Search

Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of at-risk children, associated with progression to islet autoimmunity. We analysed gene expression by RNAseq in CD4+ and CD8+ T cells, NK cells and B cells, and chromatin accessibility by ATACseq in CD4+ T cells, in five genetically at-risk children with islet autoantibodies who progressed to diabetes over a median of 3 years (‘Progressors’) compared to five children matched for sex, age and HLA-DR who had not progressed (‘Non-progressors). In Progressors, differentially expressed genes (DEGs) were largely confined to CD4+ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 Progressors and 11 Non-progressors. Flow cytometry confirmed progression was associated with expansion of CD4+ cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4+ cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4+ T cells play a role in promoting progression to type 1 diabetes.

The risk for autoimmunity and subsequently type 1 diabetes is 10-fold higher in children with a first-degree family history of type 1 diabetes (FDR children) than in children in the general population (GP children). We analyzed children with high-risk HLA genotypes (n = 4,573) in the longitudinal TEDDY birth cohort to determine how much of the divergent risk is attributable to genetic enrichment in affected families. Enrichment for susceptible genotypes of multiple type 1 diabetes–associated genes and a novel risk gene, BTNL2, was identified in FDR children compared with GP children. After correction for genetic enrichment, the risks in the FDR and GP children converged but were not identical for multiple islet autoantibodies (hazard ratio [HR] 2.26 [95% CI 1.6–3.02]) and for diabetes (HR 2.92 [95% CI 2.05–4.16]). Convergence varied depending upon the degree of genetic susceptibility. Risks were similar in the highest genetic susceptibility group for multiple islet autoantibodies (14.3% vs .12.7%) and diabetes (4.8% vs. 4.1%) and were up to 5.8-fold divergent for children in the lowest genetic susceptibility group, decreasing incrementally in GP children but not in FDR children. These findings suggest that additional factors enriched within affected families preferentially increase the risk of autoimmunity and type 1 diabetes in lower genetic susceptibility strata.

Children’s plasma metabolome, especially lipidome, reflects gene regulation and dietary exposures, heralding the development of islet autoantibodies (IA) and type 1 diabetes (T1D). The Environmental Determinants of Diabetes in the Young (TEDDY) study enrolled 8,676 newborns by screening of HLA-DR-DQ genotypes at six clinical centers in four countries, profiled metabolome, and measured concentrations of ascorbic acid, 25-hydroxyvitamin D [25(OH)D], and erythrocyte membrane fatty acids following birth until IA seroconversion under a nested case-control design. We grouped children having an initial autoantibody only against insulin (IAA-first) or GAD (GADA-first) by unsupervised clustering of temporal lipidome, identifying a subgroup of children having early onset of each initial autoantibody, i.e., IAA-first by 12 months and GADA-first by 21 months, consistent with population-wide early seroconversion age. Differential analysis showed that infants having reduced plasma ascorbic acid and cholesterol experienced IAA-first earlier, while early onset of GADA-first was preceded by reduced sphingomyelins at infancy. Plasma 25(OH)D prior to either autoantibody was lower in T1D progressors compared with nonprogressors, with simultaneous lower diglycerides, lysophosphatidylcholines, triglycerides, and alanine before GADA-first. Plasma ascorbic acid and 25(OH)D at infancy were lower in HLA-DR3/DR4 children among IA case subjects but not in matched control subjects, implying gene expression dysregulation of circulating vitamins as latent signals for IA or T1D progression.

We examined the association between plasma 25-hydroxyvitamin D [25(OH)D] concentration and islet autoimmunity (IA) and whether vitamin D gene polymorphisms modify the effect of 25(OH)D on IA risk. We followed 8,676 children at increased genetic risk of type 1 diabetes at six sites in the U.S. and Europe. We defined IA as positivity for at least one autoantibody (GADA, IAA, or IA-2A) on two or more visits. We conducted a risk set sampled nested case-control study of 376 IA case subjects and up to 3 control subjects per case subject. 25(OH)D concentration was measured on all samples prior to, and including, the first IA positive visit. Nine polymorphisms in VDR, CYP24A, CYP27B1, GC, and RXRA were analyzed as effect modifiers of 25(OH)D. Adjusting for HLA-DR-DQ and ancestry, higher childhood 25(OH)D was associated with lower IA risk (odds ratio = 0.93 for a 5 nmol/L difference; 95% CI 0.89, 0.97). Moreover, this association was modified by VDR rs7975232 (interaction P = 0.0072), where increased childhood 25(OH)D was associated with a decreasing IA risk based upon number of minor alleles: 0 (1.00; 0.93, 1.07), 1 (0.92; 0.89, 0.96), and 2 (0.86; 0.80, 0.92). Vitamin D and VDR may have a combined role in IA development in children at increased genetic risk for type 1 diabetes.

