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To investigate Armadillo repeat-containing 5 (ARMC5) mutations in Chinese patients with familial and sporadic primary bilateral macronodular adrenal hyperplasia (PBMAH), we performed clinical data collection and ARMC5 sequencing for three PBMAH families and 23 sporadic PBMAH patients. ARMC5 pathogenic germline mutations were identified in all 3 PBMAH families. Secondary ARMC5 somatic mutations were found in two adrenal nodules from two PBMAH family members with ARMC5 germline mutations. PBMAH family members with ARMC5 pathogenic germline mutations displayed various clinical manifestations. ARMC5 pathogenic germline mutations were identified in 5 sporadic PBMAH patients among whom one patient displayed both hypercortisolism and primary aldosteronism. We detected a total of 10 ARMC5 pathogenic mutations, of which 8 had not been previously reported. Our results suggest that ARMC5 pathogenic germline mutations are common in familial and sporadic Chinese PBMAH patients, and demonstrate the importance of ARMC5 screening in PBMAH family members to detect patients with insidious PBMAH. [ABSTRACT FROM AUTHOR]

Transforming growth factors beta (TGF-β) are multi-functional cytokines capable of inducing apoptosis in epithelial cells, including glomerular podocytes. We and others have previously shown that podocyte-selective genetic deletion of the microRNA (miR)-processing enzyme, Dicer, caused glomerulosclerosis that was associated with podocyte apoptosis, and the miR-30 family was implicated in the process. Here, we report that apoptosis-associated genes were highly enriched among the predicted targets of miR-30 when compared with randomly selected miRs (26% vs. 4.5 ± 2.1%) or with the known TGF-β-regulated miR-192 (6%), miR-216a (5.1%), and miR-217 (0%). miR-30 family members were abundantly expressed in podocytes in normal mice but were downregulated in albumin/TGF-β transgenic mice with podocyte apoptosis and glomerulosclerosis. In vitro, TGF-β downregulated miR-30s in wildtype and Smad3-deficient, but not Smad2- or Smad2/Smad3-deficient, podocytes. The TGF-β-induced activation of caspase 3 and an increase in TUNEL-positive nuclei were significantly inhibited by the lentivirus-mediated overexpression of miR-30d, but not by a scrambled control miR, in podocytes. TGF-β stimulated the phosphorylation of pro-apoptotic p53 in podocytes with lentiviral expression of a scrambled miR, but not in podocytes expressing miR-30d. In contrast, miR-30d had no effect on the phosphorylation of pro-apoptotic p38 MAP kinase induced by TGF-β. Thus, we report that Smad2-dependent inhibition of miR-30s in podocytes is required for the activation of p53 and the induction of apoptosis by TGF-β. These results demonstrate a novel functional role for miR-30 in podocyte survival and indicate that the loss of miR-30 survival signaling is a novel and specific mechanism of TGF-β-induced podocyte apoptosis during glomerulosclerosis. We propose the therapeutic replacement of miR-30 as a novel strategy to prevent the podocyte apoptosis that is characteristic of progressive glomerular diseases. [ABSTRACT FROM AUTHOR]

