is a reversal in its use : first , the nanolouse was accompanied by text explaining what the image depicted , but later , such explanations were not needed anymore ; the image could speak for itself and came to stand for the future of nanomedicine . in the case of the nanogear image there was a general appreciation of the image in the 1990s , and a widespread uptake which made it iconic according to our definition . but with the downturn in the standing of the drexlerian vision , it is not generally accepted anymore as standing for nanotechnology . the nanogear image builds on ideas that atoms can be used to engineer products from the bottom - up , and is used as standing for molecular manufacturing , the drexlerian vision . the image was designed by the foresight ( nanotech ) institute to carry that message , and was used almost as a logo . when the vision of molecular manufacturing lost its general standing in the early / mid 2000s , at least within the nanotechnology establishment , the use of the nanogear image as standing for nanotechnology became contested . but it continued to perform outside the immediate world of nanotechnology , up to appearing in official publications and brochures of research institutes wanting to show they were into nanotechnology as well.10 our third iconic image , the ibm nanologo ( see fig . [ it ] became visually compelling evidence of the human capacity to manipulate the world atom - by - atom and to build various molecular devices in the future (: 54 ; cf . ) . the ibm logo seems to suggest progression toward the full potential of nanotech (: 272 ) , and stimulated imaginations of the discovery and conquest of new spaces . writing of the ibm logo with the use of the american stars and stripes flag to mark territory.11 other images produced by ibm almaden like the elliptical corral of cobalt atoms on a copper substrate , with its judicious use of colours to create an aesthetically pleasing image ( fig . 7 ) , were used as illustrations , but never became iconic . fig . ring of atoms are shown in light shade of green , in which the ripples ( colored in blue ) are wave patterns of some of the electrons trapped in the corral . ring of atoms are shown in light shade of green , in which the ripples ( colored in blue ) are wave patterns of some of the electrons trapped in the corral . source : http://www.almaden.ibm.com/vis/stm/images/stm_small.gif a large variety of images of nanotechnology circulates inside as well as outside the world of nanotechnology , but most of them do not become iconic . that depends on their reception and if and how they take on a life of their own , including the struggles linked to their being the three images that did become iconic all depict actual or imagined technoscientific objects , and are widely seen as representing technoscientific achievements , even when they only offer a promise . the entanglement of imaging and imagining occurs all the time , and it can stabilize into specific patterns and established rules . this is very clear in practices of visualizing the nanoscale . while imaging is what these practices are about , imagining is an integral part , both in expectations about how the nanoscale could / should look like , and in the highlighting of the images , e.g. with colours , to enhance their visual appeal within the world of nanotechnology and for wider audiences . how to do this is broadly accepted , even if there are some debates . in concrete situations , there will always be choices to be made which require judgment rather than following a stabilized rule . incorporating the future in the present occurs in various ways , already in how images are used to visualize a design that might / should be realized . while there are debates about the plausibility of the futures that are imagined , there is widespread acceptance of such imagining , e.g. in the use of the nanolouse image . the cross - traffic between the world of nanotechnology and the wider world is premised on promises , and images embodying such promises are an integral part of this cross - traffic . while there is interest from both sides , there is little stabilization ( and perhaps there should nt be ) . seen from the world of nanotechnology , art is peripheral , so there is no pressure to stabilize the entanglements . this is different from the situation of visualization of the nanoscale , where the entanglement of imaging and imagining is addressed daily , and stabilization is necessary to work productively . for the entanglement of the present and the future , there is some pressure to stabilize , but also opportunism : if images like the nanolouse appear to work , they are used even if they could be ( and had been ) criticized . there are tensions in the entanglements and their stabilization . already within scientific practices , struggles become clearly visible when images are used to reach out to broader audiences . because the future and the present are entangled in nanotechnology and its images , there are always possibilities to emphasize one side over the other , in terms of simple actor s categories of the example of the nanolouse image shows that its fictional character is often downplayed in order to profit from the associations ( like magic bullet ) elicited by the image . this went as far as the image being featured in the documents of the european technology platform nanomedicine . at some moment , there may be a backlash , however , when actors in the world of nanomedicine get frustrated by the expectations that were induced by the wide use of the nanolouse image . for nano and art , there may be tensions between nano - actors thinking in terms of aesthetics , while artists focus on creating a new reality.12 there are no antagonisms , because the worlds of artists and of nanoscientists are far apart , and the interactions are seen merely as intriguing curiosities . there is a gray area where graphic designers produce images , and nanoscientists like chris ewels dabble in graphic design . at the moment , there is space for all of these ventures . the umbrella term this will change when images ( of whatever provenance ) might come to stand for nanotechnology as a whole.13 then there is something at stake : nanotechnology is not something given it lives on promises . so , the struggle will not be about what nanotechnology is now ( even if that is one element ) , but about what nanotechnology could be and should be . these struggles occur in a variety of arenas , like funding programs , national science and technology priority setting , and eligibility of who is allowed to speak for nanotechnology . images play a role as resources in these struggles , but also as agents in their own right when they have a life of their own . not as an active force , but as an affordance or a repertoire which will shape what happens without being determinant . it is the entangled imag(in)ing which creates such affordances and repertoires , not the force of images as such .

images , ranging from visualizations of the nanoscale to future visions , abound within and beyond the world of nanotechnology . rather than the contrast between imaging , i.e. creating images that are understood as offering a view on what is out there , and imagining , i.e. creating images offering impressions of how the nanoscale could look like and images presenting visions of worlds that might be realized , it is the entanglement between imaging and imagining which is the key to understanding what images do . three main arenas of entanglement of imag(in)ing and the tensions involved are discussed : production practices and use of visualizations of the nanoscale ; imag(in)ing the future and the present ; and entanglements of nanoscience and art . in these three arenas one sees struggles about which images might stand for nanotechnology , but also some stabilization of the entanglement of imag(in)ing , for example in established rules in the practices of visualizing the nanoscale . three images have become iconic , through the combination of their wide reception and further circulation . all three , the ibm logo , the foresight institute s nanogear image , and the so - called nanolouse , depict actual or imagined technoscientific objects and are thus seen as representing technoscientific achievements while marking out territory .

Introduction Imag(in)ing the Invisible Nanoscale Practices of Entangling the Future and the Present Entanglements of Nanoscience and Arts Images Standing for Nanotechnology In Conclusion

neuromyelitis optica ( nmo ; formerly known as devic disease ) is an inflammatory demyelinating disease of the central nervous system which is characterized by severe relapsing episodes of transverse myelitis and optic neuritis . in the early stages , other parts of the central nervous system are spared , but we now know that later in the course of the disease other parts of the brain can be involved . for a long time , nmo was considered as a specific variant of multiple sclerosis , but it can now be distinguished as a different disease entity by specific mri findings and the detection of anti - aquaporin 4 antibodies ( anti - aqp4 ) or igg serum antibodies against the myelin oligodendrocyte glycoprotein [ 1 , 2 ] . anti - aqp4 antibodies are found in patients with classic nmo , asian opticospinal multiple sclerosis , isolated longitudinally extensive transverse myelitis , isolated optic neuritis , and , in rare cases , isolated brainstem encephalitis . anti - aqp4 antibodies are not detectable in multiple sclerosis and have a 76% sensitivity and 9499% specificity for a nmo spectrum disorder ( nmosd ) . a 28-year - old woman was admitted to our hospital with short - term memory problems , confusion , incoherent and delusional thinking , and self - neglect since 2 weeks . there was also loss of appetite and she had lost 7 kg of weight in the last 2 months . she was of mixed european / african ethnicity , and her previous medical history mentioned an attention deficit hyperactivity disorder for which she used methylphenidate 10 mg daily . her psychiatric history showed a short period of paranoid features when she was 20 years old , possibly induced by overuse of methylphenidate . on physical examination there was a body temperature of 34.8c , an rr of 95/57 mm hg , and a pulse rate of 80 beats / min . she had symptoms of bradyphrenia , bradykinesia , and slowed , halting speech and was disorientated in time and place . laboratory testing showed a leukocytosis of 27.7 10/l , a c - reactive protein level of 2.1 mg / l , and a mild hypernatremia of 148 mmol / l . she was admitted to our neurology ward , but after 2 days she was transferred to our medical psychiatric ward because of progressive confused behavior . the psychiatrist suspected a first psychotic episode with catatonic features ; methylphenidate was discontinued and she was treated with lorazepam 0.5 mg twice a day . an antipsychotic drug was not yet started , because a somatic cause of her behavior had not been ruled out completely . she then developed hypothermia ( 32.6c ) and hypercapnia and was therefore transferred to the intensive care unit because of a suspected autonomic dysregulation . on the intensive care unit , a brain mri showed an abnormal hyperintense signal on t2-weighted images and fluid - attenuated inversion recovery images of the hypothalamus ( fig . 1 ) and , to a lesser extent , of the right optic nerve , with enhancement after gadolinium . a cerebrospinal fluid examination revealed a mild pleocytosis of 60 cells/l , a normal protein level of 0.45 g / l , and a normal glucose level of 3.6 mmol / l . the differential diagnosis at that moment included hypothalamic encephalitis either caused by an autoimmune disorder or of viral origin , a systemic autoimmune disease , or a paraneoplastic disorder . a ct scan of the thorax and abdomen showed no signs of sarcoidosis or an underlying malignancy . anti - dsdna and anti - sm antibodies were absent , ruling out systemic lupus erythematosus . anti - sjgren syndrome - related antigen b ( anti - ssb ) antibodies were positive , but in combination with a negative test for anti - ssa and normal findings at a lip biopsy , sjgren syndrome was considered highly unlikely . the patient was treated with intravenous methylprednisolone pulse therapy for 3 days , at 1,000 mg per day , which resulted in resolution of the hypothermia and hypercapnia . considering all the normal results on testing and the prompt reaction to steroids , an nmosd was considered and was confirmed by positive tests for serum anti - aqp4 antibody . after the 3-day course of intravenous methylprednisolone , we continued treatment with oral prednisolone at 30 mg twice a day and azathioprine at 50 mg twice a day . because she developed apraxia and hypomanic features such as hypersexuality and overeating in combination with the preexisting cognitive and behavioral problems , she was retransferred to the medical psychiatric ward and treated with haloperidol at 2.5 mg and with lorazepam . an ophthalmologist confirmed vision of 0.5/200 and 20/25 , respectively , in the right and the left eye . on treatment with haloperidol and lorazepam , the hypomanic symptoms and after 6 weeks , she was discharged to a rehabilitation center with severe memory and visual problems . oral prednisolone was continued in a 22-month tapering schedule ; azathioprine was continued at 50 mg twice a day . after a couple of weeks in rehabilitation , she was again transferred to a psychiatric unit with a compulsive eating disorder and aggressive behavior probably due to a combination of treatment with prednisolone and a brain injury caused by the initial brain lesions . a repeat mri scan 3 months later showed almost complete recovery of the hypothalamic lesions , and there were no signs of new disease activity . our patient presented with bilateral hypothalamic lesions and concomitant optic neuritis as a first episode of an nmosd . there are only a few cases describing patients with hypothalamic lesions as an initial symptom , all of them presenting with excessive daytime sleepiness as a first symptom . our patient presented with cognitive impairment , autonomic failure , and symptoms of psychosis , but no sleep disturbances . most of the nmosd patients were young women between 20 and 45 years of age [ 7 , 8 , 9 , 10 , 11 ] . remarkably , all patients were of asian ethnicity , whereas our patient was of mixed caucasian / negroid origin . it is well described that nmo is more common in nonwhite populations , in contrast to the low incidence of multiple sclerosis in nonwhite persons . all patients were treated with intravenous methylprednisolone , some followed by oral prednisolone , with variable outcomes : no symptoms at follow - up , relapses of sleepiness , and memory disturbances , as well as symptoms matching new brain or spinal cord lesions were reported [ 7 , 8 , 9 , 10 , 11 ] . whereas in multiple sclerosis , spontaneous remission may have an equal effect to high - dose immunosuppressive medication on the long - term neurological outcome , in nmosd repetitive episodes will lead to severe damage , and the current consensus is to treat every relapse to prevent severe long - term damage . the recommended treatment for an acute episode of nmo is high - dose methylprednisolone intravenously for 5 days followed by an oral steroid taper for 28 weeks depending on the severity of the attack . if high - dose corticosteroids have no or only a minimal effect , a plasma exchange is considered to be effective . white matter brain lesions are reported in up to 5085% of patients with nmosd , and 4370% of these brain mri abnormalities are reported at onset . usually , the lesions are asymptomatic and most often located in areas with high aqp4 expression , such as the hypothalamus , but also subcortical and deep white matter lesions are common [ 1 , 12 ] . a recent study by gao et al . investigated the presence of hypothalamic lesions in inflammatory diseases including nmosd , multiple sclerosis , and acute disseminated encephalomyelitis , as well as in lupus , sarcoidosis , behet disease , clippers ( chronic lymphocytic inflammation with pontocerebellar perivascular enhancement responsive to steroids ) , and hypertrophic pachymeningitis in all , 172 of the 429 study subjects were aqp4 positive and met the criteria for nmosd defined by wingerchuk et al . ( which have recently been updated ) . in 42 subjects ( 24.4% ) with nmosd there were hypothalamic lesions on t2-weighed mr images , of which 90% were bilateral ; 10 out of 257 apq4-negative subjects had hypothalamic lesions . nmosd is associated with other autoimmune disorders , especially connective tissue disorders like sjgren syndrome and systemic lupus erythematosus . the exact relationship between nmosd and other autoimmune disorders is not quite fully established , but there might be a link between aqp4 seropositivity and other connective tissue autoimmune disorders [ 1 , 15 ] . our patient had a positive anti - ssb serologic test , but a lip biopsy showed no signs of a coexisting sjgren syndrome , and also additional tests for systemic lupus erythematosus were negative , which is why we treated her as a patient with an nmosd . physicians should consider performing an mri and lumbar puncture in young women presenting with hypothalamic symptoms or atypical psychosis . early treatment with high - dose methylprednisolone is recommended to prevent severe long - term damage . integrated neurological and psychiatric care for these patients is recommended .

we present the unusual case of a patient with an aquaporin 4 antibody - seropositive neuromyelitis optica spectrum disorder who presented with autonomic dysregulation , cognitive impairment , and symptoms of psychosis . only a few previous cases have been described with similar psychiatric symptoms . brain mri showed an abnormal hyperintense t2 signal of the hypothalamus and , to a lesser extent , a minor hyperintense signal of the right optic nerve . her symptoms and mr abnormalities improved after high - dose methylprednisolone .

Background Case Presentation Discussion Statement of Ethics Disclosure Statement

until recently , primary headaches such as migraine and cluster headache ( ch ) were described as vasomotor headaches . initial positron emission tomography ( pet ) images shed light on the origins of these syndromes , documenting activation in the mesencephalic and pons regions for migraine , and in the grey hypothalamus for ch and others of trigeminal autonomic cephalalgias ( tacs ) [ 24 ] . tacs include ch , paroxysmal headache and short - lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing ( sunct syndrome ) . it is interesting to note that the common clinical aspects of this primary headaches , where posterior hypothalamic activation has been reported , concern the important cranial autonomic symptoms . the concept of tacs indicates a possible common physiopathological base for these syndromes , not shared with other primary headaches , such as migraine or tension - type headache . pet and functional magnetic resonance imaging ( f - mri ) may be regarded as of little or no importance within a clinical context , but they offer great potential for the exploration of headache physiopathology and the effects of pharmacological treatment . in recent years , f - mri studies have been conducted in tacs , but to date , no studies dealing with ch are available . in the present study we have investigated the cerebral activation centre during a ch attack in four patients with episodic ch , using f - mri . we enroled four patients ( 45 4 years ) diagnosed with episodic ch in accordance with the international headache society ( ihs ) classification ( code 3.1.1 ) . all patients were in the active phase of the illness , and had an average frequency of two episodes a day . in three patients , one of the two attacks had a very regular recurrence ( 2 p.m. ) , while in the other patient it happened at night ( 11 p.m. ) . the average headache duration , without recourse to treatment , was 110 12 min . the patients reported no cardiovascular ( hypertension and/or ischaemic cardiopathy ) , cerebrovascular risk factors , or internal pathological symptoms . a prerequisite for patient enrolment , established on the basis of medical history , was a dramatic response to subcutaneous sumatriptan administration ( 6 mg ) . the functional study of each individual patient took place in a single continuative session , without interruptions . a priori , the duration of the functional examination was established ( the same amount of time for each patient ) to be long enough to allow registration of the asymptomatic state , the onset of pain and the pain relief - state ( provided by administration of drugs ) . the patients in the study presented attacks at around the same time each day , and this allowed us to place them in the scanner , 15 min before the onset . when pain occurred they should communicate the onset by pressing buttons on a nonmagnetic button - box . after 5 min , in order to avoid movements of the patients due to the pain and to induce the state of pain - relief , sumatriptan was subcutaneously injected . functional datasets were obtained by means of a t2 * weighted gradient echo - epi pulse sequence ( tr / te / fa 3,000 ms/60 ms/30 , tr delay 20 ms , fov 20 cm , matrix 64 64 , voxel 3.125 3.125 5 mm , gap 1.25 mm ) . we acquired 20 oblique axial slices , parallel to the anterior - posterior commissure plane , extending from the foramen magnum to the vertex . anatomical datasets were obtained using a t1 mp - rage 3d pulse sequence ( tr / ti / te / fa 2,160 ms/1,100 ms/3.93 ms/10 , voxel 1 1 1.4 mm , gap 0.7 mm ) . first , functional data was processed in order to increase the snr ratio and reduce artefacts . we performed slice scan time correction and 3d motion correction by means of rigid body transformations ( the estimated displacement in each direction was less than 3 voxels in each subject ) and spatial smoothing with a gaussian kernel of 4 mm fwhm ( full - width - half - maximum ) . anatomical data was re - sampled to obtain isometric voxels of 1 1 1 mm then normalised to the talairach and tournaux standard space to allow intersession statistical analysis . functional and anatomical datasets were co - registered by translation , rotation and zoom operations on the x , y and z axis . condition specific effects ( pain vs. no pain ) were estimated using a boxcar approach connected with the hemodynamic response function . processed functional data obtained from the patients underwent single - subject and multi - subject general linear model ( gml ) analysis . a statistical map was generated as t - contrast with a threshold value of p < 0.001 . the functional study of each individual patient took place in a single continuative session , without interruptions . a priori , the duration of the functional examination was established ( the same amount of time for each patient ) to be long enough to allow registration of the asymptomatic state , the onset of pain and the pain relief - state ( provided by administration of drugs ) . the patients in the study presented attacks at around the same time each day , and this allowed us to place them in the scanner , 15 min before the onset . when pain occurred they should communicate the onset by pressing buttons on a nonmagnetic button - box . after 5 min , in order to avoid movements of the patients due to the pain and to induce the state of pain - relief , sumatriptan was subcutaneously injected . functional datasets were obtained by means of a t2 * weighted gradient echo - epi pulse sequence ( tr / te / fa 3,000 ms/60 ms/30 , tr delay 20 ms , fov 20 cm , matrix 64 64 , voxel 3.125 3.125 5 mm , gap 1.25 mm ) . we acquired 20 oblique axial slices , parallel to the anterior - posterior commissure plane , extending from the foramen magnum to the vertex . anatomical datasets were obtained using a t1 mp - rage 3d pulse sequence ( tr / ti / te / fa 2,160 ms/1,100 ms/3.93 ms/10 , voxel 1 1 1.4 mm , gap 0.7 mm ) . first , functional data was processed in order to increase the snr ratio and reduce artefacts . we performed slice scan time correction and 3d motion correction by means of rigid body transformations ( the estimated displacement in each direction was less than 3 voxels in each subject ) and spatial smoothing with a gaussian kernel of 4 mm fwhm ( full - width - half - maximum ) . anatomical data was re - sampled to obtain isometric voxels of 1 1 1 mm then normalised to the talairach and tournaux standard space to allow intersession statistical analysis . functional and anatomical datasets were co - registered by translation , rotation and zoom operations on the x , y and z axis . condition specific effects ( pain vs. no pain ) were estimated using a boxcar approach connected with the hemodynamic response function . processed functional data obtained from the patients underwent single - subject and multi - subject general linear model ( gml ) analysis . a statistical map was generated as t - contrast with a threshold value of p < 0.001 . during execution of functional mri ( three afternoon sessions and one night session ) all the patients suffered from ch attacks , and the administration of sumatriptan brought pain relief after 4 1 min ( average values and standard deviation ) . in all attacks ( single - subject gml study ) , comparing the pain state with the pain - free state , we detected significant cerebral activation ( p < 0.05 , corrected for multiple comparisons ) in the ipsilateral hypothalamic grey matter ( fig . 1 ) . the coordinates and the z - score of the activation zones figure 2 shows the temporal course of the bold signals in the hypothalamus ipsilaterally to the pain - side in patient 2 . additionally , with a less conservative threshold of p < 0.001 uncorrected , trends of activation were observed in the pre - frontal cortex , anterior cingulate cortex , contralateral thalamus , ipsilateral basal ganglia and bilaterally in the insula and the cerebellar hemispheres . however , these areas were not subject to more detailed analysis because the study was not designed to investigate the activation of the pain matrix ( stereotactic co - ordinates were not shown).fig . 1multi - subject gml analysis shows cerebral activation area in the posterior grey hypothalamus , ipsilateral to the pain side ( p < 0.001 uncorrected for descriptive purposes ) , superimposed on a t1-weighted anatomical reference mr image in axial ( a ) and coronal ( b ) planestable 1bold - signal increases during spontaneous ch attack compared with the pain - free state ( p < 0.05 , corrected for multiple comparisons)talairach coordinatesz - score p < 0.05 ( corrected)xyzpatient 161014.89patient 23814.83patient 35644.91patient 44724.32multi - subject study5816.93fig . 2temporal course of the bold signals in the hypothalamus ipsilaterally to the pain - side in patient 2 . the number of the 400 ge - epi acquisitions , is indicated multi - subject gml analysis shows cerebral activation area in the posterior grey hypothalamus , ipsilateral to the pain side ( p < 0.001 uncorrected for descriptive purposes ) , superimposed on a t1-weighted anatomical reference mr image in axial ( a ) and coronal ( b ) planes bold - signal increases during spontaneous ch attack compared with the pain - free state ( p < 0.05 , corrected for multiple comparisons ) temporal course of the bold signals in the hypothalamus ipsilaterally to the pain - side in patient 2 . this study documents the activation of cerebral areas normally attributed to the analysis of painful stimuli ( pre - frontal cortex , basal ganglia , thalamus , cingulate cortex , insula and cerebellum ) as reported in a comprehensive review and of the hypothalamic region ipsilaterally to the pain - side as documented in previous functional studies carried out using pet methods in ch . to date , f - mri has only been used in the study of patients affected by other tacs . previously , in short - lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing , f - mri studies have shown both hypothalamic activation ipsilateral to the pain side and bilateral activation of the hypothalamus . in one case of atypical tac , activation of the hypothalamus , ipsilateral to the pain side , has been reported using f - mri . overall , we have demonstrated the anatomical location of central nervous system activation with the first f - mri study during a ch attack . the diencephalon - hypothalamic region seems to be an activation area specific to ch , and for this reason , the possible role of attack generator centres has been suggested . as suggested by clinical , biohumoral and electrophysiological data , there is great need to investigate ch and other related idiopathic headache syndromes using f - mri which offers a good balance of spatial and temporal resolution . the f - mri presents major advantages in comparison with the pet : it does not use radioisotope administrative tracers ( intravenously ) , but endogenous tracers ( deoxy - haemoglobin ) . furthermore f - mri allows to effect anatomical and functional acquisitions in the same recording session . this study method seems appropriate for investigation of pathogenetic aspects in the primary headaches , particularly for analysis of functional data immediately prior to attack onset .

we have investigated the cerebral activation centre in four patients with episodic cluster headache ( ch ) with functional magnetic resonance imaging ( f - mri ) . the patients underwent mri scans for anatomical and functional data acquisition in the asymptomatic state , during a headache attack and after subcutaneous administration of sumatriptan . anatomical images were acquired by means of 3d - mprage sequences and f - mri images were obtained by means of echo - planar imaging . data was analysed using the brainvoyager qx version 1.7.81 software package . in all patients , the data showed significant hypothalamic activation of the hypothalamus ipsilateral to the pain side , attributable to a headache attack . overall , we have demonstrated the anatomical location of central nervous system activation by means the first f - mri study in ch patients . f - mri offers a good balance of spatial and temporal resolution , and this method of study appears appropriate for investigating the pathogenetic aspects of primary headaches . positron emission tomography and f - mri may be regarded as little or no importance in a clinical context , they do , however , offer great potential for the exploration of headache physiopathology and the effects of pharmacological treatment .

Introduction Materials and methods Study design Data acquisition Pre-processing of MRI data Results Discussion

