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Aims/hypothesis: Diabetes diagnosed at <6 months of age is usually monogenic. However, 10-15% of affected infants do not have a pathogenic variant in one of the 26 known neonatal diabetes genes. We characterised infants diagnosed at <6 months of age without a pathogenic variant to assess whether polygenic type 1 diabetes could arise at early ages., Methods: We studied 166 infants diagnosed with type 1 diabetes at <6 months of age in whom pathogenic variants in all 26 known genes had been excluded and compared them with infants with monogenic neonatal diabetes (n = 164) or children with type 1 diabetes diagnosed at 6-24 months of age (n = 152). We assessed the type 1 diabetes genetic risk score (T1D-GRS), islet autoantibodies, C-peptide and clinical features., Results: We found an excess of infants with high T1D-GRS: 38% (63/166) had a T1D-GRS >95th centile of healthy individuals, whereas 5% (8/166) would be expected if all were monogenic (p < 0.0001). Individuals with a high T1D-GRS had a similar rate of autoantibody positivity to that seen in individuals with type 1 diabetes diagnosed at 6-24 months of age (41% vs 58%, p = 0.2), and had markedly reduced C-peptide levels (median <3 pmol/l within 1 year of diagnosis), reflecting rapid loss of insulin secretion. These individuals also had reduced birthweights (median z score -0.89), which were lowest in those diagnosed with type 1 diabetes at <3 months of age (median z score -1.98)., Conclusions/interpretation: We provide strong evidence that type 1 diabetes can present before the age of 6 months based on individuals with this extremely early-onset diabetes subtype having the classic features of childhood type 1 diabetes: high genetic risk, autoimmunity and rapid beta cell loss. The early-onset association with reduced birthweight raises the possibility that for some individuals there was reduced insulin secretion in utero. Comprehensive genetic testing for all neonatal diabetes genes remains essential for all individuals diagnosed with diabetes at <6 months of age. Graphical abstract.

Objective: The first-appearing β-cell autoantibody has been shown to influence risk of type 1 diabetes (T1D). Here, we assessed the risk of autoantibody spreading to the second-appearing autoantibody and further progression to clinical disease in The Environmental Determinants of Diabetes in the Young (TEDDY) study., Research Design and Methods: Eligible children with increased HLA-DR-DQ genetic risk for T1D were followed quarterly from age 3 months up to 15 years for development of a single first-appearing autoantibody (GAD antibody [GADA], insulin autoantibody [IAA], or insulinoma antigen-2 autoantibody [IA-2A]) and subsequent development of a single second-appearing autoantibody and progression to T1D. Autoantibody positivity was defined as positivity for a specific autoantibody at two consecutive visits confirmed in two laboratories. Zinc transporter 8 autoantibody (ZnT8A) was measured in children who developed another autoantibody., Results: There were 608 children who developed a single first-appearing autoantibody (IAA, n = 282, or GADA, n = 326) with a median follow-up of 12.5 years from birth. The risk of a second-appearing autoantibody was independent of GADA versus IAA as a first-appearing autoantibody (adjusted hazard ratio [HR] 1.12; 95% CI 0.88-1.42; P = 0.36). Second-appearing GADA, IAA, IA-2A, or ZnT8A conferred an increased risk of T1D compared with children who remained positive for a single autoantibody, e.g., IAA or GADA second (adjusted HR 6.44; 95% CI 3.78-10.98), IA-2A second (adjusted HR 16.33; 95% CI 9.10-29.29; P < 0.0001), or ZnT8A second (adjusted HR 5.35; 95% CI 2.61-10.95; P < 0.0001). In children who developed a distinct second autoantibody, IA-2A (adjusted HR 3.08; 95% CI 2.04-4.65; P < 0.0001) conferred a greater risk of progression to T1D as compared with GADA or IAA. Additionally, both a younger initial age at seroconversion and shorter time to the development of the second-appearing autoantibody increased the risk for T1D., Conclusions: The hierarchical order of distinct autoantibody spreading was independent of the first-appearing autoantibody type and was age-dependent and augmented the risk of progression to T1D., (© 2020 by the American Diabetes Association.)

Objective: Higher gluten intake, frequent gastrointestinal infections and adenovirus, enterovirus, rotavirus and reovirus have been proposed as environmental triggers for coeliac disease. However, it is not known whether an interaction exists between the ingested gluten amount and viral exposures in the development of coeliac disease. This study investigated whether distinct viral exposures alone or together with gluten increase the risk of coeliac disease autoimmunity (CDA) in genetically predisposed children., Design: The Environmental Determinants of Diabetes in the Young study prospectively followed children carrying the HLA risk haplotypes DQ2 and/or DQ8 and constructed a nested case-control design. From this design, 83 CDA case-control pairs were identified. Median age of CDA was 31 months. Stool samples collected monthly up to the age of 2 years were analysed for virome composition by Illumina next-generation sequencing followed by comprehensive computational virus profiling., Results: The cumulative number of stool enteroviral exposures between 1 and 2 years of age was associated with an increased risk for CDA. In addition, there was a significant interaction between cumulative stool enteroviral exposures and gluten consumption. The risk conferred by stool enteroviruses was increased in cases reporting higher gluten intake., Conclusions: Frequent exposure to enterovirus between 1 and 2 years of age was associated with increased risk of CDA. The increased risk conferred by the interaction between enteroviruses and higher gluten intake indicate a cumulative effect of these factors in the development of CDA., Competing Interests: Competing interests: HH is a shareholder and chairman of the board of Vactech Ltd, which develops vaccines against picornaviruses., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Children at increased genetic risk for type 1 diabetes (T1D) after environmental exposures may develop pancreatic islet autoantibodies (IA) at a very young age. Metabolic profile changes over time may imply responses to exposures and signal development of the first IA. Our present research in The Environmental Determinants of Diabetes in the Young (TEDDY) study aimed to identify metabolome-wide signals preceding the first IA against GAD (GADA-first) or against insulin (IAA-first). We profiled metabolomes by mass spectrometry from children's plasma at 3-month intervals after birth until appearance of the first IA. A trajectory analysis discovered each first IA preceded by reduced amino acid proline and branched-chain amino acids (BCAAs), respectively. With independent time point analysis following birth, we discovered dehydroascorbic acid (DHAA) contributing to the risk of each first IA, and γ-aminobutyric acid (GABAs) associated with the first autoantibody against insulin (IAA-first). Methionine and alanine, compounds produced in BCAA metabolism and fatty acids, also preceded IA at different time points. Unsaturated triglycerides and phosphatidylethanolamines decreased in abundance before appearance of either autoantibody. Our findings suggest that IAA-first and GADA-first are heralded by different patterns of DHAA, GABA, multiple amino acids, and fatty acids, which may be important to primary prevention of T1D., (© 2020 by the American Diabetes Association.)

Aims/hypothesis: We studied the association of plasma ascorbic acid with the risk of developing islet autoimmunity and type 1 diabetes and examined whether SNPs in vitamin C transport genes modify these associations. Furthermore, we aimed to determine whether the SNPs themselves are associated with the risk of islet autoimmunity or type 1 diabetes., Methods: We used a risk set sampled nested case-control design within an ongoing international multicentre observational study: The Environmental Determinants of Diabetes in the Young (TEDDY). The TEDDY study followed children with increased genetic risk from birth to endpoints of islet autoantibodies (350 cases, 974 controls) and type 1 diabetes (102 cases, 282 controls) in six clinical centres. Control participants were matched for family history of type 1 diabetes, clinical centre and sex. Plasma ascorbic acid concentration was measured at ages 6 and 12 months and then annually up to age 6 years. SNPs in vitamin C transport genes were genotyped using the ImmunoChip custom microarray. Comparisons were adjusted for HLA genotypes and for background population stratification., Results: Childhood plasma ascorbic acid (mean ± SD 10.76 ± 3.54 mg/l in controls) was inversely associated with islet autoimmunity risk (adjusted OR 0.96 [95% CI 0.92, 0.99] per +1 mg/l), particularly islet autoimmunity, starting with insulin autoantibodies (OR 0.94 [95% CI 0.88, 0.99]), but not with type 1 diabetes risk (OR 0.93 [95% Cl 0.86, 1.02]). The SLC2A2 rs5400 SNP was associated with increased risk of type 1 diabetes (OR 1.77 [95% CI 1.12, 2.80]), independent of plasma ascorbic acid (OR 0.92 [95% CI 0.84, 1.00])., Conclusions/interpretation: Higher plasma ascorbic acid levels may protect against islet autoimmunity in children genetically at risk for type 1 diabetes. Further studies are warranted to confirm these findings., Data Availability: The datasets generated and analysed during the current study will be made available in the NIDDK Central Repository at https://www.niddkrepository.org/studies/teddy.

