Evidence that nasal insulin induces immune tolerance to insulin in adults with autoimmune diabetes.

OBJECTIVE: Insulin in pancreatic ß-cells is a target of autoimmunity in type 1 diabetes. In the NOD mouse model of type 1 diabetes, oral or nasal administration of insulin induces immune tolerance to insulin and protects against autoimmune diabetes. Evidence for tolerance to mucosally administered insulin or other autoantigens is poorly documented in humans. Adults with recent-onset type 1 diabetes in whom the disease process is subacute afford an opportunity to determine whether mucosal insulin induces tolerance to insulin subsequently injected for treatment. RESEARCH DESIGN AND METHODS: We randomized 52 adults with recent-onset, noninsulin-requiring type 1 diabetes to nasal insulin or placebo for 12 months. Fasting blood glucose and serum C-peptide, glucagon-stimulated serum C-peptide, and serum antibodies to islet antigens were monitored three times monthly for 24 months. An enhanced ELISpot assay was used to measure the T-cell response to human proinsulin. RESULTS: ß-Cell function declined by 35% overall, and 23 of 52 participants (44%) progressed to insulin treatment. Metabolic parameters remained similar between nasal insulin and placebo groups, but the insulin antibody response to injected insulin was significantly blunted in a sustained manner in those who had received nasal insulin. In a small cohort, the interferon-γ response of blood T-cells to proinsulin was suppressed after nasal insulin. CONCLUSIONS: Although nasal insulin did not retard loss of residual ß-cell function in adults with established type 1 diabetes, evidence that it induced immune tolerance to insulin provides a rationale for its application to prevent diabetes in at-risk individuals.

Measurement of islet cell antibodies in the Type 1 Diabetes Genetics Consortium: efforts to harmonize procedures among the laboratories.

BACKGROUND: and PURPOSE: Three network laboratories measured antibodies to islet autoantigens. Antibodies to glutamic acid decarboxylase (GAD65 [GADA]) and the intracellular portion of protein tyrosine phosphatase (IA-2(ic) [IA-2A]) were measured by similar, but not identical, methods in samples from participants in the Type 1 Diabetes Genetics Consortium (T1DGC). METHODS: All laboratories used radiobinding assays to detect antibodies to in vitro transcribed and translated antigen, but with different local standards, calibrated against the World Health Organization (WHO) reference reagent. Using a common method to calculate WHO units/mL, we compared results reported on samples included in the Diabetes Autoantibody Standardization Program (DASP), and developed standard methods for reporting in WHO units/mL. We evaluated intra-assay and inter-assay coefficient of variation (CV) in blind duplicate samples and assay comparability in four DASP workshops. RESULTS: Values were linearly related in the three laboratories for both GADA and IA-2A, and intra-assay technical errors for values within the standard curve were below 13% for GADA and below 8.5% for IA-2A. Correlations in samples tested 1-2 years apart were >97%. Over the course of the study, internal CVs were 10-20% with one exception, and the laboratories concordantly called samples GADA or IA-2A positive or negative in 96.7% and 99.6% of duplicates within the standard curve. Despite acceptable CVs and general concordance in ranking samples, the laboratories differed markedly in absolute values for GADA and IA-2A reported in WHO units/mL in DASP over a large range of values. LIMITATIONS: With three laboratories using different assay methods (including calibrators), consistent values among them could not be attained. CONCLUSIONS: Modifications in the assays are needed to improve comparability of results expressed as WHO units/mL across laboratories. It will be essential to retain high intra- and inter-assay precision, sensitivity and specificity and to confirm the accuracy of harmonized methods.

Autoimmunity to both proinsulin and IGRP is required for diabetes in nonobese diabetic 8.3 TCR transgenic mice.

T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8(+) T cells specific for IGRP(206-214) (NOD8.3 mice) develop accelerated diabetes that requires CD4(+) T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP(206-214)-specific CD8(+) T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP(206-214)-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4(+) T cell help to activate IGRP(206-214)-specific T cells.

Responses against islet antigens in NOD mice are prevented by tolerance to proinsulin but not IGRP.

Type 1 diabetes (T1D) is characterized by immune responses against several autoantigens expressed in pancreatic beta cells. T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) can induce T1D in NOD mice. However, whether immune responses to multiple autoantigens are caused by spreading from one to another or whether they develop independently of each other is unknown. As cytotoxic T cells specific for IGRP were not detected in transgenic NOD mice tolerant to proinsulin, we determined that immune responses against proinsulin are necessary for IGRP-specific T cells to develop. On the other hand, transgenic overexpression of IGRP resulted in loss of intra-islet IGRP-specific T cells but did not protect NOD mice from insulitis or T1D, providing direct evidence that the response against IGRP is downstream of the response to proinsulin. Our results suggest that pathogenic proinsulin-specific immunity in NOD mice subsequently spreads to other antigens such as IGRP.

Development of autoantibodies to islet antigens during childhood: implications for preclinical type 1 diabetes screening.

OBJECTIVE: Serum islet antibodies signify increased risk for type 1 diabetes (T1D). Knowledge of the relationship between age and seroconversion would guide screening for at-risk individuals. We aimed to determine the effectiveness of islet antibody screening in early childhood, in particular the proportion of negative children who subsequently seroconverted. METHODS: We identified 554 children with a first-degree relative with T1D who had tested negative for islet cell antibodies (ICA) and insulin autoantibodies (IAA) when first screened at a mean age of 7.2 yr. Of 423 who were eligible, 350 consented to re-testing for ICA and IAA and antibodies to glutamic acid decarboxylase (GADAb) and tyrosine phosphatase-like insulinoma antigen IA-2 (IA2Ab) at a mean age of 11.1 yr. GADAb and IA2Ab were measured in 239 of the initial stored samples. RESULTS: Of the 350 children who tested negative at first screening, 12 (3.4%) subsequently seroconverted, becoming positive for ICA (n = 4), IAA (n = 7), GADAb (n = 6) or IA2Ab (n = 2). Of 239 initially negative for ICA and IAA, 8/239 (3.3%) now tested positive for GADAb (n = 7) or IA2Ab (n = 1). Four of these children were positive for GADAb in both tests; the one child initially positive for IA2Ab only was positive for all four antibodies 4.6 yr later and developed diabetes. CONCLUSION: Screening for ICA and IAA failed to identify 2-3% of genetically at-risk children who subsequently developed islet antibodies. Testing for GADAb and IA2Ab would not have avoided this. Maximizing the sensitivity of detecting risk for T1D requires repeat screening for islet antibodies throughout childhood.