parental obesity increases the risk of obesity in offspring through genetic , biological , and environmental influences [ 13 ] . maternal obesity , weight gain , and increased body mass index ( bmi ) between pregnancies and gestational diabetes increase risks of offspring obesity , type 2 diabetes mellitus ( t2 dm ) , and cardiovascular disease ( cvd ) in the offspring [ 47 ] . consistent with the chronic low - grade inflammatory state of obesity , elevated concentrations of interleukin-6 ( il-6 ) and c - reactive protein ( crp ) have been observed in obese pregnant women . higher maternal levels of il-6 were predictive of the increased growth and adiposity in the offspring . maternal diet - induced obesity in sheep resulted in higher fetal triglyceride ( tg ) levels and upregulated inflammatory signaling . offspring of rats injected with il-6 throughout pregnancy had an altered inflammatory profile associated with greater body fat mass and reduced insulin sensitivity . epigenetic processes are important mediators of early - life environment , metabolism , and body composition in offspring . epigenetic changes in genes involved in the inflammatory response have been observed in response to maternal diet and adiposity . differences in methylation levels of the peroxisome proliferator - activated receptor alpha ( ppara ) and glucocorticoid receptor promoters have been observed in the liver of offspring of rats fed with protein - restricted diet during pregnancy [ 13 , 14 ] . methylation status of retinoid x receptor alpha ( rxra ) promoter in umbilical cord tissue dna was correlated with increased adiposity in children of mothers with lower carbohydrate intake . epigenetic mechanisms have been involved in inflammation and associated disorders including obesity [ 15 , 16 ] . bariatric surgery is known to improve glucose and lipid metabolism [ 1719 ] and prevent arterial hypertension and type 2 diabetes [ 1921 ] . bariatric surgery also decreases the low - grade inflammation associated with obesity . in previous studies , we demonstrated that children born after maternal bariatric surgery ( ams ) exhibited lower prevalence of severe obesity , greater insulin sensitivity , and improved lipid profile in comparison to siblings born before maternal surgery ( bms ) [ 23 , 24 ] . in addition , ams children demonstrated differences in a marker of chronic low - grade inflammation , namely the plasma c - reactive protein ( crp ) . this study was designed to analyze the relationship between maternal biliopancreatic diversion surgery and methylation levels of genes involved in immunologic and inflammatory pathways in bms and ams offspring . correlation analyses between gene methylation , expression , and plasma crp levels were conducted for the il-8 pathway . women from qubec city and surrounding areas ( administrative regions of capitale - nationale , mauricie , and chaudire - appalaches ) who had given birth before and after biliopancreatic diversion with duodenal switch for severe obesity were eligible for the current study . a subset of 20 unrelated mothers was recruited along with 50 siblings born before and after surgery ( 25 bms and 25 ams offspring ) . mothers and offspring attended the qubec heart and lung institute ( qubec city , qubec , canada ) or a regional hospital for assessment between july and october 2010 . there were 17 mothers with siblings born before and after surgery ( 23 bms and 24 ams ) , two with bms offspring only ( 2 bms ) , and one mother with only one ams offspring . this study was approved by the qubec heart and lung institute ethics committee . written informed consent was obtained from adults and mothers with assents from minors . percent body fat was determined for individuals at 6 years or more ( bms , n = 23 ; ams , n = 17 ) using bioelectric impedance analysis ( tanita , arlington heights , il , usa ) . weight , height , and resting systolic ( sbp ) and diastolic ( dbp ) blood pressure were obtained using standardized procedures and measured at the interview . bmi was calculated for mothers , and bmi percentile for children was obtained from the national health and nutrition examination survey 2000 charts . bmi z - score was calculated for children using charts from the centers for disease control and prevention . severe obesity in offspring was defined using age- and sex - corrected adiposity measurements ( bmi z - score > 3 ; bmi percentile > 98% ) . blood samples were collected from mothers and offspring after an overnight fast into bd vacutainer tubes containing edta and paxgene blood rna collection tubes ( qiagen , valencia , ca , usa ) . plasma tg , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , total cholesterol ( total - c ) , total - c / hdl - c ratio , glucose , and insulin concentrations were measured as previously described . the homeostatic model of insulin resistance ( homa - ir ) index was calculated as glucose insulin/22.5 . genomic dna was isolated from the blood buffy coat using the genelute blood genomic dna kit ( sigma , st . louis , mo , usa ) and quantified using both nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) and picogreen dna methods . dna ( 1 g ) was bisulfite converted , and quantitative genome - wide methylation analysis was conducted using the infinium humanmethylation450 beadchip ( illumina , san diego , ca , usa ) . arrays were processed at the mcgill university and gnome qubec innovation centre ( montral , canada ) according to the manufacturer 's instructions ( illumina , san diego , ca , usa ) . the infinium humanmethylation450 beadchip array was designed for genome - wide methylation analysis with coverage targeted across gene regions with sites in the promoter region , 5utr , first exon , gene body , and 3utr . the beadchip interrogates more than 485 000 methylation sites at single - nucleotide resolution . visualization and analysis of methylation data were conducted using the genomestudio software version 2011.1 ( illumina inc . ) and the methylation module . methylation levels ( beta values ; ) were estimated as the ratio of signal intensity of the methylated alleles to the sum of methylated and unmethylated intensity signals of the alleles ( value = c/(t + c ) ) . internal control probe pairs were used for data correction ( background subtraction and normalization ) , and cpg sites with a detection p value > 0.05 were removed from analysis . differences in methylation levels between bms and ams groups ( mean values ) were tested using illumina custom model . false - discovery - rate - corrected ( fdr - corrected ) p values and diffscores were computed . diffscore is a differential methylation score calculated from p values and differences in ( delta ) between the two groups and was used here as the main statistical results for comparison of bms and ams siblings . differential methylation analysis between bms and ams siblings revealed 5698 genes ( 14 466 probes ) with significant differences in methylation levels ( fdr - corrected diffscore |13 distribution of differentially methylated probes obtained from comparison between bms and ams offspring is provided in supplementary table 1 available online at http://dx.doi.org/10.1155/2013/492170 . total rna was isolated and purified from whole blood for 46 offsprings ( 23 bms and 23 ams ) using paxgene blood rna kit ( qiagen ) following the manufacturer 's recommendations . the integrity of the purified rna was analyzed using both nanodrop ( thermo scientific , wilmington , de , usa ) and 2100 bioanalyzer ( agilent technologies , cedar creek , tx , usa ) . expression levels were measured using the humanht-12 v4 expression beadchip ( illumina inc . ) which contains more than 47 000 probes derived from ncbi refseq release 38 . microarray experiments were carried out using 250 ng of total rna and processed according to the manufacturer 's instructions at the mcgill university and gnome qubec innovation centre ( montral , canada ) . expression data were visualized and analyzed using the flexarray software ( version 1.6 ) , and the lumi algorithm was used for expression data analysis and normalization . probe detection analysis was conducted using the flexarray filter algorithm to generate a list of expressed probes . to be considered as significantly expressed , a probe had to show a detection p value 0.05 in at least 25% of the samples of a group . ( sam ) algorithm with unequal - variance ( welch 's ) t - statistic was used to assess differences between bms and ams siblings for significantly expressed probes . the sam algorithm uses permutations of the data set to generate an empirical null distribution and to assess the significance of observed effects , rather than using the hypothetical t - distribution . in addition , a cutoff of fold change 1.2 or 0.83 ( symmetrical fold change 1.2 or 1.2 ) was used . p values obtained from permutations and fold change cutoff values were then used to minimize the chances of false positives . differential expression analysis revealed a total of 862 probes differentially expressed with a symmetrical fold change |1.2| ( p 0.05 ; fold change < 0.83 or > 1.2 ) . gene expression microarray results were validated using real - time polymerase chain reaction ( rt - pcr ; applied biosystems gene expression assays ; applied biosystems , foster city , ca , usa ) . spearman correlations were computed for expression levels assessed by microarray and rt - pcr methods in a subset of genes ( bcl2 , nm_000633 ; ccnd2 , nm_001759 ; ncf2 , nm_000433 ; pik3r1 , nm_181523 ; prkch , nm_006255 ) for whom correlations between gene expression and methylation levels were observed . samples were analyzed in duplicate using predesigned probes ( hs00608023_m1 , hs00153380_m1 , hs01084940_m1 , hs00933163_m1 and hs00178933_m1 ) and calibrated to the actb housekeeping gene ( endogenous control ; actb : hs99999903_m1 ) . relative quantification estimations were performed on an applied biosystems 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) . analysis of potentially altered pathways was conducted using the knowledge base of the ingenuity pathway analysis ( ipa ) system . the lists of 14 466 differentially methylated and 862 differentially expressed probes produced from differential methylation and differential expression analysis , respectively , were submitted to ipa . ipa measured the likelihood that these genes participate in a particular pathway and calculated p values using a right - tailed fisher 's exact test for each pathway . clinical data were expressed as mean sd . the effect of bariatric surgery in mothers was assessed using a within - subject paired t - test . differences in anthropometric data between bms and ams siblings were tested using analysis of variance ( general linear model , type iii sum of squares ) with adjustments for the effects of sex and puberty . in the absence of tanner scores , we arbitrarily defined puberty as 12 years for girls and 14 for boys . differences in severe obesity between bms and ams siblings were evaluated using bmi percentile and bmi z - score and tested using fisher 's exact test . p values for crp were adjusted for the effects of sex , puberty and bmi percentile , an age- and sex - corrected adiposity measurement . pairwise pearson correlations between methylation , expression , and plasma crp were computed for the il-8 signaling pathway that was overrepresented in both differential methylation and expression analyses . partial pearson correlations were also computed for the il-8 signaling pathway after adjustments for age and sex . statistical analyses were conducted using the sas software version 9.2 ( sas institute inc . ) . a total of 20 mothers ( 41.0 5.3 years ; mean sd ) who had undergone biliopancreatic diversion were recruited . mean postoperative follow up was 12 years and 2 months at the time of the study . bariatric surgery induced dramatic weight loss in mothers . on average , the women weighted 121.5 19.2 kg ( bmi = 45.0 7.2 ) at the time of the surgery and 74.8 11.9 kg ( bmi = 27.6 4.8 ) at the recruitment interview . the mean weight loss of 46.7 16.1 kg was associated with significant improvements in plasma lipids ( p 0.01 for tg , hdl - c , ldl - c , total - c , and total - c / hdl - c ratio ) and reductions in insulin resistance ( homa - ir index ; p 0.001 ) and blood pressure ( sbp and dbp ; p 0.001 ) with a trend toward lower plasma glucose levels ( p = 0.06 ) . the 50 bms and ams offspring of 20 mothers were aged between 2 years and 8 months and 24 years and 11 months with similar sex distribution ( 40% males in both bms and ams ) . bms offspring were born 3 years and 5 months before and ams offspring were born 3 years and 7 months after maternal bariatric surgery . bms siblings were older ( mean age 14.9 y versus 9.6 y ) than ams siblings ( table 1 ) . following adjustments for the effects of sex and puberty , ams offspring showed a trend toward lower body fat percent ( p = 0.07 ) . there was a significantly lower prevalence of severe obesity in ams using bmi percentile ( p = 0.01 ) or bmi z - score ( p = 0.05 ) as indicators . ams offspring had improved fasting insulin levels ( p = 0.03 ) , lower homa - ir index ( p = 0.03 ) , and blood pressure . plasma crp levels were different in bms versus ams offspring ( 5.14 7.90 versus 3.58 11.09 ; p = 0.03 ) even after taking into account the sex effect ( p = 0.03 ) . these differences in plasma crp levels were no longer significant after further adjustments for the effects of puberty , and bmi ( bmi percentile ) . from the list of 5698 differentially methylated genes between bms and ams offspring , ipa revealed 160 pathways significantly overrepresented ( p < 0.05 ) . similar analysis conducted from the list of 862 differentially expressed genes between bms and ams offspring identified 68 overrepresented pathways . the list of the top 20 overrepresented inflammatory and immune pathways identified from differential methylation analysis along with corresponding results obtained from gene expression analysis is presented in table 2 . among them , 5 pathways were found to be overrepresented in both methylation and expression : il-8 signaling , icos - icosl signaling in t - helper cells , role of nfat in regulation of the immune response , b - cell receptor signaling , and glucocorticoid receptor signaling . among the 5 pathways listed above it was thus selected for further analyses to highlight the potential link between gene methylation , expression , and inflammation . among the 193 genes assigned to the il-8 signaling pathway by ipa , 70 genes represented by 214 methylation probes were found to be differentially methylated in bms versus ams ( supplementary table 2 ) . corresponding expression data were available for 102 of these probes ( 46 genes ) . among the 46 genes analyzed , correlations between dna methylation and expression were significant for 17 genes ( 23 methylation probes ; table 3 ) . further analyses were then focused on these 17 genes to correlate gene methylation and expression with crp levels . there were significant correlations between gene methylation and plasma crp levels for 16 genes . gene expression levels of 5 genes were found to correlate with plasma crp levels ( table 3 ) . all 5 genes with significant correlations between gene expression and plasma crp levels ( bcl2 , ccnd2 , ncf2 , pik3r1 , and prkch ) also demonstrated correlations between methylation and expression and between gene methylation and plasma crp levels ( table 3 ) . following adjustments for the effects of age and sex , all 17 genes initially showing correlation between gene methylation and expression levels demonstrated significant correlation after adjustments . in addition , the rhoh gene ( methylation probe cg26163153 ) reached significance level while initially showing a trend toward significant correlation ( p = 0.09 ) . from the 16 genes correlated with crp levels only one ( pik3r1 ) of the five genes with significant correlations between gene methylation , expression , and crp levels remained significant after adjustments , while trends toward significant correlation were found for bcl2 , ccnd2 , and ncf2 . validation of gene expression microarray data for these 5 genes demonstrated significant correlations between microarray and rt - pcr results for 4 of the genes assessed ( ccnd2 , r = 0.358 , p = 0.02 ; ncf2 , r = 0.574 , p < 0.0001 ; pik3r1 , r = 0.349 , p = 0.02 ; prkch , r = 0.462 , p = 0.001 ) , while bcl2 did not reach significance ( r = 0.223 , p = 0.14 ) . we demonstrated here for the first time that surgical treatment of severe maternal obesity results in sustained differences in the methylome and transcriptome of genes involved in inflammatory pathways in ams offspring compared to bms siblings . the proportion of 34.7% of differentially methylated genes ( 1976 of 5698 genes ) being identified by two probes or more strengthen , the validity of our results arguing for a regulation of methylation levels in offspring . differential analysis of gene methylation and expression levels revealed important differences between the sibling groups . pathway analysis from differentially methylated genes in bms versus ams revealed important regulation of inflammatory and immunity pathways : 29 inflammatory- or immunity - related pathways being identified among the top 50 overrepresented pathways ( data not shown ) . pathway analysis from differentially expressed genes between bms and ams revealed 5 overrepresented pathways among the top overrepresented pathways from differential methylation data ( table 2 ) . overrepresentation of genes from the il-8 signaling pathway was identified from both methylation and expression data , and significant correlations were found between gene methylation and expression levels . moreover , 5 genes from the il-8 pathway also demonstrated correlation of gene methylation and expression levels with plasma crp levels . despite the fact that the impact of dna methylation on gene expression seems to be site and location dependent , results obtained here for the il-8 pathway generally comply to the known phenomenon that intragenic methylation ( gene body , 5utr and 3utr ) correlates with increased gene expression , while promoter methylation is associated with decreased expression [ 30 , 31 ] . gene expression profiling in obese patients who had undergone weight loss or bariatric surgery demonstrated a regulation of inflammation - related transcripts and changes in inflammatory marker levels [ 32 , 33 ] . in the present study , we extend these data by reporting that metabolic improvements in mothers following biliopancreatic diversion surgery are reflected in gene methylation and expression levels of immunologic and inflammatory genes in offspring , indeed reinforcing the involvement of epigenetic differences in immunity and inflammatory genes in the determination of the offspring phenotypes . a limited number of epigenetic studies have evaluated the impact of maternal nutrition and obesity on methylation of immune and inflammatory genes in offspring . recently , human studies have revealed the implication of immune and inflammatory gene methylation in children with respect to adiposity . methylation levels of the rxra gene in umbilical cord tissue dna of healthy neonates were associated with maternal carbohydrate intake in early pregnancy and childhood adiposity at 9 years old . relton and collaborators reported associations between anthropometric indices ( bmi , fat mass , lean mass , and height ) in 9 years old children and methylation levels of selected immune and inflammatory genes in cord blood samples . with a different design involving siblings born under different maternal obesity condition , our study supports the role of epigenetic factors in immunity and inflammatory genes in the determination of offspring phenotypes . transcriptomic data revealed that improvements seen in mothers following bariatric surgery are reflected in gene expression levels of immunologic and inflammatory genes in offspring . animal studies have demonstrated a similar impact of maternal obesity and nutrition on immune and inflammatory genes in the offspring [ 3537 ] . these results are consistent with those obtained from the present gene expression analysis in bms offspring versus ams siblings and may provide potential mechanisms for higher levels of systemic inflammation found in bms offspring . we used dna extracted from blood as it is more convenient and acceptable than biopsies of target tissues , especially in children . although epigenetic signatures have been shown to differ between tissues , the overall impact is minor given the known similarities in methylation patterns . gene expression levels are also known to be tissue - or cell - type dependent but also correlated [ 4042 ] thus justifying the use of blood in clinical studies . differential gene expression analysis led to a limited number of differentially expressed genes ( 862 ) in comparison with the 5698 differentially methylated genes identified . this discrepancy may be attributable to differences in statistical algorithms , in corrections for multiple testing , or in the different cutoffs used ; a cutoff fold change 1.2 or 0.83 was applied for gene expression analysis . nonetheless , pathway analyses conducted from differentially methylated and expressed genes are concordant and demonstrate an overrepresentation of immune and inflammatory pathways . we did not stratify for sex in these gene methylation and expression analyses ; several studies did not find sex - specific autosomal methylation patterns [ 43 , 44 ] . owing to the before - after design of the study , we were unable to fully correct for the age difference between the sibling groups . this design and the young age of the ams offspring ( 80% being younger than 12 years old ) do not allow discussing the long - term effects in children . longitudinal studies are thus needed to further assess long - term effects of maternal bariatric surgery in the offspring . age - related differential methylation has been reported but was shown to be site- and location dependent [ 4547 ] and represented a very small proportion of cpg sites . furthermore , longitudinal studies in humans demonstrated relative stability of dna methylation over time . in regards to the general conclusion drawn from our results , the potential impact of maternal weight regain on offspring is limited in the current study considering the sustained weight loss achieved with the biliopancreatic diversion with duodenal switch [ 19 , 48 , 49 ] . analysis of maternal clinical files along with the lack of inflammation - related perioperative complications in biliopancreatic diversion with duodenal switches argues against the potential impact of specific inflammatory condition in mothers . the current study was not designed to analyze methylation and expression changes induced in mothers following bariatric surgery . however , based on a recent meta - analysis demonstrating a reduction in low - grade inflammation associated with obesity following bariatric surgery , it would be tempting to speculate that these changes may be accompanied by similar changes in methylation and expression levels . improvements in inflammatory profiles in mothers following bariatric surgery may be potentially responsible for the inflammatory effects seen in offspring through modification of the in utero environment and improved methylation profiles in ams offspring . using analysis of gene methylation and expression data , we reveal that improvements seen in mothers following bariatric surgery reflect on the expression and methylation levels of genes involved in immune and inflammatory pathways in the offspring . these results argue for a beneficial effect of maternal weight loss before pregnancy and provide potential mechanisms of action for physiological improvements through regulation of methylation and expression of genes involved in inflammatory and immune pathways . since inflammation is recognized as an independent risk factor for cardiovascular diseases ( cvd ) and is also associated with the development of the metabolic syndrome , insulin resistance , hypertension , and obesity [ 50 , 51 ] , it would be important to conduct longitudinal studies to examine the long - term effect of maternal bariatric surgery on offspring .

background . maternal obesity , excess weight gain and overnutrition during pregnancy increase risks of obesity , type 2 diabetes mellitus , and cardiovascular disease in the offspring . maternal biliopancreatic diversion is an effective treatment for severe obesity and is beneficial for offspring born after maternal surgery ( ams ) . these offspring exhibit lower severe obesity prevalence and improved cardiometabolic risk factors including inflammatory marker compared to siblings born before maternal surgery ( bms ) . objective . to assess relationships between maternal bariatric surgery and the methylation / expression of genes involved in the immune and inflammatory pathways . methods . a differential gene methylation analysis was conducted in a sibling cohort of 25 bms and 25 ams offspring from 20 mothers . following differential gene expression analysis ( 23 bms and 23 ams ) , pathway analysis was conducted . correlations between gene methylation / expression and circulating inflammatory markers were computed . results . five immune and inflammatory pathways with significant overrepresentation of both differential gene methylation and expression were identified . in the il-8 pathway , gene methylation correlated with both gene expression and plasma c - reactive protein levels . conclusion . these results suggest that improvements in cardiometabolic risk markers in ams compared to bms offspring may be mediated through differential methylation of genes involved in immune and inflammatory pathways .

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

in order to determine whether the microprocessor is involved in the processing of cellular rnas ( other than mirnas ) that contain hairpin structures , we focused on the identification of endogenous rna targets of dgcr8 . for this , we used the cross - linking immunoprecipitation protocol coupled to high - throughput sequencing ( hits - clip ) . this technique relies on ultraviolet irradiation to induce protein covalent crosslinks in protein - rna complexes in situ . this allows the use of highly stringent immunoprecipitation and washing conditions so that only those rnas directly bound to the protein of interest are selected ( reviewed by ) . we performed two replicate hits - clip experiments on uv - irradiated hek293 t cells . each replicate comprised two independent experiments : one involved immunoprecipitation ( ip ) of endogenous dgcr8 protein ( supplementary fig 1a , left ) whereas the second one involved ip of a transiently transfected epitope - tagged dgcr8 protein ( pcg t7-dgcr8 ) ( supplementary fig 1a , right and 1b ) . we focused our analyses on the second replicate , since it gave higher depth of coverage and better mapping to the genome . 37 million reads for the endogenous dgcr8 protein , and 36 million reads for the epitope - tagged t7-dgcr8 and 93% and 98% of the reads were mapped to the genome , respectively ( supplementary table 1 ) . after removing read duplicates , uniquely mapped reads from each of the datasets were clustered according to their genomic positions . in order to identify significant clusters we applied a modified false discovery rate ( mfdr ) to each cluster , as previously described . we assessed the reproducibility of the dgcr8 targets identified in clip experiments for both endogenous and overexpressed dgcr8 proteins . analysis of the reads of these two samples revealed a strong correlation ( pearson correlation co - efficient r > 0.8 ) ( fig . in order to describe dgcr8 bona fide targets , we focused on overlapping significant clusters between these two samples ( for a complete list go to http://regulatorygenomics.upf.edu/data/dgcr8 ) . in this way , we found 8,196 common targets ( harboring one or more significant clusters ) at the genomic level that were distributed as follows : 45% mapped to intergenic regions , 43% to protein coding genes and 5% to long non - coding rnas ( lncrnas ) ( fig . we also found that 30% of the genomic clusters mapped to repetitive elements , mainly line-1 and ltr retroelements , but these were excluded from the graphical representation . when analyzing protein - coding genes , we observed that the majority of the clusters were located in introns ( 26% , including mirnas ) , followed by coding exons ( 12% ) . as expected , due to the canonical function of the microprocessor , we identified 117 mirnas ( fig . we also identified the only so far described non - canonical rna substrate for the microprocessor , the dgcr8 mrna ( fig . 1d , right).we also observed clip tags mapping to other non - coding rnas , such as rrna , snrnas , and snornas ( fig . in addition for each of the clusters , an rna secondary structure was predicted in order to select those clusters overlapping with structures resembling that of pri - mirnas ( see http://regulatorygenomics.upf.edu/data/dgcr8 and supplementary methods ) . importantly , we found that more than 60% of the dgcr8 binding sites were overlapping with stable rna secondary structures ( supplementary table 1 ) . in addition , we observed that between 11 - 20% of the dgcr8 binding sites overlap with small rnas harboring a 5phosphorylated end , which are proposed to be generated by endonucleolytic activity . malat1 ( metastasis - associated lung adenocarcinoma transcript 1 , also known as neat2 ) is a mammalian abundant , long non - coding rna that was initially identified due to its elevated expression in several cancers . we found that dgcr8 binds to malat1 and several other long non - coding rnas ( fig . we further confirmed this interaction by immunoprecipitating overexpressed and endogenous dgcr8 protein followed by analysis of the associated rna by semi - quantitative rt - pcr ( fig . in order to study the effect of microprocessor binding to the malat1 rna , we determined its steady - state levels upon sirna - mediated depletion of drosha in hela cells . we observed a two - fold increase in the levels of malat1 rna when drosha levels were reduced ( fig . 2c ) . 2e ) , suggesting that the microprocessor controls malat1 rna levels , independently of the activity of mirnas . in order to test direct cleavage of the malat1 mrna in vivo , we fused the 5end of malat1 , which harbors significant dgcr8 clusters ( fig . 2a , indicated with a box ) to a luciferase reporter gene and transfected this construct into hela cells . reduction of drosha levels resulted in an increased expression of this reporter , suggesting that malat1 can be cleaved in vivo by the microprocessor ( fig 2f ) . in agreement , we observed small rna reads mapping to the majority of dgcr8 clusters present on the malat1 sequence ( small rna dataset from ) , also suggesting that dgcr8 binding could indeed induce rna cleavage ; however , it still remains unknown whether small rnas generated from these loci behave like mirnas ( fig . another class of rnas bound by dgcr8 corresponds to small nucleolar rnas ( snornas ) ( fig . these small rnas are responsible for guiding many chemical modifications of other rnas , such as rrna , trna and snrnas . first , we confirmed the binding of endogenous and overexpressed dgcr8 to snornas observed by clip using immunoprecipitation followed by qrt - pcr in hek 293 t cells ( fig . this reported binding to snornas has a physiological consequence as seen by the accumulation of u17a , u16 and u92 , which represent different subclasses of snornas , in mouse cells lacking dgcr8 ( fig . 3b , left panel ) , as well as in hela cells knocked - down for dgcr8 ( fig . 3b , middle panel ) or when overexpressing a dominant negative form of dgcr8 in hek293 t cells ( supplementary fig . importantly , mature snorna levels did not change in the absence of dicer ( fig . thus , dgcr8 can affect the stability of the mature forms of an h / aca box snorna ( u17a ) , a c / d box snorna ( u16 ) and a small cajal rna ( scarna u92 ) ( fig . their biogenesis involves trimming of the host introns from the 5 and 3 end following pre - mrna splicing and release of the mature snorna form that is being protected from degradation by the core snornp components ( reviewed by ) . we determined that dgcr8 affects the levels of mature snornas but not of their precursor forms ( fig . 3c ) , indicating that dgcr8 acts at a post - synthesis level . to further elucidate the role of the microprocessor in snorna stability in human cells , we studied the effect of either depleting drosha or overexpressing a dominant negative form of drosha . to our surprise we could not detect any significant change in mature snorna levels in these situations ( fig . 2d ) . these data suggested a new dgcr8 function that operates independently of drosha , strongly indicating that dgcr8 might associate to another nuclease / s to cleave and destabilize snornas . to test this hypothesis we incubated radiolabeled mature snornas with immunoprecipitated t7-dgcr8 and observed the appearance of smaller bands , corresponding to digested products ( fig . importantly , the addition of immunoprecipitated flag - drosha did not result in the formation of these subproducts ( fig . 3e , lanes 5 and 10 ) , despite drosha being active in the processing of a pri - mirna precursor ( pri - mir-24 - 2 ) ( fig . this dgcr8-mediated effect was specific for snornas , since u1 snrna was not cleaved by the dgcr8 complex ( fig . in vitro cleavage analyses revealed the precise location of the snorna cleavages , which occur towards their 3end ; potentially generating 3end fragments of 20nt long small rnas for c / d box snornas ( u16 ) and 40nt long for h / aca ( u92 ) ( supplementary fig . furthermore , we observed stabilization of the 70nt long fragment when overexpressing dominant negative forms of dgcr8 in human cells ( supplementary fig . this observation agrees with previous bioinformatic analyses of dgcr8 ko and dicer ko small rna libraries whereby snorna - encoded small rnas ( sdrnas ) are affected by the loss of dgcr8 and dicer . here , we observed that the loss of dgcr8 decreases the amount of small rnas originated from both the 5 and the 3 ends for both c / d box and h / aca snornas ( supplementary fig . 3c , d ) and these sites do not coincide with the cleavage sites previously described for h / aca box snornas ( at the h box ) , which are required for the production of mirna - like molecules in a drosha - independent and dicer - dependent pathway . collectively , these data demonstrates a role for dgcr8 in controlling the abundance of mature snornas , in a drosha - independent manner . this strongly suggests that dgcr8 associates to another endonuclease / s to specifically destabilize snornas and/or to produce new types of small rnas we obtained dgcr8 clip - tags mapping to 2,256 different mrnas , comprising 3,610 significant clusters , indicating that some mrnas harbor more than one dgcr8 binding site . we also found dgcr8 binding sites at the 5utr and first exon of the dgcr8 mrna and in agreement with previous data , these clusters overlapped hs - mir-1306 ( fig . we confirmed by ip rt - pcr that both dgcr8 and drosha bind to the dgcr8 mrna and that drosha knock - down increases steady - state levels of dgcr8 mrna ( supplementary fig . next , we chose 6 different mrnas with exonic dgcr8 clusters in both clip samples ( endogenous and overexpressed ) and harboring a predicted rna secondary structure that resembles that of pri - mirnas ( fig . first , we confirmed binding of these mrnas by ip - rt - pcr to both endogenous and overexpressed dgcr8 ( fig . si - rna mediated depletion of drosha and/or dgcr8 in hela cells resulted in an increase of their mrna levels , suggesting that these mrnas could be direct targets of the microprocessor complex ( fig . , the upregulation observed in microprocessor - deficient cells ( dgcr8 ko ) was not detected in dicer ko cells , suggesting a direct role of the microprocessor in processing these mrnas ( supplementary fig . ( comprising regions of those mrnas that contain the dgcr8 binding sites ) with immunopurified microprocessor ( flag - drosha ) resulted in the cleavage of hoxa7 , dlg5 and snx12 ( fig . 4 g , lanes 4 , 6 and 8) , as well as of the positive control , pri - mir-30c-1 ( fig 4 g , lane 2 ) . analysis of the hits - clip data revealed dgcr8 binding to several predicted cassette exons ( n=241 ) ( fig . this would suggest the interesting possibility that microprocessor - mediated cleavage of these cassette exons would affect the relative abundance of alternatively spliced ( as ) isoforms . a global analysis of alternative splicing by exon junction arrays in mouse cells lacking dgcr8 revealed 318 changes in cassette exons , as well as 40 alternative 5/3splice site ( ss ) events and 18 changes in mutually exclusive exons ( supplementary fig . we first focused on four cassette exons shown to be bound by dgcr8 in the clip experiments that also display an rna secondary structure resembling pri - mirnas ( supplementary fig . an uv - cross - linking assay revealed that dgcr8 binding to each of these cassette exons was affected when the predicted secondary structures were disrupted by mutation ( fig . 5b ) . importantly , those isoforms bound by dgcr8 were stabilized in human cells depleted of drosha or in mouse cells lacking dgcr8 ( fig . 5f ) , strongly suggesting that the microprocessor specifically cleaves and destabilizes mrna isoforms containing dgcr8 binding sites leading to an altered ratio of the alternatively spliced isoforms . accordingly , we also observed an overlap of these dgcr8 alternatively spliced exons with small rna libraries ( fig 5c , d , lower panels ) , confirming that cleavage can occur at these locations , which could help to explain the change in the relative abundance of those isoforms containing the dgcr8 bound exon . in addition , in those cases that we tested , we could confirm that no changes in alternative splicing were observed in the absence of dicer , indicating that these effects are independent from the presence of mirnas ( tcf3 and csnk1d ) ( fig 5f and supplementary fig . 5 g ) . altogether these data suggest that dgcr8 binding to cassette exons may act to influence the relative abundance of alternatively spliced isoforms . in order to determine whether the microprocessor is involved in the processing of cellular rnas ( other than mirnas ) that contain hairpin structures , we focused on the identification of endogenous rna targets of dgcr8 . for this , we used the cross - linking immunoprecipitation protocol coupled to high - throughput sequencing ( hits - clip ) . this technique relies on ultraviolet irradiation to induce protein covalent crosslinks in protein - rna complexes in situ . this allows the use of highly stringent immunoprecipitation and washing conditions so that only those rnas directly bound to the protein of interest are selected ( reviewed by ) . we performed two replicate hits - clip experiments on uv - irradiated hek293 t cells . each replicate comprised two independent experiments : one involved immunoprecipitation ( ip ) of endogenous dgcr8 protein ( supplementary fig 1a , left ) whereas the second one involved ip of a transiently transfected epitope - tagged dgcr8 protein ( pcg t7-dgcr8 ) ( supplementary fig 1a , right and 1b ) . we focused our analyses on the second replicate , since it gave higher depth of coverage and better mapping to the genome . 37 million reads for the endogenous dgcr8 protein , and 36 million reads for the epitope - tagged t7-dgcr8 and 93% and 98% of the reads were mapped to the genome , respectively ( supplementary table 1 ) . after removing read duplicates , uniquely mapped reads from each of the datasets were clustered according to their genomic positions . in order to identify significant clusters we applied a modified false discovery rate ( mfdr ) to each cluster , as previously described . we assessed the reproducibility of the dgcr8 targets identified in clip experiments for both endogenous and overexpressed dgcr8 proteins . analysis of the reads of these two samples revealed a strong correlation ( pearson correlation co - efficient r > 0.8 ) ( fig . in order to describe dgcr8 bona fide targets , we focused on overlapping significant clusters between these two samples ( for a complete list go to http://regulatorygenomics.upf.edu/data/dgcr8 ) . in this way , we found 8,196 common targets ( harboring one or more significant clusters ) at the genomic level that were distributed as follows : 45% mapped to intergenic regions , 43% to protein coding genes and 5% to long non - coding rnas ( lncrnas ) ( fig . we also found that 30% of the genomic clusters mapped to repetitive elements , mainly line-1 and ltr retroelements , but these were excluded from the graphical representation . when analyzing protein - coding genes , we observed that the majority of the clusters were located in introns ( 26% , including mirnas ) , followed by coding exons ( 12% ) . as expected , due to the canonical function of the microprocessor , we identified 117 mirnas ( fig . we also identified the only so far described non - canonical rna substrate for the microprocessor , the dgcr8 mrna ( fig . 1d , right).we also observed clip tags mapping to other non - coding rnas , such as rrna , snrnas , and snornas ( fig . in addition for each of the clusters , an rna secondary structure was predicted in order to select those clusters overlapping with structures resembling that of pri - mirnas ( see http://regulatorygenomics.upf.edu/data/dgcr8 and supplementary methods ) . importantly , we found that more than 60% of the dgcr8 binding sites were overlapping with stable rna secondary structures ( supplementary table 1 ) . in addition , we observed that between 11 - 20% of the dgcr8 binding sites overlap with small rnas harboring a 5phosphorylated end , which are proposed to be generated by endonucleolytic activity . malat1 ( metastasis - associated lung adenocarcinoma transcript 1 , also known as neat2 ) is a mammalian abundant , long non - coding rna that was initially identified due to its elevated expression in several cancers . we found that dgcr8 binds to malat1 and several other long non - coding rnas ( fig . we further confirmed this interaction by immunoprecipitating overexpressed and endogenous dgcr8 protein followed by analysis of the associated rna by semi - quantitative rt - pcr ( fig . in order to study the effect of microprocessor binding to the malat1 rna , we determined its steady - state levels upon sirna - mediated depletion of drosha in hela cells . we observed a two - fold increase in the levels of malat1 rna when drosha levels were reduced ( fig . 2c ) . 2e ) , suggesting that the microprocessor controls malat1 rna levels , independently of the activity of mirnas . in order to test direct cleavage of the malat1 mrna in vivo , we fused the 5end of malat1 , which harbors significant dgcr8 clusters ( fig . 2a , indicated with a box ) to a luciferase reporter gene and transfected this construct into hela cells . reduction of drosha levels resulted in an increased expression of this reporter , suggesting that malat1 can be cleaved in vivo by the microprocessor ( fig 2f ) . in agreement , we observed small rna reads mapping to the majority of dgcr8 clusters present on the malat1 sequence ( small rna dataset from ) , also suggesting that dgcr8 binding could indeed induce rna cleavage ; however , it still remains unknown whether small rnas generated from these loci behave like mirnas ( fig . another class of rnas bound by dgcr8 corresponds to small nucleolar rnas ( snornas ) ( fig . these small rnas are responsible for guiding many chemical modifications of other rnas , such as rrna , trna and snrnas . first , we confirmed the binding of endogenous and overexpressed dgcr8 to snornas observed by clip using immunoprecipitation followed by qrt - pcr in hek 293 t cells ( fig . this reported binding to snornas has a physiological consequence as seen by the accumulation of u17a , u16 and u92 , which represent different subclasses of snornas , in mouse cells lacking dgcr8 ( fig . 3b , left panel ) , as well as in hela cells knocked - down for dgcr8 ( fig . 3b , middle panel ) or when overexpressing a dominant negative form of dgcr8 in hek293 t cells ( supplementary fig . importantly , mature snorna levels did not change in the absence of dicer ( fig . thus , dgcr8 can affect the stability of the mature forms of an h / aca box snorna ( u17a ) , a c / d box snorna ( u16 ) and a small cajal rna ( scarna u92 ) ( fig . their biogenesis involves trimming of the host introns from the 5 and 3 end following pre - mrna splicing and release of the mature snorna form that is being protected from degradation by the core snornp components ( reviewed by ) . we determined that dgcr8 affects the levels of mature snornas but not of their precursor forms ( fig . 3c ) , indicating that dgcr8 acts at a post - synthesis level . to further elucidate the role of the microprocessor in snorna stability in human cells , we studied the effect of either depleting drosha or overexpressing a dominant negative form of drosha . to our surprise we could not detect any significant change in mature snorna levels in these situations ( fig . these data suggested a new dgcr8 function that operates independently of drosha , strongly indicating that dgcr8 might associate to another nuclease / s to cleave and destabilize snornas . to test this hypothesis we incubated radiolabeled mature snornas with immunoprecipitated t7-dgcr8 and observed the appearance of smaller bands , corresponding to digested products ( fig . importantly , the addition of immunoprecipitated flag - drosha did not result in the formation of these subproducts ( fig . 3e , lanes 5 and 10 ) , despite drosha being active in the processing of a pri - mirna precursor ( pri - mir-24 - 2 ) ( fig . this dgcr8-mediated effect was specific for snornas , since u1 snrna was not cleaved by the dgcr8 complex ( fig . 3e , lane 13 ) . in vitro cleavage analyses revealed the precise location of the snorna cleavages , which occur towards their 3end ; potentially generating 3end fragments of 20nt long small rnas for c / d box snornas ( u16 ) and 40nt long for h / aca ( u92 ) ( supplementary fig . furthermore , we observed stabilization of the 70nt long fragment when overexpressing dominant negative forms of dgcr8 in human cells ( supplementary fig . this observation agrees with previous bioinformatic analyses of dgcr8 ko and dicer ko small rna libraries whereby snorna - encoded small rnas ( sdrnas ) are affected by the loss of dgcr8 and dicer . here , we observed that the loss of dgcr8 decreases the amount of small rnas originated from both the 5 and the 3 ends for both c / d box and h / aca snornas ( supplementary fig . 3c , d ) and these sites do not coincide with the cleavage sites previously described for h / aca box snornas ( at the h box ) , which are required for the production of mirna - like molecules in a drosha - independent and dicer - dependent pathway . collectively , these data demonstrates a role for dgcr8 in controlling the abundance of mature snornas , in a drosha - independent manner . this strongly suggests that dgcr8 associates to another endonuclease / s to specifically destabilize snornas and/or to produce new types of small rnas we obtained dgcr8 clip - tags mapping to 2,256 different mrnas , comprising 3,610 significant clusters , indicating that some mrnas harbor more than one dgcr8 binding site . we also found dgcr8 binding sites at the 5utr and first exon of the dgcr8 mrna and in agreement with previous data , these clusters overlapped hs - mir-1306 ( fig . we confirmed by ip rt - pcr that both dgcr8 and drosha bind to the dgcr8 mrna and that drosha knock - down increases steady - state levels of dgcr8 mrna ( supplementary fig . next , we chose 6 different mrnas with exonic dgcr8 clusters in both clip samples ( endogenous and overexpressed ) and harboring a predicted rna secondary structure that resembles that of pri - mirnas ( fig . first , we confirmed binding of these mrnas by ip - rt - pcr to both endogenous and overexpressed dgcr8 ( fig . si - rna mediated depletion of drosha and/or dgcr8 in hela cells resulted in an increase of their mrna levels , suggesting that these mrnas could be direct targets of the microprocessor complex ( fig . accordingly , the upregulation observed in microprocessor - deficient cells ( dgcr8 ko ) was not detected in dicer ko cells , suggesting a direct role of the microprocessor in processing these mrnas ( supplementary fig . ( comprising regions of those mrnas that contain the dgcr8 binding sites ) with immunopurified microprocessor ( flag - drosha ) resulted in the cleavage of hoxa7 , dlg5 and snx12 ( fig . 4 g , lanes 4 , 6 and 8) , as well as of the positive control , pri - mir-30c-1 ( fig 4 g , lane 2 ) . analysis of the hits - clip data revealed dgcr8 binding to several predicted cassette exons ( n=241 ) ( fig . 5a ) . this would suggest the interesting possibility that microprocessor - mediated cleavage of these cassette exons would affect the relative abundance of alternatively spliced ( as ) isoforms . a global analysis of alternative splicing by exon junction arrays in mouse cells lacking dgcr8 revealed 318 changes in cassette exons , as well as 40 alternative 5/3splice site ( ss ) events and 18 changes in mutually exclusive exons ( supplementary fig . we first focused on four cassette exons shown to be bound by dgcr8 in the clip experiments that also display an rna secondary structure resembling pri - mirnas ( supplementary fig . an uv - cross - linking assay revealed that dgcr8 binding to each of these cassette exons was affected when the predicted secondary structures were disrupted by mutation ( fig . 5b ) . importantly , those isoforms bound by dgcr8 were stabilized in human cells depleted of drosha or in mouse cells lacking dgcr8 ( fig . 5f ) , strongly suggesting that the microprocessor specifically cleaves and destabilizes mrna isoforms containing dgcr8 binding sites leading to an altered ratio of the alternatively spliced isoforms . accordingly , we also observed an overlap of these dgcr8 alternatively spliced exons with small rna libraries ( fig 5c , d , lower panels ) , confirming that cleavage can occur at these locations , which could help to explain the change in the relative abundance of those isoforms containing the dgcr8 bound exon . in addition , in those cases that we tested , we could confirm that no changes in alternative splicing were observed in the absence of dicer , indicating that these effects are independent from the presence of mirnas ( tcf3 and csnk1d ) ( fig 5f and supplementary fig . 5 g ) . altogether these data suggest that dgcr8 binding to cassette exons may act to influence the relative abundance of alternatively spliced isoforms . in this study , we present a comprehensive strategy aimed at the identification of endogenous rna targets for the microprocessor . we found that dgcr8 binds to many different types of rnas involved in different cellular pathways . we identified most of the mirnas known to be expressed in these cells , which was expected . other dgcr8 targets comprise mrnas , non - coding rnas including snornas , long non - coding rnas and retrotransposons . our analysis also revealed that dgcr8 binds to more than 2,000 mrnas and in many cases depletion of microprocessor components leads to an up - regulation of these mrnas , suggesting that they are direct targets of the microprocessor . for some of these mrnas that were up - regulated upon depletion of drosha or dgcr8 , we could indeed confirm drosha - dependent cleavage in vitro ( fig . this extends a previous observation showing that a hairpin localized in the 5 utr of the dgcr8 pre - mrna is targeted by the microprocessor . nevertheless , it remains elusive whether any of these cleavage products are also dicer substrates that would lead to the generation of mirna - like small rnas . however , we observed that a large proportion of dgcr8 binding sites also overlap with small rnas harboring a 5phosphorylated end , which have been proposed to be produced by the action of endonucleases . the fact that the microprocessor destabilizes mrnas containing dgcr8 binding sites in cassette exons suggested the interesting possibility that the microprocessor might have a role in the processing of these alternative exons , which would impact on alternative splicing regulation . we confirmed that dgcr8 can modulate the relative abundance of alternatively spliced isoforms and that binding to these sequences depends on rna secondary structure ( fig . 5 and supplementary fig . 5 ) functional studies of the mirna processing machinery have shown that drosha , dgcr8 and dicer deficiencies generate similar phenotypes , resulting in early embryonic lethality , strongly suggesting that mirnas are essential for normal development . furthermore , several lines of evidence indicated additional functions for drosha and/or dicer , although initial global studies suggested dgcr8 mrna as the only non - canonical substrate for the microprocessor . first , microarray profiling of mefs derived from drosha and dicer knock - out mice showed poor correlation between the populations of mrnas affected by these two endonucleases . secondly , drosha was shown to recognize and cleave many protein - coding messenger rnas ( mrnas ) with secondary stem - loop structures in early - stage thymocytes , as well as viral rnas to directly regulate viral expression . finally , drosha - dependent mrna cleavage events that functionally regulate mrna levels in mescs , were recently described . a simple explanation for these observations is that non - canonical functions for the microprocessor and/or dicer could be responsible for these effects . the comparison of these reports with our data only resulted in a partial overlap , probably due to the use of different cell lines for these studies ( t cells versus hek 293 t cells ) . it also remains possible that binding of dgcr8 might not always lead to changes in relative mrna expression . what we provide here is a genome - wide view of dgcr8 binding sites in hek 293 t cells . importantly , our studies show more alternative functions for the microprocessor component dgcr8 , implicating this protein in the control of non - coding rnas stability ( lncrnas and snornas ) and on the relative abundance of alternatively spliced isoforms .. in vitro processing assays with immunoprecipitated dgcr8 showed cleavage of the mature snorna species in a drosha - independent manner , suggesting the involvement of dgcr8 in cellular complexes with other endonucleases ( fig . 3 and supplementary fig . 2 - 3 ) . a dgcr8-independent role for drosha in pre - rrna processing while drosha is required for the processing of 12s pre - rrna , dgcr8 ko mouse es cells did not display evident defects in ribosomal rna processing , suggesting alternative complexes to the canonical microprocessor . the presence of mirna - like molecules encoded by h / aca snornas independent of drosha function but involving a dicer - dependent cleavage activity was previously reported . bioinformatics analyses of small rnas encoded from snornas showed that are dependent on both , dgcr8 and dicer , although the role of drosha was not assessed . from an evolutionary point of view their structure conservation as well as the retained capacity to bind dyskerin and fibrillarin , two of the core components from snornp particles , suggests a common origin . our work proposes a new complex between dgcr8 and another endonuclease / s that would specifically cleave snornas and probably other classes of cellular rnas . although it is well known how snornas are synthesized , very little is known about their turnover and decay , and it would be of great interest to further study the implication of dgcr8 in this process . these findings raise the interesting possibility that there could be alternative dgcr8 complex / es using different nucleases to process a variety of cellular rnas . significantly , all the new functions described here for the microprocessor complex could be useful to reinterpret the phenotypes observed for drosha or dgcr8 deficient cells that were mainly attributed to the deficiency of mirnas . importantly , the dgcr8 gene is present in the deleted genomic region ( 22q11.2 ) in digeorge syndrome patients that expands around 30 genes . mouse models for this deletion showed a minor alteration of mirnas , but still exhibit behavioral and cognitive deficits and cardiac abnormalities that resemble the human condition . interestingly , the dgcr8 heterozygous mouse still showed some of the traits present in digeorge patients , such as altered short - term plasticity that has been linked to schizophrenia . although it is clear that in this situation the mirna biogenesis pathway is affected , it would be of great interest to determine how important are the new alternative dgcr8 functions for the origin and development of the disease .

the drosha - dgcr8 complex ( microprocessor ) is required for microrna ( mirna ) biogenesis . dgcr8 recognizes the rna substrate , whereas drosha functions as the endonuclease . high - throughput sequencing and crosslinking immunoprecipitation ( hits - clip ) was used to identify rna targets of dgcr8 in human cells . unexpectedly , mirnas were not the most abundant targets . dgcr8-bound rnas also comprised several hundred mrnas as well as snornas and long non - coding rnas . we found that the microprocessor controls the abundance of several mrnas as well as of malat-1 . by contrast , dgcr8-mediated cleavage of snornas is independent of drosha , suggesting the involvement of dgcr8 in cellular complexes with other endonucleases . interestingly , binding of dgcr8 to cassette exons , acts as a novel mechanism to regulate the relative abundance of alternatively spliced isoforms . collectively , these data provide new insights in the complex role of dgcr8 in controlling the fate of several classes of rnas .

