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Thymic CD4 + FoxP3 + regulatory T (Treg) cells are critical for the development of immunological tolerance and immune homeostasis and requires contributions of both thymic dendritic and epithelial cells. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune cell development and, in particular, whether or how they contribute to the Treg cellular thymic compartment. Herein, using both phenotypic and functional approaches, we demonstrate that thymic B cells contribute to the maintenance of thymic Treg cells and, using an in vitro culture system, demonstrate that thymic B cells contribute to the size of the thymic Treg compartment via cell–cell MHC II contact and the involvement of two independent co-stimulatory pathways that include interactions between the CD40/CD80/CD86 co-stimulatory molecules. Our data also suggest that thymic B cells promote the generation of thymic Treg cell precursors (pre-Treg cells), but not the conversion of FoxP3 + Treg cells from pre-Treg cells. In addition, thymic B cells directly promote the proliferation of thymic Treg cells that is MHC II contact dependent with a minimal if any role for co-stimulatory molecules including CD40/CD80/CD86. Both pathways are independent of TGFβ. In conclusion, we rigorously define the critical role of thymic B cells in the development of thymic Treg cells from non-Treg to precursor stage and in the proliferation of mature thymic Treg cells. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is one of the most common gynaecological endocrine disorders, and more than 60% of PCOS patients have varying degrees of insulin resistance (IR). The regulatory role of microRNAs (miRNAs) at post-transcriptional levels in human cumulus cells relating to IR in PCOS remains unclear. In this case–control study, 26 PCOS patients with IR (PCOS-IR) and 24 patients without IR (PCOS-control) were enrolled. We determined the differentially expressed miRNA and mRNA using next-generation sequencing technology, and these miRNAs and mRNAs were validated by quantitative real-time polymerase chain reaction (PCR). These miRNA regulating pathways (e.g., MAPK pathway) were analysed by bioinformatics analysis, and the Rap1b was demonstrated to be targeted by miR-612 based on quantitative real-time PCR, western blot and luciferase activity assay. A total of 59 known miRNAs and 617 differentially expressed genes were identified that differentially expressed between PCOS-IR and PCOS-control cumulus cells. Moreover, the potential regulating roles of miRNAs and their targeting genes in pathophysiology of IR and PCOS were analysed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, and several key processes were enriched, such as MAPK activity. Furthermore, Rap1b, a regulator of the MAPK pathway, was demonstrated to be suppressed directly by miR-612 in PCOS-IR cumulus cells based on negative expression correlation validation, dual luciferase activity assay and reduction of Rap1b expression after miR-612 mimics transfection. Our results suggested that miRNAs and their targeted pathways in ovarian cumulus cells may play important roles in the aetiology and pathophysiology of PCOS with IR. [ABSTRACT FROM AUTHOR]

Problem: The role of vitamin D (VD) in IVF outcome and immune parameters has not been elucidated well. Method of study: Women undergoing IVF treatment with GnRH agonist (Agonist) and progestin‐primed ovarian stimulation (PPOS) protocols were divided into VD lower (VDL, 25(OH)VD ≤20 ng/mL) and VD higher (VDH, 25(OH)VD >20 ng/mL) groups. Follicular fluid (FF) VD level, IVF outcomes, and peripheral blood immunophenotypes by flow cytometry were analyzed. Results: FF VD levels of the whole subjects were positively correlated with peripheral blood VD level (r = 0.86, P < 0.001). The number of mature oocytes and the blastocyst formation rate were significantly higher in women with VDH group as compared with those of VDL group in both Agonist and PPOS groups (P < 0.05, respectively). In women with PPOS protocol, peripheral blood NK and B‐cell proportions and T helper/T cytotoxic (Th/Tc) cell ratios of VDL group were significantly higher than those of VDH group (P < 0.05, respectively). In women with Agonist protocol, peripheral blood B‐cell proportion and Th/Tc ratios of VDL group were significantly higher than those of VDH group (P < 0.05, respectively). Conclusion: VD level is associated with IVF outcomes possibly derived by T‐cell immunity, particularly Th/Tc ratios. [ABSTRACT FROM AUTHOR]

ABSTRACT A novel molecularly imprinted polymers (MIPs) for caffeine based on konjac glucomannan (KGM) was prepared using grafted copolymer of KGM/acrylic acid as a functional monomer, N, N′-methylenebisacrylamide as a cross-linker, and caffeine as a template. The recognition sites in MIPs were confirmed by Fourier transform infrared spectroscopy and the morphology of MIPs was characterized by scanning electron microscopy. Compared with nonimprinted polymers (NIPs), the obtained MIPs showed high adsorption capacity and good selectivity for caffeine. The maximum adsorption ( Qmax) of MIPs and NIPs for caffeine was 62.97 and 21.67 mg/g, respectively. The selective factor (α) of MIPs and NIPs was 2.50 and 0.91, respectively. Adsorption isotherm of MIPs followed the Langmuir equation. In addition, the obtained materials were packed on solid-phase extraction (SPE) cartridges and evaluated as a sorbent for selective trapping and preconcentration of caffeine from real beverage samples. Under the optimized extraction conditions, recoveries of caffeine ranging from 91.3 to 99.2% was achieved. [ABSTRACT FROM AUTHOR]

