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OBJECTIVE-The TrialNet Study Group evaluated rituximab, a B-cell-depleting monoclonal antibody, for its effect in new-onset patients with type 1A diabetes. Rituximab decreased the loss of C-peptide over the first year of follow-up and markedly depleted B lymphocytes for 6 months after administration. This article analyzes the specific effect of rituximab on multiple islet autoantibodies. RESEARCH DESIGN AND METHODS-A total of 87 patients between the ages of 8 and 40 years received either rituximab or a placebo infusion weekly for four doses close to the onset of diabetes. Autoantibodies to insulin (IAAs), GAD65 (GADAs), insulinoma-associated protein 2 (IA2As), and ZnT8 (ZnT8As) were measured with radioimmunoassays. The primary outcome for this autoantibody analysis was the mean level of autoantibodies during follow-up. RESULTS-Rituximab markedly suppressed IAAs compared with the placebo injection but had a much smaller effect on GADAs, IA2As, and ZnT8As. A total of 40% (19 of 48) of rituximab-treated patients who were IAA positive became IAA negative versus 0 of 29 placebo-treated patients (P , 0.0001). In the subgroup (n = 6) treated within 50 days of diabetes, IAAs were markedly suppressed by rituximab in all patients for 1 year and for four patients as long as 3 years despite continuing insulin therapy. Independent of rituximab treatment, the mean level of IAAs at study entry was markedly lower (P = 0.035) for patients who maintained C-peptide levels during the first year of follow-up in both rituximab-treated and placebo groups. CONCLUSIONS-A single course of rituximab differentially suppresses IAAs, clearly blocking IAAs for .1 year in insulin-treated patients. For the patients receiving insulin for .2 weeks prior to rituximab administration, we cannot assess whether rituximab not only blocks the acquisition of insulin antibodies induced by insulin administration and/or also suppresses preformed insulin autoantibodies. Studies in prediabetic non-insulin-treated patients will likely be needed to evaluate the specific effects of rituximab on levels of IAAs. [ABSTRACT FROM AUTHOR]

A series of recent studies in humans and the NOD mouse model have highlighted the central role that autoimmunity directed against insulin, in particular the insulin B chain 9-23 peptide, may play in the pathogenesis of type 1 diabetes. Both pathogenic and protective T-cell clones recognizing the B:9-23 peptide have been produced. This report describes the successful creation of BDC12-4.1 T-cell receptor (TCR) transgenic mice with spontaneous insulitis in F1 mice (FVB x NOD) and spontaneous diabetes in NOD.RAG-/- (backcross 1 generation). Disease progression is heterogeneous and is modified by a series of genetic factors including heterozygosity (H-2g7/H-2q) versus homozygosity for H-2g7, the presence of additional T-/B-cell receptor-rearranged genes (RAG+ versus RAG-/-), and the insulin 2 gene knockout (the insulin gene expressed in the NOD thymus). Despite lymphopenia, 40% of H-2g7/g7 BDC12-4.1 TCR+ RAG-/- Ins2-/- mice are diabetic by 10 weeks of age. As few as 13,500 transgenic T-cells from a diabetic TCR+ RAG-/- mouse can transfer diabetes to an NOD.scid mouse. The current study demonstrates that the BDC12-4.1 TCR is sufficient to cause diabetes at NOD backcross 1, bypassing polygenic inhibition of insulitis and diabetogenesis. Diabetes 55: 1978-1984, 2006 [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is an autoimmune disease in which pathogenic lymphocytes target autoantigens expressed in pancreatic islets, leading to the destruction of insulin-producing β-cells. Zinc transporter 8 (ZnT8) is a major autoantigen abundantly present on the β-cell surface. This unique molecular target offers the potential to shield β-cells against autoimmune attacks in T1D. Our previous work showed that a monoclonal antibody (mAb43) against cell-surface ZnT8 could home in on pancreatic islets and prevent autoantibodies from recognizing β-cells. This study demonstrates that mAb43 binds to exocytotic sites on the β-cell surface, masking the antigenic exposure of ZnT8 and insulin after glucose-stimulated insulin secretion. In vivo administration of mAb43 to NOD mice selectively increased the proportion of regulatory T cells in the islet, resulting in complete and sustained protection against T1D onset as well as reversal of new-onset diabetes. The mAb43-induced self-tolerance was reversible after treatment cessation, and no adverse effects were exhibited during long-term monitoring. Our findings suggest that mAb43 masking of the antigenic exposure of β-cells suppresses the immunological cascade from B-cell antigen presentation to T cell–mediated β-cell destruction, providing a novel islet-targeted and antigen-specific immunotherapy to prevent and reverse clinical T1D. Article Highlights: mAb43, a zinc transporter 8 (ZnT8)–specific monoclonal antibody, provides lasting protection against autoimmune diabetes in NOD mice. The in vivo islet specificity of mAb43 enables targeted therapy to enhance safety. High-affinity binding of mAb43 to the extracellular surface of ZnT8 shields β-cells from antigenic exposure. β-Cell masking results in a localized increase in regulatory T cells in the pancreatic islet. Prolonged mAb43 treatment clears destructive insulitis, preserves β-cell mass, and reverses seroconversion of insulin autoantibodies in NOD mice. The immunological processes bridging the masking of specific cell-surface autoantigens and the broad suppression of polyspecific T-cell autoimmunity are still unclear. [ABSTRACT FROM AUTHOR]

The article discusses a study concerning patients with diabetes associated autoantibodies (DAA). The study involved 987 diabetic subjects who were tested for DAA. Results show that 768 were positive for antibody (Ab+) while 220 were antibody negative (Ab-). Ab+ subjects were younger during the onset age of diabetes, had lower body mass index (BMI)-Z score and no difference in hemoglobin A1c (HbA1c). It notes that diabetes mellitus (DM) in the Ab- group may be caused by type 2 diabetes.

Type 1 diabetes (T1D) is a disease in which autoimmune attack is directed to the insulin-producing β-cell in the pancreatic islet. Autoantigens on the β-cell surface membrane are specific markers for molecular recognition and targets for engagement by autoreactive B lymphocytes, which produce islet cell surface autoantibody (ICSA) upon activation. Here, we report the cloning of an ICSA (mAb43) that recognizes a major T1D autoantigen, ZnT8, with a subnanomolar binding affinity and conformation specificity. We demonstrate that cell-surface binding of mAb43 protects the extracellular epitope of ZnT8 against immunolabeling by serum ICSA from a patient with T1D. Further, mAb43 exhibits in vitro and ex vivo specificity for islet cells, mirroring the exquisite specificity of islet autoimmunity in T1D. Systemic administration of mAb43 yields a pancreas-specific biodistribution in mice and islet homing of a mAb43-linked imaging payload through the pancreatic vasculature, thereby validating the in vivo specificity of mAb43. Identifying ZnT8 as a major antigenic target of ICSA allows for research into the molecular recognition and engagement of autoreactive B cells in the chronic phase of T1D progression. The in vivo islet specificity of mAb43 could be further exploited to develop in vivo imaging and islet-specific immunotherapies. [ABSTRACT FROM AUTHOR]

