We identified autoantibodies (AAb) reacting with a variant IA-2 molecule (IA-2var) that has three amino acid substitutions (Cys27, Gly608, and Pro671) within the full-length molecule. We examined IA-2var AAb in first-degree relatives of type 1 diabetes (T1D) probands from the TrialNet Pathway to Prevention Study. The presence of IA-2var–specific AAb in relatives was associated with accelerated progression to T1D in those positive for AAb to GAD65 and/or insulin but negative in the standard test for IA-2 AAb. Furthermore, relatives with single islet AAb (by traditional assays) and carrying both IA-2var AAb and the high-risk HLA-DRB1*04-DQB1*03:02 haplotype progress rapidly to onset of T1D. Molecular modeling of IA-2var predicts that the genomic variation that alters the three amino acids induces changes in the three-dimensional structure of the molecule, which may lead to epitope unmasking in the IA-2 extracellular domain. Our observations suggest that the presence of AAb to IA-2var would identify high-risk subjects who would benefit from participation in prevention trials who have one islet antibody by traditional testing and otherwise would be misclassified as “low risk” relatives.

We describe a protective effect on autoimmune diabetes and reduced destructive insulitis in NOD.scid recipients following splenocyte injections from diabetic NOD donors and sorted CD19+ cells compared with NOD.scid recipients receiving splenocytes alone. This protective effect was age specific (only CD19+ cells from young NOD donors exerted this effect; P < 0.001). We found that the CD19+IgM+ cell is the primary subpopulation of B cells that delayed transfer of diabetes mediated by diabetogenic T cells from NOD mice (P = 0.002). Removal of IgM+ cells from the CD19+ pool did not result in protection. Notably, protection conferred by CD19+IgM+ cotransfers were not dependent on the presence of Tregs, as their depletion did not affect their ability to delay onset of diabetes. Blockade of IL-10 with neutralizing antibodies at the time of CD19+ cell cotransfers also abrogated the therapeutic effect, suggesting that IL-10 secretion was an important component of protection. These results were strengthened by ex vivo incubation of CD19+ cells with IL-5, resulting in enhanced proliferation and IL-10 production and equivalently delayed diabetes progression (P = 0.0005). The potential to expand CD19+IgM+ cells, especially in response to IL-5 stimulation or by pharmacologic agents, may be a new therapeutic option for type 1 diabetes.

OBJECTIVE The characterization of diverse subtypes of diabetes is a dynamic field of clinical research and an active area of discussion. The objective of this study was to identify new antigenic determinants in the neuroendocrine autoantigen IA-2 (ICA512) and assess whether circulating autoantibodies directed to new IA-2 epitopes identify autoimmune diabetes in young and adult populations with diabetes. RESEARCH DESIGN AND METHODS Clinically diagnosed patients with type 2 diabetes (n = 258; diabetes duration: 0.01–31 years) were evaluated using a new biomarker detecting autoantibodies directed to the extracellular domain of the neuroendocrine autoantigen IA-2 (IA-2ec). The proportion of IA-2ec autoantibodies was also evaluated in newly diagnosed patients with type 1 diabetes (n = 150; diabetes duration: 0.04–0.49 years). In addition, IA-2 (intracellular domain), GAD65, and zinc transporter 8 autoantibodies were assayed. RESULTS IA-2ec autoantibodies were detected in patients with type 1 diabetes and, surprisingly, in 5% of patients with type 2 diabetes without serologic responses to other IA-2 antigenic epitopes or other islet autoantigens. We also assessed the ability of IA-2ec–derived peptides to elicit CD4+ T-cell responses by stimulating peripheral blood mononuclear cells from patients with type 1 diabetes (n = 18) and HLA-matched healthy subjects (n = 13) with peptides and staining with the peptide/DQ8-specific tetramers, observing disease-associated responses to previously unreported epitopes within IA-2ec. CONCLUSIONS We developed a new antibody biomarker identifying novel antigenic determinants within the N terminus of IA-2. IA-2ec autoantibodies can be detected in patients with type 1 diabetes and in a subgroup of adult autoimmune patients with type 2 diabetes phenotype negative for conventional islet autoantibody testing. These observations suggest that islet autoimmunity may be more common in clinically diagnosed type 2 diabetes than previously observed.

