Whole genome microarray meta-analyses of 1030 kidney, heart, lung and liver allograft biopsies identified a common immune response module (CRM) of 11 genes that define acute rejection (AR) across different engrafted tissues. We evaluated if the CRM genes can provide a molecular microscope to quantify graft injury in acute rejection (AR) and predict risk of progressive interstitial fibrosis and tubular atrophy (IFTA) in histologically normal kidney biopsies.Computational modeling was done on tissue qPCR based gene expression measurements for the 11 CRM genes in 146 independent renal allografts from 122 unique patients with AR (n = 54) and no-AR (n = 92). 24 demographically matched patients with no-AR had 6 and 24 month paired protocol biopsies; all had histologically normal 6 month biopsies, and 12 had evidence of progressive IFTA (pIFTA) on their 24 month biopsies. Results were correlated with demographic, clinical and pathology variables.The 11 gene qPCR based tissue CRM score (tCRM) was significantly increased in AR (5.68 ± 0.91) when compared to STA  and pIFTA , with greatest significance for CXCL9 and CXCL10 in AR (p <0.001) and CD6 (p<0.01), CXCL9 (p<0.05), and LCK (p<0.01) in pIFTA. tCRM was a significant independent correlate of biopsy confirmed AR . Gene expression modeling of 6 month biopsies across 7/11 genes  significantly (p = 0.037) predicted the development of pIFTA at 24 months.Genome-wide tissue gene expression data mining has supported the development of a tCRM-qPCR based assay for evaluating graft immune inflammation. The tCRM score quantifies injury in AR and stratifies patients at increased risk of future pIFTA prior to any perturbation of graft function or histology. Kidney transplantation is the preferred modality for treatment of end-stage renal disease by any cause  and leadAll patients included in the study gave written informed consent to participate in the research, in full adherence to the Declaration of Helsinki. The study was approved by the institutional review board at Stanford University and University of California San Francisco. 146 renal allograft biopsies from 122 unique renal transplant patients were collected between 1 month– 10 years post-transplant as protocol biopsies or as indicated by acute graft dysfunction from pediatric and adult renal transplant patients with stable renal function (no-AR), AR, and pIFTA  and no-AR (n = 92). 24 demographically matched patients with no-AR had 6 and 24 month paired protocol biopsies. Among these 24 patients all of them had histologically normal biopsies in 6 month post-transplantation time. However, only 12 of the 24 patients had evidence of progressive IFTA (pIFTA) on their 24 month biopsies. The remaining 12 patients were normal in their 24 month post-transplantation. The cross sectional samples were randomly split into two groups of 27 AR and 22 no-AR for the purpose to determine and validate a tCRM threshold for detection of AR. Of this latter group demographic variables were matched. For the 24 patients with paired samples everyone had normal kidney function and graft histology on the 6 month biopsies. Twelve patients in this group had pIFTA injury on their 24 month protocol biopsies . The remaining 12 patients had histologically normal 24 month protocol biopsies  . All theT) method [Total RNA was extracted from each biopsy using TRIzol Reagent . RNA integrity was ensured using the RNA 6000 NanoLab Chip Kit (Agilent Technologies). cDNA synthesis was performed using 25 ng of extracted quality total RNA from the biopsy samples using SuperScript VILO Master Mix  as per the manufacturer’s protocol. Briefly, specific target amplification was performed using 3.125 ng relative amount of cDNA using a pooled individual TaqMan real-time assays for the 11 genes investigated in multiplex with TaqMan PreAmp Master mix Life Technologies) to 10 μl final volume, for 18 cycles in a thermal cycler , then diluted 1:20 with sterile water . The qPCR was performed on the QuantStudio 6 Flex System (Life Technologies) using 5μl of the diluted sample from the specific target amplification, along with the TaqMan Gene Expression Master Mix under standard conditions  using TaqMan gene expression assays (Life Technologies) for each of the 11 genes investigated: BASP1, CD6, CXCL10, CXCL9, INPP5D, ISG20, LCK, NKG7, PSMB9, RUNX3, TAP1. The relative amount of mRNA expression in each sample was calculated using the comparative threshold cycle (C) method . Ribosom2 test for categorical variables, the two-way analysis of variance (ANOVA) or t-test for normally distributed data, and the nonparametric Kruskal–Wallis, Welch’s correction or Mann–Whitney U test for non-normally distributed variables. Bivariate correlation analyses were done using Pearson or Spearman tests for non-parametric variables. Sensitivity/specificity Receiver Operating Characteristic (ROC) curve analyses were performed to evaluate the most precise cut-off of the tissue CRM (tCRM) gene scores assessed at the time of biopsy, predicting the advent of acute rejection (AR). All predictive values were determined by calculating the area under the curve with SPSS software. Binary linear logistic regression analysis was performed to determine the independent correlation of several independent variables with the presence of AR. The statistical significance level was defined as p<0.05. Gene expression, tCRM scores and clinical variables were examined by multivariate analysis to determine predictive value. Cross sectional analysis was done to determine the significant increase in gene expression at event. Longitudinal studies were done to determine changes in gene expression over 2 years in patients with progressive IFTA (pIFTA).All data are presented as mean ± standard error of the mean (SEM). Groups were compared using the χThe individual expressions of all 11 genes of the tCRM score showed a significant increase in AR, with the most significant increase seen in CXCL9 and CXCL10 (p = 0.0001 in both comparisons) between AR and no-AR . The tCRWe evaluated whether the tCRM score was associated with the extent of the AR lesions observed in the matched biopsies. As observed in Biopsies from patients with chronic allograft changes as defined by the presence of IFTA were examined at 24 months, and in addition their biopsy pairs were also examined from their 6 month protocol biopsies. Significantly increased fold changes of all genes except BASP1 and CXLC10 were seen at both 6 and 24 months between pIFTA (P) and no-AR (NP) patients. While the tCRM score across all 11 genes was significant between pIFTA (P) and no-AR (NP) (p = 0.05), we found by using an adjusted R score analysis, that among the 11 genes, the greatest influence on pIFTA was from a subset of 7 genes , with maximal influence from CD6. A modified tCRM score across these 7 genes showed significantly greater scores in the 6 month biopsies of patients destined to develop pIFTA patients over time (p = 0.037) .et al, in submission), which supports data from previous reports of significant overlap in the molecular disturbances from the overlapping intragraft infiltrates in AR and BK viral nephritis [There is an urgent need in transplant medicine for developing reliable and non-invasive monitoring tools that may help transplant clinicians predict the risk of alloimmune-mediated allograft injury, preferentially before allograft damage has already been established. While a number of transcriptional biomarkers have been associated to AR –19, manyephritis , 27–30, ephritis . We and ephritis , 33, genephritis –36 and pephritis , and uriephritis , 38–41;


The orexin-hypocretin system is important for translating peripheral metabolic signals and central neuronal inputs to a diverse range of behaviors, from feeding, motivation and arousal, to sleep and wakefulness. Orexin signaling is thus an exciting potential therapeutic target for disorders of sleep, feeding, addiction, and stress.2), using a randomized, double-blind, placebo-controlled, within-subject crossover design. Alprazolam, a licensed benzodiazepine anxiolytic, was used as a positive comparator, as it has previously been validated using the insulin tolerance test (ITT) model in humans.Here, we investigated the low dose pharmacology of orexin receptor antagonist, SB-649868, on neuroendocrine, sympathetic nervous system, and behavioral responses to insulin-induced hypoglycemic stress, in 24 healthy male subjects , the peak electromyography (p < 0.0001) and galvanic skin response  to acoustic startle, the resting GSR (p = 0.01), and increased appetite following ITT (p < 0.0005). SB-649868 pre-treatment produced no significant results.Of the primary endpoints, ITT induced defined increases in pulse rate, plasma cortisol, and adrenocorticotropic hormone in the placebo condition, but these responses were not significantly impacted by alprazolam or SB-649868 pre-treatment. Of the secondary endpoints, ITT induced a defined increase in plasma concentrations of adrenaline, noradrenaline, growth hormone (GH), and prolactin in the placebo condition. Alprazolam pre-treatment significantly reduced the GH response to ITT  contains supplementary material, which is available to authorized users. The orexin-hypocretin system is fundamentally important in regulating a diverse range of behaviors from feeding, motivation and arousal Sakurai , to sleeSince the early studies of narcolepsy in animals . Insulin-induced hypoglycemia has been shown to increase ACTH, cortisol, growth hormone (GH), and catecholamine release in a number of studies , was used as a positive comparator. It has been previously validated using the ITT model in a placebo-controlled experimental medicine study, and was shown to attenuate increases in ACTH, GH, and adrenaline  and