Mediators Of Beta-cell Destruction Research Articles

Abstract #1401677: Pembrolizumab Induced Autoimmune Diabetes Mellitus

Immune checkpoint inhibitors (ICIs) are integral in the treatment of a myriad of malignancies. While ICIs have significantly improved outcomes and cancer survivorship, they have also been implicated in immune related adverse events (irAEs), particularly autoimmune endocrinopathies. Common ICI associated endocrine disorders include hypothyroidism, hyperthyroidism, and primary adrenal insufficiency but rarely diabetes mellitus (DM). ICI induced DM is defined as severe and persistent insulin deficiency after treatment with an ICI. Here, we present a rare case of pembrolizumab induced DM presenting with hyperglycemic hyperosmolar state (HHS). A 77-year-old male with a past medical history of prediabetes on no medical therapy and melanoma of the right upper extremity was admitted with progressive polydipsia, polyuria and acute encephalopathy. Notably, the patient completed two cycles of pembrolizumab two weeks prior to admission. Initial workup demonstrated a glucose of 837 mg/dL (65-99), anion gap of 10 (3-11), small amounts of serum acetone and beta hydroxybutyrate, and a serum osmolality of 369 mOsm/kg (279-295). Venous blood gas revealed a pH of 7.33 (7.31-7.41), pCO2 45 mmHg (41-51), HCO3 24 mEq/L (23-29). Objective data supported the diagnosis of HHS. Hemoglobin A1c prior to initiation of pembrolizumab was 6.1% (< 5.7%) and on admission was 9.1%. He improved with intravenous insulin and fluids. After transition to basal/bolus insulin the patient’s fasting glucose was 126 mg/dL (65-99) with a c-peptide level of 0.41 ng/mL (0.80-3.85), consistent with insulin insufficiency. Glutamic acid decarboxylase 65 autoantibodies were positive, while islet cell and IA-2 autoantibodies were negative. Pembrolizumab was discontinued after the diagnosis of ICI induced DM was established. Our case highlights the unique course of a patient with prediabetes on pembrolizumab who developed ICI induced DM. Pembrolizumab (anti PD-1 monoclonal antibody) can cause an abnormal cytotoxic T cell (CD8+ T cell) mediated beta cell destruction and development of ICI induced DM. ICI induced DM shares characteristics of Type 1 DM with positive autoantibodies and insulin insufficiency. Insulin deficiency often precipitates diabetic ketoacidosis which was not seen in our patient. Given low but detectable c-peptide levels, our patient was likely insulin insufficient and not yet insulin deficient. This case highlights the importance of closely monitoring patients on ICIs, especially those with a predisposition to DM. In patients receiving immunotherapy, early identification of adverse events and risk stratification is essential in reducing morbidity and providing comprehensive care.

Presumption of innocence for beta cells: why are they vulnerable autoimmune targets in type 1 diabetes?

It is increasingly appreciated that the pathogenic mechanisms of type 1 diabetes involve both the autoimmune aggressors and their beta cell targets, which engage in a conflicting dialogue within and possibly outside the pancreas. Indeed, autoimmune CD8+ T cells, which are the final mediators of beta cell destruction, circulate at similar frequencies in type 1 diabetic and healthy individuals. Hence a universal state of 'benign' islet autoimmunity exists, and we hypothesise that its progression to type 1 diabetes may at least partially rely on a higher vulnerability of beta cells, which play a key, active role in disease development and/or amplification. We posit that this autoimmune vulnerability is rooted in some features of beta cell biology: the stress imposed by the high rate of production of insulin and other granule proteins, their dense vascularisation and the secretion of their products directly into the bloodstream. Gene variants that may predispose individuals to this vulnerability have been identified, e.g. MDA5, TYK2, PTPN2. They interact with environmental cues, such as viral infections, that may drive this genetic potential towards exacerbated local inflammation and progressive beta cell loss. On top of this, beta cells set up compensatory responses, such as the unfolded protein response, that become deleterious in the long term. The relative contribution of immune and beta cell drivers may vary and phenotypic subtypes (endotypes) are likely to exist. This dual view argues for the use of circulating biomarkers of both autoimmunity and beta cell stress for disease staging, and for the implementation of both immunomodulatory and beta cell-protective therapeutic strategies. Graphical abstract.