Type 1 diabetes (T1D) associated genetic factors affect risk of islet autoantibodies (IA), but non-genetic factors show conflicting associations. Mechanistic interactions may play a role. Under a sufficient-component causal framework, we used a rule-based discovery method to investigate if genetic factors, early environment and first appearing IA (IAA or GADA) act synergistically to mark different disease mechanisms. TEDDY children (n=7512) were followed until age 6 years for IA development (n=518). Rules differentiating IAA-first (n=258) from GADA-first (n=243) were identified by a rule discovery algorithm (RuleFit) and examined in logistic regression models. Rule components were assessed for additive interaction on IAA-first and GADA-first separately using Relative Excess Risk due to Interaction (RERI) calculated from Cox regression models. Here we show 2 of the 5 top rules, the first involving the child having CTLA4-AA (rs231775) and mother with a gestational respiratory but no skin infection (rule1, OR=5.6, 95% CI=2.6-12.0, p Figure 1. Gene-environmental interaction with first appearing IA may mark different mechanisms. Disclosure K.F. Lynch: None. T. Feng: None. X. Qian: None. W. Hagopian: Research Support; Self; Novo Nordisk A/S. Å. Lernmark: None. A. Ziegler: None. J. Toppari: None. M. Rewers: None. J. She: None. D. Schatz: None. B. Akolkar: None. J. Krischer: None. S. Huang: None. K. Vehik: None. 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; National Institute of Environmental Health Sciences; Centers for Disease Control and Prevention; JDRF; University of Florida (UL1TR000064); University of Colorado (UL1TR001082)

Background: There is an unmet need for accurate cost-effective estimation of future T1D risk. Methods: We derived a combined T1D prediction model using 7883 children followed closely from birth for a median of 9 yr, considering T1D genetic risk score (GRS), family T1D history (FH), standard islet autoantibodies (IA), race, birth circumstances, early growth and nutrient status. T1D developed in 326. Results: Machine learning and traditional methods (Cox models) performed equivalently as measured by time-dependent AUC ROC. Future T1D was accurately predicted by combining only 3 variables: GRS, FH and IA status. Accuracy of combined scores increased with age at scoring. By age 2, they were highly predictive for T1D in the next 5 years (AUC >0.91, 95% CI 0.88-0.95) (see X on Figure). A 2-yr-old with 2 IA, positive FH and high GRS (>12) would have T1D risk over the next 1, 3 and 5 years of 14% (8-19%), 36% (25-45%) and 51% (39%-61%) respectively. A 2-yr-old with 1 IA, no FH and moderate GRS (10-11) has T1D risk of 0.8% (0.6-1.2%), 2.6% (1.9-3.2%) and 4.3% (3.4%-5.2%) respectively. After newborn genetic screening, only simple venous sampling in routine healthcare settings is required. Conclusion: This approach allows updated individual risk estimates by age, and in the future may enable release of low risk individuals from surveillance long after initial newborn screening for more cost-efficient population based pediatric T1D prediction. Disclosure L.A. Ferrat: None. K. Vehik: None. S.A. Sharp: None. Å. Lernmark: None. A. Ziegler: None. M. Rewers: None. J. She: None. J. Toppari: None. B. Akolkar: None. J. Krischer: None. M.N. Weedon: None. S.S. Rich: None. R.A. Oram: Other Relationship; Self; Randox Laboratories Ltd. W. Hagopian: Research Support; Self; Novo Nordisk A/S. 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; National Institute of Environmental Health Sciences; JDRF; Centers for Disease Control and Prevention

This study investigates two-phase growth patterns in early life and their associations with development of islet autoimmunity (IA) and type 1 diabetes (T1D). There were 7521 genetically high-risk children from Sweden, Finland, U.S. and Germany followed from birth for a median of 9.0 (IQR 5.7-10.6) years with available growth data. Of these, 761 children developed IA and 290 children progressed to T1D. Bayesian two-phase piecewise linear mixed models with a random change point were used to evaluate distinct growth phases in early life (Figure 1). Cox proportional hazard models were used to assess growth phase effects on risk of IA and progression to T1D. A higher rate of weight gain (kg/year) in the first phase was associated with an increased IA risk (HR =1.10, 95% CI 1.02, 1.18). A height growth pattern with a slower growth rate (cm/year) in the first phase, a higher growth rate in the second phase and younger age (months) at the phase transition was associated with an increased risk of progression from IA to T1D (HR=0.80, 95% CI 0.70, 0.91; HR=1.47, 95% CI 1.21, 1.78; HR=0.69, 95% CI 0.52, 0.93, respectively). A higher rate of weight gain in the second phase was associated with an increased risk of progression from IA to T1D (HR=2.54, 95% CI 1.36, 4.74) in children with first appearing GADA-only. Considering growth by phases in early life better clarified how specific growth phases contribute to the risk of IA and T1D. Disclosure X. Liu: None. K. Vehik: None. Y. Huang: None. H. Elding Larsson: None. J. Toppari: None. A. Ziegler: None. J. She: None. M. Rewers: None. W. Hagopian: Research Support; Self; Novo Nordisk A/S. B. Akolkar: None. J. Krischer: None. 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; National Institute of Environmental Health Sciences; Centers for Disease Control and Prevention; JDRF; University of Florida (UL1TR000064); University of Colorado (UL1TR001082)