neuroendocrine responses to hypoglycemia. The idea was to investigate whether SB-649868 could have effects on these systems by inhibiting abnormally activated orexin signaling, such as is thought to occur in anxiety or panic disorders  used a randomized, double-blind, placebo-controlled, within-subject crossover design. The study was conducted in two cohorts with an interim analysis separating cohorts 1 and 2. Each subject attended up to three testing sessions, each separated by a minimum 2-week washout period. At each session, subjects received either of the following: SB-649868 10 mg, alprazolam 0.02 mg/kg . However, when no significant treatment effects were demonstrated by the interim analysis, we opted, per protocol, to increase the sample size to N = 24, still testing for effects at a dose of 10 mg, to mitigate the risk of type II error. When no significant treatment effects were demonstrated by analysis of the full sample, we opted, per protocol, to stop the study.The study was specifically designed to test the effects of SB-649868 at sub-hypnotic doses. This was motivated partly by concerns about potential toxicity and pharmacokinetic variability of this molecule at high doses, and partly by the intention to investigate the possibility that SB-649868 might have benefits for patients with abnormally activated orexin signaling associated with increased sympathetic tone and/or hypercortisolemia, such as is thought to occur in anxiety or panic disorders  was administered , then every 15 min until t = 270 min (3 hours after the start of the ITT). At t = 210 min, the ITT was concluded and participants were offered an ad libitum choice of food and drink. Cardiac monitoring continued until normalization of blood glucose concentrations, and less frequent venous blood sampling until 1600 h. A summary of the testing procedure can be found in Fig. Testing sessions started between 0845 and 0915 h following an overnight fast of 9 hours. Thirty to 60 min prior to dosing, subjects were cannulated and began a schedule of continuous cardiac monitoring, and venous blood sampling for glucose, study endpoints, and pharmacokinetic assays. Subjects were dosed at time The protocol was reviewed and approved by the Welwyn Ethics Committee, and the study was conducted in accordance with ICH Good Clinical Practice guidelines, and the guiding principles of the 2008 Declaration of Helsinki. All subjects provided written informed consent prior to testing.Primary endpoints were divided into markers of HPA axis activation (plasma ACTH (pmol/L) and cortisol (nmol/L) concentrations), and markers of SNS activation (pulse rate (beats/min) and mean arterial pressure ). Secondary neuroendocrine markers included plasma glucose (mmol/L), adrenaline (nmol/L), noradrenaline , growth hormone , prolactin (μg/L), and luteinizing hormone  concentrations. Corresponding secondary SNS markers included resting galvanic skin response  over 4 min, respiratory rate (breaths/min), and acoustic startle response using GSR (μS) and surface electromyography . The acoustic startle comprised 10 pseudo-randomized bursts of white noise, at 100–110 dB, lasting 50 ms each, over the course of 15 min.2) were initially recruited in two separate cohorts of 12 subjects each. The final cohort comprised 33 subjects, including 9 replacements (see Section 3.1). Eligible participants underwent pre-study screening 30 days prior to the first dose to rule out any previous, or current, medical or psychiatric condition which may have compromised subject or investigator safety, and/or the interpretation of results. Additionally for this study, the following endocrine criteria were set: fasting plasma insulin <60 pmol/L, fasting plasma glucoses <7.0 mmol/L, and morning plasma cortisol >100 nmol/L.Twenty-four healthy male subjects  until the end of data collection for that endpoint. Statistical analysis was conducted using standard parametric ANOVA or ANCOVA. Data are plotted ± 95 % confidence intervals (95 % CI). Statistical comparison of side effects between groups was conducted using Fisher’s exact test.All endpoints in figures are shown as adjusted group means (adjusted for group size and baseline differences) of absolute values representing maximum change from pre-ITT baseline (∆max), or as areas under the curve (AUC) calculated by integration of values from the pre-ITT baseline  and SB-649868 (p = 0.008), whereas fatigue was reported more frequently in the SB-649868 and alprazolam conditions, but was not significantly different between any of the groups at p = 0.05 . In contrast, headache and lethargy were reported more frequently in the placebo and SB-649868 conditions , and dizziness only with alprazolam ; however, these effects were not significantly different between the conditions at p = 0.05.Subjects experienced typical hypoglycemic symptoms in response to the ITT, such as sweating, increased appetite, and increased heart rate. In addition, a number of drug-related side effects were also reported. These included somnolence, headache, fatigue, dizziness, and lethargy. Somnolence had a significantly higher incidence with alprazolam compared to both placebo (t = 0 min) of all neuroendocrine markers were not significantly different between drug conditions. In addition, the glucose response to ITT was similar regardless of the drug administered , where subjects were pre-treated with placebo . There was no significant difference to this AUC or ∆max response (see Methods section) when subjects were pre-treated with either SB-649868 or alprazolam , and consequently, plasma concentrations of cortisol and ACTH at subsequent time points were significantly lower in the alprazolam condition than in the placebo condition . Pre-treatment with alprazolam or SB-649868 had no significant impact on prolactin or LH responses to ITT , a marked increase in plasma concentration was observed in response to ITT for both GH and prolactin, but not LH . In addition, the pre-ITT baseline levels of GH and prolactin were significantly increased by alprazolam, but not SB-649868  of primary and secondary SNS markers were not significantly different between drug conditions.As with the neuroendocrine markers, the pre-dose baseline levels (t = 0 min), reaching a maximum 30 min after insulin administration, in the placebo condition . Pre-treatment with alprazolam or SB-649868 resulted in no significant change from the placebo responses to ITT, nor did they significantly change pre-ITT baseline values of adrenaline and NA Fig. . Similart = −45 min) of AUC GSR  and peak EMG  in response to startle, after ITT, in the placebo condition  and after (t = 120 min) ITT, in any condition, and likewise no significant difference between conditions, pre-treatment with alprazolam resulted in a significant post-ITT reduction in AUC from the pre-dose baseline resting GSR AUC value, where the effect of pre-treatment with either placebo or SB-649868 was to marginally increase, or not affect, resting GSR after ITT . There was no significant difference between pre-treatment conditions for any other behavioral endpoints ; Appetite ; Hypoglycemic symptoms (VAS); Hunger, Craving, and Fullness Questionnaire (HCFQ); and the quantity and rate of food consumption upon completion of the ITT. Pre-treatment with alprazolam significantly decreased appetite post-ITT, compared to placebo, as measured by the appetite VAS, at all time points analyzed  model for studying orexin antagonist activity in response to hypoglycemic stress in healthy adult humans. A summary of the effect sizes of pre-treatment with the positive control, alprazolam, and the study compound, SB-649868, on neuroendocrine and sympathetic nervous system responses to ITT can be found in Table In the placebo condition, ITT markedly increased plasma concentrations of most neuroendocrine hormones , and most SNS markers , suggesting that the ITT model may be of great utility for analyzing the pharmacological efficacy of compounds acting directly or indirectly on these systems.t = 90 min (the start of ITT), mean levels of cortisol, ACTH, and MAP, were significantly lower than in the placebo condition, and conversely, plasma concentrations of GH, prolactin, and LH were significantly higher. These findings are consistent with a previous experimental study on the effects of alprazolam on neuroendocrine responses to ITT  as a reference point. For endpoints where pre-ITT measurements were not taken, and pre-dose values were used as baselines , this confound was not apparent, and alprazolam pre-treatment was found to significantly reduce post-ITT responses in all of these markers, compared to placebo. The fact that alprazolam was able to effect changes in baseline levels of neuroendocrine and SNS markers suggests that the ITT model, which stimulated increases in these markers, may be useful for investigating the efficacy of alprazolam, or more generally, the role of GABA receptors in the pituitary and adrenal medulla; however, the experimental design may need to be modified to account for the effects observed immediately after compound administration. Furthermore, the results reported in our study following 0.02 mg/kg alprazolam administration were broadly, but not entirely, consistent with those reported in Giordano et al. , nor did low dose SB-649868 have a significant effect on any endpoint, and therefore the ITT model could not be validated with regard to investigating pharmacotherapeutic effects on orexinergic pathways. These findings may be particularly relevant in light of recent FDA decisions to only grant approval for low dose studies of a similar dual OxR1 and OxR2 antagonist, Suvorexant.Supplementary Fig. 1(DOC 61 kb)Supplementary Fig. 2(DOCX 40 kb)Below is the link to the electronic supplementary material.