Long-term studies of children at high genetic risk for type 1 diabetes (T1D) have identified groups of children with diabetes related autoantibodies. The ability to differentiate subjects at the highest risk for progression to T1D will be important for planned prevention studies. DAISY follows two high risk cohorts (young first degree relatives of subjects with T1D and neonates from the general population with moderate or high risk HLA genotypes). Subjects are screened at 9, 15 and 24 months of age and then annually for autoantibodies to insulin (IAA), GAD65 and ICA-512. Subjects who are identified as positive for any autoantibody are then tested every 3-6 months. Positive subjects were classified as follows: transiently positive (+for ≥ 1 antibody (confirmed by blinded duplicate sample testing) but negative at the most recent visit, n=82), persistently positive with no T1D (+ for ≥ 1 antibody and + at the most recent visit, n=65) and progressed to diabetes (regardless of autoantibody status, n=30). We hypothesized that characteristics of autoantibody positivity would differentiate the persistently positive children from those progressing to diabetes. The persistent and diabetes groups had a higher proportion with multiple antibodies positive at the 1st + test compared to the transient group (transient 1.2%, persistent 22%, diabetes 33% multiple antibodies at first positive, p<0.0001). Children who developed diabetes, compared with the persistent group, had more often multiple antibodies (70% vs. 35%, p<0.0001) and IAA (66% vs. 35%, p=0.003) at their last visit. To analyze the effect of IAA positivity over time, the area under the curve of IAA levels was calculated and divided by duration of the follow-up (AUC/duration). Subjects who developed diabetes had greater IAAAUC/duration of follow-up compared with persistent or transient subjects (ANOVA: p<0.0001). Cox proportional hazard modeling was used to determine the effect of IAA AUC/duration in the persistant and diabetes groups, controlling for family history and the number of positive antibodies at the last non-diabetic visit. Predictors of progression to diabetes included: HLA DR, DQ (Hazard Ratio (HR) 1.75, p = 0.02), age first positive (HR=0.609, p<0.0001) and IAAAUC/duration (HR=3.57 p=0.02). Therefore, IAA AUC may be useful for identifying subjects at high risk for progression to T1D and suggests that IAA levels are related to progression. [ABSTRACT FROM AUTHOR]

Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2-based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies. [ABSTRACT FROM AUTHOR]

Certain HLA class II genes increase the risk for type 1 diabetes (T1D) development while others provide protection from disease development. HLA class II alleles encode MHC proteins on antigen-presenting cells, which function to present peptides and activate CD4 T cells. The DRB1*15:01 (DR15)-DQA1*01:02-DQB1*06:02 (DQ6) haplotype provides dominant protection across all stages of T1D and is a common haplotype found in Caucasians. However, it is present in <1% of people with T1D. Knowing which metabolic, immunologic, and genetic features are unique to individuals who fail genetic protection and develop T1D is important for defining the underlying mechanisms of DQB1*06:02-mediated protection. We describe a T1D cohort with DQB1*06:02 (n = 50) and compare them to individuals with T1D and without DQB1*06:02 (n = 2,759) who were identified over the last 26 years at the Barbara Davis Center for Diabetes. The age at diagnosis was similar between the cohorts and normally distributed throughout childhood and early adulthood. The average hemoglobin A1c was 10.8 ± 2.8% (95 ± 7 mmol/mol) at diagnosis in those DQB1*06:02 positive. The majority of T1D DQB1*06:02+ individuals were positive for one or more islet autoantibodies; however, there was a greater proportion who were islet autoantibody negative compared with those T1D DQB1*06:02- individuals. Interestingly, DQB1*03:02, which confers significant T1D risk, was present in only those DQB1*06:02+ individuals with islet autoantibodies. This is one of the largest studies examining patients presenting with clinical T1D in the presence of DQB1*06:02, which provides a population to study the mechanisms of failed genetic protection against T1D. [ABSTRACT FROM AUTHOR]

Electrochemiluminescence (ECL) assays are high-affinity autoantibodies (Abs) that are more specific than radiobinding assay (RBA) Abs for the prediction of type 1 diabetes (T1D) in natural history studies screening for risk and progression to T1D. We analyzed 603 subjects from the TrialNet Pathway to Prevention Study who were positive for at least one RBA diabetes related Ab and for whom ECL and HLA data were available. We sought to characterize ECL-GADA and ECL-IAA positivity and levels (z-scores) by HLA genotype and haplotypes. Mean age at initial visit was 19.4 ± 13.7 years; 345 (57.2%) were female and 104 (17.2%) carried the high-risk HLA- DR3/4*0302 genotype. At initial visit 425 (70%) of subjects were positive for either ECL-GADA or ECL-IAA, with 207 (34.3%) ECL-IAA positive subjects and 367 (60.9%) ECL-GADA positive subjects. For subjects with high-risk HLA-DR3/4 genotype (n=104), 80 (76.9%) were positive for ECL-GADA, while 42 (40.4%) were positive for ECL-IAA. ECL and RBA-GADA z-scores were higher in subjects with HLA-DR3 haplotypes, while ECL-IAA (but not RBA-IAA) z-scores were associated with HLA-DR4 haplotypes. Associations of ECL and RIA Abs with high-risk HLA haplotypes are shown in the Table. In conclusion, ECL and RBA-GADA positivity are associated with both HLA-DR3 and DR4 haplotypes, whereas only ECL-IAA (but not RBA-IAA) positivity and levels are associated with HLA-DR4 haplotype. Disclosure: T.M. Triolo: None. L. Pyle: None. L. Yu: None. P. Gottlieb: Advisory Panel; Self; Janssen Scientific Affairs, LLC., Tolerion, Inc., Viacyte, Inc. Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Janssen Pharmaceuticals, Inc., JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Mercia/Nova Laboratories, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc. Stock/Shareholder; Self; IM Therapeutics. A. Steck: None. Funding: American Diabetes Association (1-14-CD-17 to A.S.); National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Allergy and Infectious Diseases; Eunice Kennedy Shriver National Institute of Child Health and Human Development; JDRF [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Autoimmune disorders such rheumatoid arthritis (RA) harbor autoantibodies against a variety of post-translationally modified (PTM) proteins. We aimed to investigate whether PTM epitopes of IA-2, a major autoantigen related to type 1 diabetes (T1D), are targeted by autoantibody responses. The extracellular domain of IA-2 (IA-2-ec) was synthesized incorporating deamidated amino acid residues (IA-2ec-PTM) previously found to elicit T cell responses in T1D patients. IA-2ec-PTM was then subjected to immunoprecipitation with sera from T1D patients. Notably, autoantibody reactivity towards IA-2ec-PTM was present in 28% of T1D patients as compared to ~9% of serum reactivity against the native IA-2ec. The area under the ROC curve (ROC AUC) of IA-2ec-PTM autoantibodies revealed that these autoantibodies can adequately distinguish between T1D patient and control groups (ROC AUC: 0.7132; P < 0.0001). Relatives of T1D probands (from the TrialNet Pathway to Prevention Study) carrying both IA-2ec-PTM AAb and the high-risk HLA-DRB1*04-DQB1*03:02 haplotype progressed rapidly to onset of T1D (Stage 3) compared to those relatives who did not carry these immunologic abnormalities (P = 0.003). Understanding the mechanisms underlying PTMs of islet autoantigens, such as IA-2ec-PTM, is critical to develop new research to determine the ability of modified epitopes to induce islet autoimmunity, their potential role as biomarkers of islet cell injury as well as breaking immune tolerance to islet cell antigens through neo-antigen formation. Disclosure: M.J. Acevedo-Calado: None. S. Pietropaolo: None. L. Yu: None. A.W. Michels: Stock/Shareholder; Self; IM Therapeutics. M. Pietropaolo: None. Funding: National Institutes of Health [ABSTRACT FROM AUTHOR]