Type 1 diabetes, a disease defined by absolute insulin deficiency, is considered a chronic autoimmune disorder resulting from the destruction of insulin-producing pancreatic β-cells. The incidence of childhood-onset type 1 diabetes has been increasing at a rate of 3%–5% per year globally. Despite the introduction of an impressive array of therapies aimed at improving disease management, no means for a practical “cure” exist. This said, hope remains high that any of a number of emerging technologies (e.g., continuous glucose monitoring, insulin pumps, smart algorithms), alongside advances in stem cell biology, cell encapsulation methodologies, and immunotherapy, will eventually impact the lives of those with recently diagnosed or established type 1 diabetes. However, efforts aimed at reversing insulin dependence do not address the obvious benefits of disease prevention. Hence, key “stretch goals” for type 1 diabetes research include identifying improved and increasingly practical means for diagnosing the disease at earlier stages in its natural history (i.e., early, presymptomatic diagnosis), undertaking such efforts in the population at large to optimally identify those with presymptomatic type 1 diabetes, and introducing safe and effective therapeutic options for prevention.

The gut microbiome is suggested to play a role in the pathogenesis of autoimmune disorders such as type 1 diabetes. Evidence of anti-islet cell autoimmunity in type 1 diabetes appears in the first years of life; however, little is known regarding the establishment of the gut microbiome in early infancy. Here, we sought to determine whether differences were present in early composition of the gut microbiome in children in whom anti-islet cell autoimmunity developed. We investigated the microbiome of 298 stool samples prospectively taken up to age 3 years from 22 case children in whom anti-islet cell autoantibodies developed, and 22 matched control children who remained islet cell autoantibody–negative in follow-up. The microbiome changed markedly during the first year of life, and was further affected by breast-feeding, food introduction, and birth delivery mode. No differences between anti-islet cell autoantibody–positive and –negative children were found in bacterial diversity, microbial composition, or single-genus abundances. However, substantial alterations in microbial interaction networks were observed at age 0.5 and 2 years in the children in whom anti-islet cell autoantibodies developed. The findings underscore a role of the microbiome in the pathogenesis of anti-islet cell autoimmunity and type 1 diabetes.

Women with gestational diabetes mellitus (GDM) have a high risk of developing postpartum type 2 diabetes. Strategies to prevent postpartum type 2 diabetes are important to reduce the epidemic of diabetes and its societal impact. Breastfeeding was reported to improve early postpartum glucose tolerance and reduce the subsequent risk of type 2 diabetes. To investigate whether breastfeeding influences short- and long-term postpartum diabetes outcomes, women with GDM (n = 304) participating in the prospective German GDM study were followed from delivery for up to 19 years postpartum for diabetes development. All participants were recruited between 1989 and 1999. Postpartum diabetes developed in 147 women and was dependent on the treatment received during pregnancy (insulin vs. diet), BMI, and presence/absence of islet autoantibodies. Among islet autoantibody-negative women, breastfeeding was associated with median time to diabetes of 12.3 years compared with 2.3 years in women who did not breastfeed. The lowest postpartum diabetes risk was observed in women who breastfed for >3 months. On the basis of these results, we recommend that breastfeeding should be encouraged among these women because it offers a safe and feasible low-cost intervention to reduce the risk of subsequent diabetes in this high-risk population. Diabetes 61:3167-3171, 2012

OBJECTIVE The incidence of type 1 diabetes is increasing. Delivery by cesarean section is also more prevalent, and it is suggested that cesarean section is associated with type 1 diabetes risk. We examine associations between cesarean delivery, islet autoimmunity and type 1 diabetes, and genes involved in type 1 diabetes susceptibility. RESEARCH DESIGN AND METHODS Cesarean section was examined as a risk factor in 1,650 children born to a parent with type 1 diabetes and followed from birth for the development of islet autoantibodies and type 1 diabetes. RESULTS Children delivered by cesarean section (n = 495) had more than twofold higher risk for type 1 diabetes than children born by vaginal delivery (hazard ratio [HR] 2.5; 95% CI 1.4–4.3; P = 0.001). Cesarean section did not increase the risk for islet autoantibodies (P = 0.6) but was associated with a faster progression to diabetes after the appearance of autoimmunity (P = 0.015). Cesarean section–associated risk was independent of potential confounder variables (adjusted HR 2.7;1.5–5.0; P = 0.001) and observed in children with and without high-risk HLA genotypes. Interestingly, cesarean section appeared to interact with immune response genes, including CD25 and in particular the interferon-induced helicase 1 gene, where increased risk for type 1 diabetes was only seen in children who were delivered by cesarean section and had type 1 diabetes–susceptible IFIH1 genotypes (12-year risk, 9.1 vs. CONCLUSIONS These findings suggest that type 1 diabetes risk modification by cesarean section may be linked to viral responses in the preclinical autoantibody-positive disease phase.