AR-V bound to the promoter of its specific target without AR-FL, but co-occupied the promoter of canonical AR target with AR-FL in a mutually-dependent manner. AR-V expression attenuated both androgen and enzalutamide modulation of AR-FL activity/cell growth, and mitigated the in vivo antitumor efficacy of enzalutamide. Furthermore, ARv567es levels were upregulated in xenograft tumors that had acquired enzalutamide resistance. Collectively, this study highlights a dual function of AR-Vs in mediating castration resistance. In addition to trans-activating target genes independent of AR-FL, AR-Vs can serve as a “rheostat” to control the degree of response of AR-FL to androgen-directed therapy via activating AR-FL in an androgen-independent manner. The findings shed new insights into the mechanisms of AR-V-mediated castration resistance and have significant therapeutic implications.Upregulation of constitutively-active androgen receptor splice variants (AR-Vs) has been implicated in AR-driven tumor progression in castration-resistant prostate cancer. To date, functional studies of AR-Vs have been focused mainly on their ability to regulate gene expression independent of the full-length AR (AR-FL). Here, we showed that AR-V7 and AR Androgen deprivation therapy, which disrupts androgen receptor (AR) signaling through androgen ablation or AR antagonists, is the first-line treatment for disseminated prostate cancer. While this regimen is effective initially, progression to the presently incurable and lethal stage, termed castration-resistant prostate cancer (CRPC), invariably occurs ,2. Resurv567es are two major AR-Vs expressed in clinical specimens [v567es have a significantly shorter survival than other CRPC patients [AR-Vs are upregulated in most CRPCs compared to hormone-naïve cancers ,7,13-17.pecimens . Strikinv567es confers castration-resistant growth of LNCaP xenograft tumors [Preclinical studies have pointed to an important role of AR-Vs in mediating castration resistance. Ectopic expression of AR-V7 or ARt tumors ,15,20. Ct tumors . AR-Vs ht tumors ,11. Redut tumors . Thus, Av567es has been shown to coimmunoprecipitate with AR-FL and facilitate AR-FL nuclear localization in the absence of androgen [Gene expression profiling showed that AR-Vs regulate the expression of both canonical androgen-responsive genes and a distinct set of targets enriched for cell-cycle function ,13,15. Tv567es and AR-V7 can reside constitutively in the nucleus [v567es has been shown to facilitate AR-FL nuclear localization in the absence of androgen [v567es-TurboFP in the AR-null COS-7 cells. Consistent with previous reports [Both AR nucleus ,15,18, aandrogen . To asse reports ,15,18, av567es  and TMPRSS2 (2.6-fold vs. 1.4-fold), and enzalutamide was very effective in blocking the induction. Conversely, ectopic expression of AR-V7 or ARv567es in LNCaP cells dose-dependently induced basal PSA and TMPRSS2 expression and diminished the degree of response of PSA and TMPRSS2 to androgen  and AR-V-specific targets (CCNA2 and UBE2C) in 22Rv1 cells in response to AR-FL or AR-V7 knockdown Figure . While kn Figure . Taken tn Figure , the pren Figure . Collectvs. 1.3-fold in control cells). Consequently, the knockdown cells were more responsive to enzalutamide growth inhibition than the control cells. We next inoculated AR-V7-knockdown cells or control cells in nude mice, and characterized the response of the ensuing tumors to enzalutamide. As shown in Figure We proceeded to characterize the effect of AR-V7 knockdown on androgen and enzalutamide modulation of the growth of 22Rv1 cells. Congruent with the mRNA data, after AR-V7 knockdown, the cells became more sensitive to DHT induction of growth  were upregulated in enzalutamide-resistant tumors, while the levels of AR-V4 or AR-FL transcript did not differ . All cell lines were tested and authenticated by the method of short tandem repeat profiling. Enzalutamide was purchased from Selleck Chemicals , and the purity of >99% was confirmed by Nuclear Magnetic Resonance. The following antibodies were used in Western blot analysis: anti-glyceraldehyde-3-phosphate dehydrogenase , anti-AR , and anti-AR-V7 (Precision Antibody). Cell growth was determined by the Sulforhodamine assay.v567es plasmids were generated by cloning the cDNA fragments for AR-V7 and ARv567es, respectively, into the pCMV-TurboFP635 vector. COS-7 or PC-3 cells were transfected with indicated plasmids and cultured in phenol red-free RPMI-1640 supplemented with 10% charcoal-stripped fetal bovine serum. At 40 hr after transfection, cells were pre-treated with or without 10 μM enzalutamide for 2 hr, followed by treatment with or without 1 nM R1881 for 3 hr. The COS-7 cells were then fixed with 2% paraformaldehyde, and the nuclei stained with 2.5 μM DRAQ5 . The PC-3 cells were then fixed with 70% ethanol, and the nuclei stained with DAPI. Confocal images were obtained by using a Leica TCS SP2 system with a 63X oil-immersion objective on a Z-stage, and an average of 6 fields with ~10 cells per field was captured for each group. Data quantitation was performed as described [AR subcellular localization is detected by confocal fluorescence microscopy. The pTurboFP-AR-V7 and pTurboFP-ARqRT-PCR was performed as described . The qPCet al. [et al. [ChIP and Re-ChIP were performed as described . The folet al.  and the  [et al.  were use6) infected with lentivirus encoding AR-FL or 22Rv1 cells infected with lentivirus encoding control shRNA or AR-V7 shRNA were inoculated into castrated or intact nude mice (Charles River), respectively. The cells were mixed with 50% Matrigel and inoculated subcutaneously on the right dorsal flank. Tumor volume was calculated as 2 × length0.524 × width [3, the mice were randomized to daily treatment with vehicle or 10 mg/kg/day enzalutamide through oral gavage as described [Xenograft studies were conducted essentially as described ,32. LNCa× length . When th3 pieces of the tumors were transplanted into castrated nude mice. When the tumor bits grew to 100~200 mm3, the mice started to receive 10 mg/kg/day enzalutamide through oral gavage. The tumors were harvested when they reached ~800 mm3 and serially passaged in castrated nude mice following the same protocol. The second to fourth passages of tumors were considered as enzalutamide-resistant. All animal procedures were approved by the Tulane University Institutional Animal Care and Use Committee.For the development of enzalutamide-resistant tumors, two LNCaP tumors that relapsed after enzalutamide treatment were resected, and ~20 mmStudent's two-tailed t test was used to determine the mean differences between two groups. P < 0.05 is considered significant. Data are presented as mean ± SEM.The


AR) gene were associated with differences in the clinical or humoral immune manifestations of lupus in a cohort of female subjects. We found that shorter AR CAG repeat lengths in lupus subjects correlated with a higher Systemic Lupus Erythematosus Disease Activity Index score, higher ANA levels, and expression of a broader array of IgG autoantibodies. Our findings of more severe clinical manifestations and more exuberant humoral autoimmunity in women with a shorter AR exon 1 CAG repeat length suggest a role for genetically determined sensitivity to androgens as a modulator of autoimmune processes.Clinical and experimental evidence support a role for gonadal steroids in modulating the expression and course of autoimmune diseases such as lupus. Whether or not inherited variation in sensitivity to circulating androgenic hormones could influence the manifestations of such disease is, however, unknown. We sought to determine whether differences in androgen sensitivity conferred by variation in the exon 1 CAG repeat region of the androgen receptor ( Androgenic gonadal steroid hormones exert effects on both human autoimmune disease and the corresponding animal models. In an experimental mouse model of human systemic lupus erythematosus, females acquire fatal renal disease while males are generally spared; treatment of females with androgens prevents the progression of nephritis while castration of males results in disease progression and mortality AR) gene affects androgen action. Deleterious mutations in AR can result in syndromes ranging from mild abnormalities to total failure of normal male phenotypic development AR gene also includes a CAG repeat region of variable length  that encodes a polyglutamate tract in the N-terminal region of the protein n sequence in the protein) is inversely related to the capacity of the receptor to activate a target gene in vitroAR signaling capacity in vitro diminishes by an average of 1.7% for each additional CAG repeat within the range from 16 to 35 AR CAG repeat lengths has been found to be associated with phenotypic features of men with Klinefelter's syndrome AR CAG repeats were associated with more exuberant expression of IgG autoantibodies in males with lupus AR CAG repeat length and disease activity in women with rheumatoid arthritis Inherited variation in the androgen receptor ), and assessments of the overall breadth of humoral autoimmune reactions by autoantigen microarrays.We reasoned that, as women have much lower circulating levels of endogenous androgens, inherited variation in sensitivity to these hormones might play an even more significant role in modulation of immune reactivity when compared with men. We genotyped a cohort of women with lupus for variation at the Study samples and de-identified clinical data are obtained from normal female volunteers and from female patients seen in clinics at the Penn State Milton S Hershey Medical Center. The study was approved by the Institutional Review Board of the Penn State College of Medicine and the M S Hershey Medical Center, and all subjects gave informed consent. De-identified subject samples included serum for autoantibody analyses and DNA for genotyping. Clinical data on individuals with lupus included age, number and type of lupus diagnostic criteria Genomic DNA was isolated from peripheral whole blood or from buffy coat cells using the QIAamp DNA Mini Kit (Qiagen) following the manufacturer's protocol. DNA was quantitated using a Nanodrop 2000c spectrophotometer.AR exon 1 CAG repeat length was measured for both alleles in each subject using a PCR-based technique as described previously xl Genetic Analyzer in the Penn State College of Medicine Molecular Genetics Core Facility. Determination of the sizes of the PCR products was performed using the GeneMapper Software (Applied Biosystems).The AR allele using the CpG methylation-sensitive restriction endonucleases HpaII and HhaI Methylation analysis was carried out to detect the degree of inactivation of each The percentage of each allele that was unmethylated (active) was determined by calculating the ratio of the respective peak areas in the enzyme-digested samples. As individual alleles usually do not amplify with equal efficiency, we used a correction based on the amplification of each allele in the undigested samples AR CAG repeat length was calculated A weighted mean (a) peak area of allele 1 in digested samples(b) peak area of allele 2 in digested samples(c) peak area of allele 1 in undigested samples(d) peak area of allele 2 in undigested samples(e) number of CAG repeats in allele 1(f) number of CAG repeats in allele 2These variables were used in the following equations:Fractional inactivation of allele 1:Fractional inactivation of allele 2:AR CAG repeat length:Weighted mean AR CAG repeat length was the average of these two determinations.Samples were analyzed in this fashion with both HpaII and HhaI (Promega). The results were uniformly concordant and the final weighted mean Levels of ANAs were measured in serum samples using the ELISA technique . A positive result in this assay is defined as >20 ELISA units. ANA results using this technique correlate with those obtained by immunofluorescence assays s.d. of the values observed. For the lupus subjects, values above this level were scored as positive tests for each respective autoantibody.Individual serum samples (5 μl) were analyzed for IgG and IgM autoantibodies directed against the components of an 84-component autoantigen array described previously AR CAG repeat lengths for alleles and for weighted mean AR CAG repeat values based on methylation analysis were determined in the study populations by a t-test. The Gaussian curve fitting for the frequency distribution of AR CAG repeat lengths was done using the GraphPad Prism 6 Software . The same software was used to determine linear correlations between individual AR CAG repeat lengths and measures of disease activity. Hierarchical