Progression to clinical type 1 diabetes varies among children who develop β-cell autoantibodies. Differences in autoantibody patterns could relate to disease progression and etiology. Here we modeled complex longitudinal autoantibody profiles by using a novel wavelet-based algorithm. We identified clusters of similar profiles associated with various types of progression among 600 children from The Environmental Determinants of Diabetes in the Young (TEDDY) birth cohort study; these children developed persistent insulin autoantibodies (IAA), GAD autoantibodies (GADA), insulinoma-associated antigen 2 autoantibodies (IA-2A), or a combination of these, and they were followed up prospectively at 3- to 6-month intervals (median follow-up 6.5 years). Children who developed multiple autoantibody types (n = 370) were clustered, and progression from seroconversion to clinical diabetes within 5 years ranged between clusters from 6% (95% CI 0, 17.4) to 84% (59.2, 93.6). Children who seroconverted early in life (median age <2 years) and developed IAA and IA-2A that were stable-positive on follow-up had the highest risk of diabetes, and this risk was unaffected by GADA status. Clusters of children who lacked stable-positive GADA responses contained more boys and lower frequencies of the HLA-DR3 allele. Our novel algorithm allows refined grouping of β-cell autoantibody-positive children who distinctly progressed to clinical type 1 diabetes, and it provides new opportunities in searching for etiological factors and elucidating complex disease mechanisms. [ABSTRACT FROM AUTHOR]

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B-chain amino acids 9-23 (B:9-23), which are known to be a critical self-antigen-driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment. [ABSTRACT FROM AUTHOR]

The molecular signaling mechanisms between glomerular cell types during initiation/progression of diabetic kidney disease (DKD) remain poorly understood. We compared the early transcriptome profile between DKD-resistant C57BL/6J and DKD-susceptible DBA/2J (D2) glomeruli and demonstrated a significant downregulation of essential mitochondrial genes in glomeruli from diabetic D2 mice, but not in C57BL/6J, with comparable hyperglycemia. Diabetic D2 mice manifested increased mitochondrial DNA lesions (8-oxoguanine) exclusively localized to glomerular endothelial cells after 3 weeks of diabetes, and these accumulated over time in addition to increased urine secretion of 8-oxo-deoxyguanosine. Detailed assessment of glomerular capillaries from diabetic D2 mice demonstrated early signs of endothelial injury and loss of fenestrae. Glomerular endothelial mitochondrial dysfunction was associated with increased glomerular endothelin-1 receptor type A (Ednra) expression and increased circulating endothelin-1 (Edn1). Selective Ednra blockade or mitochondrial-targeted reactive oxygen species scavenging prevented mitochondrial oxidative stress of endothelial cells and ameliorated diabetes-induced endothelial injury, podocyte loss, albuminuria, and glomerulosclerosis. In human DKD, increased urine 8-oxo-deoxyguanosine was associated with rapid DKD progression, and biopsies from patients with DKD showed increased mitochondrial DNA damage associated with glomerular endothelial EDNRA expression. Our studies show that DKD susceptibility was linked to mitochondrial dysfunction, mediated largely by Edn1-Ednra in glomerular endothelial cells representing an early event in DKD progression, and suggest that cross talk between glomerular endothelial injury and podocytes leads to defects and depletion, albuminuria, and glomerulosclerosis. [ABSTRACT FROM AUTHOR]

An allelic variant of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), PTPN22(R620W), is strongly associated with type 1 diabetes (T1D) in humans and increases the risk of T1D by two- to fourfold. The NOD mouse is a spontaneous T1D model that shares with humans many genetic pathways contributing to T1D. We hypothesized that the introduction of the murine orthologous Ptpn22(R619W) mutation to the NOD genome would enhance the spontaneous development of T1D. We microinjected CRISPR-Cas9 and a homology-directed repair template into NOD single-cell zygotes to introduce the Ptpn22(R619W) mutation to its endogenous locus. The resulting Ptpn22(R619W) mice showed increased insulin autoantibodies and earlier onset and higher penetrance of T1D. This is the first report demonstrating enhanced T1D in a mouse modeling human PTPN22(R620W) and the utility of CRISPR-Cas9 for direct genetic alternation of NOD mice. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is caused by autoreactive T cells that recognize pancreatic islet antigens and destroy insulin-producing β-cells. This attack results from a breakdown in tolerance for self-antigens, which is controlled by ectopic antigen expression in the thymus and pancreatic lymph nodes (PLNs). The autoantigens known to be involved include a set of islet proteins, such as insulin, GAD65, IA-2, and ZnT8. In an attempt to identify additional antigenic proteins, we performed an expression-based genome-wide association study using microarray data from 118 arrays of the thymus and PLNs of T1D mice. We ranked all 16,089 protein-coding genes by the likelihood of finding repeated differential expression and the degree of tissue specificity for pancreatic islets. The top autoantigen candidate was vitamin D-binding protein (VDBP). T-cell proliferation assays showed stronger T-cell reactivity to VDBP compared with control stimulations. Higher levels and frequencies of serum anti-VDBP autoantibodies (VDBP-Abs) were identified in patients with T1D (n = 331) than in healthy control subjects (n = 77). Serum vitamin D levels were negatively correlated with VDBP-Ab levels in patients in whom T1D developed during the winter. Immunohistochemical localization revealed that VDBP was specifically expressed in α-cells of pancreatic islets. We propose that VDBP could be an autoantigen in T1D. [ABSTRACT FROM AUTHOR]

The HLA-DRB1*15:01-DQA1*01:02-DQB1*06:02 haplotype is linked to protection from the development of type 1 diabetes (T1D). However, it is not known at which stages in the natural history of T1D development this haplotype affords protection. We examined a cohort of 3,358 autoantibody-positive relatives of T1D patients in the Pathway to Prevention (PTP) Study of the Type 1 Diabetes TrialNet. The PTP study examines risk factors for T1D and disease progression in relatives. HLA typing revealed that 155 relatives carried this protective haplotype. A comparison with 60 autoantibody-negative relatives suggested protection from autoantibody development. Moreover, the relatives with DRB1*15:01-DQA1*01:02-DQB1*06:02 less frequently expressed autoantibodies associated with higher T1D risk, were less likely to have multiple autoantibodies at baseline, and rarely converted from single to multiple autoantibody positivity on follow-up. These relatives also had lower frequencies of metabolic abnormalities at baseline and exhibited no overall metabolic worsening on follow-up. Ultimately, they had a very low 5-year cumulative incidence of T1D. In conclusion, the protective influence of DRB1*15:01-DQA1*01:02-DQB1*06:02 spans from autoantibody development through all stages of progression, and relatives with this allele only rarely develop T1D. [ABSTRACT FROM AUTHOR]