RESULTS HITS-CLIP identifies novel targets of the Microprocessor The Microprocessor regulates the abundance of MALAT1 DGCR8 regulates snoRNA stability independently of Drosha The Microprocessor cleaves the mRNA of protein-coding genes Modulation of alternative spliced isoforms by the Microprocessor DISCUSSION Supplementary Material

recently , the focus of management in schizophrenia has been shifting to early detection and intervention of the disorder . the primary prevention is to eliminate etiological risk factors or to strengthen individual resistibility to the morbid risk ; however , these have not been shown to be effective in schizophrenia to date . instead , many researchers have been interested in secondary early intervention , which is preventing or postponing the onset of psychosis , reducing the severity of illness , and enhancing treatment outcomes . during the past decade , clinical studies on first - episode schizophrenia have revealed that the duration of untreated psychosis ( dup ) may be associated with short - term and/or long - term outcomes , that is , a longer dup is associated with a poorer outcome of schizophrenia ( 1 , 2 ) . also , according to mcgorry et al . ( 3 ) , early therapeutic intervention during the prodromal phase of schizophrenia may influence the overall course of the disease . the onset of psychotic symptoms observed in schizophrenia is frequently preceded by nonspecific changes in behavior , emotional state , and/or cognitive state ( 4 ) , with common signs and symptoms of the prepsychotic stage of schizophrenia including sleep disturbance , anxiety , anger / irritability , depressed mood , deterioration in functioning , social withdrawal , poor concentration , suspiciousness , loss of motivation , and low energy ( 5 , 6 ) . therefore , it is critical that these signs and symptoms should be detected as early as possible to reduce the duration of the prodromal phase . according to goldstein and lewine ( 7 ) , males with schizophrenia generally have an earlier onset of illness and a more severe form of the disease as reflected in greater cognitive or social impairment , while female patients are more likely to suffer from anxiety and affective symptoms ( 8) . this sexual difference is regarded as a consequence of different neuroendocrine and affective arousal systems ( 9 ) . thus , the early symptoms and signs experienced and observed during the prodromal stage of schizophrenia may differ between males and females with regard to their frequency and relationship with other clinical characteristics . however , relatively little work examined the gender difference of clinical manifestation in this population ( 10 ) , although several prospective studies examined gender as a predictive variable of conversion to psychosis and found mixed results ( 11 , 12 ) . therefore , in the present study , we examined gender differences in the prodromal symptoms experienced by the patients or observed by caregivers before the onset of first psychotic symptoms in patients with first - episode schizophrenia . we hypothesized more affective symptoms in women and more cognitive or negative symptoms in men would be found during prodromal phase , as in patients with schizophrenia in previous studies . this study was performed according to regulations on the use of human subjects established by the institutional review board of seoul national university hospital . we reviewed the medical records of all patients admitted to the psychiatric inpatient ward between march 2005 and february 2007 in seoul national university hospital whose diagnoses at the time of discharge were schizophrenia . first , the admission records were complete with detailed present illness including all past psychiatric diagnoses , tentative dsm - iv multi - axial diagnoses , clinical global impression ( cgi ) and global assessment of functioning ( gaf ) scores . second , the discharge records were complete with definite dsm - iv multi - axial diagnoses , cgi and gaf scores , and signed by at least two psychiatrists . third , the patients had experienced either delusions or hallucinations for at least 1 month , regardless of the subtypes of schizophrenia they were diagnosed with . fourth , from previous medical records , it was evident that patients had never been diagnosed with schizophrenia before the admission . the authors discarded cases in which the section for present illness lacked the evidence for the dsm - iv diagnosis of schizophrenia , estimated onset of noticeable symptoms , or cgi and gaf scores . the subjects either had never taken psychotropic medication or had received pharmacotherapy for less than 1 month . patients who met the criteria for substance use disorder , mental retardation , or major general medical conditions associated with psychotic symptoms were also excluded from the study . a total of 214 patients were admitted during the study period and received a diagnosis of schizophrenia on discharge . among them , 63 patients ( 38 males , 25 females ) met these criteria . all had been admitted under the mental health act with the permission of at least one eligible caregiver . the numbers of subjects diagnosed with each subtype of schizophrenia were as follows : paranoid ( n=46 ) , undifferentiated ( n=12 ) , disorganized ( n=4 ) , and catatonic ( n=1 ) . thirty - nine patients ( 25 males , 14 females ) had been treated with psychotropic medication such as antipsychotics , antidepressants , and benzodiazepines . the onsets of the prodromal phase and the psychotic phase were estimated to the nearest 1 month according to the admission and discharge records by one of the authors . the onset of the prodromal phase was defined as the appearance of the first symptoms noticeable either to the patient him / herself or to the caregiver(s ) that were considered to indicate the appearance of the disease . the duration from the onset of prodromal symptom to the onset of psychotic symptom was considered as the duration of prodromal symptom . the onset of the psychotic phase was defined as the appearance of active psychotic symptoms . in this study , prodromal symptoms were categorized into 7 dimensions as follows : 1 ) attenuated positive symptoms ( unusual thought content , suspiciousness , unusual perceptual experiences , and disorganized speech ) , 2 ) negative symptoms ( social withdrawal , decreased energy , and flat affect ) , 3 ) cognitive changes ( difficulties with concentration and memory problems ) , 4 ) mood symptoms ( depression , elation , and irritability ) , 5 ) anxiety , 6 ) obsessive compulsive symptoms , and 7 ) somatic symptoms . these dimensions were derived from the instrument for the retrospective assessment of onset of schizophrenia ( 13 ) and criteria of prodromal syndrome ( 14 ) . the presence of symptoms from each dimension was determined 1 ) if the symptom dimension was directly mentioned in the medical record either in english or korean , i.e , for dimension of mood symptoms , phrases such as " mood disturbance " , " depression ( or depressive mood ) " , " elation ( or elated mood ) " , " irritability " or their korean equivalents or 2 ) if the authors could conclude the presence of symptoms from each dimension from the description of the subjects??thought , emotion and behavior from the medical record . differences in the frequency of prodromal symptoms between male and female patients were analyzed using a chi - square test . clinical characteristics or duration of overall and individual prodromal symptoms were compared between male and female patients using a t - test . correlational analysis was used to examine the relationships between the duration of prodromal and/or psychotic symptoms and , cgi or gaf scale score at admission in each gender . this study was performed according to regulations on the use of human subjects established by the institutional review board of seoul national university hospital . we reviewed the medical records of all patients admitted to the psychiatric inpatient ward between march 2005 and february 2007 in seoul national university hospital whose diagnoses at the time of discharge were schizophrenia . first , the admission records were complete with detailed present illness including all past psychiatric diagnoses , tentative dsm - iv multi - axial diagnoses , clinical global impression ( cgi ) and global assessment of functioning ( gaf ) scores . second , the discharge records were complete with definite dsm - iv multi - axial diagnoses , cgi and gaf scores , and signed by at least two psychiatrists . third , the patients had experienced either delusions or hallucinations for at least 1 month , regardless of the subtypes of schizophrenia they were diagnosed with . fourth , from previous medical records , it was evident that patients had never been diagnosed with schizophrenia before the admission . the authors discarded cases in which the section for present illness lacked the evidence for the dsm - iv diagnosis of schizophrenia , estimated onset of noticeable symptoms , or cgi and gaf scores . the subjects either had never taken psychotropic medication or had received pharmacotherapy for less than 1 month . patients who met the criteria for substance use disorder , mental retardation , or major general medical conditions associated with psychotic symptoms were also excluded from the study . a total of 214 patients were admitted during the study period and received a diagnosis of schizophrenia on discharge . among them , 63 patients ( 38 males , 25 females ) met these criteria . all had been admitted under the mental health act with the permission of at least one eligible caregiver . the numbers of subjects diagnosed with each subtype of schizophrenia were as follows : paranoid ( n=46 ) , undifferentiated ( n=12 ) , disorganized ( n=4 ) , and catatonic ( n=1 ) . thirty - nine patients ( 25 males , 14 females ) had been treated with psychotropic medication such as antipsychotics , antidepressants , and benzodiazepines . the onsets of the prodromal phase and the psychotic phase were estimated to the nearest 1 month according to the admission and discharge records by one of the authors . the onset of the prodromal phase was defined as the appearance of the first symptoms noticeable either to the patient him / herself or to the caregiver(s ) that were considered to indicate the appearance of the disease . the duration from the onset of prodromal symptom to the onset of psychotic symptom was considered as the duration of prodromal symptom . the onset of the psychotic phase was defined as the appearance of active psychotic symptoms . in this study , prodromal symptoms were categorized into 7 dimensions as follows : 1 ) attenuated positive symptoms ( unusual thought content , suspiciousness , unusual perceptual experiences , and disorganized speech ) , 2 ) negative symptoms ( social withdrawal , decreased energy , and flat affect ) , 3 ) cognitive changes ( difficulties with concentration and memory problems ) , 4 ) mood symptoms ( depression , elation , and irritability ) , 5 ) anxiety , 6 ) obsessive compulsive symptoms , and 7 ) somatic symptoms . these dimensions were derived from the instrument for the retrospective assessment of onset of schizophrenia ( 13 ) and criteria of prodromal syndrome ( 14 ) . the presence of symptoms from each dimension was determined 1 ) if the symptom dimension was directly mentioned in the medical record either in english or korean , i.e , for dimension of mood symptoms , phrases such as " mood disturbance " , " depression ( or depressive mood ) " , " elation ( or elated mood ) " , " irritability " or their korean equivalents or 2 ) if the authors could conclude the presence of symptoms from each dimension from the description of the subjects??thought , emotion and behavior from the medical record . differences in the frequency of prodromal symptoms between male and female patients were analyzed using a chi - square test . clinical characteristics or duration of overall and individual prodromal symptoms were compared between male and female patients using a t - test . correlational analysis was used to examine the relationships between the duration of prodromal and/or psychotic symptoms and , cgi or gaf scale score at admission in each gender . the mean age of the study population was 24.665.62 yr . the duration of prodromal symptoms and psychotic symptoms were 5.634.53 yr and 0.821.07 yr , respectively . on average , patients were admitted for 34.5725.53 days . at admission , mean cgi score and gaf scores were 5.710.58 and 35.0812.33 , respectively . a total of 17 males had axis i psychiatric diagnoses , including obsessive - compulsive disorder ( ocd ) , adjustment disorder , social phobia , and major depression , prior to the onset of psychotic symptoms , whereas 12 females had prior axis i diagnoses , including bipolar disorder , major depression , and ocd before the onset of psychotic symptoms ( table 1 ) . as shown in table 2 , no significant differences were detected between genders in terms of age , age of onset , duration of psychotic symptoms before admission , duration of admission , as well as cgi and gaf scores . overall , the most common symptoms were attenuated positive symptoms ( 89% ) and mood symptoms ( 86% ) . however , the most common symptoms in males were negative symptoms ( 97.4% ) , whereas the most common in females were attenuated positive symptoms ( 84% ) . significant differences were observed in the frequency of negative , cognitive , and obsessive - compulsive symptoms between males and females . a total of 37 males ( 97.4% ) and 14 females ( 56% ) experienced negative symptoms ( =14.16 , p=0.000 ) , and 29 males ( 76.3% ) and 10 females ( 40% ) experienced cognitive symptoms ( =6.96 , p=0.008 ) . twenty males ( 52.6% ) and 5 females ( 20% ) experienced obsessive - compulsive symptoms ( =4.55 , p=0.033 ) . duration of symptoms from each of the 7 dimensions did not significantly differ between genders , although male patients suffered from negative symptoms for a longer period than female patients did ( t=1.754 , p=0.086 ) . further analyses showed that there was a significant negative correlation between the duration of negative symptoms ( 5.324.98 yr ) and gaf score at admission in male patients ( r=-0.341 , p=0.039 ) . our findings demonstrated differences in the frequency of prodromal symptoms between male and female patients with first - episode schizophrenia . negative symptoms and attenuated positive symptoms were frequent in males , whereas attenuated positive symptoms and mood symptoms in females . in addition , male patients showed more frequent prodromal symptoms in negative , cognitive , and obsessive - compulsive symptom dimensions than female patients did . in several studies , a prolonged dup has been predictive of poor prognosis ( 2 , 15 ) and associated with sustained neuropsychological deficits , increased negative symptoms and disorganization , and unfavorable functional outcome of schizophrenia ( 16 ) . moreover ( 17 ) suggested that the process underlying toxic brain damage may be halted by administering proper doses of olanzapine . so far , therefore , there have been many efforts to detect patients presenting active psychotic symptoms and decrease dup . however , prior to the onset of psychotic symptoms , many patients are usually suffering from various nonspecific symptoms , such as depression , cognitive impairment , or negative symptoms . additionally , duration of the prodromal phase influenced grey and white matter volumes at the onset of psychosis . that is , the severity of volumetric abnormalities in first - episode psychosis was correlated with the duration of the prodromal phase ( 18 ) . in light of suffering , functional decline and brain change of patients before the onset of active psychotic symptoms , strategies for early detection and intervention during the prodromal phase will likely gain further prominence in the clinical setting . according to a retrospective examination of early course of patients with first - episode schizophrenia by hafner et al . ( 19 ) , the 10 most frequent and earliest signs of schizophrenia such as restlessness , depression , anxiety , trouble with thinking and concentration , were reported by patients with a first - episode illness . no gender differences were observed in the earliest prodromal symptoms , except worrying , and the first signs of a prodromal phase were depressive and negative symptoms . whereas they reported the frequency of initial symptoms during the prodromal phase , we emphasized on the frequency of all prodromal symptoms that patients had experienced . thus , negative , cognitive , and obsessive - compulsive symptoms were more frequent in male patients than in female patients . cognitive change and negative symptoms such as social withdrawal and decreased energy , may be associated with impaired role functioning . in the present study , the gaf score at admission was inversely correlated with the duration of negative symptoms during the prodromal phase in male patients , although there was no significant difference in the gaf score between genders . therefore , functional impairment in males may be influenced by the duration of negative symptoms even in the prodromal phase ( r=0.116 ) . ( 20 ) that showed a correlation between impaired functioning in the prepsychosis period and increased negative symptoms in schizophrenia spectrum disorders . psychiatric syndromes such as mood disorders , cannabis dependence ( 21 ) , or anxiety disorders ( 22 ) are known to be often comorbid with prodromal schizophrenia . of 63 patients included in this study , 29 ( 17 males , 12 females ) had prior psychiatric axis i diagnoses before the onset of active psychotic symptoms . interestingly , there were some differences in the frequency of psychiatric diagnoses between the genders , although a statistical power was not sufficient . diagnoses among male patients included ocd ( n=4 ) , adjustment disorder ( n=4 ) , and social phobia ( n=3 ) , whereas diagnoses among female patients comprised bipolar disorder ( n=4 ) and major depression ( n=3 ) . female patients tended to have a diagnosis of mood disorder more often . on the contrary , considering that both male and female patients had mostly attenuated positive symptoms during the prodromal phase , clinicians may need to pay attention to diagnose patients presenting mood symptoms or anxiety symptoms combined with attenuated positive symptoms . previous studies have indicated that the dup and duration of the prodromal phase differ between males and females . hafner et al . ( 23 ) reported that the prodromal period was longer in females than in males , and the dup has been shown to be significantly longer in males than in females ( 24 ) . some factors such as strength of social networks , degree of family involvement , gender difference in help - seeking behavior , and relative frequency of gender - specific stressors may lead to differences in the dup and the duration of the prodromal period between genders ( 10 , 25 ) . although no significant differences were observed in the dup and the duration of prodromal symptoms between males and females in the present study , the differences in the frequency of prodromal symptoms and prior psychiatric diagnoses , and correlation of the duration of negative symptoms with the gaf score at admission in male may have been influenced by several factors . first , females tend to express their emotions more readily and behave in a relatively unobtrusive way . thus , female patients may be more likely to be misdiagnosed , for example , to be a mood disorder ( 26 ) . second , a relative younger age at onset of the prodromal phase in males may also adversely affect their social and cognitive development , resulting in more frequent negative and cognitive symptoms and a negative correlation between the duration of negative symptoms and the gaf score at admission in male patients . additionally , these phenomenological differences may be derived from neurobiological differences between males and females . estrogen protects neural circuits by reducing the sensitivity of d2 receptors in the central nervous system and by acting as a neuromodulator with agonistic effects on other transmitter systems , such as serotonin and glutamate ( 19 , 27 ) . ( 28 ) conducted a positron emission tomography study to evaluate the striatal dopamine d2 receptor binding characteristics in 33 healthy males and in 21 healthy females and found that the female group had lower dopamine d2 receptor affinity than the male group . in addition , the prolactin response to neuroleptic medications was greater in females than in males . ( 29 ) reported that among patients receiving treatment with risperidone , females were 2.6 times more likely to develop hyperprolactinemia than males . extrapyramidal and anticholinergic reactions were more frequent among female patients , whereas sexual problems were more frequent among male patients ( 30 ) . in terms of these neurobiological and symptomatological differences , males and females could not be regarded as homogenous group , and therefore , clinicians and researchers should consider the role of gender in the evaluation and treatment of patients . the onset of prodromal and active psychotic symptoms was determined according to the information provided by patients and their family members . however , we excluded cases whose sources were lacking and chose only cases with comprehensive information . furthermore , as all of our subjects were admitted for the first time , they may be less prone to recall bias compared to recurrent or chronic patients , although diagnostic stability of these patients may be somewhat compromised . second , as all subjects in our study were hospitalized , presumably having more severe symptoms , they might represent the patient population with more severe symptoms . third , data in this study were descriptive and none pertained to the severity of prodromal and psychotic symptoms , except the gaf and cgi scores at admission . the purpose of this study was to investigate the differences in the frequency of early symptoms and signs during the prodromal phase between male and female patients . therefore , we did not have systematic data to evaluate the severity of symptoms . in conclusion , our present findings provide evidence for gender differences in the pattern of prodromal symptoms in patients with first - episode schizophrenia . although our study was retrospective and explorative in its nature , our result alone points that gender differences in prodromal symptoms should be considered when assessing patients presenting nonspecific symptoms combined with attenuated positive symptoms . further prospective studies exploring the relationship between gender differences in prodromal symptoms and long - term outcomes are warranted .

to investigate the gender difference of early symptoms appearing before the onset of the psychotic symptoms in patients with first - episode schizophrenia , we reviewed the medical records of 63 patients ( 38 males , 25 females ) , who were hospitalized for first - episode schizophrenia . the frequency and duration of prodromal and psychotic symptoms , clinical global impression scale scores , global assessment of functioning ( gaf ) scale scores at admission , and other clinical characteristics were recorded for all patients . overall , the most common prodromal symptoms were attenuated positive symptoms ( 89% ) , followed by mood symptoms ( 86% ) . negative symptoms were the most common in male patients ( 97.4% ) , whereas attenuated positive symptoms were the most common in female patients ( 84% ) . male patients demonstrated more frequent negative , cognitive , and obsessive - compulsive symptoms than female patients did and also showed a tendency of having negative symptoms for the longer period . correlational analysis showed a significant negative correlation between the duration of negative symptoms and gaf scores at admission in male patients . our findings suggest that different patterns of prodromal symptoms between male and female begin before the onset of the psychosis . further prospective studies should be needed .

INTRODUCTION MATERIALS AND METHODS Subjects Assessments Statistical analysis RESULTS DISCUSSION

the dynamics of the diabetes and hyperlipidemia are changing rapidly in low- to middle - income countries . in 2030 the number of adult with impaired glucose tolerance may rise from 344 million ( 2010 ) to 472 million by 2030 . international diabetes federation ( idf ) estimated that 80% of world diabetic population live in low- and middle - income countries in 2030 . as per idf 2011 report , china , india , and united states of america have 90.0 , 61.3 , and 23.7 million peoples were living with diabetes that may be increase up to 129.7 , 101.2 , and 29.3 million people , respectively , in 2030 . the treatment of any diseases with allopathic drugs causes moderate to severe adverse events , which could cause death . india is one of the eight important vavilovian centers of origin and crop plant diversity . a large majority of indian population is treated by traditional systems of medicine such as ayurveda , unani , and siddha . ayurveda , the indian indigenous system of medicine , has been an integral part of indian culture . although indian 's traditional medicinal system are deeply rooted in the indian psyche , today , we found that these system of medicine do not hold a there is a global resurgence in the use of herbal medicines along with a growing scientific interest in them as a source of new drugs . ( asteraceae ) is one of the common herb present throughout india , and this plant extract was commonly used for treating the cut wounds ( anti - inflammatory ) by rural indians . studies showed that t. procumbens has anti - inflammatory , hepatoprotective , wound healing , antimicrobial , antiseptic , hypotensive , and immunomodulatory properties . the previous studies on t. procumbens extract showed presence of hypoglycemic effect and antidiabetic effect against alloxan - induced diabetes in rats . alloxan is used to induce non - insulin - dependent diabetes , and the effect of t. procumbens extract on insulin - dependent diabetes is unknown . hence , the present study is undertaken to investigate the antidiabetic and antihyperlipidemic potential of an ethanolic extract of the whole plant of t. procumbens on streptozotocin ( stz)-induced insulin - dependent diabetes mellitus in rats . ( asteraceae ) plant was collected from different regions of madurai district , tamil nadu , india , in july 2011 . plant was identified and authenticated by botanist of agricultural college and research institute , madurai , tamil nadu . the whole plant was dried under the shade for a few days and grinded in electrical grinder to coarse powder . the whole plant powder of t. procumbens was packed in the soxhlet apparatus and extracted with 95% ethanol . later on , the extract was filtered , and the filtrate was concentrated to a dry mass by simple distillation . the male wistar albino rats , aged 4 months ( body weight : 180 10 g ) , were obtained from sainath enterprises , hyderabad , india . the animals were housed in large , spacious poly acrylic cages at an ambient room temperature with 12-h - light/12-h - dark cycle . rats have free access to water and rat pellets ( hindustan lever ltd . , bangalore , india ) . the study was approved by the institute animal ethics committee of ultra college of pharmacy , madurai , india . all the animal experiments were carried out according to committee for the purpose of control and supervision of experiments on animals ( cpcsea ) guidelines . biochemical assay kits for glucose , serum glutamic pyruvate transaminase ( sgpt ) , serum glutamic oxaloacetic transaminase ( sgot ) , total cholesterol , total protein , triglyceride , and glucose-6-phosphate dehydrogenase kits were procured from coral diagnostics ltd . all other chemicals used were of analytical grade obtained from sd - fine , india . the extract was subjected for phytochemical screening to screen the presence of various constituents such as sterols , alkaloids , tannins , glycosides , flavonoids and saponins/ phenolic compounds by libermann - burchard reaction , dragendroff 's reaction , ferric chloride solution test , keller - kiliani test , shinoda test and lead acetate test respectively . adult , healthy male wistar rats weighing between 180 10 g were used for the experiment . group i - normal control rats group ii - diabetic control group iii - diabetic rats treated with glibenclamide ( 0.25 mg / kg ) group iv - diabetic rats treated with ethanolic extract of the whole plant of t. procumbens ( 250 mg / kg ) group v - diabetic rats treated with ethanolic extract of the whole plant of t. procumbens ( 500 mg / kg ) . diabetes was induced in overnight - fasted rats by a single intra - peritoneal injection of freshly prepared stz 50 mg / kg b.w . followed by 120 mg / kg of nicotimanide in 0.1 m citrate buffer ( ph 4.5 ) in a volume of 0.5 ml / kg b.w . diabetes was confirmed in the stz - treated rats by measuring fasting blood glucose levels after 48 hours of stz injection using one - touch horison glucometer , with gluco - strips ( ortho - clinical diagnostics , johnson and johnson company , usa ) . after 24 h of stz + nicotinamide injection , animals were given 5% w / v of glucose solution ( 2 ml / kg b.w . ) to prevent initial drug - induced hypoglycemic mortality . rats with fasting blood glucose of more than 200 mg / dl were considered as diabetics . the diabetic rats were divided randomly into group- ii to group- v. the ethanolic extract of the whole plant of t. procumbens linn . ( asteraceae ) doses were selected form previously published reports ( i.e. , 250 and 500 mg / kg ) . the standard ( glibenclamide ) and investigational drugs were suspended in 0.5% w / w carboxymethyl cellulose ( cmc ) and administered once daily through oral gavage for 21 consecutive days . the blood sample ( few drops ) were collected on 1 , 7 , 14 , and 21 day from the tail vein of rats by pricking with sharp tip needle and immediately used for the estimation of blood glucose with glucometer . weekly body weight variations were monitored for all the experimental animals . at the end of the experiment blood sample was withdrawn from all the experimental animals through retro - orbital plexus puncture , and serum was separated . the collected blood was allowed to clot for 30 min and centrifuged at 3000 rpm for 20 min . the serum was used for estimation of biochemical parameters such as liver biomarkers ( sgot and sgpt ) and cholesterols levels ( total cholesterol , triglycerides , and hdl - cholesterol ) . the total cholesterol , triglyceride , hdl - cholesterol , sgot and sgpt levels in significance serum were analyzed using an auto - analyzer ( robonik prietest , india ) using enzymatic kits of coral diagnostics ltd . were tested using one - way anova followed by dennett 's post - hoc test . ( asteraceae ) plant was collected from different regions of madurai district , tamil nadu , india , in july 2011 . plant was identified and authenticated by botanist of agricultural college and research institute , madurai , tamil nadu . the whole plant was dried under the shade for a few days and grinded in electrical grinder to coarse powder . the whole plant powder of t. procumbens was packed in the soxhlet apparatus and extracted with 95% ethanol . later on , the extract was filtered , and the filtrate was concentrated to a dry mass by simple distillation . the male wistar albino rats , aged 4 months ( body weight : 180 10 g ) , were obtained from sainath enterprises , hyderabad , india . the animals were housed in large , spacious poly acrylic cages at an ambient room temperature with 12-h - light/12-h - dark cycle . the study was approved by the institute animal ethics committee of ultra college of pharmacy , madurai , india . all the animal experiments were carried out according to committee for the purpose of control and supervision of experiments on animals ( cpcsea ) guidelines . biochemical assay kits for glucose , serum glutamic pyruvate transaminase ( sgpt ) , serum glutamic oxaloacetic transaminase ( sgot ) , total cholesterol , total protein , triglyceride , and glucose-6-phosphate dehydrogenase kits were procured from coral diagnostics ltd . all other chemicals used were of analytical grade obtained from sd - fine , india . the extract was subjected for phytochemical screening to screen the presence of various constituents such as sterols , alkaloids , tannins , glycosides , flavonoids and saponins/ phenolic compounds by libermann - burchard reaction , dragendroff 's reaction , ferric chloride solution test , keller - kiliani test , shinoda test and lead acetate test respectively . adult , healthy male wistar rats weighing between 180 10 g were used for the experiment . group i - normal control rats group ii - diabetic control group iii - diabetic rats treated with glibenclamide ( 0.25 mg / kg ) group iv - diabetic rats treated with ethanolic extract of the whole plant of t. procumbens ( 250 mg / kg ) group v - diabetic rats treated with ethanolic extract of the whole plant of t. procumbens ( 500 mg / kg ) . diabetes was induced in overnight - fasted rats by a single intra - peritoneal injection of freshly prepared stz 50 mg / kg b.w . followed by 120 mg / kg of nicotimanide in 0.1 m citrate buffer ( ph 4.5 ) in a volume of 0.5 ml / kg b.w . diabetes was confirmed in the stz - treated rats by measuring fasting blood glucose levels after 48 hours of stz injection using one - touch horison glucometer , with gluco - strips ( ortho - clinical diagnostics , johnson and johnson company , usa ) . after 24 h of stz + nicotinamide injection , animals were given 5% w / v of glucose solution ( 2 ml / kg b.w . ) to prevent initial drug - induced hypoglycemic mortality . rats with fasting blood glucose of more than 200 mg / dl were considered as diabetics . the diabetic rats were divided randomly into group- ii to group- v. the ethanolic extract of the whole plant of t. procumbens linn . ( asteraceae ) doses were selected form previously published reports ( i.e. , 250 and 500 mg / kg ) . the standard ( glibenclamide ) and investigational drugs were suspended in 0.5% w / w carboxymethyl cellulose ( cmc ) and the blood sample ( few drops ) were collected on 1 , 7 , 14 , and 21 day from the tail vein of rats by pricking with sharp tip needle and immediately used for the estimation of blood glucose with glucometer . weekly body weight variations were monitored for all the experimental animals . at the end of the experiment blood sample was withdrawn from all the experimental animals through retro - orbital plexus puncture , and serum was separated . the collected blood was allowed to clot for 30 min and centrifuged at 3000 rpm for 20 min . the serum was used for estimation of biochemical parameters such as liver biomarkers ( sgot and sgpt ) and cholesterols levels ( total cholesterol , triglycerides , and hdl - cholesterol ) . the total cholesterol , triglyceride , hdl - cholesterol , sgot and sgpt levels in significance serum were analyzed using an auto - analyzer ( robonik prietest , india ) using enzymatic kits of coral diagnostics ltd . were tested using one - way anova followed by dennett 's post - hoc test . the preliminary phytochemical analysis of an ethanolic extract of the whole plant of t. procumbens showed the presence of alkaloids , tannins , flavonoids , saponins , and phenolic compounds . throughout the study , diabetic control animals showed a significant increases of blood glucose levels ( p<0.001 ) . the standard and investigational drugs treated animals showed a significant reduction in glucose levels when compare to diabetic control . at the end of the study , t. procumbens ( 500 mg/ kg ) and glibenclamide ( 0.25 mg / kg ) treated animals reversed the hyperglycemia induced by stz to normal levels . glibenclamide and ethanolic extract of the whole plant of t. procumbens ( 250 and 500 mg / kg ) showed significant antidiabetic activity after two weeks of administration . ethanolic extract of the whole plant of t. procumbens ( 500 mg / kg ) treated animals showed significant antidiabetic potential from first week of the drug administration onwards [ table 1 ] . the whole plant ethanolic extract of t. procumbens and glibenclamide did not affected the body weight of the animals , but significant reduction in body weight was observed in diabetic control animals [ figure 1 ; p < 0.001 ] . effect of an ethanolic extract of the whole plant of t. procumbens on blood glucose levels in streptozotocin - induced diabetes effect of ethanolic extract of the whole plant of t. procumbens on body weight as shown in table 2 , elevated total cholesterol , triglyceride , ldl cholesterol levels , and reduced hdl cholesterol level were observed in the diabetic control compared with that of normal controls . administration of the ethanolic extract of the whole plant of t. procumbens to stz - diabetic rats protected the alterations of lipid levels . the liver biomarkers such as sgpt , sgot found to be normal in ethanolic extract of the whole plant of t. procumbens- and glibenclamide - treated rats , but the diabetic control showed elevated sgpt , sgot levels at the end of the experiment ( p < 0.001 ) . effect of an ethanolic extract of the whole plant of t. procumbens on lipid profile in streptozotocininduced diabetes the ethanolic extract of the whole plant of t. procumbens at 250 and 500 mg / kg showed significant antidiabetic activity against stz - induced diabetes mellitus in rats , and the effect was comparable with that of the standard drug glibenclamide . loss in body weight was observed in stz - induced diabetes mellitus in rats and was controlled by treatment with ethanolic extract of the whole plant of t. procumbens . administration of an ethanolic extract of the whole plant of t. procumbens to diabetic rats resulted in an increase in body weight compared to diabetic rats . the present study findings suggested that t. procumbens treatment has positive effect on maintaining body weights in diabetic rats . the protective effect of plant fraction on body weight of diabetic rats may be due to its ability to reduce hyperglycemia . a gradual increase in body weights of glibenclamide treated groups was similar to that of normal control rats . stz - induced diabetes mellitus was characterized by severe loss of body weight due to increased muscle wasting in diabetes . stz is a glucosamine - nitrosourea derived from streptomyces achromogenes ( gram - positive bacterium ) , and it is used for the treatment of pancreatic beta cell carcinoma . alcoholic and petroleum ether extracts of leaves of t. procumbens ( 200 mg / kg ) showed significant antidiabetic activity in alloxan monohydrate - induced diabetes mellitus in rats . methanolic extract of the whole plant of t. procumbens showed significant antidiabetic activity in alloxan - induced diabetes mellitus in rats , and the antidiabetic activity might be due to individual or synergistic activity of flavonoids and other active phytoconstituents of the plant . in previous studies , alcoholic and methanolic extract of t. procumbens showed significant antidiabetic activity against alloxan - induced diabetes in rodents , and the present study result findings also suggest the same . plant which have flavonoids , terpenoids , alkaloids , and glycosides have antioxidant activity and claimed to possess antidiabetic effect . flavonoids present in the plant regenerate the damaged beta cells of pancreases , and the polyphenolic compounds and saponin present in the plants inhibit glucose transport by inhibiting sodium glucose co - transporter-1 ( s - glut-1 ) in intestine . antidiabetic effect of an ethanolic extract of the whole plant of t. procumbens might be due to presence of flavonoids , polyphenolic compounds , and saponin in the extract . on bases of the present and pervious study reports , t. procumbens has significant antidiabetic activity against both alloxan- and stz - induced diabetes mellitus in rodents . the action may be due to presence of flavonoids , and further investigation required to rollout the bioactive compound present in the plant . the ethanolic extract of the whole plant of t. procumbens exerts an antidiabetic activity against stz - induced diabetes mellitus in rats . it is also found to be effective in managing the complications associated with diabetes mellitus , such as hyperlipidaemia , and prevents the defects in lipid metabolism .