cluster analysis of normalized microarray data was carried out using Cluster 3.0 and the results were prepared for visual presentation as heat maps using Tree View 1.6. Fisher's exact test was used to analyze autoantigen microarray data as discrete variables  in individuals with short (≤19) AR CAG repeats compared with those with long (>19) AR CAG repeats.Differences in mean AR CAG repeat lengths differed between healthy individuals and subjects with lupus, we genotyped 25 healthy female volunteers and 39 women with a diagnosis of lupus who are subjects in a registry at the Milton S Hershey Medical Center. No difference was observed in the average length of the AR exon 1 CAG repeat in all alleles between the healthy control group and the lupus subjects  do not play any primary role in the predisposition to the development of autoimmunity in individuals with lupus.X chromosomal inactivation could conceivably alter effective AR alleles of greater or lesser length might be more prevalent in the lupus subjects. Skewed X inactivation can be defined by a variety of criteria – usually ranging from >70 to >90% inactivation (methylation) of one allele. We noted such skewed methylation in 7.8–32.8% of individuals  have been reported to occur more frequently in individuals with autoimmune disease ividuals  based onAR CAG repeat length might be related to differences in clinical features of lupus or in the manifestations of humoral autoimmunity that characterize the disorder. Of the 23 subjects with lupus, 13 had been treated with hydroxychloroquine, nine had received treatment with a pharmacological dose of glucocorticoids, and two had received mycophenolate mofetil. There was no difference in the weighted mean AR CAG repeat length among the groups who had received different therapies, and none of them differed from the mean for the total subject population.While there was little reason to expect that the genetic determinants of androgen insensitivity could influence overall susceptibility to autoimmune disease, we hypothesized that inherited variation in sensitivity to hormone action might affect aspects of the course, severity, or breadth of autoimmune reactivity. We examined in detail a subset of individuals  to test whether the AR CAG repeat length in the lupus subjects. The number of lupus diagnostic criteria observed for each patient at the time of entry into the registry failed to show a linear correlation with the AR CAG repeat length when analyzed as a continuous variable . The SLEDAI showed a significant inverse correlation with the weighted mean AR CAG repeat length  used to assess disease activity both showed evidence of an inverse correlation with the weighted mean AR CAG repeat length in our cohort of lupus subjects  against individual antigens and showed more numerous autoantibody specificities than did the lupus subjects with longer AR CAG repeat lengths. A similar  qualitative trend appeared for IgM autoantibodies (AR CAG length as a dichotomous variable (≤19 vs >19) revealed a statistically significant increase in the number of autoreactive IgG antibodies in individuals with a shorter AR CAG length  that are generally believed to result in diminished efficiency of hormonal signaling are associated with decreased severity of clinical and serological parameters of autoimmunity. Conversely, females with lupus who had shorter AR exon 1 CAG repeats  were found to have greater disease activity and more robust humoral autoimmune responses. Our current data are also similar to the observations in women with rheumatoid arthritis – in whom a shorter AR CAG repeat length was noted to be associated with earlier onset and more aggressive disease course AR CAG repeat length was inversely correlated with the SLICC/ACR index of disease damage AR CAG repeat variation in women do not appear to be compensated by alterations in circulating hormone levels. While longer AR CAG repeats are associated with higher levels of circulating androgens in men AR CAG repeats have previously been found to have lower testosterone levels  Since the time of Talal's seminal experiments in the NZB/W mouse, the preponderance of evidence has been that androgenic signaling in the immune system was generally suppressive AR allele CAG repeat lengths and their enrollment in the Hershey Medical Center lupus registry was not biased for any particular manifestation of disease, for severity of manifestations, or for specific therapy. The study is limited by its cross-sectional design. Our clinical data (diagnostic criteria and SLEDAI score) were based on disease manifestations present at the time of each subject's enrollment in the study and further longitudinal data on the relationship of AR genotype to the course of disease would certainly be of interest. Finally, our data are from DNA samples derived from whole blood samples or buffy coat cells. Obviously, while this would not affect the genotyping of the individual AR alleles, the presence of different patterns of X chromosomal inactivation in different cell types could affect the calculation of the weighted mean AR repeat length. It might be argued that specific subsets of B cells at different points during the lymphopoiesis pathway or even bone marrow stromal cells  Some limitations of the present study might be noted. First, a relatively small number of subjects have been evaluated, and the possibility of some unrecognized unique characteristics of our study population cannot be excluded. Our subjects exhibited normal distribution of their AR are, in fact, responsible for the observed effects, and several candidate genes on the X chromosome have been identified as susceptibility factors for the development of lupus. Interleukin 1 receptor-associated kinase 1  and toll-like receptor 7  are both such X chromosomal genes for which both human association studies and mechanistic experiments in mice support roles in lupus pathogenesis AR gene (at Xq12) would certainly seem to preclude such linkage disequilibrium. A number of other X chromosomal genes, including that encoding CD40 ligand, have been found to be overexpressed in T cells of women with lupus as a consequence of gene demethylation on an otherwise inactivated X chromosome CXCR3, OGT, and MIR421 (on X q13), are in closer proximity to the AR gene.Our current studies do not explain how attenuation of androgen action could result in amelioration of features of the autoimmune phenotype in women with lupus. It is possible that X chromosomal genes in linkage disequilibrium with AR CAG repeats, both estrogen and androgen levels are increased AR CAG repeat lengths have been reported to have lower circulating androgen levels A second possibility is that inherited differences in androgen sensitivity might alter the steroid hormonal milieu of an individual by altering feedback regulation of gonadotropins or other mechanisms. In men with long Nod1 mouse has now been shown to interact with the animal's hormonal milieu – with consequent impact on the expression of autoimmunity It is also possible that interactions between an individual and the environment might account for the relationship between inherited androgen sensitivity and the manifestations of autoimmunity. The microbiotic environment of the A variety of mechanisms have been proposed to account for the remarkable sexual dimorphism of most human autoimmune diseases


Supplemental Digital Content is available in the text The association between polymorphism of androgen receptor gene CAG (AR-CAG) and male infertility in several studies was controversial. Based on studies on association between AR-CAG repeat length and male infertility in recent years, an updated meta-analysis is needed. We aimed to evaluate the association between AR-CAG repeat length and male infertility in advantage of the data in all published reports.We searched for reports published before August 2015 using PubMed, CNKI, VIP, and WanFang. Data on sample size, mean, and standard deviation (SD) of AR-CAG repeat length were extracted independently by 3 investigators.P < 0.01; SMD = 0.36, 95% CI: 0.10–0.61, P < 0.01). AR-CAG repeat length was longer in infertile men in Asian, Caucasian, and mixed races . The overall study shows that increased AR-CAG repeat length was associated with male infertility. The subgroup study on races shows that increased AR-CAG repeat length was associated with male infertility in Asian, Caucasian, and mixed races. Increased AR-CAG repeat length was also associated with azoospermia.Forty-four reports were selected based on criteria. The overall infertile patients and azoospermic patients were found to have longer AR-CAG repeat length (standard mean difference (SMD) = 0.19, 95% confidence interval (CI): 0.10–0.28, This meta-analysis supports that increased androgen receptor CAG length is capable of causing male infertility susceptibility. About 50% infertile cases are attributed to male factor. Nevertheless, the etiology of half of male infertility cases remains obscure.2 The protein encoded by exon 1 of the AR gene is linked to AR transcriptional activity.3 The exon 1 of AR gene contains CAG and GGC trinucleotide polymorphic repeats which can respectively encode for polyglutamine and polyglycine. Previous studies have reported that the lengths of the 2 polymorphisms vary in different people.6 The usual variation of the AR-CAG trinucleotide repeat length is 11 to 36,7 and the median number is 22 in Caucasians.8 AR-CAG trinucleotide repeats were found possibly in association with male fertility in 1991 for the first time,9 and a great many studies followed to further that investigation. But the results are divergent.Androgen, mainly secreted by Leydig cells in male testis, is essential for male sex differentiation, development, spermatogenesis, and sexual behavior. It is mediated by the androgen receptor (AR), a member of the steroid hormone receptor superfamily. The gene of AR is located at chromosome Xq11-12, which has 8 exons and 7 introns.10 Other reports, however, did not provide corresponding results. Whether AR-CAG trinucleotide repeats is linked to male infertility has not been fully revealed. We hope that an updated meta-analysis based on the data obtained from recent studies and all published literature could be helpful to the deeper exploration into this field.Some studies demonstrated that longer length of AR-CAG trinucleotide repeats was associated with increased risk of male infertility, which is typical of impaired spermatogenesis with different severity. Also, it was suggested that AR-CAG tracts longer than 40 repeats give rise to Kennedy disease, a fatal neuromuscular disease accompanied by reduced sperm quality.Our study was composed of searching eligible reports, extracting data, analyzing data, and conducting sensitivity analysis and testing publication bias. We classified infertile men into different groups according their ethnic races and sperm concentration in hopes of exploring more potential factors which lead to male infertility. Ethical approval was waived because this study is a meta-analysis.To link AR-CAG trinucleotide repeat length and risk of male infertility, we searched the PubMed, China National Knowledge Infrastructure (CNKI), VIP Database for Chinese Technical Periodicals (VIP), WanFang Med Database (WanFang) to retrieve all articles available before August 2015 without language restrictions. The keywords used were a combination of “AR, CAG, male infertility.’