The role of diet in type 1 diabetes development is poorly understood. Metabolites, which reflect dietary response, may help elucidate this role. We explored metabolomics and lipidomics differences between 352 cases of islet autoimmunity (IA) and controls in the TEDDY (The Environmental Determinants of Diabetes in the Young) study. We created dietary patterns reflecting pre-IA metabolite differences between groups and examined their association with IA. Secondary outcomes included IA cases positive for multiple autoantibodies (mAb+). The association of 853 plasma metabolites with outcomes was tested at seroconversion to IA, just prior to seroconversion, and during infancy. Key compounds in enriched metabolite sets were used to create dietary patterns reflecting metabolite composition, which were then tested for association with outcomes in the nested case-control subset and the full TEDDY cohort. Unsaturated phosphatidylcholines, sphingomyelins, phosphatidylethanolamines, glucosylceramides, and phospholipid ethers in infancy were inversely associated with mAb+ risk, while dicarboxylic acids were associated with an increased risk. An infancy dietary pattern representing higher levels of unsaturated phosphatidylcholines and phospholipid ethers, and lower sphingomyelins was protective for mAb+ in the nested case-control study only. Characterization of this high-risk infant metabolomics profile may help shape the future of early diagnosis or prevention efforts.

Aims/hypothesis: The long-term effects of successful immune therapies for treatment of type 1 diabetes have not been well studied. The Autoimmunity-Blocking Antibody for Tolerance (AbATE) trial evaluated teplizumab, an Fc receptor non-binding humanised anti-CD3 monoclonal antibody in individuals with new-onset type 1 diabetes, and ended in 2011. Clinical drug-treated responders showed an increased frequency of 'partially exhausted' CD8 + T cells. We studied the clinical, immunological and metabolic status of participants after an average follow-up of 7 years., Methods: Participants with detectable C-peptide at year 2 of AbATE returned for follow-up. C-peptide responses were assessed by 4 h mixed-meal tolerance test. Autoantibodies and HbA 1c levels were measured and average daily insulin use was obtained from patient logs. Peripheral blood mononuclear cells were analysed by flow cytometry and cytokine release., Results: Fifty-six per cent of the original participants returned. Three of the original control group who did not return had lost all detectable C-peptide by the end of the 2 year trial. The C-peptide responses to a mixed-meal tolerance test were similar overall in the drug vs control group of participants but were significantly improved, with less loss of C-peptide, in drug-treated responders identified at 1 year. However, the improvements in C-peptide response were not associated with lower HbA 1c levels or insulin use. Drug-treated responders showed a significantly increased frequency of programmed cell death protein 1-positive central memory and anergic CD8 + T cells at follow-up., Conclusions/interpretation: These findings suggest there is reduced decline in C-peptide and persistent immunological responses up to 7 years after diagnosis of diabetes in individuals who respond to teplizumab., Trial Registration: ClinicalTrials.gov NCT02067923; the protocol is available at www.immunetolerance.org (ITN027AI).

Objective: We investigated the association between maternal use of vitamin D and omega-3 fatty acids (n-3 FAs) supplements during pregnancy and risk of islet autoimmunity (IA) in the offspring., Methods: The Environmental Determinants of Diabetes in the Young (TEDDY) Study is prospectively following 8676 children with increased genetic risk for type 1 diabetes in Finland, Germany, Sweden, and the United States. Blood samples were collected every 3 months between 3 and 48 months of age then every 6 months thereafter to determine persistent IA. Duration, frequency, and supplement dose during pregnancy were recalled by mothers at 3 to 4 months postpartum. Cumulative intakes of supplemental vitamin D and n-3 FAs were analyzed as continuous or binary variables. We applied time-to-event analysis to study the association between maternal supplement use and IA, adjusting for country, human leukocyte antigen-DR-DQ genotype, family history of type 1 diabetes and sex. Secondary outcomes included insulin autoantibodies (IAA) or glutamic acid decarboxylase (GADA) as the first appearing autoantibody., Results: As of February 2018, there were 747 (9.0%) children with IA. Vitamin D supplement intake during pregnancy (any vs none) was not associated with risk for IA (hazard ratio [HR] 1.11; 95% confidence interval [CI] 0.94, 1.31); neither was cumulative vitamin D supplement intake. Supplemental n-3 FA intake was similarly not associated with IA risk (HR: 1.19, 95% CI 0.98, 1.45). Similar lack of association was observed for either IAA or GADA as the first appearing autoantibody., Conclusions: The TEDDY cohort showed no evidence of benefit regarding IA risk for vitamin D or n-3 FA supplementation during pregnancy., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose of Review: The environmental triggers of islet autoimmunity leading to type 1 diabetes (T1D) need to be elucidated to inform primary prevention. The Environmental Determinants of Diabetes in the Young (TEDDY) Study follows from birth 8676 children with T1D risk HLA-DR-DQ genotypes in the USA, Finland, Germany, and Sweden. Most study participants (89%) have no first-degree relative with T1D. The primary outcomes include the appearance of one or more persistent islet autoantibodies (islet autoimmunity, IA) and clinical T1D., Recent Findings: As of February 28, 2018, 769 children had developed IA and 310 have progressed to T1D. Secondary outcomes include celiac disease and autoimmune thyroid disease. While the follow-up continues, TEDDY has already evaluated a number of candidate environmental triggers, including infections, probiotics, micronutrient, and microbiome. TEDDY results suggest that there are multiple pathways leading to the destruction of pancreatic beta-cells. Ongoing measurements of further specific exposures, gene variants, and gene-environment interactions and detailed "omics" studies will provide novel information on the pathogenesis of T1D.

The development of the microbiome from infancy to childhood is dependent on a range of factors, with microbial-immune crosstalk during this time thought to be involved in the pathobiology of later life diseases 1-9 such as persistent islet autoimmunity and type 1 diabetes 10-12 . However, to our knowledge, no studies have performed extensive characterization of the microbiome in early life in a large, multi-centre population. Here we analyse longitudinal stool samples from 903 children between 3 and 46 months of age by 16S rRNA gene sequencing (n = 12,005) and metagenomic sequencing (n = 10,867), as part of the The Environmental Determinants of Diabetes in the Young (TEDDY) study. We show that the developing gut microbiome undergoes three distinct phases of microbiome progression: a developmental phase (months 3-14), a transitional phase (months 15-30), and a stable phase (months 31-46). Receipt of breast milk, either exclusive or partial, was the most significant factor associated with the microbiome structure. Breastfeeding was associated with higher levels of Bifidobacterium species (B. breve and B. bifidum), and the cessation of breast milk resulted in faster maturation of the gut microbiome, as marked by the phylum Firmicutes. Birth mode was also significantly associated with the microbiome during the developmental phase, driven by higher levels of Bacteroides species (particularly B. fragilis) in infants delivered vaginally. Bacteroides was also associated with increased gut diversity and faster maturation, regardless of the birth mode. Environmental factors including geographical location and household exposures (such as siblings and furry pets) also represented important covariates. A nested case-control analysis revealed subtle associations between microbial taxonomy and the development of islet autoimmunity or type 1 diabetes. These data determine the structural and functional assembly of the microbiome in early life and provide a foundation for targeted mechanistic investigation into the consequences of microbial-immune crosstalk for long-term health.

Objective: Cesarean section (C-section) is associated with various immune-mediated diseases in the offspring. We investigated the relationship between mode of delivery and celiac disease (CD) and CD autoimmunity (CDA) in a multinational birth cohort., Methods: From 2004 to 2010, infants from the general population who tested positive for HLA DR3-DQ2 or DR4-DQ8 were enrolled in The Environmental Determinants for Diabetes in the Young (TEDDY) study. Children were annually screened for transglutaminase autoantibodies, if positive, they are retested after 3 to 6 months and those persistently positive defined as CDA. Associations of C-section with maternal (age, education level, parity, pre-pregnancy weight, diabetes, smoking, weight gain during pregnancy) and child characteristics (gestational age, birth weight) were examined by Fisher exact test or Wilcoxon rank-sum test. Hazard ratios (HRs) for CDA or CD were calculated by Cox proportional hazard regression models., Results: Of 6087 analyzed singletons, 1600 (26%) were born by C-section (Germany 38%, United States 37%, Finland 18%, Sweden 16%), and the remaining were born vaginally without instrumental support; 979 (16%) had developed CDA and 343 (6%) developed CD. C-section was associated with lower risk for CDA (hazard ratio [HR] = 0.85; 95% confidence interval [CI] 0.73, 0.99 P = 0.032) and CD (HR = 0.75; 95% CI 0.58, 0.98; P = 0.034). After adjusting for country, sex, HLA-genotype, CD in family, maternal education, and breast-feeding duration, significance was lost for CDA (HR = 0.91; 95% CI 0.78, 1.06; P = 0.20) and CD (HR = 0.85; 95% CI 0.65, 1.11; P = 0.24). Presurgical ruptured membranes had no influence on CDA or CD development., Conclusion: C-section is not associated with increased risk for CDA or CD in the offspring.