CD70 Modulates Thymocyte Development and Type 1 Diabetes in NOD Mice

CD70 is a tumor necrosis factor (TNF) superfamily member expressed by activated T cells, B cells, dendritic cells, NK cells and mature macrophages. It interacts with CD27, a TNF receptor family member on T cells that regulates T cell activation, proliferation and survival. Type 1 diabetes (T1D) is caused by T cell mediated beta cell destruction, however, T1D-prone NOD mice developed accelerated disease in the absence of CD70. This was associated with altered proportions of double negative vs. double positive thymocytes in CD70 knockout mice, indicating that CD70 modulates global thymopoiesis. Also affected, but in opposing directions, was the development of regulatory T cells (Treg) and natural killer T (NKT) cells, two immunoregulatory lymphocyte subsets that are functionally and numerically deficient in NOD mice. Tregs frequencies were reduced by 23% and 32% in CD70 heterozygous (CD70-/+) and homozygous (CD70-/-) knockout mice. In contrast, NKT cell frequencies were respectively increased by 48% and 61% in CD70-/+ and CD70-/- mice compared to standard NOD mice. The higher level of NKT cells in CD70 knockout mice was unexpectedly associated with reduced levels of thymic CD1d, a molecule that is required for NKT cell development. Collectively, our results show that CD70 controls the development of several T cell populations that are known to modulate T1D development in NOD mice and that, in the absence of CD70, T1D is exacerbated. These finding may facilitate the development of clinical therapeutics aimed at enhancing the stimulatory function of CD70 as a way to suppress disease. Disclosure C. Ye: None. J. Driver: None. M.V. Wiles: None. D. Serreze: None.

Antithymocyte globulin therapy for patients with recent-onset type 1 diabetes: 2year results of a randomised trial.

Aims/hypothesisType 1 diabetes results from T cell mediated destruction of beta cells. We conducted a trial of antithymocyte globulin (ATG) in new-onset type 1 diabetes (the Study of Thymoglobulin to ARrest T1D [START] trial). Our goal was to evaluate the longer-term safety and efficacy of ATG in preserving islet function at 2 years.MethodsA multicentre, randomised, double-blind, placebo-controlled trial of 6.5 mg/kg ATG (Thymoglobulin) vs placebo in patients with new-onset type 1 diabetes was conducted at seven university medical centres and one Children’s Hospital in the USA. The site-stratified randomisation scheme was computer generated at the data coordinating centre using permuted-blocks of size 3 or 6. Eligible participants were between the ages of 12 and 35, and enrolled within 100 days from diagnosis. Subjects were randomised to 6.5 mg/kg ATG (thymoglobulin) vs placebo in a 2:1 ratio. Participants were blinded, and the study design included two sequential patient-care teams: an unblinded study-drug administration team (for the first 8 weeks), and a blinded diabetes management team (for the remainder of the study). Endpoints assessed at 24 months included meal-stimulated C-peptide AUC, safety and immunological responses.ResultsFifty-eight patients were enrolled; at 2 years, 35 assigned to ATG and 16 to placebo completed the study. The pre-specified endpoints were not met. In post hoc analyses, older patients (age 22–35 years) in the ATG group had significantly greater C-peptide AUCs at 24 months than placebo patients. Using complete preservation of baseline C-peptide at 24 months as threshold, nine of 35 ATG-treated participants (vs 2/16 placebo participants) were classified as responders; nine of 11 responders (67%) were older. All participants reported at least one adverse event (AE), with 1,148 events in the 38 ATG participants vs 415 in the 20 placebo participants; a comparable number of infections were noted in the ATG and placebo groups, with no opportunistic infections nor difficulty clearing infections in either group. Circulating T cell subsets depleted by ATG partially reconstituted, but regulatory, naive and central memory subsets remained significantly depleted at 24 months. Beta cell autoantibodies did not change over the 24 months in the ATG-treated or placebo participants. At 12 months, ATG-treated participants had similar humoral immune responses to tetanus and HepA vaccines as placebo-treated participants, and no increased infections.Conclusions/interpretationA brief course of ATG substantially depleted T cell subsets, including regulatory cells, but did not preserve islet function 24 months later in the majority of patients with new-onset type 1 diabetes. ATG preserved C-peptide secretion in older participants, which may warrant further study.Trial registration:ClinicalTrials.gov NCT00515099Public data repository:START datasets are available in TrialShare www.itntrialshare.org Funding:National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH). The trial was conducted by the Immune Tolerance Network (ITN).Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-3917-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