. All reports were independently identified by 2 authors, and references cited in all original reports and review articles were examined through manual retrieval to identify other potentially eligible publications.11, includes a control group of proven or presumed fertile men, and includes sufficient allele frequency data for extraction. Studies were excluded because the design of the study is not rigorous, data of study is incomplete, repetition of the published literature, and a meta-analysis or a review. The procedure of articles screening is illustrated in a flow chart . All reports collected in Table 65The included studies should be the ones that evaluates the association between AR-CAG trinucleotide repeat length and risk of male infertility, includes a case group of male patients with male infertility, which some of them were divided into azoospermia group and oligospermia group by semen analysis according to World Health Organization guidelines24 azoospermic and oligospermic groups were specially classified and the data of each group were respectively extracted. Information of the range of AR-CAG repeat length, geographic location of the study, year of publication, ethnicity of participants was also noted. In several articles,28 we derived SD from standard error (SE): SD = SE × n, where n represents the size of case or control groups.All eligible reports were screened independently by 3 investigators  to extract available data according to the prespecified selection criteria. Disagreement was resolved by discussion with coauthors. The sample size, mean, and standard deviation (SD) of case and control group were extracted. In some studies,P <0.05 were considered to be significant. Adding to the study on the total infertile case group, we conducted studies on the group of azoospermia and oligospermia. When it comes to oligospermic group, we classified it into the mild oligospermic group (sperm concentration >5 × 106 and <20 × 106/mL) and severe oligospermic group (sperm concentration <5 × 106/mL). In some studies, the definition of mild oligospermia and severe oligospermia was different from other reports.20 Since the difference was not too wide to influence the study, so the data of those reports were also included in the study of mild or severe oligospermia. To explore the effect of race of study participants on results, we also conducted studies based on the race . The standardized mean difference (SMD) of the AR-CAG repeats length and its 95% confidence interval (95% CI) were used as statistic. Homogeneity of the included studies was tested. When P <0.1 or I2 >50%, there is a high extent of heterogeneity between studies and random-effect model was used; when P >0.1 and I2 <50%, no heterogeneity is found between studies, so fixed-effect model was used. The impact of quality of the included studies on the results was estimated by sensitivity analysis. Funnel plot studies were used to evaluate the publication bias.All statistical analyses were carried out using Stata , and 64 The characteristics of the selected studies are shown in Table 25 included studies on 2 different ethnic groups, so we consider it as 2 separate studies. Three studies24 did not provide data for the overall infertile patients. They only provided the data for subgroups such as azoospermic group or oligospermic group. So we did not include these 3 studies in our analysis for the overall study, but they were included in studies of subgroups. Data of azoospermic group were provided by 19 reports.45 Six reports62 provided data of oligospermic group without further stratification. Some reports24 classify oligospermic group according to sperm concentration. We got 3950 cases and 3684 controls for the overall study. At the same time, we got 1145 cases and 1447 controls for the study of azoospermic group, 442 cases and 664 controls for the study of severe oligospermic group, as well as 210 cases and 523 controls for the study of mild oligospermic group.We selected articles in accordance with the process demonstrated in Figure P <0.001)     .The results of this meta-analysis on the association between AR-CAG repeat length polymorphism and male infertility are illustrated in Table ) Figure . The res43 The study showed no association between AR-CAG repeat length and male infertility. In severe oligospermic group, the result showed that increased AR-CAG repeat length could be a risk for male infertility after wiping out 2 of the reports.19 These results may be caused by different inclusion standard and number of cases. No obvious publication bias was found based on the shape of funnel plot studies .Sensitivity analysis was conducted by wiping out 1 report each time to find whether the result of the study would be changed. Results remained the same in analysis on overall infertile men, Asian race, mixed race, unclarified race, azoospermic group, and mild oligospermic group. In the analysis on Caucasian race, the result was changed by wiping out 1 of the reports.66Spermatogenesis disorder has a complex pathogenesis. Androgen functions through combining with AR to stimulate or inhibit the expression of relevant gene. The number of glutamine encoded by (CAG)n is essential to the structure and function of AR molecule and its cofactors. The normal range of CAG repeat length is considered between 16 and 29.24 included in the meta-analysis is different from others. Liu et al24 using small sample size found that the AR-CAG repeat length was 30.8 ± 1.1 in group of azoospermia, which was significantly higher than that of fertile control. The study with a relatively small sample size might lack of adequate power to draw a fair conclusion. Additionally, the gene polymorphism might differ among different geography areas and race. Therefore, future studies with larger sample size in this area are needed to verify the association between AR-CAG repeat length and male infertility. Though the result of the study was different from others, it also met the strict criteria of study and thus were included in our meta-analysis.Reports on association between male infertility and gene polymorphism of androgen receptor mostly focused on Caucasian race and Asian race. Our study suggests that Asian men are more easily affected by the abnormality of AR-CAG polymorphism. Our study shows that the overall infertile population is typical of an increased AR-CAG repeat length. When it comes to racial factors, Asian, Caucasian, and mixed races all show the same result. In recent years, many studies classified infertile patients according to sperm concentration, such as azoospermia and oligospermia. We conducted analysis on those groups to explore the possible relationship between the severity of defect spermatogenesis and AR-CAG repeat length. Azoospermia was found to be associated with increased AR-CAG repeat length, but this result was not found in either severe oligospermia or mild oligospermia. That indicates oligospermia could be a result of many more complex factors and increased AR-CAG repeat length could result in severe spermatogenesis disorder. The study of Liu et al67 When the joint of CAG and GGC was taken into consideration, 2 haplotypes  could make the risk of male infertility increase.18 Whether (GGN)n polymorphism could influence the function of androgen receptor remains to be fully studied.(GGN)n polymorphism has also been analyzed but its function is still unknown. Nevertheless, many more studies revealed that there was no association between AR-GGN repeat length and male infertility.Meta-analysis is a quantitative systematic review and its result could be influenced by publication bias, database bias, inclusion criteria bias, and language bias. We followed the strict criteria to eliminate the ineligible reports to ensure the quality of included studies. Funnel plot studies were conducted. We searched both PubMed database and Chinese database such as CHKI, VIP, WanFang to get a more comprehensive set of data.68 Nevertheless, no significant association was found between erectile dysfunction and CAG repeat length.69 Increased CAG repeat was also found to be associated with depression and men with CAG repeat length ≥23 more frequently encountered decreased potency.70 Recent studies show that hypogonadal patients with shorter CAG repeat length had a more significant improvement after testosterone replacement therapy.72 Those reports indicated that failed sexual intercourse resulting from impaired sexual function could be a reason why increased CAG repeat length could lead to male infertility. Additionally, our meta-analysis suggests that AR-CAG repeat polymorphism has a relationship with male infertility, but the exact molecular mechanisms of how the CAG repeat polymorphism affects male infertility are unknown. We suppose that the secondary structure of AR mRNA sequence or the transcription factors binding site may change by the longer CAG repeat length. Our analysis suggests that further exploration of the molecular mechanism of AR-CAG repeat polymorphism and risk male infertility is demanded.AR-CAG repeat polymorphism has been studied for its influence on decreased sexual function, which could lead to infertility. Increased CAG repeat length was found to compromise sexual function.The present study had some limitations that require consideration. First, some studies with small sample size may not have enough statistical power to explore the real association. Second, our results were based on unadjusted estimates and a more precise analysis should be conducted if individual data were available, which would allow for adjustment by other covariants such as age, body mass index, smoking status, drinking status, environment factors, and lifestyle. Third, in the subgroup analyses, the number of mild oligospermia was relatively small, not having enough statistical power to investigate the association of the polymorphism with male infertility susceptibility.In conclusion, we again confirmed the association between increased AR-CAG repeat length and defected spermatogenesis. However, its association with the severity of the disease is to be fully studied. The studies on other possible factors such as race and lifestyle are also needed. AR-CAG polymorphism could be an effective way of evaluating the risk of infertility. The studies also paved the way for the gene therapy for the male infertility. Further studies are needed to clarify the association between length of CAG repeats and male infertility as well as its mechanism.


The androgen receptor (AR) is a widely expressed ligand-activated transcription factor which mediates androgen signalling by binding to androgen response elements (AREs) in normal tissue and prostate cancer (PCa). Within tumours, the amount of AR plays a crucial role in determining cell growth, resistance to therapy and progression to fatal castrate recurrent PCa in which prostate cells appear to become independent of androgenic steroids. Despite the pivotal role of the AR in male development and fertility and all stages of PCa development, the mechanisms governing AR expression remain poorly understood. In this work, we describe an active nonconsensus androgen response element (ARE) in the 5′ UTR of the human AR gene. The ARE represses transcription upon binding of activated AR, and this downregulation is relieved by disruption of the regulatory element through mutation. Also, multiple species comparison of the genomic region reveals that this ARE is specific to primates, leading to the conclusion that care must be exercised when elucidating the operation of the human AR in PCa based upon rodent promoter studies.The online version of this article (doi:10.1007/s12672-014-0185-y) contains supplementary material, which is available to authorized users. Unfortunately, the AR signalling axis can also actuate and stimulate carcinogenesis of the prostate, and prostate cancer (PCa) is now the second most common cancer in men in Western nations, with comparable figures rising in Asian countries [In male humans, androgens induce development of the prostate gland during the second and third trimester from the endodermally derived urogenital sinus through epithelial-mesenchymal interactions that lead to epithelial proliferation, invasion, and bud formation (reviewed in Prins and Putz ). Circulountries .Although multiple mechanisms contribute to the lethal progression from benign prostatic hyperplasia to metastatic cancer, AR-mediated cell signalling continues to govern cell growth and survival –5 with mThe androgen receptor exerts its influence by acting as an androgen-activated transcription factor which binds to androgen response elements (AREs)  where hicis-acting regulatory sequences involved in androgen-mediated downregulation of AR mRNA remain poorly understood with only one site in the second intron described in detail to date [Despite the fundamental importance of AR levels in all stages of PCa progression, the  to date . Convers to date ). In thi2.Human prostate carcinoma cell lines LNCaP and VCaP were obtained from the European Collection of Cell Cultures, and DU145 was from the American Type Culture Collection. VCaP and DU145 were grown in DMEM while LNCaP were maintained in RPMI containing 1 mM Na pyruvate and 10 mM HEPES. All media were supplemented with either 10 % foetal bovine serum or 10 % charcoal-stripped foetal bovine serum (both from PAA) and maintained at 37 °C without antibiotics in a humidified atmosphere containing 95 % air and 5 % COLNCaP or VCaP cells were grown in medium containing charcoal-stripped serum to approximately 70 % confluence and then cultured for a further 24 h in complete medium containing either 10 nM DHT or ethanol vehicle. Extraction of RNA and RT-PCR were carried out as described earlier . SemiquaGAGCTCTGGACAAAATT-3′ and 5′-TTCAAAAGATGCCCAGATCTTAAAA-3′, and Pfu Turbo ultrahigh fidelity DNA polymerase from Stratagene. The