Objective: To examine duration of breastfeeding and timing of complementary foods and risk of islet autoimmunity (IA)., Research Design and Methods: The Environmental Determinants of Diabetes in the Young (TEDDY) study prospectively follows 8,676 children with increased genetic risk of type 1 diabetes (T1D) in the U.S., Finland, Germany, and Sweden. This study included 7,563 children with at least 9 months of follow-up. Blood samples were collected every 3 months from birth to evaluate IA, defined as persistent, confirmed positive antibodies to insulin (IAAs), GAD, or insulinoma antigen-2. We examined the associations between diet and the risk of IA using Cox regression models adjusted for country, T1D family history, HLA genotype, sex, and early probiotic exposure. Additionally, we investigated martingale residuals and log-rank statistics to determine cut points for ages of dietary exposures., Results: Later introduction of gluten was associated with increased risk of any IA and IAA. The hazard ratios (HRs) for every 1-month delay in gluten introduction were 1.05 (95% CI 1.01, 1.10; P = 0.02) and 1.08 (95% CI 1.00, 1.16; P = 0.04), respectively. Martingale residual analysis suggested that the age at gluten introduction could be grouped as <4, 4-9, and >9 months. The risk of IA associated with introducing gluten before 4 months of age was lower (HR 0.68; 95% CI 0.47, 0.99), and the risk of IA associated with introducing it later than the age of 9 months was higher (HR 1.57; 95% CI 1.07, 2.31) than introduction between 4 and 9 months of age., Conclusions: The timing of gluten-containing cereals and IA should be studied further., (© 2018 by the American Diabetes Association.)

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., (© 2017 by the American Diabetes Association.)

Background and Objectives: Few birth cohorts have prospectively followed development of type 1 diabetes (T1D) and celiac disease (CD) autoimmunities to determine timing, extent of co-occurrence, and associated genetic and demographic factors., Methods: In this prospective birth cohort study, 8676 children at high genetic risk of both diseases were enrolled and 5891 analyzed in median follow-up of 66 months. Along with demographic factors and HLA-DR-DQ, genotypes for HLA-DPB1 and 5 non-HLA loci conferring risk of both T1D and CD were analyzed., Results: Development of persistent islet autoantibodies (IAs) and tissue transglutaminase autoantibodies (tTGAs), as well as each clinical disease, was evaluated quarterly from 3 to 48 months of age and semiannually thereafter. IAs alone appeared in 367, tTGAs alone in 808, and both in 90 children. Co-occurrence significantly exceeded the expected rate. IAs usually, but not always, appeared earlier than tTGAs. IAs preceding tTGAs was associated with increasing risk of tTGAs (hazard ratio [HR]: 1.48; 95% confidence interval [CI]: 1.15-1.91). After adjusting for country, sex, family history, and all other genetic loci, significantly greater co-occurrence was observed in children with a T1D family history (HR: 2.80), HLA-DR3/4 (HR: 1.94) and single-nucleotide polymorphism rs3184504 at SH2B3 (HR: 1.53). However, observed co-occurrence was not fully accounted for by all analyzed factors., Conclusions: In early childhood, T1D autoimmunity usually precedes CD autoimmunity. Preceding IAs significantly increases the risk of subsequent tTGAs. Co-occurrence is greater than explained by demographic factors and extensive genetic risk loci, indicating that shared environmental or pathophysiological mechanisms may contribute to the increased risk., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2017 by the American Academy of Pediatrics.)

Background & Aims: Little is known about the pathogenic mechanisms of gluten immunogenicity in patients with celiac disease. We studied temporal associations between infections and the development of celiac disease autoimmunity, and examined effects of HLA alleles, rotavirus vaccination status, and infant feeding., Methods: We monitored 6327 children in the United States and Europe carrying HLA risk genotypes for celiac disease from 1 to 4 years of age for presence of tissue transglutaminase autoantibodies (the definition of celiac disease autoimmunity), until March 31, 2015. Parental reports of gastrointestinal and respiratory infections were collected every third month from birth. We analyzed time-varying relationships among reported infections, rotavirus vaccination status, time to first introduction of gluten, breastfeeding, and risk of celiac disease autoimmunity using proportional hazard models., Results: We identified 13,881 gastrointestinal infectious episodes (GIE) and 79,816 respiratory infectious episodes. During the follow-up period, 732 of 6327 (11.6%) children developed celiac disease autoimmunity. A GIE increased the risk of celiac disease autoimmunity within the following 3 months by 33% (hazard ratio [HR], 1.33; 95% confidence interval [CI], 1.11-1.59). This risk increased 2-fold among children born in winter and introduced to gluten before age 6 months (HR, 2.08; 95% CI, 1.46-2.98), and increased 10-fold among children without HLA-DQ2 alleles and breastfed for fewer than 4 months (HR, 9.76; 95% CI, 3.87-24.8). Risk of celiac disease autoimmunity was reduced in children vaccinated against rotavirus and introduced to gluten before age 6 months (HR, 0.57; 95% CI, 0.36-0.88)., Conclusions: Gastrointestinal infections increase the risk of celiac disease autoimmunity in children with genetic susceptibility to this autoimmune disorder. The risk is modified by HLA genotype, infant gluten consumption, breastfeeding, and rotavirus vaccination, indicating complex interactions among infections, genetic factors, and diet in the etiology of celiac disease in early childhood., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Increased growth in early childhood has been suggested to increase the risk of type 1 diabetes. This study explored the relationship between weight or height and development of persistent islet autoimmunity and progression to type 1 diabetes during the first 4 years of life in 7,468 children at genetic risk for type 1 diabetes followed in Finland, Germany, Sweden, and the U.S. Growth data collected every third month were used to estimate individual growth curves by mixed models. Cox proportional hazards models were used to evaluate body size and risk of islet autoimmunity and type 1 diabetes. In the overall cohort, development of islet autoimmunity (n = 575) was related to weight z scores at 12 months (hazard ratio [HR] 1.16 per 1.14 kg in males or per 1.02 kg in females, 95% CI 1.06-1.27, P < 0.001, false discovery rate [FDR] = 0.008) but not at 24 or 36 months. A similar relationship was seen between weight z scores and development of multiple islet autoantibodies (1 year: HR 1.21, 95% CI 1.08-1.35, P = 0.001, FDR = 0.008; 2 years: HR 1.18, 95% CI 1.06-1.32, P = 0.004, FDR = 0.02). No association was found between weight or height and type 1 diabetes (n = 169). In conclusion, greater weight in the first years of life was associated with an increased risk of islet autoimmunity., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

A total of 15 SNPs within complement genes and present on the ImmunoChip were analyzed in The Environmental Determinants of Diabetes in the Young (TEDDY) study. A total of 5474 subjects were followed from three months of age until islet autoimmunity (IA: n = 413) and the subsequent onset of type 1 diabetes (n = 115) for a median of 73 months (IQR 54-91). Three SNPs within ITGAM were nominally associated (p < 0.05) with IA: rs1143678 [Hazard ratio; HR 0.80; 95% CI 0.66-0.98; p = 0.032], rs1143683 [HR 0.80; 95% CI 0.65-0.98; p = 0.030] and rs4597342 [HR 1.16; 95% CI 1.01-1.32; p = 0.041]. When type 1 diabetes was the outcome, in DR3/4 subjects, there was nominal significance for two SNPs: rs17615 in CD21 [HR 1.52; 95% CI 1.05-2.20; p = 0.025] and rs4844573 in C4BPA [HR 0.63; 95% CI 0.43-0.92; p = 0.017]. Among DR4/4 subjects, rs2230199 in C3 was significantly associated [HR 3.20; 95% CI 1.75-5.85; p = 0.0002, uncorrected] a significance that withstood Bonferroni correction since it was less than 0.000833 (0.05/60) in the HLA-specific analyses. SNPs within the complement genes may contribute to IA, the first step to type 1 diabetes, with at least one SNP in C3 significantly associated with clinically diagnosed type 1 diabetes.