ELISA Test for Analyzing of Incidence of Type 1 Diabetes Autoantibodies (GAD and IA2) in Children and Adolescents.

Introduction:Anti GAD (antibodies on glutamic acid decarboxylase) and anti-IA2 antibodies (against tyrosine phosphatase), today, have their place and importance in diagnosis and prognosis of Type 1 diabetes. Huge number of patients with diabetes mellitus type 1 have these antibodies. Insulin antibodies are of critical importance in diagnosis of diabetes mellitus type 1 for pediatric population.Materials and methods:During 2014, the samples of 80 patients from Clinical Center University Sarajevo (CCUS) Pediatrics clinic’s, Endocrinology department were analyzed on anti-GAD and IA2 antibodies. The samples of serums of all patients were analyzed with ELISA tests using Anti GAD ELISA (IgG) kites from EUROIMMUN company. These are quantitative in vitro tests for human antibodies against decarboxylase of glutamine acid (GAD) and IA2, in serum or EDTA plasm.Results:During the period of one year, in CCUS’s Organizational unit, Institute for Clinical Immunology, 80 samples of patients with anti GAD and IA2 antibodies were analyzed. Out of total number of samples, 41 were male patients, or 51% and 39 female, or 49%. The youngest patient was born in 2012, and the oldest in 1993. Age average was represented by the patients born in 2001. Share of positive results for IA2 antibodies and GAD antibodies was 37% for IA2 antibodies, and 63% for GAD antibodies.Discussion:During an autoimmune – mediated Diabetes mellitus type 1 leads to T-cell mediated destruction of beta cells of pancreatic islets, reduced production of insulin and glucose metabolism. Studies have shown that these bodies are the most intense single marker for identifying persons with increased risk for diabetes development.

Streamlined protocol for TCR sequencing, cloning, and in vivo functional analysis of islet antigen reactive human T cells (BA11P.131)

Abstract Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of insulin secreting beta cells present in the islets of Langerhans. A large body of evidence has implicated T cells as the predominant mediators of beta cell destruction leading to T1D development. The goal of our research is to develop a system for isolation and characterization of diabetogenic CD4+ T cells from the peripheral blood of early onset T1D patients. We were able to obtain a blood sample from a treatment-naïve T1D patient at the time of diagnosis. Through a novel multiplex-nest PCR protocol, we cloned corresponding alpha and beta TCR chains from individual islet antigen-specific T cells and tested them in vitro for antigenic specificity and HLA restriction. Utilizing a humanized TCR/HLA retrogenic mouse system, we are testing human islet antigen reactive TCRs for their in vivo functionality and diabetogenic potential.

Activation of autoreactive CD4+ T cells is synchronous to islet autoantibody seroconversion and shows an IFNγ signature

Islet autoantibody seroconversion is the first detectable evidence of autoimmunity preceding type 1 diabetes. Autoantibodies are markers of pre-diabetes, but T cells mediate beta cell destruction. Little is known about the timing of autoreactive T cell activation against beta cell antigens in the prediabetic period.

A new role for an old player: Do B cells unleash the self-reactive CD8+ T cell storm necessary for the development of type 1 diabetes?

