Autoimmune Destruction Of Β-cells Research Articles

Microencapsulated G3C Hybridoma Cell Graft Delays the Onset of Spontaneous Diabetes in NOD Mice by an Expansion of Gitr+ Treg Cells.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3+ Treg-cell expansion in the spleen of wild-type mice but not in Gitr-/- mice. G3C/cps also induced the expansion of nonconventional Cd4+Cd25-/lowFoxp3lowGitrint/high (GITR single-positive [sp]) Tregs. Both Cd4+Cd25+GitrhighFoxp3+ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.

Saccharomyces boulardii Tht 500101 changes gut microbiota and ameliorates hyperglycaemia, dyslipidaemia, and liver inflammation in streptozotocin-diabetic mice.

Type 1 diabetes mellitus (T1DM) is a disorder resulting from chronic autoimmune destruction of insulin-producing pancreatic β-cells, lack of insulin production and hyperglycaemia. The aim of this study was to evaluate the hypothesis that streptozotocin-diabetic mice treated with Saccharomyces boulardii THT 500101 strain present improvement of glucose and triglycerides metabolism, reduction of liver inflammation concomitant with a beneficial impact in the gut microbiota profile. C57BL/6 male mice were randomly assigned into three groups: Control, Diabetes, Diabetes+Probiotic, and were euthanised 8 weeks after probiotic chronic administration. Mice submitted to treatment presented reduced glycemia in comparison with the diabetic group, which was correlated with an increase in C-peptide level and in hepatic glycogen content. Fat metabolism was significantly altered in streptozotocin-induced diabetic group, and S. boulardii treatment regulated it, leading to a decrease in serum triglycerides secretion, increase in hepatic triglycerides storage and modulation of inflammatory profile. The phenotypic changes seen from chronic S. boulardii treatment were found to be broadly associated with the changes in microbioma of diabetic animals, with increased proportion in Bacteroidetes, Firmicutes and Deferribacteres, and a decreased proportion of Proteobacteria and Verrucomicrobia phylum. Thus, the data presented here show up a novel potential therapeutic role of S. boulardii for the treatment and attenuation of diabetes-induced complications.

Evaluating Vascularization of Heterotopic Islet Constructs for Type 1 Diabetes Using an In Vitro Platform.

Type 1 diabetes (T1D) results from the autoimmune destruction of β-cells within the pancreatic islets of Langerhans. Clinical islet transplantation from healthy donors is proposed to ameliorate symptoms, improve quality of life, and enhance the life span of afflicted T1D patients. However, post-transplant outcomes are dependent on the survival of the transplanted islets, which relies on the engraftment of the islets with the recipient's vasculature among other factors. Treatment strategies to improve engraftment include combining islets with supporting cells including endothelial cells (EC) and mesenchymal stem cells (MSC), dynamic cells capable of robust immunomodulatory and vasculogenic effects. In this study, we developed an in vitro model of transplantation to investigate the cellular mechanisms that enhance rapid vascularization of heterotopic islet constructs. Self-assembled vascular beds of fluorescently stained EC served as reproducible in vitro transplantation sites. Heterotopic islet constructs composed of islets, EC, and MSC were transferred to vascular beds for modeling transplantation. Time-lapsed imaging was performed for analysis of the vascular bed remodeling for parameters of neo-vascularization. Moreover, sampling of media following modeled transplantation showed secretory profiles that were correlated with imaging analyses as well as with islet function using glucose-stimulated insulin secretion. Together, evidence revealed that heterotopic constructs consisting of islets, EC, and MSC exhibited the most rapid recruitment and robust branching of cells from the vascular beds suggesting enhanced neo-vascularization compared to islets alone and control constructs. Together, this evidence supports a promising cell transplantation strategy for T1D and also demonstrates a valuable tool for rapidly investigating candidate cellular therapies for transplantation.

Review of Advanced Hydrogel-Based Cell Encapsulation Systems for Insulin Delivery in Type 1 Diabetes Mellitus.