Objectives: There are significant geographical differences in the prevalence and incidence of celiac disease that cannot be explained by HLA alone. More than 40 loci outside of the HLA region have been associated with celiac disease. We investigated the roles of these non-HLA genes in the development of tissue transglutaminase autoantibodies (tTGA) and celiac disease in a large international prospective cohort study., Methods: A total of 424,788 newborns from the US and European general populations and first-degree relatives with type 1 diabetes were screened for specific HLA genotypes. Of these, 21,589 carried 1 of the 9 HLA genotypes associated with increased risk for type 1 diabetes and celiac disease; we followed 8676 of the children in a 15 y prospective follow-up study. Genotype analyses were performed on 6010 children using the Illumina ImmunoChip. Levels of tTGA were measured in serum samples using radio-ligand binding assays; diagnoses of celiac disease were made based on persistent detection of tTGA and biopsy analysis. Data were analyzed using Cox proportional hazards analyses., Results: We found 54 single-nucleotide polymorphisms (SNPs) in 5 genes associated with celiac disease (TAGAP, IL18R1, RGS21, PLEK, and CCR9) in time to celiac disease analyses (10-4>P>5.8x10-6). The hazard ratios (HR) for the SNPs with the smallest P values in each region were 1.59, 1.45, 2.23, 2.64, and 1.40, respectively. Outside of regions previously associated with celiac disease, we identified 10 SNPs in 8 regions that could also be associated with the disease (P<10-4). A SNP near PKIA (rs117128341, P = 6.5x10-8, HR = 2.8) and a SNP near PFKFB3 (rs117139146, P<2.8x10-7, HR = 4.9) reached the genome-wide association threshold in subjects from Sweden. Analyses of time to detection of tTGA identified 29 SNPs in 2 regions previously associated with celiac disease (CTLA4, P = 1.3x10-6, HR = 0.76 and LPP, P = 2.8x10-5, HR = .80) and 6 SNPs in 5 regions not previously associated with celiac disease (P<10-4); non-HLA genes are therefore involved in development of tTGA., Conclusions: In conclusion, using a genetic analysis of a large international cohort of children, we associated celiac disease development with 5 non-HLA regions previously associated with the disease and 8 regions not previously associated with celiac disease. We identified 5 regions associated with development of tTGA. Two loci associated with celiac disease progression reached a genome-wide association threshold in subjects from Sweden.

Importance: Probiotics have been hypothesized to affect immunologic responses to environmental exposures by supporting healthy gut microbiota and could therefore theoretically be used to prevent the development of type 1 diabetes mellitus (T1DM)-associated islet autoimmunity., Objective: To examine the association between supplemental probiotic use during the first year of life and islet autoimmunity among children at increased genetic risk of T1DM., Design, Setting, and Participants: In this ongoing prospective cohort study that started September 1, 2004, children from 6 clinical centers, 3 in the United States (Colorado, Georgia/Florida, and Washington) and 3 in Europe (Finland, Germany, and Sweden), were followed up for T1DM-related autoantibodies. Blood samples were collected every 3 months between 3 and 48 months of age and every 6 months thereafter to determine persistent islet autoimmunity. Details of infant feeding, including probiotic supplementation and infant formula use, were monitored from birth using questionnaires and diaries. We applied time-to-event analysis to study the association between probiotic use and islet autoimmunity, stratifying by country and adjusting for family history of type 1 diabetes, HLA-DR-DQ genotypes, sex, birth order, mode of delivery, exclusive breastfeeding, birth year, child's antibiotic use, and diarrheal history, as well as maternal age, probiotic use, and smoking. Altogether 8676 infants with an eligible genotype were enrolled in the follow-up study before the age of 4 months. The final sample consisted of 7473 children with the age range of 4 to 10 years (as of October 31, 2014)., Exposures: Early intake of probiotics., Main Outcomes and Measures: Islet autoimmunity revealed by specific islet autoantibodies., Results: Early probiotic supplementation (at the age of 0-27 days) was associated with a decreased risk of islet autoimmunity when compared with probiotic supplementation after 27 days or no probiotic supplementation (hazard ratio [HR], 0.66; 95% CI, 0.46-0.94). The association was accounted for by children with the DR3/4 genotype (HR, 0.40; 95% CI, 0.21-0.74) and was absent among other genotypes (HR, 0.97; 95% CI, 0.62-1.54)., Conclusions and Relevance: Early probiotic supplementation may reduce the risk of islet autoimmunity in children at the highest genetic risk of T1DM. The result needs to be confirmed in further studies before any recommendation of probiotics use is made.

Background: Deficient soluble fiber intake has been suggested to dysregulate the immune response either directly or through alterations of the microbial composition in the gut., Objective: We hypothesized that a high intake of dietary soluble fiber in early childhood decreases the risk of type 1 diabetes (T1D)-associated islet autoimmunity., Design: We analyzed 17,620 food records collected between age 9 and 48 mo from 3358 children from the United States and Germany prospectively followed in the TEDDY (The Environmental Determinants of Diabetes in the Young) study. HRs for the development of any/multiple islet autoantibodies (242 and 151 events, respectively) and T1D (71 events) by soluble fiber intake were calculated in Cox regression models and adjusted for potential confounders., Results: There were no statistically significantly protective associations observed between a high intake of soluble fiber and islet autoimmunity or T1D. For example, the adjusted HRs (95% CIs) for high intake (highest compared with lowest quintile) at age 12 mo were 0.90 (0.55, 1.45) for any islet autoantibody, 1.20 (0.69, 2.11) for multiple islet autoantibodies, and 1.24 (0.57, 2.70) for T1D. In analyzing soluble fiber intake as a time-varying covariate, there were also no short-term associations between soluble fiber intake and islet autoimmunity development, with adjusted HRs of 0.85 (0.51, 1.42) for high intake and development of any islet autoantibody, for example., Conclusion: These results indicate that the intake level of dietary soluble fiber is not associated with islet autoimmunity or T1D in early life., (© 2015 American Society for Nutrition.)

Objectives: Tissue transglutaminase autoantibodies (tTGAs) represent the first evidence of celiac disease (CD) development. Associations of HLA-DR3-DQA1*05:01-DQB1*02:01 (i.e., DR3-DQ2) and, to a lesser extent, DR4-DQA1*03:01-DQB1*03:02 (i.e., DR4-DQ8) with the risk of CD differ by country, consistent with additional genetic heterogeneity that further refines risk. Therefore, we examined human leukocyte antigen (HLA) factors other than DR3-DQ2 for their contribution to developing tTGAs., Methods: The Environmental Determinants of Diabetes in the Young (TEDDY) study enrolled 8,676 infants at an increased HLA-DR-DQ risk for type 1 diabetes and CD into a 15-year prospective surveillance follow-up. Of those followed up, 21% (n=1,813) carried DR3-DQ2/DR3-DQ2, 39% (n=3,359) carried DR3-DQ2/DR4-DQ8, 20% (n=1701) carried DR4-DQ8/DR4-DQ8, and 17% (n=1,493) carried DR4-DQ8/DQ4. Within TEDDY, a nested case-control design of 248 children with CD autoimmunity (CDA) and 248 matched control children were genotyped for HLA-B, -DRB3, -DRB4, -DPA1, and -DPB1 genes, and the entire cohort was genotyped for single-nucleotide polymorphisms (SNPs) using the Illumina ImmunoChip. CDA was defined as a positive tTGA test at two consecutive clinic visits, whereas matching in those with no evidence of tTGAs was based on the presence of HLA-DQ2, country, and sex., Results: After adjustment for DR3-DQ2 and restriction to allele frequency (AF) ≥5%, HLA-DPB1*04:01 was inversely associated with CDA by conditional logistic regression (AF=44%, odds ratio=0.71, 95% confidence interval (CI)=0.53-0.96, P=0.025). This association of time to CDA and HLA-DPB1*04:01 was replicated with statistical significance in the remainder of the cohort using imputation for specific HLA alleles based on SNP genotyping (hazard ratio=0.84, 95% CI=0.73-0.96, P=0.013)., Conclusions: HLA-DPB1*04:01 may reduce the risk of tTGAs, an early marker of CD, among DR3-DQ2 children, confirming that additional variants in the HLA region influence the risk for CDA.

