Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase , but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies. Cholesterol is the predominant sterol in vertebrates and it is an essential component of numerous cellular processes. Consequently, cholesterol synthesis, uptake, and efflux are tightly regulated in cells . Key to Escherichia coli and then by Trueperella pyogenes and other anaerobes in vivo  responses to LPS, and live n = 144 over a 24-mo period) within 15 min of slaughter, as part of the routine operation of a commercial slaughterhouse. Postpartum cattle were not used because of the ubiquitous bacterial contamination and disruption of the epithelium that is typical of the endometrium after parturition  ready for treatment. Endometrium was collected using 8-mm diameter punch biopsy, and EVOC was performed as described previously  (T. pyogenes (isolate MS249) , followed by centrifugation at 6000 × g for 10 min at 4°C. After washing, bacteria were diluted to 1 × 103 CFU/ml (E. coli) or 1 × 108 CFU/ml (T. pyogenes) in complete medium. Ultrapure LPS from E. coli 0111:B4 was obtained from InvivoGen  and used at 100 ng/ml, because this concentration has previously been shown to be optimal for stimulating IL-6 and CXCL8 responses in endometrial cells (Cultures of e MS499)  or T. pye MS249) , previoue MS249) , 18. Theal cells . Full de570 measurements and the number of live cells was confirmed using trypan blue exclusion and counting the number of live cells using a hemocytometer.We selected small molecules  and scren = 9) or stromal (n = 9) cells or EVOCs (n = 10) were pretreated with control medium or medium containing 10 μM atorvastatin to inhibit HMGCR, 10 μM squalestatin (zaragozic acid) to inhibit FDFT1, or 25 nM dexamethasone as a positive control for 24 h. Endometrial cells were subsequently challenged with control medium or medium containing 100 ng/ml LPS for 24 h, whereas EVOCs were challenged with control medium, or medium containing 1 × 103 CFU/ml E. coli or 1 × 108 CFU/ml T. pyogenes for 24 h. The supernatants were then collected and stored at −20°C before analysis of IL-6, CXCL8, and IL-1β by ELISA. Cell viability was assessed by MTT as described earlier, and EVOC tissues were weighed to enable IL concentrations to be adjusted for tissue weight.Purified endometrial epithelial (n = 4) and stromal (n = 4) cells were pretreated for 24 h with control medium or medium containing a range of concentrations of atorvastatin (0.1–0 μM), etidronate (1–100 μM) to inhibit farnesyl diphosphate synthase (FDPS), squalestatin (0.1–10 μM), or 25 nM dexamethasone. Endometrial cells were subsequently challenged with control medium or medium containing 100 ng/ml LPS for 24 h, in the continued presence of the inhibitors. Supernatants were then collected and stored at −20°C before analysis of IL-6, and CXCL8 by ELISA. Cells were lysed and stored in radioimmunoprecipitation assay buffer (RIPA) buffer at −80°C for analysis of total cell cholesterol by enzymatic assay.Purified endometrial epithelial (n = 4) were treated with medium containing 10 μM atorvastatin, 100 μM etidronate, 10 μM squalestatin, or 10 μM CP-34086894 , for 0, 6, 12, 18, 24, or 48 h. Supernatants were discarded and cells stored in RIPA buffer at −80°C before analysis of total cell cholesterol by enzymatic assay.For time-course experiments, purified endometrial stromal cells (n = 4) were treated for 0, 1, 6, 12, or 24 h with medium containing 1 mM methyl-β cyclodextrin, which binds to cholesterol. Supernatants were discarded and cells lysed and stored in RIPA buffer at −80°C before analysis of total cell cholesterol by enzymatic assay. To assess the impact of cholesterol reduction on inflammatory responses to LPS, we treated endometrial stromal cells (n = 4) with control medium or medium containing 1 mM methyl-β cyclodextrin or 25 nM dexamethasone for 24 h. Cells were then challenged with control medium or 100 ng/ml LPS for 24 h, in the continued presence of the small molecule. Supernatants were collected and stored at −20°C before analysis of IL-6 and CXCL8 by ELISA.For the membrane cholesterol sequestration experiments, purified endometrial stromal cells , etidronate (5–200 μM), squalestatin (0.5–20 μM), or methyl-β cyclodextrin (50–2000 μM). Cell viability was assessed by the mitochondria-dependent reduction of MTT to formazan, as described previously , and in n = 4) and stromal (n = 4) cells were pretreated with control medium or medium containing CP-34086894 (0.01–100 μM), squalestatin (0.01–100 μM), the isoprenoids farnesol (0.01–1000 μM) or geranylgeraniol (0.01–1000 μM), or 25 nM dexamethasone for 24 h. Cells were subsequently challenged with 100 ng/ml LPS for 24 h in the continued presence of the small molecule, and supernatants were then collected and stored at −20°C before analysis of IL-6 and CXCL8 by ELISA.Endometrial epithelial . Concentrations of CXCL8 in cell and EVOC culture supernatants were measured by the human CXCL8/IL-8 DuoSet ELISA according to the manufacturer’s instructions , which has previously been validated for the measurement of bovine CXCL8 . To takeCholesterol concentrations were determined using the Amplex red cholesterol assay kit (Life Technologies). The intra-assay and interassay coefficients of variation were <5%, and the limit of detection was 200 nM.HMGCR and FDFT1 was performed using SYBR green–based PCR with primers designed using the Eurofins MWG Operon qPCR primer design software  and validated by BLAST analysis against the Bos taurus (taxid: 9913) Refseq mRNA database. The HMGCR and FDFT1 primers, and GAPDH and ACTB reference gene primers  with primers added in nuclease-free water to a final concentration of 0.4 mM and 2 μl cDNA. Thermal cycling parameters were: one cycle of 95°C for 5 min, followed by 40 cycles of 95°C for 30 s and 60°C for 60 s. The expression of each gene was normalized against the geometric mean of the two reference genes GAPDH and ACTB, which were validated as invariant across treatment groups using standard methods (−5) in nuclease-free water (Qiagen). For each sample, target and reference gene mRNA abundance was determined from the appropriate standard curve [quantification cycle (Cq)]. Changes in mRNA abundance between samples were then determined from the ratio of the target gene Cq to the reference gene Cq.Gene expression analysis was performed according to the MIQE guidelines . Total R primers  were obt methods , and the methods . To geneProteins from lysed cells were normalized to 1 μg/μl using the DC Assay (Bio-Rad) and separated (10 μg per lane) using 10% (v/v) SDS-PAGE, with m.w. markers run in parallel lanes (Bio-Rad). After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad); nonspecific sites were blocked using a solution of 5% (w/v) BSA (Sigma-Aldrich) in TBS overnight at 4°C with gentle agitation. Membranes were probed with Abs targeting FDFT1 , and HMGCR , using Abs selected based on recognition of immunoreactive proteins of appropriate m.w. Primary Abs were used at 1:500 dilutions in 5% (w/v) BSA in TBS for 2 h with gentle agitation. After incubation, membranes were washed three times for 5 min in TBS and 0.1% Tween 20 (pH 7.6). Membranes were then incubated in secondary HRP-conjugated Ab  in 5% (w/v) BSA in TBS for 1.5 h and washed three times for 5 min in TBS and 0.1% Tween 20 (pH 7.6). Steady-state levels of immunoreactive proteins were visualized using ECL . Protein loading was evaluated and normalized by examining β-actin protein levels using a β-actin Ab (Abcam). The average peak densities of unsaturated bands were analyzed using Quantity-one software (Bio-Rad).n = 4) were transfected with Lipofectamine RNAiMAX Reagent (Invitrogen) and small interfering RNA  targeting HMGCR and FDFT1. siRNA duplex sequences were siHMG: sense 5′-CAGCAUGGAUAUUGAACAAUU-3′, antisense 5′-UUGUUCAAUAUCCAUGCUGUU-3′; siFDFT1: sense 5′-GCGAGAAGGGAGAGAGUUUUU-3′, antisense 5′-AAACUCUCUCCCUUCUCGCUU-3′. In brief, RNAiMAX-RNAi duplex complexes were formed by adding 50 pmol siRNA to 500 μl Opti-MEM I Reduced Serum Media  in each well of a six-well plate (Helena Bioscience). For controls, 50 pmol ONTARGETplus Nontargeting siRNA #1 (Dharmacon) was used instead of the targeted siRNA. Then 7.5 μl RNAiMAX was added to each well containing the diluted RNAi molecules and left for 20 min at room temperature. Exponentially growing cells were then seeded in 2.5 ml complete medium without antibiotics per well to give ∼50% confluence (5 × 105 cells/well). All transfections were carried out in duplicate. Cells were challenged with 100 ng/ml LPS, 24 h after the addition of the siRNA, and changes in mRNA and protein expression were assessed 48 h after transfection.Primary endometrial stromal cells . In addition, dexamethasone was used as a positive control because it has a well-established anti-inflammatory effect, which was confirmed in the present screening system . Molecules of interest were defined by an inflammatory response >1 SD from the reference response to LPS. Based on the IL-6 response and cell viability data presented in We initially explored putative modes of action that might modulate inflammatory responses to LPS. Bovine endometrial stromal cell secretion of IL-6 in response to 24-h treatment with 100 ng/ml LPS is a well-established model of endometritis , 14. Prip < 0.05) and more CXCL8  in response to challenge with 100 ng/ml LPS for 24 h, as expected. However, pretreatment of epithelial cells with atorvastatin for 24 h increased IL-6 but not CXCL8 secretion in response to LPS challenge (p < 0.05) and CXCL8 (p < 0.05). Stromal cells secreted more IL-6  and more CXCL8  in response to challenge with 100 ng/ml LPS for 24 h. Pretreatment of stromal cells with atorvastatin for 24 h had no significant effect on IL-6 or CXCL8 secretion, but pretreatment with squalestatin or dexamethasone for 24 h reduced the secretion of IL-6 in response to LPS challenge (p < 0.05), and dexamethasone also reduced the secretion of CXCL8 , CXCL8 , and IL-1β . However, pretreatment of EVOCs for 24 h with squalestatin reduced the accumulation of IL-6 , CXCL8 , and IL-1β . Pretreating EVOCs with atorvastatin for 24 h before challenge had no significant effect on subsequent IL responses to live T. pyogenes. However, pretreatment with squalestatin reduced the accumulation of IL-6 , whereas the FDPS inhibitor, etidronate, had no significant effect on cholesterol concentrations  HMGCR, 2) FDPS, and 3) FDFT1 , using atrations , 4F. Strp < 0.05; p < 0.05; p = 0.07; p < 0.05; p < 0.001; To further investigate the potential role of cholesterol, we treated endometrial stromal cells with atorvastatin, etidronate, or squalestatin for 48 h and measured total cell cholesterol concentrations at several time points . Squalesp < 0.05; p < 0.05; HMGCR (siHMG) and FDFT1 (siFDFT1). The effectiveness of siRNA treatment was confirmed using qPCR and Western blot , and pretreatment of stromal cells with geranylgeraniol for 24 h significantly reduced the accumulation of CXCL8  and geranylgeranyl diphosphate (GGPP), they might be important regulators of innate immunity. To examine this concept, we treated endometrial cells with farnesol and geranylgeraniol isoprenoid alcohols, which are converted intracellularly to their respective pathway intermediate diphosphates, FPP and GGPP , 28. PreEvidence is emerging that integration of innate immunity, lipid metabolism, and cholesterol biosynthesis impact the development of uterine disease , 9. ThisInhibition of FDFT1 by squalestatin or CP34086894 was equally effective at reducing the inflammatory response to bacteria in organ cultures, or to LPS in epithelial or stromal cells. In addition, siRNA targeting FDFT1 reduced LPS-mediated IL-6 and CXCL8 secretion by endometrial cells. The more rapid and efficient reduction in cholesterol within cells treated with FDFT1 inhibitors, compared with atorvastatin or etidronate, implied that cellular cholesterol may impact innate immune responsiveness to LPS. However, a similar reduction of cholesterol using methyl-β cyclodextrin had no effect on LPS-mediated IL-6 secretion, suggesting that cellular cholesterol was not the determining factor for modulating innate immunity. Indeed, cholesterol reduction per se is also not the main mechanism of statin-driven immune modulation, which occurs primarily via disrupted intracellular signaling and trafficking brought about by a reduction of intracellular cholesterol intermediates .In this study, inhibition of HMGCR either had no effect or increased LPS-mediated endometrial innate immune responses. The implication in this study is that molecules downstream from mevalonate are capable of regulating endometrial innate immune responses to LPS. At high concentrations, statins reduce the production of isoprenoids, including FPP and GGPP, in various cell types through depletion of isoprenoid precursors within the mevalonate pathway –33. The Inhibition of FDFT1, by inhibitors or siRNA, increases intracellular concentrations of FPP and GGPP , 37. FurIn summary, we show that manipulating the mevalonate pathway modulates innate immunity in endometrial cells and tissue. Surprisingly, inhibition of FDFT1  reduced inflammatory responses to bacteria or LPS in contrast with modulating the rate-limiting enzyme, HMGCR, with statins, which had no beneficial effect. These findings are important because they uncover a role for FDFT1 and geranyl and farnesyl isoprenoids in mucosal immunity. Furthermore, these findings could be translated to use topical administration of FDFT1 inhibitors into the uterus to limit the severity of bacterial endometritis.Data Supplement