objective : to study the antidiabetic and antihyperlipidemic effects of an ethanolic extract of the whole plant of tridax procumbens ( asteraceae ) in streptozotocin - induced diabetic rats.materials and methods : the whole plant of t. procumbens was collected in different regions of madurai districts , tamil nadu . the air dried whole plant of t. procumbens was extracted with ethanol ( 95% ) in a soxhlet apparatus for 72 h. diabetes was induced in male wistar rats by streptozotocin ( 50 mg / jk , i.p . ) and nicotinamide ( 120 mg / kg , i.p ) injection . the dry mass of the extract was used for preliminary phytochemical and pharmacological analysis . diabetic rats were treated with glibenclamide ( 0.25 mg / kg , p.o . ) or t. procumbens extract ( 250 and 500 mg / k , p.o . ) for 21 consecutive days . the blood samples were collected at regular intervals to access hypoglycemic effect of an ethanolic extract of the whole plant of t. procumbens . at the end of the experiment , serum lipid profile and liver enzymes levels were analyzed for all the experimental animals and compared with diabetic control.results:the preliminary phytochemical analysis of an ethanolic extract of the whole plant of t. procumbens indicated the presence of alkaloids , tannins , flavonoids , saponins , and phenolic compounds . the ethanolic extract of the whole plant of t. procumbens at 250 and 500 mg / kg has significant antidiabetic and antihyperlipidemic activities . the diabetic control animals exhibited a significant decrease in body weight compared with control animals . t. procumbens inhibited streptozotocin - induced weight loss and significantly alter the lipid levels.conclusion:the ethanolic extract of the whole plant of t. procumbens showed significant antidiabetic and antihyperlipidemic activities against streptozotocin - induced diabetes in rats .

Introduction Materials and Methods Plant collection Extraction of leaves Animals Chemicals and reagents Phytochemical screening Anti-diabetic screening of ethanolic extract of whole plant of Biochemical analysis: Statistical analysis Result Discussion Conclusion

the first macroscopic description of tg belongs to merkel , followed and extended by krause and schaffer . the authors report that the tg composition is characterized by ganglion cells ( pseudounipolar neurons ) , satellite cells and their appendages . histological preparations visualize that the perikarya of the pseudo - unipolar neurons are tightly enveloped by small satellite cells with intimately related neurolemmas . the interior of the ganglion is pierced by a lot of connective tissue filaments , consisting mainly of collagen type iii fibers . externally the ganglia are dorsally enveloped by a multi - layer perineural sheath and an outer fibrous layer - epineurium , as described by schaffer . along with the general principles of the structure of various sensory ganglia , certain specific characteristics of tg , pertaining solely to it , have been observed . first , it has been described that in animals before birth , small islands of nerve cells appear near the neurons , having some signs of astroglial cells normally occurring in the cns . observations and experiments confirm that they are linked together through communicating contacts of the type of gap junctions . secondly , after staining with methods specific for nervous tissue , such as silver impregnation techniques , another type of cells with ultrastructural characteristics of neurons have been observed between the astroglial type of cells and also between myelin nerve fibers in the ganglion root . they have , however , characteristic morphological differences in comparison to the other pseudounipolar sensory neurons . what distinguishes them is primarily the lack of the satellite cuff around the perikaryon of the cell , their form , and last but not least , the arrangement of their cytoplasmic organelles . therefore , it is presumed that they represent central nervous system neurons which have migrated to the periphery . lastly , the tg lies on the border between the peripheral and the central nervous system and possesses the characteristics of both . the diversity in the structure of the trigeminal sensory system in the human and animals has been the subject of extensive research over a long period of time , and the accumulated morphofunctional data are a source of disagreement on various morphological and functional issues . despite a significant amount of experimental data on the normal tg cytoarchitectonics , there are still some gaps in literature . the experiments were done in strict observance of the ethical work principles , applied at the medical university of sofia and the legislative requirements for the protection of rights . the experiments were conducted on twenty pairs of tgs from 20 patients aged 21 to 82 . the material was taken during routine autopsies at the department of forensic medicine and deontology , and the department of general and clinical pathology of the medical university of sofia . after removing the whole brain , the dura mater sheath on the anterior wall of the pyramid was carefully removed and by cutting the three main branches of the trigeminal nerve , the tg was excised . the ganglia were then placed in a solution of 4% neutral formalin and after 5 days fixation the procedure continued with dehydration of the tissue pieces in an ascending series of alcohols , followed by clearing in cedar oil . the samples were embedded in paraffin and then serial cross - sections of 20 m each were cut . after they were mounted on slides some of the serial cross - sections were stained by the nissl method with cresyl violet , toluidine blue or thionine according to the landau modifications [ 710 ] , while others were stained for demonstration of myelin by a technique , modified by woelcke . consistent monitoring of the cytoarchitecture and myeloarchitecture of the structure was ensured by observing stained consecutive sections . during an observation of human tg with a small magnification of the light microscope ( lm ) three separate agglomerations , delicately separated from each other by fibers , passing between them , were seen ( figure 1 ) . each of the cell agglomerations was composed of a cluster of pseudounipolar neurons , diffusely scattered and forming the three branches of the trigeminal nerve ( figure 1 , 3 ) . it was clearly evidenced that each of these agglomerations could be subdivided into two parts , dorsomedial and ventrolateral ( figure 3 ) . the cells , located in the dorsomedial subdivision , were more tightly packed than the cells located in the ventrolateral subdivision , and visibly had a smaller size . the ventrolateral trigeminal neurons had larger perikarya and were more distinctly separated . in fact , these areas contained the cell bodies of the neurons of origin of the three main branches of the trigeminal nerve , i.e. ophthalmic , maxillary and mandibular nerves ( figure 1 , 3 ) . at the beginning of the first and second branches single perikarya enveloped with a less dense cuff of satellite cells were often observed ( figure 6 ) . in spite of the uniform cellular pattern at a low magnification ( figure 1 ) , the larger percentage ( over 80% ) of trigeminal neurons were small- and medium - sized pseudounipolar cells , while a more detailed observation at a larger magnification revealed cells with bodies of various shape : round , oval , fusiform ( polygonal ) and elongated ( figure 1 , 6 ) . the sizes of their cell bodies also widely varied ranging from 15 to 110 m in diameter ( figure 4 ) . apart from their external morphology , the trigeminal ganglion cells differed in the structural features of their nuclei and cytoplasm , as well as the nucleus cytoplasm ratio . the morphological picture revealed that dark stained nucleoli were observed in some of the nuclei ( figure 4 , 5 ) . the thorough study of serial horizontal cross - sections of the tg from rostral to caudal allowed us to differentiate the following types of trigeminal neurons according to the size of their perikaryon : large- , medium- and small - sized neurons ( figure 4 , 5 ) . depending on the intensity of staining of their cell bodies , each of these neuronal types was observed in two varieties , light and dark neurons . according to the shape of the perikarya , the tg cells could be divided into typical ( oval ) and atypical ( elongated and/or fusiform ) neurons . the location of the different tg neurons in the human was established in relation to their morphological patterns . in particular , the large light neurons ( with a diameter > 40 m ) were predominantly located at the periphery of the ganglion , near the site of origin of the three main branches of the trigeminal nerve , though they also occurred inside the tg as agglomerations ( figure 1 , 3 , 6 ) . they were typical pseudounipolar neurons with a pale cytoplasm and large hypochromic oval nuclei with dark nucleoli ( figure 4 , 6 ) . in the human tg , they were diffusely scattered in the three agglomerations of the tg ( figure 3 , 4 ) . the nucleus was centrally located and had an oval shape ( figure 4 ) . according to the density of cytoplasmic staining these neurons were divided into two subtypes : medium - sized light and medium - sized dark neurons ( figure 6 ) . the small neurons were ubiquitous and were also classified as light and dark , according to the staining of their cytoplasm ( figure 4 ) . they had a slightly oval in shape nucleus , usually located eccentrically , and a nucleolus was rarely noticed . along with the described typical oval neurons there were also neurons with a fusiform or polygonal shape . these were mostly medium in size , but also large- and small - sized were seen . these cells were usually located centrally in the neurophil , but sometimes they were found at the initial segments of the three trunks of the nerve ( figure 2 ) . near the individual well - defined agglomerations some elongated tg cells were observed . their perikarya were mostly medium- sized , though some large- and small - sized neurons were also visualized . in the ganglion , stained by the heidenhain method according to the woelcke modification , we found fibers penetrating the tg or originating from it ( figure 3 , 4 , 5 ) . these bundles of fine fibers ran in a fan - like manner in a ventrodorsal direction between the neuronal perikarya and thus they discriminated the agglomerations where the neurons of the three branches of the trigeminal nerve were mainly clustered . during an observation of human tg with a small magnification of the light microscope ( lm ) three separate agglomerations , delicately separated from each other by fibers , passing between them , were seen ( figure 1 ) . each of the cell agglomerations was composed of a cluster of pseudounipolar neurons , diffusely scattered and forming the three branches of the trigeminal nerve ( figure 1 , 3 ) . it was clearly evidenced that each of these agglomerations could be subdivided into two parts , dorsomedial and ventrolateral ( figure 3 ) . the cells , located in the dorsomedial subdivision , were more tightly packed than the cells located in the ventrolateral subdivision , and visibly had a smaller size . the ventrolateral trigeminal neurons had larger perikarya and were more distinctly separated . in fact , these areas contained the cell bodies of the neurons of origin of the three main branches of the trigeminal nerve , i.e. ophthalmic , maxillary and mandibular nerves ( figure 1 , 3 ) . at the beginning of the first and second branches single perikarya enveloped with a less dense cuff of satellite cells were often observed ( figure 6 ) . in spite of the uniform cellular pattern at a low magnification ( figure 1 ) , the larger percentage ( over 80% ) of trigeminal neurons were small- and medium - sized pseudounipolar cells , while a more detailed observation at a larger magnification revealed cells with bodies of various shape : round , oval , fusiform ( polygonal ) and elongated ( figure 1 , 6 ) . the sizes of their cell bodies also widely varied ranging from 15 to 110 m in diameter ( figure 4 ) . apart from their external morphology , the trigeminal ganglion cells differed in the structural features of their nuclei and cytoplasm , as well as the nucleus cytoplasm ratio . the morphological picture revealed that dark stained nucleoli were observed in some of the nuclei ( figure 4 , 5 ) . the thorough study of serial horizontal cross - sections of the tg from rostral to caudal allowed us to differentiate the following types of trigeminal neurons according to the size of their perikaryon : large- , medium- and small - sized neurons ( figure 4 , 5 ) . depending on the intensity of staining of their cell bodies , each of these neuronal types was observed in two varieties , light and dark neurons . according to the shape of the perikarya , the tg cells could be divided into typical ( oval ) and atypical ( elongated and/or fusiform ) neurons . the location of the different tg neurons in the human was established in relation to their morphological patterns . in particular , the large light neurons ( with a diameter > 40 m ) were predominantly located at the periphery of the ganglion , near the site of origin of the three main branches of the trigeminal nerve , though they also occurred inside the tg as agglomerations ( figure 1 , 3 , 6 ) . they were typical pseudounipolar neurons with a pale cytoplasm and large hypochromic oval nuclei with dark nucleoli ( figure 4 , 6 ) . in the human tg , they were diffusely scattered in the three agglomerations of the tg ( figure 3 , 4 ) . the nucleus was centrally located and had an oval shape ( figure 4 ) . according to the density of cytoplasmic staining these neurons were divided into two subtypes : medium - sized light and medium - sized dark neurons ( figure 6 ) . the small neurons were ubiquitous and were also classified as light and dark , according to the staining of their cytoplasm ( figure 4 ) . they had a slightly oval in shape nucleus , usually located eccentrically , and a nucleolus was rarely noticed . along with the described typical oval neurons there were also neurons with a fusiform or polygonal shape . these were mostly medium in size , but also large- and small - sized were seen . these cells were usually located centrally in the neurophil , but sometimes they were found at the initial segments of the three trunks of the nerve ( figure 2 ) . near the individual well - defined agglomerations some elongated tg cells were observed . their perikarya were mostly medium- sized , though some large- and small - sized neurons were also visualized . in the ganglion , stained by the heidenhain method according to the woelcke modification , we found fibers penetrating the tg or originating from it ( figure 3 , 4 , 5 ) . these bundles of fine fibers ran in a fan - like manner in a ventrodorsal direction between the neuronal perikarya and thus they discriminated the agglomerations where the neurons of the three branches of the trigeminal nerve were mainly clustered . disclosing the cytoarchitecture of the tg is directly dependent on the methods used . despite the numerous studies on the ganglion , performed by using the nissl staining method , and other methods less often used for visualizing pseudounipolar neurons , such as the golgi method , a full knowledge about the tg cytoarchitecture still requires additional data . for this purpose , we applied a standard approach and routine methods for its staining . preparations of human material from both sexes from fertile to senile age were used in forensic studies . the great quantity of studied material is the grounds for our claim of the comprehen - siveness and credibility of the present research . in general , the experimental data on the cytoarchitectonic charac - teristics of the human tg , obtained in this study , correlate with the results of other authors who worked on sensory ganglia in animal species . here we report considerable differences in the arrangement of the represented neuron groups , their classification and characteristics as follows . neurons of a large size are described in the works of several authors , studying the tg in various animal species and the human . this type of pseudounipolar neurons prevails in the peripheral part of the ganglion near the point of origin of the three main branches of the trigeminal nerve , and also at the most dorsal edge of the trigeminal ganglion in man . we have very rarely observed the accumulation of pigment and its content increases with age in the human tg . the perikaryal shape of this neuronal type is typically oval and characteristic of pseudounipolar neurons , but neurons with an elongated and fusiform shape , with an oval nucleus and a compact nucleolus , also occur . this is the most common neuronal type of pseudounipolar neurons observed in this study , in the three agglomerations , giving rise to the respective main branches of the trigeminal nerve , especially in the human tg . despite the numerous cytoarchitectonic studies of the tg in cats , rodents and other mammals , performed by a number of authors we did not observe a significant difference in their size and diversity , associated with the pigmentation of their cytoplasm , i.e. between neurons with lighter , and those with darker cytoplasm , which is consistent with the findings of other authors , working on the topic . neurons with darker pigmented cytoplasm prevail in sites , specifically at the origin of the three main trigeminal branches . small neurons are scattered throughout all the agglomerations , from the ventro - medial to the dorsolateral pole of the human tg . as already noted , we can divide them into neurons with a lighter and darker cytoplasm . in morphometric measurements we have found that in comparison to the lighter ones , the darker are with a smaller diameter of their perikarya . often the darker - stained neurons have an irregular , and in some cases , a fusiform cell body . small neurons are the best impregnated cell group of the pseudounipolar neurons , according to the above mentioned authors , a statement we are in agreement with . under the light microscope , in the human tg , a significant difference between small and dark neurons may be evidenced , when compared to light neurons . neurons of a large size are described in the works of several authors , studying the tg in various animal species and the human . this type of pseudounipolar neurons prevails in the peripheral part of the ganglion near the point of origin of the three main branches of the trigeminal nerve , and also at the most dorsal edge of the trigeminal ganglion in man . we have very rarely observed the accumulation of pigment and its content increases with age in the human tg . the perikaryal shape of this neuronal type is typically oval and characteristic of pseudounipolar neurons , but neurons with an elongated and fusiform shape , with an oval nucleus and a compact nucleolus , also occur . this is the most common neuronal type of pseudounipolar neurons observed in this study , in the three agglomerations , giving rise to the respective main branches of the trigeminal nerve , especially in the human tg . despite the numerous cytoarchitectonic studies of the tg in cats , rodents and other mammals , performed by a number of authors we did not observe a significant difference in their size and diversity , associated with the pigmentation of their cytoplasm , i.e. between neurons with lighter , and those with darker cytoplasm , which is consistent with the findings of other authors , working on the topic . neurons with darker pigmented cytoplasm prevail in sites , specifically at the origin of the three main trigeminal branches . small neurons are scattered throughout all the agglomerations , from the ventro - medial to the dorsolateral pole of the human tg . as already noted , we can divide them into neurons with a lighter and darker cytoplasm . in morphometric measurements we have found that in comparison to the lighter ones , the darker are with a smaller diameter of their perikarya . often the darker - stained neurons have an irregular , and in some cases , a fusiform cell body . small neurons are the best impregnated cell group of the pseudounipolar neurons , according to the above mentioned authors , a statement we are in agreement with . under the light microscope , in the human tg , a significant difference between small and dark neurons may be evidenced , when compared to light neurons .

the trigeminal ganglion ( tg ) , a cluster of pseudounipolar neurons , is located in the trigeminal impression of the temporal pyramid . it is covered by a sheath of the dura mater and arachnoid and is near the rear end of the cavernous sinus . the peripheral processes of the pseudounipolar cells are involved in the formation of the first and second branch and the sensory part of the third branch of the fifth cranial nerve , and the central ones form the sensory root of the nerve , which penetrates at the level of the middle cerebellar peduncle , aside from the pons , and terminate in the sensory nuclei of the trigeminal complex . we found that the primary sensory neurons involved in sensory innervation of the orofacial complex are a diverse group . although they possess the general structure of pseudounipolar neurons , there are significant differences among them , seen in varying intensities of staining . based on our investigations we classified the neurons into 7 groups , i.e. large , subdivided into light and dark , medium , also light and dark , and small light and dark , and , moreover , neurons with an irregular shape of their perikarya . further research by applying various immunohistochemical methods will clarify whether differences in the morphological patterns of the neurons are associated with differences in the neurochemical composition of various neuronal types .

Introduction Materials and methods Results Lightmicroscopic study of the trigeminal ganglion Discussion Large light neurons Medium-sized neurons Small neurons

cellular allograft rejection is one of the commonest causes of graft dysfunction in the early as well as late posttransplant period . chemokines induce migration of lymphocytes bearing specific chemokine receptors , which is central to allograft rejection , as has been emphasized in many experimental and human studies.[46 ] the chemokine receptor cc chemokine receptor 5 ( ccr5 ) is expressed on the alloaggressive th1 cells , which are responsible for activating macrophages and mediating cytotoxicity via fas - fas ligand in the graft . this study aimed to determine the distribution of ccr5-positive cells in the graft and correlate it with the degree of cellular rejection . in this prospective study , 28 patients who had presented with graft dysfunction and demonstrated features of acute cellular rejection ( acr ) on biopsy [ figure 1a and b ] were included . c4d immunostaining was performed in all cases to exclude a concomitant antibody - mediated rejection . cases of graft dysfunction attributable to calcineurin inhibitor toxicity or viral infection on biopsy were also excluded . ( a ) foci of tubulitis ( h and e , 200 ) ; ( b ) foci of tubulitis ( pas , 200 ) ; ( c ) lymphocytes in interstitium and foci of tubulitis ( anti - ccr5 , 200 ) ; ( d ) lymphocytes in interstitium and foci of tubulitis ( anti - cd3 , 200 ) sequential slides were subjected to immunohistochemistry for cd3 ( rabbit polyclonal abcam ab5690 1:100 ) and ccr5 ( rabbit polyclonal abd serotec ahp568 1:150 ) using conventional techniques . the slides were examined by two pathologists ( akd and rg ) . in the same area of the section , both cd3- and ccr5-positive cells were counted separately in the tubules , interstitium , and glomeruli , and expressed as number of cells per high power field ( hpf ) . banff grading of acr depends on the quantity of leukocytes infiltrating the interstititum , tubules , and arterioles . thus , we correlated ccr5-positive t lymphocytes with the total grade of rejection as well as the individual components such as tubulitis , intimal arteritis , and interstitial inflammation using statistical tools of correlation such as spearman correlation and pearsons correlation . sequential slides were subjected to immunohistochemistry for cd3 ( rabbit polyclonal abcam ab5690 1:100 ) and ccr5 ( rabbit polyclonal abd serotec ahp568 1:150 ) using conventional techniques . the slides were examined by two pathologists ( akd and rg ) . in the same area of the section , both cd3- and ccr5-positive cells were counted separately in the tubules , interstitium , and glomeruli , and expressed as number of cells per high power field ( hpf ) . banff grading of acr depends on the quantity of leukocytes infiltrating the interstititum , tubules , and arterioles . thus , we correlated ccr5-positive t lymphocytes with the total grade of rejection as well as the individual components such as tubulitis , intimal arteritis , and interstitial inflammation using statistical tools of correlation such as spearman correlation and pearsons correlation . on histopathologic examination , the cases showed acr of variable banff grades , as detailed in table 2a and b. none of the protocol biopsies showed features to suggest borderline / acr . clinical characteristics of the study population histological and immunohistochemical analysis of banff grade i cases histological and immunohistochemical analysis of banff grade ii - iii cases immunohistochemical analysis revealed that the cases had a significantly higher number of cd3-positive ( 35.16 vs. 2.82 ) and ccr5-positive cells ( 15.48 vs. 0.64 ) compared to the control group ( p = 0.010 ) , and the pattern of distribution between ccr5- and cd3-positive cells in sequential sections was closely comparable ( p < 0.01)[figure 1c and d ] . in cases with rejection , the ccr5-positive lymphocytes were observed to be significantly more in the tubulointerstitium than in the foci of glomerulitis ( 15.48 vs. 0.32 ; p = 0.007 ) and also more in the foci of tubulitis compared to the interstitium , although this did not reach levels of significance ( p = 0.351 ) . quantitation of cd3- and ccr5-positive cells infiltrating the arterial intima could not be performed due to very small numbers of infiltrating cells ; however , few infiltrating ccr5-positive cells were noted [ figure 2 ] . correlation of chemokine receptor 5-positive cells in foci of tubulitis with interval after transplant a positive correlation was found between ccr5-positive cells and grading of rejection by banff 2007 ( p < 0.05 ) , that is , higher grade of rejection had higher number of ccr5-positive cells . percent of t cells expressing ccr5 in the foci of tubulitis positively correlated with the severity of tubulitis , and also with the severity of interstitial inflammation ( p < 0.05 ) . no significant correlation with chronic features such as interstitial fibrosis and tubular atrophy was noted . no correlation was noted with the serum creatinine levels at the time of the biopsy , and the number of ccr5-positive cells did not predict response to antirejection therapy determined by follow - up serum creatinine levels ( p = 0.134 ) . thus , although the number of ccr5-positive cells correlated with the grade of rejection , it did not correlate with the degree of graft dysfunction or response to therapy within rejection cases . the patient population was divided into two groups : one containing tacrolimus and the other containing cyclosporine . patients on tacrolimus containing regimens were observed to have less total number of ccr5-positive cells , particularly in the foci of tubulitis ( p < 0.05 ) . in relation to the time interval after transplantation , the mean count of t cells expressing ccr5 in the foci of tubulitis was significantly more in rejection occurring within 6 months of transplantation than in that occurring after a period of 6 months ( p < 0.05 ) . in cases with rejection , the ccr5-positive lymphocytes were observed to be significantly more in the tubulointerstitium than in the foci of glomerulitis ( 15.48 vs. 0.32 ; p = 0.007 ) and also more in the foci of tubulitis compared to the interstitium , although this did not reach levels of significance ( p = 0.351 ) . quantitation of cd3- and ccr5-positive cells infiltrating the arterial intima could not be performed due to very small numbers of infiltrating cells ; however , few infiltrating ccr5-positive cells were noted [ figure 2 ] . correlation of chemokine receptor 5-positive cells in foci of tubulitis with interval after transplant a positive correlation was found between ccr5-positive cells and grading of rejection by banff 2007 ( p < 0.05 ) , that is , higher grade of rejection had higher number of ccr5-positive cells . percent of t cells expressing ccr5 in the foci of tubulitis positively correlated with the severity of tubulitis , and also with the severity of interstitial inflammation ( p < 0.05 ) . no significant correlation with chronic features such as interstitial fibrosis and tubular atrophy was noted . no correlation was noted with the serum creatinine levels at the time of the biopsy , and the number of ccr5-positive cells did not predict response to antirejection therapy determined by follow - up serum creatinine levels ( p = 0.134 ) . thus , although the number of ccr5-positive cells correlated with the grade of rejection , it did not correlate with the degree of graft dysfunction or response to therapy within rejection cases . the patient population was divided into two groups : one containing tacrolimus and the other containing cyclosporine . patients on tacrolimus containing regimens were observed to have less total number of ccr5-positive cells , particularly in the foci of tubulitis ( p < 0.05 ) . in relation to the time interval after transplantation , the mean count of t cells expressing ccr5 in the foci of tubulitis was significantly more in rejection occurring within 6 months of transplantation than in that occurring after a period of 6 months ( p < 0.05 ) . as the renal allograft is recognized as foreign , an allospecific immune response is triggered which is largely mediated by chemokines . an increase in concentration of the cc chemokine regulated and normal t cell expressed and secreted ( rantes ) in the allograft has been demonstrated in cases of cellular rejection.[911 ] this chemokine is secreted by activated fibroblasts , and mesangial and tubular epithelial cells in the graft , and induces the migration of cd3-positive t cells expressing the ccr5 receptor . studied the distribution of ccr5-positive cells by immunohistochemistry in cases of glomerulonephritis , interstitial nephritis , and transplant rejection . in the 18 transplant cases ( 9 acute and 9 chronic ) , ccr5-positive cells were seen in all interstitial areas , with mononuclear cell infiltrates constituting approximately 90% of the cd3-positive cells in cases of acute rejection and approximately 65% in cases of chronic rejection . seven of the nine cases of acute rejection were of banff grade i. however , no correlations with histopathology were made . in clinical correlations , they found that the mean ccr5-positive cells per cortical hpf was significantly higher in cases with creatinine more than 1.2 mg / dl ( p < 0.01 ) . in another study , segerer et al . showed an increase in mrna expression of ccr5 , ccr2b , and cxcr4 in allograft infiltrating leukocytes , compared to that in controls . compared ccr5 and cxcr3 expression in 13 cases of acute rejection and an equal number of cases without rejection in biopsies . they found significantly higher numbers of ccr5-positive cells in acute rejection ( 160.9 33.1/30 hpfs ) compared to no rejection cases ( 36.2 10.7 , p < 0.007 ) , similar to that in this present study . their cases varied from banff grade i a to ii b ; however , no histopathologic correlations were made . we also found significantly increased number of ccr5-positive cells in patients on cyclosporine compared to those on tacrolimus containing regimen . daniel seron et al . reported increased cd3- and cd68-positive allograft infiltrating cells in patients on cyclosporine compared to those on tacrolimus , similar to our study . however , ours is the first study correlating cc chemokine receptor 5 in these two groups and shows significantly more ccr5-positive cells in patients on cyclosporine containing regime compared to those on tacrolimus . one important observation in cases 1 and 8 [ table 2a ] and cases 4 , 11 , and 14 [ table 2b ] was the presence of very few or no ccr5-positive cells . a possible explanation to this could be that ccr5 , which is an early marker of rejection responsible for recruitment of the leukocytes , may not be observed in large numbers once rejection is for a longer time and well established . almost all of these cases had intimal arteritis ( except case 1 ) , pointing toward increased severity of the cases . in this study , it was again confirmed that ccr5-positive cells are significantly increased in rejecting compared to stable grafts and are found predominantly in the foci of interstitial inflammation and tubulitis . in addition , histopathologic correlations showed significant increase in the infiltrating ccr5-positive cells with worsening banff grades . early acr had significantly more ccr5-positive t cells in foci of tubulitis compared to late acr . with animal model experiments demonstrating that blockade of ccr5 receptors leads to retardation of rejection episodes , the findings of this study also indicate a future role of anti - ccr5 therapy in the treatment of rejection , particularly early episodes . in addition , as ccr5 represents the actively alloaggressive subset of t cells and increases with worsening of rejection , the authors propose that it be added to the ever - expanding list of activation markers for t cells , and future studies on cases of borderline rejection are warranted .

allospecific recruitment of t cells is primary to the pathogenesis of renal transplant rejection . chemokines and their receptors inducing a th1 cytokine response play a central role in this recruitment . renal allograft biopsies of 28 patients with acute cellular rejection and 10 protocol biopsies ( controls ) were examined in accordance with banff grading 2007 schema . immunohistochemistry for cd3 and cc chemokine receptor 5 ( ccr5 ) in sequential sections was performed and quantitatively assessed in the glomeruli , tubules , and interstitium . histopathologic and clinical correlations were carried out . cd3- and ccr5-positive cells were observed in significantly higher numbers in rejection cases than in controls ( p = 0.010 ) . a larger proportion of ccr5-positive cells were noted in the foci of tubulitis compared to the interstitial infiltrates and glomeruli in all cases , and it correlated with the grade of cellular rejection ( p = 0.010 ) . a greater number of ccr5-positive cells were seen in early rejection ( < 6 months posttransplant ) compared to late rejection . no clinical correlation with serum creatinine levels was found . ccr5-positive cells represent the alloaggressive subset of t cells in acr , and their numbers correlate with rejection severity . ccr5 may be used as a marker of early acute rejection and may be an important target for future antirejection therapies .