Type 1 Diabetes Mellitus (T1DM) is characterized by the autoimmune destruction of β-cells in the pancreatic islets. In this regard, islet transplantation aims for the replacement of the damaged β-cells through minimally invasive surgical procedures, thereby being the most suitable strategy to cure T1DM. Unfortunately, this procedure still has limitations for its widespread clinical application, including the need for long-term immunosuppression, the lack of pancreas donors and the loss of a large percentage of islets after transplantation. To overcome the aforementioned issues, islets can be encapsulated within hydrogel-like biomaterials to diminish the loss of islets, to protect the islets resulting in a reduction or elimination of immunosuppression and to enable the use of other insulin-producing cell sources. This review aims to provide an update on the different hydrogel-based encapsulation strategies of insulin-producing cells, highlighting the advantages and drawbacks for a successful clinical application.

Protein Kinase CK2-A Putative Target for the Therapy of Diabetes Mellitus?

Since diabetes is a global epidemic, the development of novel therapeutic strategies for the treatment of this disease is of major clinical interest. Diabetes is differentiated in two types: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM arises from an autoimmune destruction of insulin-producing β-cells whereas T2DM is characterized by an insulin resistance, an impaired insulin reaction of the target cells, and/or dysregulated insulin secretion. In the past, a growing number of studies have reported on the important role of the protein kinase CK2 in the regulation of the survival and endocrine function of pancreatic β-cells. In fact, inhibition of CK2 is capable of reducing cytokine-induced loss of β-cells and increases insulin expression as well as secretion by various pathways that are regulated by reversible phosphorylation of proteins. Moreover, CK2 inhibition modulates pathways that are involved in the development of diabetes and prevents signal transduction, leading to late complications such as diabetic retinopathy. Hence, targeting CK2 may represent a novel therapeutic strategy for the treatment of diabetes.

1747-P: 3D Imaging and Quantification of Pancreatic Immune Cell Infiltration during Emergence and Progression of Spontaneous Autoimmune Diabetes in Nonobese Diabetic (NOD) Mice

The non-obese diabetic (NOD) mouse develops diabetes as the result of spontaneous autoimmune insulitis and is widely used as a translational polygenic model of type 1 diabetes. Disease progression in the NOD mouse model is slow, involving a long prodromal phase of pancreatic islet infiltration before autoimmune β-cell destruction and manifestation of diabetic hyperglycemia, making the NOD mouse ideal for probing the effectiveness of potential disease prevention therapies. Improved understanding of the spatial dynamics of immune cell infiltration of the pancreas during the emergence and natural progression of autoimmune diabetes would permit further resolution of drug treatment effects in this model. The present study therefore aimed to image the spontaneous development and progression of autoimmune diabetes dynamically and at 3-dimensional cellular resolution in the NOD mouse. Female NOD mice were terminated before diabetes onset, at 14 and 18 weeks of age, and pancreata were isolated and divided into two alternating samples consisting of either the head and tail. One half was stained for insulin and CD45 using iDISCO whole-mount immunohistochemistry and subsequently scanned using a light sheet microscope. Islet infiltration was quantified using an in-house developed algorithm. The other half of each pancreas was simultaneously evaluated by flow cytometry to discriminate and quantify immune cells of lymphoid and myeloid lineage. Infiltrating CD45-positive leukocytes progressively increased in abundance over time, however, with highly variable distribution in the pancreas. Flow cytometry confirmed extensive leukocyte infiltration and revealed predominant changes in T-cell profiles. In conclusion, 3D imaging allows for visualization and unbiased quantitative analysis of the unsynchronized spreading of insulitis into different parts of the pancreas in NOD mice. Disclosure B.B. Boland: Employee; Self; Gubra, MedImmune. A. Manresa-Arraut: None. R.V. Grônlund: None. U. Roostalu: Employee; Self; Gubra. J. Hecksher-Sørensen: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. N. Vrang: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. J. Jelsing: Stock/Shareholder; Self; Gubra. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S.