Type I diabetes mellitus is an autoimmune disease mediated by a selective immune-mediated destruction of the insulin containing beta cells within the pancreatic islets of Langerhans. T cells reactive to beta cell-derived antigens are critical for the pathogenesis of type I diabetes, indeed treatments that target T cells are currently in clinical trials. CD8+ T cells may play a particularly crucial role in the onset of hyperglycaemia, as they can mediate beta cell destruction late in the pathogenesis of diabetes. However, the precise steps by which beta cell-reactive CD8+ T cells are activated are poorly understood. In this review we speculate on the possibility that B cells are essential for the activation and expansion of pathogenic CD8+ T cells that cause final beta cell destruction. We also discuss the involvement of different B cell subsets in the aetiology of diabetes.

Resistência à insulina e síndrome metabólica no diabetes melito do tipo 1

Insulin resistance (IR) plays a larger role in the type 1 diabetes mellitus (T1DM) disease process than commonly recognized. Overweight and physical inactivity have increased steadily for the last 20-30 years in children and adolescents in many populations, concurrently with a rising incidence of T1DM. The role of IR in T1DM has only recently been gaining acceptance. This review will focus on how IR influences our current understanding of disease development and metabolic syndrome (MS) in T1DM. Increases in IR by weight gain and sedentarism, associated to decreased beta cell mass by autoimmune process, may disrupt normoglycemia in pre-T1DM individuals. IR may reflect a more aggressive form of autoimmune disease mediated by immuno-inflammatory factors that also mediate beta cell destruction (TNF-alpha and IL-6). These concepts are included in the "accelerator hypothesis". Moreover, family history of T2DM and chronic hyperglycemia (glucotoxicity), occurring after T1DM diagnosis, contribute to decrease peripheral glucose uptake. The onset of diabetic nephropathy (DN) might also contribute to IR and metabolic syndrome (MS) via low-grade inflammation and increased oxidative stress. MS is found between 12 to 40% in T1DM, especially in patients with advanced DN and poor glycemic control. These findings have therapeutic and cardiovascular prognostic implications as children make the transition toward adolescence and young adulthood T1DM.

Is Mortalin a Candidate Gene for T1DM ?

Mortalin has been found to be up-regulated by 2D-protein gel analysis in isolated rodent islets exposed to cytokines. In islets from two rat strains with different sensitivity to the toxic effects of cytokines we observed a significant difference in IL-1β mediated mortalin expression. Constitutive over-expression of rat mortalin in NIH3T3 cells reduced cellular survival in accordance with mortalin being associated to cellular senescence. Hence we consider the gene encoding for mortalin at chromosome 5q31.1 a putative candidate gene in cytokine induced beta-cell destruction. We scanned the human mortalin gene for polymorphisms and identified three novel polymorphisms. Neither the SNPs individually nor as constructed haplotypes showed disease association tested by (E)TDT in a Danish type 1 diabetes (T1DM) population. Furthermore, we tested the D5S500 microsatelite located close to 5q31.1 without finding linkage to (T1DM). In conclusion, the functional data identifying a difference in mortalin expression in IL-1β stimulated islets between two rat strains and over-expression of mortalin in NIH3T3 cells associated with decreased viability suggests a functional role for mortalin in cytokine mediated beta cell destruction; however, the identified polymorphisms did not reveal any association in the presence of linkage disequilibrium of mortalin to T1DM in the Danish population.

Cytokines activate genes of the endocytotic pathway in insulin-producing RINm5F cells.