The aim of this study was to stratify risk for postpartum diabetes in women who have gestational diabetes. Women with gestational diabetes were recruited between 1989 and 1999, and 302 were followed with oral glucose tolerance tests at 9 months and 2, 5, 8, and 11 years postpregnancy. The 8-year postpartum diabetes risk was 52.7% (130 diabetic cases). Risk was increased in women with autoantibodies to GAD and/or insulinoma antigen-2 (adjusted hazard ratio 4.1; P < 0.0001), women who required insulin during pregnancy (4.7; P < 0.0001), women with BMI >30 kg/m2 (1.5; P = 0.04), and women with more than two prior pregnancies (2.5; P = 0.02). Women without these risk factors had a postpartum diabetes risk of 14% by 8 years, and risk rose incrementally to 96% by 8 years in autoantibody-positive women. Parity status, C-reactive protein concentration, a diabetes family history, maternal age, weeks of gestation, and the child’s birth weight did not significantly affect risk in multivariate analysis. Prospective diabetes assessment is indicated and intervention should be considered in women with gestational diabetes who are autoantibody positive, require insulin treatment during pregnancy, or are obese.

The natural history of autoimmune type 1 diabetes in children is associated with the appearance of islet autoantibodies early in life, which is influenced by genetic and environmental factors. Once islet autoantibodies have developed, the progression to diabetes in antibody-positive individuals is determined by the age of antibody appearance and by the magnitude of the autoimmunity, in turn related to the age of the subject. Characteristics that describe the magnitude of the autoimmunity can stage progression to type 1 diabetes in islet autoantibody–positive subjects regardless of genetic background or age.

Family history of type 1 diabetes and autoantibodies to the islet antigens insulin (IAA), glutamate decarboxylase (GADA), and the protein tyrosine phosphatase-like protein IA-2 (IA-2A) are strong predictors of type 1 diabetes, but the rate of progression to diabetes in multiple islet autoantibody-positive relatives varies widely. We asked whether detailed characterization of islet autoantibodies that included determination of titer, epitope specificity, and IgG subclass would improve diabetes prediction in a large cohort of autoantibody-positive relatives. The study shows a strong association between risk and high titer, broad antibody responses to IA-2 and insulin. The highest risks were associated with high-titer IA-2A and IAA, IgG2, IgG3, and/or IgG4 subclass of IA-2A and IAA, and antibodies to the IA-2-related molecule IA-2β. Using models based on these antibody characteristics, autoantibody-positive relatives can be classified into groups with risks of diabetes ranging from 7 to 89% within 5 years.

Diagnosis of the autoimmune disease type 1 diabetes (T1D) is preceded by the appearance of circulating autoantibodies to pancreatic islets. However, almost nothing is known about events leading to this islet autoimmunity. Previous epidemiological and genetic data have associated viral infections and antiviral type I interferon (IFN) immune response genes with T1D. Here, we first used DNA microarray analysis to identify IFN-β–inducible genes in vitro and then used this set of genes to define an IFN-inducible transcriptional signature in peripheral blood mononuclear cells from a group of active systemic lupus erythematosus patients (n = 25). Using this predefined set of 225 IFN signature genes, we investigated the expression of the signature in cohorts of healthy controls (n = 87), patients with T1D (n = 64), and a large longitudinal birth cohort of children genetically predisposed to T1D (n = 109; 454 microarrayed samples). Expression of the IFN signature was increased in genetically predisposed children before the development of autoantibodies (P = 0.0012) but not in patients with established T1D. Upregulation of IFN-inducible genes was transient, temporally associated with a recent history of upper respiratory tract infections (P = 0.0064), and marked by increased expression of SIGLEC-1 (CD169), a lectin-like receptor expressed on CD14+ monocytes. DNA variation in IFN-inducible genes altered T1D risk (P = 0.007), as exemplified by IFIH1, one of the genes in our IFN signature for which increased expression is a known risk factor for disease. These findings identify transient increased expression of type I IFN genes in preclinical diabetes as a risk factor for autoimmunity in children with a genetic predisposition to T1D.

The temporal development of autoantibodies was studied in 1,353 offspring of parents with type 1 diabetes. Islet cell antibodies (ICAs) and autoantibodies to insulin (IAAs), glutamic acid decarboxylase, and IA-2 were measured at birth, 9 months, 2 years, and 5 years of age. At birth, no offspring had islet autoimmunity other than maternally acquired antibodies, which were shown to influence antibody prevalence up to age 6 months. Antibodies detected thereafter were likely to represent a true de novo production, since prevalences were the same for offspring from mothers and fathers with diabetes, antibodies detected at 9 months were almost always confirmed in the 2-year sample and were associated with an increased likelihood of having or developing other antibodies. By 2 years of age, autoantibodies appeared in 11% of offspring, 3.5% having more than one autoantibody. IAAs were detected most frequently, and few had autoantibodies in the absence of IAAs. In 23 offspring with multiple islet autoantibodies, IAAs preceded other antibodies in 10 cases and were first detected concurrently with other antibodies in 12 and after detection of other antibodies in 1. Development of additional antibodies and changes in levels, including decline of IAAs at older age, was frequent. Nine children, all with IAAs and ICAs, developed diabetes. Overall cumulative risk for disease by 5 years of age was 1.8% (95% CI 0.2-3.4) and was 50% (95% CI 19-81) for offspring with more than one autoantibody in their 2-year sample. Autoimmunity associated with childhood diabetes is an early event and a dynamic process. Presence of IAAs is a consistent feature of this autoimmunity, and IAA detection can identify children at risk.