Introduction Subjects and Methods Immunohistochemistry Results Distribution of CC chemokine receptor 5-positive cells and histopathologic correlations Clinical correlations Discussion

edible plants ( 100 g ) were minced into 5 mm fragments and extracts were obtained with methanol ( 200 ml ) overnight at room temperature . the extracts were filtered and the residue was re - extracted under the same conditions as above . the combined filtrates were evaporated in a vacuum slightly below 40c in a rotary evaporator . the specimen was dissolved in dmso ( 1.0 ml ) . human umbilical vein endothelial cells ( no . jcrb0408 , health science research resources bank , osaka , japan ) were cultured at 37c in sfm cs - c medium ( ds pharma biomedical , osaka , japan ) supplemented with 1.5 g / ml fungizone under air - co2 ( 95:5 ) ( referred to hereafter as cs - cf medium ) . for screening study purposes , huvecs were seeded in 60 mm culture dishes ( 1.5 10 cells ) . then , huvecs were incubated with 1 g / l ( normal glucose medium ) or 4.5 g / l ( high glucose medium ) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed , one receiving 1 ) continuous 10 l meep , and the other receiving 2 ) continuous 100 l meep . determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer 's protocol ( b - bridge international , inc . , supernatants cultured with each sample were added to elisa plates to measure the esrage content . dna of huvecs was extracted from the homogenates using a dna extractor wb kit ( wako pure chemical industries , osaka , japan ) according to the protocol . the 8-ohdg level was determined by using the 8-ohdg check elisa kit ( japan institute for the control of aging , shizuoka , japan ) according to the manufacturer 's protocol . the recovery rate of 8-ohdg level was expressed as % recovery of the normal glucose medium a450 nm ( experimental a450nm / normal glucose medium a450 nm 100 ) . peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein ( dcdhf ) to 2,7-dichlorofluorescein ( dcf ) was estimated based on the method described by crow . samples or control ( dmso ) were added to 100 mm pbs ( ph 7.4 ) containing 100 m diethylenetriamine - n , n , n , n , n-pentaacetic acid ( dojindo molecular technologies , inc . , japan ) , 5 m sodium peroxynitrite and 100 m dcdhf . the peroxynitrite scavenging activity was expressed as % inhibition of the control a500 nm ( [ control a500 nm experimental a500nm]/control a500 nm 100 ) . because meep were methanolic extracts , we measured their tpc . tpc was measured with a modified version of the folin briefly , 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . we allowed the mixture to sit for 60 min before reading absorbance at 750 nm using a uv-460 spectrophotometer ( nihonbunko ltd . , the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . a statistical comparison between the groups was carried out using either anova or student 's t - test . edible plants ( 100 g ) were minced into 5 mm fragments and extracts were obtained with methanol ( 200 ml ) overnight at room temperature . the extracts were filtered and the residue was re - extracted under the same conditions as above . the combined filtrates were evaporated in a vacuum slightly below 40c in a rotary evaporator . jcrb0408 , health science research resources bank , osaka , japan ) were cultured at 37c in sfm cs - c medium ( ds pharma biomedical , osaka , japan ) supplemented with 1.5 g / ml fungizone under air - co2 ( 95:5 ) ( referred to hereafter as cs - cf medium ) . for screening study purposes , huvecs were seeded in 60 mm culture dishes ( 1.5 10 cells ) . then , huvecs were incubated with 1 g / l ( normal glucose medium ) or 4.5 g / l ( high glucose medium ) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed , one receiving 1 ) continuous 10 l meep , and the other receiving 2 ) continuous 100 l meep . determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer 's protocol ( b - bridge international , inc . , supernatants cultured with each sample were added to elisa plates to measure the esrage content . dna of huvecs was extracted from the homogenates using a dna extractor wb kit ( wako pure chemical industries , osaka , japan ) according to the protocol . the 8-ohdg level was determined by using the 8-ohdg check elisa kit ( japan institute for the control of aging , shizuoka , japan ) according to the manufacturer 's protocol . the recovery rate of 8-ohdg level was expressed as % recovery of the normal glucose medium a450 nm ( experimental a450nm / normal glucose medium a450 nm 100 ) . peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein ( dcdhf ) to 2,7-dichlorofluorescein ( dcf ) was estimated based on the method described by crow . samples or control ( dmso ) were added to 100 mm pbs ( ph 7.4 ) containing 100 m diethylenetriamine - n , n , n , n , n-pentaacetic acid ( dojindo molecular technologies , inc . , japan ) , 5 m sodium peroxynitrite and 100 m dcdhf . the peroxynitrite scavenging activity was expressed as % inhibition of the control a500 nm ( [ control a500 nm experimental a500nm]/control a500 nm 100 ) . because meep were methanolic extracts , we measured their tpc . tpc was measured with a modified version of the folin briefly , 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . we allowed the mixture to sit for 60 min before reading absorbance at 750 nm using a uv-460 spectrophotometer ( nihonbunko ltd . , the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . a statistical comparison between the groups was carried out using either anova or student 's t - test . we examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . the result showed an increase in esrage production in the following gight samples ( eggplant [ solanum melongena ] , carrot peel [ daucus carota subsp . sativus ] , young sweet corn [ zea mays saccharata ] , broad bean [ vicia faba ] , japanese radish [ raphanus sativus var . longipinnatus ] sprout , jew 's marrow [ corchorus olitorius ] and cauliflower [ brassica oleracea var . the results of the esrage production of these eight samples on huvecs are summarized in figure 1 . figure 1a shows the effects of the eight samples ( 10 l ) on esrage production in high glucose - induced huvecs . the esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . genistein ( 10 m ) increased esrage production by approximately 12% , but egcg ( 10 m ) caused no increase . the results for plant samples suggest that white radish sprout , jew 's marrow and cauliflower have esrage production potential on induced endothelial cells . in particular , white radish sprout , which had higher esrage production potential than genistein , significantly ( p < 0.01 ) increased esrage production . in addition , white radish sprout showed an esrage production potential approximately 2.4 times higher than the control ( 4.5 g / l of glucose only ) . on the other hand , in 100 l meep [ figure 1b ] , more samples had a significant influence on esrage production . the results suggest that eggplant , carrot peel , young sweet corn , broad bean , japanese radish , and white radish sprout have esrage production potential on induced endothelial cells . however , different meep seem to have different effects on esrage production in the presence of high glucose . in 100 m concentration , genistein also showed a tendency to increase slightly , but the increase was not seen in egcg . in studies to date , no plant has yet been shown to increase esrage production . therefore , the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . however , it was reported through similar experimental techniques that hibiscus sabdariffa polyphenolic extract inhibited the expression of rage . furthermore , lu et al . indicated that atorvastatin exerted a beneficial effect on diabetic nephropathy with reduced age accumulation , down - regulating rage expression and up - regulating srage in the kidney . from the above - mentioned report , the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . however , although lee and lee show that 5 m egcg down - regulates the rage expression , this may not necessarily indicate an inverse correlation between rage and esrage because in our study 10 m egcg does not increase esrage production . effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products ( esrage ) production in human umbilical vein endothelial cells cultured in high glucose . the esrage productions of control ( dimethyl sulfoxide ) and samples in human umbilical vein endothelial cells cultured in high ( 4.5 two groups of experiments were formed , one receiving continuous 10 l methanolic extracts from edible plants ( meep ) ( a ) and the other receiving continuous 100 l meep ( b ) values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . performed the study to compare concentration of srage and its ligands ( en - rage and hmgb1 ) in type 1 ( t1 dm ) and t2 dm . similarly , en - rage was significantly higher in both diabetic groups and hmgb1 concentrations were elevated in t2 dm patients . they concluded that serum srage and rage ligands concentrations reflect endothelial dysfunction developing in diabetes . therefore , the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . on the other hand , some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . on other word , showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . in addition , liu et al . showed that administration of euk134 ( peroxynitrite scavenger ) attenuated rage expression , and decreased cardiomyocyte apoptosis in diabetic mice . therefore , we examined the correlation between esrage production , and the two indicators mentioned above ( peroxynitrite and 8-ohdg levels ) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . in our experiment , the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml ( data not shown ) . the formation of 8-ohdg in calf thymus dna by 3-morpholinosydnomine n - ethylcarbamide ( sin-1 ; peroxynitrite donor ) was also inhibited by egcg . in our experiment , egcg ( 100 m ) showed 8-ohdg recovery rates of approximately 90% , but genistein ( 100 m ) caused 8-ohdg recovery rates of approximately 60% . the calculated 8-ohdg recovery rates for white radish sprout , broad bean , young sweet corn , japanese radish , eggplant , jew 's marrow , cauliflower , and carrot peel were 61.9% , 47.7% , 68.9% , 55.5% , 43.8% , 62.0% , 60.2% , and 77.1% , respectively in comparison to the normal glucose medium [ figure 2 ] indicating that the samples are potent inhibitors of 8-ohdg . in particular , carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein , significantly ( p < 0.05 ) decreased 8-ohdg level . in addition , carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . figure 3 shows the correlation between esrage production and 8-ohdg level of eight plant extracts . the results show that the 8-ohdg level does not correlate with esrage production ( r = 0.086 ) . the 8-ohdg recovery rates of control ( normal glucose medium ) and samples ( high glucose medium ) are indicated by unshaded and shaded columns , respectively . values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls . the correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants figure 4 shows the calculated peroxynitrite scavenging activities of eight extracts . because peroxynitrite has a short half - life , the measurement of the intracellular scavenging activity is difficult . therefore , we performed the scavenging activity measurement of the sample in the test tube . amongst the eight extracts , white radish sprout revealed the highest scavenging activity ( 44.8% ) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . the peroxynitrite level significantly correlated with esrage production ( r = -0.706 , p < 0.10 ) . wu et al . showed that treatment of sinoaortic denervated rats with srage abated aortic oxidative stress . this was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the correlation between esrage production and tpc was analyzed [ figure 6 ] . the correlation coefficient for esrage production was found to be greater than 0.6 ( r = 0.660 , p < 0.10 ) . a positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . the correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . however , the seven meep except for eggplant contain a lot of quercetin . because there was a positive correlation between tpc and esrage [ figure 6 ] , it is very likely to be quercetin that had an effect . the effect of these seven plants may resemble the mechanism that zhang et al . showed . hyperoside , quercetin-3-o - galactoside , is a flavonoid isolated from many medicinal plants . in their studies , quiescent ecv304 ( human endothelial - like ) cells were treated in vitro with advanced glycation end products ( ages ) in the presence or absence of hyperoside . the results demonstrated that ages induced c - jun n - terminal kinases ( jnk ) activation and apoptosis in ecv304 cells . furthermore , hyperoside significantly inhibited rage expression in age - stimulated ecv304 cells , whereas knockdown of rage inhibited age - induced jnk activation . these results suggested that ages may promote jnk activation , leading to viability inhibition of ecv304 cells via the rage signaling pathway . furthermore , from the above - mentioned report , the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes . we examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . the result showed an increase in esrage production in the following gight samples ( eggplant [ solanum melongena ] , carrot peel [ daucus carota subsp . sativus ] , young sweet corn [ zea mays saccharata ] , broad bean [ vicia faba ] , japanese radish [ raphanus sativus var . longipinnatus ] sprout , jew 's marrow [ corchorus olitorius ] and cauliflower [ brassica oleracea var . the results of the esrage production of these eight samples on huvecs are summarized in figure 1 . figure 1a shows the effects of the eight samples ( 10 l ) on esrage production in high glucose - induced huvecs . the esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . genistein ( 10 m ) increased esrage production by approximately 12% , but egcg ( 10 m ) caused no increase . the results for plant samples suggest that white radish sprout , jew 's marrow and cauliflower have esrage production potential on induced endothelial cells . in particular , white radish sprout , which had higher esrage production potential than genistein , significantly ( p < 0.01 ) increased esrage production . in addition , white radish sprout showed an esrage production potential approximately 2.4 times higher than the control ( 4.5 g / l of glucose only ) . on the other hand , in 100 l meep [ figure 1b ] , more samples had a significant influence on esrage production . the results suggest that eggplant , carrot peel , young sweet corn , broad bean , japanese radish , and white radish sprout have esrage production potential on induced endothelial cells . however , different meep seem to have different effects on esrage production in the presence of high glucose . in 100 m concentration , genistein also showed a tendency to increase slightly , but the increase was not seen in egcg . in studies to date , no plant has yet been shown to increase esrage production . therefore , the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . however , it was reported through similar experimental techniques that hibiscus sabdariffa polyphenolic extract inhibited the expression of rage . furthermore , lu et al . indicated that atorvastatin exerted a beneficial effect on diabetic nephropathy with reduced age accumulation , down - regulating rage expression and up - regulating srage in the kidney . from the above - mentioned report , the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . however , although lee and lee show that 5 m egcg down - regulates the rage expression , this may not necessarily indicate an inverse correlation between rage and esrage because in our study 10 m egcg does not increase esrage production . effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products ( esrage ) production in human umbilical vein endothelial cells cultured in high glucose . the esrage productions of control ( dimethyl sulfoxide ) and samples in human umbilical vein endothelial cells cultured in high ( 4.5 two groups of experiments were formed , one receiving continuous 10 l methanolic extracts from edible plants ( meep ) ( a ) and the other receiving continuous 100 l meep ( b ) values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . performed the study to compare concentration of srage and its ligands ( en - rage and hmgb1 ) in type 1 ( t1 dm ) and t2 dm . similarly , en - rage was significantly higher in both diabetic groups and hmgb1 concentrations were elevated in t2 dm patients . they concluded that serum srage and rage ligands concentrations reflect endothelial dysfunction developing in diabetes . therefore , the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . on the other hand , some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . on other word , showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . in addition , liu et al . showed that administration of euk134 ( peroxynitrite scavenger ) attenuated rage expression , and decreased cardiomyocyte apoptosis in diabetic mice . therefore , we examined the correlation between esrage production , and the two indicators mentioned above ( peroxynitrite and 8-ohdg levels ) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . in our experiment , the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml ( data not shown ) . the formation of 8-ohdg in calf thymus dna by 3-morpholinosydnomine n - ethylcarbamide ( sin-1 ; peroxynitrite donor ) was also inhibited by egcg . in our experiment , egcg ( 100 m ) showed 8-ohdg recovery rates of approximately 90% , but genistein ( 100 m ) caused 8-ohdg recovery rates of approximately 60% . the calculated 8-ohdg recovery rates for white radish sprout , broad bean , young sweet corn , japanese radish , eggplant , jew 's marrow , cauliflower , and carrot peel were 61.9% , 47.7% , 68.9% , 55.5% , 43.8% , 62.0% , 60.2% , and 77.1% , respectively in comparison to the normal glucose medium [ figure 2 ] indicating that the samples are potent inhibitors of 8-ohdg . in particular , carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein , significantly ( p < 0.05 ) decreased 8-ohdg level . in addition , carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . figure 3 shows the correlation between esrage production and 8-ohdg level of eight plant extracts . the results show that the 8-ohdg level does not correlate with esrage production ( r = 0.086 ) . the 8-ohdg recovery rates of control ( normal glucose medium ) and samples ( high glucose medium ) are indicated by unshaded and shaded columns , respectively . values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls . the correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants because peroxynitrite has a short half - life , the measurement of the intracellular scavenging activity is difficult . therefore , we performed the scavenging activity measurement of the sample in the test tube . amongst the eight extracts , white radish sprout revealed the highest scavenging activity ( 44.8% ) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . the peroxynitrite level significantly correlated with esrage production ( r = -0.706 , p < 0.10 ) . wu et al . showed that treatment of sinoaortic denervated rats with srage abated aortic oxidative stress . this was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the tpc of the eight meep significantly correlated with esrage production . the correlation coefficient for esrage production was found to be greater than 0.6 ( r = 0.660 , p < 0.10 ) . a positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . the correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . because there was a positive correlation between tpc and esrage [ figure 6 ] , it is very likely to be quercetin that had an effect . the effect of these seven plants may resemble the mechanism that zhang et al . showed . hyperoside , quercetin-3-o - galactoside , is a flavonoid isolated from many medicinal plants . in their studies , quiescent ecv304 ( human endothelial - like ) cells were treated in vitro with advanced glycation end products ( ages ) in the presence or absence of hyperoside . the results demonstrated that ages induced c - jun n - terminal kinases ( jnk ) activation and apoptosis in ecv304 cells . furthermore , hyperoside significantly inhibited rage expression in age - stimulated ecv304 cells , whereas knockdown of rage inhibited age - induced jnk activation . these results suggested that ages may promote jnk activation , leading to viability inhibition of ecv304 cells via the rage signaling pathway . furthermore , from the above - mentioned report , the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes .

background : in diabetic populations , endogenous secretory receptor for advanced glycation end products ( esrage ) levels may be related to the degree of diabetic complications or to the protection from diabetic complications.objective:we investigated the impact of 29 methanolic extracts from edible plants on esrage production in human umbilical vein endothelial cells ( huvecs ) cultured in high ( 4.5 g / l ) glucose.materials and methods : edible plants were minced , and extracts were obtained with methanol overnight . the methanolic extracts from 29 edible plants were evaporated in a vacuum . for screening study purposes , huvecs were seeded in culture dishes ( 1.5 105 cells ) . then , huvecs were incubated with 1 g / l or 4.5 g / l of glucose in sfm cs - c medium treated with methanolic extracts from edible plants ( meep ) for 96 h. determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa . the 8-hydroxydeoxyguanosine ( 8-ohdg ) level was determined by using the 8-ohdg check elisa kit . peroxynitrite - dependent oxidation of 2 , 7-dichlorodihydrofluorescein to 2 , 7-dichlorofluorescein was estimated based on the method described by crow . because meep were methanolic extracts , we measured their total phenolic content ( tpc ) . tpc was measured with a modified version of the folin ciocalteu method.results:the results showed eight extracts increased esrage production . the extract from white radish sprouts showed the highest esrage production activity , and then eggplant , carrot peel , young sweet corn , jew 's marrow , broad bean , japanese radish and cauliflower . in order to understand the mechanism of esrage production , the eight extracts were examined for dna damage , peroxynitrite scavenging activity , and tpc in correlation with their esrage production . the results showed esrage production correlates with the peroxynitrite level and tpc.conclusion:this study supports the utilization of these eight extracts in folk medicine for improved treatment of diabetic complications .

Materials and Methods Preparation of methanolic extracts from 29 edible plants Cell culture Measurement of endogenous secretory receptor for advanced glycation end products concentrations in culture media Measurement of 8-hydroxydeoxyguanosine in human umbilical vein endothelial cells DNA Measurement of peroxynitrite level Determination of total phenolic content in methanolic extracts from edible plants Statistical analysis Results and Discussion Effects of methanolic extracts from edible plants on endogenous secretory receptor for advanced glycation end products production in high glucose-induced human umbilical vein endothelial cells Assessment of the relationship between 8-hydroxydeoxyguanosine level and endogenous secretory receptor for advanced glycation end products production Assessment of the relationship between peroxynitrite level and endogenous secretory receptor for advanced glycation end products production Assessment of the relationship between total phenolic content and endogenous secretory receptor for advanced glycation end products production

over - the - counter ( otc ) and prescription drug misuse are the nonmedical use of medication , which is used without a prescription or for the feeling caused by the drug . otc and prescription drugs are fast becoming the drugs of choice for adolescents , with the national center on addiction and substance abuse reporting a 212% increase in prescription drug misuse among adolescents between the 1992 and 2003 period . the national survey on drug use and health and the drug abuse warning network have also documented an increase in otc drug misuse since 1992 . compton and volkow have reported that pain medication is second only to marijuana in being the most popular drug of choice for adolescents . given that parent and peer influence have been highlighted as major predictors of substance use in adolescents and that pain medications are the most widely abused prescription drugs , we hypothesize that parent and peer influence will affect pain medication misuse , specifically misuse to get high ( recreational use ) , in the same way these influences affect marijuana use . very little is known about what is responsible for the increase in prevalence and incidence rates of prescription drug misuse . it has been suggested that adolescents ' and young adults ' otc and prescription drug misuse could be explained by their perception that such drugs are safer than illicit drugs , by the ease of access to drugs and by lower societal stigma [ 1117 ] . additionally , because otc drugs are easily purchased , they can be used as an alternative when a prescription drug misuser is unable to access their preferred drug as well as mixed with alcohol to achieve a some researchers have shown that there is a strong relationship between illicit drug use and prescription drug misuse [ 13 , 1924 ] . additionally , ford highlighted the importance of family and school bonds as protective factors against misuse . ford also suggested that adolescents whose parents and peers disapprove of substance use and whose peers use drugs are more likely to engage in prescription drug misuse . marijuana use , on the other hand , has been thoroughly studied , and clear correlates of marijuana use in adolescents have been identified across the literature . specifically , sensation - seeking , risk perception of use , parent and peer attitudes towards use , adolescent and peers ' delinquent behavior , peer use , adolescent tobacco and alcohol use , and demographic variables including gender and ethnicity [ 2732 ] have all been shown to be related to adolescent marijuana use . to better understand the relationship between illicit drug use and pain medication recreational use , we explored the extent to which three known predictors of marijuana use were similarly predictive of otc and prescription pain medication recreational use . specifically , we explored the extent to which risk perception , parent disapproval , and peer disapproval were predictive of recreational use and whether risk perception mediated the effects of parent and peer disapproval . parent and peer disapproval have been strongly linked to risk perception and use in the marijuana literature [ 9 , 28 , 31 , 33 ] , hence their inclusion in the model ( see figure 1 ) . additionally , risk perception has been indicated in the health risk behaviors literature and alluded to in the prescription drug literature as a contributing variable in adolescents ' decisions to engage in these behaviors [ 12 , 14 , 27 , 34 ] . we examined the extent to which these relationships were gender dependent as the marijuana use literature has suggested that different variables are predictive of males ' and females ' use . additionally , because illicit drug use is correlated with otc and prescription drugs misuse [ 13 , 20 , 21 ] , we propose that the relationship between risk perception , parent and peer disapproval , and otc and prescription pain medication recreational use will differ for marijuana users and nonusers . this study differs from ford , in that our measures of parents ' and peers ' disapproval of misuse ( specifically recreational use ) are specific to otc and prescription drug recreational use independent of other substance use . this is especially important since previous research has shown that perceptions of prescription drug misuse often differ from perceptions of illicit drug use [ 15 , 17 ] , and these differing perceptions will subsequently affect peers ' and possibly parents ' level of disapproval . additionally , the inclusion of risk perception as a predictor variable provides some clinical utility to the study , in that it investigates the role of an easily targetable variable for intervention to prevent or decrease misuse . the inclusion of separate path analyses for otc pain medication recreational use , prescription pain medication recreational use , and marijuana use also provides for a comparison of predictors of use of the different drugs . the sample consisted of 465 college students between the ages of 18 and 24 years ( mage = 18.57 , sd = 0.86 ) . the majority of participants ( 91% ) were 18 or 19 years of age ; the remaining 9% were ages 2024 years . nineteen percent of participants ( n = 88 ) reported some ( nonzero ) lifetime otc or prescription drug recreational use with 13% ( n = 60 ) and 11% ( n = 50 ) reporting otc and prescription pain medication recreational use , respectively . twenty - nine percent of participants ( n = 134 ) reported nonzero lifetime marijuana use . risk perception / perceived susceptibility was measured by perceived personal risk , which was the extent to which participants felt they would be at risk of getting sick or hurt if they recreationally used otc pain medication , prescription pain medications , or marijuana , respectively . risk perception was measured on a 7-point evaluation scale ranging from no risk at all ( 1 ) to very much at risk ( 7 ) . to assess for parents ' and peers ' disapproval , participants were asked to report on how they felt their parents and peers would feel about them engaging in the recreational use of otc pain medication , prescription pain medication , or marijuana on a 5-point likert scale ranging from strongly approve ( 1 ) to strongly disapprove ( 5 ) . recreational use or misuse of otc and prescription pain medication was defined as use of medications to get high . participants were asked about their lifetime misuse of otc pain medication , prescription pain medication , and marijuana use on a 5-point frequency scale ( see table 1 ) . responses included never ( 1 ) , 15 times ( 2 ) , 619 times ( 3 ) , 2040 times ( 4 ) , and more than 40 times ( 5 ) . otc pain medication recreational use included the misuse of otc tylenol , motrin , advil , aleve , ibuprofen , and aspirin . prescription pain medication recreational use included the misuse of vicodin , codeine , oxycontin , and percocet . participants also reported on their recreational use of other classifications of otc and prescription drugs including tranquilizers , stimulants , sedatives , steroids , and cough and cold syrup , although these items were not included in the analyses . data was collected as part of a larger project , media influences on health risk behaviors and prescription drug use in young adults . data was collected using a survey developed by the authors specifically for the project . participants were invited to complete an online survey at computer workstations separated by desk partitions . the survey was administered via http://www.surveymonkey.com/. the online survey took approximately 3550 minutes to complete , and participants received research credit for their class upon completion of the survey . to ensure participant privacy , descriptive statistics are shown in table 2 ( for otc pain medication and prescription pain medication variables ) and table 3 ( for marijuana variables ) . path models were estimated separately for each set of variables for one of the three drugs : otc pain medication , prescription pain medication , and marijuana . parent and peer disapproval of misuse of a drug were used to predict risk perception for that drug . both disapproval variables and risk perception misuse of the drug was entered using two variables , one representing whether a respondent reported zero misuse of the drug versus nonzero misuse and the other representing amount of use for respondents who reported a nonzero amount . this model allowed for the testing of whether risk perception mediated the effects of the disapproval variables on the misuse variables , which was done using the joint significance test . for each drug , the path model was first estimated using data from all participants . it was then of interest to test whether any of the associations among the variables differed as a function of gender or as a function of use or nonuse of the other drugs . each path model was reestimated first with gender as a grouping variable and second with misuse of marijuana ( for otc and prescription pain medication models ) or otc and prescription pain medication misuse ( for the marijuana model ) . in order to be used as grouping variables , misuse variables were dichotomized into zero use versus nonzero use . because otc and prescription pain medication misuse were combined into a single grouping variable , a zero represented no misuse of either type and respondents reporting any use of either type , were scored as nonzero . the misuse - dependent variables were each entered using two variables in the model by way of the twopart feature of mplus , which scores all participants on a dichotomous use variable depending on whether they reported zero or nonzero misuse and also score amount of misuse as a separate continuous variable . amount of misuse is scored as missing for respondents who have zero use , but data from these participants was still able to be included in the models because mplus can estimate models using full information maximum likelihood , which can include partially complete data . as the amount of misuse was skewed , the mlr estimator , a variation of maximum likelihood that is more robust to nonnormal data than the more typical maximum likelihood , was used to estimate the models . regarding descriptive statistics , parent disapproval was positively correlated with peer disapproval and risk perception for marijuana and otc pain and prescription pain medication misuse variables . parent disapproval for otc pain medication was also negatively correlated with marijuana use and lifetime use . among marijuana use variables , parent disapproval was negatively correlated with lifetime use ; however , this was only true for participants who did not report any pain medication misuse . peer disapproval was positively correlated with risk perception and negatively correlated with lifetime use for marijuana and otc pain and prescription pain medication misuse variables . peer disapproval of otc pain and prescription pain medication misuse was also negatively correlated with marijuana use . risk perception was positively correlated with gender and negatively correlated with lifetime use and marijuana use for both types of pain medication variables . for marijuana variables , risk perception however , risk perception was only positively correlated with gender among participants who reported no pain medication misuse . lifetime use was positively correlated with marijuana use and negatively correlated with gender for the prescription pain variables . because mplus uses numerical integration to estimate models with dichotomous - dependent variables , it does not report the usual fit statistics or fit indices for such models . fit of the models was not an issue , though , as they included all possible associations among the variables ( note that no association between the two misuse variables could be included because the continuous variable only took on nonmissing values for respondents scoring one on the dichotomous variable ) and so were effectively saturated . note that because the data is cross - sectional , the term predictor is used for statistical purposes rather than to indicate causality . results for the path models are shown in table 4 . for otc pain medication misuse , peer disapproval ( = .516 , p < .05 ) significantly predicted risk perception , with higher levels of peer disapproval predicting higher levels of risk perception . both peer disapproval ( = .401 , p < .05 ) and risk perception ( = .221 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . because peer disapproval predicted risk perception and risk perception predicted dichotomous misuse , risk perception was a significant mediator of the effect of peer disapproval on dichotomous misuse . there were no significant predictors of amount of misuse for respondents who reported a nonzero amount . multiple groups analyses revealed no significant differences in path coefficients as a function of either gender groups ( (8 ) = 3.44 , p > .05 ) or groups defined by zero or nonzero marijuana misuse ( (8 ) = 7.60 , p > .05 ) . for prescription pain medication misuse , both parent disapproval ( = .183 , p < .05 ) and peer disapproval ( = .425 , p < .05 ) , significantly predicted risk perception , with higher levels of parent and peer disapproval predicting higher levels of risk perception . both peer disapproval ( = .424 , p < .05 ) and risk perception ( = .188 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . because peer disapproval predicted risk perception and risk perception predicted dichotomous misuse , risk perception was a significant mediator of the effect of peer disapproval on dichotomous misuse . parent disapproval ( = .286 , p < .05 ) significantly predicted amount of misuse for respondents who reported a nonzero amount , but in an unexpected direction : higher levels of disapproval predicted greater amounts of misuse . multiple groups analyses revealed no significant differences in path coefficients as a function of either gender groups ( (8 ) = 12.35 , p > .05 ) or groups defined by zero or nonzero marijuana misuse ( (8 ) = 3.13 , p > .05 ) . for marijuana misuse , both parent disapproval ( = .162 , p < .05 ) and peer disapproval ( = .493 , p < .05 ) significantly predicted risk perceptions , with higher levels of parent and peer disapproval predicting higher levels of risk perception . peer disapproval ( = .356 , p < .05 ) and risk perception ( = .409 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . parent disapproval also significantly predicted dichotomous misuse , but this effect varied depending on whether respondents were in the zero misuse or nonzero misuse group for otc and prescription pain medications , so it is discussed below . both peer disapproval ( = .262 , p < .05 ) and risk perception ( = .454 , p < .05 ) were significant predictors of amount of misuse for respondents who reported a nonzero amount , with higher levels of disapproval and risk perception predicting smaller amounts of use . because both parent and peer disapproval predicted risk perception and risk perception predicted both dichotomous misuse and amount of misuse , risk perception was a significant mediator of the effect of both parent and peer disapproval on both dichotomous misuse and amount of misuse . multiple group analyses revealed no significant differences in path coefficients as a function of gender groups ( (8 ) = 10.02 , p > .05 ) . there was a significant difference in path coefficients as a function of groups defined by zero or nonzero use of otc or prescription pain medications ( (8 ) = 18.81 , p < .05 ) . tests of invariance of the individual paths revealed that the only path to differ significantly was for parent disapproval predicting dichotomous misuse . for respondents reporting zero otc or prescription pain medication misuse , this association was significant and negative ( = .131 , p < .05 ) , so higher levels of disapproval predicted lower likelihoods of nonzero misuse . for respondents reporting nonzero otc or prescription pain misuse , this association was significant and positive ( = .282 , p < .05 ) , so higher levels of disapproval predicted higher likelihoods of nonzero misuse . to add to the understanding of otc and prescription pain medication misuse and its relationship with other substance abuse in adolescents and young adults , we explored the relationship between parent and peer disapproval of recreational use , risk perception of recreational use , and reported recreational use . the results of this study indicate that similar to marijuana use , risk perception of otc and prescription pain medication recreational use are influenced by parent and peer disapproval of recreational use , and use itself is influenced by peer disapproval and risk perception of recreational use . noteworthy was the ability of peer disapproval of misuse to predict dichotomous misuse over and above the mediated effect through risk perception , for all three drugs : otc and prescription pain medication misuse and marijuana use . this is consistent with findings in the substance abuse literature [ 31 , 33 ] and suggests that peer subculture may either encourage or act as a protective variable against adolescents engaging in the recreational use of all substances including prescription drugs . this also highlights the role of development in adolescents ' decisions to engage in health risk behaviors ; that is , they are in a period in their lives where peers are their key counterplayers and they may follow the norms outlined by their subgroup ; hence , peer disapproval has more influence on their perceptions of harm and use . these results also suggest that in order to increase risk perception and reduce recreational use of otc and prescription pain medication , program planners should work towards reducing the acceptability of otc and prescription pain medication misuse among adolescents , since they influence each others ' perceptions . the office of national drug policy and partnership for a drug - free america launched a prescription drug abuse media campaign targeting parents and health and school professionals ; however , no ads were directly marketed toward adolescents and young adults . based on the significant influence of peer disapproval on risk perception and use , future campaigns should target adolescents and young adults . parent disapproval was predictive of risk perception of prescription pain medication recreational use and marijuana use . we speculate that adolescents may take their parents ' opinions into consideration when formulating their risk perceptions on both marijuana and prescription pain medication as opposed to otc pain medication because of the restrictive access to these substances ; however , this hypothesis should be explored in future studies . parent disapproval of misuse failed to directly predict dichotomous misuse of otc and prescription pain medications ( although it did have a mediated effect through risk perception for prescription pain medications ) . this highlights the need for parents and by extension other adults working with adolescents to focus less on preventing behavior via direct methods ( e.g. , just say no campaigns ) and focus more on educating adolescents on the risks associated with the behavior . the goal of redirecting adults ' focus on educating adolescents on the risks of otc and prescription pain misuse is to influence other correlates of use , such as peer disapproval and risk perception , in order to prevent or decrease use . parent disapproval predicting dichotomous misuse of marijuana is consistent with the marijuana use literature [ 39 , 40 ] . however , the finding of reverse relationships when the sample was divided into otc and prescription pain drugs zero and nonzero misusers provides some additional data about the relationship of use for the top two drugs of choice for adolescents . specifically , adolescents who engaged in otc and prescription pain misuse were more likely to be marijuana users the more their parents disapproved of marijuana use , whereas those who were zero otc and prescription pain misusers were less likely to engage in marijuana use if their parents disapproved of marijuana use . these results are suggestive of a deviant and high sensation - seeking subculture trend in adolescents who engage in both pain medication and marijuana use and this subculture is seen across the substance abuse literature [ 9 , 31 , 33 ] . it is possible that among this subculture , parent interventions may encourage adolescents to engage in substance use , providing further need for parents to focus less on preventing behavior and more on highlighting the risks associated with the behaviors . another explanation may be that of reverse causality , whereby the more the adolescents engage in use , the more their parents disapprove of their behavior . although risk perception was significantly predictive of use in the general models , the regression weights and significance values suggest that it was a weaker predictor for otc and prescription pain medication recreational use than for marijuana use . these findings suggest that although risk perception may play an important role in adolescents ' decision making regarding substance use and other health risk behaviors [ 28 , 34 , 41 , 42 ] , risk perception is less of an important variable in adolescents ' decision - making regarding recreational use of otc and prescription pain medication . additional variables that are uncommon to the substance abuse literature , such as lower societal stigma , and comparative risk perception to street drugs may be more important in adolescents ' decision making regarding prescription drug recreational use [ 12 , 14 ] . additionally , risk perception may interact with other variables not studied here to explain adolescents ' decision making regarding use ( e.g. , general attitudes toward prescription drug use , frequency of family and friends ' medical use of prescription drugs ) . future studies should explore how other correlates of recreational use of otc and prescription pain medication interact with risk perception to influence misuse . this study was restricted to recreational users of otc and prescription pain medication ; these users do not encompass all nonmedical use ; therefore , generalizations about misuse are limited . another limitation regarding generalization is the use of a convenience sample of college students ; future studies should test this model on a community sample . another limitation of this study is that the psychosocial characteristics of the sample were not considered . adolescents and young adults are highly influenced by developmental characteristics and their external environment and future studies should explore the extent to which developmental characteristics interact with these environmental variables to predict risk perception and use . additionally , future studies should retest these paths with a larger sample ( preferably including more participants who report nonzero use ) to check for consistency of the results across samples . future studies should also compare otc and prescription pain medication misuse to other illicit drug use and other health risk behaviors . finally , the present study was cross - sectional , so temporal precedence can not be established for the assumed causal relationships between variables . despite these limitations , this study provides evidence that suggests that predictors of otc and prescription pain medication recreational use are similar to predictors of marijuana use in adolescents in that peer disapproval is a significant predictor of risk perception and misuse . additionally , both peer and parent disapproval are significant predictors of risk perception of prescription pain medication misuse . unlike marijuana use , otc and prescription pain medication misuse in adolescents are relatively less explained by risk perception suggesting that other variables may be responsible for adolescents ' decision to engage in the misuse of otc and prescription pain medication .

parent and peer disapproval were examined as potential predictors of recreational use of over - the - counter ( otc ) and prescription pain medication . risk perception was studied as a potential mediator of the effects of parent and peer disapproval . four hundred and sixty - five college students ( mage = 18.57 , sd = 0.86 ) were recruited between september 2009 and september 2010 . participants completed an online survey about their recreational medication use , other substance use , and correlates of use . path analyses showed that predictors of otc and prescription pain medication recreational use are largely similar to predictors of marijuana use in college students such that risk perception mediated both the effect of parent and peer disapproval on dichotomous misuse , and peer disapproval had a significant direct effect on dichotomous misuse . prevention interventions for recreational use of pain medication should target risk perception and peer disapproval .