A subset of children develops persistent insulin autoantibodies (IAA; almost always as the only islet autoantibody) without evidence of progression to diabetes. The aim of the current study was the development and characterization of the performance of a nonradioactive fluid phase IAA assay in relation to standard IAA radioassay. We developed a nonradioactive IAA assay where bivalent IAA cross-link two insulin moieties in a fluid phase. The serum samples positive for anti-islet autoantibodies from 150 newly diagnosed patients with diabetes (Barbara Davis Center plus Diabetes Autoantibody Standardization Program [DASP] workshop) and 70 prediabetic subjects who were followed to diabetes were studied. In addition, sequential samples from 64 nondiabetic subjects who were persistently IAA(+) were analyzed. With 99th percentile of specificity, the new assay with the technology from Meso Scale Discovery Company (MSD-IAA) detects as positive 61% (61 of 100) of new-onset patients and 80% (56 of 70) of prediabetic patients compared with our current fluid phase micro-IAA radioassay (mIAA; 44 and 74%, respectively). In addition, MSD-IAA demonstrated better sensitivity than our mIAA from blinded DASP workshop (68 vs. 56% with the same 99% specificity). Of 64 IAA(+) nondiabetic subjects, 25% (8 of 32) who had only IAA and thus the low risk for progression to diabetes were positive with MSD-IAA assay. In contrast, 100% (32 of 32) high-risk children (IAA plus other islet autoantibodies) were positive with MSD-IAA. The IAA detectable by radioassay, but not MSD-IAA, were usually of lower affinity compared with the IAA of the high-risk children. These data suggest that a subset of IAA with current radioassay (not MSD-IAA) represents biologic false positives in terms of autoimmunity leading to diabetes. We hypothesize that factors related to the mechanism of loss of tolerance leading to diabetes determine high affinity and MSD-IAA reactivity. [ABSTRACT FROM AUTHOR]

OBJECTIVE--A major goal in genetic studies of type 1A diabetes is prediction of anti-islet autoimmunity and subsequent diabetes in the general population, as >85% of patients do not have a first-degree relative with type 1A diabetes. Given prior association studies, we hypothesized that the strongest candidates for enhancing diabetes risk among DR3-DQBI*0201/DR4-DQBI*0302 individuals would be alleles of DP and DRB1*04 subtypes and, in particular, the absence of reportedly protective alleles DPB1*0402 and/or DRB1*0403. RESEARCH DESIGN AND METHODS--We genotyped 457 DR3-DQBI*0201/DR4-DQBI*0302 Diabetes Autoimmunity Study of the Young (DAISY) children (358 general population and 99 siblings/offspring of type 1 diabetic patients) at the DPB1, DQB1, and DRB1 loci using linear arrays of immobilized sequence-specific oligonucleotides, with direct sequencing to differentiate DRB1*04 subtypes. RESULTS--By survival curve analysis of DAISY children, the risk of persistently expressing anti-islet autoantibodies is ∼55% for relatives (children with a parent or sibling with type 1 diabetes) in the absence of these two protective alleles vs. 0% (P = 0.02) with either protective allele, and the risk is 20 vs. 2% (P = 0.000) for general population children. Even when the population analyzed is limited to DR3-DQBI*0201/DR4-DQBI*0302 children with DRB1*0401 (the most common DRB1*04 subtype), DPB1*0402 influences development of anti-islet autoantibodies. CONCLUSIONS--The ability to identify a major group of general population newborns with a 20% risk of anti-islet autoimmunity should enhance both studies of the environmental determinants of type 1A diabetes and the design of trials for the primary prevention of anti-islet autoimmunity. Diabetes 56: 2405-2409, 2007 [ABSTRACT FROM AUTHOR]

Diabetic kidney disease (DKD) is the leading single cause of end-stage renal disease in the United States. Approximately 30% of diabetics develop DKD with comparable blood glucose levels, indicating a significant genetic contribution for susceptibility. However, the underlying mechanisms that contribute to differential susceptibility are poorly understood. The glomerulus is the primary site of injury with hypertrophy and podocyte depletion being the hallmarks for progressive DKD. We have demonstrated that mitochondrial oxidative damage accumulation in glomerular endothelial cells, leads to podocyte loss via endothelial-to-podocyte crosstalk in DKD susceptible DBA/2J (D2) mice compared to DKD resistant C57BL/6J (B6) mice. To map genetic loci associated with podocyte depletion after long-term diabetes (6mth), we used the 39 strains of BXD, and parental B6 and D2 mice. We identified a significant cis-acting variant in the promoter of xanthine oxidoreductase (XOR). XORs catalyze the oxidation of purine substrates produce uric acid and reactive oxygen species. XOR expression in the kidney and circulating activity were significantly higher in diabetic D2 compared to B6. XOR inhibition significantly reduced albuminuria, oxidative damage in glomeruli and prevented podocyte depletion in diabetic D2 mice. We used CRISPR/Cas9 to knock-in the XOR risk variant into DKD-resistant B6 mice and generated mutant B6-XORem1 mice with significantly higher XOR activity than B6. This risk variant is a transcription factor binding site to C/EBPβ, we show higher C/EBPβ expression in glomerular endothelium from diabetic B6-XORem1 mice but not in diabetic B6. B6-XORem1 mice had increased mitochondrial oxidative stress in endothelial cells, podocyte depletion, basement membrane thickening, albuminuria, glomerulosclerosis and tubular injury, and these changes were prevented with XOR inhibition. Our data suggest that the identified promoter variant is causal for DKD susceptibility via the C/EBPβ-XOR axis. Disclosure: Q. Wang: None. H. Qi: None. S. Li: None. G. Casalena: None. L. Yu: None. I. S. Daehn: None. Funding: National Institutes of Health [ABSTRACT FROM AUTHOR]

Background: A cohort of children with single ZnT8 autoantibody (ZnT8A) positivity were identified in two large clinical trials, Autoimmunity Screening for Kids (ASK) and Diabetes Autoimmunity Study in the Young (DAISY), and their risk of type 1 diabetes (T1D) have never been studied. Methods: Three cohorts were analyzed using a high-affinity electrochemiluminescence (ECL) ZnT8A assay including 302 new onset T1D patients, 135 children positive for ZnT8A by standard radio-binding assay (RBA) from 23400 children screened in ASK study, and 123 children with multiple autoantibodies or single ZnT8A by RBA from 2547 children in DAISY study. Results: ECL-ZnT8A had a similar sensitivity with RBA-ZnT8A in the cohort of 302 new onset T1D patients (62.3% vs. 61.9%). In ASK, 135/23400 of general population children were positive for RBA-ZnT8A and 64/135 were found as single ZnT8A only without other islet autoantibodies. Of 64 children with single ZnT8A, only 14.1% (9/64) were detectable by ECL-ZnT8A. Antibody-affinity analysis revealed that the ZnT8A in RBA but not confirmed by ECL were of low affinity and they were easily disappeared during the follow-up behaving as 'transient' positivity. In terms of T1D classification from stage 1 to stage 3, positive predictive value (PPV) of ECL-ZnT8A (61/70 [87.1%]) was significantly higher than that of RBA-ZnT8A (73/137 [53.3%]; P<0.0001). The cumulative risk of clinical T1D development with ZnT8A confirmed by ECL (16/66) was significantly enhanced during a short time of follow-up, compared with ZnT8A not confirmed by ECL (1/43; p=0.023). Similarly in DAISY participants, 11/2547 children were identified as single ZnT8A by RBA while only three who developed T1D were detected ECL-ZnT8A. Conclusions: Large proportion of ZnT8A by current standard RBA are single ZnT8A with majority at low risk in population-based screening while ECL-ZnT8A can discriminate high risk from low risk and are highly predictive for T1D development. Disclosure: X. Jia: None. L. He: None. D. Miao: None. K. Waugh: None. C. Geno rasmussen: None. F. Dong: None. M. Rewers: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; RTI, Research Support; Self; Janssen Research & Development, LLC, JDRF. L. Yu: None. Funding: Diabetes Research Center (P30DK116073); JDRF (2-SRA-2018-533-S-B, 2-SRA-2020-965-S-B, 1-SRA-2016-208-S-B); National Institutes of Health (DK32083) [ABSTRACT FROM AUTHOR]