Soluble interleukin-7 (IL-7) receptor α (sCD127) is implicated in the pathogenesis of autoimmune diseases. We show that serum sCD127 concentrations are increased at the onset of type 1 diabetes (T1D; n = 390) as compared with concentrations in age-matched islet autoantibody–negative first-degree relatives of patients (n = 392; P = 0.00001). sCD127 concentration in patients was influenced by islet autoantibody status (P = 0.003) and genotype of the rs6897932 single nucleotide polymorphism within the IL-7RA gene (P = 0.006). Release of sCD127 in vitro was strongly upregulated by activation of T lymphocytes and affected by exposure to cytokines. sCD127 bound IL-7 and was antagonistic to IL-7 signaling and IL-7–mediated T-cell proliferation, suggesting a regulatory feedback mechanism on T-cell expansion. Remarkably, high glucose led to a glycated form of sCD127 that was ineffective as an IL-7 antagonist. The finding of glycated sCD127 in the circulation of patients at onset of T1D suggested that physiological regulation of IL-7–mediated T-cell survival and expansion by sCD127 may be compromised in T1D. The findings indicate that genetic, immunologic, and metabolic factors contribute to a dysregulation of the IL-7/IL-7 receptor pathway in T1D and identify a novel hyperglycemia-mediated interference of immune regulatory networks.

In IDDM, T-cells are postulated to mediate the destruction of pancreatic β-cells. We analyzed peripheral blood mononuclear cell (PBMC) responses to human insulin, glutamate decarboxylase GAD65, tyrosine phosphatase ICA512, glucagon, membrane preparations of RIN cells and human pancreas, and three control antigens (La = nuclear cell antigen, tetanus toxoid, and phytohemagglutinin). A total of 28 patients with newly diagnosed IDDM, 9 antibody-positive (Ab+) first-degree relatives, and 16 healthy control subjects were included. Increased proliferative responses to pancreatic islet cell antigens were observed in diabetic patients and in Ab+ relatives compared with control subjects, whereas T-cell reactivity to nonpancreatic control antigens was similar between the study groups. The highest differences in the magnitude of proliferative responses were seen for ICA512, followed by membrane preparations of RIN cells, GAD65, and human pancreas. Few subjects reacted with insulin or glucagon. Interestingly, Ab+ relatives showed higher T-cell reactivity with respect to stimulation indexes and prevalences than newly diagnosed diabetic patients, and as many as 89% of Ab+ relatives showed proliferation to more than one islet cell antigen preparation in comparison to 43% of newly diagnosed diabetic patients and none of the control subjects. Statistical analysis revealed significant positive correlation of insulin autoantibody levels with the levels of insulin-specific T-cells in Ab+ relatives, but no relation of PBMC responses to age, sex, or HLA-DR haplotypes. Our results demonstrate the simultaneous existence of various autoreactive T-cells specific for islet cell antigens in the prediabetic period. These T-cells may play a significant role in the pathogenesis of the disease.

We established a T-cell line and 20 CD4+ T-cell clones from the peripheral blood lymphocytes of a type I diabetic patient using a membrane preparation of a rat insulinoma cell line (beta membrane antigen [BMA]) as a source of antigen. The T-cell line and three selected clones proliferated specifically to stimulation with BMA and to membranes prepared from human islets, rat pancreas, and NOD pancreas, but not to control antigens. Proliferation-inhibition studies using human leukocyte antigen (HLA)-specific monoclonal antibodies revealed HLA-DQw1-restricted recognition of BMA. An analysis of the T-cell receptor (TCR) repertoire of the T-cell line after 8 and 40 days of culture showed a prominent usage of the V alpha 1 and V alpha 12 gene families. Although sequencing of the TCR V alpha and V beta chains of the three clones demonstrated that each used different V alpha and V beta gene segments, structural similarities were found in complementary-determining region 3 (CDR3), the region that is postulated to interact with the peptide component of the TCR ligand. When we compared these TCR sequences with published sequences of disease-inducing T-cells of NOD mice, highly related TCR V beta families were detected. Furthermore, stretches of identical amino acids within the CDR3 region were found between two pairs of human and mouse T-cells. If one considers the species differences in TCR genes and sequence differences in the restriction elements for these cells (HLA-DQ vs. H-2 I-A nod), these sequence similarities are striking and may be useful for pinpointing T-cells of primary importance in the development of disease.