Cytokines are important humoral mediators of beta cell destruction in autoimmune diabetes. The aim of this study was to identify novel cytokine-induced genes in insulin-producing RINm5F cells, which may contribute to beta cell death or survival. A global gene expression profile in cytokine-exposed insulin-producing RINm5F cells was achieved by automated restriction fragment differential display PCR. The expression of selected candidate genes was confirmed by real-time RT-PCR analysis. Exposure of RINm5F cells to IL-1beta or to a cytokine mixture (IL-1beta, TNF-alpha, IFN-gamma) for 6 h resulted in the differential expression of a functional gene cluster. Apart from the well-known up-regulation of the cytokine-responsive genes iNOS, NF-kappaB, MnSOD and Hsp70, several genes that belong to the functional cluster of the endocytotic pathway were identified. These endocytotic genes comprised: clathrin, megalin, synaptotagmin and calcineurin, which were up-regulated by IL-1beta or the cytokine mixture. In contrast, the expression of the calcineurin inhibitor CAIN and of the GDP/GTP exchange protein Rab3 was down-regulated by cytokines. Other up-regulated cytokine-responsive genes were: agrin, murine adherent macrophage protein mRNA ( MAMA) and transport-associated protein ( TAP1/MTP), whereas the plasma membrane calcium ATPase ( PMCA) 2 and PMCA 3 genes were down-regulated by cytokines. Our results indicate that genes of the endocytotic pathway are regulated by pro-inflammatory cytokines. This might affect the density of cytokine receptors at the beta cell surface and concomitantly the sensitivity of the cells to cytokine toxicity. A better understanding of the functional cross-talk between endocytotic and cytokine signalling pathways could further the development of novel strategies to protect pancreatic beta cells against toxic effects of pro-inflammatory cytokines.

Genetic differentiation of Poly I:C from B:9-23 peptide induced experimental autoimmune diabetes

Type 1 diabetes is an immune-mediated disease, in which T cells of the adaptive immune system mediate beta cell destruction. Recently the innate immune system has been linked to etiopathogenesis of several autoimmune diseases including type 1 diabetes, as innate effector cells (e.g. dendritic cells, monocytes/macrophages and NK cells) can prime and promote or regulate (auto)immune responses. We have previously developed an experimental autoimmune diabetes (EAD) model with insulin peptide B:9-23 immunization in transgenic H-2 dmice expressing the costimulatory molecule B7.1 in their islets (under the Rat Insulin Promotor, RIP). We compared the induction of diabetes with polyinosinic–polycytidylic acid (Poly I:C), a mimic of double stranded viral RNA versus insulin B:9-23 peptide in mice following backcrossing of the B7.1 transgene on to BALB/c mice from original B7.1 C57Bl/6 mice. We find that diabetes induction by Poly I:C is C57Bl/6 associated, whereas B:9-23 peptide induced diabetes and induction of insulin autoantibodies (IAA) are dependent on BALB/c genes. This B:9-23 peptide induced diabetes is consistent with MHC class II H-2 dbeing necessary for the response to this peptide. Of note Poly I:C induction of diabetes was lost while B:9-23 induction was retained with backcrossing to BALB/c mice. Interaction of genes and environment (antigenic epitope and viral mimic) can be important in the pathogenesis of immune mediated diabetes and activation of the innate immune system (e.g. Poly I:C) may be one key determinant.

Immunohistochemical study of caspase-3-expressing cells within the pancreas of non-obese diabetic mice during cyclophosphamide-accelerated diabetes

During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1beta-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.

Fas and Fas ligand immunolocalization in pancreatic islets of NOD mice during spontaneous and cyclophosphamide-accelerated diabetes.

During insulin-dependent diabetes mellitus, immune cells which infiltrate pancreatic islets mediate beta cell destruction over a prolonged asymptomatic prediabetic period. The molecular mechanisms of beta cell death in vivo remain unresolved. At least two major molecular processes of destruction have been proposed. One involves the Fas-FasL (Fas-Fas ligand) system and the other, the perforin pathway. Here, dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of Fas and FasL in the NOD mouse, during spontaneous diabetes (days 21, 40 and 90) and following acceleration of diabetes with cyclophosphamide (days 0, 4, 7, 11 and 14 after cyclophosphamide administration). The expression of the proteins was correlated with advancing disease. FasL was expressed constitutively in most beta cells but not in glucagon or somatostatin cells or islet inflammatory cells and paralleled the loss of insulin immunolabelling with advancing disease. It was also expressed in beta cells of non-diabetes prone CD-1 and C57BL/6 mice from a young age (day 21). Strong immunolabelling for Fas was first observed in extra-islet macrophages and those close to the islet in NOD and non-diabetes-prone mice. During spontaneous and cyclophosphamide diabetes, it was observed in a higher proportion of islet infiltrating macrophages than CD4 and CD8 T cells, concomitant with advancing insulitis. In cyclophosphamide-treated mice, the proportion of Fas-positive intra-islet CD4 and CD8 T cells at day 14 (with and without diabetes) was considerably higher than at days 0, 4, 7 and 11. At days 11 and 14, a proportion of Fas-positive intra-islet macrophages co-expressed interleukin-1beta and inducible nitric oxide synthase. Fas was not detectable in beta cells and other islet endocrine cells during spontaneous and cyclophosphamide induced diabetes. Our results show constitutive expression of FasL in beta cells in the NOD mouse and predominant expression of Fas in intra-islet macrophages and to a lesser extent in T cells prior to diabetes onset. Interleukin-1beta in intra-islet macrophages may induce Fas and inducible nitric oxide synthase expression in an autocrine and paracrine manner and mediate beta cell destruction or even death of some macrophages and T cells. However, other mechanisms of beta cell destruction during spontaneous and cyclophosphamide-accelerated diabetes and independent of Fas-FasL, require examination.