However, most experiments have been conducted in established cell lines subjected to specific p53-inducing stimuli, both factors potentially biasing the results.Many recent studies using ChIP-seq approaches cross-referenced to trascriptome data and also to potentially unbiased p53retriever, a pattern search algorithm that maps p53 response elements (REs) and ranks them according to predicted transactivation potentials in five classes. Besides canonical, full site REs, we developed specific pattern searches for non-canonical half sites and 3/4 sites and show that they can mediate p53-dependent responsiveness of associated coding sequences. Using ENCODE data, we also mapped p53 REs in about 44,000 distant enhancers and identified a 16-fold enrichment for high activity REs within those sites in the comparison with genomic regions near transcriptional start sites (TSS). Predictions from our pattern search were cross-referenced to ChIP-seq, ChIP-exo, expression, and various literature data sources. Based on the mapping of predicted functional REs near TSS, we examined expression changes of thirteen genes as a function of different p53-inducing conditions, providing further evidence for PDE2A, GAS6, E2F7, APOBEC3H, KCTD1, TRIM32, DICER, HRAS, KITLG and TGFA p53-dependent regulation, while MAP2K3, DNAJA1 and potentially YAP1 were identified as new direct p53 target genes.We developed p53retriever is available as an R package at: http://tomateba.github.io/p53retriever.We provide a comprehensive annotation of canonical and non-canonical p53 REs in the human genome, ranked on predicted transactivation potential. We also establish or corroborate direct p53 transcriptional control of thirteen genes. The entire list of identified and functionally classified p53 REs near all UCSC-annotated genes and within ENCODE mapped enhancer elements is provided. Our approach is distinct from, and complementary to, existing methods designed to identify p53 response elements. The online version of this article (doi:10.1186/s12864-015-1643-9) contains supplementary material, which is available to authorized users. The p53 tumor suppressor is certainly one of the most studied sequence-specific transcription factor to date. Yet, much has still to be learned to fully describe its transcriptional regulatory network, both in terms of the crosstalk with other transcription factors and in terms of the entire spectrum of regulated transcriptional target genes, that can be both up-regulated or down-regulated –6.Recently, several genome-scale techniques such as ChIP-on-chip, ChIP-seq, and, more recently, ChIP-exo, have provided us with different and largely non-overlapping maps of p53 bound sites in the human genome in response to specific stimuli –17. Corrin vitro biochemical assays and in vivo occupancy measurements 11], all eriments . Howevereriments , 27, 44.eriments , 28, 62,eriments , yet remeriments , suggesteriments .We have coded in p53retriever sequence and structural features of p53 REs impacting on transactivation potential that were revealed in the past several years using our yeast-based transactivation assay , 65, 66.We mapped and ranked functional REs near TSS for all annotated transcripts in UCSC  REs and none of them failed to be classified by our tool Fig. . InsteadFinally, we decided to validate a few of the predictions from the pattern search, particularly for non-canonical 3Q sites using cell lines as a model. 13 genes with mapped functional REs were chosen. Overall, despite our algorithm doesn’t consider the system complexity of transcriptional regulation in living cells and the response variability upon each different p53 stimulus, results support p53-dependent transactivation for the majority of them. Based on the combined qPCR and ChIP results we conclude that DNAJA1, MAP2K3, and potentially YAP1 can be considered new direct p53 target genes, linking p53 to yet additional potential biological outcomes. Furthermore, our data further establish the very recent findings of PDE2A, GAS6, E2F7, APOBEC3H, KCTD1, TRIM32, HRAS, KITLG and TGFA as p53 target genes.http://tomateba.github.io/p53retriever/). p53retriever contains a main function that identifies potential REs. This function needs as input an arbitrary DNA sequence, and returns a table containing information about the identified REs, such as position, sequence, spacer length, mismatch label and grade. The format of the output is similar to Additional file We implemented the set of manually curated rules  considering, for each transcript with a distinct TSS, the 20 kB region surrounding the transcription binding site (genome build GRCh37/hg19). The final dataset consists of 23,541 promoter sequences, associated to distinct UCSC identifiers and corresponding to 18,355 HGNC genes.Human promoter sequences were extracted from the UCSC database (Encode DNase-seq regulatory regions (genome build GRCh37/hg19) were obtained from the following cell lines: Gm12878, H1hesc, Helas3, Hepg2, Hmec, Hsmm, Hsmmt, Huvec, K562, Monocd14, Nha, Nhdfad, Nhek, Nhlf, Osteobl, Hsmmfshd, Lncap, Nb4, Nt2d1,Panc1. The consensus was defined as the merge of all the regions that were present in at least two cell lines. Only distal regions, with more than 10 kb from the nearest annotated TSS, were kept in the dataset, in order to avoid overlap with promoter regions. The final dataset consists of 43,787 regions, with a mean length of 673.3 bases.Sets of scrambled promoter sequences were generated by local permutations (bin size = 500 nt) of human promoter sequences . This allowed to preserve the local GC content in the random model; p53 REs were then identified and classified with p53retriever. The random simulation was run ten times, and the results were compared to REs identified in real human promoters.Set of random sequences were generated, with the same number and the same GC content (44 %) of human DNA sequences; p53 REs were then identified and classified with p53retriever. The random simulation was run ten times, and the results were compared to REs identified in human distal DNase regions promoters.www.ingenuity.com). Only direct interactions were considered in the setting parameters.All pathways analyses were performed using IPA  and IPA, or from previous publications, referenced in the main text. These lists were used to select populations of genes among our dataset of human promoters, and analyze the grade of the REs identified by p53retriever . One PWM, MA0106.1, is built on SELEX data, while the second, MA0106.2, is built on ChIPseq data. The original values of the downloaded PWMs were based on nucleotide frequencies and therefore more similar to Positional Frequency Matrices. These frequency values were transformed in log2 probability ratio values with the PWM function implemented in the Bioconductor Biostring package, using a multinomial model with a Dirichlet conjugate prior to calculate the estimated probability of base b at position i. The final score of a match ranges from 0 to 1. All REs identified by p53retriever in the set of human promoters were scored with JASPAR PWMs: the comparison with MA0106.2 is shown in Fig. Two PWMs for p53 were downloaded from the JASPAR database (+/+) cell line and its p53−/− derivative were a gift from B. Vogelstein . MCF7 cells stably expressing an shRNA targeting p53 (MCF7shp53) or control cells (MCF7vector) were kindly provided by Dr. Agami . Cells were normally maintained in DMEM or RPMI  supplemented with 10 % FCS, antibiotics (100 units/ml penicillin plus 100 mg/ml streptomycin) and 2 mM glutamine. Puromycin  was used to maintain the selection, at 0.5 μg/mL as final concentration.The human breast adenocarcinoma-derived MCF7 cell line (p53 wild type) was obtained from the InterLab Cell Line Collection bank, ICLC  while the colon adenocarcinoma HCT116  while Nutlin-3A was obtained from Alexis Biochemicals . After 8 h, 16 h or 24 h of treatment cells were harvested and total RNA was extracted using RNeasy Mini Kit  according to the manufacturer’s instructions. In-column DNAse treatment  was performed to remove DNA contamination during the extraction. Purity of RNAs  and concentration were measured using the Nanodrop spectrophotometer.q combined with absence of primer dimer formation. Reaction volumes were set at 10 μl. SYBR Green assays contained 5X KAPA SYBR FAST qPCR Mastermix , 400 nM each primer  and 25 ng of cDNA. Initial thermal cycling conditions were 1 cycle of 95 °C for 3 mins, followed by 40 cycles of 95 °C for 30 s, 60 °C for 20 s, 72 °C for 60 s. At the end a melt curve analysis was performed. Post-run relative mRNA quantification was obtained using the comparative Cq method (ΔΔCq), where glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-2microglobulin (B2M) served as reference genes.cDNA was generated starting from 1 μg of RNA by using the RevertAidTM First Strand cDNA Synthesis Kit  in 20 μL as final volume following manufacturer’s instructions. Primers were designed by Primer-BLAST performing in silico analysis as well as standard curves to define assay specificity and efficiency .The data sets supporting the results of this article are included within the article and its additional files. p53retriever source and binary files are available on Github, at (