1696-P: PBMC of Recent-Onset Patients with Type 1 Diabetes Present a Differential Gene Expression Profile

Introduction: Type 1 diabetes is characterized by the autoimmune destruction of insulin-secreting β-cells, mediated by T auto reactive infiltrating cells, inflammatory cytokines and immunological mechanisms. However, little is known about the expression of genes and pathways dysregulated in peripheral blood mononuclear cells of patients with T1D. Objective: to investigate the gene expression profiles of circulating PBMC in recent-onset T1D patients (up to 6 months of diagnosis) in comparison with controls in the context of putative disease-related pathobiological processes and pathways. Methods: expression of mRNA from PBMC of 20 T1D patients, 15.8±8.6 years and age-matched 20 healthy controls, 14.7±6.7 years, (p>0.05) was evaluated with the Whole Human Genome Microarray Agilent and analyzed by GeneSpring with a fold change cutoff of 1.5 and adjusted P value <0.05; pathways analysis was performed with the software IPA. Results: 326 genes were differentially expressed between T1D patients and controls, being 204 (63%) upregulated, and 122 (37%) downregulated in T1D. Interactions between the differentially expressed genes evidenced expression patterns of 24 networks. The most significantly enriched canonical pathways were namely ErbB (erythroblastic leukemia viral oncogene) signaling, micropinocytosis signaling, savage pathways of pyrimidine ribonucleotides and hepathocyte growth factor in descending order of significance. The up-regulated protein targets within these signaling pathways were NEK2, PLK1, ATM, PRKCD, CCND1, NRG2 and those down regulated were AREG, CDC42 and CDADC1. Conclusion: The majority of upregulated genes of patients with T1D was related to cell cycle control, mitotic progression, proliferation and DNA repair, probably related to changes in PBMC metabolism and function due to DNA damage associated with hyperglycemia. The lower expression of AREG and CDC42 can be related to reduce immunosuppressive activity of regulatory T cells and B cell functions. Disclosure A. Santos: None. C. Chevillard: None. N. Gonfinetti: None. F. Bertonha: None. C. Moreira-Filho: None. J. Kalil: None. E. Cunha-Neto: None. M. Silva: None. Funding European Foundation for the Study of Diabetes; São Paulo Research Foundation

1727-P: A Novel Islet-Peripheral Blood Mononuclear Cell Coculture Model for Studying Type 1 Diabetes

Type 1 diabetes is a heterogeneous group of disorders majorly characterized by autoimmune destruction of pancreatic β-cells, resulting in absolute insulin deficiency. Current research models lack many functions critical for understanding the onset and progression of this disease in humans. Although isolated primary islets are considered the standard tool for diabetes research, their experimental use is challenging due to inherent heterogeneity in size, cellular composition and purity, as well as rapid decline in their functionality and viability ex vivo. Here we investigate a novel co-culture platform of peripheral blood mononuclear cells (PBMCs) and a uniform 3D islet model. Human islet microtissues, produced by optimized dissociation and controlled reaggregation of primary islet cells, were cultured in a one-islet per well format in 96-well plates. They displayed uniform, long-term (>28 days), and robust viability and function enabling high-throughput and longitudinal study of immune cell-endocrine cell interactions and β-cell function. PBMCs in their naïve form or following T-cell specific activation, were combined in various ratios with healthy or stressed islets - preconditioned with a mild cytokine cocktail. Co-culturing of naïve PBMCs and islet microtissues had only a minor impact on β-cell function. Whereas, combination of activated PBMCs and islet microtissues resulted in a PBMC-number dependent decline in islet health shown by increased basal insulin release and decreased glucose-stimulated insulin secretion, insulin content and PDX-1 positive nuclei within each microtissue. The observed destruction correlated with the amount of CD3+ cells infiltrating the microtissues and was significantly augmented by cytokine preconditioning. The established islet-PBMC co-culture platform represents a novel in vitro model to study the autoimmune component of T1D and to screen for compounds that could prevent immune-cell mediated destruction of pancreatic islets. Disclosure B. Yesildag: None. N. Perdue: None. J. Mir-Coll: Employee; Self; InSphero. Employee; Spouse/Partner; Roche Pharma. A. Biernath: None. A. Neelakandhan: None. F. Forschler: Employee; Self; InSphero. M.G. von Herrath: None. J.D. Wesley: Employee; Self; Novo Nordisk A/S.

