The Trial to Reduce IDDM in the Genetically at Risk (TRIGR) study: recruitment, intervention and follow-up.

AIMS/HYPOTHESIS: The Trial to Reduce IDDM in the Genetically at Risk (TRIGR) study was designed to establish whether weaning to a highly hydrolysed formula in infancy subsequently reduces the risk of type 1 diabetes. METHODS: The study population comprises newborn infants who have first-degree relatives with type 1 diabetes and meet the increased risk HLA inclusion, but not exclusion criteria. The study is being performed in 15 countries in three continents. First-degree relatives of patients with type 1 diabetes were identified from diabetes clinics, diabetes registries, and from other endocrinology or obstetrics offices and websites. HLA typing was performed at birth from cord or heel stick blood, and the results sent to the study's Data Management Unit within 2 weeks for communication of eligibility to the clinical study centre. All mothers recruited were encouraged to breastfeed. The intervention lasted for 6 to 8 months, and weaning formulas based on hydrolysed casein and standard cow's milk were compared. RESULTS: TRIGR recruited 5,606 infants, of whom 2,160 were enrolled as eligible participants, 6% more than the target of 2,032. Of those enrolled, 80% were exposed to the study formula. The overall retention rate over the first 5 years is 87%, with protocol compliance at 94%. The randomisation code will be opened when the last recruited child turns 10 years of age, i.e. in 2017. CONCLUSIONS/INTERPRETATION: The TRIGR experience demonstrates the feasibility and successful implementation of an international dietary intervention study. TRIGR is the first ever primary prevention trial for type 1 diabetes and, if completed successfully, will provide a definite answer to the research question. TRIAL REGISTRATION: ClinicalTrials.gov NCT00179777 FUNDING: The study was funded by the National Institute of Child Health and Development (NICHD) and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) (grant numbers HD040364, HD042444 and HD051997), Canadian Institutes of Health Research, the Juvenile Diabetes Research Foundation International and the Commission of the European Communities (specific RTD programme 'Quality of Life and Management of Living Resources', contract number QLK1-2002-00372 'Diabetes Prevention'. Other funding came from the EFSD/JDRF/Novo Nordisk Focused Research Grant, Academy of Finland, Dutch Diabetes Research Foundation and Finnish Diabetes Research Foundation).

Feasibility and compliance in a nutritional primary prevention trial in infants at increased risk for type 1 diabetes.

AIM: The international Trial to Reduce IDDM in the Genetically at Risk (TRIGR) was launched to determine whether weaning to a highly hydrolysed formula in infancy reduces the incidence of type 1 diabetes in children at increased genetic disease susceptibility. We describe here the findings on feasibility and compliance from the pilot study. METHODS: The protocol was tested in 240 children. The diet of the participating children was assessed by self-administered dietary forms, a structured questionnaire and a food record. Blood samples were taken and weight and height measured at birth and at 3, 6, 9, 12, 18 and 24 months. RESULTS: A majority of the subjects (84%) were exposed to the study formula at least for 2 months. Linear growth or weight gain over the first 2 years of life was similar in the two study groups. The levels of IgA and IgG antibodies to cow's milk and casein were higher in the cow's milk-based formula group than in the hydrolysed formula group during the intervention period (p<0.05), reflecting the difference in the intake of cow's milk protein. CONCLUSION: This randomized trial on infant feeding turned out to be feasible, and dietary compliance was acceptable. Valuable experience was gained for the planning and sample size estimation of the study proper.

HLA-DQB1-defined genetic susceptibility, beta cell autoimmunity, and metabolic characteristics in familial and nonfamilial insulin-dependent diabetes mellitus. Childhood Diabetes in Finland (DiMe) Study Group.