Background and Aims: Recent studies have proposed that if a severe caloric restriction (SCR) is initiated at the earliest period of postnatal life, it can lead to beneficial cardiac adaptations later on. We investigated the effects of SCR in Wistar rats from birth to adult age on risk factors for cardiac diseases (CD), as well as cardiac function, redox status, and HSP72 content in response to ischemia/reperfusion (I/R) injury.Methods and Results: From birth to the age of 3 months, CR50 rats were fed 50% of the food that the ad libitum group (AL) was fed. Food intake was assessed daily and body weight were assessed weekly. In the last week of the SCR protocol, systolic blood pressure and heart rate were measured and the double product index was calculated. Also, oral glucose and intraperitoneal insulin tolerance tests were performed. Thereafter, rats were decapitated, visceral fat was weighed, and blood and hearts were harvested for biochemical, functional, tissue redox status, and western blot analyzes. Compared to AL, CR50 rats had reduced the main risk factors for CD. Moreover, the FR50 rats showed increased cardiac function both at baseline conditions (45% > AL rats) and during the post-ischemic period (60% > AL rats) which may be explained by a decreased cardiac oxidative stress and increased HSP72 content.Conclusion: SCR from birth to adult age reduced risk factors for CD, increased basal cardiac function and protected hearts from the I/R, possibly by a mechanism involving ROS. Cardiac diseases (CD) are the main leading cause of mortality worldwide accounting for up to 30% of all deaths  are well-documented for a long-term moderate CR (20–40% reduction)  submitted to a 50% CR since the first weeks of age presented increased basal heart function, increased cardiomyocyte density and number as well as increased levels of phosphorylated AKT . The experimental protocols were approved by Ethics Committee on Animal Use/Federal University of Jequitinhonha and Mucuri Valleys, Diamantina, MG, Brazil, protocol #04/2015.n = 8), approximately 90 days of age, were housed in individual cages and maintained in a room with controlled temperature (22 ± 2°C) and a 12 h light–12 h dark cycle, with free access to food and water. After they gave birth, half of the mothers (n = 4) were assigned to ad libitum-fed group, and the other half (n = 4) was assigned to the calorie-restricted group. Both groups had free access to water. In the mothers assigned to the calorie-restricted group, the amount of food provided on a daily basis was equivalent to 50% of the amount consumed by the mothers in the ad libitum-fed group, and each mother suckled 8 newborn rats for 21 days. After weaning, rats were housed in individual cages and received the same treatment as their mothers up to the age of 90 days. The weaned rats were divided into rats fed ad libitum  and rats subjected to long-term SCR .Female pregnant Wistar rats . Tail systolic blood pressure and heart rate were measured at the last week prior to the end of the experiment. Additionally, we calculated the double product index using systolic blood pressure and heart rate values, an indicative of cardiac work  and carbon dioxide production (VCO2) within a period of 60 min. The measurements were taken by a computer-monitored indirect calorimeter  coupled to a metabolic chamber (air flow = 1.0/min), which accommodated the rats. The calorimetric parameters were measured using a respiratory-based software program .After an overnight fast (12 h), the resting metabolic rate was assessed by the average oxygen consumption . Blood glucose levels were determined by small clipping of the rat tail, immediately before (0 min) the glucose challenge, as well as at 30, 60, and 120 min thereafter. Blood glucose levels were determined using an ACCU-CHEK .OGTT, the animals were fasted for 8 h, then an intraperitoneal injection of insulin (1 IU/kg body weight) was administered. Blood glucose levels were determined following the same OGTT protocol.Forty-eight hours after the −1: NaCl, 118.40; KCl, 4.70; KH2PO4, 1.17; MgSO4, 1.17; CaCl2, 2.50; glucose, 11.65; and NaHCO3, 26.30) at 37 ± 1°C, with constant pressure (65 mm Hg) and oxygenation (5% CO2 and 95% O2). A water-filled latex balloon coupled to a pressure transducer  was inserted into the left ventricular cavity via the left auricle to record pressure. The rate of pressure development (± dP/dt) was obtained by calculating derivatives from the pressure variation recordings, using the Labchart8 software. After 20 min of stabilization in the perfusion system, the hearts were evaluated at baseline cardiac function for 20 min and then subjected to global ischemia  for 20 min. After induction of ischemia, the hearts were reperfused for an additional 20 min to evaluate the post-ischemic cardiac function. All the ± dP/dt measurements were normalized to heart weight.The animals were anesthesized with ketamine (10 mg/kg) and xylazine (5 mg/kg) and decapitated 10–15 min after an intraperitoneal injection of 400 IU heparin. The hearts were perfused in a Langendorff apparatus  and left ventricular pressure (± dP/dt) was continuously recorded according to the Langendorff technique. Briefly, the thorax was opened and the heart was carefully dissected and rapidly cannulated and retrogradely perfused with Krebs–Ringer solution ] y et al. .Blood was centrifuged to obtain plasma, and then aliquoted in eppendorf tubes and kept at −80°C until analyses. Total plasma cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels were determined using commercial kits (LabTest®), according to the specifications of the manufacturer and using a semi-automatic biochemical analyzer . The low-density lipoprotein cholesterol (LDL-C) level was also estimated by using the Friedewald equation , pH 7.2, extensively washed to remove blood and stored at −80°C until the time of assay. At the time of assay, samples were then homogenized  for 3 min at 0–4°C. After this, the homogenate was centrifuged for 10 min at 10,000 xg , and the supernatant was used for the biochemical assays described below. Protein concentration for all assays was determined according to Bradford  using boThe assay to determine SOD activity (EC 1.15.1.1) was performed according to Marklund and Marklund  with som−1× mg−1 protein). This procedure avoids the possible interference associated with glutathione peroxidase activity since the necessary cofactors are not present in the reaction media.Catalase (EC 1.11.1.6) activity was measured in the supernatants as described by Nelson and Kiesow . BrieflyAs an index of lipid peroxidation, we used the formation of thiobarbituric acid-reactive substance (TBARS) during an acid-heating reaction . The level of significance was set at p < 0.05.All data are expressed as means ± standard deviation. Statistical significance was estimated using Student's The characteristics of the groups are presented in Table Next, we evaluated the effects of 50% CR on key risk factors for developing CD Table . We founn = 9): 1310 ± 96, CR50 (n = 11): 2448 ± 177 mmHg/s-1/g-1, Figure n = 9): 660 ± 56, CR50 (n = 11): 1310 ± 125 mmHg/s-1/g-1. Figure n = 9): 220 ± 16, CR50 (n = 11): 231 ± 13 bpm, Figure n = 8): 384 ± 55, CR50 (n = 9): 1001 ± 132 mmHg/s-1/g-1, Figure n = 8): 236 ± 34, CR50 (n = 9): 585 ± 33 mmHg/s-1/g-1. Figure n = 8): 100 ± 14, CR50 (n = 9): 131 ± 57 bpm, Figure To address if reductions of cardiometabolic risk factors in CR50 rats were followed by an improvement of direct cardiac function, we performed Langendorff analysis Figure . Confirmn = 8): 6.3 ± 0.9, CR50 (n = 9): 16.8 ± 3.4 U/mg protein, Figure n = 8): 0.6 ± 0.05, CR50 (n = 9): 1.4 ± 0.2 ΔE/min/mg protein, Figure In order to further understand the mechanisms involved in the cardioprotective effects of our CR protocol in I/R injury, we assessed cardiac oxidative stress by measuring lipid peroxidation, catalase and superoxide dismutase activities in post-ischemic hearts. As shown in Figure In the current study, we evaluated the cardioprotective effects of SCR since birth to the age of 3 months in Wistar rats. First, we found that CR50 rats presented a reduction in main cardiometabolic risk factors, as shown by (i) diminished adiposity index and visceral fat weight; (ii) improvement of the plasma lipid profile and atherogenic index; (iii) improvement of the glucose tolerance and insulin response, indicating a higher insulin sensitivity; and (iv) reduced systolic blood pressure, cardiac double product index and basal metabolic rate. Moreover, CR50 rats showed increased cardiac function both at baseline levels (45% > AL rats) and during post-ischemic period (60% > AL rats) which may be explained by a decreased cardiac oxidative stress and increased HSP72 content.The effects of SCR on cardiac function have been poorly investigated with some controversial results. Although most studies supported the notion that SCR induces positive effects , but also during the post-ischemic period compared to AL hearts. The post-ischemic function of CR50 hearts actually was similar to the basal levels of AL hearts and 60% higher than the post-ischemic levels of AL hearts. These findings are supported by other studies showing that a long-term SCR protects heart function during the post-ischemic period in adult (Broderick et al., A possible mechanism involved in these benefits, especially the preservation of the cardiac function at the post-ischemic period, may be a lower increase in ROS generation in response to an ischemic insult. Low levels of ROS regulate a variety of key mechanisms involved in the homeostasis of heart function (Hancock et al., The HSP family, especially HSP72, is believed to provide myocardial protection against increased ROS generation during I/R events (Suzuki et al., In conclusion, our findings revealed that SCR from birth to adult age reduces the main cardiometabolic risk factors and directly improves cardiac function at baseline and after an I/R insult. This latter effect may be a result of a decrease in ROS damage through increased antioxidant responses which could be triggered by an increase in HSP72 content.Conceived and designed the experiments: DM, MD; performed the experiments: DM, LC, CS, BM, and KC; analyzed the data: DM; contributed reagents/materials/analysis tools: CS, EV, FM, EE; wrote the paper: DM, MD, AF, and SG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective. Vernal keratoconjunctivitis (VKC) is a chronic recurrent bilateral inflammation of the conjunctiva associated with atopy. Several inflammatory and tissue remodeling factors contribute to VKC disease. The aim is to provide a chip-based protein analysis in tears from patients suffering from quiescent or active VKC. Methods. This study cohort included 16 consecutive patients with VKC and 10 controls. Participants were subjected to clinical assessment of ocular surface and tear sampling. Total protein quantification, total protein sketch, and protein array (sixty protein candidates) were evaluated. Results. An overall increased Fluorescent Intensity expression was observed in VKC arrays. Particularly, IL1β, IL15, IL21, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, ICAM2, βNGF, NT4, BDNF, βFGF, SCF, MMP1, and MMP2 were increased in quiescent VKC. Of those candidates, only IL1β, IL15, IL21, βNGF, SCF, MMP2, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, and ICAM2 were increased in both active and quiescent VKC. Finally, NT4, βFGF, and MMP1 were highly increased in active VKC. Conclusion. A distinct “protein tear-print” characterizes VKC activity, confirming some previously reported factors and highlighting some new candidates common to quiescent and active states. Those candidates expressed in quiescent VKC might be considered as predictive indicators of VKC reactivation and/or exacerbation out-of-season.  Vernal keratoconjunctivitis (VKC) is a multifactorial eye disease associated with atopy, characterized by a chronic recurrent bilateral inflammation of the conjunctiva . This chγ, IL2) and Th2  subgroups have been detected in tears and conjunctival impression cytologies (IC) from VKC patients [ in vitro studies [Current knowledge indicates that several inflammatory and tissue remodeling factors contribute to signs and symptoms of VKC . Infiltrpatients –10. In a studies –13. Adva studies , 15.To date, different clinical features and therapeutic outcome in VKC suggest the need for predictive approaches, the crosstalk between a new grading approach and suitable biomarkers –19. TherAuthorization to carry out the study was provided by the intramural Ethical Committee at the University Hospital Campus Bio-Medico . The approval included patient management, tear sampling, and full experimental procedures. Furthermore, all procedures of handling human samples were conducted in accordance with guidelines established by the Association for Research in Vision and Ophthalmology and adhered to the tenets of the Declaration of Helsinki with respect to human subjects.Unless specified in the text, sterile plastic-ware and analytical grade reagents were from NUNC , SERVA , ICN , Euroclone , and Sigma-Aldrich . Ultrapure MilliQ-Grade water was daily provided .After an accurate explanation of the study design and the description of the potential information arising from the study, the participants (patients and controls or their parents) provided written informed consent to proceed to tear sampling. 