Mass Cytometry Identifies Distinct Subsets of Regulatory T Cells and Natural Killer Cells Associated With High Risk for Type 1 Diabetes.

Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin producing β-cells. The time from onset of islet autoimmunity to manifest clinical disease can vary widely in length, and it is fairly uncharacterized both clinically and immunologically. In the current study, peripheral blood mononuclear cells from autoantibody-positive children with high risk for T1D, and from age-matched healthy individuals, were analyzed by mass cytometry using a panel of 32 antibodies. Surface markers were chosen to identify multiple cell types including T, B, NK, monocytes, and DC, and antibodies specific for identification of differentiation, activation and functional markers were also included in the panel. By applying dimensional reduction and computational unsupervised clustering approaches, we delineated in an unbiased fashion 132 phenotypically distinct subsets within the major immune cell populations. We were able to identify an effector memory Treg subset expressing HLA-DR, CCR4, CCR6, CXCR3, and GATA3 that was increased in the high-risk group. In addition, two subsets of NK cells defined by CD16+ CD8+ CXCR3+ and CD16+ CD8+ CXCR3+ CD11c+ were also higher in the same subjects. High-risk individuals did not show impaired glucose tolerance at the time of sampling, suggesting that the changes observed were not the result of metabolic imbalance, and might be potential biomarkers predictive of T1D.

SAT-164 Cornus Officinalis Significantly Improves Oxidative Capacity and Promotes the Calcium-Dependent Transcription Factor, NFATC2, in Human 1.1B4 Pancreatic Cell Line

Type 1 diabetes (T1D) is an autoimmune disease resulting in the destruction of pancreatic β cells (β-cells) and subsequent loss of insulin production. The only treatment for T1D is using exogenous insulin coupled with continual glucose monitoring following significant autoimmune destruction of β-cells. Novel interventional therapies are needed that can preserve and protect existing pancreatic β cells in individuals with early identified T1D autoimmunity. Our initial in-vitro evidence indicates Cornus officinalis (CO) may be able to serve in this function. What sets ethnopharmacology apart from conventional medicine is the simultaneous targeting of multiple mechanisms using a single herb due to the composition of numerous bioactive ingredients. CO has been used in TCM (traditional Chinese medicine) for over 2 millennia for the therapeutic effect of improved glucose tolerance and has also demonstrated efficacy in animal models but rarely examined in the context of T1D. We hypothesize that CO treatment may provide a β cell restorative and protective therapy for T1D and inhibit progressive cytokine-mediated β cell loss while enhancing existing β cells. Our preliminary evidence demonstrated a dose-dependent exposure of 1.1B4 cells to CO increased proliferation and protected against cytokine-induced cell death. We examined the metabolic effect of CO using the Agilent Seahorse XF Analyzer and at a two-hour time point there was a remarkable increase in maximal respiratory and glycolytic capacity following CO treatment. However, the molecular mechanism in which CO is inducing a proliferative and metabolic effect in 1.1B4 cells has not been elucidated. Therefore, we employed transcriptomics by RNA-Seq to analyze the early initiators of this increased metabolic effect. Our strongest and most significantly differentially expressed transcript was calcium-dependent transcription factor, NFATc. Expression of NFATc was validated by qPCR, which displayed a 2-fold increase in gene expression. NFATc is an essential transcription factor for β cell proliferation, endocrine function, and insulin secretion and may be a mediator of CO induced biological effects. Lastly, to date, 300 compounds have been elucidated from CO; therefore we wanted to analyze the components within our CO extracts via HPLC/MS in order to elucidate the bioactive ingredients. We found over 300 compounds, and of those, we found known bioactive ingredients such as, loganin and morroniside, while also finding novel extracts with potential bioactive properties. Altogether, CO increased β cell metabolism while inducing the NFAT pathway to signal for increased proliferation and endocrine function. Further experiments will examine the anti-diabetic effects of individual CO constituents that were identified via HPLC/MS, and the full molecular mechanism related to the NFAT signaling pathway.