Familial aggregation of insulin-dependent diabetes mellitus (IDDM) is a common phenomenon, but the reasons behind it are poorly understood. To investigate whether there is heterogeneity between familial and nonfamilial forms of IDDM we compared genetic, immunological, and clinical characteristics of diabetic children with and without an affected first-degree relative in a population-based series of Finnish children with IDDM. The frequencies of HLA-DQB1 genotypes known to be associated with high (DQB1*0302/0201) or moderate (*0302/x) IDDM risk in the Finnish population were increased, while the proportions of DQB1 genotypes associated with low or decreased risk for IDDM were reduced in the 121 familial cases as compared with the 574 nonfamilial cases (32.7 vs. 21.3%, 41.3 vs. 35.9%, 18.3 vs. 31.4%, and 7.7 vs. 11.4%, respectively; P = 0.002). The frequencies and serum concentrations of islet cell antibodies, insulin autoantibodies, and antibodies to the 65-kD isoform of glutamic acid decarboxylase were similar at diagnosis in the familial and nonfamilial cases. The 31 first-affected cases in the multiple case families were younger at diagnosis than the nonfamilial cases (6.9 vs. 8.5 yr; P < 0.05). The 90 second-affected familial cases had less severe metabolic decompensation at diagnosis than either the first-affected familial or nonfamilial cases. In conclusion, familial aggregation of IDDM in Finland is at least partly explained by a higher frequency of IDDM susceptibility genes in families with multiple affected individuals. The lack of differences in autoantibody levels between the familial and nonfamilial cases indicates homogeneity rather than heterogeneity in the pathogenetic process of beta cell destruction.

Prediction of insulin-dependent diabetes mellitus in siblings of children with diabetes. A population-based study. The Childhood Diabetes in Finland Study Group.

An unselected population of 755 siblings of children with insulin-dependent diabetes mellitus (IDDM) was studied to evaluate the predictive characteristics of islet cell antibodies (ICA), antibodies to the IA-2 protein (IA-2A), antibodies to the 65-kD isoform of glutamic acid decarboxylase (GADA), insulin autoantibodies (IAA), and combinations of these markers. We also evaluated whether the histochemical ICA test could be replaced by the combined detection of other markers. 32 siblings progressed to IDDM within 7.7 yr of the initial sample taken at or close to the diagnosis of the index case (median follow-up, 9.1 yr). The positive predictive values of ICA, IA-2A, GADA, and IAA were 43, 55, 42, and 29%, and their sensitivities 81, 69, 69, and 25%, respectively. In contrast to the other three antibody specificities, GADA levels were not related to the risk for IDDM. The risk for IDDM in siblings with four, three, two, one, or no antibodies was 40, 70, 25, 2, and 0.8%, respectively. Combined screening for IA-2A and GADA identified 70% of all ICA-positive siblings, and all of the ICA-positive progressors were also positive for at least one of the three other markers. The sensitivity of the combined analysis of IA-2A and GADA was 81%, and the positive predictive value was 41%. In conclusion, combined screening for IA-2A and GADA may replace the ICA assay, giving comparable sensitivity, specificity, and positive predictive value. Accurate assessment of the risk for IDDM in siblings is complicated, as not even all those with four antibody specificities contract the disease, and some with only one or no antibodies initially will progress to IDDM.

Identification and characterization of glima 38, a glycosylated islet cell membrane antigen, which together with GAD65 and IA2 marks the early phases of autoimmune response in type 1 diabetes.

Immunoprecipitating IgG autoantibodies to glutamic acid decarboxylase, GAD65, and/or a tyrosine phosphatase, IA2, are present in the majority of individuals experiencing pancreatic beta cell destruction and development of type 1 diabetes. Here we identify a third islet cell autoantigen, a novel 38-kD protein, which is specifically immunoprecipitated with sera from a subset of prediabetic individuals and newly diagnosed type 1 diabetic patients. The 38-kD autoantigen, named glima 38, is an amphiphilic membrane glycoprotein, specifically expressed in islet and neuronal cell lines, and thus shares the neuroendocrine expression patterns of GAD65 and IA2. Removal of N-linked carbohydrates results in a protein of 22,000 Mr. Glima 38 autoantibodies were detected in 16/86 (19%) of newly diagnosed patients, including three very young children, who had a rapid onset of disease, and in 6/44 (14%) of prediabetic individuals up to several years before clinical onset. The cumulative incidence of GAD65 and glima 38 antibodies in these two groups was 83 and 80%, respectively, and the cumulative incidence of GAD65, glima 38, and IA2 antibodies in the same groups was 91 and 84%, respectively. GAD65, IA2, and glima 38 represent three distinct targets of immunoprecipitating IgG autoantibodies associated with beta cell destruction and type 1 diabetes.