cloned hAR genomic DNA was digested with SacI and BglII (sites underlined in the PCR primers) and ligated into the SacI and BglII sites in the vector.The luciferase reporter plasmid phAR1.6Luc, in which luciferase expression is driven by the promoter and 5′ UTR of the human androgen receptor gene, was created using the promoterless firefly luciferase vector pGL4.17 (Promega). The region of the human AR gene spanning between −741 and +842 was amplified by PCR using human male placental genomic DNA template (Cambio), the oligonucleotides 5′-GTTTACACTGTCCTCTGTTTTCCCCCAC-3′ followed by AREm2 5′-CACGCGGAGACTGTCCTCGCAGTTCCCCCACTCTCTCTCC-3′. The integrity of all constructs was confirmed by DNA sequencing.Both half sites within a potential androgen response element (ARE) in the hAR 5′ UTR of phAR1.6Luc were mutated using the QuikChange II Mutagenesis kit (Agilent Technologies) according to the manufacturer’s protocol. Mutagenesis was performed in two sequential rounds to create phAR1.6Luc-AREm using the following oligonucleotides with their reverse complements (mutated bases shown in bold font): AREm1, 5′-GGTTAGGCTGCACGCGGAGA4 cells/cm2, while DU145 cells were seeded at a density of 1.2 × 104 cells/cm2. The cells were cultured in complete medium for 24 h, then transfected with 440 ng/well of either firefly luciferase reporter plasmid alone or cotransfected with pSVARo human androgen receptor expression plasmid (2:1 ratio) using jetPEI polyethylenimine transfection reagent (Polyplus Transfection) according to the manufacturer’s protocol. After 24 h, the medium was replaced and the cells were cultured for a further 48 h.Twenty-four-well plates were seeded with LNCaP and VCaP cells at a density of 5 × 10Plasmid transfection was performed in quadruplicate and luciferase activity was measured in duplicate by using a GloMax 96 Microplate luminometer (Promega) and normalised for protein concentration as previously described .3VO4) using the method of Dignam et al. [Nuclear extracts were prepared from LNCaP cells in the presence of protease inhibitors (complete protease inhibitor cocktail from Roche plus 1.0 mM PMSF) and protein phosphatase inhibitors  plus DNA-binding domain (DBD) or DBD alone  were expressed and purified as described previously; the GST tags were removed by digestion with thrombin  . The proEither 10 μg LNCaP cell nuclear extract or 200 nM recombinant hAR-DBD or hAR-NTD-DBD proteins were incubated with 20 fmol biotin 3′ end-labelled double-stranded DNA oligonucleotides using previously described conditions. The forward sequences of the oligonucleotides were as follows: ARE, 5′-ACGCGGAGAGAACCCTCTGTTTTCCCCCAC-3′; AREm, 5′-ACGCGGAGACTGTCCTCGCAGTTCCCCCAC-3′; and PSA-ARE-III, 5′-ACTCTGGAGGAACATATTGTATCGATTGTC-3′. Unlabelled versions of these oligonucleotides, along with a random oligonucleotide (RO), 5′-CGAGCACCCTTCACCCTCCAGGCTTAACGG-3′, containing no regulatory elements were used for competition assays in which they were added 15 min prior to the labelled probe. Similarly, AR441 antibody against human androgen receptor  was added 15 min prior to the addition of labelled probe for supershift assays.The resulting DNA:protein products were resolved in cooled 6 % nondenaturing polyacrylamide gels run in 0.5× TBE buffer, pH 8.3  and detected using Pierce LightShift Chemiluminescent reagents (Thermo Scientific) according to the manufacturer’s protocol. Figures were compiled using autorads of electrophoretic mobility shift assay (EMSA) gels with the order of lanes within some gels being altered to aid clarity and facilitate comparisons. Digital integration of the DNA:protein complexes was carried out using a Vilber Loumat Fusion SL cooled CCD sensor with care being taken to ensure that no pixel saturation occurred.A detailed account of the chromatin immunoprecipitation (ChIP) methodology is presented in t-test analysis of variance was employed for all other comparisons between complementary data.The statistical significance of differences in data sets of DNA:protein complex formation in EMSA experiments was determined using two-way ANOVA, and paired p < 0.01). Conversely, treatment with androgen reduced AR mRNA in VCaP to a much greater degree than in LNCaP with values of 80 and 48 %, respectively.Autoregulation of the AR gene by androgens is likely to play an important role during development and in conditions where circulating androgen levels have been reduced. To confirm earlier observations that androgens downregulate AR gene expression, LNCaP and VCaP cells were treated with 10 nM DHT followed by isolation of the RNA. Figure Bioinformatic analysis of publically available DNA sequences was used to identify possible AREs in the promoter and adjacent proximal and distal sequences of the human androgen receptor (hAR) gene. Only a previously described suggested nonconsensus ARE (AGAACCctcTGTTTT) at position 611 bp in the 5′ UTR of exon 1  was reveThe possibility that hAR binds to the nonconsensus ARE was examined by electrophoretic mobility shift assays (EMSAs). In initial experiments, purified hAR protein encoding the N-terminal domain (NTD) and DNA-binding domain (DBD), i.e. amino acids 1 to 645 was incubated with labelled oligonucleotide probe (ARE) containing the putative 5′ UTR ARE. Electrophoretic resolution of the resulting products showed a single high molecular weight DNA:protein complex near the top of the gel Fig. , lane 1.Incubation of nuclear extract prepared from the AR expressing prostate cancer cell line LNCaP with either ARE or PSA-RE-III oligonucleotides produced several bands with virtually identical electrophoretic motilities, but not with the mutated AREm . A similar finding was obtained using the hAR DBD . Together these results show that hAR binds to the nonconsensus 5′ UTR ARE; however, it does so with lower affinity than to ARE-III in the PSA enhancer.The EMSA in Fig. p < 0.001 in both instances). The lower reduction seen in VCaP compared to LNCaP may reflect the presence of multiple androgen insensitive and constitutively active splice variants in the former cell line [p > 0.85); however, cotransfection with the hAR expression plasmid pSVARo led to 48 % DHT-induced downregulation (p < 0.001). Treatment of DU145 cells expressing the hAR with the antiandrogens, bicalutamide (Bic) or enzalutamide (Enz) failed to repress luciferase activity  was cloned into the pGL4.17 promoterless luciferase reporter plasmid to create phAR1.6Luc Fig. . This reell line . In contp < 0.001). While LNCaP cells express AR but not glucocorticoid receptor (GR), the converse occurs in DU145 cells [p > 0.34), thus confirming that no new regulatory element had been created, and that GR does not interact with this ARE. However, cotransfection of DU145 cells with the pSVARo hAR expression plasmid resulted in the 5′ UTR ARE mutation raising transcriptional activity by 25 % . Together, these results show that AR binds to the 5′ UTR ARE to downregulate transcription and mutation of the site leads to a release of this repression.The next step was to mutate the putative ARE to create the reporter construct phAR1.6Luc-AREm Fig.  and dete45 cells , and in p < 0.01). Interestingly, from Fig. p < 0.01 in both instances), albeit to a much lesser degree.Lastly, the role of the 5′ UTR ARE in contributing to androgen auto-downregulation of the hAR promoter was confirmed by looking at the effect of the site’s mutation on DHT-induced repression in LNCaP and VCaP cells. The results presented in Fig. psa gene which contains three active AREs, and the results are shown in Supplemental Fig. Because the conditions of EMSA incubations cannot always reflect the chromatin environment, ChIP assays were undertaken to look at AR binding to the endogenous hAR 5′ UTR ARE site. In order to study the regulatory element in its native state and to compare the effects of its mutation, LNCaP cells were used directly or transiently transfected with either phAR1.6Luc or phAR1.6Luc-AREm containing the WT or mutated ARE, respectively, and treated with either vehicle or 10 nM DHT. Initial experiments to confirm the specificity of the PG21 anti-hAR antibody were performed using the well-characterised promoter and enhancer regions of the Chromatin and plasmid were digested with NheI and PvuII in order to isolate the region of the 5′ UTR under study in the ChIP experiments from a previously described potential AR binding site proximal to the 5′ UTR ARE , and solp < 0.05). PCR amplification of precipitated DNA with oligonucleotide primers encompassing the active ARE-III site in the human PSA upstream enhancer  in the presence of androgen. On the other hand, hAR failed to bind to the 5′ UTR ARE of the luciferase reporter plasmid in which both half sites of the ARE had been mutated (phAR1.6Luc-AREm), regardless of the presence of hormone .Anti-hAR antibody, but not preimmune Ig, immunoprecipitated the 5′ UTR ARE region of the AR gene in LNCaP cells that had been treated with either vehicle or DHT Fig. . This recer Fig.  confirmeEnduring expression of the androgen receptor in PCa contributes to tumour survival and proliferation as well as facilitating progression to fatal CRPC status. Therefore, it is vital to understand the molecular mechanics of human AR gene regulation; especially the negative feedback loop whereby ligand-activated AR downregulates transcription of its own gene, since androgen deprivation therapy (ADT) is the principal strategy of advanced PCa treatment regimes. In this study, we have identified an ARE in the 5′ UTR of the human AR gene and confirmed binding of AR to it by EMSA using both purified AR and LNCaP nuclear extract and ChIP assays. Importantly, luciferase measurements of transcriptional activity in PCa cell lines established that the 5′ UTR ARE downregulates expression in response to androgens, and disruption of this ARE alleviates repression in an AR-dependent manner. Furthermore, the clinically relevant antiandrogens, bicalutamide and enzalutamide were unable to mediate receptor-dependent repression. These observations were made independently of Vismara et al.  who suggThe adverse nature of the AR’s influence on PCa progression is manifested from the several lines of evidence. Androgen activation of AR limits proliferation of prostate epithelial cells in keeping with its role of promoting terminal differentiation , 42. HowIn order to maintain AR levels within narrow constraints in normal cells, the AR gene is under the control of multiple regulatory elements. In terms of hAR gene autoregulation, activated AR can stimulate expression through an exonic enhancer about 170 kbp distal from the promoter where specificity for AR is dependent upon the structure of the receptor’s NTD . AndrogeInterestingly, while mutation of the 5′ UTR ARE in the reporter plasmid, which did not contain the intronic repressor ARE, significantly lessened AR-dependant downregulation in the PCa cell lines examined, this repression was not completely abolished Fig. . Our datOne of the routes by which AR regulates target gene expression is through epigenetic remodelling of chromatin. An example is the recruitment of lysine-specific demethylase 1 (LSD1) by activated AR to its associated ARE where it can behave as a corepressor or coactivator . Indeed,Another significant finding was that the 5′ UTR ARE is confined to primates with the equivalent region in other species, especially the rodents, rat and mouse, displaying particularly low homology. This strongly suggests that caution should be exercised when using rodent models to investigate regulation of the AR gene.In conclusion, pertinacious AR signalling is implicated in the progression to CRPC with the levels of the receptor mediating the life and death of tumours. The repressor ARE we have characterised in the 5′ UTR lends valuable insight into the control of AR expression and will provide targets for novel therapeutic agents against CRPC. The finding that AR expression is downregulated by androgens through multiple sites raises the question of whether ADT can on occasion be counterproductive as a consequence of transcriptional repression being alleviated. In addition, DHT can have an indirect protective role as it has recently been found to inhibit the induction of autoimmune and inflammatory responses in human prostatic stromal cells . Thus, tSupplemental Fig. 1(GIF 125 kb)High resolution image (TIFF 122 kb)Supplemental Fig. 2(GIF 112 kb)High resolution image (TIFF 105 kb)Supplemental Fig. 3(GIF 75 kb)High resolution image (TIFF 29 kb)Supplemental Fig. 4(GIF 72 kb)High resolution image (TIFF 20 kb)Supplementary Methods and Figure legends (DOCX 37 kb)Below is the link to the electronic supplementary material.