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4 −/− Tg). Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4 −/− Tg mice with established disease at 8–9 weeks of age with C57BL/6 control mice. Such parabiotic “twins” had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4 −/− Tg and CD8 −/− mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8 + T effector cells in the presence of wild type CD4 cells was noted. In conclusion, “correcting” the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis. [ABSTRACT FROM AUTHOR]

IFN-γ is a signature Th1 cell associated cytokine critical for the inflammatory response in autoimmunity with both pro-inflammatory and potentially protective functions. IL-17A is the hallmark of T helper 17 (Th17) cell subsets, produced by γδT, CD8+ T, NK and NKT cells. We have taken advantage of our colony of IL-2Rα−/− mice that spontaneously develop both autoimmune cholangitis and inflammatory bowel disease. In this model CD8+ T cells mediate biliary ductular damage, whereas CD4+ T cells mediate induction of colon-specific autoimmunity. Importantly, IL-2Rα−/− mice have high levels of interferon γ (IFN-γ), and interleukin-17A (IL-17A). We produced unique double deletions of mice that were either IL-17A−/−IL-2Rα−/− or IFN-γ−/−IL-2Rα−/− to specifically address the precise role of these two cytokines in the natural history of autoimmune cholangitis and colitis. Of note, deletion of IL-17A in IL-2Rα−/− mice led to more severe liver inflammation, but ameliorated colitis. In contrast, there were no significant changes in the immunopathology of double knock-out IFN-γ−/− IL-2Rα−/− mice, compared to single knock-out IL-2Rα−/− mice with respect to cholangitis or colitis. Furthermore, there was a significant increase in pathogenetic CD8+ T cells in the liver of IL-17A−/−IL-2Rα−/− mice. Our data suggest that while IL-17A plays a protective role in autoimmune cholangitis, it has a pro-inflammatory role in inflammatory bowel disease. These data take on particular significance in the potential use of anti-IL-17A therapy in humans with primary biliary cirrhosis. [ABSTRACT FROM AUTHOR]

Abstract: The IL-12 family modulates T cell mediated autoimmune diseases and GWAS in PBC have suggested a critical role of IL-12 and its subunits in modulating portal inflammation. We have taken advantage of an aggressive model of portal inflammation and colitis in IL-2Rα−/− mice to study the specific role of IL-12 and, in particular, the immunobiology of p40−/−IL-2Rα−/− mice. Colonies of IL-2Rα+/−, IL-2Rα−/− and p40−/−IL-2Rα−/− mice were studied for the natural history of immunopathology in liver and colon using histology and immunohistochemistry. Further, to focus on mechanisms, liver, spleen and mesenteric lymph node flow cytometry was employed to identify specific phenotypes; cytokine analysis on inflammatory cell populations was compared between groups. Finally, Real-Time PCR was used to focus on the genes involved in hepatic fibrosis. Surprisingly, p40−/−IL-2Rα−/− mice manifest more severe portal inflammation and bile duct damage, including signs of portal hypertension and liver fibrosis, but a significant reduction in colitis. Indeed, p40−/−IL-2Rα−/− mice reveal a profound hepatic CD8+ T cell infiltrate, whose major component are effector memory cells as well as enhanced hepatic Th1 but reduced Th17 responses. These observations were confirmed by Real-Time PCR analysis of fibrosis-related genes in the liver. Distinct from its canonical effects, IL-12p40 plays a critical role in autoimmune cholangitis, including hepatic fibrosis. These data take on striking significance for any proposed human trials that modulate the IL-12p40 pathway in human PBC. [Copyright &y& Elsevier]

Abstract: There have been major advances in defining the immunological events associated with fibrosis in various chronic liver diseases. We have taken advantage of this data to focus on the mechanisms of action of a unique multi-kinase inhibitor, coined sorafenib, on CCl4-induced murine liver fibrosis, including the effects of this agent in models of both acute and chronic CCl4-mediated pathology. Importantly, sorafenib significantly attenuated chronic liver injury and fibrosis, including reduction in liver inflammation and histopathology as well as decreased expression of liver fibrosis-related genes, including α-smooth muscle actin, collagen, matrix metalloproteinases and the tissue inhibitor of metalloproteinase-1. Furthermore, sorafenib treatment resulted in translocation of cytoplasmic STAT3 to the nucleus in its active form. Based on this observation, we used hepatocyte-specific STAT3 knockout (STAT3Hep−/−) mice to demonstrate that hepatic STAT3 was critical for sorafenib-mediated protection against liver fibrosis, and that the upregulation of STAT3 phosphorylation was dependent on Kupffer cell-derived IL-6. In conclusion, these data reflect the clinical potential of the multi-kinase inhibitor sorafenib for the prevention of fibrosis as well as the treatment of established liver fibrosis and illustrate the immunological mechanisms that underlie the protective effects of sorafenib. [Copyright &y& Elsevier]