The Environmental Determinants of Diabetes in the Young (TEDDY) study prospectively follows 8,677 children enrolled from birth who carry HLA-susceptibility genotypes for development of islet autoantibodies (IA) and type 1 diabetes (T1D). During the median follow-up time of 57 months, 350 children developed at least one persistent IA (GAD antibody, IA-2A, or micro insulin autoantibodies) and 84 of them progressed to T1D. We genotyped 5,164 Caucasian children for 41 non-HLA single nucleotide polymorphisms (SNPs) that achieved genome-wide significance for association with T1D in the genome-wide association scan meta-analysis conducted by the Type 1 Diabetes Genetics Consortium. In TEDDY participants carrying high-risk HLA genotypes, eight SNPs achieved significant association to development of IA using time-to-event analysis (P < 0.05), whereof four were significant after adjustment for multiple testing (P < 0.0012): rs2476601 in PTPN22 (hazard ratio [HR] 1.54 [95% CI 1.27-1.88]), rs2292239 in ERBB3 (HR 1.33 [95% CI 1.14-1.55]), rs3184504 in SH2B3 (HR 1.38 [95% CI 1.19-1.61]), and rs1004446 in INS (HR 0.77 [0.66-0.90]). These SNPs were also significantly associated with T1D in particular: rs2476601 (HR 2.42 [95% CI 1.70-3.44]). Although genes in the HLA region remain the most important genetic risk factors for T1D, other non-HLA genetic factors contribute to IA, a first step in the pathogenesis of T1D, and the progression of the disease., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Background: Early childhood environmental exposures, possibly infections, may be responsible for triggering islet autoimmunity and progression to type 1 diabetes (T1D). The Environmental Determinants of Diabetes in the Young (TEDDY) follows children with increased HLA-related genetic risk for future T1D. TEDDY asks parents to prospectively record the child's infections using a diary book. The present paper shows how these large amounts of partially structured data were reduced into quantitative data-sets and further categorized into system-specific infectious disease episodes. The numbers and frequencies of acute infections and infectious episodes are shown., Methods: Study subjects (n = 3463) included children who had attended study visits every three months from age 3 months to 4 years, without missing two or more consecutive visits during the follow-up. Parents recorded illnesses prospectively in a TEDDY Book at home. The data were entered into the study database during study visits using ICD-10 codes by a research nurse. TEDDY investigators grouped ICD-10 codes and fever reports into infectious disease entities and further arranged them into four main categories of infectious episodes: respiratory, gastrointestinal, other, and unknown febrile episodes. Incidence rate of infections was modeled as function of gender, HLA-DQ genetic risk group and study center using the Poisson regression., Results: A total of 113,884 ICD-10 code reports for infectious diseases recorded in the database were reduced to 71,578 infectious episodes, including 74.0% respiratory, 13.1% gastrointestinal, 5.7% other infectious episodes and 7.2% febrile episodes. Respiratory and gastrointestinal infectious episodes were more frequent during winter. Infectious episode rates peaked at 6 months and began declining after 18 months of age. The overall infectious episode rate was 5.2 episodes per person-year and varied significantly by country of residence, sex and HLA genotype., Conclusions: The data reduction and categorization process developed by TEDDY enables analysis of single infectious agents as well as larger arrays of infectious agents or clinical disease entities. The preliminary descriptive analyses of the incidence of infections among TEDDY participants younger than 4 years fits well with general knowledge of infectious disease epidemiology. This protocol can be used as a template in forthcoming time-dependent TEDDY analyses and in other epidemiological studies.

Objective: Gut microbiome dysbiosis is associated with numerous diseases, including type 1 diabetes. This pilot study determines how geographical location affects the microbiome of infants at high risk for type 1 diabetes in a population of homogenous HLA class II genotypes., Research Design and Methods: High-throughput 16S rRNA sequencing was performed on stool samples collected from 90 high-risk, nonautoimmune infants participating in The Environmental Determinants of Diabetes in the Young (TEDDY) study in the U.S., Germany, Sweden, and Finland., Results: Study site-specific patterns of gut colonization share characteristics across continents. Finland and Colorado have a significantly lower bacterial diversity, while Sweden and Washington state are dominated by Bifidobacterium in early life. Bacterial community diversity over time is significantly different by geographical location., Conclusions: The microbiome of high-risk infants is associated with geographical location. Future studies aiming to identify the microbiome disease phenotype need to carefully consider the geographical origin of subjects., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Objective: The Environmental Determinants of Diabetes in the Young (TEDDY) study is designed to identify environmental exposures triggering islet autoimmunity and type 1 diabetes (T1D) in genetically high-risk children. We describe the first 100 participants diagnosed with T1D, hypothesizing that (i) they are diagnosed at an early stage of disease, (ii) a high proportion are diagnosed by an oral glucose tolerance test (OGTT), and (iii) risk for early T1D is related to country, population, human leukocyte antigen (HLA)-genotypes and immunological markers., Methods: Autoantibodies to glutamic acid decarboxylase (GADA), insulinoma-associated protein 2 (IA-2) and insulin (IAA) were analyzed from 3 months of age in children with genetic risk. Symptoms and laboratory values at diagnosis were obtained and reviewed for ADA criteria., Results: The first 100 children to develop T1D, 33 first-degree relatives (FDRs), with a median age 2.3 yr (0.69-6.27), were diagnosed between September 2005 and November 2011. Although young, 36% had no symptoms and ketoacidosis was rare (8%). An OGTT diagnosed 9/30 (30%) children above 3 yr of age but only 4/70 (5.7%) below the age of 3 yr. FDRs had higher cumulative incidence than children from the general population (p < 0.0001). Appearance of all three autoantibodies at seroconversion was associated with the most rapid development of T1D (HR = 4.52, p = 0.014), followed by the combination of GADA and IAA (HR = 2.82, p < 0.0001)., Conclusions: Close follow-up of children with genetic risk enables early detection of T1D. Risk factors for rapid development of diabetes in this young population were FDR status and initial positivity for GADA, IA-2, and IAA or a combination of GADA and IAA., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Protégé was a phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety of teplizumab immunotherapy at 2 years and to identify characteristics associated with therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years of follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean area under the curve (AUC), a prespecified secondary end point, at 2 years versus placebo. In analyses of prespecified and post hoc subsets at entry, U.S. residents, patients with C-peptide mean AUC >0.2 nmol/L, those randomized ≤6 weeks after diagnosis, HbA1c <7.5% (58 mmol/mol), insulin use <0.4 units/kg/day, and 8-17 years of age each had greater teplizumab-associated C-peptide preservation than their counterparts. Exogenous insulin needs tended to be reduced versus placebo. Antidrug antibodies developed in some patients, without apparent change in drug efficacy. No new safety or tolerability issues were observed during year 2. In summary, anti-CD3 therapy reduced C-peptide loss 2 years after diagnosis using a tolerable dose.

Trials of immune therapies in new-onset type 1 diabetes (T1D) have shown success, but not all subjects respond, and the duration of response is limited. Our aim was to determine whether two courses of teplizumab, an Fc receptor-nonbinding anti-CD3 monoclonal antibody, reduces the decline in C-peptide levels in patients with T1D 2 years after disease onset. We also set out to identify characteristics of responders. We treated 52 subjects with new-onset T1D with teplizumab for 2 weeks at diagnosis and after 1 year in an open-label, randomized, controlled trial. In the intent to treat analysis of the primary end point, patients treated with teplizumab had a reduced decline in C-peptide at 2 years (mean -0.28 nmol/L [95% CI -0.36 to -0.20]) versus control (mean -0.46 nmol/L [95% CI -0.57 to -0.35]; P = 0.002), a 75% improvement. The most common adverse events were rash, transient upper respiratory infections, headache, and nausea. In a post hoc analysis we characterized clinical responders and found that metabolic (HbA1c and insulin use) and immunologic features distinguished this group from those who did not respond to teplizumab. We conclude that teplizumab treatment preserves insulin production and reduces the use of exogenous insulin in some patients with new-onset T1D. Metabolic and immunologic features at baseline can identify a subgroup with robust responses to immune therapy.