A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis.

Apoptosis is likely to be the main form of beta-cell death in immune-mediated diabetes mellitus in rodents and possibly in humans. Clarification of the regulation of beta-cell death could indicate novel sites for therapeutic intervention in Type I (insulin-dependent) diabetes mellitus. We review the molecular effectors and signal transduction of immune-mediated beta-cell apoptosis. Data obtained on non-obese diabetic (NOD) mice suggest that macrophages and CD4+ T-cells are the main cellular effectors, whereas CD8+ T-cells are more important initiators of the immune process leading to beta-cell death. Perforin could be the effector molecule utilized by CD8+ T-cell initiation, whereas CD4+ mediated beta-cell destruction is mostly dependent on Fas/FasL and the cytokines IFNgamma and TNF-alpha. The macrophage cytokine IL-1beta in combination with IFN-gamma and TNF-alpha, plays an important role for beta-cell dysfunction and death. Signal transduction by these cytokines involves: (i) binding to specific receptors, (ii) signal transduction by cytosolic kinases (especially the so-called mitogen- and stress-activated protein kinases) and/or phosphatases, (iii) mobilization of diverse transcription factors - with nuclear factor kappaB (NF-kappaB), AP-1 and STAT-1 probably playing key roles for beta-cell apoptosis; (iv) up-regulation or down-regulation of gene transcription. Recent data obtained by microarray and proteomic analysis suggest that the process of beta-cell apoptosis depends on the parallel and/or sequential up-regulation and down-regulation of considerable numbers of genes, which can be grouped in gene modules or patterns according to their functions. A detailed characterization of these "gene modules", and of the signalling pathways and transcription factors regulating them could allow us to understand the ultimate mechanisms leading to beta-cell apoptosis.

Prevention of beta cell dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I.

Interleukin-1beta is a potent pro-inflammatory cytokine that has been shown to inhibit islet beta cell function as well as to activate Fas-mediated apoptosis in a nitric oxide-dependent manner. Furthermore, this cytokine is effective in recruiting lymphocytes that mediate beta cell destruction in IDDM onset. The insulin-like growth factor I (IGF-I) has been shown to block IL-1beta actions in vitro. We hypothesized that gene transfer of the insulin-like growth factor I to intact human islets could prevent IL-1beta-induced beta cell dysfunction and sensitization to Fas-triggered apoptosis activation. Intact human islets were infected with adenoviral vectors encoding IGF-I as well as beta-galactosidase and enhanced green fluorescent protein as controls. Adenoviral gene transfer of human IGF-I prevented IL-1beta-mediated nitric oxide production from human islets in vitro as well as the suppression of beta cell function as determined by glucose-stimulated insulin production. Moreover, IGF-I gene transfer prevented IL-1beta-induced, Fas-mediated apoptosis. These results suggest that locally produced IGF-I from cultured islets may be beneficial in maintaining beta cell function and promoting islet survival before and following islet transplantation as a potential therapy for type I diabetes.

Tumor necrosis factor-alpha-activated cell death pathways in NIT-1 insulinoma cells and primary pancreatic beta cells.