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

the first case occurred in a 40-year - old woman from kuranda , queensland , with a 1-week history of fever , myalgia , headache , and dry cough . a provisional diagnosis of leptospirosis was made , and she was treated with intravenous penicillin . urea and creatinine levels continued to increase over 3 days to 14.8 mmol / l and 300 mol / l , respectively , despite administration of intravenous fluids . the patient was discharged on day 7 and given a 5-day course of oral doxycycline , 100 mg , twice a day . a diagnosis of qtt was made retrospectively on the basis of serologic results .. * by immunofluorescence . the second case occurred in a 69-year - old woman from innisfail , queensland , who was hospitalized with a 2-week history of fever , myalgia , neck pain , and confusion . she was febrile ( temperature 39.2c ) and had tachycardia ( 140 beats / min ) . multiorgan failure , purpura fulminans , and digital necrosis developed over a 2-day period , and she was transferred to an intensive care unit . prothrombin time was 22 s ( normal range 814 s ) , activated partial thromboplastin time was 53 s ( normal range 2538 s ) , and fibrinogen level was 1.6 results of a pcr for neisseria meningitidis in blood and an extensive screen for primary vasculitides and prothrombotic disorders were negative . a skin biopsy specimen showed vessel thrombosis and no evidence of vasculitis , which is consistent with purpura fulminans . tissue from the skin biopsy specimen was tested by pcr with primers against the rickettsial 17-kda gene ( 2 ) , and immunohistochemical analysis was performed with polyclonal rabbit antisera against spotted fever group ( sfg ) rickettsiae . the third case occurred in a 45-year - old man from deeral , queensland , who was seen with headache , malaise , and vomiting 10 days after a tick bite . he was febrile ( temperature 38.2c ) and had tachycardia ( 145 beats / min ) and tachypnea ( 40 breaths / min ) . a chest radiograph showed bilateral interstitial infiltrates ( figure 2 ) . over the ensuing hours , respiratory failure developed , and he required intubation and ventilation . twelve hours after admission , his arterial blood gas results were ph 7.32 , po2 59 mmhg , and pco2 55 mmhg on 100% oxygen while ventilated . mol / l over the first 2 days and then slowly returned to normal . he was given broad - spectrum antimicrobial drugs , including doxycycline , on the day of admission . he was ventilated for 16 days and spent 3 weeks in the intensive care unit . the first case occurred in a 40-year - old woman from kuranda , queensland , with a 1-week history of fever , myalgia , headache , and dry cough . a provisional diagnosis of leptospirosis was made , and she was treated with intravenous penicillin . urea and creatinine levels continued to increase over 3 days to 14.8 mmol / l and 300 mol / l , respectively , despite administration of intravenous fluids . the patient was discharged on day 7 and given a 5-day course of oral doxycycline , 100 mg , twice a day . a diagnosis of qtt was made retrospectively on the basis of serologic results .. * by immunofluorescence . the second case occurred in a 69-year - old woman from innisfail , queensland , who was hospitalized with a 2-week history of fever , myalgia , neck pain , and confusion . she was febrile ( temperature 39.2c ) and had tachycardia ( 140 beats / min ) . multiorgan failure , purpura fulminans , and digital necrosis developed over a 2-day period , and she was transferred to an intensive care unit . prothrombin time was 22 s ( normal range 814 s ) , activated partial thromboplastin time was 53 s ( normal range 2538 s ) , and fibrinogen level was 1.6 . results of a pcr for neisseria meningitidis in blood and an extensive screen for primary vasculitides and prothrombotic disorders were negative . a skin biopsy specimen showed vessel thrombosis and no evidence of vasculitis , which is consistent with purpura fulminans . tissue from the skin biopsy specimen was tested by pcr with primers against the rickettsial 17-kda gene ( 2 ) , and immunohistochemical analysis was performed with polyclonal rabbit antisera against spotted fever group ( sfg ) rickettsiae . the third case occurred in a 45-year - old man from deeral , queensland , who was seen with headache , malaise , and vomiting 10 days after a tick bite . he was febrile ( temperature 38.2c ) and had tachycardia ( 145 beats / min ) and tachypnea ( 40 breaths / min ) . a chest radiograph showed bilateral interstitial infiltrates ( figure 2 ) . over the ensuing hours , respiratory failure developed , and he required intubation and ventilation . twelve hours after admission , his arterial blood gas results were ph 7.32 , po2 59 mmhg , and pco2 55 mmhg on 100% oxygen while ventilated . mol / l over the first 2 days and then slowly returned to normal . he was given broad - spectrum antimicrobial drugs , including doxycycline , on the day of admission . he was ventilated for 16 days and spent 3 weeks in the intensive care unit . the clinical features of qtt have been described in 2 reviews ( 1,3 ) . one review of 62 cases included patients from flinders island in the bass strait , an area now known to be endemic for a new rickettsia in the sfg , rickettsia honei ( 4 ) . an eschar is seen in up to half the cases and lymphadenopathy in < 70% . less common clinical manifestations include joint pain , splenomegaly , cough , conjunctivitis , sore throat , nausea , abdominal pain , and photophobia . one fatal case of qtt has been described in a 68-year - old man from northern queensland ( 5 ) . his illness was clinically marked by progressive renal failure , bilateral pulmonary infiltrates , acidosis , abnormal liver function test results , thrombocytopenia , and hypoprothrombinemia . the first patient described in this report had moderate renal impairment , which is commonly associated with leptospirosis . her renal dysfunction did not improve with rehydration but had fully resolved when her condition was reevaluated 3 weeks after hospital discharge . rocky mountain spotted fever ( rmsf ) , which is caused by r. rickettsii , is associated with a multifocal perivascular interstitial nephritis . renal dysfunction is believed to be a consequence of hypovolemia secondary to rickettsial disease induced alterations in capillary permeability ( 6,7 ) . renal failure has also been recorded as a complication of infection with r. conorii ( mediterranean spotted fever ) ( 8) . n. meningitidis was considered a possible cause of her illness . however , her 2-week illness before deterioration was not typical . purpura fulminans has been described in rmsf . in a review of cases of gangrene complicating this infection , 6 cases with remarkable similarities to our second case pulmonary involvement has been described in rmsf , in which the pathology changes are thought to be related to noncardiogenic pulmonary edema consequent to capillary endothelial damage ( 10,11 ) . rmsf is associated with a mortality rate of 7% , even with treatment ( 5 ) , and r. conorii has been associated with severe disease and fatal cases . the 3 cases described here were seen over a 4-year period at cairns base hospital in northern queensland . we are aware of 2 other cases seen at other hospitals , 1 complicated by renal failure , confusion , abnormal coagulation test results , and impaired gas exchange , and the other with severe pneumonia ( p. marshall , r. miller , pers . although qtt is a mild disease in most patients , its diagnosis should now be considered in patients who reside in or visit the rickettsial disease endemic area of eastern coastal australia and are hospitalized with renal failure or impaired pulmonary function . delays in seeking treatment may have contributed to illness severity in 2 of our patients . given the serologic cross - reactivity between members of the sfg rickettsia , it can not be assumed that all cases described were caused by r. australis . another rickettsia of the sfg has been recently described in northern queensland ( 13 ) , and distinguishing between species will be important in future studies .

we report 3 cases of spotted fever group rickettsial infection ( presumed queensland tick typhus ) in residents of northern queensland , australia , who had unusually severe clinical manifestations . complications included renal failure , purpura fulminans , and severe pneumonia . clinical illness caused by rickettsia australis may not be as benign as previously described .

The Cases Case 1 Case 2 Case 3 Conclusions

breast cancer is a leading cause of mortality and morbidity all over the world . in 2008 , close to 1.4 million cases were diagnosed with breast cancer worldwide . the incidence varies among different populations with high rates seen in developed countries compared to developing countries [ 2 , 3 ] . in general , breast cancer rates are highest in white european and lowest in east asian populations [ 1 , 4 , 5 ] . the estimated incidence rate for women living in the south - east asia region of world health organization ' is 26.1 per 100000 population and this figure is 89.7 for women living in western europe . the established risk factors of breast cancer are , mainly , early age at menarche , late age at menopause , nulliparity , number of live birth , and age at first live birth . contrary to the large variations seen in incidence between population of europe and asia , the prevalence of the established risk factors is not very much different between the two populations [ 5 , 6 ] . a higher risk of breast cancer among american and european women has been blamed for so - called western lifestyle characterized [ 7 , 8 ] by the combination of early menarche , decreased parity , delayed childbearing , and a sedentary lifestyle . studies of migrants have confirmed the relative importance of environment and lifestyle in the etiology of breast cancer [ 911 ] . the so - called western lifestyle is now very common in asian countries such as japan , korea , taiwan , and hong kong and is spreading fast in the economic booming region of east asia . in addition to the major differences in magnitude of rates between asians and europeans , there is a distinct difference in the shape of age specific rates between the two populations . in asian population , the age specific incidence curve peaks at 4550 and then plateaus and even slightly decreases so that the rates after 60 years are less than or close to the rates at age group 4555 . in european population , the age specific curve increases steadily with no change of pace around age 50 and the increase continues up to age 80 with a peak around 65 years . a lower risk and the distinct pattern of age specific incidence rates among the asian population ( even in countries with great extent of similar lifestyle with western population such as japan , taiwan , and hong kong ) have been a challenging issue among epidemiologists and cancer scientists to the extent that some have labeled breast cancer in the population of asia as a different disease . a recent symposium in montreal , canada , specifically addressing the same topic , highlighted the younger age at onset as one of the chief characteristics of breast cancer natural history in asian population . what contributes to the peculiar phenomenon of younger age at onset has been the subject of inertest to epidemiologists , and it has been hypothesized that a cohort effect among asian population causes this phenomenon . the aim of this study was to use the incidence rates reported in the cancer incidence in five continents for the two populations of asia and europe in order to address the nature of the age specific rate differences between the two populations using age - period - cohort analysis . registered cases of female breast cancer and corresponding person years were ascertained from the ci5plus , cancer incidence in five continents annual dataset ( an online data repository of international agency on research on cancer , iarc ) for 29 registries in europe and 9 registries in asia for a duration from 1953 to 2002 . cases from europe included cases registered for the period from 1953 to 2002 and cases from asia included cases registered for the period from 1963 to 2002 . cases and their corresponding person years were pooled for each population to make two distinct populations , referred to hereafter as asian and european . age specific rates were addressed both cross - sectionally ( period wise ) and longitudinally ( generational birth cohort wise ) . for period wise age specifics , the rates were constructed and described based on five years period ( 1955 , 1960 , 1965 , 2000 ) . for the cohort wise , age specific rates were estimated from age - period - cohort analysis constructed over each five years cohort . in addition , the trends in rates expressed as annual percentage change ( apc ) and their 95% confidence intervals were estimated using the age - period - cohort model . for analytical part , the age - period - cohort model was used . for this , the periods and cohorts were constructed in intervals of 5 years . the five - year age groups were truncated to age more than 25 years with the last interval ( 85 and over ) included all cases more than 85 years ( 13 five - year age groups were constructed ) . the constrained generalized linear model ( cglm ) , the most utilized approach in the epidemiology literature dealing with age - period - cohort analysis , was used . for this purpose , a log - linear model with the general form that includes a ( age ) , p ( period ) , and c ( cohort ) was applied as follows : ( 1)log{(a , p)}=f(a)+g(p)+h(c ) , where a , p , and c represent the mean age , period , and cohort and f , g , and h are parametric functions fitted to the data . in this model , in addition to estimating the main effect of age , other components contributing to magnitude of rates specially the secular changes of rate across study periods and birth cohorts are estimated . the secular change or net drift corresponds , interchangeably , to hazard due to period or cohort , and it has been used to estimate the annual percent changes of rates over a period of time [ 14 , 15 ] . as the purpose of our study was to tackle the difference between the two populations ' age specific rates , it was assumed that mainly the cohort effect explains the changing of rates across aging intervals during the study period in both populations . with this assumption , the model estimates the age function presented as the log of the age specific rates for the reference cohort ( longitudinal age specific or age specifics across cohorts ) and the cohort effect as log of rate ratio relative to a reference cohort while period effect constrained to be zero on average with zero slopes . the estimated logs of age specific rates were transformed to rate scale ( number per 100000 population ) for better realization . in the model , the cohort born during 1970 was considered as the reference cohort and the period of 1970 was considered as the reference period . the longitudinal age specific raters were estimated and reported for cohort born on 1885 , 1910 , 1930 , 1950 , and 1970 . for details of the modeling please refer to age - period - cohort models for the lexis diagram by carstensen . data were analyzed using the r 2.14.1 statistical software utilizing epi 1.1.9 package ( r development core team , 2009 ) . a total of 236,851 cases of breast cancer registered in the 29 european registries and a total of 188,630 cases registered in the 9 asian registries were included in the analysis ( table 4 presents details of the included registries for the two populations ) . there was a constant increasing of rates for both populations during the last 50 years with an estimated annual percent of change 1.03 ( with 95% ci of 1.029 , 1.031 ) for asians and 1.016 ( 95% ci of 1.015 , 1.017 ) for europeans . the incidence rates across all age groups in europeans were higher than asians , especially in older age groups . during the study period , the magnitude of rates increased for both populations for each succeeding five - year period for all age groups . the shape of the age specific rates ( period wise ) showed basic differences between the two populations . for asian population , the age specific rates for all periods peaked around 50 years and then decreased and plateaued afterward ( figure 1 ) . for the european population , the age specific rates increased up to the last age group for periods ending 1985 and for the periods after 1985 , the age specific rates peaked between 55 to 75 years and then slightly decreased ( figure 1 ) . the fitting of the age - period - cohort model to data indicated that the model that included all the main effects ( age , period , and cohort ) has the greatest reduction of deviance , indicating the best model to explain the observed rates in both populations ; table 1 presents the goodness of fit of the models along with their parameters . there were cohort effects present in incidence rates of both populations during the study period ; however , the cohort effects in asians were much stronger than european . in the asian population , the rate ratios presenting the cohort effects ranged from a low of 0.06 ( 95% ci 0.05 , 0.08 ) for those born in 1870 to 0.94 ( 95% ci , 0.93 , 0.96 ) for those born in 1965 . in the european population , the rate ratios presenting the cohort effect ranged from 0.33 ( 95% ci , 0.32 , 0.35 ) for cohort born in 1865 to 1.03 ( 95% ci , 1.02 , 1.04 ) for the cohort born in 1965 ( table 2 and figure 2 ) . there were residual period effects in the asian population around 1975 ( rate ratio of 0.89 and 95% ci 0.86 , 0.92 ) and 1985 ( rate ratio of 1.12 95% ci of 1.09 , 1.13 ) , figure 2 . for both populations , the estimated age specific rates expressed as longitudinal age specific indicated the same pattern for both populations ; the age specific rates increased sharply before the age of 50 , and the increase slowed down pace with the last age groups ( over 75 years ) , still the groups with highest incidence rates . the pattern of longitudinal age specific rate is presented in figure 2 along with the other effects , cohorts , and periods . the estimated longitudinal age specific rates ( in an increment of 20 years ) and their corresponding confidence intervals are presented numerically in table 3 and graphically in figure 3 . as table 3 and figure 3 indicate , the estimated age specific rates have steadily increased in all age groups for both populations but the increase is more in asians compared to europeans . there is a large difference in the magnitude of rates between the two populations in early cohort ( 1890 ) when they are compared with the most recent cohort ( 1970 ) , table 3 and figure 3 . the difference in age specific rates between early and late cohorts is indicative of the cohort effects that cause the distinct pattern of age specific rates observed between the two populations . in addition , comparing the magnitude of the cohort effects between the two populations ( figure 2 ) , they indicate that , though , the cohort effects are decreasing along succeeding cohorts for both populations , but the decrease in cohort effects in asians is far larger than those of europeans . this difference in decreasing rates of cohort effects between the two populations indicates that both populations may experience similar rates in the future if there are no other major changes to the underlying cause of the disease in future years . our study proved a steady increase of breast cancer rate with similar pace during the last 50 years for both populations . we demonstrated that there is no difference between the patterns of age specific rates between the two populations when rates are measured as longitudinal age specific rates . it was demonstrated that a strong cohort effect contributes to the differences in pattern of age specific rates between the two populations . the difference in breast cancer rates with low rates for asians versus high rates for europeans has been documented since registries in asia started reporting population rates [ 17 , 18 ] . while several studies have demonstrated marked differences in magnitude and the pattern of age specific rates among different countries of europe and asia , no study systematically and collectively has addressed the age specific rate differences in the two populations as our study did . an overall increasing trend of morbidity from breast cancer has been reported for all populations of the world and the increase has been attributed to ageing and increasing median age of women [ 15 , 17 , 1922 ] . this similarity in slope of increase indicates that despite the fact that the two populations are basically different in terms of culture , ethnicity , lifestyle , and social attributes , the breast cancer epidemic enforces its own pace of epidemic projection . any increase in incidence of breast cancer rates is due to either changing of risk factors or implementation of mass screening ( especially mammographic screening ) . the difference in the pattern of the age specific rates between the two populations is well recognized and several studies have addressed this discrepancy ; a study comparing the shape of the age specific rates between taiwanese and caucasian american reported that the age specific rates of breast cancer differed between the two populations and the study concluded that the difference is due to a cohort effect presented in taiwanese . another study comparing breast cancer rates among populations of singapore and sweden attributed the difference in rates to a large cohort effect and concluded that this effect will decrease in future generations causing similar incidence rates between the two population in coming decades . in addition to comparative studies , it has been demonstrated that the pattern of age specific rates for the populations of japan , korea , china , singapore , thailand , and philippine has changed in the recent years attributing this change to changing of life style toward more westernization and implementation of mammographic screening . the pattern and magnitude of age specific rates of breast cancer have been affected by screening mammography specially in the european population , and this effect has been mainly presented as increase of incidence in the age group of 50 to 70 [ 2628 ] . this is compatible with our finding as it was demonstrated in figure 1 that age specific rates for european population increased in that age group 50 to 70 and the change happened after 1985 when the wide spread use of mammographic screening started [ 27 , 29 ] . mammographic screening started in asia in late 1990 [ 30 , 31 ] and its effect on the shape of age specific incidence can not be assessed in our study . compatible with previous studies comparing the age specific rates of breast cancer between population of europe and asia , our study proved that a large cohort effect in asian population rates plays a major role in the differences in age specific rates between the two populations . the age specific rates can be defined both cross - sectionally ( period wise ) and longitudinally ( cohort wise ) . if there are no cohort or period effects , the two definitions will show similar magnitude and pattern of age specific rates . the distinct pattern of age specific rates ( cross - sectional rates ) between the two populations is in fact due to the cohort effect that was demonstrated in our study . in the other world , what contributes to the observed pattern of period wise age specific rates in asian population is the additive nature of the cross - sectional definition of age specific rates in the presence of decreasing cohort effects . since the rates for two sequential age groups come from two different cohorts , when the older cohort has lower risk compared to younger cohort , the cross sectional patterns of age specifics will decrease . the strong cohort effect that exists in the asian breast cancer is responsible for a distinct pattern of cross - sectional age specifics seen in the asian population . in applying the cglm model , the choice of constraint ( period or cohort ) is based on an external knowledge of the underlying cause of change of rates in a population . our choice of cohort instead of period as constraint was based on previous studies that attributed the changes of the rates to a cohort effect [ 15 , 19 , 32 ] . the period effect has been mainly attributed when change of health policy ( e.g. , introduction of more sensitive detection techniques or availability of certain diagnostic procedures ) causes the changing of rates . in the light of the nature of breast cancer risk factors that are mainly of hormonal and sociobehavioral nature this study enjoyed data of adequate quality as the data were utilized from the registries that met acceptable degree of validity and reliability to be published in the international agency in research on cancer ( iarc ) official report . in addition , the quality of the data for both populations is comparable owing to the efforts and quality assurances and control protocols that iarc requires for different registries contributing data to the cancer incidence in five continents reports ; one may ask . we would have been able to draw the same conclusion on analyzing just the data of asian population without the need of european population . the main reason for using european for comparison was that the fact that the specificity attributed to asian breast cancer age specific rates has been defined as it has contrasted to the well - sestablished breast cancer epidemiology in europe and western countries . the methodology we used is a very established and routine way of analyzing rates at population level when the data of calendar time exist . the age - period - cohort analysis has been a major tool in the hands of demographers and , in recent decades well utilized by epidemiologists . the methodology , while very common in use , suffers major problem especially when it is utilized to attribute the underlying cause of changes of a rate to period versus cohort ( the nonidentifiability problem ) . in our study , this problem was not a concern as our assumption was that the nature of risk factors in breast cancer would translate into cohort effect other than period effect . it was concluded that no differences in the pattern of age specific rates exist between the two populations when the age specifics are measured cohort wise , and the difference seen in the period wise age specific rate is due to a strong cohort effect present in the asian population rates .

introduction . there is an established fact that asian breast cancer patients are , on average , younger than their european counterparts . this study aimed to utilize the data from the cancer incidence in five continents i through xiii ( published by the international agency for research on cancer ) to examine what contributes to the younger age at onset in the asian population . material and methods . data ( number of breast cancer cases and corresponding population figures ) for 29 registries in europe and 9 registries in asia for the period of 19532002 was accessioned and pooled to form two distinct populations , asia and europe . the age specific rates were defined and analyzed cross - sectionally ( period wise ) and longitudinally ( cohort wise ) . the magnitude and the pattern of age specific rates were analyzed using the age - period - cohort analysis . the constrained generalized linear model with a priority assumption of cohort effect as contributing factor to changing rates was used to analyze the data . result . during the last 50 years , the rate of breast cancer increased for both populations with an estimated annual percent change of 1.03% ( with 95% ci of 1.029 , 1.031 ) for asia and 1.016% ( 95% ci of 1.015 , 1.017 ) for europe . there were stronger cohort effects in the magnitude of rates among the asian population compared to the european population . the cohort effects , expressed as the rate ratio with cohort born in 1970 as reference , ranged from 0.06 ( 95% ci 0.05 , 0.08 ) to 0.94 ( 95% ci 0.93 , 0.96 ) for asians and 0.35 ( 95% ci 0.33 , 0.36 ) to 1.03 ( 95% ci 1.02 , 1.04 ) for europeans . the estimated longitudinal age specific rates ( adjusted for cohort and period effects ) showed similar patterns between the two populations . conclusion . it was concluded that a strong cohort effect contributes to the younger age at onset among asian breast cancer patients .

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

reversible phosphorylation of serine , threonine , and tyrosine residues is one of the most common and important regulatory posttranslational modifications of proteins and plays a crucial role in many biological processes , including cellular signal transduction , regulation of enzyme activities , metabolic regulation , molecular recognition and biomolecular interaction , protein localization , cell differentiation and proliferation [ 15 ] . therefore , identifying phosphorylated proteins is an important step to elucidate the structural and functional role of the protein 's phosphorylation regulation . up to now , mass spectrometry , including matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof - ms ) and electrospray ionization mass spectrometry ( esi - ms ) have been key technologies in characterizing protein phosphorylation and phosphoproteome , and each approach presented strengths and limitations [ 615 ] . however , because of the low and dynamic stoichiometry of phosphorylation on proteins [ 16 , 17 ] and the low ionization efficiency of phosphopeptides , it remains challenging to thoroughly characterize phosphorylation sites using mass spectrometry . considerable effort has been made recently to improve the ionization efficiency of phosphopeptides by selecting and optimizing appropriate matrices with maldi - tof - ms . several materials have been employed in phosphopeptide analysis , including 2 , 5-dihydroxybenzoic acid ( dhb ) [ 1921 ] , 2 , 4 , 6-trihydroxyacetophenone ( thap ) , and ionic liquid matrices . in addition , ammonium salts , including diammonium hydrogen citrate ( dahc ) [ 2224 ] , ammonium acetate and monoammonium phosphate , and phosphoric acid [ 1921 ] have been used as matrix additives and have shown some ability to enhance phosphopeptide ionization during maldi - tof - ms . 2 , 6-dihydroxyacetophenone ( dhap ) was first used by gorman et al . as a matrix for maldi analysis of fragile peptides , disulfide bonding , and small proteins , including phosphopeptides . in recent years , xu et al . reported that the dhap / dahc matrix combination was used as an effective maldi matrix in detecting methyl esterified phosphopeptides by maldi - tof - ms [ 26 , 27 ] . after systematic optimization in this study , we found that contrary to previous reports , the matrix forms at a lower ratio of dhap / dahc and that this ratio shows greater efficiency in improving ionization ability of phosphopeptides . we investigated the characteristics of an optimal dhap / dahc matrix , and analyzed morphology and detection sensitivity of phosphopeptides . using tryptic digests of - and -casein , we demonstrated a cid ms / ms analysis of phosphopeptides by maldi - tof / tof - ms with an optimized dhap / dahc matrix . finally , we used this approach to analyze phosphorylation sites on human histone h1 treated with cyclin - dependent kinase-1 ( cdk1 ) . the chemicals 2 , 6-dihydroxyacetophenone ( dhap ) , 2 , 5-dihydroxy - benzoic acid ( dhb ) , diammonium hydrogen citrate ( dahc ) , ammonium bicarbonate , trifluoroacetic acid ( tfa ) , alkaline phosphatases , -casein and -casein ( both from bovine milk , purity by electrophoresis ) were all purchased from sigma aldrich ( st . louis , mo , usa ) . cyclin - dependent kinase-1(cdk1 ) and human histone h1 were purchased from new england biolabs , inc . the standard peptides vnqigptlsesik ( pt , purity > 95% ) and sgslhripythqs ( py , purity > 85% ) were synthesized in beijing scilight biotechnology ltd . sequencing grade modified trypsin ( porcine ) was purchased from promega co. ( madison , wi , usa ) . hplc - grade acetonitrile and acetic acid were purchased from mallinckrodt baker , inc , ( phillipsburg , nj , usa ) . ethanol and ammonium hydroxide solutions ( nh3 h2o ) were purchased from beijing chemical company ( beijing china ) ; ultrapure water was obtained from a milli - q system ( millipore , bedford , ma , usa ) . the dhap matrix stock solution was prepared by dissolving 10 mg of dhap in 1 ml of anhydrous ethanol . a series of modified dhap / dahc matrix solutions were prepared by mixing dhap stock solution with dahc stock solution at different ratios from 10 : 1 to 1 : 80 ( v / v ) . the dhb / phosphoric acid ( pa ) matrix was prepared as previously reported ; we modified this previous protocol by dissolving 20 mg of dhb in 1 ml of 50% aqueous acetonitrile containing 1% phosphoric acid . -casein and -casein ( 100 nm in 50 mm ammonium bicarbonate buffer at ph 8.3 ) were digested overnight at 37c with trypsin at an enzyme / substrate ratio of 1 : 25 ( w / w ) . after digestion , tfa was added into the sample solution at 1.0% to quench the reaction . the digest was diluted with 60% acetonitrile containing 0.1% tfa to produce a series of sample solutions at concentrations of 1 pmol/l , 200 fmol/l , 50 fmol/l , 20 fmol/l , and 10 fmol/l . a stock solution containing standard peptides pt ( 1 mm ) and py(1 mm ) was prepared using ultra pure water . the solution was diluted with 60% acetonitrile containing 0.1% tfa to 1 pmol/l , 200 fmol/l , 50 fmol/l , 20 fmol/l , and 10 fmol/l . all of the peptide solutions prepared for dhb / pa measurements were diluted with 1% phosphoric acid . briefly , the reaction solution was prepared with 3 l 10 cdk1 reaction buffer , 0.15 l cdk1 ( 150 u ) , 4 l histone h1 ( 1 g/l ) , 100 m atp , and 13 l water , then incubated at 30c for 45 minutes . the phosphorylated histone h1 was then separated using sds - page ( 12% ) and stained with coomassie brilliant blue using microwave - assisted methods . the in - gel digestion of phosphorylated histone h1 was performed as follows : the band of histone h1 was excised from the polyacrylamide gel , washed twice with water , and destained with 40% acetonitrile/50 mm nh4hco3 . the gel pieces were dehydrated with 100% acetonitrile and dried for 5 min using a speedvac . disulfide bonds were reduced with dtt ( 10 mm , 56c , 45 minutes ) , and the free sulfhydryl groups were alkylated with iodoacetamide ( 55 mm , 25c , 60 minutes in the dark ) . gel pieces were washed with 50 mm nh4hco3 , 50% acetonitrile/50 mm nh4hco3 , and dehydrated with 100% acetonitrile . after drying with a speedvac , the gel was rehydrated using 100 ng/l trypsin ( 50 mm nh4hco3 , ph 8.3 ) on ice for 30 minutes , and the digestion was carried out at 37c for 60 minutes and then quenched with 1.0% tfa . the tryptic peptides were extracted twice with 60% acetonitrile containing 0.1% tfa , and then the combined digest solution was concentrated to 10 l under vacuum . maldi - tof - ms analysis of peptides was performed using an axima - cfr plus maldi - tof mass spectrometer ( shimadzu / kratos , manchester , uk ) equipped with a pulsed nitrogen laser operated at 337 nm . positive / negative ion maldi mass spectra were acquired in the reflectron mode under the following parameters : ion source , 20/20 kv , lens , 6.3/6.3 kv , pulsed extraction , 2.5/2.5 kv , reflection , 25/25 kv . high - energy cid ms / ms was performed with the axima - tof maldi mass spectrometer ( shimadzu / kratos , manchester , uk ) , equipped with the same pulsed 337-nm nitrogen laser . operation parameters were : ion source , 20.0 kv ; lens , 6.5 kv ; pulsed extraction , 2.5 kv ; and reflectron , 24.4 kv . to prepare for maldi - tof - ms analysis , 1 l of peptide solution was mixed with 3 l of matrix solution in an eppendorf tube , and 1 l of the peptide / matrix solution was spotted onto the maldi sample plate and then crystallized either under vacuum ( when dhap / dahc was used as matrix ) or in the air ( when dhb / pa was used as matrix ) . measurements using dhap / dahc were performed by detecting 1520 different positions on the broad outer zone ( see discussion below ) for each sample to accumulate a total of 200 profiles ( 5 shots per profile ) . for measurements with dhb / pa , a total of 200 profiles ( 5 shots per profile ) were accumulated by searching for 1015 hot spots for each sample . the standard peptide pt ( 250 fmol on target ) was used to determine sample homogeneity of the matrix . on each sample spot , 196 ( 14 14 ) positions were selected manually to ensure a full overview over the sample preparation . for each position , 50 profiles ( 5 shots per profile ) were accumulated in positive ion mode . mass spectrometry data were processed with launchpad 2.7.1 software ( shimadzu / kratos , manchester , uk ) . the data of the cid ms / ms spectra of phosphopeptides from histone h1 were compared to the swissprot 56.6 protein sequence database ( homo sapiens ) using the on - line mascot database search engine ( matrix science , london , uk ) with the following parameters : trypsin with three missed cleavages ; average mass values ; peptide mass tolerance : 0.8 da ; fragment mass tolerance : 1.0 da ; fixed modifications : carbamidomethyl ( c ) , variable modifications : oxidation ( m ) , phosphor ( st ) . only the peptides with ions score above 38 the solution composition of the matrix is a factor that significantly influences the ionization efficiency and the quality of maldi mass spectra . therefore , we prepared a series of different ratios of dhap ( 10 mg / ml ) and dahc ( 20 mg / ml ) matrix solutions ( 10 : 1 , 5 : 1 , 1 : 1 , 1 : 4 , 1 : 8 , 1 : 16 , 1 : 20 , 1 : 30 , 1 : 40 , 1 : 50 , 1 : 60 , and 1 : 80 v / v ) and investigated these matrix combinations using the tryptic digest of -casein , which contains nine phosphopeptides ( t1-t9 ) and some nonphosphopeptides ( such as t4 , t10 , t11 , t18 ) ( see table 1 ) . figure 1 shows a comparison of the maldi - tof - ms analysis of the tryptic digest of -casein ( 250 fmol on target ) using matrix dhap / dahc at different ratios in positive ion mode . the signal intensity of phosphopeptides ( especially for multiple phosphorylated peptides ) changed dramatically with the ratio of dhap / dahc . it was clear that all phosphopeptides could be easily detected with a low dhap / dahc matrix ratio in the range of 1 : 30 to 1 : 60 . in an optimal matrix solution , the concentration of dhap was 1.63.3 mg / ml , which is lower than the concentration of 1015 mg / ml used by other groups [ 24 , 26 , 27 ] . once the dhap / dahc ratio was down to 1 : 80 , most multiple phosphorylated peptides could no longer be detected , likely because there were not sufficient numbers of dhap molecules to transfer the laser energy to the peptides to be protonated . another interesting phenomenon apparent in figure 1 : the signal intensity of nonphosphopeptides was stable ( t3 ) , enhanced ( t10 ) or weakened ( t11 , t4 , t18 ) with the change of the dhap / dahc ratio . we calculated the bull and breese index ( bb index ) of these peptides , an index generally used to reflect the hydrophobic / hydrophilic nature of peptides . higher bb index values are associated with more hydrophilic peptides . for the phosphopeptide , we calculated the bb index by replacing the phosphoserine or phosphothreonine residues with glutamic acid . by comparing the bb index of peptides listed in table 1 , we found that the phosphopeptides were nearly hydrophilic peptides with high bb indices and that the nonphosphopeptides for which signal intensity was weakened at low dhap / dahc ratio were highly hydrophobic with lower bb index ( t4 : 5530 , t11 : 6480 , t18 : 6900 ) . the nonphosphopeptide t10 for which the ion signal was enhanced at lower ratios of dhap / dahc was found to be slightly hydrophobic with a bb index of 750 . these findings indicated that the optimal dahc / dhap matrix tended to detect more hydrophilic peptides . most phosphopeptides are hydrophilic , so we propose that the low ratio of dhap / dahc matrix could increase cocrystallization between matrix and phosphopeptides and further improve ionization efficiency of phosphopeptide in maldi ms . in this study , the phosphopeptide pt ( 250 fmol on target ) was used to investigate the sample homogeneity of optimized dhap / dahc matrix , and for a comparison , we chose matrix combination of 2,5-dhb with 1% phosphoric acid , a currently popular matrix used for detection of phosphopeptide [ 3032 ] . it was apparent from the photographs of the maldi sample spots that there was a dramatic difference between the optimized dhap / dahc ( figure 2(a ) ) and dhb / pa ( figure 2(b ) ) . small crystals formed in a dhap / dahc sample spot while larger , needle - like crystals formed in a dhb / pa sample spot . furthermore , 196 ( 14 14 ) positions on the target were measured covering the crystalline layer for a full overview ( figure 2(e ) ) , and the ms signal intensities of pt were monitored . the majority of the outer zones of the crystalline samples exhibited high signal intensities , except for a small center zone when dhap / dahc used as matrix ( figure 2(c ) ) . in comparison , the crystalline dhb / pa matrix exhibited the hot - spot phenomenon , signals were primarily observed in the narrow , inhomogeneous ring at the edge of the sample layer , and the inner position almost did not exhibit any signal ( figure 2(d ) ) . therefore , the samples prepared with dhap / dahc were more homogeneous than those prepared with a dhb / pa matrix , reducing the need for time - consuming hot - spot searches . based on the sample homogeneity of dhap / dahc and dhb / pa , in the next experiments , the data acquisition with dhap / dahc was carried out by randomly measuring the outer zone of the sample layer , while data were acquired from the dhb / pa matrix by searching for hot spots of sample . in order to further evaluate the measurement sensitivity of phosphopeptides with the optimal dhap / dahc matrix , the tryptic digest of -casein and -casein with 2.5250 fmol on the target were detected by maldi - tof ms in both positive and negative ion mode . figure 3 shows that all eleven phosphopeptides from -casein and -casein could be detected with signal - to - noise ( s / n ) ratios greater than 3 at the low amount of 12.5 fmol in the positive mode ( a ) and ( b ) and detected at a lower amount of 5 fmol in the negative mode ( c ) and ( d ) . the monophosphopeptides ( t1 and t1 ) could still be observed at 2.5 fmol on target in negative ion mode ( figure 3(c ) ) . phosphopeptides generally exhibited better s / n ratios in the ms spectra in negative ion mode than that in positive ion mode , although , consistent with previous reports , the absolute signal intensity of phosphopeptide was usually lower in negative ion mode . because all tryptic phosphopeptides from -casein and -casein are serine - phosphorylated peptides , a threonine - phosphorylated peptide pt and a tyrosine - phosphorylated peptide py ( 2.5250 fmol on target ) were used to investigate whether the dhap / dahc matrix had a generic nature in detection of phosphopeptides . as expected , pt and py could be readily detected down to 5 fmol on the target in both positive and negative modes ( figure 4 ) . we further directly compared optimized dhap / dahc to dhb / pa . tryptic digests of -casein and -casein ( 12.5250 fmol on target ) were detected using these two matrices ( figure 5 ) . when using the dhap / dahc matrix , all the phosphopeptides could readily be detected at three different sample amounts . by comparison , when dhb / pa was used as the matrix , although all the phosphopeptides in 250 fmol digest ( on target ) could be detected with dhb / pa , the amount of digest went down to 50 fmol ( on the target ) , and only t1 , t2 , t3 , t6 , t1 , and t2 could be observed . furthermore , when the amount of digest went down to 12.5 fmol , only t1 and t1 were detected , with weak signals . comparing these results , it was clear that compared with dhb / pa , the optimized dhap / dahc had comparative sensitivity in the detection of singly phosphorylated peptides ( t1 and t1 ) , but greater sensitivity in the detection of multiply phosphorylated peptides , such as t2 ( 4p ) and t4 ( 5p ) . herein , we suggest that sample should be analyzed immediately after sample preparation when optimized dhap / dahc matrix used , as the crystals might not last too long under atmospheric conditions and in vacuo . we employed the axima - tof maldi mass spectrometer ( shimadzu / kratos , manchester , uk ) to perform high energy cid ms / ms of phosphopeptides to characterize phosphorylation sites . figure 6 shows the ms / ms spectra of three phosphopeptides t1 , t3 , and t2 from -casein and -casein ( 250 fmol on target ) with dhap / dahc as matrix . the number of [ mh-98 ] or [ mh-80 ] peaks in the ms / ms spectra indicated the number of phosphate groups in each phosphorylated peptide , and the fragment ions including a , b , and y ions clearly show the phosphorylation sites of the phosphopeptides t1 , t3 , and t2 . these results demonstrated the feasibility of maldi - tof / tof ms with the dhap / dahc matrix for the analysis of the phosphorylation sites of phosphopeptides , including singly and multiply phosphorylated peptides . further investigating the practicality of the dhap / dahc matrix experimentally , phosphorylated histone h1 was separated by sds - page and in - gel digested by trypsin . in order to generate appropriate peptide sizes for maldi - ms analysis , since the sequence of histone proteins is known to contain many lysine and arginine residues , the trypsin digestion time was optimized and reduced to 1 hour . figure 7(a ) shows the results of the maldi - tof - ms analysis of the tryptic digest of the phosphorylated histone h1 without the desalting step , directly detected with the optimized dhap / dahc matrix in positive ion mode . after performing data processing using the on - line mascot search engine , 31 peptides , including 2 possible phosphopeptides ( in figure 7(a ) labeled with * ) , were assigned to human histone h1 , and 81% of the sequence was covered . these two peptides corresponded to the two monophosphopeptides of histone h1 , comprising amino acids 117127 ( vatpkkaskpk , 1234.7 m / z ; possible phosphorylation sites indicated with underlined letters ) and 133145 ( aptkkpkatpvkk , 1473.9 m / z ) . the tryptic sample of h1 was also analyzed with dhb / pa , but the two peaks of the phosphopeptide were lower than those in figure 7(a ) ( see figure s1 ) . to validate the mascot searching result for the two phosphorylated peptides , we compared the mass spectra of tryptic peptides of h1 before and after treatment with alkaline phosphatase , which can cleave the phosphate group from phosphopeptides . it was clear that the two peptide peaks ( marked with asterisks in figure 7(a ) ) had disappeared and two new peaks with a mass shift of 80 da ( hpo3 = 80 da ) were visible ( figure 7(b ) ) . this demonstrated that the two peptides ( marked with asterisks in figure 7(a ) were singly phosphorylated peptides . to identify the phosphorylation sites , we used maldi - tof / tof ms to perform ms / ms analysis of the two peptides ( figure 7(c ) and 7(d ) ) . only one neutral - loss peak corresponding to [ mh - h3po4 ] indicated that both phosphopeptides were monophosphopeptides . after a mascot search , the phosphorylation sites of the two phosphopeptides were determined to be aptkkpkaptpvkk ( pt indicating the phosphothreonine ) and vaptpkkaskpk with ion scores of 53 and 50 , respectively ( scores exceeding 38 were accepted as significant matches ) . in general , the protein phosphorylation sites by a particular protein kinase shared a set of consensus sequence , which is necessary and sufficient for recognition by the kinase . to further validate the phosphorylation sites of histone h1 identified with maldi - tof / tof ms , we compared the two phosphorylated peptide sequences with the consensus sequence ps / pt - p - x - r / k , most frequently recognized by cdk1 [ 34 , 35 ] , and found that both phosphopeptide sequences were consistent with the consensus sequence . these results thus demonstrate that the dhap / dahc matrix was robust and effective in analyzing the protein phosphorylation of the biological sample by maldi - ms . by optimizing the matrix solution composition , we found that a low ratio of dhap / dahc formulation was more efficient in detecting phosphopeptides than earlier protocols . compared with dhb / pa , the optimized dhap / dahc exhibited higher sample homogeneity , and the phosphopeptides in tryptic digests of - and -casein without desalting and phosphopeptide enrichment could be measured with a higher sensitivity . further , the optimized dhap / dahc showed high energy cid ms / ms analysis of the phosphorylation sites of phosphopeptides , including multiple phosphorylated peptides , and we successfully applied this method to the characterization of the phosphorylation sites of cdk1-treated human histone h1 .