Background: The vast array and quantity of longitudinal samples collected in The Environmental Determinants of Diabetes in the Young study present a series of challenges in terms of quality control procedures and data validity. To address this, pilot studies have been conducted to standardize and enhance both biospecimen collection and sample obtainment in terms of autoantibody collection, stool sample preservation, RNA, biomarker stability, metabolic biomarkers and T-cell viability., Research Design and Methods: The Environmental Determinants of Diabetes in the Young is a multicentre, international prospective study (n = 8677) designed to identify environmental triggers of type 1 diabetes (T1D) in genetically at-risk children from ages 3 months until 15 years. The study is conducted through six primary clinical centres located in four countries., Results: As of May 2012, over three million biological samples and 250 million total data points have been collected, which will be analysed to assess autoimmunity status, presence of inflammatory biomarkers, genetic factors, exposure to infectious agents, dietary biomarkers and other potentially important environmental exposures in relation to autoimmunity and progression to T1D., Conclusions: Detailed procedures were utilized to standardize both data harmonization and management when handling a large quantity of longitudinal samples obtained from multiple locations. In addition, a description of the available specimens is provided that serve as an invaluable repository for the elucidation of determinants in T1D focusing on autoantibody concordance and harmonization, transglutaminase autoantibody, inflammatory biomarkers (T-cells), genetic proficiency testing, RNA lab internal quality control testing, infectious agents (monitoring cross-contamination, virus preservation and nasal swab collection validity) and HbA1c testing., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: For cost-effective population-based diabetes prediction and confirmation, islet autoantibody assays must be made more economical., Methods: We evaluated glutamic acid decarboxylase (GAD)-Ruc (renilla luciferase) and IA2ic (also known as ICA512ic)-Ruc (renilla luciferase) fusion protein constructs in high-throughput islet antibody assay formats., Results: Antigen production via transfection onto COS cells in 100 mm culture dishes yielded sufficient antigen to assay 375 and 535 serum samples for GAD and IA2ic per dish, respectively. Antigen was usably stable after -80 °C storage for 40-80 days after which luciferase activity decreased. The mean signal-to-noise ratios for luciferase-based immunoprecitation system (LIPS) GAD and LIPS IA2ic were 88±24 and 219±89, respectively, comparing favourably to radio-binding assays (RBA) in the same format. However, the coefficient of variation among triplicate wells was higher for IA2ic than for GAD in LIPS, similar to findings in RBA format. Correlation coefficients between autoantibody indices determined from the RBA and LIPS methods were only R2=0.79 and R2=0.75 for GAD and IA2ic, respectively, raising the possibility that different epitopes were favoured in the two different assay formats. Nevertheless, overall concordance for the two assay types was high, at 228/240=95.0% for GAD and 494/521=94.8% for IA2ic. Using optimal cutoffs, Diabetes Autoantibody Standardization Program (DASP) 2010 sensitivity/specificity was 80/99% for GAD RBA, 80/99% for GAD LIPS, 70/98% for IA2 RBA, and 72/99% for IA2 LIPS., Conclusion: The LIPS assays for islet autoantibodies to GAD and IA2ic performed as well as RBA in DASP 2010. With further refinements in expression and storage, these assays may be more economical than current methods to measure islet autoantibodies in type 1 diabetes., (Copyright © 2011 John Wiley & Sons, Ltd.)

Anti-CD3 mAbs may prolong beta cell function up to 2 years in patients with new onset Type 1 diabetes (T1DM). A randomized open label trial of anti-CD3 mAb, Teplizumab, in T1DM was stopped after 10 subjects because of increased adverse events than in a previous trial related with higher dosing of drug. Teplizumab caused transient reduction in circulating T cells, but the recovered cells were not new thymic emigrants because T cell receptor excision circles were not increased. There was a trend for reduced loss of C-peptide over 2 years with drug treatment (p=0.1), and insulin use was lower (p<0.001). In 4 drug-treated subjects followed up to 60 months, C-peptide responses were maintained. We conclude that increased doses of Teplizumab are associated with greater adverse events without improved efficacy. The drug may marginate rather than deplete T cells. C-peptide levels may remain detectable up to 5 years after treatment.

Background: In experimental models, Type 1 diabetes T1D can be prevented by adoptive transfer of CD4+CD25+ (FoxP3+) suppressor or regulatory T cells. Recent studies have found a suppression defect of CD4+CD25+(high) T cells in human disease. In this study we measure apoptosis of CD4+CD25+(high) T cells to see if it could contribute to reduced suppressive activity of these cells., Methods and Findings: T-cell apoptosis was evaluated in children and adolescent 35 females/40 males subjects comprising recent-onset and long-standing T1D subjects and their first-degree relatives, who are at variable risk to develop T1D. YOPRO1/7AAD and intracellular staining of the active form of caspase 3 were used to evaluate apoptosis. Isolated CD4+CD25+(high) and CD4+CD25- T cells were co-cultured in a suppression assay to assess the function of the former cells. We found that recent-onset T1D subjects show increased apoptosis of CD4+CD25+(high) T cells when compared to both control and long-standing T1D subjects p<0.0001 for both groups. Subjects at high risk for developing T1D 2-3Ab+ve show a similar trend p<0.02 and p<0.01, respectively. On the contrary, in long-standing T1D and T2D subjects, CD4+CD25+(high) T cell apoptosis is at the same level as in control subjects p = NS. Simultaneous intracellular staining of the active form of caspase 3 and FoxP3 confirmed recent-onset FoxP3+ve CD4+CD25+(high) T cells committed to apoptosis at a higher percentage 15.3+/-2.2 compared to FoxP3+ve CD4+CD25+(high) T cells in control subjects 6.1+/-1.7 p<0.002. Compared to control subjects, both recent-onset T1D and high at-risk subjects had significantly decreased function of CD4+CD25+(high) T cells p = 0.0007 and p = 0.007, respectively., Conclusions: There is a higher level of ongoing apoptosis in CD4+CD25+(high) T cells in recent-onset T1D subjects and in subjects at high risk for the disease. This high level of CD4+CD25+(high) T-cell apoptosis could be a contributing factor to markedly decreased suppressive potential of these cells in recent-onset T1D subjects.

Environmental factors appear to play an important role in the pathogenesis of childhood-onset type 1 diabetes (T1D). The most important factors are thought to be infectious, dietary, perinatal, and psychosocial. Enteroviruses (especially Coxsackie B virus), breastfeeding, the early presence or lack of certain foods, birth weight, childhood over-nutrition, maternal islet autoimmunity, and negative stress events have been shown to be related to the prevalence of T1D. However, clear conclusions to date are limited because most studies lacked power to detect exposure/disease associations, were not prospective or long-term, did not start in infancy, had imprecise or infrequent exposure estimates, had confounding exposures, and failed to account for genetic susceptibility. In addition to the identification of specific antigenic triggers, several more general hypotheses, including the accelerator and hygiene hypotheses, are testable approaches worth pursuing.

Despite advances in understanding autoimmune diabetes in animal models, there has been little progress in altering the natural course of the human disease, which involves progression to insulin deficiency. Studies with immunosuppressive agents have shown short-term effectiveness, but they have not induced tolerance, and continuous treatment is needed. We studied the effects of hOKT3gamma1(Ala-Ala), a humanized Fc mutated anti-CD3 monoclonal antibody, on the progression of type 1 diabetes in patients with recent-onset disease in a randomized controlled trial. In general, the drug was well tolerated. A single course of treatment, within the first 6 weeks after diagnosis, preserved C-peptide responses to a mixed meal for 1 year after diagnosis (97 +/- 9.6% of response at study entry in drug-treated patients vs. 53 +/- 7.6% in control subjects, P < 0.01), with significant improvement in C-peptide responses to a mixed meal even 2 years after treatment (P < 0.02). The improved C-peptide responses were accompanied by reduced HbA(1c) and insulin requirements. Clinical responses to drug treatment were predicted by an increase in the relative number of CD8(+) T-cells in the peripheral blood after the lymphocyte count recovered 2 weeks after the last dose of drug. We conclude that treatment with the anti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala) results in improved C-peptide responses and clinical parameters in type 1 diabetes for at least 2 years in the absence of continued immunosuppressive medications.

