A TCR-like antibody against a proinsulin-containing fusion peptide ameliorates type 1 diabetes in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by destruction of insulin-producing ß cells. The response of autoreactive T cells to ß cell antigens plays a central role in the development of T1D. Recently, fusion peptides composed by insulin C-peptide fragments and other proteins were reported as ß cell target antigens for diabetogenic CD4+ T cells in non-obese diabetic (NOD) mice. In this study, we generated a T cell-receptor (TCR)-like monoclonal antibody (mAb) against a fusion peptide bound to major histocompatibility complex (MHC) class II component to elucidate the function of the fusion peptides in T1D. In addition, we developed a novel NFAT-GFP TCR reporter system to evaluate the TCR-like mAb. The NFAT-GFP reporter T cells expressing the diabetogenic TCR were specifically activated by the fusion peptide presented on the MHC class II molecules. By using the NFAT-GFP reporter T cells, we showed that the TCR-like mAb blocks the diabetogenic T cell response against the fusion peptide presented on the MHC class II molecules. Furthermore, the development of T1D was ameliorated when pre-diabetic NOD mice were treated with this mAb. These findings suggest that NFAT-GFP reporter T cells are useful to assess the function of specific TCR and the recognition of fusion peptides by T cells is crucial for the pathogenesis of T1D.

Affimers as an alternative to antibodies for protein biomarker enrichment.

INTRODUCTION: Assessing the specificity of protein binders is an essential first step in protein biomarker assay development. Affimers are novel protein binders and can potentially replace antibodies in multiple protein capture-based assays. Affimers are selected for their high specificity against the target protein and have benefits over antibodies like batch-to-batch reproducibility and are stable across a wide range of chemical conditions. Here we mimicked a typical initial screening of affimers and commercially available monoclonal antibodies against two non-related proteins, IL-37b and proinsulin, to assess the potential of affimers as alternative to antibodies. METHODS: Binding specificity of anti-IL-37b and anti-proinsulin affimers and antibodies was investigated via magnetic bead-based capture of their recombinant protein targets in human plasma. Captured proteins were analyzed using SDS-PAGE, Coomassie blue staining, Western blotting and LC-MS/MS-based proteomics. RESULTS: All affimers and antibodies were able to bind their target protein in human plasma. Gel and LC-MS/MS analysis showed that both affimer and antibody-based captures resulted in co-purified background proteins. However, affimer-based captures showed the highest relative enrichment of IL-37b and proinsulin. CONCLUSIONS: For both proteins tested, affimers show higher specificity in purifying their target proteins from human plasma compared to monoclonal antibodies. These results indicate that affimers are promising antibody-replacement tools for protein biomarker assay development.

Inhibition of preproinsulin gene expression by leptin induction of suppressor of cytokine signaling 3 in pancreatic beta-cells.

Leptin inhibits insulin secretion and preproinsulin gene expression in pancreatic beta-cells, but signal transduction pathways and molecular mechanisms underlying this effect are poorly characterized. In this study, we analyzed leptin-mediated signal transduction and preproinsulin gene regulation at the molecular level in pancreatic beta-cells. Leptin stimulation led to janus kinase (JAK)2-dependent phosphorylation and nuclear translocation of the transcription factors signal transducer and activator of transcription (STAT)3 and STAT5b in INS-1 beta-cells. Leptin also induced mRNA expression of the JAK-STAT inhibitor suppressor of cytokine signaling (SOCS)3 in INS-1 beta-cells and human pancreatic islets in vitro and in pancreatic islets of ob/ob mice in vivo. Transcriptional activation of the rat SOCS3 promoter by leptin was observed with concomitant leptin-induced STAT3 and STAT5b DNA binding to specific promoter regions. Unexpectedly, SOCS3 inhibited both basal and STAT3/5b-dependent rat preproinsulin 1 gene promoter activity in INS-1 cells. These results suggest that SOCS3 represents a transcriptional inhibitor of preproinsulin gene expression, which is induced by leptin through JAK-STAT3/5b signaling in pancreatic beta-cells. In conclusion, although SOCS3 is believed to be a negative feedback regulator of JAK-STAT signaling, our findings suggest involvement of SOCS3 in a direct gene regulatory pathway downstream of leptin-activated JAK-STAT signaling in pancreatic beta-cells.