Tumor necrosis factor-alpha (TNFalpha) is a potential mediator of beta cell destruction in insulin-dependent diabetes mellitus. We have studied TNF-responsive pathways leading to apoptosis in beta cells. Primary beta cells express low levels of the type I TNF receptor (TNFR1) but do not express the type 2 receptor (TNFR2). Evidence for TNFR1 expression on beta cells came from flow cytometry using monoclonal antibodies specific for TNFR1 and TNFR2 and from RT-PCR of beta cell RNA. NIT-1 insulinoma cells similarly expressed TNFR1 (at higher levels than primary beta cells) as detected by flow cytometry and radio-binding studies. TNF induced NF-kappaB activation in both primary islet cells and NIT-1 cells. Apoptosis in response to TNFalpha was observed in NIT-1 cells whereas apoptosis of primary beta cells required both TNFalpha and interferon-gamma (IFNgamma). Apoptosis could be prevented in NIT-1 cells by expression of dominant negative Fas-associating protein with death domain (dnFADD). Apoptosis in NIT-1 cells was increased by coincubation with IFNgamma, which also increased caspase 1 expression. These data show that TNF-activated pathways capable of inducing apoptotic cell death are present in beta cells. Caspase activation is the dominant pathway of TNF-induced cell death in NIT-1 cells and may be an important mechanism of beta cell damage in insulin-dependent diabetes mellitus.

An immunohistochemical study of macrophage influx and the co-localization of inducible nitric oxide synthase in the pancreas of non-obese diabetic (NOD) mice during disease acceleration with cyclophosphamide.

Cyclophosphamide has been used to accelerate and synchronize diabetes in non-obese diabetic (NOD) mice. It was injected to 70-day-old female NOD mice and its effect on the progression of insulitis studied at days 0, 4, 7, 11 and at onset of diabetes. Pancreatic sections were also examined for the influx of CD4 and CD8 T cells and macrophages following immunofluorescence staining. The kinetics of macrophage immunoreactive cells in the exocrine and intra-islet areas were also investigated. Light and confocal microscopy were-employed to examine the expression and co-localization of inducible nitric oxide synthase following dual- and triple-label immunofluorescence histochemistry. After cyclophosphamide administration, the severity of insulitis remained similar from days 0 to 4 but began to rise at day 7 and markedly by day 11 and at onset of diabetes. At these two later stages, the insulitis scores were close to 100% while in age-matched control groups the insulitis scores were considerably lower. Immunohistochemical staining showed increasing numbers of CD4 and CD8 T cell subsets and macrophages within the islets and in exocrine, sinusoidal and peri-vascular regions. At onset of diabetes, several islets contained prominent clusters of macrophage immunoreactive cells. Macrophage influx into the islets increased sharply from day 7 (mean number per islet: 119 +/- 54 SEM), peaked at day 11 (mean number per islet: 228 +/- 42), and then declined at onset of diabetes (mean number per islet: 148 +/- 49). Several cells with immunolabelling for inducible nitric oxide synthase were detectable from day 7 onwards until the onset of diabetes. Dual- and triple-label immunohistochemistry showed that a significant proportion of macrophages and only a few beta cells contained the enzyme. Macrophages positive for the enzyme were located as clusters or occasionally contiguously, in the peri-islet and intra-islet areas but rarely in the exocrine region. Islets with minimal distribution of macrophages in the peri-islet areas were not positive for inducible nitric oxide synthase. Beta cells positive for the enzyme were observed in islets with significant macrophage infiltration in locations close to macrophages. The present results show that cyclophosphamide administration to female NOD mice results in a rapid influx of CD4 and CD8 cells and macrophages. The marked up-regulation of inducible nitric oxide synthase in a selective proportion of macrophages, within the islets, immediately preceding and during the onset of diabetes suggests that nitric oxide released by islet macrophages may be an important molecular mediator of beta cell destruction in this accelerated model of insulin-dependent diabetes mellitus.

In vitro response to interleukin-1 beta and streptozotocin in pancreatic islets isolated from male and female nonobese diabetic mice.