Oxygen consumption by isolated mitochondria is generally measured during state 4 respiration (no ATP production) or state 3 (maximal ATP production at high ADP availability). However, mitochondria in vivo do not function at either extreme. Here we used ADP recycling methodology to assess muscle mitochondrial function over intermediate clamped ADP concentrations. In so doing, we uncovered a previously unrecognized biphasic respiratory pattern wherein O2 flux on the complex II substrate, succinate, initially increased and peaked over low clamped ADP concentrations then decreased markedly at higher clamped concentrations. Mechanistic studies revealed no evidence that the observed changes in O2 flux were due to altered opening or function of the mitochondrial permeability transition pore or to changes in reactive oxygen. Based on metabolite and functional metabolic data, we propose a multifactorial mechanism that consists of coordinate changes that follow from reduced membrane potential (as the ADP concentration in increased). These changes include altered directional electron flow, altered NADH/NAD+ redox cycling, metabolite exit, and OAA inhibition of succinate dehydrogenase. In summary, we report a previously unrecognized pattern for complex II energized O2 flux. Moreover, our findings suggest that the ADP recycling approach might be more widely adapted for mitochondrial studies. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is increasing in incidence and predictable with measurement of serum islet autoantibodies (iAb) years prior to clinical disease onset. Identifying iAb positive individuals reduces diabetic ketoacidosis and identifies individuals for T1D prevention trials. However, large scale screening for iAb remains challenging as assays have varying sensitivities and specificities, insulin autoantibodies remain difficult to measure and venipuncture is generally required to obtain serum. We developed an approach to reliably measure all four major iAb, including insulin autoantibodies, from dried blood spots (DBS) on filter-paper. By spiking iAb positive serum into iAb negative whole blood in a dose titration, we optimized the conditions for autoantibody elution from filter paper as measured by fluid phase radioimmunoassays. After assessing stability of measuring iAb from DBS over time, we then screened iAb from DBS and the corresponding serum in new-onset T1D (n = 52), and controls (n = 72) which included first-degree relatives of T1D patients. iAb measured from eluted DBS in new-onset T1D strongly correlated with serum measurements (R2 = 0.96 for mIAA, GADA = 0.94, IA-2A = 0.85, ZnT8A = 0.82, p<0.01 for each autoantibody). There were no false positives in control subjects, and 5/6 with previously unknown iAb positivity in sera were detected using DBS. With further validation, measuring iAb from DBS can be a reliable method to screen for T1D risk. [ABSTRACT FROM AUTHOR]

The prevalence of celiac disease autoimmunity or tissue transglutaminase autoantibodies (TGA) amongst patients with type 1 diabetes (T1D) and autoimmune thyroid disease (AITD) in the Chinese population remains unknown. This study examined the rate of celiac disease autoimmunity amongst patients with T1D and AITD in the Chinese population. The study included 178 patients with type 1 diabetes and 119 with AITD where 36 had both T1D and AITD, classified as autoimmune polyglandular syndrome type 3 variant (APS3v). The study also included 145 patients with type 2 diabetes (T2D), 97 patients with non-autoimmune thyroid disease (NAITD), and 102 healthy controls. Serum islet autoantibodies, thyroid autoantibodies and TGA were measured by radioimmunoassay. TGA positivity was found in 22% of patients with either type 1 diabetes or AITD, much higher than that in patients with T2D (3.4%; p< 0.0001) or NAITD (3.1%; P < 0.0001) or healthy controls (1%; p<0.0001). The patients with APS3v having both T1D and AITD were 36% positive for TGA, significantly higher than patients with T1D alone (p = 0.040) or with AITD alone (p = 0.017). T1D and AITD were found to have a 20% and 30% frequency of overlap respectively at diagnosis. In conclusion, TGA positivity was high in the Chinese population having existing T1D and/or AITD, and even higher when both diseases were present. Routine TGA screening in patients with T1D or AITD will be important to early identify celiac disease autoimmunity for better clinical care of patients. [ABSTRACT FROM AUTHOR]

Diabetes mellitus occurs spontaneously in dogs. Although canine diabetes shares many features with human type-1 diabetes, there are differences that have cast doubt on the immunologic origin of the canine disease. In this study, we examined whether peripheral immune responses directed against islet antigens were present in dogs with diabetes. Routine diagnostics were used to confirm diabetic status, and serum samples from dogs with (N = 15) and without (N = 15) diabetes were analyzed for the presence of antibodies against islet antigens (insulin, glutamic acid decarboxylase, insulinoma-associated protein tyrosine phosphatase, and islet beta-cell zinc cation efflux transporter) using standard radioassays. Interferon-γ production from peripheral blood T cells stimulated by porcine insulin and by human insulin was tested using Elispot assays. Anti-insulin antibodies were detectable in a subset of diabetic dogs receiving insulin therapy. Pre-activated T cells and incipient insulin-reactive T cells in response to porcine or human insulin were identified in non-diabetic dogs and in dogs with diabetes. The data show that humoral and cellular anti-insulin immune responses are detectable in dogs with diabetes. This in turn provides support for the potential to ethically use dogs with diabetes to study the therapeutic potential of antigen-specific tolerance. [ABSTRACT FROM AUTHOR]

