AimsType 1 diabetes (T1D) is characterized by autoimmune depletion of insulin-producing pancreatic beta cells. We showed previously that deletion of the 12/15-lipoxygenase enzyme (12/15-LO, Alox15 gene) in NOD mice leads to nearly 100 percent protection from T1D. In this study, we test the hypothesis that cytokines involved in the IL-12/12/15-LO axis affect both macrophage and islet function, which contributes to the development of T1D.Methods12/15-LO expression was clarified in immune cells by qRT-PCR, and timing of expression was tested in islets using qRT-PCR and Western blotting. Expression of key proinflammatory cytokines and pancreatic transcription factors was studied in NOD and NOD-Alox15null macrophages and islets using qRT-PCR. The two mouse strains were also assessed for the ability of splenocytes to transfer diabetes in an adoptive transfer model, and beta cell mass.Results12/15-LO is expressed in macrophages, but not B and T cells of NOD mice. In macrophages, 12/15-LO deletion leads to decreased proinflammatory cytokine mRNA and protein levels. Furthermore, splenocytes from NOD-Alox15null mice are unable to transfer diabetes in an adoptive transfer model. In islets, expression of 12/15-LO in NOD mice peaks at a crucial time during insulitis development. The absence of 12/15-LO results in maintenance of islet health with respect to measurements of islet-specific transcription factors, markers of islet health, proinflammatory cytokines, and beta cell mass.ConclusionsThese results suggest that 12/15-LO affects islet and macrophage function, causing inflammation, and leading to autoimmunity and reduced beta cell mass.

Aims/hypothesis IL-12 is an important cytokine in early inflammatory responses and is implicated in the immunemediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells. Methods In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ). Results The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines. Conclusions/interpretation These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.

Cole BK, Kuhn NS, Green-Mitchell SM, Leone KA, Raab RM, Nadler JL, Chakrabarti SK. 12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response.

The objective of these studies was to identify differentially expressed peptides/proteins in the culture media of embryos grown during in vitro fertilization (IVF) treatment to establish their value as biomarkers predictive of implantation potential and live birth. Micro-droplets of embryo culture media from IVF patients (conditioned) and control media maintained under identical culture conditions were collected and frozen at -80°C on Days 2-3 of in vitro development prior to analysis. The embryos were transferred on Day 3. The peptides were affinity purified based on their physico-chemical properties and profiled by mass spectrometry for differential expression. The identified proteins were further characterized by western blot and ELISA, and absolute quantification was achieved by multiple reaction monitoring (MRM). We identified up to 14 differentially regulated peptides after capture using paramagnetic beads with different affinities. These differentially expressed peptides were used to generate genetic algorithms (GAs) with a recognition capability of 71-84% for embryo transfer cycles resulting in pregnancy and 75-89% for those with failed implantation. Several peptides were further identified as fragments of Apolipoprotein A-1, which showed consistent and significantly reduced expression in the embryo media samples from embryo transfer cycles resulting in viable pregnancies. Western blot and ELISA, as well as quantitative MRM results, were confirmatory. These results demonstrated that peptide/protein profiles from the culture medium during early human in vitro development can discriminate embryos with highest and lowest implantation competence following uterine transfer. Further prospective studies are needed to establish validated thresholds for clinical application.

Purpose The aim of this study was to use on- tissue reduction followed by MALDI-IMS to identify an m/z 5812.85 peak which is over-expressed in healthy human pancreatic tissue compared to Type one Diabetes (T1D) tissue. Experimental design A major constraint of MALDI-IMS is identification of compounds with m/z ≥ 4000. On-tissue reduction using tris (2-carboxyethyl) phosphine (TCEP) breaks the inter-domain disulphide bonds generating low molecular weight peptides amenable to direct MS/MS analysis. Pancreatic tissues from healthy (n=1) and diabetic subjects (n=1) were profiled by MALDI-IMS with/without reduction. Results On-tissue reduction resulted in the loss of the over expressed 5812.85 m/z peak and the simultaneous appearance of a 3430.664 m/z peak in healthy tissue. The latter peak presumably derived from the 5812.85 m/z peak was identified as the insulin B chain by MS/MS. MALDI-IMS images show that both the 5812.85 insulin peak before reduction and the 3430.664 peak after reduction co-localized with the healthy pancreatic islets. Conclusion and clinical relevance On-tissue reduction followed by MALDI-IMS resulted in the identification of insulin and localization of pancreatic islets of langerhans. The approach will be useful in the future identification of novel therapeutic molecular targets to beta-cells lost during T1D.

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