An association between complement-fixing cytoplasmic islet cell antibodies and endogenous insulin secretion in children with insulin-dependent diabetes mellitus.

Cytoplasmic islet cell antibodies and endogenous insulin secretion were studied in 184 children and adolescents having insulin-dependent diabetes mellitus (IDDM) in a cross-sectional study. The mean age of the subjects was 12.3 yr (range: 2.8-19.2 yr), and the mean duration of diabetes was 4.6 yr (range: 0.1-15.6 yr). Islet cell antibodies (ICA) were determined by both the indirect immunofluorescence (IF-ICA) and the complement-fixing (CF-ICA) methods. Forty-four patients (23.9%) were positive with respect to both IF- and CF-ICA, 54 patients (29.3%) had only IF-ICA, and 86 patients had no ICA. The patients having CF-ICA had a significantly higher endogenous insulin secretion in comparison with the patients who were only IF-ICA positive. The difference between the groups remained significant even when the age at onset of diabetes and the duration of the disease were taken into account. This finding, revealing an association between CF-ICA and endogenous insulin secretion, suggests that complement-fixing antibodies are seen only if the beta-cell mass is sufficiently preserved. The result contradicts the hypothesis, based on studies in vitro, that CF-ICA should be involved in the selective beta-cell damage in IDDM.

HLA-DQB1 alleles and absence of Asp 57 as susceptibility factors of IDDM in Finland.

It has been proposed that negatively charged aspartic acid at position 57 of the HLA-DQ beta-chain determines resistance to development of insulin-dependent diabetes mellitus (IDDM), whereas genetic susceptibility to IDDM correlates with a neutral amino acid residue. The disease rate is very low in Oriental populations with high frequencies of Asp 57. This raises a question whether the high incidence of IDDM in Finland could be explained by the distribution of this disease marker. In this study, the polymerase chain reaction products of 86 diabetic patients and 115 nondiabetic control subjects were analyzed with seven sequence-specific oligonucleotide probes. Only 25.5% of the diabetic subjects were phenotyped as Asp 57+ compared to 82% of control subjects, which suggests that Asp 57 negativity is a definite risk marker for developing IDDM in Finnish patients. However, the susceptibility conferred by various non-Asp and Asp haplotypes was not equally strong: DQw8 was the most important risk marker and DQw6 the most protective one. The frequency of Asp 57+ DQw4 was similar in diabetic patients and control subjects. The highest genotype-associated relative risk was defined by DQw2/DQw8 heterozygosity (RR 91), whereas it was 13 for non-Asp homozygosity. In the control subjects, the frequency of Asp 57+ phenotypes was higher than in several white populations with lower IDDM incidence figures. We conclude that the disease risk in Finland appears to be most strongly related to specific Asp 57- alleles, although other HLA- or non-HLA-associated genes may also contribute to IDDM susceptibility in this population.

Relation between insulin antibody and complement-fixing islet cell antibody at clinical diagnosis of IDDM.