The aim of the Finland-United States Investigation of NIDDM Genetics (FUSION) study is to identify genes that predispose to type 2 diabetes or are responsible for variability in diabetes-related traits via a positional cloning and positional candidate gene approach. In a previously published genome-wide scan of 478 Finnish affected sibling pair (ASP) families (FUSION 1), the strongest linkage results were on chromosomes 20 and 11. We now report a second genome-wide scan using an independent set of 242 Finnish ASP families (FUSION 2), a detailed analysis of the combined set of 737 FUSION 1 + 2 families (495 updated FUSION 1 families), and fine mapping of the regions of chromosomes 11 and 20. The strongest FUSION 2 linkage results were on chromosomes 6 (maximum logarithm of odds score [MLS] = 2.30 at 95 cM) and 14 (MLS = 1.80 at 57 cM). For the combined FUSION 1 + 2 families, three results were particularly notable: chromosome 11 (MLS = 2.98 at 82 cM), chromosome 14 (MLS = 2.74 at 58 cM), and chromosome 6 (MLS = 2.66 at 96 cM). We obtained smaller FUSION 1 + 2 MLSs on chromosomes X (MLS = 1.27 at 152 cM) and 20p (MLS = 1.21 at 20 cM). Among the 10 regions that showed nominally significant evidence for linkage in FUSION 1, four (on chromosomes 6, 11, 14, and X) also showed evidence for linkage in FUSION 2 and stronger evidence for linkage in the combined FUSION 1 + 2 sample.

The frequency of type 1 diabetes mellitus (T1DM)-associated HLA DQ alleles in the U.S. Pacific Northwest is as high as in Scandinavia, which has the highest T1DM incidence in the world. The high regional rate of islet autoimmunity observed among DPT-1 relatives supports this notion. Fortunately, Washington State archives dried blood spots after legislature-mandated newborn screening. The Diabetes Evaluation in Washington (DEW-IT) study aims to show that population-based prospective prediction of T1DM by HLA genotype screening followed by autoantibody surveillance can be performed within the public health infrastructure.

The aim of the study was to describe 5-year changes in meal-stimulated pancreatic insulin reserve in adults with normal and impaired glucose tolerance (NGT, IGT) and diabetes, with or without islet-related antibodies. This was a 5-year follow-up of 270 residents of Wadena, MN, of northern European origin, with good kidney function, defined as creatinine clearance greater than 60 mL/min/1.73 m(2). The subjects comprised a population-based sample originally studied in 1986 to 1987. Urine C-peptide (CP), in a 260-minute collection, was the integrated measure of insulin secretion; Ensure-Plus (Ross, Columbus, OH) was the liquid meal. Islet cytoplasmic antibodies (ICA), insulin autoantibodies (IAA), and glutamate decarboxylase antibodies (GAD65ab) were measured. In 182 subjects with NGT, there was no mean within-subject change in urine CP over 5 years (P =.34). In 41 subjects with impaired GT (IGT), there was a moderate, but nonsignificant, increase in mean CP, and 6 (15%) subjects increased. In 37 type 2 diabetic subjects not taking insulin (type 2-No Ins), who had a mean diabetes duration at the 5-year examination of 9.6 +/- 6.3 years, there was a 21% decrease in mean urine CP (P =.012), attributable mostly to a major drop in 8 of the 37 subjects (22%). Islet-related antibody tests were mostly negative; GAD65ab positivity was related to CP decline only among insulin-taking subjects. In summary, in Wadena adults, meal-stimulated urine CP was stable or increased over 5 years in subjects with NGT and IGT, but CP decreased significantly in about one fifth of type 2-No Ins subjects, with no relation to antibody test results., (Copyright 2002 by W.B. Saunders Company)

Complex protein antigens contain multiple potential T cell recognition epitopes, which are generated through a processing pathway involving partial antigen degradation via proteases, binding to MHC molecules, and display on the APC surface, followed by recognition via the T cell receptor. We have investigated recognition of the GAD65 protein, one of the well-characterized autoantigens in type I diabetes, among individuals carrying the HLA-DR4 haplotypes characteristic of susceptibility to IDDM. Using sets of 20-mer peptides spanning the GAD65 molecule, multiple immunostimulatory epitopes were identified, with diverse class II DR molecules functioning as the restriction element. The majority of T cell responses were restricted by DRB1 molecules; however, DRB4 restricted responses were also observed. Antigen-specific T cell clones and lines were derived from peripheral blood samples of pre-diabetic and IDDM patients and T cell recognition and response were measured. Highly variable proliferative and cytokine release profiles were observed, even among T cells specific for a single GAD65 epitope.

We evaluated the role of the humoral arm of the immune response in causing or contributing to virus-induced diabetes. Transgenic mice expressing the nucleoprotein (NP) or glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter (RIP) in pancreatic beta cells (RIP-LCMV) and RIP-LCMV mice with genetic dysfunction of B cells (RIP-LCMV x microMT/microMT) were compared for development of diabetes after challenge with LCMV. After inoculation with LCMV, B and T lymphocytes and macrophages infiltrated into pancreatic islets in RIP-LCMV mice, and over 50% of these mice generated Abs against host insulin or glutamate decarboxylase. However, neither B cells nor the autoantibodies played a direct role in the initiation, kinetics, or severity of the virus-induced diabetes as judged by comparing disease in RIP-LCMV mice to littermates whose functional B cells were genetically eliminated. Furthermore, the quality and quantity of T lymphocyte and macrophage infiltration was similar in the B cell-deficient and non-B cell-deficient RIP-LCMV mice. Although the development of autoantibodies to islet Ags had no direct influence on the pathogenesis of insulin-dependent (type 1) diabetes mellitus, it served as a prediabetes marker, as such autoantibodies were often elevated before the onset of disease. Hence, the RIP-LCMV model is not only useful for understanding the pathogenetic mechanisms of how islets are destroyed and spared but also for evaluating therapeutic strategies before onset of clinical diabetes.

We are conducting a genome scan at an average resolution of 10 centimorgans (cM) for type 2 diabetes susceptibility genes in 716 affected sib pairs from 477 Finnish families. To date, our best evidence for linkage is on chromosome 20 with potentially separable peaks located on both the long and short arms. The unweighted multipoint maximum logarithm of odds score (MLS) was 3.08 on 20p (location, chi = 19.5 cM) under an additive model, whereas the weighted MLS was 2.06 on 20q (chi = 57 cM, recurrence risk,lambda(s) = 1. 25, P = 0.009). Weighted logarithm of odds scores of 2.00 (chi = 69.5 cM, P = 0.010) and 1.92 (chi = 18.5 cM, P = 0.013) were also observed. Ordered subset analyses based on sibships with extreme mean values of diabetes-related quantitative traits yielded sets of families who contributed disproportionately to the peaks. Two-hour glucose levels in offspring of diabetic individuals gave a MLS of 2. 12 (P = 0.0018) at 9.5 cM. Evidence from this and other studies suggests at least two diabetes-susceptibility genes on chromosome 20. We have also screened the gene for maturity-onset diabetes of the young 1, hepatic nuclear factor 4-a (HNF-4alpha) in 64 affected sibships with evidence for high chromosomal sharing at its location on chromosome 20q. We found no evidence that sequence changes in this gene accounted for the linkage results we observed.

Background: We sought to identify novel islet-cell autoantigens to better understand the pathogenesis, prediction, and immunotherapy of type 1 diabetes., Materials and Methods: Macaque and human islet cDNA libraries expressed in mammalian cells were screened with human diabetes sera. A positive clone was sequenced directly and after 5' rapid amplification of cDNA ends (RACE). Northern blotting and in situ hybridization revealed the tissue distribution of the corresponding protein. Antigen, expressed by in vitro translation, and tryptic peptides were analyzed by SDS-PAGE. For the immunoprecipitations, 183 diabetic, 60 prediabetic, and 91 control sera were used. Truncated antigens were used in immunoprecipitations for epitope mapping. Recombinant antigen expressed in transfected fibroblasts was used in competition assays., Results: Sequencing yielded an 111-kDa, 1,013 amino acid, transmembrane protein (M1851) containing consensus protein tyrosine phosphatase (PTPase) sequence. M1851 was 77% identical in the intracellular domain, but only 31% identical extracellularly, to the islet-cell autoantigen ICA512. mRNA localized to brain, prostate, pancreatic islets, and adrenal medulla. After limited trypsinization, the in vitro translated antigen was 37 kDa. M1851 was recognized by 47% of prediabetes sera, 31% of new diabetes sera, but only 1% of healthy controls. Only 1/73 sera binding M1851 failed to bind ICA512, whereas 42/114 binding ICA512 did not bind M1851. M1851 reactivity was not fully displaced by ICA512 in 24/49 sera. Removing the C-terminal 27, 80, or 160 amino acids of M1851 decreased reactivity by 70%, 90%, and 100%, respectively., Conclusions: This new islet-cell PTPase is likely to be the precursor to the 37-kDa tryptic fragment antigen. It is structurally related to ICA512 but has distinct diabetes autoantibody epitopes located at the C terminus.