Kin selection, which can lead organisms to behave altruistically to their genetic relatives, works differently when—as is often the case in human societies—altruism can be boosted by social pressure. Here I present a model of social norms enforced by indirect reciprocity. In the model there are many alternative stable allocations of rewards ; a stable norm is stable in the sense that each player is best off following the norm if other players do the same. Stable norms vary widely in how equally they reward players with unequal abilities. In a population of mixed groups  with modest within-group coefficients of relatedness, selection within groups favors those who compromise, and selection between groups favors generous generalized reciprocity rather than balanced reciprocity. Thus evolved social norms can amplify kin altruism, giving rise to a uniquely human mode of kin-based sociality distinct from spontaneous altruism among close kin, or cooperation among non-kin. Natural selection favors this altruistic act as long as Hamilton’s Rule is satisfied, r ⋅ b > c, where r is the coefficient of relatedness of recipient to donor  is uniform. Kinship predicates the axiom of amity, the prescriptive altruism exhibited in the ethic of generosity. … [T]he rule posits … that “kinsfolk” have irresistible claims on one another’s support and consideration … simply by reason of the fact that they are kin. Kinsfolk must ideally share … and they must, ideally, do so without putting a price on what they give. individual nepotism) is motivated by benefactors’ feelings of attachment to their beneficiaries. It is largely directed to close kin . The second variety  is governed by conventions about how kin ought to be treated, and is motivated by social pressure and concern for one’s standing in a moral community. It can extend even to distant kin. “[T]he most convincing evidence … of kinship amity [is found in] situations where kinship is so tenuous as to be only nominal, as when persons seek out remote clansfolk … and without further ado claim and receive hospitality and protection.”  over x ≥ 0, y ≥ 0. This function dictates how much benefit a player with ability x ought to get from a player with ability y. If i and j have abilities αi and αj, and j follows the norm, then bij = bA. Suppose however that j cannot assess i’s ability directly, but must act according to an ascribed ability, or reputation, for i. To achieve this, the function bA must made to operate in two directions, determining both the benefits j produces for other players and the reputation others assign her. First, if j knows her own ability, αj, and ascribes ability si to player i, she can reward i according to bij = bA. Second, if some player k knows what benefits j has produced for other players, and has ascribed abilities for them, she can work backward to assign a new reputation, sj, to j, where sj is implicitly given by bA (as spelled out below). This means that reputation is defined recursively: the reputation of each player depends on the benefits she bestows on others and on their reputations. The result, at equilibrium, is that reputation equals ability (sj = αj) and players receive benefits according to bij = bA and pay costs according to i and j.We are interested in indirect reciprocity, where player  of play –25. Who bA is allowed, including a norm of pure altruism, “from each according to his ability, to each according to his need”  Penalty for impersonation. For each active player j, her summed net benefits must be greater if she honestly advertises her ability, αj, than if she “impersonates” a player of lesser or greater ability, y:y ≠ αj. So summed net benefits must increase, neither too slowly nor too quickly, with donor ability.(ii) Penalty for inconsistency. Player j might be less generous to some players and more generous to others than is consistent with honestly advertising her ability aj or impersonating a player with ability y: there might not be any one y such that bij = bA for all i. In this case, j is assigned an intermediate weighted average reputation, sj, chosen so that bij < bA for some i and bij > bA for other i. The aim is not to make sj an accurate estimate of αj, but to penalize j for behaving inconsistently. For example, letsj as an implicit function of the benefits j produces, bij, and the reputations of her beneficiaries, si. Suppose ω = 1. Then the ω term drops out, and j’s actual cost, sj. So inconsistent play gets her the same reputation, for the same cost, as consistent play. But suppose instead that 0 < ω < 1. Then j pays the same cost as before, but her reputation is now lower, because subpar bij’s are more heavily weighted, and reduce sj more than excessive bij’s increase it. So we make 0 < ω < 1: either j is consistent, and gets the appropriate sj for honest advertising or consistent impersonation, or j is inconsistent, and pays a reputational penalty.(iii) not stable. But within these limits, there is a wide range of stable norms, including the two below, which differ in how well they treat weak players, and are meant to capture the anthropologist’s distinction between balanced and generalized reciprocity.With a distributional norm and a reputation function that satisfy these conditions, consistent honest advertising is the best strategy for each player, as long as others follow the norm. So the honest advertising constraints set limits on what distributional norms are stable among self-interested players—for example, the norm matching the payoffs under pure altruism is bA = bA for all x and y. Players get only as much they give, and “there ain’t no such thing as a free lunch.” We can pick one member of this family by allowing the weaker player to set bA = bA to her preferred value, x ≤ y. This norm is stable under direct reciprocity, and also under indirect reciprocity where players estimate reputations according to One family of distributional norms assigns equal payoffs in both directions, dynamically stable. If players start somewhere other than at equilibrium, and estimate one another’s abilities based on past play, then reputations and benefits produced don’t converge on the equilibrium. Even small perturbations are destabilizing. The underlying problem is familiar from the Iterated Prisoner’s Dilemma game: two tit-for-tat players don’t get to the cooperative equilibrium if they aren’t already there. In the prisoner’s dilemma case, this can be fixed with a forgiving amendment to the tit-for-tat strategy  slightly higher than bA for x < y, with the difference adjusted to satisfy bB  match the extra benefits. Discarding the principle that exchanges must balance allows for a more even distribution of benefits and greater total net benefits. Consider the norm that maximizes group net benefits, subject to the honest advertising constraints. With this norm, a strong player gives substantial help to weak players, even entirely helpless players with j some knowledge, not just of the benefits i produces, but of the costs i pays. In this case, a weak i helping others at some cost to herself can receive more credit than a stronger player providing the same benefits at less cost [bG  or Generalized Reciprocity (G), is stable if everyone agrees to it. But what happens if players don’t agree? Evolutionary considerations can help answer this question.j can be assigned two reputations, sB reputations and type G players reward according to sG (r + pB − r ⋅ pB), and a G player finds herself in a group where type B numbers (n−1)(pB − r ⋅ pB)  is expected to have higher fitness in most groups. Selection within groups, favoring the common type, is stronger than selection between groups, favoring G. Whichever distributional norm starts out at high frequency goes to fixation.Now consider a large population of groups, each of size Yet the survival of the commonest is not the end of the evolutionary story. The equilibrium where some group members play B and others G is not a Nash equilibrium. Individuals of either type can do better if they don’t stick to their preferred strategy, but adapt to the presence of the other type. Such accommodation within a mixed group can take various forms.w0, with 0 < w0 < 1, such that the summed net benefit received by a player with ability y is a weighted mean of the summed net benefits received by a y player under norms B and G, with weights w0 and 1−w0. For a given w0, a benefit function bW can be defined such that the corresponding norm W is stable, giving each player an incentive to honestly advertise her abilities as long as others follow the norm. Here we allow each group to settle on the w0 for which bWτ1/ gives a least-squares best fit to First, all players can compromise on an intermediate strategy. A compromise distributional norm  can be defined by a parameter w0 than weak players. In groups where more of the strong players are type B, the B’s “vote” greater net benefits for the strongest players, themselves; where more of the strong players are type G, the G’s “vote” greater net benefits for the average player. But G can still prevail: with positive within-group relatedness, type G players are more likely to find themselves in groups with extra type G players The work here extends earlier findings that indirect reciprocity depends on reputation and reputation must be defined recursively, so that each player’s reputation depends both on the benefits she provides and on the reputations of the beneficiaries. In the model here, reputation is an index of ability, and, like ability, can vary continuously. So the model faces the problem of assigning a numerical reputation to each player based on the reputations of other players. This is more complicated than simply choosing to cooperate or not based on whether the other player is in good or bad standing. It is close to the problem of designing a page-ranking algorithm that ranks a collection of web pages by assigning a score to each page based on the scores of the pages that link to it . Some ofincentive compatible): a player of whatever ability should do better sticking to the norm, rather than impersonating a stronger or weaker player or playing inconsistently, as long as other players do the same. All these conditions rule out many distributional norms. For example, strict balanced reciprocity is dynamically unstable, and pure altruism is unenforceable. But the conditions also allow a wide range of distributional norms with very different distributions of benefits, including the two we concentrate on here, Almost Balanced Reciprocity and Generalized Reciprocity.The model must satisfy a further set of conditions. We require that a distributional norm be enforceable Click here for additional data file.S1 Figαj. As in (PDF)Click here for additional data file.S2 Figw0, this shows how a player with ability αj should weight her costs between bB and bG to achieve a compromise distributional norm satisfying Condition 13 for stability Click here for additional data file.