To test the hypothesis that insulin-binding antibodies (IBAs) appearing in the circulation before insulin treatment are markers of beta-cell damage, we studied the prevalence of IBAs and both conventional (IF-ICAs)-and complement (CF-ICAs)-fixing cytoplasmic islet cell antibodies in 60 newly diagnosed diabetic children with a mean age of 9.5 yr. Seventeen (28.3%) had an insulin binding exceeding the upper range (2.8%) of that observed in 68 age-matched controls. The IBA-positive subjects were characterized by a younger age of onset [6.2 +/- 4.0 (SD) vs. 10.8 +/- 3.2 yr; P less than 0.001], lower glycosylated hemoglobin A1 levels (14.1 +/- 3.1 vs. 16.0 +/- 3.0%; P less than 0.05), lower serum C-peptide concentrations (0.12 +/- 0.07 vs. 0.20 +/- 0.17 nmol/L; P less than 0.05), and an increased frequency of HLA-Dw4 (9/13 vs. 11/37; P less than 0.05). There was no significant relation between IBAs and serum C-peptide concentrations after age adjustment by multiple regression analysis. Forty-three children (75%) were positive for IF-ICA and 38 (63.3%) for CF-ICA. Twelve IBA-positive diabetics (70.6%) had IF-ICA as well as CF-ICA in their serum. No association could be observed between IBA and either IF-ICA or CF-ICA, however. The results suggest that IBAs developing before diagnosis serve as an indicator of clinical and genetic heterogeneity within IDDM rather than as a marker of autoimmune beta-cell destruction.

Intracellular enzymes of collagen biosynthesis in rat kidney in streptozotocin diabetes.

The activities of the four enzymes catalyzing intracellular post-translational modifications of the collagen polypeptide chains were assayed in the kidneys of rats with streptozotocin diabetes. When the changes in the four enzyme activities were expressed per milligram of protein in the 15,000 X g supernatant of the kidney homogenates, there were no changes in any of the enzyme activities at four weeks and only slight increases in the prolyl and lysyl hydroxylase activities at 12 weeks after the induction of diabetes. When the changes were expressed as total enzyme activities per two kidneys, again no changes were found in any enzyme activity at four weeks, but at 12 weeks significant increases were found in all four enzyme activities, namely prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase, and collagen glucosyltransferase. The data would be consistent with an increased collagen synthesis in diabetic kidneys, but they do not support the hypothesis that there might be specific changes in some of these enzyme activities or in the level of certain posttranslational modifications of the collagen polypeptide chains in this disease.

Dynamic pattern of disease-associated autoantibodies in siblings of children with type 1 diabetes: a population-based study.

To study the dynamics of disease-associated humoral immune responses, we analyzed autoantibodies to the IA-2 protein (IA-2A), glutamic acid decarboxylase (GADA), and insulin (IAA) and also islet cell antibodies (ICA) in a population-based, prospective, representative series of 710 siblings (<20 years of age) of children with type 1 diabetes. Positivity for single autoantibodies was observed in 8-13% of these siblings during an average follow-up of 4 years. The overall incidence rates per 1,000 years (number of cases/person-years in parentheses) for positive seroconversion of IA-2A were nine (19/2,123), followed by six (12/2,049) for GADA, 19 (40/2,111) for IAA, and 16 (31/1965) for ICA. Positive seroconversions seemed to be associated with a young age of the sibling, HLA DR3/DR4 heterozygosity, HLA identity, and a high initial number of detectable autoantibodies. The overall incidence rates per 1,000 years (number of cases/person-years in parentheses) for inverse seroconversion of IA-2A were 76 (12/157), followed by 42 (10/237) for GADA, 460 (32/70) for IAA, and 27 (9/331) for ICA. No consistent risk factor for inverse seroconversions was present, although seroconversions were most frequent in siblings with older age, male sex, HLA phenotypes other than DR3/DR4, a small family size, and no other autoantibodies detectable at seroconversion. Altogether, these observations indicate that beta-cell autoimmunity may be induced at any age in childhood and adolescence. HLA-conferred genetic disease susceptibility is a strong determinant of persistent beta-cell autoimmunity, but environmental factors may also contribute to such autoimmunity.

Effect of cow's milk exposure and maternal type 1 diabetes on cellular and humoral immunization to dietary insulin in infants at genetic risk for type 1 diabetes. Finnish Trial to Reduce IDDM in the Genetically at Risk Study Group.