Novel cancer therapies and their association with diabetes.

Over the last decade, there has been a shift in the focus of cancer therapy from conventional cytotoxic drugs to therapies more specifically directed to cancer cells. These novel therapies include immunotherapy, targeted therapy and precision medicine, each developed in great part with a goal of limiting collateral destruction of normal tissues, while enhancing tumor destruction. Although this approach is sound in theory, even new, specific therapies have some undesirable, 'off target effects', in great part due to molecular pathways shared by neoplastic and normal cells. One such undesirable effect is hyperglycemia, which results from either the loss of immune tolerance and autoimmune destruction of pancreatic β-cells or dysregulation of the insulin signaling pathway resulting in insulin resistance. These distinct pathogenic mechanisms lead to clinical presentations similar to type 1 (T1DM) and type 2 (T2DM) diabetes mellitus. Both types of diabetes have been reported in patients across clinical trials, and data on the mechanism(s) for developing hyperglycemia, prevalence, prognosis and effect on cancer mortality is still emerging. With the rapidly expanding list of clinical indications for new cancer therapies, it is essential to understand the impact of their adverse effects. In this review, we focus on hyperglycemia and diabetes related to cancer therapies, describe what is known about mechanism(s) leading to dysregulated glucose metabolism and provide a guide to management of complex oncology patients with a new diagnosis of diabetes.

Prevalence of islet autoantibodies in Thai juvenile-onset type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of islet β-cells of the pancreas. There are overlapping phenotypes in a significant proportion of youth with type 1 and 2 diabetes. Thus, positive pancreatic autoantibodies are helpful to diagnose T1DM. Zinc transporter 8 antibody (ZnT8A) is a recently identified autoantibody in T1DM and no data on ZnT8A in the Thai population have been reported. The aim of this study was therefore to estimate the prevalence of ZnT8A in Thai juvenile-onset T1DM and to evaluate its diagnostic value relative to glutamic acid decarboxylase and insulinoma-2 antigen antibodies (GADA and IA2A). In this cross-sectional study, patients with T1DM diagnosed before age 15years, and disease duration <10years were enrolled. Serum ZnT8A, GADA, and IA2A were measured using commercial enzyme-linked immunosorbent assay kits. The subjects consisted of 81 youths (30 boys, 51 girls) aged 12.3 ± 4.5years with T1DM. The median diabetes duration was 3years (range, 0-10years). The prevalence of ZnT8A, GADA, and IA2A was 54.3%, 75.3%, and 45.7%, respectively. ZnT8A were detected in 16% of T1DM patients negative for both GADA and IA2A. A combination of ZnT8A, GADA and IA2A could detect 80.2% of patients with T1DM. Combined use of ZnT8A and GADA identified 100% of antibody-positive patients. The prevalence of ZnT8A in Thai juvenile-onset T1DM appears to be higher than in previous studies from Asia. ZnT8A could replace IA2A as an autoimmunity marker in Thai pediatric T1DM patients, with better diagnostic performance.

Type 1 Diabetes Development Is Modulated by Select Lipid Signaling Derived from Beta Cells and Immune Cells