Background. The beta-2 adrenergic receptor is expressed by neoplastic cells and is correlated with a wide spectrum of tumor cell mechanisms including proliferation, apoptosis, angiogenesis, migration, and metastasis. Objectives. The present study aimed to analyze the expression of the beta-2 adrenergic receptor (β2-AR) in tumor-free surgical margins of oral squamous cell carcinomas (OSCC) and at the invasive front. Sixty-two patients diagnosed with OSCC, confirmed by biopsy, were selected for the study. The clinicopathological data and clinical follow-up were obtained from medical records and their association with β2-AR expression was verified by the chi-square test or Fischer's exact test. To verify the correlation of β2-AR expression in tumor-free surgical margins and at the invasive front of OSCCs, Pearson's correlation coefficient test was applied. Results. The expression of β2-AR presented a statistically significant correlation between the tumor-free surgical margins and the invasive front of OSCC . The immunohistochemical distribution of β2-AR at the invasive front of OSCC was also statistically significant associated with alcohol (p = 0.038), simultaneous alcohol and tobacco consumption (p = 0.010), and T stage (p = 0.014). Conclusions. The correlation of β2-AR expression in OSCC and tumor-free surgical margins suggests a role of this receptor in tumor progression and its expression in normal oral epithelium seems to be constitutive. These c ligands , 4. Onceunctions . A growitastases , 2, 6–25β2-AR signaling upregulates interleukin-6 (IL-6) mRNA, a cytokine that is involved in angiogenesis and tumor progression process, increasing proliferation and invasion of the tumor [β2-AR immunoexpression in oral cancer is significantly correlated with age, tumor size, clinical stage, and cervical lymph node metastasis in OSCC patients, suggesting a role of β2-AR in the metastasis of oral cancer [β2-AR expression by malignant epithelial cells, presented higher survival rates compared with weak/negative β2-AR expression [Particularly, investigations in oral cancer cell lines have demonstrated that he tumor , 18. Furl cancer . In contpression .β2-AR is present in normal oral tissues, the exact differences between β2-AR expression levels in normal oral epithelium and oral cancer are not so clear [β2-AR was highly expressed in OSCC tissue when compared to adjacent normal oral mucosa [β2-AR mRNA levels in normal oral mucosa and OSCC specimens [Although so clear , 18, 21;l mucosa . In turnβ2-AR in OSCC, the present study aimed to analyze the expression of β2-AR in the tumor-free surgical margin and at the invasive front of a large sample of OSCC to verify if β2-AR expression is correlated with tumor-free surgical margins and the invasive front of OSCCs and to explore associations between β2-AR expression levels and clinicopathological features.In order to contribute with recent studies and investigate the role of The present retrospective study was based on the analysis of 62 patients previously studied by Bravo-Calderón et al. . All pat μm thick section of tumor tissue was selected and paraffin-embedded for hematoxylin and eosin staining and immunohistochemistry analysis of β2-AR. Three observers  analyzed the tumor sections without knowledge of the clinical data. Histopathological malignancy grade of OSCC was determined according to the Bryne et al. system [A formalin-fixed 3. system . Tumor i mM citrate buffer (pH 6.0) in a domestic pressure cooker  for 4 min, and then the endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 30 min. Tumor sections were incubated for 18 hours at 4°C in a humid chamber with the anti-beta-2 adrenergic receptor primary antibody  and diluted 1 : 50 in phosphate buffered saline (PBS) with bovine serum albumin solution  to block nonspecific reactions. Next, the tumor sections were sequentially incubated with Post Primary Block  for 30 min, followed by incubation with the Polymer from the same kit. The antigen-antibody reactions were revealed using 3.3′ diaminobenzidine tetrahydrochloride  for 5 min in the dark. Tumor sections were counterstained with Harris hematoxylin before being dehydrated and prepared with a cover slip. The vascular smooth muscle within the sections served as the positive internal control. For a negative control, the primary antibody was omitted during the immunohistochemical staining. All the OSCC specimens were immunostained in a single session.Sections of the 62 OSCC specimens were deparaffinized in xylene and hydrated using graded alcohol/water baths. Antigen retrieval was performed using 10β2-AR. Images were obtained digitally with a camera  attached to a light microscope  and saved in a computer program system . The immunohistochemical expression of β2-AR was evaluated as previously described [β2-AR expression were categorized according to the following RGB channel value ranges: R (red) channel was from 90 to 194; G (green) channel was from 50 to 140; B (blue) channel was from 45 to 147; the R value should be greater than the B value; the G value should be greater than the B value.Ten microscopic fields of the tumor-free margins were captured using 400x magnification to analyze the immunohistochemical expression of escribed . Brieflyβ2-AR immunopositive regions). After performing this computer-assisted immunohistochemistry analysis, the average of the β2-AR expression levels in ten surgical margin images was calculated. Next, the averages of 62 OSCC specimens, previously obtained by Bravo-Calderón et al. [β2-AR expression. This measurement of β2-AR expression by epithelial cells of surgical margins was then subjectively confirmed by three investigators  without knowledge of the histopathological features and patient clinical status. In case of discordance between these analyzers, the criterion of the subjective evaluation was retained because this assessment involved the entire specimen.Ten images captured from the surgical margins of each OSCC were automatically segmented using the MATLAB computing language-based software according to the criteria listed above. This software measures, based on the number of pixels, each segmented area . Correlation between β2-AR in parakeratinized stratified squamous epithelium of all tumor-free surgical margins, and 50 (80.6%) demonstrated strong expression of β2-AR levels. β2-AR immunostaining was detected at the cytoplasm and at the plasma membrane of normal epithelial cells in tumor-free surgical margins. Similarly, the same pattern of β2-AR expression (cytoplasmic and membranous) was identified in malignant epithelial cells at the invasive front of tumors.Immunohistochemical analysis of the 62 OSCC specimens revealed a positive expression of β2-AR expression levels in tumor-free surgical margins and at the invasive front of tumors   . Effectilso high .β2-AR levels in the tumor-free margins and at the invasive front of tumors, as shown in β2-AR expression levels when compared to their respective tumor-free surgical margins  OSCC samples presented no correlation between  margins . On the β2-AR expression, in this sample, was verified in most OSCC specimens, being positively immunoexpressed in 87.5% of the tumor-free surgical margins and in 55.5% of the front of invasion. In the basal and corneum layers of the surgical margins or in the keratin pearls of oral cancers, no immunoexpression of β2-AR was detected. Furthermore, the cytoplasmic and membranous expressions of β2-AR were confirmed in oral epithelial cells adjacent to the tumor, as well as in the malignant epithelial cells of OSCC.An additional analysis was performed to confirm our findings in formalin-fixed, paraffin-embedded tissue material by the evaluation of 19 new OSCC frozen specimens. β2-AR expression in oral cancer and the associations between this protein expression and the clinicopathological features of OSCC patients, chi-square or Fischer's exact tests were performed.To determine the possible clinical significance of β2-AR in the tumor-free surgical margins and clinical variables evaluated , simultaneous consumption of alcohol, and tobacco (p = 0.010) and with the clinical T stage (p = 0.014), as shown in β2-AR at the invasive front of tumor exhibited alcohol consumption or alcohol and tobacco consumption. In addition, strong β2-AR expression by malignant epithelial cells was more frequently detected in patients with early clinical stage of T1/T2.No statistically significant associations were found regarding immunohistochemical expression of valuated . On the β2-AR expression and the histopathological characteristics was shown, including grade of malignancy, lymph nodes involvement (pN+), vascular embolization and perineural invasion, and muscular or bone infiltration   . Despiteand OSCC . Contradand OSCC  reportedg et al. , and acceriments . Moreoveβ2-AR can influence the progression of a wide variety of malignant tumors [β2-AR in the progression of oral cancer is not well established. Thus, the scientific literature has demonstrated that β2-AR immunoexpression by malignant cells is significantly correlated with age, tumor size, clinical stage, and cervical lymph node metastasis in OSCC patients [β2-AR expression at the invasive tumor front (β2-AR was presented in all tumor-free surgical margins, it was expected that no statistically significant associations would be found regarding immunohistochemical expression in the normal oral epithelial cells and clinicopathological variables evaluated (Tables Biological evidence has demonstrated that chronic psychological stress through catecholamine-induced activation of t tumors , 2, 6–25patients . Neverthor front . In addid Tables  and 2.β2-AR expression by tumoral cells may decrease (β2-AR expression and low survival rates, compared with strong β2-AR expression [β2-AR increases cell migration and invasion in normal prostate cells and that weak expression of this protein was associated with metastases and worst survival rates in prostate cancer patients. Considering the present results, other studies with β2-AR expression should be performed to further elucidate the role of this receptor. In vitro analyses are suggested with the stimulatory and inhibitory factors for β2-AR.On the other hand, the evidence that, in certain OSCCs, decrease , as prevpression , reinforpression . Yu et aβ2-AR is constitutive in normal oral epithelial cells and is positively correlated with the expression levels of β2-AR by OSCC cells, further clinical, cellular, and animal studies are needed to elucidate the role of β2-AR in oral cancer, specially, in relation to the importance of its decrease on tumor progression.Thereby, in light of these results we can conclude that although the present study reinforces that