Type 1 diabetes is considered to be a T-cell-mediated autoimmune disease in which insulin-producing beta-cells are destroyed. Immunity to insulin has been suggested to be one of the primary autoimmune mechanisms leading to islet cell destruction. We have previously shown that the first immunization to insulin occurs by exposure to bovine insulin (BI) in cow's milk (CM) formula. In this study, we analyzed the development of insulin-specific T-cell responses by proliferation test, emergence of insulin-binding antibodies by enzyme immunoassay, and insulin autoantibodies by radioimmunoassay in relation to CM exposure and family history of type 1 diabetes in infants with a first-degree relative with type 1 diabetes and increased genetic risk for the disease. The infants were randomized to receive either an adapted CM-based formula or a hydrolyzed casein (HC)-based formula after breast-feeding for the first 6-8 months of life. At the age of 3 months, both cellular and humoral responses to BI were higher in infants exposed to CM formula than in infants fully breast-fed (P = 0.015 and P = 0.007). IgG antibodies to BI were higher in infants who received CM formula than in infants who received HC formula at 3 months of age (P = 0.01), but no difference in T-cell responses was seen between the groups. T-cell responses to BI at 9 months of age (P = 0.05) and to human insulin at 12 (P = 0.014) and 24 months of age (P = 0.009) as well as IgG antibodies to BI at 24 months of age (P = 0.05) were lower in children with a diabetic mother than in children with a diabetic father or a sibling, suggesting possible tolerization to insulin by maternal insulin therapy. The priming of insulin-specific humoral and T-cell immunity occurs in early infancy by dietary insulin, and this phenomenon is influenced by maternal type 1 diabetes.

Glutamate decarboxylase-reactive peripheral blood lymphocytes from patients with IDDM express gut-specific homing receptor alpha4beta7-integrin.

Migration of lymphocytes to the pancreas is a prerequisite for insulitis in IDDM. Mucosal vascular addressin (MAdCAM-1), involved in the recirculation of lymphocytes to the gut, has been found in the inflamed islets in NOD mice. In humans, triggers of the gut immune system (e.g., early exposure to cow's milk proteins in infancy, exposure to enteroviral infections) have been associated with IDDM. To study the possible link between the gut immune system and IDDM, we tested the expression of the alpha4beta7-integrin, a homing receptor for MAdCAM-1, on GAD65-reactive lymphocytes. Using immunomagnetic cell sorting, we depleted the lymphocytes with high expression of alpha4beta7-integrin in the peripheral blood mononuclear cell population from IDDM patients and patients with autoimmune polyendocrine disease type 1 (APD-I). The depletion led to a marked decrease (mean 70%) in the cellular response against GAD65 in three of six IDDM patients and in one subject at high risk for IDDM. A decrease of 37% in the GAD response was observed after depletion in the case of one APD-I patient who also had IDDM. Cellular response to tetanus toxoid increased in the majority of patients as well as in three control subjects studied. We demonstrated that a remarkable population of islet cell antigen-reactive lymphocytes express the gut-specific homing receptor, which emphasizes the role of gut immunity in IDDM. The manipulation of the gut immune system is therefore proposed as a tool for modulation of the autoimmunity against pancreatic beta-cells in IDDM.

Cellular immune response to cow's milk beta-lactoglobulin in patients with newly diagnosed IDDM.

Elevated levels of antibodies to cow's milk proteins, i.e., beta-lactoglobulin (BLG) and bovine serum albumin (BSA), have been associated with IDDM. We observed enhanced cellular immune response by a proliferation test of peripheral blood mononuclear cells to BLG in 22 of 40 (55%) patients with newly diagnosed IDDM compared with 7 of 32 healthy children (22%) (P = 0.004, chi 2 test). The median stimulation index to BLG was 3.3 in patients and 1.5 in healthy children (P = 0.003, Mann-Whitney U test). No difference was found in cellular reactivity to other cow's milk proteins, such as BSA or alpha-casein, or to a dietary immunogenic protein, ovalbumin. Cellular responsiveness to BLG was not associated with HLA-DQB1* risk alleles of IDDM, which suggests that immune response to the protein does not only reflect the accumulation of these HLA alleles in the patients with IDDM. We suggest that enhanced cellular immune response to dietary BLG may reflect a disturbance in the regulation of immune response to oral antigens in IDDM. This kind of defect may play a fundamental role in the development of beta-cell autoimmunity in IDDM.