0-quiescent, absence of ocular symptoms; 1-mild, presence of ocular symptoms but not photophobia; 2-moderate, presence of symptoms and photophobia; 3-severe, presence of ocular symptoms and mild to moderate superficial punctuate keratitis (SPK); 4-very severe, presence of diffuse superficial keratopathy and/or corneal ulcer [A total of 16 consecutive patients suffering from VKC  were included in the study . Enrollment criteria included a positive history of VKC and the absence of topical/systemic therapy. VKC diagnosis was based on ocular surface inflammation characterized by itching, photophobia, tearing/mucous discharge, the presence of a giant papillary reaction on the upper tarsal conjunctiva, and/or at the limbus associated with the presence of eosinophils in conjunctival scrapings . ExclusiTen sex/age matched healthy control volunteers with no signs/symptoms of conjunctivitis or ocular surface disease and not receiving systemic/local medications  were enrolled for appropriate comparisons .µL sterile Balanced Salt Solution  and a quick collection of tears with sterile single-wrapped plastic micropipettes (Sigma) [At the end of ophthalmic examination, the subjects underwent nonanesthetized tear collection. Briefly, tears were sampled according to the standardized “eye-flush” procedure implying the addition of 50  (Sigma) –22. The  (Sigma) , 21. AllµL samples were analyzed with the A280 program . Tear samples showing low protein amounts were from patients/subjects having symptoms of ocular dryness and reduced mucus production. Protein separation was performed under reducing conditions on 4–12% mini-SDS polyacrylamide gels  and the bands were transferred to Hybond membranes  under semidry conditions . Prestained broad weight marker was run in parallel . Bands were visualized by Ponceau S. Samples having high albumin/IgGs protein profiles were cleared .Tear samples were diluted in cold lysis buffer , briefly sonicated , and clarified by centrifugation (13000 rpm/7 min). For protein quantification, 3 µL/well) were diluted and hybridized in subarrays. VKC/control samples were processed simultaneously and the procedures of sample dilution, incubation/washing, detection, and labeling were according to the manufacturer's recommendation. Spin-dried slides were scanned in a GenePix 4400 Microarray platform . The specific area  was first manually spotted and then automatically adjusted by the software, and the capturing conditions were routinely applied to all glass slides. Images were uniformly adjusted for size, brightness, contrast, and chip-to-chip comparisons by the software, and provided as 8-bit Tiff converted format . Interassay normalization was guaranteed by the presence of multiple internal controls for each subarray. The minimum sensitivity range of the array was 3.8–56 pg/mL.Protein analysis was performed on chip-based arrays provided on glass slides, each comprising 14 identical subarrays containing 60 factors (antibody spots in duplicate) retrospectively selected (literature search) for custom-built array chips . Briefly, normalized protein extracts . As cut-offs, ≥ 2-fold changes  with respect to control) and p ≤ 0.05 or p < 0.00083  for multiple testing with the Bonferroni correction were considered. Correlations between differentially expressed candidates and clinical findings were calculated by Spearman correlation rank test (rho ≥ 0.5 and p ≤ 0.05 or p < 0.00083). Volcano plots and Venn diagrams were used to illustrate microarray data sets and results [The pooled-samples choice was not considered in this cohort study, guaranteeing good statistical power and biological sensitivity/variance. The Fluorescent Intensity (FI) values (spot) were obtained by subtracting the background signal (GenePix Pro 6.0 software). Single FI values were entered into a Microsoft Excel database  and duplicate spots outside the 10% coefficient of variability were refused from the statistical analysis. FDR value of 0.01 was set. FI averages (means ± SD) were automatically calculated from replicates (2 spots) of not-pooled tear samples. Comparisons between VKC and control groups or within VKC groups were performed by using the two-sided unpaired  results , 24.quiescent VKC) and 7 out of 16 patients referred to our Clinical Unit in-season, showing signs and symptoms of disease (active VKC). The Total Symptom Score (TSyS) and the Total Sign Score (TSS) were, respectively, 7.33 ± 4.51 and 8.67 ± 3.05 (±SD). The mean overall scores were 0-1 in quiescent and 3-4 in active groups. A 1.27-fold increase of total protein concentration was quantified in quiescent samples while a 2.83-fold increase was detected in active VKC samples . Although a trend toward a decrease in total protein amount was detected in tears from the left eye, with respect to right one (p > 0.05), no significant intragroup changes were observed between active and quiescent total protein amounts. A representative total protein sketch is depicted in The entire study population included 16 consecutive VKC patients and 10 normal subjects (see M&M). The clinical and biochemical data of VKC patient group and subgroups are summarized in To recognize VKC proteins of prognostic value in a whole array of potential candidates, both quiescent and active VKC tears were subject to a chip-based protein array evaluation and appropriate statistical analysis. Proteins were selected from a literature search and antibodies were thereafter assembled. Both quiescent and active VKC tear samples were hybridized on chip-based arrays followed by a case-control statistical analysis. As shown in Figures p < 0.0001) differential expression  1α and 3α); 16 out of 60 candidates had a ≥ 3.00-fold (p < 0.005) differential expression  and 8 out of 16 candidates showed a ≥ 3.00-fold (p < 0.01) differential expression . With respect to the expression of enzymes involved in the ECM metabolism, the MMP1 (5.81-fold with p < 0.0001) and MMP13 , the MMP2 , and the MMP7  were highly increased in active VKC tears, with respect to controls. While TIMP1/2 tissue inhibitors were not increased in active subgroup, the tissue inhibitor TIMP4 showed a significant increase in active VKC tear samples , as compared to controls. The Volcano plot underlying the complete protein expression in active VKC tears is shown in From this active VKC versus control comparison, 4 out of 16 candidates showed a ≥ 4.00-fold . MMP1 and MMP2 were found slightly increased in quiescent VKC tears . As above, a 3.94-fold increase was detected for TIMP4, although slightly significant (p = 0.051). Finally, IL33 expression was particularly high in both active  and quiescent  VKC tears, with respect to controls. The Volcano plot displaying the complete protein expression in quiescent VKC tears is shown, respectively, in From the quiescent VKC versus control comparison, some candidates were also increased in quiescent VKC tears (IL12 expression). The scatter plot indicates that the majority of factors common to both VKC states are displayed in the lower region (low fold expression). Those factors highly expressed are mainly of active tear root , βNGF, NT4, BDNF, SCF (growth factors), MMP1, MMP2 (tissue proteases) and Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, and ICAM2 (chemokines and adhesion molecules). A further analysis of quiescent versus active VKC indicated that three out of those 16 candidates . Albeit not significant, MIP1β was extremely high in both active and quiescent specimens .The whole distribution of quiescent/active candidate fold changes in tear is shown in β, IL15, IL21, βNGF, BDNF, SCF, MMP2, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, and ICAM2, as shown in the Venn diagram in Currently in force for a wide-range biomolecular investigation of disease-linked profiles, the microarray platform represents an excellent high-throughput technology that facilitates the simultaneous detection of more than a few “biomarker or gene/oligo/protein candidates” , 15, 25.β, IL15, IL21, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, ICAM2, βNGF, NT4, BDNF, βFGF, SCF, MMP1, MMP2, and sTNFRI/II is in line with literature, as reported in previous studies carried out on VKC tears, ICs, and conjunctival biopsies [α, IL6, and IL10 expression and slight IFNγ increase in active VKC might be consistent with the absence of corneal involvement and tissue remodeling, as provided by the VKC score [Firstly, the widespread protein expression in active VKC with respect to control tears is in line with the inflammatory and tissue remodeling process occurring at the ocular surface. Cytokines, chemokines, and adhesion molecules, growth factors, and some soluble receptors play a major role in chronic inflammatory disorders, through a tidy regulation of cell influx leading to an exacerbation of the local inflammation and the promotion of corneal lesions . The obsbiopsies . Particubiopsies , 27; IL7biopsies ; IL17 habiopsies ; IL18 habiopsies , and IL1biopsies . The uncKC score , 27, 32.KC score .β, IL15, and IL21 were monitored in quiescent VKC. Overexpressed in active VKC, Eotaxin2 detection in quiescent tears might be indicative of a forthcoming eosinophil recruitment, according to in vitro/in vivo chemoattractant studies [α, MIP3α, TACE, NCAM1, and ICAM2 might represent new potential candidates for their well-known cell homing effects [α-MIP3α has been recently implicated in the epithelial cell activation as well as eosinophil, lymphocyte, and neutrophil tissue-homing and, more appropriately, MIP3α has been recently prospected as a serum prognostic factor at least for systemic inflammatory diseases [Although at a relatively low level, the common expression of some candidates in quiescent VKC tears would imply that these factors might provide long-lasting conditions suitable for disease reactivation and therefore might be proposed as a potential indicator of forthcoming reactivation. Of those cytokines significantly expressed in active VKC tears , only IL1 effects . It is ndiseases . NCAM1 adiseases , 34–37. diseases .β1 changes in active VKC might be due to production by activated eosinophil, mast cell, and T cells [βFGF expression in quiescent VKC is actually an open question [A cross-talk between neuronal and nonneuronal cells has been described in VKC and a bidirectional (neuro)protective and/or anti-inflammatory cross-talk between immune and structural cells might be prospected under quiescent conditions , 38, 39. T cells , 41, thequestion . Some nequestion .α values (a major MMP9 modulator) and might be supported by the absence of superficial corneal involvement or ulcers in this study population [β1, and NGF) [Several tissue proteases/inhibitors (MMPs/TIMPs) trigger long-lasting ECM metabolism in active VKC, while playing homeostatic effects under normal states , 2. The pulation . As repopulation . The sigand NGF) , 45. Witβ, IL15, IL21, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, ICAM2, βNGF, NT4, BDNF, βFGF, SCF, MMP1, and MMP2, all quantified in acute VKC and detected also in quiescent VKC tears. Moreover the eye-flush tear sampling technique appears appropriate as it allows an easy collection and a good volume (diluted tears) for the chip-based array. The specificity of this way of sampling is provided by the observation that only a few of the significantly changed proteins were identified.Overall, VKC is a self-limiting eye disease characterized by a chronic inflammatory process  and overt ECM remodeling –6. None Understanding the potential candidates might be helpful for prognostic purposes as well as for developing appropriate strategies to counteract VKC either outside or during the season, and with respect to other VKC-associated conditions .