Type 1 diabetes (T1D) is a consequence of autoimmune destruction of β-cells and we find that the Ca2+-independent phospholipase A2β (iPLA2β) is induced in β-cells and immune cells and mitigation of iPLA2β attenuates β-cell death and T1D incidence. Activation of iPLA2β leads to generation of pro-inflammatory or anti-inflammatory bioactive lipids and we previously reported that iPLA2β activation favors M1 pro-inflammatory macrophage phenotype. Here, we utilized UPLC ESI-MS/MS protocols to assess the lipid profile generated by macrophages as a means to identify select lipids that are associated with T1D development. Macrophages were treated with DMSO (vehicle), IFNγ+LPS (to induce M1 polarization), or IL-4 (to induce M2 polarization) and at 16h, the media was collected and processed for assessment of eicosanoid class of bioactive lipids. Comparison of spontaneous diabetes-resistant C57BL/6J and spontaneous diabetes-prone NOD mice revealed that nearly all pro-inflammatory eicosanoids were elevated in NOD during the prediabetic phase, relative to C57. Theabundances increased in the NOD between 4-8 and decreased by 15 weeks, correlating with onset of insulitis. These findings are consistent with a higher inflammatory status in the NOD vs. C57. To assess contribution of iPLA2β to the lipid profile, macrophage responses in NOD were compared with those in NOD.iPLA2β-/+. Such analyses revealed that production of select pro-inflammatory and anti-inflammatory lipids were decreased and increased, respectively, from macrophages and islets of NOD.iPLA2β-/+, relative to age-matched NOD-WT, and this correlated with reduced T1D incidence in the NOD.iPLA2β-/+. We further find that iPLA2β-derived lipids (iDLs) play a critical role in alternative splicing events that favor generation of pro-apoptotic variants of several factors (Bcl-x, capsase-9, and RAGE). These findings raise the possibility that iDLs are candidates for targeting to counter T1D development. Disclosure A. Nelson: None. M.A. Park: None. C. Chalfant: None. S. Ramanadham: None.

Phosphatidylserine-Liposomes Promote Tolerogenic Features on Dendritic Cells in Human Type 1 Diabetes by Apoptotic Mimicry.

Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing β-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow β-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic β-cells arrested autoimmunity to β-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)—the main signal of the apoptotic cell membrane—and β-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity.

Apoptosis of pancreatic β-cells in type 1 diabetes: The role of Caspase-3 in β-cell apoptosis

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic β-cells after asymptomatic period over years. Insulitis activates antigen presenting cells, which trigger activating DC4+ helper-T cells, releasing cytokines. Cytokines activate CD8+ cytotoxic –T cells and lead to β-cell destruction. Apoptosis pathway consists of extrinsic and intrinsic pathway. Extrinsic pathway includes Fas pathway to CD4+-CD8+ interaction whereas intrinsic pathway includes mitochondria-driven pathway at balance between anti-apoptotic Bcl-2 and Bcl-xL and pro-apoptotic proteins. Activated cleaved caspase-3 (ACC) is the converging point between extrinsic and intrinsic pathway. ACC may be used as a marker for β-cell apoptosis: inT1DM islets weakly insulin-positive β-cells are present with increased ACC positive β-cells whereas β-cell absent islets, which exclusively consist of α-cells and PP-cells with decreased ACC-cells, represent the end-stage of regenerating β-cells. Weakly insulin-positive β-cells could be rejuvenated to supply endogenous insulin. Apoptosis takes place only when pro-apoptotic protein exceeds anti-apoptotic proteins. Since concordance rate of T1D in the identical twins is about 50%, environmental factors are involved in development of T1D, opening a door to means to prevent autoimmune β-cell destruction for therapeutic application by transfecting β-cells with Casp3-/- genes or pan-caspase inhibitor therapy. Prudent glucose control prevents ongoing hyperglycemia-induced β-cell apoptosis.

Brakes on the accelerator: the journey of accelerator hypothesis from “the missing link” to “an evolving concept”

&lt;p&gt;The incidence of Type 1 diabetes (T1D) has increased significantly over the past few decades but the causes for this increase are poorly understood and hence the strategies for preventing T1D are difficult to design. T1D is characterised by autoimmune destruction of pancreatic β-cells resulting in insulin deficiency as opposed to Type 2 diabetes (T2D) characterised by weight driven insulin resistance (IR). The accelerator hypothesis (AH), proposed by the late Prof Terence Wilkin in 2001 offers an alternative mechanism for T1D and a different approach to prevention of T1D. This hypothesis considers both T1D and T2D as one and proposes that obesity driven IR is the key factor that may lead to either type of diabetes. It thus offers an easy explanation for the increasing worldwide incidence of childhood diabetes which is paralleled by the increase in childhood obesity rates. However, one of the key predictions of AH that the obesity related IR accelerates the onset of diabetes and hence heavier children should develop diabetes at a younger age, has remained a matter of debate since the hypothesis was first proposed. Since the inception of AH, the results of a number of studies which aimed at testing the hypothesis in diverse patient populations have shown support or opposed this key prediction. This article discusses the relevance of AH in the context of data from these studies.&lt;/p&gt;