Non-class II HLA gene associated with type 1 diabetes maps to the 240-kb region near HLA-B.

Several studies provide evidence that in addition to the DQ-DR genes, HLA contains another uncharacterized gene or genes associated with type 1 diabetes. Our aim was to investigate the effect of this gene independently of the DQ-DR genes and to localize it with a matched case-control study. More than 1,400 patients and 30,000 control individuals from Finland were studied. They were first genotyped for the selected alleles of the HLA-DQB1, -DQA1, and -DRB1 genes. For the DR3/4(0404) genotype, 75 patients and 181 control subjects were stratified, and 241 patients and 354 controls were stratified for the DR3/4(0401) genotype. Ten microsatellite markers in the HLA class III and I regions (D6S273, TNFa, C12A, STR MICA, MIB, C125, C143, C245, C3211, and MOGc) and selected alleles of the HLA-A and HLA-B genes were studied. In the DR3/4(0404)-stratified group, we found that markers located between C12A and C143 near the HLA-B gene confer a strong additional diabetes association. This was confirmed by the population differentiation test in both DR3/4(0404)- and DR3/4(0401)-stratified groups. Our data indicate that an additional gene associated with type 1 diabetes is located in the 240-kb region near HLA-B. We excluded STR MICA polymorphism as a mutation responsible for diabetes association.

Cow's milk consumption, HLA-DQB1 genotype, and type 1 diabetes: a nested case-control study of siblings of children with diabetes. Childhood diabetes in Finland study group.

The evidence for the putative role of cow's milk in the development of type 1 diabetes is controversial. We studied infant feeding patterns and childhood diet by structured questionnaire (n = 725) and HLA-DQB1 genotype by a polymerase chain reaction-based method (n = 556) in siblings of affected children and followed them for clinical type 1 diabetes. In a nested case-control design in a population who had both dietary and genetic data available, we selected as cases those siblings who progressed to clinical diabetes during the follow-up period (n = 33). For each case, we chose as matched control subjects siblings who fulfilled the following criteria: same sex, age within 1 year, not from the same family, the start of the follow-up within 6 months of that of the respective case, and being at risk for type 1 diabetes at the time the case presented with that disease (n = 254). The median follow-up time was 9.7 years (range 0.2-11.3). Early age at introduction of cow's milk supplements was not significantly associated with progression to clinical type 1 diabetes (relative risk adjusted for matching factors, maternal education, maternal and child's ages, childhood milk consumption, and genetic susceptibility markers was 1.60 [95% CI 0.5-5.1]). The estimated relative risk of childhood milk consumption for progression to type 1 diabetes was 5.37 (1.6-18.4) when adjusted for the matching and aforementioned sociodemographic factors, age at introduction of supplementary milk feeding, as well as for genetic susceptibility markers. In conclusion, our results provide support for the hypothesis that high consumption of cow's milk during childhood can be diabetogenic in siblings of children with type 1 diabetes. However, further studies are needed to assess the possible interaction between genetic disease susceptibility and dietary exposures in the development of this disease.

Genetic markers, humoral autoimmunity, and prediction of type 1 diabetes in siblings of affected children. Childhood Diabetes in Finland Study Group.