Background: Recent genome-wide association (GWA) studies in Caucasians identified multiple single nucleotide polymorphisms (SNPs) associated with coronary artery disease (CAD). The associations of those SNPs with myocardial infarction (MI) have not been replicated in Asian populations. Among those previously identified SNPs, we selected nine (rs10953541, rs1122608, rs12190287, rs12413409, rs1412444, rs1746048, rs3798220, rs4977574, rs579459, in or near genes 7q22, LDLR, TCF21, CYP17A1, LIPA, CXCL12, LPA, CDKN2A, ABO, respectively) because of the relatively high minor allele frequencies in Chinese individuals and tested the associations of the SNPs with MI and MI related risk factors in Chinese population. Methods and Results: We conducted a case–control association study on a cohort of 2365 MI patients and 2678 unrelated controls from the Chinese population. Genotyping of 9 SNPs were performed by the TaqMan Real Time PCR method. After age, sex, and BMI adjustment, we observed the SNPs rs12190287, rs12413409, rs1412444, rs1746048 and rs4977574, were significantly associated with MI in additive models and rs12190287, rs12413409, rs4977574 were significantly associated with phenotypes of MI at the same time. We also found three SNPs rs1122608, rs3798220 and rs579459 were significantly associated with risk factors of MI, although they had no association with MI in Chinese population. Conclusion: Results of this study indicate that 5 SNPs were associated with MI and 3 SNPs were associated with associated with lipoprotein levels but not with MI in a Chinese population. The present study supports some CAD-related genes in Caucasian as important genes for MI in a Chinese population. [ABSTRACT FROM AUTHOR]

Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 (Chr7) copy number variation (CNV) in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNV commonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7, as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established glioma cell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic glioma cultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 mis-segregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvement of chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor cell types. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneity could be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations. Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumor heterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers. [ABSTRACT FROM AUTHOR]

Objectives: Follicular helper T (Tfh) cells exert an important role in autoimmune diseases. Whether it might be involved in type 1 diabetes (T1D) is unknown. Our aim was to investigate the role of Tfh cells in patients with T1D and the effect of anti-CD20 monoclonal antibody (rituximab) on Tfh cells from T1D patients. Patients and Methods: Fifty-four patients with T1D and 37 healthy controls were enrolled in the current study. 20 of those patients were treated with rituximab. The frequencies of circulating CD4+CXCR5+ICOS+T cells were analyzed by flow cytometry. The serum autoantibodies were detected by radioligand assay. The levels of IL-21, IL-6 and BCL-6 were assessed using ELISA and/or real-time PCR. Results: Increased frequencies of circulating Tfh cells together with enhanced expression of IL-21 were detected in patients. The correlation between the frequencies of circulating Tfh cells and the serum autoantibodies or C-peptide level was comfirmed. After rituximab therapy, follow-up analysis demonstrated that the frequencies of circulating Tfh cell and serum IA2A were decreased. The levels of IL-21, IL-6 and Bcl-6 mRNA were decreased after treatment. Furthermore, beta cell function in 10 of 20 patients was improved. Conclusions: These data indicate Tfh cells may participate in the T1D-relatede immune responses and B cells might play a role in the development of Tfh responses in the disease progression. [ABSTRACT FROM AUTHOR]

Background: In this study, we investigated whether PKR protein expression is correlated with mRNA levels and also evaluated molecular biomarkers that are associated with PKR, such as phosphorylated PKR (p-PKR) and phosphorylated eIF2α (p-eIF2α). Methodology and Findings: We determined the levels of PKR protein expression and mRNA in 36 fresh primary lung tumor tissues by using Western blot analysis and real-time reverse-transcriptase PCR (RT-PCR), respectively. We used tissue microarrays for immunohistochemical evaluation of the expression of p-PKR and p-eIF2α proteins. We demonstrated that PKR mRNA levels are significantly correlated with PKR protein levels (Spearman's rho = 0.55, p<0.001), suggesting that PKR protein levels in tumor samples are regulated by PKR mRNA. We also observed that the patients with high p-PKR or p-eIF2α expression had a significantly longer median survival than those with little or no p-PKR or p-eIF2α expression (p = 0.03 and p = 0.032, respectively). We further evaluated the prognostic effect of combined expression of p-PKR plus PKR and p-eIF2α plus PKR and found that both combinations were strong independent prognostic markers for overall patient survival on stage I and all stage patients. Conclusions: Our findings suggest that PKR protein expression may controlled by transcription level. Combined expression levels of PKR and p-PKR or p-eIF2α can be new markers for predicting the prognosis of patients with NSCLC. [ABSTRACT FROM AUTHOR]