Mycobacterium neoaurum strain DSM 44074T, a nontuberculosis species responsible for opportunistic infections in immunocompromised patients. The strain described here is composed of 5,536,033 bp, with a G+C content of 66.24%, and carries 5,274 protein-coding genes and 72 RNA genes.We report the draft genome sequence of  Mycobacterium neoaurum is an environmental, rapidly growing mycobacterium that is rarely reported as an opportunistic pathogen responsible for bloodstream infections, cutaneous infections, and pneumonia in immunocompromised patients  at 37°C in an atmosphere enriched with 5% CO2. Genomic DNA was then sequenced using two high-throughput next-generation sequencing technologies: Roche 454 (M. neoaurum consists of 10 scaffolds of 45 contigs containing 5,504,703 bp and has an estimated size of 5,536,033 bp, including gaps. The G+C content of this genome is 66.24%.Genomic DNA was isolated from oche 454 , 7 whileoche 454  and Operoche 454  and thenoche 454  to reducNoncoding genes and miscellaneous features were predicted using RNAmmer , ARAGORNM. neoaurum (This report illustrates the genomic variability within neoaurum , a featuM. neoaurum DSM 44074T strain genome sequence has been deposited at EMBL under the accession numbers LK021337 to LK021346. The whole-genome shotgun master numbers are CCDR010000001 to CCDR010000045.The


The quantitative ELISA-like immunohistochemistry was a feasible and reproducible procedure to detect the high FGF23 expression in formalin-fixed and paraffin-embedded biopsies or specimens. Since TIO was often delay-diagnosed or misdiagnosed, clinicians and pathologists should be aware of TIO and PMTMCT, respectively.Tumor-induced osteomalacia (TIO) is a rare acquired paraneoplastic disorder and fibroblast growth factor 23 (FGF23) plays a key role in its pathogenesis. This study was conducted to describe a novel FGF23 detecting procedure and describe clinical features of the disease. Fourteen TIO cases were retrieved and FGF23 expression was measured by quantitative ELISA-like immunohistochemistry using formalin-fixed and paraffin-embedded tissues. As summarized from 14 TIO cases, clinical features of TIO were long-standing history of osteomalacia, hypophosphatemia, and urinary phosphate wasting. The associated tumors were mostly benign phosphaturic mesenchymal tumors mixed connective tissue variant (PMTMCT) which could be located anywhere on the body, and most of them could be localized by conventional examinations and octreotide scanning. By quantitative ELISA-like immunohistochemistry, all the 14 TIO cases had high FGF23 expression (median 0.69, 25%–75% interquartile 0.57–1.10, compared with 26 non-TIO tumors of median 0.07, 25%–75% interquartile 0.05–0.11,  Tumor-induced osteomalacia (TIO) or oncogenic osteomalacia (OOM) is reported to be a rare acquired paraneoplastic disorder caused by renal phosphate wasting and characterized by hypophosphatemia and osteomalacia –5. SinceThe causative tumors of TIO are mostly located in bones, soft tissues, and craniofacial sinuses . PatholoBy inhibition of renal tubular epithelial phosphate transport, phosphaturic hormone of fibroblast growth factor 23 (FGF23) plays a key role in the pathogenesis of TIO , 5. SeruTIO cases were retrieved from the Clinical Database of the Chinese PLA General Hospital from 2003 to 2012. Either diagnosed TIO/PMTMCT cases or cases with the following characteristics were included: (1) clinical symptoms of osteomalacia such as proximal muscle weakness and bone pain, (2) absence of personal and familial history of hypophosphatemic disorders, and (3) typical preoperative biochemical parameters such as hypophosphatemia and renal phosphate wasting which returned normal after complete tumor resection , 13, 14.p < 0.05 were considered of statistical significance.We collected the demographic data, clinical features, biochemical results, intraoperative findings, and postoperative outcomes of the included cases. Maximum tubular reabsorption of phosphorus factored for glomerular filtration rate was calculated according to the reported method . (TmP/GFμm in thickness) were trimmed into a core (1 cm in diameter) on slides by scalpel and then deparaffinized and rehydrated. Epitope retrieval was performed in citrate buffer (pH 6.0) at 120°C for 2.5 min. Then the slides were exposed to 3% hydrogen peroxide for 20 min and washed by PBS buffer 5 min for 3 times. A circle was drawn with an ImmEdge hydrophobic barrier pen on the slides around the tissues and further blocking was performed with normal goat serum for 30 min. The component of horseradish peroxidase (HRP) conjugated FGF23 antibody (Cat. # 40-6120) of the Human FGF23 ELISA kit  was used. The purified goat polyclonal FGF23 antibody was conjugated to enzyme HRP and could detect epitopes within carboxyl-terminal portion of FGF23. After incubation of the HRP conjugated FGF23 antibody with the sample, slides were washed and incubated with ELISA HRP substrate and then measured in a spectrophotometric microtiter plate reader. The detailed procedure was as follows: 20 μL HRP conjugated FGF23 antibody diluted by 10 μL PBS was added to the sample, sealed, and incubated at room temperature for 4 hours on a horizontal rotator. Then the slides were washed by PBS for 5 min and repeated 5 times. The ImmEdge hydrophobic barrier pen circle was drawn again and 90 μL ELISA HRP substrate (tetramethylbenzidine with hydrogen peroxide) was added and resealed and incubated at room temperature for 30 min on a horizontal rotator. Then transfer 30 μL of the reaction solution to a 96 well plate, and read the absorbance at 620 nm against a blank HRP substrate taken as wavelength correction. Stop the reaction with 0.36 M sulfuric acid and read the absorbance at 450 nm against a reagent blank. Each specimen was measured thrice at different times.Quantitative ELISA-like immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissues. Sections  according to manufacturer's instructions. The housekeeping gene of phosphoglycerokinase (189 bp) was coamplified to check the RNA quality [The FGF23 mRNA transcription was detected by RT-PCR assay both in TIO specimens and the control tumors. Total RNA was extracted from paraffin-embedded sections with TRIzol Reagent kit  following a previous reported procedure . For RT Between 2003 and 2010, there were about 400,000 hospitalized patients at our institution. From the clinical database, we retrieved 5 cases of diagnosed TIO or phosphaturic mesenchymal tumor (PMT). Other 11 cases with typical clinical and laboratory characteristics were also retrieved, two of which were excluded for no surgical specimen available. Eventually there were 14 cases included, 6 males and 8 females, with a mean age of 39.0 ± 10.8 years (18–55 years) at the time of surgery (case  11 was previously reported elsewhere ).Detailed in Six of the 14 TIO tumors were localized in bones, 6/14 in soft tissues and 2/14 in craniofacial sinuses. They were measured at 1.0–14.0 cm in the maximum dimension . During the postoperative follow-up periods of 0.7–8.0 years , renal phosphate wasting and clinical symptoms of osteomalacia for 11/14 cases had resolved. Bone pain had disappeared at 1.0–2.0 months (median 1.5 months) and they had walked again at 0.5–3.5 months (median 2.0 months). No recurrence occurred for the 11/14 cases and their chemical examinations were normal. As shown in As detailed in As presented in The histopathologic feature of the 13/14 benign TIO tumors was similar from case to case wherever the tumor was located. As presented in p < 0.001, Mann-Whitney test). The results of non-TIO tumors were as follows: hemangiopericytomas 0.10 ± 0.05, giant cell tumors of tendon sheath 0.10 ± 0.02, giant cell tumors of bone 0.06 ± 0.02, osteosarcomas 0.10 ± 0.04, osteoblastomas 0.06 ± 0.01, chondroblastomas 0.09 ± 0.07, chondromyxoid fibromas 0.05 ± 0.03, and aneurysmal bone cysts 0.05 ± 0.03.Besides the 14 PMTMCTs, 26 mesenchymal tumors where PMTMCTs were usually misdiagnosed were also included, including 4 hemangiopericytomas, 4 giant cell tumors of tendon sheath, 3 giant cell tumors of bone, 3 osteosarcomas, 3 osteoblastomas, 3 chondroblastomas, 3 chondromyxoid fibromas, and 3 aneurysmal bone cysts. As presented in As presented in p < 0.001). The results were also confirmed by the previous reported RT-PCR assay. As mRNA is usually degraded in paraffin-embedded tissues and RT-PCR experiments usually fail, the quantitative ELISA-like immunohistochemistry for FGF23 expression could be a more practical way for the diagnosis of TIO.TIO is a rare acquired paraneoplastic disorder characterized by hypophosphatemia and osteomalacia –5, 8, 9.Summarized by the 14 cases, TIO patients were characterized by a long-standing history of osteomalacia (3.9 ± 2.5 years) in most cases (13/14). The initial symptom was usually nonspecific bone pain which would progress gradually. Bone pain of the whole body, walking difficulty, muscle weakness, and osteoporosis would be developed in 2.0–3.0 years. Osteoporotic fractures were common and eventually patients were bedridden. The biochemical manifestations included low serum phosphate level (0.52 ± 0.09 mmol/L), high serum ALP, normal or mild low serum calcium, normal or high intact PTH, high urine phosphate, and low TmP/GFR (0.46 ± 0.10 mmol/L). After complete tumor resection, the renal phosphate wasting and clinical symptoms would be resolved. Bone pain would disappear at 1.0–2.0 months (1.3 ± 0.3 months) and patients usually could walk again at 0.5–3.5 months (1.8 ± 1.9 months). Of the 12/14 patients who underwent intact tumor resection, only 1 patient developed local recurrence during the follow-up period of 0.7–8.0 years (3.0 ± 2.1 years).While most of the TIO tumors are small slow-growing tumors which frequently situated in unusual sites, identification of the responsible tumor is of challenge and conventional examination usually fails , 20. As PMTMCTs are usually misdiagnosed as other tumors, such as vascular tumors (hemangiopericytoma and hemangioma), giant cell tumors, osteoblastoma, chondromyxoid fibroma, chondroblastoma, and osteosarcoma –4. As shFGF23 is an important phosphaturic factor that inhibits inorganic phosphate reabsorption at the renal proximal tubule . As abunThe present study reported a large series of TIO cases from a single center. Besides the comprehensive description of clinical, laboratory, and pathological characteristics of the 14 cases, we documented a novel FGF23 detecting procedure. Because the commercial antibody of FGF23 does not work well for conventional immunohistochemistry staining (including the antibody used in the present study) and mRNA is usually degraded for RT-PCR procedure, the technique of quantitative ELISA-like immunohistochemistry could be a useful technique to detect the high FGF23 expression in paraffin-embedded tumor specimens. We simplified the quantitative ELISA-like immunohistochemistry on the slide and made it more convenient. The technique could also be generalized to many other situations in surgical pathology.The study had several limitations. First, there was no corresponding fresh specimen, no preoperative and postoperative serum available (only available in 1 patient and previously reported elsewhere : the preQuantitative ELISA-like immunohistochemistry was a feasible and reproducible procedure to detect and quantify the high FGF23 expression in formalin-fixed and paraffin-embedded biopsies or specimens and it could provide new insights into the diagnosis and differential diagnosis of TIO associated tumors. Since TIO was often delay-diagnosed or misdiagnosed, clinicians and pathologists should be aware of TIO and PMTMCT, respectively.