The relationships between genetic markers and disease-associated autoantibodies were studied in an unselected population of 701 siblings of children with type 1 diabetes, and the predictive characteristics of these markers over a period of 9 years were determined. Increased prevalences of all the antibodies were closely associated with HLA identity to the index case, the DR4 and DQB1*0302 alleles, and the DR3/4 phenotype and the DQB1*02/0302 genotype. Antibodies to GAD (GADA) were also associated with the DR3 and DQB1*02 alleles. Siblings carrying the protective DR2 and DQB1*0602-3 alleles were characterized by lower frequencies of islet cell antibodies (ICA), antibodies to IA-2 (IA-2A), and GADA. Higher levels of ICA were related to HLA identity, the DR4 and DQB1*0302 alleles, and the susceptible DQB1 genotypes, while no significant differences were observed in the levels of IA-2A, GADA, or insulin autoantibodies among siblings with different HLA risk markers. The DR2 or DQB1*0602-3 alleles were not related to the levels of any antibody specificity. A combination of the genetic markers and autoantibodies increased the positive predictive values of all autoantibodies substantially, which may have clinical implications when evaluating the risk of developing type 1 diabetes at the individual level or when recruiting high-risk individuals for intervention trials. However, because such combinations also resulted in reduced sensitivity, autoantibodies alone rather than in combination with genetic markers are recommended as the first-line screening in siblings. Finally, not all siblings with a broad humoral autoimmune response or high-risk genetic markers present with type 1 diabetes, while some with a low genetic risk and weak initial signs of humoral autoimmunity may progress to disease.

Disease-associated antibodies in offspring of mothers with IDDM.

We studied 20 infants of mothers with IDDM participating in a pilot study for a dietary intervention trial, testing the hypothesis that avoidance of cow's milk proteins early in life will reduce the risk of subsequent IDDM. The aim was to evaluate the elimination of IDDM-associated antibodies from the peripheral circulation of the infants, the possible emergence of autoantibodies indicating beta-cell destruction, and the influence of the dietary intervention and genetic disease susceptibility on the development of these autoantibodies. Transplacentally transferred islet cell antibodies (ICAs) and antibodies to the 65-kDa isoform of glutamic acid decarboxylase (GAD65As) disappeared from the peripheral circulation of most infants over the first few months of life and in all infants before the age of 9 months. Insulin antibodies were eliminated before the same age in all cases but one. The higher the initial antibody level was, the longer the time required for elimination. Four infants tested positive for insulin autoantibodies (IAAs) on at least one occasion during the first year of life, and 5 out of 16 unaffected subjects (31%) had IAAs at the age of 2 years. One infant became positive for IAA before the age of 6 months, with increasing levels later, seroconverted to positivity for ICAs and GAD65As between 6 and 9 months and presented with clinical IDDM at the age of 14 months. He had the HLA DQB1*0302/x genotype, which predisposes carriers to IDDM, and had been given the casein hydrolysate formula as supplementary milk. There were no significant differences in the levels of various autoantibodies between two groups of subjects defined either on the type of dietary intervention or the degree of genetic susceptibility. The findings indicate that transplacentally transferred antibodies related to IDDM are usually eliminated from the peripheral circulation of infants before 9 months of age and that IDDM-associated autoantibodies may emerge before the age of 6 months. Our results also illustrate that avoidance of cow's milk proteins over the first 9 months of life does not provide total protection against IDDM.

Human leukocyte antigen identity and DQ risk alleles in autoantibody-positive siblings of children with IDDM are associated with reduced early insulin response. Childhood Diabetes in Finland (DiMe) Study Group.

To investigate the relationship between human leukocyte antigen (HLA)-associated genetic factors and the development of beta-cell dysfunction, we performed sequential intravenous glucose tolerance tests (IVGTTs) on 81 islet cell antibody (ICA)-positive and/or insulin autoantibody-positive healthy siblings of children with newly diagnosed insulin-dependent diabetes mellitus (IDDM). A lower glucose disappearance rate (Kg) (P < 0.5) and decreased first-phase insulin response (FPIR) (P < 0.05) were observed on multiple occasions in HLA-identical siblings compared with the haploidentical or nonidentical ones. Siblings carrying the DQB1*0302/0201, -0302/x, or -0201/x genotype also had lower FPIRs (P < or = 0.05) at several time points than those with no DQB1 risk genotype. When all IVGTTs were taken into account, DQB1*0302/0201 heterozygous siblings had an abnormally low FPIR (< 45 mU/l; 3rd percentile) in at least one test more often than did siblings with no DQB1 risk genotype (50.0% vs. 6.1%; P = 0.001). Siblings carrying either the DQB1*0602 or the DQB1*0603 protective allele had lower serum peak ICA and glutamic acid decarboxylase (GAD)65 antibody levels (P = 0.023 and 0.007, respectively) and higher FPIRs on several occasions (P < 0.05) than those with the DQB1 risk genotypes. Progression to IDDM was related to both HLA identity and the presence of the DQB1*0302/0201 genotype. Normal Kg and FPIR levels were observed in siblings who were positive for only insulin autoantibody, and none of them developed IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of HLA-B allele on the IDDM risk defined by DRB1*04 subtypes and DQB1*0302.