Summary The Network for Pancreatic Organ donors with Diabetes (nPOD) programme was developed in response to an unmet research need for human pancreatic tissue obtained from individuals with type 1 diabetes mellitus and people at increased risk [i.e. autoantibody (AAb)-positive] for the disease. This necessitated the establishment of a type 1 diabetes-specific AAb screening platform for organ procurement organizations (OPOs). Assay protocols for commercially available enzyme-linked immunosorbent assays (elisas) determining AAb against glutamic acid decarboxylase (GADA), insulinoma-associated protein-2 (IA-2A) and zinc transporter-8 (ZnT8A) were modified to identify AAb-positive donors within strict time requirements associated with organ donation programmes. These rapid elisas were evaluated by the international islet AAb standardization programme (IASP) and used by OPO laboratories as an adjunct to routine serological tests evaluating donors for organ transplantation. The rapid elisas performed well in three IASPs (2011, 2013, 2015) with 98-100% specificity for all three assays, including sensitivities of 64–82% (GADA), 60–64% (IA-2A) and 62–68% (ZnT8A). Since 2009, nPOD has screened 4442 organ donors by rapid elisa; 250 (5·6%) were identified as positive for one AAb and 14 (0.3%) for multiple AAb with 20 of these cases received by nPOD for follow-up studies (14 GADA+, two IA-2A+, four multiple AAb-positive). Rapid screening for type 1 diabetes-associated AAb in organ donors is feasible, allowing for identification of non-diabetic, high-risk individuals and procurement of valuable tissues for natural history studies of this disease.

There is a paucity of literature describing metabolic and histological data in adult-onset autoimmune diabetes. This subgroup of diabetes mellitus affects at least 5% of clinically diagnosed type 2 diabetic patients (T2DM) and it is termed Latent Autoimmune Diabetes in Adults (LADA). We evaluated indexes of insulin secretion, metabolic assessment, and pancreatic pathology in clinically diagnosed T2DM patients with and without the presence of humoral islet autoimmunity (Ab). A total of 18 patients with at least 5-year duration of clinically diagnosed T2DM were evaluated in this study. In those subjects we assessed acute insulin responses to arginine, a glucose clamp study, whole-body fat mass and fat-free mass. We have also analyzed the pancreatic pathology of 15 T2DM and 43 control cadaveric donors, using pancreatic tissue obtained from all the T2DM organ donors available from the nPOD network through December 31, 2013. The presence of islet Ab correlated with severely impaired β-cell function as demonstrated by remarkably low acute insulin response to arginine (AIR) when compared to that of the Ab negative group. Glucose clamp studies indicated that both Ab positive and Ab negative patients exhibited peripheral insulin resistance in a similar fashion. Pathology data from T2DM donors with Ab or the autoimmune diabetes associated DR3/DR4 allelic class II combination showed reduction in beta cell mass as well as presence of autoimmune-associated pattern A pathology in subjects with either islet autoantibodies or the DR3/DR4 genotype. In conclusion, we provide compelling evidence indicating that islet Ab positive long-term T2DM patients exhibit profound impairment of insulin secretion as well as reduced beta cell mass seemingly determined by an immune-mediated injury of pancreatic β-cells. Deciphering the mechanisms underlying beta cell destruction in this subset of diabetic patients may lead to the development of novel immunologic therapies aimed at halting the disease progression in its early stage.

We used the LEW1.WR1 rat model of Kilham Rat Virus (KRV)-induced type 1 diabetes (T1D) to test the hypothesis that disease mechanisms are linked with beta cell infection and intra-islet immune activation prior to insulitis. KRV induces genes involved in type I and type II interferon pathways in islet cell lines in vitro and in islets from day-5-infected animals in vivo via mechanisms that do not involve insulitis, beta cell apoptosis, or impaired insulin expression. Immunohistochemistry studies indicated that KRV protein is expressed in beta cells 5 days following infection. KRV induces the phosphorylation of Janus Kinase 1/2 (JAK1/2) and signal transducer and activator of transcription 1 (STAT-1) in islet cells via a mechanism that could involve TLR9 and NF-κB pathways. These data demonstrate for the first time that KRV-induced islet destruction is associated with beta cell infection and intra-islet innate immune upregulation early in the disease process.

AimMetabolic syndrome affects a large proportion of the population and increases cardiovascular disease risk. Because metabolic syndrome often co-exists clinically with atherosclerosis, it is difficult to distinguish the respective contributions of the components to vascular abnormalities. Accordingly, we utilized a porcine dietary model of metabolic syndrome without atherosclerosis to investigate early abnormalities of vascular function and signaling.MethodsThirty-two Yucatan micropigs were fed either a high-fat, high-simple-sugar, high-calorie (HFHS) or standard chow diet (STD) for 6months. Neither diet contained added cholesterol. Blood pressure and flow-mediated vasodilatation were assessed at baseline and 6months. Aortas were harvested at 6months to assess histology, insulin signaling, and endothelial nitric oxide (eNOS) phosphorylation.ResultsHFHS pigs developed characteristics of metabolic syndrome including obesity, dyslipidemia, and insulin resistance, but without histologic evidence of atherosclerosis. Although arterial intima-media thickness did not differ between groups, vascular dysfunction in HFHS was manifest by increased blood pressure and impaired flow-mediated vasodilation of the femoral artery. Compared with STD, aortas from HFHS exhibited increased p85α expression and Ser307 IRS-1 phosphorylation, and blunted insulin-stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase activity. In the absence of insulin stimulation, aortic Akt Ser473-phosphorylation was greater in HFHS than in STD. With insulin stimulation, Akt phosphorylation increased in STD, but not HFHS. Insulin-induced Ser1177-phosphorylation of eNOS was decreased in HFHS, compared with STD.ConclusionsPigs with metabolic syndrome develop early vascular dysfunction and aortic insulin signaling abnormalities, and could be a useful model for early human vascular abnormalities in this condition.