Background: Type 1 diabetes (T1D), a major chronic autoimmune disease starting mostly from childhood, is now predictable with the measurement of four major islet autoantibodies (IAbs) including IAA, GADA, IA-2A and ZnT8A. Currently, there are approximately 1.4 million people affected with T1D in the United States and the incidence has doubled in the last 20 years, especially in young children. Mass screening for eligible subjects and the general population remains inefficiency and laborious bottleneck. Methods: With the platform of our extensively validated high affinity electrochemiluminescence (ECL) assay, we have developed a simple multiplexed ECL assay to combine all four major IAb assays in a single well. Two cohorts were studied including 80 samples from newly diagnosed patients with T1D and 1,140 from Autoimmunity Screening for Kids (ASK) study. Both multiplex ECL assay and standard radio-binding assay (RBA) were performed in parallel. Results: With >99th percentile of specificity for both assays in over 1,000 samples from general population, sensitivity of multiplex ECL assay in new onset patients with T1D were similar to RBA, GADA 75% vs. 71%; IA-2A 91% vs. 90%; IAA 61% vs. 65%; ZnT8A 90% vs. 87%. In ASK cohort, multiplex ECL assay was concordant with RBA among children with multiple IAbs (42/44, 96%). In contrast, only 39% (21/54, p<0.0001) of children with single IAb by RBA were positive by multiplex ECL assay. Conclusion: Multiplex ECL assay illustrated its high sensitivity/specificity as RBA in T1D patients. In general population screening, multiplex ECL assay, similar to single ECL assay, is capable to discriminate high risk IAbs from low risk IAbs generated by RBA. With a multiplexing feature, it provides a high throughput tool with low cost and low sample volume for large scale of population screening for T1D. Disclosure: L. He: None. X. Jia: None. D. Miao: None. C. Geno rasmussen: None. M. Rewers: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; RTI, Research Support; Self; Janssen Research & Development, LLC, JDRF. L. Yu: None. Funding: JDRF (2-SRA-2018-533-S-B, 1-SRA-2016-208-S-B); National Institutes of Health (DK32083); Diabetes Research Center (P30DK116073) [ABSTRACT FROM AUTHOR]

Background: The TrialNet Immune Effects of Oral Insulin (OI) in Relatives at Risk for Type 1 Diabetes Mellitus (TN20) study (2-arm, randomized, open-label clinical trial for Stages 1-2 T1D) observed, in a subset of participants (pts), a decline in insulin autoantibody (Ab) titers associated with an increase in islet-specific CD4+ T cell frequencies in Arm A (OI 67.5 mg/d) but not Arm B (500 mg/2 wks). Here, we compared the 2 arms to assess whether metabolic effects also occur in the 67.5 mg/d treatment arm. Methods: High-risk Ab+ pts (mean age 6.4±3.1 yrs; range 3-16 yrs) were randomized, treated for 6 months, and assessed at 0, 6 and 12 months. We compared the 2 arms for changes of mean glucose and C-peptide response curves (GCRCs) during OGTTs in pts with Diabetes Prevention Trial Risk Score (DPTRS) ≥6.75 (a subset where a metabolic effect of OI has been shown). Results: GCRC shapes and centroids (central point of GCRC shape) in each arm are shown in the Figure. The C-peptide/glucose ratio of centroid coordinates changed little in Arm A but decreased in Arm B (1.03±8.71 vs. -4.39±7.01; p<0.05 adjusted for baseline ratio, age and BMI z-score). Frequencies of Ab+ and HLA DR3/DR4 did not differ between arms. Conclusions: The better metabolic outcome of 67.5 mg/d of OI than 500 mg/2 wks is consistent with the prior finding that immunologic change is associated with 67.5 mg/d, suggesting a linkage of immune and metabolic effects. Disclosure: M. J. Redondo: Advisory Panel; Self; Provention Bio, Inc. Å. Lernmark: None. P. Gottlieb: Advisory Panel; Self; Janssen Research & Development, LLC, Tolerion, Inc., Viacyte, Inc., Other Relationship; Self; ImmunoMolecular Therapeutics, Inc., Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Mercia Pharma Inc., National Institute of Diabetes and Digestive and Kidney Diseases, Precigen, Inc., Tolerion, Inc. J. Sosenko: None. E. K. Sims: None. D. D. Cuthbertson: None. E. A. James: Consultant; Self; Provention Bio, Inc., Research Support; Self; Bristol-Myers Squibb Company, Janssen Pharmaceuticals, Inc., Novartis AG, Sanofi. S. Long: None. W. Kwok: None. L. Yu: None. A. W. Michels: Stock/Shareholder; Self; IM Therapeutics. Funding: National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Allergy and Infectious Diseases; Eunice Kennedy Shriver National Institute of Child Health and Human Development; JDRF [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing β-cells in the pancreatic islets. Islet autoantibodies (IAbs) are indicators of islet autoimmunity. However, IAbs do not fully meet the need for the prediction of future hyperglycemia and the rate of disease progression. We assumed that functional proteins associated with autoimmune cellular process are great sources for biomarkers. In an attempt to identify additional biomarkers, we performed an expression-based genome-wide association study (eGWAS) using microarray data from 169 arrays of the pancreatic islets of T1D rodents. We ranked all 16,099 protein-coding genes by the likelihood of finding repeated differential expression in the pancreatic islets. Our top 20 secreted proteins were screened using the serum samples from newly diagnosed children with diabetes. First, we screened 20 selected candidates with 170 sera including 100 T1D, and 50 type 2 diabetes (T2D) and 20 age matched healthy children. With 7 proteins showing significance, we further did validation study using the second set of 400 samples from newly diagnosed children with diabetes and age matched controls including 200 T1D, 100 T2D, and 100 age-matched controls. We identified 2 serum proteins were significantly changed in T1D vs. both control and T2D and 5 serum proteins were significantly changed in both T1D and T2D vs. control. Serum Osteopontin (OPN) levels were uniquely higher in T1D (p< 0.0001) with no difference between T2D and healthy control subjects. Serum Interleukin 1 receptor antagonist (IL-1RA) levels were lower in T1D compared with both T2D (p<0.001) and healthy subjects (p< 0.0001). Our results suggest that OPN and IL1RA could be the useful biomarkers of T1D in children. Disclosure: X. Jia: None. K. Toda: None. L. He: None. D. Miao: None. S. Yamada: None. L. Yu: None. K. Kodama: None. Funding: JDRF (2-SRA-2019-695-S-B) [ABSTRACT FROM AUTHOR]