Ictalurus punctatus) is an important agricultural fish that has been plagued by Streptococcus iniae (S. iniae) infections in recent years, some of them severe. C5a peptidase is an important virulent factor of S. iniae. In this study, the subunit vaccine containing the truncated part of C5a peptidase (pSCPI) was mixed with aluminum hydroxide gel (AH), propolis adjuvant (PA), and Freund’s Incomplete Adjuvant (FIA). The immunogenicity of the pSCPI was detected by Western-blot in vitro. The relative percent survival (RPS), lysozyme activity, antibody titers, and the expression of the related immune genes were monitored in vivo to evaluate the immune effects of the three different adjuvants. The results showed that pSCPI exerted moderate immune protection (RPS = 46.43%), whereas each of the three adjuvants improved the immune protection of pSCPI. The immunoprotection of pSCPI + AH, pSCPI + PA, and pSCPI + FIA was characterized by RPS values of 67.86%, 75.00% and, 85.71%, respectively. Further, each of the three different adjuvanted pSCPIs stimulated higher levels of lysozyme activity and antibody titers than the unadjuvanted pSCPI and/or PBS buffer. In addition, pSCPI + FIA and pSCPI + PA induced expression of the related immune genes under investigation, which was substantially higher than the levels stimulated by PBS. pSCPI + AH significantly stimulated the induction of MHC II β, CD4-L2, and IFN-γ, while it induced slightly higher production of TNF-α and even led to a decrease in the levels of IL-1β, MHC I α, and CD8 α. Therefore, we conclude that compared with the other two adjuvants, FIA combined with pSCPI is a more promising candidate adjuvant against S. iniae in channel catfish.Channel catfish ( Ictalurus punctatus) is an important agricultural fish, especially in China where its yield was 150,000 tons in 2008 [Streptococcus iniae (S. iniae) has seriously damaged fish farming in recent years [S. iniae, a β-hemolytic Streptococcus species, has been an important fish pathogen with a broad host range, such as the hybrid striped bass (Morone chrysops × M saxatilis), tilapia (Oreochromis spp.), flounder , rainbow trout (Oncorhynchus mykiss), channel catfish, and salmon  [Channel catfish  ,7,8. Furo salar) ,9.S. iniae infection, vaccines are a good choice. In comparison with the inactivated and/or attenuated live vaccines, subunit vaccines composed of conserved proteins are safer and more serotype-independent [Fortunately, as green interventions to prevent ependent . Howeverependent ,12,13. Fependent , oil-basependent ,12,15,16ependent ,18,19 adependent ,21. Freuependent , due to S. iniae in the hybrid striped bass had equal degrees of similarity  to the C5a peptidases of GAS and GBS [S. iniae might act as a valuable subunit vaccine for protection of fish against S. iniae infection.C5a peptidase is a highly conserved, multifunctional surface protein. C5a peptidase of group A streptococci (GAS) has proven efficacy in humans and mice against GAS infections ,24. LikeHowever, since C5a peptidase is a macromolecule with a molecular weight of 123.4 kDa, its expression was difficult. Based on the results of previous investigations, in the present study, we truncated a part of C5a peptidase containing most of the B-cell epitopes (30–695 aa) and the conserved domains (designated pSCPI) to obtain high expression of recombinant protein.in vitro. Channel catfish were individually vaccinated to evaluate their immune protection in vivo. Subsequently, the lysozyme activity, serum bactericidal activity, antibody titer, and immune-related gene expression were used to assess the effect of the three adjuvants. All the experimental results indicated that FIA is a more promising candidate adjuvant combined with pSCPI against S. iniae in channel catfish.The aim of this work was to investigate the immune enhancement of three different adjuvants against pSCPI. We used Western-blot analysis to confirm the presence of the pSCPI protein pSCPI proteins were quantified by SDS-PAGE and transferred to a PVDF membrane. Rabbit anti-pSCPI and negative sera were employed as primary antibodies for Western blotting. A specific band of approximately 87 KDa was determined in the rabbit anti-pSCPI experimental group, which was missing in the negative group , tumor necrosis factor-α (TNF-α), major histocompatibility complex (MHC) class I α (MHC I α) and II β (MHC II β), CD4-L2, CD8 α and interferon-γ (IFN-γ) was examined by qRT-PCR. Compared with the expression in PBS, pSCPI + FIA and pSCPI + PA significantly induced the production of all the investigated genes. The treatment with pSCPI + AH considerably stimulated the induction of MHC II β, CD4-L2, and IFN-γ, while it promoted only a slightly higher production of TNF-α and even caused a decline in the levels of IL-1β, MHC I α, and CD8 α. pSCPI alone substantially enhanced the expression of the genes encoding TNF-α, MHC II β, CD4-L2, and IFN-γ, whereas it stimulated a moderately higher production of IL-1β, MHC I α, and CD8 α (The expression of genes encoding interleukin 1β (nd CD8 α .S. iniae (SCPI) isolated in the hybrid striped bass had equal degrees of similarity to the C5a peptidases of GAS and GBS [The highly conserved streptococcal C5a peptidase (SCP) is an important surface virulence protein and a prime vaccine candidate of Group A streptococci (SCPA) and Group B streptococci (SCPB) in humans and mice ,24,25. S and GBS , we conc and GBS . Therefo and GBS  or SCPB Edwardsiella tarda in Japanese flounder vaccines [Three different adjuvants were used in this study. Although FCA has been employed as the most effective adjuvant in the largest range of animals, investigation of its role in immunostimulation in fish has been limited . Moreovevaccines , AH in ovaccines , and PA vaccines .Due to their ectothermic nature and evolutionary status, fish predominantly manifest non-specific immunity, which usually precedes the specific immune response and activation, and its role in the maintenance of homeostasis . LysozymIt is well known that a subunit vaccine has weak immunogenicity. Further, the immune system of fish is slower to react than the immune systems of higher vertebrates . TherefoACTB and GAPDH) for normalizing mRNA expression in rats [et al. have reported that 18S rRNA is the best choice for normalizing real-time PCR data collected from channel catfish tissues experimentally [18S rRNA to be a housekeeping gene to normalize the real-time PCR data. Aluminum adjuvants are known to induce only a Th2 response [MHC II, which enables extracellular antigen presentation in conjunction with CD4+ T helper (Th) cells [MHC I α and CD8 α are involved in the cellular immunity, and CD4-L2 participates in humoral immunity, IFN-γ exerts an important role in both innate and cellular immune responses [IL-1β and TNF-α are generally related to the inflammatory response and were also expressed higher in the fish vaccinated with both pSCPI + PA and pSCPI + FIA compared to controls. However, while the aluminum adjuvant functioned predominantly in humoral immunity, the expression of IFN-γ was enhanced considerably, which might contribute to innate immunity.Ribosomal RNAs constitute between 85% and 90% of the total cellular RNA. As their expression has been shown to be fairly stable under unfavorable conditions, they have been widely used as quantitative references for gene expression . Moreove in rats , human [ in rats , and mou in rats  tissues mentally . Thereforesponse ,21, but response  and FIA h) cells , was stiesponses . On the RPS is an important parameter to evaluate the effects of vaccines. In the present study, the subunit vaccine pSCPI exhibited moderate immunoprotective efficacy, while the levels of immunoprotection mediated by pSCPI + AH, pSCPI + PA, and pSCPI + FIA were higher and manifested RPS values of 67.86%, 75.00%, and 85.71%, respectively. Thus, FIA manifested the strongest RPS among the three adjuvants, which also indicated that FIA is a more effective adjuvant than PA and AH.S. iniae strain DGX07 was isolated from channel catfish in China and stored in our laboratory  [S. iniae strain DGX07 was cultured in brain-heart infusion medium ) at 28 °C, and Escherichia coli BL21 (DE3)  was cultured in Luria-Bertani (LB) medium at 37 °C. Ampicillin  was supplemented at a final concentration of 100 mg/mL.The , China) . The pET, China) . The S. S. iniae DGX07. Fish were anaesthetized with MS222  prior to the experiments, which involved manipulations such as injections and serum collection. All the animal experiments complied with the ethical standards set by the Ethical Committee of the Faculty of Veterinary Medicine .To obtain specific antisera, rabbits weighting 2–2.5 kg were acclimated in an animal room for 2 weeks and fed daily with commercial feed. Channel catfish (60.0 ± 5.0 g) were purchased from a fish farm in PuJiang  and were acclimatized in a concrete culture tank (1.5 m × 1.0 m × 1.5 m) for 2 weeks before experimental manipulation. The fish were fed a commercial diet daily, and the water was partly replaced every day, maintaining a temperature of 25 °C ± 3 °C. Agglutination test results showed no reaction between the serum and Escherichia coli BL21 (DE3) strains transformed with pET-32a (+)-pSCPI were cultured in LB medium with agitation at 37 °C. When the OD600nm reached 0.6, IPTG  was added to a final concentration of 0.1 mM at 37 °C, and the samples in the conical flask were cultured for 4 h. After the induction period, the cultures were centrifuged at 8000× g for 10 min at 4 °C and were suspended in 20 mM Tris-HCl buffer. The crude extracts were obtained from sonication using a Sonic Dismembrator Model 500 and examined by 12.5% SDS-PAGE. The inclusion bodies from the insoluble fractions were purified by Ni-NTA-Sefinose Column  after dissolution in 8 M urea solutions and passage through 0.22-µm filters. The refolding of the purified proteins was carried out via transition passage from 6 M urea solutions to PBS  via gradient dialysis at 4 °C, and then analyzed by 12.5% SDS-PAGE. The quantity was determined using a BIO-RAD Smart Spec Plus  according to the manufacturer’s instructions. Purified proteins were stored at −20 °C.g for 15 min. The serum was prepared and stored at −20 °C. Immunoglobulin G (IgG) was purified by the ammonium sulfate precipitation method [New Zealand white rabbits were divided into an experimental group and a control group. The purified pSCPI (1.2 mg/mL) was used