selecting an appropriate matrix solution is one of the most effective means of increasing the ionization efficiency of phosphopeptides in matrix - assisted laser - desorption / ionization time - of - flight mass spectrometry ( maldi - tof - ms ) . in this study , we systematically assessed matrix combinations of 2 , 6-dihydroxyacetophenone ( dhap ) and diammonium hydrogen citrate ( dahc ) , and demonstrated that the low ratio dhap / dahc matrix was more effective in enhancing the ionization of phosphopeptides . low femtomole level of phosphopeptides from the tryptic digests of -casein and -casein was readily detected by maldi - tof - ms in both positive and negative ion mode without desalination or phosphopeptide enrichment . compared with the dhb / pa matrix , the optimized dhap / dahc matrix yielded superior sample homogeneity and higher phosphopeptide measurement sensitivity , particularly when multiple phosphorylated peptides were assessed . finally , the dhap / dahc matrix was applied to identify phosphorylation sites from -casein and -casein and to characterize two phosphorylation sites from the human histone h1 treated with cyclin - dependent kinase-1 ( cdk1 ) by maldi - tof / tof ms .

1. Introduction 2. Experimental 3. Results and Discussion 4. Conclusion

epidemiological studies have established that periodontitis is a risk factor for cardiovascular diseases , lung diseases , renal diseases , and low birth weight in children . accordingly , it may be assumed that dental plaque bacteria not only influence the oral cavity locally , but may also contribute to the development of some serious systemic diseases . the prevalence of cardiovascular diseases in patients with periodontitis is 2550% higher than in healthy individuals . poor self - reported oral health ( as a possible risk factor for periodontitis ) and tooth loss ( as a possible consequence of periodontitis ) are positively associated with a coronary atherosclerotic burden . severe tooth loss ( likely to be due to periodontal disease ) may be a predictor of cerebrovascular disease - silent cerebral infarct . an association between oral health and cardiovascular disease has been proposed for more than a century . recently , the possible links between periodontitis and atherosclerosis have intensified and are being investigated for possible association and causality . common risk factors for these diseases include increasing age , smoking , alcohol abuse , ethnicity , educational and socioeconomic status , being male , diabetes mellitus , and obesity [ 7 , 8 ] . reviewed observational studies to date support an association between periodontitis and atherosclerosis but do not support a direct causative relationship . this extensive review illustrates an important general trend towards periodontal treatment - induced suppression of systemic inflammation and improvement in noninvasive markers of atherosclerosis and endothelial function . in the late 1990s , periodontitis - atherosclerosis syndrome ( pas ) was described and the number of articles devoted to pas has increased every year . in 1998 , there were only 4 articles on this subject , 73 articles in 2007 and at present there are 3928 articles focusing on pas in the literature . the periodontium . it is the most frequent cause of tooth loss in the adult population . the prevalence of the disease is high , with the moderate form affecting 50% and the progressive form 515% of the adult population . periodontitis is a multifactorial disease and as such the significant elements are not only the presence of pathogenic bacteria and the immune mechanism , but also the genetic predisposition of the patient . the origin and progress of the inflammatory reaction in the periodontium are a result of the altered interplay of the defense mechanisms in the periodontal tissue to respond to the activity of dental plaque bacteria . the causes of the onset and progress of periodontitis have been investigated for hundreds of years . the first records concerning the disease now called periodontitis date back to the ninth and tenth centuries a.d . with arabian physicians already ascribing the disease to soft plaque on teeth . the assumption that dental plaque was one of the significant etiological factors was confirmed as recently as the 1960s [ 12 , 13 ] . during this time , the first articles appeared in which the authors demonstrated that patients ' blood serum had enhanced levels of antigens reacting with dental plaque bacteria . a low redox potential , supply of nutrients in the crevicular fluid , and limited amount of oxygen in the periodontal pocket characterize the optimal conditions for the occurrence of gram - negative anaerobic bacteria . among the bacteria involved in pathogenesis of the disease are porphyromonas gingivalis , prevotella intermedia , fusobacterium nucleatum , tannerella forsythia , treponema denticola , and others . most of these pathogenic bacteria belong to gram - negative bacteria that contain the lipopolysaccharides ( lps ) , a potent activator of b lymphocytes . porphyromonas gingivalis is one of the most important pathogenic bacteria due to the production of a protease which breaks and deactivates il-1beta . it additionally contains a cysteine protease called gingipain which is specifically split by cd14 molecule , a receptor for lps . although the presence of subgingival microbiota is a necessary condition for the disease to progress , it is not the only cause . a genetically dependent effect in the immune mechanism or a modified immune reaction on the presence of pathogenic bacteria may be also involved in disease progression . the amount of evidence corroborating the association between specific bacteria and coronary diseases developing as a result of atherosclerosis has increased over the last two decades . the original classical hypotheses about the development of atherosclerosis did not include inflammation as a primary factor of the pathogenesis . one of the hypotheses assumed that changes of endothelium as a consequence of mechanical damage with subsequent contamination by toxins , metal ions , and free radicals lead to the formation of atherosclerotic plates [ 1619 ] . another hypothesis assumed that lipoproteins of low density caused the atherogenesis , transported by endothelium to the intima , where they oxidize and act as chemoattractant of monocytes / macrophages , leading to the formation of foam cells [ 2024 ] . in contrast , other studies [ 25 , 26 ] have illustrated the significance of proliferation of smooth muscles in the intima . in the last decade , it has been demonstrated that atherosclerosis begins as an inflammatory reaction against endothelial cells and other components of the artery wall . the inflammation sites attract accumulation of macrophages , t and b lymphocytes , and mast cells . the blood vessel walls are also covered by deposited fats which led to occlusion of the vessels . are circulating lipoproteins ( hypercholesterolemia ) , genetic predisposition , hypertension , smoking , obesity , and diabetes . epidemiological studies further indicate that infection by various types of bacteria , including periodontopathic ones ( chlamydia pneumoniae , helicobacter pylori , porphyromonas gingivalis , prevotella intermedia , and aggregatibacter actinomycetemcomitans ) and the presence of products of these bacteria ( lps , heat shock protein ( hsp ) ) in serum contributed to the development of atherosclerosis . a genetically conditioned reaction to bacterial stimulation may play a certain role in pathogenesis of atherosclerosis . patients who survived myocardial infarction exhibited a higher frequency of allele t(-260 ) in the promoter of gene for the cd14 receptor than controls . patients with verified atherosclerotic changes further exhibited enhanced serum levels of antibodies against hsp 60 . experiments performed on animal models show that infection may lead to enhanced induction of antibodies against hsp 60 . these antibodies may bond to hsp 60 expressed on endothelium of blood vessels at sites of their bifurcation where the endothelium is in stress . the binding of antibodies to the endothelium surface may be the triggering mechanism of inflammatory autoimmune disease . a recent review dealt with the epidemiological and etiopathogenetic association between chronic periodontitis and stroke . it specifically reviewed the relationship between oral infection caused by dental plaque bacteria and the stimulation of proatherogenetic mechanisms as atherosclerosis of the cerebral vessels and ischemic stroke are most frequent causes of acute stroke . there are several mechanisms by which dental plaque bacteria may initiate or worsen atherosclerotic processes : activation of innate immunity , bacteremia related to dental treatment , direct involvement of mediators activated by dental plaque antigens in atheroma processes , involvement of cytokines and heat shock proteins from dental plaque bacteria , common predisposing factors influencing both diseases . activation of innate immunity , bacteremia related to dental treatment , direct involvement of mediators activated by dental plaque antigens in atheroma processes , involvement of cytokines and heat shock proteins from dental plaque bacteria , common predisposing factors influencing both diseases . the oral cavity is permanently exposed to the activity of bacteria colonizing it . the epithelium forms not only a physiological barrier but also interacts with an innate immune response resulting in the production of antimicrobial peptides . important components of innate immunity at sites of contact with microorganisms are alkaline antimicrobial peptides that contain less than 100 amino acids and are phylogenetically very stable ; they exist in both plant and animal life . in mammals , they are present in phagocyte granules , are produced by the epithelium , and are present in bodily fluids and secretions ; the most important are defensins and cathelicidins . of particular importance for defense in the oral cavity are defensins , calprotectin , histatin and , only in humans , cathelicidin - ll37/hcap18 . defensins and histatin present in the phagocyte granules are produced by mucosal epithelium and by salivary glands . it binds lps , neutralizes endotoxine activity and acts chemotactically on neutrophils , monocytes , t lymphocytes , and mast cells and exhibits bactericidal activity . its presence in large quantities in the junctional epithelium as a result of the migration of neutrophils is of great importance for the defense of the oral cavity . functional defects of neutrophils are a risk factor of the development of aggressive periodontitis [ 33 , 34 ] . both the gram - positive and gram - negative bacteria of dental plaque contain many structural and secretory components that either directly damage the periodontal tissue or stimulate the immune system of the host . the cell walls of gram - negative bacteria are formed of peptidoglycans , polysaccharides , proteins , lipids , lipopolysaccharides , and lipoproteins . the walls of gram - positive bacteria consist of peptidoglycans , teichoic acid , and polysaccharides . lps influences the immune reaction by binding to toll - like receptor-4 ( lps of escherichia coli and aggregatibacter actinomycetemcomitans ) or to toll - like receptor-2 ( lps of porphyromonas gingivalis ) . lps also stimulates expression of costimulatory molecules cd80/cd86 , via binding to toll - like receptor-4 ; furthermore , it stimulates molecules of the major histocompatibility complex mhc - ii which are important for activation of t - cells . peptidoglycans activate the cells through binding to the toll - like receptor-2 ; they are recognized by the complement as well as by specific receptors and they also participate in the activation of the complement system . the immune response , directed against an infection , also leads to further destruction of the tissue . it was confirmed in in vitro experiments that cells of the junctional epithelium activated by porphyromonas gingivalis produce tnf- and il-1 and express surface molecules icam-1 and vcam-1 . porphyromonas gingivalis also produces proteases which cleave and inactivate il-1 and the cysteine protease - gingipain , which specifically cleaves cd14 ( receptor for lps ) . gingipain produced by porphyromonas gingivalis degrades proteins to generate free arginine or lysine ; the primary goal of the degradation is to obtain the peptides and amino acids necessary for survival of the bacteria . however , it also degrades many important molecules on the surface of cells or in its environment and thus it protects p. gingivalis against the immune reaction . this enzyme also degrades il-8 , il-1 , il-6 [ 39 , 40 ] , surface molecules icam-1 on epithelium cells , cd14 , lipopolysaccharide binding protein ( lbp ) , molecules on the surface of monocytes and fibroblasts , components of the complement , and also immunoglobulins . among the markers of developing inflammation crp belongs to the highly conservative pentraxin family of proteins significant to the innate immune reaction . crp is bound to apoptotic cells , oxidized low density lipoprotein ( ox - ldl ) and oxidized phospholipids , but do not bind to native low density lipoprotein . it is assumed that crp is involved in modulation of developing atherosclerosis , because crp and ox - ldl are present in atherosclerotic lesions . another sign of an activated innate immune system is an enhanced level of neopterin in the patient 's serum . a high concentration of neopterin corresponds to a high degree of activation of the immune reaction in acute coronary syndrome . bacteria of dental plaque and their components in the periodontal tissues may penetrate into the circulation system and exhibit pathogenic potential . increased incidence of bacteremia by gram - negative bacteria and infectious endocarditis was described more than 30 years ago . this synoptic review summarizes the cases of infectious endocarditis caused by bacteria of dental plaque . the following bacteria were proven as etiological factors : aggregatibacter actinomycetemcomitans , eikenella corrodens , streptococcus species , capnocytophaga , neisseria , and lactobacillus . dental infection affecting the periodontium can spread into the systemic circulation by dental treatment procedures or teeth brushing and can induce bacteremia . patients with untreated adult periodontitis are at greater risk of bacteremia after periodontal probing than patients with chronic gingivitis . following dental extraction , the most frequently identified bacteria in the positive blood cultures were the streptococcus species . high incidence of bacteremia was found in patients without antibiotic prophylaxis after conservative and surgical dental treatment . low incidence of bacteremia was demonstrated after orthodontic banding and debanding [ 53 , 54 ] . dental surgical procedures were a cause of bacterial endocarditis in children . in these cases , viridans streptococci were mainly detected [ 56 , 57 ] . in another study , an increased level of dental plaque bacteria in blood circulation after dental plaque removal and tooth extraction were described . on the other hand , the ability of porphyromonas gingivalis to actively invade aortic and heart endothelial cells is an example of the relationship between periodontitis and atherosclerosis . the presence of porphyromonas gingivalis and streptococcus sanguis in atherosclerotic plaques in samples of veins after surgical reconstruction of venous system was established . using pcr reaction , microbial ribosomal rna ( rrna ) and dna from porphyromonas gingivalis , aggregatibacter actinomycetemcomitans , and prevotella intermedia were detected in atherosclerotic plaques [ 6366 ] . a possible link between periodontal disease and abdominal aortic aneurysm resected specimens from abdominal aortic aneurysm were positive for periodontal bacterial dna in 86% of cases . the presence of bacteria was demonstrated in the intima layer of the atherosclerotic occlusive aorta but not in control specimens . in 31 carotid endarterectomy specimens , porphyromonas gingivalis was detected in 52% , fusobacterium nucleatum in 34% , tannerella forsythia in 34% , prevotella intermedia in 41% , and aggregatibacter actinomycetemcomitans in 17% . in a recent review , the results of 16 studies investigating the presence of oral bacteria in atheromatous plaque were compared , identifying aggregatibacter actinomycetemcomitans and porphyromonas gingivalis as the most frequently occurring bacteria . the relationship between atherosclerosis and porphyromonas gingivalis the development of periodontitis and atherosclerosis was induced by oral inoculation by porphyromonas gingivalis . over the course of 4 months , the mice exhibited lipid stripes in which the presence of porphyromonas gingivalis was detected , and in mice with periodontitis , higher serum levels of il-6 and vcam-1 in aorta were detected . study on the influence of microbiota showed that both conventional and germ - free kept mice with apoe/ deficit on a high cholesterol diet had lesions of heavy atherosclerosis in thoracic and ventral aortas . in some cases , these lesions completely obstructed the vessel . in germ - free mice on a diet with high cholesterol content , histopathological evaluation of removed tissue samples displayed greater damage of organs than that found in conventionally kept mice . after stimulation by bacteria and their components ( lps , peptidoglycans ) , the periodontal tissue produces inflammatory cytokines ( il-1 , tnf- , il-6 , inf- , il-12 , il-10 ) , chemokines ( mcp-5 , il-8 , mip-1 ) , prostaglandine pge2 , and no [ 72 , 73 ] . lps from aggregatibacter actinomycetemcomitans significantly enhances expression of 2 integrins and l - selectins . peptidoglycans are components of the bacterial walls and , like lps , they contribute to activation of immune cells via binding to tlr-2 receptor . in addition , peptidoglycans are recognized by the complement system and specific receptors resulting in production of tnf- , il-1 , il-6 , il-8 , and mip-1 [ 74 , 75 ] and no in macrophages . in comparison with lps , peptidoglycans are not so strong stimulators of immune reaction . the presence of circulating oral bacteria or bacterial components may stimulate blood cells to produce cytokines . higher levels of il-6 were detected in the sera of patients with periodontitis compared to healthy controls . heat shock proteins ( hsps ) are known to be the most immunogenic antigens of bacteria . the extensive homology between human and bacterial hsps may play a role in the activation of atherosclerotic changes . decreased proliferative responses of peripheral blood cells to hsp in periodontitis patients compared to control patients were found . decreased production of ifn- after stimulation of peripheral blood mononuclear cells with hsp 60 and hsp 70 was observed in periodontitis patients as compared to control patients . these findings support the hypothesis of suppressed th1 response in periodontitis patients that may lead to increased susceptibility for development of aggressive periodontitis . antibodies against human hsp 60 and antibodies against porphyromonas gingivalis ( groel ) in sera and inflamed gingival tissues were found in periodontitis patients . a quantitative analysis of serum antibodies demonstrated significantly increased levels in periodontitis patients as compared to controls . anti - porphyromonas gingivalis groel antibodies were detected in all samples of inflamed gingival tissues of periodontitis patients . enhanced levels of antibodies against hsp 60 were also found in the serum of patients with positive atherosclerotic changes . molecular mimicry between groel of the periodontopathic porphyromonas gingivalis and autologous human hsp 60 may play a role in immune mechanisms . experiments performed on animal models show that bacterial infection may lead to enhanced production of antibodies against hsp 60 expressed on the endothelium of blood vessels at sites of their bifurcation where the endothelium is in stress . the binding of antibodies to the endothelium surface may be a triggering mechanism of inflammatory autoimmunity disease . several studies have demonstrated that the immune response to hsp 60 may be involved in the pathogenesis of both atherosclerosis and chronic periodontitis . antibody levels to human as well as to porphyromonas gingivalis hsp 60s were the highest in patients with atherosclerosis in comparison to healthy controls . clonal analysis of the t cells clearly demonstrated the presence of both human and porphyromonas gingivalis hsp groel - reactive t - cells in the peripheral circulation of patients with atherosclerosis . these results suggest that t - cell clones with the same specificity may be involved in the pathogenesis of the different diseases . analysis of the nucleotide sequences of the t - cell receptor ( tcr ) demonstrated that human hsp 60-reactive t - cell clones and t - cells have the same receptors infiltrating periodontitis lesions . analysis of the cytokine profile demonstrated that hsp 60-reactive peripheral blood mononuclear cells produced significant levels of ifn- in periodontitis patients , whereas porphyromonas gingivalis groel did not induce type 1 or type 2 cytokine profiles . in control subjects , no significantly increased expression of ifn- or il-4 was induced . these results suggest that periodontitis patients have human hsp 60-reactive t - cells with a type 1 cytokine profile . in another study , groel specific t - cell lines from peripheral blood and groel human hsp 60 and porphyromonas gingivalis specific t - cell lines from atherosclerotic plaques were characterized in their cross - reactivity . the cytokine profiles of arterial t - cell lines specific for groel , human hsp 60 , and porphyromonas gingivalis were th2 cd4 cells predominantly . cross - reactivity between bacterial cells , including periodontal pathogens , with endothelial cells expressing hsp 60 may explain an association between atherosclerosis and periodontal disease . the nature of inflammatory infiltrate and the presence of hsp and groel were examined in 31 carotid endarterectomy specimens . human hsp 60 was detected on surface of endothelial cells , smooth muscle cells , and lymphocytes ; groel and bacteria were detected within intimal cells . endogenous hsp is also a target of autoantibodies in autoimmune disorders , atherosclerosis and vascular diseases . hsp is one of the endothelial cell autoantigens able to trigger cytotoxic and apoptotic response by related autoantibodies [ 84 , 85 ] . marked atherogenic effects of repeated immunizations with porphyromonas gingivalis , bacterial and host hsp , were examined in apoe/ deficient mice . the development of lesions in proximal aorta correlated with the levels of hsp 60 and groel antibodies and may be explained as molecular mimicry between groel and hsp 60 in porphyromonas gingivalis immunized apoe/ mice . the presence of porphyromonas gingivalis and tannerella forsythia increased in patients with myocardial infarction and periodontitis with periodontal pockets deeper than 4 mm . the authors of this study also reported a correlation between these bacteria and increased levels of corresponding antibodies against hsp 60 in patients ' sera . long - term studies indicate that patients with periodontitis have a 2025% higher risk of myocardial infarction [ 89 , 90 ] and a 17% higher risk of brain stroke . the first lipid stripes can appear in blood vessels of 10-year - old children , and foam cells have been found in children less than 1 year old . it remains an open question whether periodontitis is the cause or a supporting factor of atherosclerosis . the relationship between periodontitis and atherosclerosis has been a subject of many research activities with the number of publications focusing on this relationship rapidly increasing in recent years . the actual number of studies which focus on this subject is 3928 according to a review from this year . periodontitis and atherosclerosis are multifactorial diseases with an onset in early childhood , although first symptoms may appear in adulthood . since foam cells have been found in early childhood and lipid stripes in 10-year - old children , we do not think that in these cases , periodontitis is the cause of changes in blood vessels . increased prevalence of obesity in children and young people is a risk factor that may influence lipid stripes development [ 95 , 96 ] . however , endothelial damage by formation of lipid stripes in early childhood may lead to the capture of bacteria of dental plaque origin that penetrate into blood circulation after treatment procedures in oral cavity of children as well as of patients with aggressive or chronic periodontitis . preatheroma and atheroma are usually diagnosed in patients aged 2030 years similar to the age which aggressive periodontitis ( early onset periodontitis ) is diagnosed . fibroatheroma is diagnosed in patients aged 40 years and over , in the similar age group where periodontitis is diagnosed in more than 50% of patients . when solving the question whether periodontitis is the cause or consequence of developing atherosclerosis , we may conclude that circulating microorganisms or their products ( hsp ) may promote pathogenesis and enhance local inflammatory changes in vessel walls that may promote clotting and clot formation . prevention programs focused on monitoring patients with chronic periodontitis in relation to the risk of developing cardiovascular diseases should be emphasized . in patients with early inflammatory symptoms in the periodontium , a dental examination should be supplemented with a laboratory examination of cardiovascular markers . timely therapy of both diseases diminishes the risk of developing initial as well as serious changes at a later time . since bacteremia develops after dental treatment and the first lipid stripes arise in early childhood , research of early phases of atherosclerosis and a more precise specification of the risk factors of the disease could lead to an enhanced quality of preventive care and treatment of patients at a later age . antibodies in the patients ' sera reacting with ox - ldl and hsp 60 and hsp 65 may be detected in very young patients with aggressive periodontitis and may be a predicative factor in the development of serious systemic diseases .

over the last two decades , the amount of evidence corroborating an association between dental plaque bacteria and coronary diseases that develop as a result of atherosclerosis has increased . these findings have brought a new aspect to the etiology of the disease . there are several mechanisms by which dental plaque bacteria may initiate or worsen atherosclerotic processes : activation of innate immunity , bacteremia related to dental treatment , and direct involvement of mediators activated by dental plaque and involvement of cytokines and heat shock proteins from dental plaque bacteria . there are common predisposing factors which influence both periodontitis and atherosclerosis . both diseases can be initiated in early childhood , although the first symptoms may not appear until adulthood . the formation of lipid stripes has been reported in 10-year - old children and the increased prevalence of obesity in children and adolescents is a risk factor contributing to lipid stripes development . endothelium damage caused by the formation of lipid stripes in early childhood may lead to bacteria penetrating into blood circulation after oral cavity procedures for children as well as for patients with aggressive and chronic periodontitis .