Aims: It is important to differentiate the two major phenotypes of adult-onset diabetes, autoimmune type 1 diabetes (T1D) and non-autoimmune type 2 diabetes (T2D). Serum GAD65 autoantibodies (GADA) are the most sensitive biomarker for adult-onset autoimmune T1D, but the clinical value of GADA by current standard radio-binding assays (RBA) remains questionable. The present study focused on the clinical utility of GADA differentiated by a new electrochemiluminescence (ECL) assay in patients with adult-onset diabetes. Methods: Two cohorts of individuals recently diagnosed with diabetes at ≥20 years of age were analyzed including 771 patients 30-70 years olds from the Action LADA study and 2,063 patients 20-45 year olds from the Diabetes in Young Adults (DiYA) study. Clinical characteristics including requirement of early insulin treatment, BMI, and development of multiple islet autoantibodies were analyzed according to the status of RBA-GADA and ECL-GADA, respectively. Results: GADA was the most prevalent and predominant autoantibody, >90% in both cohorts with any positive autoantibody. GADA positivity by either RBA or ECL assay significantly discriminated clinical T1D from T2D. However, patients with ECL-GADA in both cohorts were at greatest risk of insulin treatment, multiple islet autoantibodies and lower BMI (all p<0.0001). Among patients GADA positive by RBA, 32.4% (90/278) in the Action LADA study and 25.6% (32/125) in the DiYA study were negative by ECL assay. Patients with GADA detectable by RBA, not confirmed by ECL, had a phenotype more similar to T2D and these RBA-GADA had lower affinity compared to GADA confirmed by ECL assay. Conclusion: The ECL-GADA assay offers advantages over the RBA-GADA in identification of adult-onset diabetes patients at higher risk of early insulin dependency. Disclosure: X. Jia: None. L. Yu: None. R. D. Leslie: None. The action lada consortium: n/a. The diabetes in young adults (diya) study group: n/a. Y. Gu: None. T. Vartak: None. D. Miao: None. F. Dong: None. S. T. Jerram: None. M. Rewers: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; RTI, Research Support; Self; Janssen Research & Development, LLC, JDRF. A. Ferrara: None. J. M. Lawrence: None. Funding: JDRF (2-SRA-2019-695-S-B); National Institutes of Health (DK32083); 5th Framework Program of the European Union; Centers for Disease Control and Prevention (1U18DP006289) [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) results from the T cell-mediated destruction of insulin-producing beta-cells within pancreatic islets. T cell responses to post-translationally modified hybrid insulin peptides (HIPs) have been implicated in T1D pathogenesis, and we hypothesized that CD4 T cell responses to HIPs precede clinical diabetes onset. Therefore, we measured longitudinal T cell responses to native and hybrid insulin peptides in a cohort of genetically at-risk individuals from the Diabetes AutoImmunity Study in the Young (DAISY). Using cytokine ELISPOT assays, both pro-inflammatory (IFN-γ) and anti-inflammatory (IL-10) cytokine responses to HIPs were more robust than to native peptides. Three of the 38 autoantibody negative individuals (med. age 18.7 yrs) developed islet autoimmunity during 2 years of follow-up, and the C-peptide-islet amyloid polypeptide-2 (C:IAPP-2), C-peptide-neuropeptide Y (C:NPY), and C-peptide-insulin A chain (C:A chain) HIPs induced more frequent pro-inflammatory T cell responses compared to those that did not develop autoantibodies. Five of the 25 autoantibody positive individuals (med. age 18.7 yrs) progressed to clinical T1D during the study, and the C:IAPP-2 HIP more frequently induced a pro-inflammatory response compared to those that did not progress (p=0.037). Additionally, length of time from autoantibody seroconversion correlated with proinflammatory T cell responses to the insulin B:9-23 (B22E) HIP (p=0.007) and the native B:9-23 peptide (p=0.022). T cell responses to the B22E HIP also strongly correlated with insulin autoantibody levels (p=0.0001). Furthermore, responses to the C:IAPP-2 HIP correlated with worsening measurements of blood glucose (A1c %, p=0.046; glucose area under the curve, p=0.054). Monitoring T cell responses to post-translationally modified antigens prior to T1D onset holds promise for determining which individuals are likely to progress to clinical disease and may benefit from immune intervention to prevent T1D onset. Disclosure: A. M. Mitchell: None. M. Rewers: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; RTI, Research Support; Self; Janssen Research & Development, LLC, JDRF. A. W. Michels: Stock/Shareholder; Self; IM Therapeutics. A. Alkanani: None. K. Mcdaniel: None. L. Pyle: None. K. Waugh: None. A. Steck: None. M. Nakayama: None. L. Yu: None. P. Gottlieb: Advisory Panel; Self; Janssen Research & Development, LLC, Tolerion, Inc., Viacyte, Inc., Other Relationship; Self; ImmunoMolecular Therapeutics, Inc., Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Mercia Pharma Inc., National Institute of Diabetes and Digestive and Kidney Diseases, Precigen, Inc., Tolerion, Inc. Funding: National Institutes of Health (DK108868, DK110845, DK032083, DK032493, DK116073); JDRF (3-PDF-2020-943-A-N); Children's Diabetes Foundation; Colorado Clinical and Translational Science Institute (TR002535) [ABSTRACT FROM AUTHOR]

The prevalence of diabetes in many South Asian countries is above 10%, but type 1 diabetes (T1D) is not fully characterized due to a lack of widespread access to islet autoantibody (IA) testing. Determining the prevalence of IA positivity in new and recent onset cases of childhood diabetes in these countries will allow for improved classification of diabetes and may impact treatment. We measured IA directed against glutamic acid decarboxylase 65 (GADA), insulinoma antigen-2 (IA-2A), insulin (IAA) and zinc transporter 8 (ZnT8A) using fluid-phase radiobinding assays from dried blood spot eluents in children with diabetes from the Republic of Maldives (n=64), Bangladesh (n=103) and Pakistan (n=54). We compared the results to youth from our large diabetes Center in Colorado (n=132). South Asian children with clinically diagnosed T1D have a lower rate of positivity for each IA when compared to youth with T1D from Colorado (p<0.0001) (Figure). The majority of youth with T1D in each country were diagnosed at a young age, not overweight, and required insulin treatment. Interestingly, 52% of Bangladeshi youth are antibody negative (p<0.0001 compared to Colorado), yet 92% require insulin treatment, thus highlighting the need to investigate other forms of diabetes such as monogenic causes. Studying the phenotype of diabetes in youth across the globe provides an opportunity to dissect the heterogeneity within childhood onset diabetes. Disclosure: K. Simmons: Other Relationship; Self; Provention Bio, Inc. E. M. Youngkin: None. A. Condie: None. A. Alkanani: None. K. Mcdaniel: None. L. Yu: None. G. D. Ogle: None. A. W. Michels: Stock/Shareholder; Self; IM Therapeutics. Funding: National Institutes of Health (K12DK094712-08) [ABSTRACT FROM AUTHOR]