nPOD actively promotes a multidisciplinary and unbiased approach toward a better understanding of T1D and identify novel therapeutic targets, through its focus on the study of human samples. Unique to this effort is the coordination of collaborative efforts and real-time data sharing. Studies supported by nPOD are providing direct evidence that human T1D isa complex and heterogeneous disease, in which a multitude of pathogenic factors may be operational and may contribute to the onset of the disease. Importantly, the concept that beta cell destruction is almost completed and that the autoimmune process is almost extinguished soon after diagnosis is being challenged. nPOD investigators are exploring the hypothesis that beta cell dysfunction may also be a significant cause of hyperglycemia, at least around the time of diagnosis, and are uncovering novel molecules and pathways that are linked to the pathogenesis and etiology of human T1D. The validation of therapeutic targets is also a key component of this effort, with recent and future findings providing new strategic direction for clinical trials.

agreed, as detailed in the text box.

Diabetes mellitus has been tradition ally classified as Type I and Type 2 on the basis ofseveral criteria that generally reflect either insulin deficiency or functional defects in insulin secret ion. In this chapter, we propose a new classification diabetes based on age of onset, body mass index and biomarkers such as islet autoantibodies and DR HLA alleles. In the second part of this chapter, we briefly discuss some novel hypotheses on the possibility of beta-cell regeneration in diabetes in relation to the islet pathology of the disease.

Background The Network for Pancreatic Organ Donors with Diabetes (nPOD) was established to recover and characterize pancreata and related organs from cadaveric organ donors with various risk levels for type 1 diabetes (T1D). These biospecimens are available to investigators for collaborative studies aimed at addressing questions related to T1D natural history and pathogenesis. Research design and methods Organ donors included T1D patients (new onset to long term), non-diabetic autoantibody-positive subjects, non-diabetic controls and individuals with disorders relevant to beta-cell function. Pancreas recovery and transport met transplant-grade criteria. Additional samples recovered included serum, whole blood, spleen and pancreatic and non-pancreatic lymph nodes. Biospecimens were processed for cryopreserved cells, fixed paraffin and fresh frozen blocks and snap frozen samples. T1D autoantibodies, C-peptide levels and high-resolution HLA genotyping for risk alleles were also determined. Results Over 160 donors have been enrolled (ages of 1?day to >90?years). Standard operating procedures were established along with a quality management system. Donor demographics, laboratory assays and histopathological characterizations were shared through an open online informatics system. Biospecimens were distributed to more than 60 investigators. Conclusions The nPOD programme provides access to high quality biospecimens without cost to investigators. Collaborations and open data sharing are emphasized to maximize research potential of each donor. On the basis of initial successes, the nPOD programme is expanding to recover additional organs relevant to T1D pathogenesis and complications from European countries (PanFin network). Copyright (C) 2012 John Wiley & Sons, Ltd.

The issue of beta cell regeneration in human pancreas is probably one of the most controversial aspects of type 1 diabetes research. In this review, we will first describe the known mechanisms underlying beta cell development and expansion in normal human pancreatic development because it is likely that such mechanisms might also play a role in beta cell regeneration. The sensu strictiori definition of beta cells implies replacement of lost beta cell mass by new beta cells. In our discussion, however, we will use the term in a more general way, defining as regeneration the formation of new beta cells, whether or not a loss of beta cells has actually occurred. The potential mechanisms of beta cell regeneration in the human pancreas will be discussed in the second part of this review. In particular, we will analyze beta cell regeneration through proliferation of beta cells, neogenesis from non-beta cell precursors, and transdifferentiation from alpha cells. In the third part of this review, we will explore the arguments for and against the ability of the human pancreas to regenerate functional beta cells in the context of type 1 diabetes and in other pathological conditions.

A seeming majority of research articles pertaining to Type I diabetes (TID) begin by noting that the disorder results from an autoimmune destruction of the insulin-secreting pancreatic beta cells. This concept finds its basis in decades-old observations that at the symptomatic onset of T ID, a chronic inflammatory infiltrate affecting primarily the insulin-containing islets is present in most subjects. This infiltrate consists predominantly of T-lymphocytes, with lesser numbers of B-lymphocytes and macrophages. Apoptotic beta cells, enhanced beta cell expression of Fas, T-lymphocyte expression of Fas-L, hyperexpression of class I MHC on all endocrine cells in insulin-containing islets, and interferon alpha production by beta cells are also noteworthy occurrences often seen with this lesion. The subsequent observation of islet cell autoantibodies in the serum of patients with TID led to its definition as an autoimmune disease (Type 1A diabetes). In this chapter, we will review the pathology of Type 1A diabetes and discuss its possible pathogenesis on the basis of evidence obtained from pancreatic specimens. Finally, we will briefly discuss nonautoimmune forms of TID.