Insulin-dependent diabetes mellitus (IDDM) is linked to HLA factors on human chromosome 6 and strongly associated with the presence of autoantibodies against the glutamic acid decarboxylase isoform GAD65. These autoantibodies, GAD65Ab are detected both before and at the time of clinical diagnosis. Molecular sequencing of HLA alleles and PCR-based genotyping have improved our understanding of the linkage between HLA and IDDM. At the same time, the molecular cloning of human islet GAD65 and the development of precise and reproducible GAD65Ab assays with recombinant human GAD65 has given new insights to the problem of to what extent HLA control the development of a GAD65 immune response or to the development of IDDM. Recent data are briefly reviewed. In new onset IDDM patients GAD65Ab were associated with the DQ2/8 or DQ2/X genotype. However, in patients with an older age at onset the association was particularly pronounced with the DQ2/8 genotype. The DQ5/8 genotype was significantly decreased among GAD65Ab positive patients. Certain DQ genotypes, therefore, seem permissive for the formation of GAD65Ab in IDDM. Studies of the general population is needed to determine if the DQ2, 8 or both alleles predispose to GAD65 autoreactivity. This is important since other factors may control the development of IDDM in only a fraction of GAD65 antibody positive individuals detected following a screening of the general population.

Glutamate decarboxylase autoantibodies (GAD65Ab) and beta-cell function were evaluated at and 3 years after diabetes onset in consecutive subjects over 15 years of age. At onset, 21/32 (66%) insulin-treated patients (mean age 43, range 16-79 years) had GAD65Ab; all GAD65Ab persisted 3 years later. At onset, 20/82 (24%) non-insulin-treated patients (mean age 56, range 20-79 years) had GAD65Ab. Of those with persistent GAD65Ab, 8 non-insulin-treated and 11 insulin-treated patients consented to follow-up glucose and glucagon stimulation tests. For non-insulin-treated patients, quantitative GAD65Ab index at onset correlated inversely with 1 + 3 min C-peptide response to glucose (r = -0.68, P < 0.05) and to glucagon (r = -0.79, P < 0.05) 3 years later. Those with high (> 0.50) initial GAD65Ab index had lower C-peptide (fasting, 1 + 3 min after glucose and after glucagon) 3 years later, versus those with low (< 0.50) initial GAD65Ab index (P < 0.05). In conclusion, not only did GAD65Ab presence predict future insulin dependence, but higher GAD65Ab levels may mark more rapid decline in beta-cell function in apparent non-insulin-dependent diabetes.

ICA and GAD65 autoantibody profiles and HLA-DR and DQ analysis were performed on 43 Black juvenile onset IDDM patients and 34 unrelated Black controls from Tennessee, USA. 75% of patients were positive for GAD65 autoantibodies but only 53% had ICA; 39% both ICA and GAD65 antibodies. The strongest HLA association was with the DR3 haplotype DRB1*03 DQA1*0501 DQB1*0201 (63% of patients v 12% of controls RR = 13.0, p < 0.00002). DRB1*04 DQA1*0301 DQB1*0302, associated with IDDM in Caucasians but rare in Negroids, occurred in 27% of patients and 6% of controls (RR = 5.9, p < 0.04). All patients carried DQB1*0302 or DQB1*0201. DQB1*0602 was significantly reduced in patients (2.4% v 41%, RR = 0.036, p < 0.008) and DRB1*1501 was absent in patients (0% v 35%). The frequency of GAD65 autoantibodies in Black American IDDM patients is comparable to that in Caucasians; however ICA positivity is reduced. GAD65 antibodies may therefore be a more sensitive serological test to identify individuals in the Black American general population for markers associated with increased risk of developing IDDM. Current screening methods for predicting preclinical IDDM in Caucasians relies on a combination of immune and HLA markers of IDDM; studies of these markers in the Black Americans will make it possible to extend these options to additional genetically diverse populations.

Insulin-dependent diabetes mellitus (IDDM) is associated with autoreactivity against GAD but the diagnostic sensitivity (positivity in disease) and specificity (negativity in health) of isoform-specific GAD antibodies have yet to be defined in assay systems suitable for screening large number of samples. One set of IDDM patient (n = 10) and control (n = 50) standard sera were used to develop quantitative antibody assays with in vitro synthesized recombinant 35S-methionine-labelled GAD65 and GAD67, respectively, and protein A-Sepharose to separate free from antibody-bound ligand. Binding levels were not normally distributed (p < 0.0001) and therefore, the diagnostic accuracy of GAD antibodies was analysed by the ROC plots in population-based, consecutively-diagnosed, recent onset, 0-14 year-old patients (n = 105), and matched, healthy control subjects (n = 157). The ROC plots showed that the diagnostic sensitivity of GAD65 antibodies was 77% and the specificity 92% compared with 8% and 98%, respectively for GAD67 antibodies. In the IDDM sera, GAD65 and GAD67 antibodies were concordant in 7% (6 of 81) and GAD65 antibodies and ICA in 89% (72 of 81) without a correlation between the autoantibody levels. Autoantibodies to recombinant human islet GAD65 are specific and sensitive markers for childhood IDDM in this immunoassay with in vitro synthesized 35S-methionine-labelled recombinant GAD.

Stiff-man syndrome is a neurologic disorder characterized by progressive rigidity of skeletal muscles. Deficiency of the neurotransmitter gamma-aminobutyric acid and autoantibodies to glutamic acid decarboxylase (GAD), the enzyme synthesizing gamma-aminobutyric acid, are closely associated with the disorder, although the relevant antigenic epitopes have not been identified. In the present study, sera from two patients with SMS was used in an immunoblotting assay with recombinant GAD67 (M(r) 67,000) and GAD65 (M(r) 65,000) isoforms to test whether SMS sera can recognize specific epitopes. We found that both SMS sera recognized the GAD65, but not the GAD67, isoform. Using 13 different synthetic GAD peptides to block the autoantibodies, two GAD65 epitopes were identified. One epitope recognized by both patients' sera, was blocked by the peptide representing amino acid residues 354-368. In one patient only, blocking was also observed by a peptide representing residues 390-402, which includes the binding site of the GAD cofactor, pyridoxal 5'-phosphate. A single amino acid substitution in GAD65 at position 401 (leucine to proline) and representing the analogous GAD67 sequence in this region significantly reduced the peptide's inhibitory effect. These findings suggest that SMS GAD autoantibodies share distinct GAD65 linear epitopes and that some SMS patients' autoantibodies may block the active site, explaining SMS GABA deficiency.

Insulin-dependent diabetes mellitus is associated with a growing number of immune abnormalities. At the time of clinical onset, most patients developing the disease as children or young adults have autoantibodies reactive with islet beta cells. Current autoantibody markers for IDDM are not sufficient to predict the disease in the general population. Studies in first-degree relatives indicate the presence of a subclinical disease characterized by beta cell dysfunction, which may or may not progress to overt IDDM. Although IDDM is genetically linked to certain HLA-DQ class II molecules, it needs to be clarified whether these molecules determine the propensity to react to certain antigens, the failure to maintain tolerance, or the ability to produce disease-associated autoantibodies. Circumstantial evidence suggests that yet another gene outside the HLA complex on chromosome 6 is more important. The interaction with the environment needs to be clarified, and the etiologic role of viruses has not been substantiated. An underlying systemic autoimmune propensity may influence environmental insults and perpetuate islet beta cell destruction. Until these mechanisms are understood, clinicians should periodically check their patients with IDDM for other organ-specific as well as non--organ-specific autoimmune diseases. Our understanding of these phenomena is poor, which may explain why clinical trials with immunosuppressive agents have been of limited success. Further studies on the molecular biology of the immune response against islet beta cell-specific antigens are necessary for the development of both predictive tests and novel measures to prevent IDDM.

A patient who developed Pneumocystis carinii hepatitis and choroiditis despite receiving prophylactic pentamidine therapy by aerosol is described. Liver biopsy showed histology typical of Pneumocystis hepatitis, but his respiratory status was stable and his lungs were free of P carinii organisms on BAL. Thus, inhaled pentamidine prophylaxis did not prevent extrapulmonary pneumocystosis. Patients receiving pentamidine prophylaxis with unexplained symptoms should undergo investigation for possible extrapulmonary P carinii infection.