Islet autoantibody (IAb) assays for a general population screening should ideally be high-troughput, low-cost, and offer high-sensitivity and high positive predictive value in a low-risk population. The Autoimmunity Screening for Kids (ASK) study has compared the performance of the 'gold standard' radiobinding assays (RBAs) and a multiplex electrochemiluminesence assay (ECL) in a large unselected pediatric population. Single IAb by RBA but negative by ECL have been previously shown to be low-affinity and of low predictive value for progression to clinical diabetes. From 2017-2019, ASK screened for IAbs 23,400 Colorado children ages 1-17 y. A high-throughput multiplex ECL assay combining IAbs to insulin, GAD and IA-2 in one single well was used in parallel with standard non-multiplex RBAs. At the initial screening with RBA, 3.0% (697/23400) of children were positive for at least one IAb, including 0.4% (98/23400) for multiple IAbs and 2.6% (599/23400) for a single IAb. Among children with ≥2 IAbs, ECL assay results were 89.8% congruent with RBA. In contrast, only 21.5% (129/599 p<0.0001) of children with single IAb by RBA were ECL positive (i.e., high-affinity). During a median follow-up of 0.5 y (range 0.1-2.5 y), 90% (98/109) of children with a single IAb by both RBA and ECL remained persistently positive and 9.2% of those progressed to ≥2 IAbs. In contrast, 74% (242/328, p=0.0003) of children with a single IAb by RBA but ECL negative remained persistently positive; only 1.8% (p=0.001) of those converted to ≥2 IAbs (half became ECL positive). ASK results suggest that applying the ECL assay as the confirmation test may help to remove nearly 80% of subjects with single IAb from further follow-up and monitoring, with little loss of sensitivity. Alternatively, the multiplex ECL could be used as the primary screening tool to leverage its high throughput and low cost, compared to RBAs. Disclosure: X. Jia: None. L. He: None. D. Miao: None. K. Waugh: None. B.I. Frohnert: None. A. Steck: None. M. Rewers: None. L. Yu: None. C.R. Geno Rasmussen: None. [ABSTRACT FROM AUTHOR]

Human leucocyte antigen (HLA) class II genes confer significant genetic risk for type 1 diabetes (T1D) development and protection. The dominant protection afforded by the DRB1*15:01 (DR15)-DQA1*01:02-DQB1*06:02 (DQ6) haplotype across all stages of T1D is well documented. This is a common haplotype found in Caucasians but present in <1% of people with T1D. Knowing which metabolic, immunologic and genetic features are unique to individuals who fail genetic protection and develop T1D is important for defining the underlying mechanisms of DQB1*06:02-mediated protection. We describe a T1D cohort with DQB1*06:02 (n=50) and compare them to a T1D cohort lacking DQB1*06:02 (n=2,759), which were both identified over the last 26 years at the Barbara Davis Center, a large tertiary referral center in the Rocky Mountain Region. As expected, the average A1C was 10.8 ± 2.8% at diagnosis in 0602+ individuals. The age at diagnosis was similar between the 0602+ and 0602- participants (10.8 ± 6.0 vs. 12.5 ± 8.7 years, respectively, p=0.069) and normally distributed throughout childhood and early adulthood. The majority of 0602+ individuals with T1D were positive for one or more islet autoantibodies; however, there was a greater proportion that were islet autoantibody negative compared to 0602- individuals with T1D (p=0.004). The presence of autoantibodies directed against insulinoma antigen-2 (IA-2) was less common in 0602+ individuals (p=0.005), while the percentage of antibodies directed against GAD65, insulin and zinc transporter 8 were similar between 0602+ and 0602- individuals. Interestingly, the DQ allele on the other chromosome, DQB1*03:02 (DQ8) which confers significant T1D risk, was present in 0602+ individuals with islet autoantibodies and not in individuals without autoantibodies (p=0.002). In conclusion, studying the clinical, immunologic and genetic features of individuals that failed genetic protection holds promise for understanding mechanisms that ultimately lead to beta cell destruction in T1D. Disclosure: K. Simmons: None. A.M. Mitchell: None. A. Alkanani: None. K. McDaniel: None. E. Baschal: None. T. Armstrong: None. L. Pyle: None. L. Yu: None. A.W. Michels: Other Relationship; Self; ImmunoMolecular Therapeutics, LLC. Funding: National Institutes of Health (DK095995, DK108868, DK110845, DK032083, K12DK094712-08); JDRF/Children's Diabetes Foundation (2-SRA-2016-202-5-B); Colorado Clinical and Translational Science Institute (TR001082) [ABSTRACT FROM AUTHOR]

The objective of this study was to determine if islet autoantibodies (IA) can develop de novo in youth ≥10 y. We also explored the risk of progression to T1D in youth seroconverting to IA after 10 y. From the prospective DAISY birth cohort (n=2547), we excluded those who were ever previously IA positive, disenrolled prior to age 10, or had >24 month gap in annual IA testing, leaving 917 for analysis. Seroconversion was defined as IA+ by radiobinding assays for insulin, GAD, IA-2 or ZnT8 autoantibodies, confirmed within 3-6 months. The same autoantibodies were also measured by electrochemiluminescence (ECL) assays, to identify high-affinity IA. IA developed after age 10 at a higher rate (p=0.049) in first-degree relatives than in general population youth with increased-risk HLA (Figure 1); there was no difference by sex or race/ethnicity. Of the 35 IA+ seroconverters, 6 progressed to T1D, during a median follow-up of 5.04 years (IQR: 3.56-8.45). This included 2 of 5 individuals who were multiple IA+ at seroconversion, with at least one IA confirmed as high-affinity by ECL. Among those with a single persistent IA+ at seroconversion, 4/9 youth with high-affinity IA (ECL+) progressed to T1D, compared to 0/21 of the youth with low-affinity IA (ECL-). In conclusion, seroconversion continues after age 10. In our genetically at-risk cohort, all who progress to T1D had high-affinity IA confirmed by ECL. Disclosure: B.I. Frohnert: None. F. Dong: None. K. Waugh: None. A. Steck: None. J.M. Norris: None. L. Yu: None. M. Rewers: None. Funding: JDRF (5-ECR-2017-388-A-N); National Institutes of Health (R01DK32493) [ABSTRACT FROM AUTHOR]