Influence of long term cyclosporine therapy on insulin and its precursors secretion in patients after heart transplantation.

Diabetes mellitus is a very well recognized risk factor for coronary artery disease in non-transplant patients. With the introduction of new immunosuppressive agents in solid organ recipients, there is an interest in medical complications of immunosuppressive therapy. An influence of long-term cyclosporine-A (CyA) therapy on glucose metabolism was analyzed in a group of 122 heart transplant recipients who developed hyperglycemia after heart transplantation. Based on WHO criteria for diagnosis of diabetes two groups were identified: group 1 (102 pts) included pts with impaired glycemic control and group 2 (20 pts) with clinical diabetes. Fasting insulin, proinsulin, C-peptide, HbA1c and cyclosporine-A trough levels were determined 12-18 months post surgery in clinically stable period without transplant rejection. The immunosuppressive treatment in both groups was the same and consisted of cyclosporine A, azathioprine and prednisone. We observed a statistically significant negative correlation between CyA concentration and insulin in both groups, a statistically significant negative correlation between CyA concentration and proinsulin, C-peptide blood level in group 1 and statistically significant positive correlation between CyA and glucose blood level in both groups.

Interleukin-1 beta inhibits proinsulin conversion in rat beta-cells via a nitric oxide-dependent pathway.

Exposure of pancreatic beta-cells to interleukin-1 beta (IL-1 beta) alters their protein expression and phenotype. Previous work has shown that IL-1 beta inhibited proinsulin conversion in rat islets, but the mechanism of this inhibition remained unknown. To investigate this phenomenon further, we examined purified rat beta-cells for IL-1 beta-induced inhibition of proinsulin conversion and nitric oxide (NO)-dependency of this inhibitory process. Rat beta-cells were cultured for 24 h with or without IL-1 beta and the inducible-nitric-oxide-synthase (iNOS) inhibitor N(G)-methyl-L-arginine (NMA). Exposure to IL-1 beta suppressed proinsulin-1 and proinsulin-2 synthesis by more than 50 %. Conversion of both proinsulin isoforms was also delayed. The suppressive effects of IL-1 beta on proinsulin synthesis and conversion were prevented by addition of NMA. Exposure to IL-1 beta also decreased the expression of the proinsulin convertase (PC) PC2. This decrease in PC2 expression was NO-dependent. In conclusion, IL-1 beta inhibition of proinsulin conversion in rat beta-cells occurs via an NO-mediated pathway.

Cortisol increases the susceptibility of rat islets to interleukin 1.

Interleukin 1 beta has been proposed to play an essential role in the pathogenesis of IDDM by direct interaction with the pancreatic beta-cell. Glucocorticoids are widely used as immunosuppressive agents and have been suggested to interfere both with the production and action of interleukin 1. The aim of the present study was to evaluate the interaction between cortisol and interleukin-1 on the pancreatic beta-cell function in vitro. Newborn rat islets were precultured for seven days before they were exposed to interleukin 1 with or without addition of cortisol. The release of insulin to the culture medium was followed and the insulin content and biosynthesis were measured after one week in culture. Cortisol, 10(-6) mol/l, resulted in a 50% lower rate of release during the culture period and a similar reduction in storage and biosynthesis of insulin in the islets at the end of the culture period. In the control islets interleukin 1, 0.5 microgram/l, resulted in an early increase in insulin release followed by a marked reduction. In the cortisol-treated islets interleukin 1 increased the release up to 72 h followed by a moderate decrease. In the control islets, interleukin 1 reduced the insulin content to about 50%, whereas in the cortisol-treated islets interleukin 1 resulted in an even greater reduction, to about 30%. This additional effect of cortisol seems not to be due to an augmented cytotoxic effect of interleukin 1 as indicated by the DNA content of the islets and the viability of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Loss of immunological and biological activity of insulin with a human muscle extract.

The isolation of a human muscle extract that degrades insulin to products incapable of any immunological or biological activity is reported. The extract Km value for human insulin is 0.79, and 1.43 for porcine proinsulin. It does not degrade Human Growth Hormone at the concentrations tested, it is inactivated by N-ethyl-maleimide, Trasylol, and heat, and it does not require glutathione for its action. Human muscle, therefore can contain an enzyme similar to "insulin-specific-protease" present in rat muscle.