Biochemical and cellular properties of insulin receptor signalling.

The mechanism of insulin action is a central theme in biology and medicine. In addition to the rather rare condition of insulin deficiency caused by autoimmune destruction of pancreatic β-cells, genetic and acquired abnormalities of insulin action underlie the far more common conditions of type 2 diabetes, obesity and insulin resistance. The latter predisposes to diseases ranging from hypertension to Alzheimer disease and cancer. Hence, understanding the biochemical and cellular properties of insulin receptor signalling is arguably a priority in biomedical research. In the past decade, major progress has led to the delineation of mechanisms of glucose transport, lipid synthesis, storage and mobilization. In addition to direct effects of insulin on signalling kinases and metabolic enzymes, the discovery of mechanisms of insulin-regulated gene transcription has led to a reassessment of the general principles of insulin action. These advances will accelerate the discovery of new treatment modalities for diabetes.

The innate immune receptor MDA5 limits rotavirus infection but promotes cell death and pancreaticinflammation.

Melanoma differentiation-associated protein 5 (MDA5) mediates the innate immune response to viral infection. Polymorphisms in IFIH1, the gene coding for MDA5, correlate with the risk of developing type 1 diabetes (T1D). Here, we demonstrate that MDA5 is crucial for the immune response to enteric rotavirus infection, a proposed etiological agent for T1D. MDA5 variants encoded by minor IFIH1 alleles associated with lower T1D risk exhibit reduced activity against rotavirus infection. We find that MDA5 activity limits rotavirus infection not only through the induction of antiviral interferons and pro-inflammatory cytokines, but also by promoting cell death. Importantly, this MDA5-dependent antiviral response is specific to the pancreas of rotavirus-infected mice, similar to the autoimmunity associated with T1D. These findings imply that MDA5-induced cell death and inflammation in the pancreas facilitate progression to autoimmune destruction of pancreatic β-cells.

Differential regulation of pro-inflammatory cytokine signalling by protein tyrosine phosphatases in pancreatic β-cells.

Type 1 diabetes (T1D) is characterized by the destruction of insulin-producing β-cells by immune cells in the pancreas. Pro-inflammatory including TNF-α, IFN-γ and IL-1β are released in the islet during the autoimmune assault and signal in β-cells through phosphorylation cascades, resulting in pro-apoptotic gene expression and eventually β-cell death. Protein tyrosine phosphatases (PTPs) are a family of enzymes that regulate phosphorylative signalling and are associated with the development of T1D. Here, we observed expression of PTPN6 and PTPN1 in human islets and islets from non-obese diabetic (NOD) mice. To clarify the role of these PTPs in β-cells/islets, we took advantage of CRISPR/Cas9 technology and pharmacological approaches to inactivate both proteins. We identify PTPN6 as a negative regulator of TNF-α-induced β-cell death, through JNK-dependent BCL-2 protein degradation. In contrast, PTPN1 acts as a positive regulator of IFN-γ-induced STAT1-dependent gene expression, which enhanced autoimmune destruction of β-cells. Importantly, PTPN1 inactivation by pharmacological modulation protects β-cells and primary mouse islets from cytokine-mediated cell death. Thus, our data point to a non-redundant effect of PTP regulation of cytokine signalling in β-cells in autoimmune diabetes.

AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice

ObjectiveType 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties. MethodsTransgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28–30 weeks. ResultsIn transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice. ConclusionsLocal expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.