Biomarkers are needed to characterize heterogeneity within populations at risk for type 1 diabetes (T1D). The ratio of proinsulin to C-peptide (PI:C ratio), a biomarker of beta cell dysfunction, has been associated with progression to T1D, but relationships between PI:C ratios and autoantibody (Ab) type and number have not been explored. We sought to characterize PI:C ratios in multiple islet Ab positive, single Ab positive and Ab negative relatives of individuals with T1D. We measured PI:C ratios and Abs with both electrochemiluminescence (ECL) assays (ECL-IAA, ECL-GADA and ECL-IA2)and radio-binding (RBA) assays (mIAA, GADA, IA2A and ZnT8A) in 98 relatives of persons with T1D followed in the Pathway to Prevention Study at the Barbara Davis Center. Of these subjects, 31 were multiple Ab positive, 37 were single Ab positive and 22 were Ab negative at the most recent visit. Eight subjects progressed to T1D over a mean follow-up of 5.5±3.6 years. In cross-sectional analyses, there were no significant differences in PI:C ratios between T1D and/or multiple Ab positive subjects (4.16±4.06) compared to single Ab positive subjects (4.08±4.34) and negative Ab subjects (3.72±3.78) (p=0.92) overall or after adjusting for age, sex and BMI. Higher PI:C ratios were associated with ECL-IA2A (p=0.03) and ECL-IAA (p=0.03) levels, but not with mIAA, GADA, ECL-GADA, IA2A nor ZnT8A levels. In mixed-effects longitudinal models, the trajectories of PI:C ratio over time were not significantly different between the 4 groups, overall or after adjusting for sex, age, and BMI. In conclusion, higher PI:C ratios were associated with ECL-IAA and ECL-IA2A levels, which have previously been associated with progression to T1D. Disclosure: T.M. Triolo: None. L. Pyle: None. S. Seligova: None. L. Yu: None. K. Simmons: None. P. Gottlieb: Consultant; Self; Bristol-Myers Squibb Company, Eli Lilly and Company, Kamada, Tolerion, Inc. Research Support; Self; Intrexon, Janssen Pharmaceuticals, Inc., Mercia/NovaImmune, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc. Stock/Shareholder; Self; IM Therapeutics. Other Relationship; Self; Viacyte, Inc. C. Evans-Molina: None. A. Steck: None. Funding: American Diabetes Association (1-14-CD-17 to A.S.) [ABSTRACT FROM AUTHOR]

Little is known about the prevalence of diabetes autoantibodies (DAA) among young adults newly diagnosed with diabetes (DM). Our objective was to identify patient characteristics associated with presence of DAA among 20-45-year-olds with new-onset DM from 2.3 million Kaiser Permanente California members. Patients underwent research testing for GADA, ZnT8A, IA2A and IAA. IAA results were not counted for patients with prior insulin use. Demographic and clinical data were from electronic health records. Models predicting any DAA positivity were trained on 70% of cases and validated on the remaining 30%. The best predictive logistic regression model was based on area under the receiver operating characteristic curve (AUC), parsimony and interpretability. Of 7,862 patients with incident DM (mean age 36.8±6.0 years, BMI 37.1 ±8.7), 1,669 (mean age 36.9 ±6.0 years, BMI 37.2 ±9.2) were tested for DAA ∼9.5 (±3.6) months post-diagnosis; 130 (7.8%) were positive (5.4% single DAA, 2.4% multiple DAA). Prevalence of GADA was 6.1%, IA2A 2.0%, IAA 1.9%, and ZnT8A 1.7%. The best model (Table) yielded AUCs of 0.74 and 0.68 in training and validation datasets, respectively. Association with DAA was positive for younger age, lower BMI, and glucagon prescription and negative for Asian race. These findings provide insights into patient characteristics associated with antibody positivity in young adults. Disclosure: J.M. Lawrence: None. J. Slezak: None. X. Li: None. L. Yu: None. M. Rewers: None. H.S. Takhar: None. S.B. Sridhar: None. L.I. Vega-Daily: None. J.G. Alexander: None. J. Ritchie: None. S. Saydah: None. G. Imperatore: None. A. Ferrara: None. Funding: Centers for Disease Control and Prevention (1U18DP006289); JDRF (2-SRA-2018-533-S-B) [ABSTRACT FROM AUTHOR]

Previous studies demonstrate ZnT8 is displayed on the β-cell surface upon insulin secretion and IgG from ZnT8A-positive T1D patient sera stains the surface of live pancreatic β-cells. To further identify autoantibodies (AAb) to the ZnT8 extracellular epitope(s) (ZnT8ec) and to develop a ZnT8ec AAb assay, we first stabilized its native structure without the N-terminal domain (a.a 66-369; R325 or W325) by forming ZnT8-intracellular domain-Ab (ZnT8ic) complexes prior to release from liposome membranes. Sulfo-tagged ZnT8-Ab complexes were used as antigen in our electrochemiluminescence (ECL) AAb assay to analyze 130 sera (96 T1D patients, 22 T2D patients and 12 normal controls). Autoantibodies to ZnT8ec were identified in T1D patient sera by cross-reactivity of sera previously identified as exclusively ZnT8R or W positive, and ZnT8A negative sera. The prevalence of ZnT8ec AAbs in T1D patients was 21% (22/96: 5/37 of R+ sera, 7/24 of W+ sera and 8/35 of ZnT8A- sera) compared to 1/22 T2D patients and 0/12 normal controls. These are the first biochemically defined AAbs from T1D sera reported to target the β-cell surface. Further analysis of the role of these autoantibodies in β-cell autoimmunity and T1D etiopathology will be interesting and important for future studies. Disclosure: Y. Gu: None. C. Merriman: None. J.M. Wenzlau: None. L. Yu: None. D. Fu: None. [ABSTRACT FROM AUTHOR]

Hybrid insulin peptides (HIPs) are unique post-translationally modified antigens whereby derivatives of proinsulin (PI) form peptide bonds with endogenous granule peptides. HIPs have been identified in mouse β-cell extracts and human islets by mass spectrometry. Diabetogenic CD4 T cell clones in NOD mice and CD4 T cells from T1D patients are stimulated by HIP ligands and imply that HIPs may also serve as epitopes for humoral immunity. To identify and characterize antibodies (Abs) targeting HIPs among T1D individuals and evaluate their utility as biomarkers, a sensitive and specific ECL-based HIP Ab assay was adapted from the hallmark ECL-IAA protocol. The key determinant for the HIP-Ab assay was design and implementation of a conformational HIP protein probe reflecting derivatives of the HIPs capable of eliciting an antigenic response in CD4 T cell lines from T1D individuals and NOD mice. Sera from a new onset T1D cohort using a PI/IAPP HIP probe showed the presence of anti-PI/IAPP Abs (32.2%, 58/180). However unexpected prevalence was measured among control (18.8%, 18/96) and T2D (25.9%, 29/112) sera samples even when the data was stratified according to HLA genotype. These HIP-Ab epitopes are both conformation and HIP-sequence specific as they are pre-absorbed only with refolded PI/IAPP, but not alternate or random HIPs, linear PI/IAPP HIPs or cyclic PI/IAPP peptides. In sera from prediabetic subjects from the DAISY cohort, 7 of 22 (31.8%) display a peak of HIP-Abs that appears prior to the peak of IAA autoAbs in most cases (87.5%, 7/8). Large number of individuals have PI/IAPP-specific Abs in their circulation, irrespective of T1D disease state or risk. Although HIP-Abs are pervasive and emerge early in the disease process in prospectively followed at risk prediabetic individuals, they are not predictive of disease. As the appearance of PI/IAPP HIP-Abs typically precedes IAAs, it is possible that Abs to other HIPs may serve as earlier biomarkers for ensuing disease. Disclosure: Y. Gu: None. J.M. Wenzlau: None. A.W. Michels: Stock/Shareholder; Self; IM Therapeutics. M. Rewers: None. K. Haskins: None. L. Yu: None. [ABSTRACT FROM AUTHOR]