Sera from 114 first-degree relatives of insulin-dependent diabetes mellitus (type I diabetes) patients and 81 healthy individuals living in Germany were analyzed for antibodies to rat brain glutamic acid decarboxylase (GAD-ab) using an immunoprecipitation assay. The determination of GAD-ab in the 81 islet cell antibody (ICA) and insulin autoantibody (IAA) negative healthy individuals established a normal range (mean ± 2 SD); 2 healthy individuals (2.5%) possessed GAD-ab levels above this range, but became negative on follow-up. None of 86 ICA−/IAA− first-degree relatives had GAD-ab; whereas, 42.9% of 28 ICA+ and/or IAA+ relatives were positive for GAD-ab. Presence of GAD-ab was negatively correlated with IAA (P = 0.02) and positively with ICA (P = 0.0006). Follow-up samples were obtained from 25 of 28 ICA+ and/or IAA+ relatives with a mean (± SD) follow-up period of 20.6 ± 12.1 months. In these 25 relatives, GAD-ab were positive in 70% ICA+/IAA−, 0% ICA−/IAA+, and 57.1% ICA+/IAA+ relatives in the first sample and in 57.1% ICA+/IAA−, 0% ICA−/IAA+, and 70% ICA+/IAA+ relatives in the last sample. GAD-ab, once detected, persisted in 9 of 11 GAD-ab+ relatives. Of the relatives, 2 converted to GAD-positivity, concomitant with the appearance of ICA, and 2 others lost GAD-ab during follow-up. Of the 28 ICA+ and/or IAA+ relatives, 6 progressed to overt type I diabetes on follow-up, and GAD-ab were detectable in 4 of these relatives. In conclusion, this study indicates that GAD-ab in first-degree relatives are significantly correlated with ICA, are independent of the appearance of IAA, and are present in 66.7% of relatives who subsequently developed type I diabetes.

OBJECTIVE Islet autoimmunity precedes type 1 diabetes and often initiates in childhood. Phenotypic variation in islet autoimmunity relative to the age of its development suggests heterogeneous mechanisms of autoimmune activation. To support this notion, we examined whether serum metabolite profiles differ between children with respect to islet autoantibody status and the age of islet autoantibody development.RESEARCH DESIGN AND METHODS The study analyzed 29 metabolites of amino acid metabolism and 511 lipids assigned to 12 lipid clusters in children, with a type 1 diabetic parent, who first developed autoantibodies at age 2 years or younger (n = 13), at age 8 years or older (n = 22), or remained autoantibody-negative, and were matched for age, date of birth, and HLA genotypes (n = 35). Ultraperformance liquid chromatography and mass spectroscopy were used to measure metabolites and lipids quantitatively in the first autoantibody-positive and matched autoantibody-negative serum samples and in a second sample after 1 year of follow-up.RESULTS Differences in the metabolite profiles were observed relative to age and islet autoantibody status. Independent of age-related differences, autoantibody-positive children had higher levels of odd-chain triglycerides and polyunsaturated fatty acid–containing phospholipids than autoantibody-negative children and independent of age at first autoantibody appearance (P < 0.0001). Consistent with our hypothesis, children who developed autoantibodies by age 2 years had twofold lower concentration of methionine compared with those who developed autoantibodies in late childhood or remained autoantibody-negative (P < 0.0001).CONCLUSIONS Distinct metabolic profiles are associated with age and islet autoimmunity. Pathways that use methionine are potentially relevant for developing islet autoantibodies in early infancy.Islet autoantibodies precede the development of type 1 diabetes and can appear throughout childhood (1). In prospective studies of offspring of parents with type 1 diabetes, we have observed a peak incidence of islet autoantibody appearance at the age of ∼1 year, followed by a decline through age 2 to 5 years and a subsequent rise in incidence toward puberty together with the rise in incidence of developing other autoantibodies such as thyroid peroxidase antibodies (2–4). The characteristics of the islet autoantibodies that develop in the first 2 years are not the same as those that develop later (4). Early antibodies frequently start with insulin autoantibodies (IAA), are high affinity, and spread to multiple targets, whereas children who develop islet autoantibodies late start with IAA or GAD autoantibodies (GADA) that are less likely to spread to other targets (4,5). These observations suggest age-dependent differences in the events that lead to islet autoimmunity or in the immune response to the event.Metabolic phenotypes have been used to identify heterogeneity between subjects. The metabolomic profile has been shown to differ in a manner that is associated with genetics, environment, feeding, and disease (6–9). In type 1 diabetes, specific differences are present in islet autoantibody-positive children before islet autoantibody development (10). These could reflect early environmental exposures that influence the autoimmunization process. With respect to the heterogeneity in islet autoimmunity, we reasoned that if the age-related differences in islet autoantibody appearance reflected different immunizing events, we would observe differences in metabolomic profiles in early versus late developers of islet autoantibodies. Here we tested this by analyzing metabolomic profiles in children who developed islet autoantibodies in the first (age 1 to 2 years) and second (age ≥8 years) age peaks and in matched islet autoantibody-negative control subjects. We found differences that are dependent on age, islet autoantibody positivity, and the age of islet autoantibody development that support our hypothesis.

We studied in detail the anti-insulin autoantibodies in 29 nondiabetic relatives of patients with type I diabetes. The affinity of the autoantibodies for [125I]human insulin was high (1.34 × 109–20.71 × 109 L/mol), and the capacity was low (0.84 × 10−12–37.80 × 10−12 M). The product of affinity × capacity of each relative's antibodies directly correlated (r = 0.99) with the level of antibodies determined in our standard radioassay. The autoantibodies from each of the subjects studied had the same rank order of affinities for insulin from different species. Guinea pig, fish insulin, and insulin containing Trp rather than Leu in position 13 of the A-chain inhibited minimally the human insulin binding. Human proinsulin, insulin containing Gln rather than Glu in position 17 of the A-chain, and desoctapeptide insulin (des B23–30) all inhibited binding effectively. Insulin autoantibodies in relatives of patients with type I diabetes share common epitope(s), which suggests a common pathogenic mechanism for production of such antibodies. The epitopes from this initial analysis appear to include amino acids B1–B3 and A8–A13. The region recognized can be distinguished from the insulin receptor binding domain.