as the antigen and mixed with an equal volume of FCA . This mixture was injected into the rabbits in the experimental group, followed by three booster shots of pSCPI + FIA by subcutaneous injection at one-week intervals. The rabbits immunized with PBS were utilized as a control. Three days after the final injection with pSCPI + FIA, blood was collected from the rabbits in both groups, which was subsequently clarified, and centrifuged at 3000× n method .The purified proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane electrically at 150 V for 4 h. After pre-blocking with TBST (containing 3% BSA) for 1 h at room temperature, the membrane was incubated with rabbit anti-pSCPI/negative sera diluted 1:100 in TBST (containing 0.5% BSA) for 12 h at 4 °C. After washing with TBS, the membrane was incubated with goat-anti-rabbit IgG (H + L)-HRP  diluted 1:5000 in TBST (containing 3% BSA) for 1 h. The reaction was visualized using DAB (Sigma) for 5 to 15 min, and was terminated by rinsing with distilled water .w/v). The mixtures were incubated at 25 °C for 24 h with stirring and were sterilized by passing through 0.22-µm filters. Finally, the mixed solutions were resuspended in PBS to 30 mg/mL. To obtain pSCPI + PA, the purified recombinant pSCPI was mixed in equal volumes with the propolis adjuvants.The purified pSCPI (1.2 mg/mL) admixed with propolis adjuvants (pSCPI + PA) was prepared based on the following procedure : 10 g pr2(SO4)3 and 5% NaOH were filter-sterilized, as described above. The solutions were incubated at 60 °C for 30 min. Five volumes of 5% Al2(SO4)3 and two volumes of 5% NaOH were mixed with stirring, followed by centrifugation at 10,000× g for 5 min. After washing twice with sterile PBS, the mixtures were resuspended in PBS to 0.2 mg/mL. The purified recombinant pSCPI was mixed with an equal volume of aluminum hydroxide adjuvant to obtain pSCPI + AH.The purified pSCPI adjuvanted with aluminum hydroxide (pSCPI + AH) was prepared as follows : 5% Al2(The purified pSCPI (1.2 mg/mL) adjuvanted with FIA (pSCPI + FIA) was prepared by mixing pSCPI and FIA  in equal volumes until a stable emulsion (a homogeneous white mixture) was formed .All the adjuvanted vaccines above were stored at 4 °C until use (no more than three days).Three hundred channel catfish were divided into five groups of 60 fish each: four vaccine-treated groups and one control group. The individual fish in the four vaccine groups, namely, pSCPI + FIA, pSCPI + AH, pSCPI + PA, and pSCPI, were injected intraperitoneally with 200 µL pSCPI + FIA, pSCPI + AH, pSCPI + PA, and pSCPI, respectively. The control (PBS) group received an identical dose of PBS. 2 weeks post-vaccination, the fish in the four vaccine groups were boosted with 200 µL purified pSCPI.S. iniae strain DGX07 at 6 × 107 CFU/mL [On the 28th day after booster vaccination, 20 fish were randomly selected from each treatment group, and challenged with 200 µL of the 7 CFU/mL  by intra7 CFU/mL .The 40 remaining fish were sampled at week 3 to 8 during the trial. Serum samples from five individuals in each group were collected for assessment of lysozyme activity and ELISA. Individual sera from each group were pooled to conduct the above-mentioned assays, which were repeated three times for all samples. Head-kidney samples from five fish were taken for qRT-PCR 48 h post-challenge.Micrococcus lysodeikticus cells (Sigma) was performed. M. lysodeikticus (150 µL) with a concentration of 0.2 mg/mL  was added to 15 µL sera in a 96-well U-bottom microtiter plate. The initial optical density (OD) was detected at 450 nm instantly after adding M. lysodeikticus, and then, the final OD was measured after incubation for 1 h at 37 °C. Lyophilized hen egg-white lysozyme (Sigma) was used to develop a standard curve. The lysozyme levels were expressed as units·mL−1, with a decrease in absorbance of 0.001 min−1 representing one unit.The lysozyme activity was assessed using the previously described method ,53. BrieSpecific antibody titers for pSCPI were determined by ELISA . In brieCt−ΔΔ method). All data were presented in terms of relative mRNA and expressed as means ± SE.Head-kidney samples were obtained from the vaccinated fish at 48 h post-challenge. Total RNA was extracted from the kidney by the RNAprep Tissue/Bacteria Kit , following the manufacturer’s instructions. The cDNA was synthesized using a RevertAid First Strand cDNA Synthesis kit  according to the manufacture’s guidelines. The qRT-PCR was performed with the ABI PlusOne System  with primers . All results were shown as mean ± SE, and the differences were considered statistically non-significant and significant when S. iniae infection in channel catfish. Our findings invitro revealed that the three different adjuvants  triggered an immune response and improved the immune protection of pSCPI. Of the three adjuvants, FIA exhibited the maximum immunostimulation, followed by PA and AH. Therefore, compared to the other two adjuvants used in our study, FIA is a more promising candidate adjuvant, in combination with pSCPI, against S. iniae infections in channel catfish.In conclusion, the subunit vaccine pSCPI possesses immunogenicity and exerts moderate immune protection against


Surgery-induced changes in cognitive dysfunction and changes in Aβ1–42 and TGFβ/Smad signaling were prevented by the administration of IL17A monoclonal antibody. In addition, IL17A-stimulated TGFβ/Smad activation and Aβ1–42 expression were reversed by IL17A receptor small interfering RNA and a TGFβ receptor inhibitor in cultured astrocytes. Our findings suggest that surgery can provoke IL17A-related hippocampal damage, as characterized by activation of astrocytes and TGFβ/Smad pathway dependent Aβ1–42 accumulation in old subjects. These changes likely contribute to the cognitive decline seen in POCD.Although postoperative cognitive dysfunction (POCD) is relatively common in elderly patients who have undergone major surgery, the mechanisms underlying this postoperative complication are unclear. Previously, we have investigated the role of cytokine-mediated hippocampal inflammation in the development of POCD in a rat model. Here, we sought to determine in mice the role of cytokine interleukin17A (IL17A) in POCD and to characterize the associated signaling pathways. Old mice underwent hepatectomy surgery in the presence or absence of IL17A monoclonal antibody, and cognitive function, hippocampal neuroinflammation, and pathologic markers of Alzheimer’s disease (AD) were assessed. We found that the level of IL17A in the hippocampus was increased in hepatectomy mice and that cognitive impairment after surgery was associated with the appearance of certain pathological hallmarks of AD: activation of astrocytes, β-amyloid Postoperative cognitive dysfunction (POCD) is a subtle disorder of perception, memory, and information processing after surgery . A multiAlthough it was previous suggested that the type of anesthesia may contribute to the development of POCD, a study on the relationship between POCD and anesthesia showed that the incidence of POCD was similar after regional and general anesthesia, indicating that anesthesia does not appear to contribute to the development of POCD . More rein vivo and in vitro and to promote Aβ formation by a transcriptional mechanism involving the signaling mother against decapentaplegic peptide 3 (Smad3) signaling pathway in astrocytes [TGFβ, a cytokine overexpressed in the brain of AD patients , was shotrocytes . The TGFtrocytes , multipltrocytes , and AD trocytes . To dateHere, we describe a new role for cytokine IL17A, a cytokine produced by Th17 cells, that plays key roles in tissue inflammation and autoimmune responses . IL-17 h1–42 (Western blot) were purchased from Cell Signaling Technology . Antibodies for Smad2/3 and pSmad2/3  were purchased from Upstate Biotechnology , and antibodies for pSmad2/3 (Immunofluorescence), GFAP, Aβ1–42 (immunohistochemistry) were from Santa Cruz Biotechnology . The pharmacological inhibitors for TGFβ receptorI/II  were purchased from Selleck Chemicals , and IL17RA siRNA was from Wanlei life sciences .IL17A monoclonal antibody was purchased from R&D Systems , and rat anti mouse IgG2a was from Abcam . Antibodies for IL17A, IL6, TGFβ, GFAP, APP, and Aβad libitum. Experiments were approved by the China Medical University Animal Care and Use Committee and adhered to the guidelines for care and use of laboratory animals from the Chinese Academy of Science.C57BL/6 14-mo-old male mice were housed in polypropylene cages and maintained at 23°C under a 12-h light/dark cycle in a humidity controlled environment with food and water available Mice were trained for five consecutive days on the Morris water maze (MWM) test and for 3 days on the T maze test, then randomly assigned to one of three groups: 1) Control group , mice received no intervention; 2) Anesthesia group , mice received general anesthesia with 50mg/Kg sodium pentobarbital injected intraperitoneally (i.p.); And 3) Anesthesia-plus-surgery group , mice underwent partial hepatectomy under general anesthesia. For hepatectomy, the liver was exposed through a 1–2 cm midline abdominal incision. The left lateral lobes of the liver (30% of the organ) were excised. The wound was then infiltrated with 0.25% bupivacaine and closed by sterile suture. For the IL17A blockade test, there were four groups: 1) Control group , mice received no intervention; 2) Surgery group , mice only underwent partial hepatectomy under general anesthesia; 3) Surgery plus IL17A monoclonal antibody group , mice were treated as in the SA group plus administration of IL17A monoclonal antibody during the surgery; and 4) Surgery plus IgG2a control group , mice were treated as in the SI group but rat anti mouse IgG2a was administered in place of IL17A monoclonal antibody.Mice were prepared with an intracerebroventricular (ICV) cannula, as previously described . In brieThe MWM is a hippocampal-related test of spatial learning for rodents . A circuHippocampal tissues from mice in each group were rapidly dissected and stored at −70°C until they were used for messenger RNA (mRNA) and Western blot studies. Remaining animals in each group were sacrificed and transcardially perfused with heparinized saline followed by 300 ml of ice-cold 4% paraformaldehyde in PBS (pH 7.4). Brains were dissected and post-fixed in 4% paraformaldehyde in PBS for 4 h and subsequently embedded in paraffin and