The genes encoding the HLA-DQ heterodimer molecules, DQB1 and DQA1, have been found to have the strongest association with IDDM risk, although there is cumulative evidence for the effect of other gene loci within the major histocompatibility complex gene region. After the HLA-DQ locus, the HLA-DR locus has been suggested most often as contributing to the disease susceptibility. In this study we analyzed at the population level the effect of DR4 subtypes and class I, HLA-B alleles, on IDDM risk when the influence of the DQ locus was stratified. In all three populations studied (Estonian, Latvian, and Russian), DQB1*0302 haplotypes most frequently carried DRB1*0401 or DRB1*0404. DRB1*0401 was the most prevalent subtype in IDDM patients, whereas DRB1*0404 was decreased in frequency. DRB1*0402 was also prevalent among Russian haplotypes, but was not associated with IDDM risk. When HLA-B alleles were analyzed, strong associations between the presence of specific B alleles and DRB1*04 subtypes were detected. The HLA-B39 allele was found significantly more often in DRB1*0404-DQB1*0302-positive patients than in healthy control subjects positive for this haplotype: 27 of 54 (50%) vs. 4 of 49 (8.2%) (P < 0.0001). The results demonstrate that DQ and DR genes cannot explain all of the HLA-linked susceptibility to IDDM, and that the existence of a susceptibility locus telomeric to DR is probable.

Cow's milk formula feeding induces primary immunization to insulin in infants at genetic risk for type 1 diabetes.

Insulin autoantibodies (IAAs) often appear as the first sign of islet cell autoimmunity in prediabetic children. Because cow's milk contains bovine insulin, we followed the development of insulin-binding antibodies in children fed with cow's milk formula. Bovine insulin- and human insulin-binding antibodies by enzyme immunoassay and IAA by radioimmunoassay were analyzed in 200 infants carrying HLA-DQB1*0302 but no protective alleles who participated in a Finnish population-based birth-cohort study. Based on the prospectively registered information, the first 100 infants enrolled in the study who were exposed to cow's milk formula before age 12 weeks and the first 100 infants enrolled in the study who were exclusively breast-fed for longer than their first 12 weeks of life were selected for the present study. Also, 11 children from the birth cohort who developed at least two diabetes-associated autoantibodies, 98 children with newly diagnosed type 1 diabetes, and 92 healthy children were studied. We found that the amount of IgG-antibodies binding to bovine insulin was higher at age 3 months in infants who were exposed to cow's milk formula than in infants who were exclusively breast-fed at that age (median 0.521 vs. 0.190; P < 0.0001). The antibodies binding to bovine insulin cross-reacted with human insulin. None of these infants tested positive for IAA. The levels of bovine insulin-binding antibodies declined in both groups at ages 12 and 18 months, whereas in the 11 children with at least two diabetes-associated autoantibodies the levels increased during the follow-up period (P < 0.0001). IgG antibodies correlated with IgG2 antibodies binding to bovine insulin (r = 0.43, P = 0.004) and IAA (r = 0.27, P = 0.02) in diabetic children, but not in healthy children. Cow's milk feeding is an environmental trigger of immunity to insulin in infancy that may explain the epidemiological link between the risk of type 1 diabetes and early exposure to cow's milk formulas. This immune response to insulin may later be diverted into autoaggressive immunity against beta-cells in some individuals, as indicated by our findings in children with diabetes-associated autoantibodies.