1. Introduction 2. Periodontitis 3. Atherosclerosis 4. Dental Plaque Bacteria and Atherosclerotic Processes 5. Conclusion

heart failure is a chronic , progressive disease affecting approximately 12% of all the adult population , increasing to more than 10% in the population segment over 70 years ; its incidence rose with the extension of life - span and the aging of the population [ 13 ] . its prognosis is poor , the mortality is higher than that of some cancers and the morbidity is marked by a low quality of life and frequent rehospitalizations . death occurs in months to years from the onset , but in an unpredictable manner [ 410 ] . its evolution is the result of the overexpression of a number of biologically active molecules with negative influence on the cardiovascular system , with chronic activation of signaling pathways secondary to inadequate tissue perfusion . the compensatory mechanisms described until now are the activation of the adrenergic and renin - angiotensin systems ( raas ) with the purpose of maintaining the cardiac output but with long - term deleterious effects . balancing these systems , there are other molecular pathways with key roles in the physiology of the cardiovascular system and which are now the principal subject of research in this field . the gene of apelin is located on the long arm of x chromosome ; the apelin is syntethized as preproapelin ( 77 aminoacids ) which is then cleaved by an angiotensin - converting enzyme to shorter active fragments ( c - terminal peptides : apelin-13 , -16 , -17 , -19 , -36 ) . it was discovered in 1998 from bovine stomach extracts and named apelin : apj endogenous ligand . the most active of all is apelin-13 , the shorter peptide , with its pyroglutamated form representing the principally biological active ligand ; its activity is 8 and 60 folds higher than apelin-17 and apelin-36 , respectively . apelin s receptor , apj , identified in 1993 by homologous cloning in the human genome project , was initially considered an orphan until the discovery of apelin in 1998 . apj has 31% sequence homology with angiotensin ii type 1 receptor ( at1r ) and shares its tissue distribution , but it can not bind angiotensin ii ( ang ii ) . it is coupled with gi protein , and probably gq protein too , and it is desensitized differently by different fragments of apelin . the shorter fragments activate the receptor for a short period of time because it is rapidly internalized in the cellular membrane , returning to the surface within an hour and becoming available for activation again . in contrast , the longer fragments of apelin dissociate slowly from the receptor , blocking it in an active state for a longer period of time and lowering the recycling rate . at cellular level , the apelin activates the proteinkinase b ( akt ) , phosphatidil - inositol-3 kinase ( pi3k ) and the extracellular signal - regulated kinases ( erk ) pathway , which are involved in controlling apoptosis and cellular migration and proliferation . the apelin and its receptor can be found in a variety of tissues ( central nervous system , enterochromaffin - like gastric cells , pancreatic islet cells , osteoblasts , t - lymphocytes , adipose tissue ) , with higher concentrations in the lungs , cardiovascular system and spleen . it is synthesized locally in the endothelium and it is found in the cellular organelles as endoplasmic reticulum , golgi apparatus and secretory vesicles . later studies showed that the most important site of apelin - apj expression and action is the cardiovascular system , especially the vascular endothelium , vascular smooth muscle cells , endocardial endothelium , and in a lesser extend , the myocardium . this explains the higher concentration in the lungs and spleen , paralleling the level of vascularization [ 2025 ] . in the atrial tissue the apelin concentration is 200 folds higher than in the ventricular tissue and it appears to be correlated to the plasma level . although we do not know the exact source of the plasma apelin , it may be generated from the atrium . in some other tissues as the kidney and adrenal gland it is expressed only in the blood vessels , although its effects on these tissues are complex and cover multiple sites . apelin is a diuretic , but its effects on the kidneys are not limited only to the renal tissue . apelin is synthesized in the brain and inhibits the secretion of vasopresin from the neurons , favoring water excretion ; also it acts on the renal microcirculation and probably on the tubular function to validate its diuretic effect [ 2830 ] . the apelin / apj system counter - regulates the raas by antagonizing the activity of ang ii , its major effector . describe this mechanism as a result of direct , physical interaction between the apj and atr1 receptors , dimerization and sending atr1 into a low affinity state , thus decreasing its interaction with ang ii . apelin induces this physical interaction and increases the density of apj as compared to atr1 ( by increasing its expression and its availability at the membrane level ) , but the density of both receptors were not modified by the exogenous ang ii in this study . this may be the explanation for the development of cardiovascular diseases in the presence of dysfunctional apelin / apj system that can not balance the overactivated , injurious raas . also , recent studies demonstrated a relationship between apelin / apj system and the angiotensin converting enzyme type 2 ( ace2 ) which has a key role in counterbalancing the raas by catalyzing the transformation of ang ii into angiotensin 17 ( ang 17 ) , a molecule with opposing effects to ang ii . these findings stand for the theory than apelin / apj is a beneficial molecular pathway in the physiology of cardiovascular system . the apelin / apj system has many physiologic effects on water balance , glycemic control , nutritional behavior , immunity , but its principal target is the cardiovascular system . latest studies have showed that apelin / apj system has a key role in its normal function and in the development of vascular and heart diseases such as atherosclerosis , coronary heart disease , heart failure , systemic and pulmonary arterial hypertension and ischemic - reperfusion lesion . its action seems to be mainly autocrine and paracrine , the ligand expression paralelling the receptor expression , but its plasma concentration also corresponds to a circulating hormone . plasma concentration is about 10 g / ml with a half time of less than 5 minutes ; its slow , sustained , inotropic effect is validated in a subnanomolar concentration ( ec50=33 pmol / l ) , being the most potent endogenous inotropic molecule , overcoming the adrenomedulin and endothelin . in the experimental studies conducted so far the effect reached its maximum in 2030 min - completely different from mimetics which act within seconds - and last longer ; it was valid even when the no production was blocked , and the endothelin , the ang ii and receptors were inhibited . the undelying mechanism for apelin s inotropy is the activation of phospholipase c , proteinkinase c , na - h sarcolemmal exchange ( nhe ) and na - ca exchange ( ncx ) pathways , without involving the l - type ca - channels and voltage - activated k - channels and without inducing myocardial hypertrophy . describe that even after inhibition of both nhe and ncx , the inotropic effect of apelin continues in a proportion of about 40% ; this fact suggests the presence of additional , unclear , mechanism for inotropism . in the study of dai et al . results showed that the inotropic effect of apelin is due mostly to an increase in ca availability in the myocardial cell and not to a sensitization of myofilaments ; the mechanism is not yet completely understood . apelin - apj receptor interaction at the vascular level induces vasodilation through no release from endothelial cells ( in arteries and veins ) with consequent decrease in systemic vascular resistance , lowering the pre- and the afterload of the left ventricle and improving its filling pattern . the apj receptor is present in the vascular smooth muscle and it stimulates its contraction as was demonstrated in experimental condition on endothelial denuded vein . the vasoconstricion induced by apelin on denuded vessels and the vasodilation produced in the presence of normal functioning endothelium suggest the complexity of apelin s role on the circulatory system , the fine tuning of the vascular tone and the easily disturbed apelin s effects in the presence of injured endothelium . the studies conducted so far showed the importance of apelin / apj pathway in the development of heart failure and the fact that its downregulation in the myocardial cell contributes to the structural and functional alteration of the heart . in experimental models of iwanaga and colleagues ( dahl salt - sensitive rats with left ventricular hypertrophy and secondary heart failure ) , the cardiac apelin and apj receptor were down - regulated in the lv dysfunction stage , associated with an up - regulation of ace2 ( the breakdown enzyme for apelin peptides ) , which may contribute to further lowering the plasma apelin , which is already downregulated . in their study , the rats were treated with angiotensin receptor blocker ( arb ) , metalloproteinase ( mmp ) inhibitor and a betablocker , and although there was functional improvement in all groups , only in the arb group the apelin and apj expression increased ; this is a strong proof of the fact that the beneficial effects of acei / arbs are based on the modulation of apelin . further , the expression of apelin was downregulated after infusion of ang ii , both in the pressor and the subpressor dose , effect that was prevented with the at1r blockade , sustaining furthermore the interrelation between the raas and the apelin / apj system . similar results about the tissue expression of apelin and its marn were obtained by wang and colleagues on dogs with heart failure , but levels of apj ( and its marn ) were equivalent to normal dogs . this difference may be due to the characteristics of the species used in the experiments , but further investigation is needed . used as an exogenous vasoactive agent , apelin showed a unique combination of inotropic and vasodilatory properties of apelin , which leads to a rise of cardiac output and to a decrease of systemic vascular resistance without significant arterial hypotension and tachycardia , regarding the dose . this study brings additional proof about the importance of apelin and its receptor in the development of heart failure and the potential favorable effect of treatment with exogenous apelin in patients with systolic dysfunction , and it is backed - up by other , recent studies . in a study on genetic apelin deficiency in mice ( apelin ) the results led to the conclusions that apelin is a crucial peptide for mantaining the systolic function of the aging heart in the conditions of chronic pressure overload . szokodi et al also found low levels of apelin marn in rats myocardium exposed to mechanical stretch and chronic pressure overload . wang and colleagues used similar experimental models in 2013 ( apelin and apelin ) and observed an aggravated postinfarction remodeling , neovascularization and impaired functional recovery of apelin deficient mice . the lack of apelin compromises the activation of prosurvival akt / pi3k and erk pathway , both in vitro and in vivo , thus amplifying the myocardial damage and lowering the global cardiac performance . more recent studies that used apelin analogues confirmed these results and demonstrated their capacity of lowering the myocardial ishemic / reperfusion injury in vivo . zhang et al . in 2013 experimented the effects of exogenous apelin on rats with myocardial infarction induced through left anterior artery ( lad ) ligation ; their results showed exquisite effects of apelin on microcirculation and angiogenesis by stimulating the migration of endothelial cells to ischemic regions , enhanced healing and new vessel formation . also , apelin decreased the vascular permeability in the ischemic region with possible effects on postischemic edema and inflammation . these findings can be extrapolated to ischemic heart disease with consequent heart failure , suggesting the potential benefit of apelin treatment , both short and long term . in 2012 a study using exogenous apelin on dahl salt - sensitive rats with end - stage heart failure showed an improvement of lv dysfunction and remodeling , a lowering of the oxidative stress ( through inhibition of nadph oxidase ) , a regulation of the akt / enos pathways and apelin / apj expression in the rat hearts after apelin treatment . this study showed the correlation of apelin with the oxidative damage and gave the premise for the potential benefit of the apelin treatment in the end - stage heart failure . exogenous apelin showed benefit also upon the myocardial hypertrophy and fibrosis ( secondary to increased afterload ) through inhibition of fibroblasts tgf--induced differentiation into myofibroblasts and diminishing the fibrosis process . the already formed myocardial hypertrophy and fibrosis were reduced and the lv dysfunction was prevented . the studies published so far bring evidence for down - regulation of apelin and its receptor during the process of myocardial remodeling ( hypertensive or ischemic ) with consequent reduction of its inotropic , antifibrotic and cytoprotective effects . the treatment with apelin restores the position of apj within the membrane and revives the mechanism of inotropy , both in failing and normal hearts . the exogenous apelin has benefic effects on the cardiac dysfunction secondary to other causes too , besides hypertensive , ischemic or idiopapathic cardiomyopathy . in humans the studies conducted so far led to conflicting results : although some research showed no significant difference of apelin plasma level between idc patients and normal controls , larger studies have showed that apelin rises in early , mild to moderate heart failure , and lowers significantly in severe , end - stage heart failure . plasma apelin increases in early heart failure and lowers in the final stage but did not correlate with nt - probnp and , with the information we have at this moment , it can not be used as a diagnostic tool , nor as a prognostic marker [ 5456 ] . also , plasma apelin appears not to be correlated with age , sex , body mass index , etiology of heart failure and renal function . in patients with heart failure plasma levels of apelin ( from peripheral venous puncture ) is reduced compared to controls , regarding of nyha class and the severity of systolic dysfunction . chandrasekaran and colleagues measured the level of apelin in three different sites ( coronary sinus , aorta and renal vein ) in patients with heart failure ( of different nyha class ) and controls . they found a significant reduction of apelin in the coronary sinus of heart failure patients as comparedto controls and a reduction of apelin in the aorta of controls in comparison with the coronary sinus that was not seen in heart failure patients . also , the heart failure patients had no apelin gradient between the three sites as was seen in control patients . left ventricular apelin marn increases in chronic heart failure secondary to ischemic heart disease or idiopathic dilated cardiomyopathy ( idc ) . also , in idc the apj receptor marn is significantly decreased , although the plasma apelin was no different from the healthy controls as was demonstrated by another study . in 2010 piktin et al analyzed samples of human myocardial tissue collected from cardiac transplant recipients ( for dilated cardiomyopathy and ischemic heart disease ) and found extreme down - regulation of apj in all samples with unchanged apelin levels , which may be the explanation for the progression of the cardiac failure , the apelin having no receptors to manifest its inotropic effect . it appears that the mechanical stretch is one of the stimuli for down - regulation of apj receptor , with secondary up - regulation after mechanical offloading of the left ventricle ( after left ventricle assisting device implantation ) in chen et al . cardiac resynchronization , another method of improving the mechanic and electric properties of a failing heart , had a good influence over the plasmatic apelin at nine months after implantation of the device . it is already well - known that wall stress and activation of raas augment in heart failure and these pathways are feasible of altering the apelin - apj expression . also , the ang ii negatively influence the level of apj in the cardiomyocites . the studies conducted so far showed the importance of apelin / apj pathway in the development of heart failure and the fact that its downregulation in the myocardial cell contributes to the structural and functional alteration of the heart . in experimental models of iwanaga and colleagues ( dahl salt - sensitive rats with left ventricular hypertrophy and secondary heart failure ) , the cardiac apelin and apj receptor were down - regulated in the lv dysfunction stage , associated with an up - regulation of ace2 ( the breakdown enzyme for apelin peptides ) , which may contribute to further lowering the plasma apelin , which is already downregulated . in their study , the rats were treated with angiotensin receptor blocker ( arb ) , metalloproteinase ( mmp ) inhibitor and a betablocker , and although there was functional improvement in all groups , only in the arb group the apelin and apj expression increased ; this is a strong proof of the fact that the beneficial effects of acei / arbs are based on the modulation of apelin . further , the expression of apelin was downregulated after infusion of ang ii , both in the pressor and the subpressor dose , effect that was prevented with the at1r blockade , sustaining furthermore the interrelation between the raas and the apelin / apj system . similar results about the tissue expression of apelin and its marn were obtained by wang and colleagues on dogs with heart failure , but levels of apj ( and its marn ) were equivalent to normal dogs . this difference may be due to the characteristics of the species used in the experiments , but further investigation is needed . used as an exogenous vasoactive agent , apelin showed a unique combination of inotropic and vasodilatory properties of apelin , which leads to a rise of cardiac output and to a decrease of systemic vascular resistance without significant arterial hypotension and tachycardia , regarding the dose . this study brings additional proof about the importance of apelin and its receptor in the development of heart failure and the potential favorable effect of treatment with exogenous apelin in patients with systolic dysfunction , and it is backed - up by other , recent studies . in a study on genetic apelin deficiency in mice ( apelin ) the results led to the conclusions that apelin is a crucial peptide for mantaining the systolic function of the aging heart in the conditions of chronic pressure overload . szokodi et al also found low levels of apelin marn in rats myocardium exposed to mechanical stretch and chronic pressure overload . wang and colleagues used similar experimental models in 2013 ( apelin and apelin ) and observed an aggravated postinfarction remodeling , neovascularization and impaired functional recovery of apelin deficient mice . the lack of apelin compromises the activation of prosurvival akt / pi3k and erk pathway , both in vitro and in vivo , thus amplifying the myocardial damage and lowering the global cardiac performance . more recent studies that used apelin analogues confirmed these results and demonstrated their capacity of lowering the myocardial ishemic / reperfusion injury in vivo . zhang et al . in 2013 experimented the effects of exogenous apelin on rats with myocardial infarction induced through left anterior artery ( lad ) ligation ; their results showed exquisite effects of apelin on microcirculation and angiogenesis by stimulating the migration of endothelial cells to ischemic regions , enhanced healing and new vessel formation . also , apelin decreased the vascular permeability in the ischemic region with possible effects on postischemic edema and inflammation . these findings can be extrapolated to ischemic heart disease with consequent heart failure , suggesting the potential benefit of apelin treatment , both short and long term . in 2012 a study using exogenous apelin on dahl salt - sensitive rats with end - stage heart failure showed an improvement of lv dysfunction and remodeling , a lowering of the oxidative stress ( through inhibition of nadph oxidase ) , a regulation of the akt / enos pathways and apelin / apj expression in the rat hearts after apelin treatment . this study showed the correlation of apelin with the oxidative damage and gave the premise for the potential benefit of the apelin treatment in the end - stage heart failure . exogenous apelin showed benefit also upon the myocardial hypertrophy and fibrosis ( secondary to increased afterload ) through inhibition of fibroblasts tgf--induced differentiation into myofibroblasts and diminishing the fibrosis process . the already formed myocardial hypertrophy and fibrosis were reduced and the lv dysfunction was prevented . the studies published so far bring evidence for down - regulation of apelin and its receptor during the process of myocardial remodeling ( hypertensive or ischemic ) with consequent reduction of its inotropic , antifibrotic and cytoprotective effects . the treatment with apelin restores the position of apj within the membrane and revives the mechanism of inotropy , both in failing and normal hearts . the exogenous apelin has benefic effects on the cardiac dysfunction secondary to other causes too , besides hypertensive , ischemic or idiopapathic cardiomyopathy . in humans the studies conducted so far led to conflicting results : although some research showed no significant difference of apelin plasma level between idc patients and normal controls , larger studies have showed that apelin rises in early , mild to moderate heart failure , and lowers significantly in severe , end - stage heart failure . plasma apelin increases in early heart failure and lowers in the final stage but did not correlate with nt - probnp and , with the information we have at this moment , it can not be used as a diagnostic tool , nor as a prognostic marker [ 5456 ] . also , plasma apelin appears not to be correlated with age , sex , body mass index , etiology of heart failure and renal function . in patients with heart failure plasma levels of apelin ( from peripheral venous puncture ) is reduced compared to controls , regarding of nyha class and the severity of systolic dysfunction . chandrasekaran and colleagues measured the level of apelin in three different sites ( coronary sinus , aorta and renal vein ) in patients with heart failure ( of different nyha class ) and controls . they found a significant reduction of apelin in the coronary sinus of heart failure patients as comparedto controls and a reduction of apelin in the aorta of controls in comparison with the coronary sinus that was not seen in heart failure patients . also , the heart failure patients had no apelin gradient between the three sites as was seen in control patients . left ventricular apelin marn increases in chronic heart failure secondary to ischemic heart disease or idiopathic dilated cardiomyopathy ( idc ) . also , in idc the apj receptor marn is significantly decreased , although the plasma apelin was no different from the healthy controls as was demonstrated by another study . in 2010 piktin et al analyzed samples of human myocardial tissue collected from cardiac transplant recipients ( for dilated cardiomyopathy and ischemic heart disease ) and found extreme down - regulation of apj in all samples with unchanged apelin levels , which may be the explanation for the progression of the cardiac failure , the apelin having no receptors to manifest its inotropic effect . it appears that the mechanical stretch is one of the stimuli for down - regulation of apj receptor , with secondary up - regulation after mechanical offloading of the left ventricle ( after left ventricle assisting device implantation ) in chen et al . cardiac resynchronization , another method of improving the mechanic and electric properties of a failing heart , had a good influence over the plasmatic apelin at nine months after implantation of the device . it is already well - known that wall stress and activation of raas augment in heart failure and these pathways are feasible of altering the apelin - apj expression . also , the ang ii negatively influence the level of apj in the cardiomyocites . apelin / apj is a crucial molecular pathway in the protection of the heart from the injuries caused by hemodynamic overload or structural damage , regardless of the cause , and thus temporary preventing the development of heart failure . apelin is a counter - regulator of the raas and keeps its activation in a balance . intense and prolonged raas activation disable the apelin cardioprotection and puts the system into withdrawal . experimental administration of apelin in heart failure showed unique combination of inotropism and vasodilation effects with no short - term deleterious effects . this is the premise for the potential benefit of apelin in heart failure , but further studies are needed .

heart failure is a chronic , progressive disease in which the overexpression of biologically active molecules and neurohomonal activation are the key factors of the evolution and natural history . the apelin - apj system is a newly discovered molecular pathway and the raas counterbalance is its principal effect . the apelin is a potent inotrope , vasodilator and diuretic with crucial cardioprotective effects against angiotensin and aldosterone injuries . intense and prolonged raas induces the downregulation of the apelin and its receptor at myocardial level and cancels their protection . compared to the vasoactive agents used in the treatment of acute heart failure , exogen apelin has unique intropic and vasodilatory effects without deleterious consequences , being a promising therapeutic option .

Introduction Physiological effect of apelin/APJ receptor pathway on the cardiovascular system The role of apelin/APJ system in heart failure Experimental animal studies Human studies Conclusions

diabetes mellitus is the most common metabolic disease and becomes a heavy burden of public health systems . in china , deterioration of beta cell function and insulin resistance are two fundamental pathophysiologic defects of type 2 diabetes mellitus ( t2 dm ) . it has been proven that at the time when t2 dm was established , the loss of beta cell function was shown to reduce by 50% and this decline of beta cell function progressed over time although traditional antihyperglycemic therapy had been applied . in order to postpone the progress of disease , new therapies are required to persistently act on beta cell failure and insulin resistance . in our previous studies , intensive insulin interventions , especially continuous subcutaneous insulin infusion ( csii ) , induced near - normoglycemia over 1 year without antihyperglycemic agents in nearly half of the patients with newly diagnosed t2 dm with favorable recovery of beta cell function [ 3 , 4 ] . the reason for glycemic remission in these patients was considered to be alleviation of glucotoxicity , lipotoxicity , and insulin resistance [ 5 , 6 ] . however , the therapy , which lasted for only 2 - 3 weeks , had its limitations in covering the multiple pathophysiological defects in the long term . in another trial investigating the effect of combination of metformin or rosiglitazone with csii , the combination of metformin for 3 months had better effects on insulin secretion function measured by acute insulin response ( air ) and homa - b while the combination with rosiglitazone better improved muscle insulin resistance . since the two medicines used in that study mainly were targeted at insulin resistance , it would be of great interest whether combining csii with medicine intervening beta cell failure , the critical pathophysiology mechanism of t2 dm , might provide better clinical outcomes compared with short - term csii alone . liraglutide , a glucagon - like peptide-1 ( glp-1 ) analog with a 97% homology with endogenous glp-1 , lowers blood glucose by enhancing glucose - dependent insulin secretion of beta cells and suppressing glucagon secretion of alpha cells . in some rodent studies , liraglutide reduced beta cell apoptosis and promoted its proliferation , which might potentially modify the progression of t2 dm [ 9 , 10 ] . moreover , liraglutide also reduced body weight in a dose dependent manner , ameliorated lipid profiles , lowered blood pressure , and reduced cardiovascular risk markers such as adipokines and proinflammatory factors , all of which are favorable in management of t2 dm . we hypothesized that combining csii with liraglutide might have better effects over csii alone . therefore , we conducted this randomized controlled trial investigating whether liraglutide in combination with short - term csii therapy has better effect over csii alone on beta cell function and sustained glycemic control . thirty - nine newly diagnosed t2 dm patients diagnosed according to the 1999 world health organization diagnostic criteria , without previous usage of antihyperglycemic and antihyperlipidemic medication , were enrolled . the included patients were between 20 and 65 years of age and had a body mass index of 2035 kg / m , with fasting plasma glucose ( fpg ) between 7.0 and 16.7 mmol / l . patients were excluded if they had severe acute or severe chronic diabetic complications and severe intercurrent illness and were positive for autoimmune antibodies against islets or with a recent history of being treated with corticosteroid , immunosuppressing drugs , or cytotoxic drugs . all patients were admitted to the hospitals after a 35-day run - in period and assigned to one of the following two groups by sequentially opening sealed , opaque envelopes arranged in a computer - generated random order . during hospitalization , patients in csii alone group received insulin aspart ( novorapid , novo nordisk , bagsvrd , denmark ) or insulin lispro ( humalog , eli lilly , usa ) with an insulin pump ( minimed 712 , medtronic , northridge , ca ) as csii therapy , while the csii + lira group received liraglutide ( victoza , novo nordisk , bagsvaerd , denmark ) 0.6 mg per day in addition to aforementioned csii regimen . the initial insulin dosage was 0.50.7 iu / kg / d , with the total daily dosage divided into 50/50 as basal and bolus infusion . in order to achieve euglycemia , basal rates and premeal boluses of insulin were adjusted every day according to capillary blood glucose values which were monitored at least 7 times per day . the glycemic goal was defined as fasting blood glucose less than 6.0 mmol / l and postprandial blood glucose less than 8.0 mmol / l . after the glycemic targets were achieved , csii treatments were maintained for additional 14 days . after being discharged from the hospital , all patients were guided with diet and physical exercise . patients in csii + lira group continued to use liraglutide 1.2 mg per day until the 12-week treatment period was finished . all recruited patients provided written informed consent for participation , and the study protocol was approved by the medical research and ethics committee of the first affiliated hospital of sun yat - sen university ( guangzhou , china ) . baseline anthropometric data such as blood pressure , height , weight , and waist and hip circumferences were measured , while fasting blood samples were collected for measurements of fpg and hba1c . an intravenous glucose tolerance test ( ivgtt ) using 25 g of glucose ( 50 ml of 50% glucose ) was conducted to assess air which was used to estimate the first - phase beta cell insulin secretion . serum insulin levels before and 1 , 2 , 4 , 6 , and 10 min after glucose injection were measured , and air was calculated as the incremental trapezoidal area during the first 10 min of the ivgtt . homeostasis model assessment was used to estimate insulin resistance ( homa - ir ) and beta cell function ( homa - b ) . after csii suspension , all baseline measurements were repeated at least 15 hours after cessation of insulin infusion and before liraglutide injection for csii + lira group . at the 12-week visit , the assessments were performed after 12 weeks of csii suspension for csii alone group or 7 days after liraglutide suspension . normally distributed data were presented as mean sd , and nonnormally distributed variables ( triglyceride , air , homa - b , and homa - ir ) were expressed as median ( interquartile range ) . the differences of normally distributed data between two groups were compared by independent - sample t - tests , while the comparisons of nonnormally distributed variables were using mann - whitney u tests . paired - sample t - tests or wilcoxon signed ranks tests were performed to estimate the changes before and after intervention . the tests were applied to analyze the differences of proportions . a 2-sided value of p < 0.05 was defined statistically significant . the enrolled patients were 45.91 8.7 years in age , with a bmi of 25.7 2.8 kg / m , fpg of 11.4 3.2 mmol / l , ppg of 17.4 5.9 mmol / l , and hba1c of 10.7 2.2% . they were assigned to csii alone group ( n = 19 ) and csii + lira group ( n = 20 ) and finished csii therapy . at the subsequent 12-week visit 8 patients ( 20.5% , 4 in csii alone group , 4 in csii + lira group ) dropped out due to withdrawal of consent . at baseline there were no significant differences in clinical characteristics , fpg , and hba1c between two groups except for ppg , which was slightly higher in csii + lira group ( 15.2 6.1 mmol / l versus 14.4 4.1 mmol / l , p = 0.025 ) . markers of beta cell function ( air and homa - b ) and insulin sensitivity ( homa - ir ) were also comparable ( table 1 ) . patients in csii + lira group reached target glycemic control in less time than those in csii alone group ( 2 ( 3 ) days versus 1 ( 0 ) days , for csii alone group and csii + lira group , resp . the 14 days of csii for maintaining euglycemia was divided into three stages : early stage ( days 15 ) , medium stage ( days 610 ) , and late stage ( days 1114 ) . average daily insulin dosage was similar in both groups , while the proportions of daily bolus dosage in total daily insulin dosage were lower in csii + lira group throughout the csii therapy ( figure 1 ) . air was restored in all patients after short - term csii therapy compared with baseline . at csii suspension , air improved from 6.60 ( 26.2 ) umin / ml to 52.05 ( 100.55 ) umin / ml in csii alone group and from 6.98 ( 21.71 ) umin / ml to 168.62 ( 350.95 ) umin / ml in csii + lira group . the increment of air was significantly higher in csii + lira group than that in csii alone group ( 177.58 ( 351.57 ) umin / ml versus 58.15 ( 51.30 ) umin / ml , p < 0.001 ) . however , after withdrawal of liraglutide after the 12-week treatment , the improvement in air rapidly disappeared in csii + lira group ( 168.62 ( 350.95 ) umin / ml versus 50.43 ( 70.40 ) umin / ml , for csii suspension and 12-week visit , resp . , p < 0.001 ) . therefore , air between two groups at the end of follow - up was similar ( p = 0.921 ) ( figure 2(a ) ) . in both groups , homa - b was ameliorated significantly after csii treatment compared with baseline . similar to air , homa - b in csii + lira group was higher than that in csii alone group at the end of csii ( 67.64 ( 46.31 ) versus 40.00 ( 35.53 ) , p = 0.007 ) , but the improvement was not sustained after stop of liraglutide at 12-week visit ( 41.28 ( 21.62 ) , p = 0.003 , compared with that after csii suspension ) and became similar to csii alone group ( 55.65 ( 56.27 ) , p = 0.110 ) ( figure 2(b ) ) . but at 12-week visit , homa - ir was significantly elevated from csii suspension in both groups ( figure 2(c ) ) . hba1c level was slightly lower in csii + lira group at the end of the 12-week follow - up compared with csii alone group but did not reach statistical significance ( 6.0 0.5% versus 6.3 0.7% , p = 0.325 ) , with similar proportions of patients who achieved hba1c 6.5% ( 73% ( 11/15 ) versus 94% ( 15/16 ) , for csii alone group and csii + lira group , resp . considerable reduction in fpg and ppg from baseline was observed at csii suspension . however , at 12-week visit there was a slight but statistically significant elevation of fpg in csii + lira group from csii suspension ( from 6.1 0.9 mmol / l to 6.9 1.1 mmol / l , p = 0.01 ) , which was not seen in csii alone group ( figure 3(a ) ) . there was a tendency of higher hyperglycemia relapse rate ( > 7.0 mmol / l ) in csii + lira group at 12-week visit ( 20% ( 3/15 ) versus 43.75% ( 7/16 ) , for csii alone group and csii + lira group , resp . , suspension , certain body weight loss was recorded in both groups ( 1.6 2.0 kg versus 1.2 2.3 kg , for csii alone group and csii + lira group , resp . continuous decline of body weight during the 12-week visit was recorded in csii + lira group ( 69.8 7.5 kg versus 66.2 9.3 kg , for csii suspension and 12-week visit , resp . , p = 0.005 ) but not in csii alone group ; however , the reduction of body weight during the 12-week visit in the two groups did not reach statistical significance ( 1.6 3.5 kg versus 3.3 4.1 kg , for csii alone group and csii + lira group , resp . during short - term csii therapy phase , the incidence of hypoglycemia which was defined as capillary blood glucose level < 3.9 mmol / l was similar in csii alone group and csii + lira group ( 4 ( 5 ) versus 2 ( 3 ) times per patient , p = 0.120 ) . most of the hypoglycemic episodes were mild and could be corrected after ingestion of carbohydrate . gastrointestinal symptoms happened in 35% of patients in csii + lira group in the first few days of liraglutide injections , and most of these symptoms were well tolerated . intensive insulin treatment was introduced in the management of newly diagnosed t2 dm since 1997 . by fast correction of glucotoxicity and lipotoxicity , intensive insulin treatment is able to induce long - term glycemic remission and thereby be suggested by the latest chinese guideline for t2 dm [ 3 , 4 , 15 ] . in this study , a glp-1 analog , liraglutide , was used as an add - on therapy of csii and lasted for additional 12 weeks . as expected , liraglutide facilitated the achievement of euglycemia by shortening the time required for insulin dose titration before reaching glycemic targets . liraglutide was also reported to reduce the daily insulin requirement in patients with more advanced t2 dm treated with insulin . although the total daily insulin dosage throughout csii treatment did not significantly differ between the two treatment groups , liraglutide significantly decreased the proportion of daily premeal bolus . furthermore , there was also a tendency of better average glycemic control in csii + lira group during the 12-week extended therapy phase , as indicated by a lower hba1c level than that in csii alone group at the end of the follow - up . these findings were probably attributed to a better amelioration of beta cell function in csii + lira group compared with that in csii alone group . these data were in accordance with previous reports on liraglutide , which showed that it reduced hyperglycemia , especially postprandial glycemic fluctuation , by glucose - dependent insulinotropic effect . however , to our surprise , shortly after the suspension of liraglutide , its effect on beta cell function rapidly faded with 1 week , leading to an elevation of fasting blood glucose . recently retnakaran et al . reported that 48 weeks of liraglutide administration in patients with mean diabetes duration of 2 - 3 years after 4 weeks of insulin therapy also robustly increased beta cell function measured by issi-2 . similar to this study , they also found a rapid deterioration of beta cell function shortly after cessation of liraglutide . however , an earlier observation showed that , in patients whose blood glucose was insufficiently controlled by metformin , a prolonged treatment with exenatide for 3 years had a slight but statistical significant benefit in beta cell function 4 weeks after stopping the medicine , which was not seen in the 1-year follow - up . there are several possible explanations for the discrepancy between short - term and long - term glp-1 analogs therapies . firstly , because of the beneficial effects of glp-1 analogs on beta cell proliferation and apoptosis from rodent models , liraglutide was expected to further improve functional beta cell mass [ 9 , 10 ] . however , the renewal rate of beta cells in human islets was so slow that a prolonged therapy targeted at pancreatic beta cells might be necessary for an overt change in islet architecture . less than 1 year , according to the results from both retnakaran et al . and secondly , part of the effects of glp-1 analogs is attributed to their effect on body weight which could help to relieve insulin resistance and restore beta cell function . in lead-3 monostudy , the maximum weight loss in 1.8 mg liraglutide treatment group existed in 20 weeks . although ongoing weight loss was observed in csii + lira group rather than csii alone group in this study , the difference of weight loss between the two groups was not statistically significant in a relatively short treatment period ( 12 weeks ) in a lower dose ( 1.2 mg / d ) . thirdly , it has been well documented that glp-1 analogs could suppress inappropriate secretion of glucagon from alpha cells . however , despite certain controversy , there are some reports showing that incretin therapy may induce hyperplasia of pancreatic alpha cells in human and rodent models [ 1921 ] . the importance and clinical consequence of alpha cell hyperplasia are largely unknown due to lack of data , but it is not impossible that , after stopping liraglutide , the previous suppressed glucagon secretion could rebound , resulting in relapse of hyperglycemia . previous observations suggested that persistent improvement in insulin sensitivity was critical for long - term maintenance of near - normoglycemia [ 5 , 6 ] . our previous studies also showed that combining csii with insulin sensitizers , that is , metformin or rosiglitazone , increases short - term remission rate by improving both insulin sensitivity and beta cell function patients with newly diagnosed t2 dm . as shown in this study , 12-week treatment with liraglutide was not sufficient to cause prominent effect on insulin resistance . enhancement of insulin action may decrease insulin demand and subsequent beta cell overload , endoplasmic reticulum stress , or oxidative stress , leading to a longer duration of glycemic remission [ 22 , 23 ] . in this point of view , insulin sensitizer , other than insulin secretagogues , should be tested as combination therapy to csii in future studies . first of all , as a pilot study , the relatively small sample size may reduce the statistical power when analyzing some clinical parameters . second , ivgtt and homeostasis model were used to evaluate beta cell function and insulin resistance . using clamp technique as well as physiologic challenge tests such as ogtt or mix - meal test could facilitate the achievement of glycemic targets and further improve beta cell function in patients with newly diagnosed t2 dm . rapid waning of beneficial effects of liraglutide implied that a prolonged treatment period might be required to obtain a sustained favorable outcome .

the objective of this paper is to investigate the effects of liraglutide in combination with short - term continuous subcutaneous insulin infusion ( csii ) therapy on glycemic control and beta cell function in patients with newly diagnosed type 2 diabetes mellitus ( t2 dm ) . thirty - nine eligible newly diagnosed t2 dm patients were recruited and randomized to receive either of two therapies : short - term csii alone ( csii alone group ) or csii in combination with liraglutide ( csii + lira group ) for 12 weeks . blood glucose control , homeostasis model assessment ( homa ) indices , and acute insulin response ( air ) were compared between the two groups . the patients in csii + lira group achieved euglycemia with equivalent insulin dosage in shorter time ( 1 ( 0 ) versus 2 ( 3 ) days , p = 0.039 ) . hba1c at the end of study was comparable between two groups ( 6.3 0.7% versus 6.0 0.5% , for csii alone group and csii + lira group , resp . , p = 0.325 ) . the increment of air was higher in csii + lira group ( 177.58 ( 351.57 ) umin / ml versus 58.15 ( 51.30 ) umin / ml , p < 0.001 ) . however , after stopping liraglutide , its effect on beta cell function disappeared completely . liraglutide combined with short - term csii was effective in further improving beta cell function , but the beneficial effects did not sustain after suspension of the therapy .

1. Introduction 2. Subjects and Methods 3. Statistical Analyses 4. Results 5. Safety Issues 6. Discussion