sectioned for histopathological studies.The T-maze, which has been used in a variety of ways to assess the cognitive ability of an animal , was madPrimary astrocyte cultures from wild-type C57BL/6 mice were established from neonatal cerebra, as described previously . Cells w-ΔΔct).Total RNA was extracted from tissue specimens using Trizol Reagent . cDNA was synthesized with the QuantiTect Reverse Transcription Kit with integrated removal of genomic DNA  according to the manufacturer’s protocol. Quantitative real time PCR was performed using an ABI 7500 PCR system, and levels of mRNA for IL17A, IL6, TGFβ, Smad2, Smad3, and APP from all groups were assayed according to the manufacturer's protocols. The amplification conditions were 8 min at 95°C, followed by 45 cycles of 95°C for 5 s, 60°C for 34 s, and 72°C for 15 s. Specific primers were designed using Primer5 . For norg for 10 min at 4°C. The supernatant was collected, and the protein concentration was determined by the bicinchoninic acid  method using bovine serum albumin as the standard. Protein samples were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidenedifluoride (PVDF) membrane . Membranes were blocked in 5% skim milk and incubated overnight at 4°C with primary antibodies against IL17A (1:100), IL6 (1:100), TGFβ (1:100), GFAP (1:100), APP (1:100), Aβ1–42 (1:100), Smad2/3 (1:100) and pSmad2/3 (1:100). Membranes were incubated with HRP-labeled secondary antibodies (1:5000), and proteins were detected by enhanced chemiluminescence  according to the manufacturer’s instructions. Signals were quantitatively analyzed with Quantity-One software (Bio-Rad). B-actin was used as a protein-loading control.Hippocampal tissue samples were homogenized in ice-cold lysis buffer containing protease inhibitors and centrifuged at 12,000 Sections (5 μm) were cut from paraffin embedded hippocampal tissue samples for immunohistochemistry. In brief, sections were incubated in 0.3% hydrogen peroxide-methanol solution, blocked in 5% bovine serum albumin, and incubated overnight at room temperature with rabbit polyclonal anti- GFAP (1:100), rabbit polyclonal anti- Aβ antibody (1:100), or rat immunoglobulin IgG (1:100) as the negative control. After washing with PBS, biotin-conjugated secondary antibodies were applied (1:100) at 37°C for 30 min. Sections were washed with PBS and incubated with diaminobenzidine (DAB) for detection. Finally, the sections were mounted on gelatin-coated slides, dehydrated in an ascending alcohol series, cleared in xylene, and photographed under bright field microscopy . For quantification, the positive cells in the hippocampus were estimated by using mean integrated optical density (OD). MetaMorph software system  was used for image analysis.Cells were fixed in 4% paraformaldehyde for 15 min and washed in PBS. After cells were blocked with normal rabbit serum for one h at room temperature, they were incubated with rabbit anti pSmad2/3 (1:50) and rat anti mouse GFAP (1:50) at 4°C overnight. Following washes, cells were incubated with secondary antibodies containing a mixture of Cy3-conjugated goat anti rabbit IgG (1:100) and fluorescein isothiocyanate (FITC)-conjugated goat anti rat IgG (1:100) at room temperature for one h. Cell nuclei were counterstained with 4',6-diamidine-2'- phenylindoledihydrochloride  at room temperature for five min. The cells were then examined under fluorescence with the Nikon Eclipse E800 microscope  and the Spot advanced digital camera . Fluorescence images were merged using Andor IQ software .P < 0.05 was considered significant.All data are presented as mean±SEM. Statistical analysis was performed with analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple comparison test for numerical data. Unpaired Student t test was used for comparisons between two groups. P>0.05, P<0.01, P<0.01; day five: P<0.05, P>0.05, P<0.05 or 0.01, respectively; P<0.01; day 7: P<0.05, P<0.05, P>0.05).To elucidate the effect of partial hepatectomy on learning and memory, we conducted the MWM test, a highly sensitive and well validated test of cognitive function for rodents. It is particularly sensitive at testing hippocampus-dependent learning and memory formation. In our tests, mice in all groups were able to find the platform, and the escape latency gradually decreased over the five consecutive training days. By the fifth training day, the latency was reduced to 51%, 46%, and 50% of the first day of training in control group (group C), anesthesia group (group A), and anesthesia-plus-surgery group (group SA), respectively P>0.05, . On postP>0.05). However, on postoperative day 1 and 3, the levels of IL17A, IL6, and TGFβ mRNA expression were elevated in group SA, peaking on day postoperative 3 (P<0.01). Likewise, protein expression of IL17A, IL6, and TGFβ was also markedly elevated on postoperative days 1 and 3 . Immunohistochemistry was also used to detect Aβ1–42 expression, and similar results were obtained  signaling in surgery triggered hippocampus pathological injury, mice were administered either IL17A monoclonal antibody (group SI) or IgG2a (group SC) during surgery. In the MWM test, compared with group SC or group SA mice, group SI mice demonstrated a significant decrease in escape latency time and swim distance on postoperative day 3 P<0.01, . Consist P<0.01, .P<0.05 or 0.01, respectively; IL17R is expressed in most tissues examined to date, importantly in CNS glial cells , 31. We 1–42 expression, we prepared stimulated primary astrocyte cultures, as described above, and measured APP mRNA levels by quantitative RT-PCR. IL17A stimulation significantly upregulated astrocytic APP mRNA levels relative to control, and 24 h treatment with IL17 R siRNA attenuated this effect  treated cells, little GFAP and pSmad 2/3 were present in the cytoplasm and nucleus . These rPOCD is one of the most common complications after cardiac and non-cardiac surgery and is independently associated with poor long-term outcome and even permanent dependence on social care systems in some patients , 35. Cli1–42 in the hippocampus. Neither cognitive dysfunction nor Aβ1–42 accumulation was detected in mice subjected to anesthesia alone.In the present study, we investigated the relationship between IL17A-triggered inflammatory response in the hippocampus and the development of POCD. After hepatectomy surgery under general anesthesia, mice exhibited memory impairment that was associated with activation of astrocytes and accumulation of AβSurgery promote expression of proinflammatory cytokines, including IL1β, IL6 and TNFα in the hippocampus, and these changes may be involved with the development of POCD , 6, 7, 81–42 was induced conspicuously on postoperative day 3, and Aβ1–42 levels remained elevated on postoperative day 7. When IL17A was blocked, surgery-triggered IL17A inflammation, which resulted in astrocyte activation, TGFβ/Smad activation, AD-like pathologic changes, and cognitive dysfunction, was abolished. These results suggested that in our surgical model, surgery triggered in hippocampus an immediate expression of IL17A, a gradual activation of astrocytes, and TGFβ/Smad molecular upregulation. Both our in vivo and in vitro studies are the first to show that cytokine IL17A-mediated POCD may share a common molecular mechanism with AD.We found that hippocampal IL17A expression was significantly increased on postoperative day 1 and 3, with peak cognitive dysfunction on day 3, the day linked with activation of astrocytes. Meanwhile, on postoperative day 3, TGFβ expression was prominently enhanced, and Smad2/3 protein phosphorylation was increased in hippocampus. Target protein production of APP and Aβ1–42. We postulated that POCD may be caused by the development of these postoperative pathologic changes induced by hippocampal IL17A. Several studies demonstrated that Aβ is directly toxic to cultured neurons, inducing cell degeneration and death that is apoptotic in nature [In AD, the inflammatory response is localized primarily within the vicinity of amyloid plaques. There is mounting evidence suggesting that inflammation in the central nervous system, particularly in the hippocampus, is mediated by the activation of glial cells and the production of inflammatory mediators (such as cytokines). These changes may contribute to the neuropathogenesis of AD , 42, 43.n nature , 45, 46.1–42 accumulation. The molecular mechanisms, however, by which IL17A increased the level of Aβ1–42 in astrocytes are unknown. Nevertheless, the observed increase in astrocytic TGFβ and pSmad levels following IL17A stimulation suggested that activated TGFβ and Smad signaling was at least partially responsible.Cytokine IL1β has a wide array of reported effects on cognitive dysfunction, ranging from enhancement of glutamate neurotoxicity  to the p1–42 levels in POCD, we pretreated primary astrocyte cultures with the TGFβ/Smad inhibitor followed by stimulation with IL17A. Not only were astrocytic Aβ1–42 levels reduced with TβRI, but there was a significant effect on astrocytic pSmad levels. Here, we demonstrated a direct link between TGFβ/Smad pathway activation and IL17A-triggered Aβ1–42 accumulation in mice after surgery.TGF-βs belong to a family of peptides that play important roles in intercellular signal transduction . They tr1–42 processing. Inflammatory cytokine IL17A stimulates: 1. astrocyte activation to produce more cytokines; 2. continued activation of TGFβ/Smad pathway; 3. increased levels of APP and secreted Aβ1–42 from astrocytes; and 4. further elevation of hippocampus Aβ1–42 levels, promoting production of more cytokines and Aβ1–42. There is some evidence in favor of the existence of this cycle. Aβ is capable of stimulating astrocytes to secrete inflammatory cytokines, and conversely, cytokine combinations, such as IL1, IL6, TNFα, and TGFβ, are capable of promoting astrocytic Aβ secretion. When working together, these events lead to a feedforward loop in the progression of the disease [In POCD, a positive feedback mechanism may operate to elevate and sustain astrocytic Aβ disease , 57, 58.1–42 in the hippocampus. Strategies designed to attenuate this feed-forward mechanism may represent a means of preventing postoperative cognitive dysfunction.In summary, we have demonstrated that surgery triggers a transient neurocognitive decline in a mouse model that is associated with IL17A-induced astrocyte activation and consequent TGFβ/Smad pathway-dependent secretion of Aβ