The aim of the present study was to examine if the islet donor gender influences the beta-cell sensitivity to possible mediators of beta-cell destruction in insulin-dependent diabetes mellitus (IDDM). We have currently addressed this issue by comparing the action of the cytokine interleukin-1 beta (IL-1 beta) and the alkylating agent streptozotocin (STZ), on isolated pancreatic islets derived from prediabetic female and male nonobese diabetic (NOD) mice. In this mouse strain the females have a much higher incidence of spontaneous IDDM than the males. Pancreatic islets were isolated from female and male mice of the same litter, cultured for 6 days and thereafter either 5-50 U/ml IL-1 beta were added for 48 h or 1.8 mM STZ for 30 min. The cytokine exposure caused a strong increase in the medium insulin accumulation in the cultures of islets obtained from both males and females. The insulin and DNA contents were similar when comparing female and male islets before and after cytokine exposure. The male pancreatic islets exhibited a higher rate of islet glucose oxidation than islets obtained from females after IL-1 beta addition. Non-cytokine treated islets from males showed a higher insulin content compared with corresponding female islets. After IL-1 beta addition male and female islets had a similar inhibition of insulin secretion following stimulation by glucose. On the other hand, islets of both genders were equally sensitive to the inhibitory action of STZ, when studied 6 days after STZ incubation, as assessed by glucose oxidation and insulin release experiments. In conclusion the data of the present study did not demonstrate a clear gender difference in the islet beta-cell sensitivity to immune mediated suppression by IL-1 beta or alkylation damage in IDDM-prone NOD mice. However, it cannot be excluded that in vivo, under the influence of sex hormones, a decisive alteration in beta-cell sensitivity to damage might develop.

Inducible nitric oxide synthase in pancreatic islets of the non-obese diabetic mouse: a light and confocal microscopical study of its ontogeny, co-localization and up-regulation following cytokine administration.

Nitric oxide has been shown to mediate beta-cell destruction in rodent islets exposed to interleukin 1 beta in culture. The inhibitory effect is potentiated by tumour necrosis factor-alpha and interferon-gamma. Cytokine stimulation leads to gene transcription and translation of inducible nitric oxide synthase, the biosynthetic enzyme of nitric oxide. In the non-obese diabetic mouse, progressive invasion of pancreatic islets by immune cells may lead to local production of inflammatory cytokines, resulting in inducible nitric oxide synthase expression within the islets. In this study, the ontogeny of this enzyme and its cellular expression were examined in pancreatic sections of female non-obese diabetic mice by double-label immunofluorescence. Light and confocal microscopy were employed to study the up-regulation, co-localization and immunocytoplasmic distribution of the enzyme in female non-obese, diabetic and Swiss mice following cytokine treatment. From day 40 to day 220 a small number of beta-cells and a proportion of macrophages, usually in peri-islet and exocrine areas, expressed the enzyme. At onset of diabetes, an increasing number of macrophages within and surrounding the islets were positive for the enzyme. Treatment of day 60 female non-obese diabetic mice with interleukin 1 beta alone and in combination with tumour necrosis factor-alpha and/or interferon-gamma resulted in a significant influx of macrophages into the pancreas, while this was lower in female Swiss mice treated similarly. Cytokine administration led to intense but sometimes eccentric immunocytoplasmic labelling for the enzyme in a considerable proportion of macrophages and beta-cells. Macrophages positive for inducible nitric oxide synthase were located in peri- and intra-islet areas, being distal and adjacent to enzyme-positive and negative beta-cells. Treatment with tumour necrosis factor-alpha and/or interferon-gamma did not lead to enzyme up-regulation. These results show that in the non-obese diabetic mouse there is low and sustained expression of islet inducible nitric oxide synthase in the prediabetic period, which is followed by an increase around onset. However, treatment of female non-obese diabetic and Swiss mice with interleukin-1 beta, alone or together with tumour necrosis factor-alpha and/or interferon-gamma leads to a marked expression of this enzyme within macrophages and beta-cells.