To assess a possible HLA association with anti-insulin autoantibodies (IAAs) in human insulin-dependent (type I) diabetes, 51 newly diagnosed type I diabetic patients (mean age 22 ± 8 yr) were typed for HLA-DR and HLA-DQ and studied for IAAs before exogenous insulin therapy with a competitive radioimmunoassay (normal range ≤49 nU/ml). The level of IAAs in 16 patients exceeded our upper limit of normal, and 18 had high-titer islet cell antibodies (ICAs; ≥40 Juvenile Diabetes Foundation U). A striking association with HLA-DR4 (DQw3) in both the prevalence and the level of IAAs was found (IAA positivity in patients with DR4/4 vs. DR4 heterozygous vs. non-DR4: 90 vs. 29%, corrected [c] P < 0.01, vs. 5%, Pc < 0.0001; IAA positivity in patients with DR4 vs. non-DR4: 50 vs. 5%, Pc < 0.005; IAA level in patients with DR4/4 vs. DR4 heterozygous vs. non-DR4: 111 vs. 17 nU/ml, Pc < 0.01, vs. 20 nU/ml, Pc < 0.0001; IAA level in patients with DR4 vs. non-DR4: 45 vs. 20 nU/ml, Pc < 0.01). In contrast, none of the DR3+ subjects had IAAs above normal range, except in conjunction with DR4 (DR3 vs. non-DR3: 12 vs. 42%, Pc < 0.05). However, there was no significant relationship between DR3 and IAAs after correcting for the number of DR4 alleles. No relationship was seen between age of onset, IAA level, and HLA typing in our population, and no relationship was found between ICA positivity and HLA antigens. These data suggest that humoral in predominantlu DR4+ patients responding to insulin before exogenous insulin treatment.

OBJECTIVE—Autoantibodies to insulin and GAD are features of preclinical type 1 diabetes in children. For insulin autoantibodies, the antibody affinity and epitope specificity predict which children progress to diabetes. We asked whether autoantibodies to GAD (GADAs) are heterogeneous in affinity and epitope recognition and whether diabetes-related GADA are restricted to high-affinity responses. RESEARCH DESIGN AND METHODS—GADA affinity was measured by competitive binding experiments with [125I]-labeled and -unlabeled recombinant human GAD65 in the first GADA-positive sample from 95 children with a type 1 diabetes family history who were prospectively followed from birth and in follow-up samples from 65 of these children. RESULTS—At first GADA appearance, affinity ranged from 107 to 1010 l/mol. Affinity was higher in multiple islet autoantibody-positive children (P < 0.0001) and in HLA DR3–positive children (P = 0.006). GADA affinities were >109 l/mol in 52 of 53 multiple autoantibody-positive children. In contrast, children who were single GADA positive often had lower affinity GADA and/or GADA with specificities that were restricted to minor NH2-terminal GAD65 epitopes. At follow-up, affinity increased from low to high in 3 of 65 children. All 24 children who developed diabetes had high-affinity GADAs before diabetes onset. CONCLUSIONS—Children develop discrete, heterogeneous antibody responses to GAD that could arise from distinct immunization events, only some of which are diabetes relevant. Subtyping the GADA responses using affinity measurement will improve type 1 diabetes risk assessment.

A new radiobinding assay for the simultaneous detection of antibodies to GAD and the tyrosine phosphatase IA2 has been recently described in patients with newly diagnosed type 1 diabetes. Here we assessed sensitivity and predictive value of this GADIA2-combi test in first-degree relatives of type 1 diabetic patients compared with islet cell antibody (ICA) and insulin autoantibody (IAA) screening. Of 1,606 relatives, 77 (4.8%) had elevated GADIA2-combi titers above the 99th percentile of 105 nondiabetic control subjects, and results were confirmed by testing these samples for GAD antibody (GADA) and tyrosine phosphatase IA2 antibody (IA2A) in the single antibody test (29 GADA+/IA2A+, 44 GADA+/IA2A-, and 4 IA2A+/GADA-). A further 9 of 1,606 relatives had detectable ICA (1) or IAA (8), but they were negative in the GADIA2-combi assay as well as in the single test for GADA or IA2A. Twenty-four relatives progressed to IDDM within a median follow-up time of 5.6 years (range 0.5-8.2). The sensitivity of antibody determination in relatives with progression to IDDM was 92% for the GADIA2-combi assay, 96% for the combined testing of IAA and GADIA2-combi antibodies, and 83, 67, 67, and 79%, respectively, for GADA, IA2A, IAA, or ICA testing alone. The cumulative life-table risk of antibody-positive relatives was related to GADIA2-combi titers (5-year risk: >50 U, 51% [95% CI 30-73]; >10 to 50 U, 12% [1-24]; 15 years) or relation to proband (offspring, sibling, parent). Relatives with GADIA2-combi antibodies >10 U and the additional presence of IAA had a slightly higher diabetes risk than relatives without IAA (5-year: IAA+, 46% [23-68]; IAA-, 19% [6-32]; P=0.07). Furthermore, low first-phase insulin release after intravenous glucose tolerance test was associated with risk in relatives with GADIA2-combi antibodies (P=0.01). These results indicate that the GADIA2-combi test is a valuable marker for first-line screening and risk assessment of type 1 diabetes in relatives. It can be used for venous as well as capillary blood samples.

IDDM results from immune-mediated destruction of insulin-producing pancreatic (β-cells in individuals genetically susceptible for the disease. There is evidence that the 65-kDa isoform of GAD plays a critical role in the induction of autoimmune diabetes in NOD mice. In humans, it is still unclear when and to what β-cell antigens autoreactive lymphocytes become activated during early disease. We conducted a prospective study from birth, BABY-DIAB, among children of mothers with IDDM or gestational diabetes or fathers with IDDM, and we investigated the temporal sequence of antibody responses to islet cells (ICA), insulin (IAA), GAD (GADA), and the protein tyrosine phosphatase IA-2/ICA512 (IA-2A). Of 1,019 children included at birth, we have currently followed 513 to the age of 9 months, 214 to the age of 2 years, and 37 to the age of 5 years. At birth, all antibody specificities were frequent in newborns of diabetic mothers but not fathers and are suggested to be transplacentally acquired because they are strongly correlated with antibody levels in their diabetic mothers. In early childhood, antibody levels were

Cytoplasmic islet cell antibody-negative (ICA-; less than 20 Juvenile Diabetes Foundation units, n = 1670) and ICA+ (n = 42) first-degree relatives of type I (insulin-dependent) diabetic individuals were studied for competitive insulin autoantibodies (CIAAs) with a radioassay. Overall, 3.7% of first-degree relatives (64 of 1712) were CIAA+. Of ICA- relatives, 2.7% (45 of 1670) exceeded the upper limit of our normal CIAA range (greater than 39 nU/ml), and 45% (19 of 42) of ICA+ relatives exceeded this normal range. Follow-up serums for repeat CIAA determination have been obtained from 16 of the nondiabetic CIAA+/ICA- individuals (time between samples, 0.4-5.8 yr). Fourteen of these 16 (87%) CIAA+/ICA- relatives were found to still be positive on follow-up, and 2 of the relatives who were positive on the first determination were negative on their follow-up test. With a mean follow-up of approximately 2 yr, 4 of 45 (9%) of the CIAA+/ICA- relatives, 5 of 23 (22%) of the ICA+/CIAA- relatives, and 12 of 19 (63%) of the CIAA+/ICA+ relatives developed diabetes. Life-table analysis indicated that, overall, 53% of CIAA+ relatives become diabetic after 5 yr of follow-up versus 65% of ICA+ relatives. Also by life-table analysis, the predicted risk after 5 yr of follow-up for progression to diabetes is 17% for CIAA+/ICA- relatives, 42% for ICA+/CIAA- relatives, and 77% for CIAA+/ICA+ relatives. The highest rate of progression to diabetes was found in ICA+ relatives with CIAA levels greater than 150 nU/ml (100% projected to be diabetic within 5 yr, P less than .008 vs. ICA+/CIAA- relatives).

In an initial cross-sectional study, 29 female and 25 male nondiabetic weaned nonobese diabetic (NOD) mice of various ages (age range 30–300 days, mean 108 ± 10 days) and 11 unweaned NOD pups were evaluated for competitive insulin autoantibodies (CIAAs) with a fluid-phase radioassay. Eleven of 54 (20%) weaned NOD mice had CIAA levels above the range (>39 nU/ml) of 81 control mice. The group of NOD mice that progressed to diabetes had a significantly higher level of CIAAs than NOD mice that did not progress to diabetes (NOD mice progressing to diabetes: CIAA 63 ± 12 nU/ml; NOD mice not progressing to diabetes: CIAA 8 ± 4 nU/ml; P < .02). Seven of 11 (64%) NOD mice having CIAA concentrations exceeding the normal range progressed to diabetes, whereas only 4 of 43 (9%) NOD mice progressed to diabetes without detection of elevated CIAAs (Fisher's exact test, P < .0005). The relative risk of progressing to overt diabetes with CIAA levels >39 nU/ml was therefore 17 (P < .005), giving a positive predictive value of 64%, a negative predictive value of 91%, and an overall accuracy of 85%. None of 11 unweaned NOD pups had CIAA levels above the normal range (mean −9.4 ± 4.9 nU/ml). At 6 wk of age, 37% of female NOD mice were CIAA+, whereas none of the male animals exceeded the normal range at this age (38 ± 13 vs. 5 ± 6 nU/ml, P < .05). Results of testing two samples during the follow-up of NOD mice indicated that individual NOD mice positive or negative for CIAAs generally remain positive or negative, and CIAA concentrations often increased in initially positive animals. Fourteen NOD mice were included in a prospective study, and CIAAs were evaluated at 9-wk intervals over a follow-up of 8 mo. Four of these NOD mice had elevated CIAA levels, and 3 of 4 CIAA+ mice developed overt diabetes, whereas none of the NOD mice with CIAAs