Exposing the oral mucosa to antigen may stimulate immune tolerance. It is unknown whether treatment with oral insulin can induce a tolerogenic immune response in children genetically susceptible to type 1 diabetes. OBJECTIVE To assess the immune responses and adverse events associated with orally administered insulin in autoantibody-negative, genetically at-risk children. DESIGN, SETTING, AND PARTICIPANTS The Pre-POINT study, a double-blind, placebo-controlled, dose-escalation, phase 1/2 clinical pilot study performed between 2009 and 2013 in Germany, Austria, the United States, and the United Kingdom and enrolling 25 islet autoantibody-negative children aged 2 to 7 years with a family history of type 1 diabetes and susceptible human leukocyte antigen class II genotypes. Follow-up was completed in August 2013. INTERVENTIONS Children were randomized to receive oral insulin (n = 15) or placebo (n = 10) once daily for 3 to 18 months. Nine children received insulin with dose escalations from 2.5 to 7.5 mg (n = 3), 2.5 to 22.5 mg (n = 3), or 7.5 to 67.5 mg (n = 3) after 6 months; 6 children only received doses of 22.5 mg (n = 3) or 67.5 mg (n = 3). MAIN OUTCOMES AND MEASURES An immune response to insulin, measured as serum IgG and saliva IgA binding to insulin, and CD4 + T-cell proliferative responses to insulin. RESULTS Increases in IgG binding to insulin, saliva IgA binding to insulin, or CD4 + T-cell proliferative responses to insulin were observed in 2 of 10 (20% [95% CI, 0.1%-45%]) placebo-treated children and in 1 of 6 (16.7% [95% CI, 0.1%-46%]) children treated with 2.5 mg of insulin, 1 of 6 (16.7%[ 95% CI, 0.1%-46%]) treated with 7.5 mg, 2 of 6 (33.3% [95% CI, 0.1%-71%]) treated with 22.5 mg, and 5 of 6 (83.3% [ 95% CI, 53%-99.9%]) treated with 67.5 mg (P = .02). Insulin-responsive T cells displayed regulatory T-cell features after oral insulin treatment. No hypoglycemia, IgE responses to insulin, autoantibodies to glutamic acid decarboxylase or insulinoma-associated antigen 2, or diabetes were observed. Adverse events were reported in 12 insulin-treated children (67 events) and 10 placebo-treated children (35 events). CONCLUSIONS AND RELEVANCE In this pilot study of children at high risk for type 1 diabetes, daily oral administration of 67.5 mg of insulin, compared with placebo, resulted in an immune response without hypoglycemia. These findings support the need for a phase 3 trial to determine whether oral insulin can prevent islet autoimmunity and diabetes in such children.

Very little is known about the role of the innate immune system in the course of human type 1 diabetes. Here we investigated neutrophil numbers along with other leukocyte populations in patients at diagnosis of type 1 diabetes and during prediabetes. Complete and differential blood counts were analyzed from 107 adult patients with newly diagnosed type 1 diabetes, 21 children with persistent islet autoantibodies and a family history of type 1 diabetes, and 1 238 age and gender matched control subjects, all individuals without any signs of acute infection.Adult patients with newly diagnosed type 1 diabetes had significantly lower total WBC (p<1×10⁻⁶), neutrophil (p<1×10⁻⁶), basophil (p<1×10⁻⁶), monocyte (p=4×10⁻⁶) and lymphocyte (p<1×10⁻⁶) counts compared to control subjects. Erythrocyte, eosinophil and platelet counts did not differ between groups. Similarly, children with persistent islet autoantibodies had decreased WBC (p=0.001), neutrophils (p=0.003), and lymphocytes (p=0.006) in comparison to control children. Our findings demonstrate a perturbation of leukocyte homeostasis at and prior to onset of type 1 diabetes suggesting a general involvement of the innate immune system in the pathogenesis of type 1 diabetes.

ObjectiveType 1 diabetes can be identified by the presence of beta-cell autoantibodies that often arise in the first few years of life. The purpose of this perspective is to present the case for primary prevention of beta-cell autoimmunity and to provide a study design for its implementation in Europe.MethodsWe examined and summarized recruitment strategies, enrollment rates, and outcomes in published TRIGR, FINDIA and BABYDIET primary prevention trials, and the TEDDY intensive observational study. A proposal for a recruitment and implementation strategy to perform a phase II/III primary prevention randomized controlled trial in infants with genetic risk for developing beta-cell autoimmunity is outlined.ResultsInfants with a family history of type 1 diabetes (TRIGR, BABYDIET, TEDDY) and infants younger than age 3 months from the general population (FINDIA, TEDDY) were enrolled into these studies. All studies used HLA genotyping as part of their eligibility criteria. Predicted beta-cell autoimmunity risk in the eligible infants ranged from 3% (FINDIA, TEDDY general population) up to 12% (TRIGR, BABYDIET). Amongst eligible infants, participation was between 38% (TEDDY general population) and 97% (FINDIA). Outcomes, defined as multiple beta-cell autoantibodies, were consistent with predicted risks. We subsequently modeled recruitment into a randomized controlled trial (RCT) that could assess the efficacy of oral insulin treatment as adapted from the Pre-POINT pilot trial. The RCT would recruit infants with and without a first-degree family history of type 1 diabetes and be based on general population genetic risk testing. HLA genotyping and, for the general population, genotyping at additional type 1 diabetes susceptibility SNPs would be used to identify children with around 10% risk of beta-cell autoimmunity. The proposed RCT would have 80% power to detect a 50% reduction in multiple beta-cell autoantibodies by age 4 years at a two-tailed alpha of 0.05, and would randomize around 1160 infants to oral insulin or placebo arms in order to fulfill this. It is estimated that recruitment would require testing of between 400,000 and 500,000 newborns or infants.ConclusionIt is timely and feasible to establish a platform for primary prevention trials for type 1 diabetes in Europe. This multi-site European infrastructure would perform RCTs, supply data coordination and biorepository, provide cohorts for mechanistic and observational studies, and increase awareness for autoimmune diabetes.

Leptin receptor-deficient db/db mice are a commonly used research model and it is known that the genetic background, on which the mutation is bred, modulates the phenotype. While diabetes-resistant strains sustain near normal glycemia and hyperinsulinemia, susceptible backgrounds develop overt hyperglycemia and islet involution. We hypothesized that genetically-determined differences in the proliferative capacity and the apoptotic frequency of pancreatic beta cells contribute to this phenotypic disparity. We studied C57BLKS/J (BKS; diabetes-susceptible) and C57BL/6 (B6; diabetes-resistant) db/db mice and heterozygous controls from 5 to 12 weeks of age. Body weight, fasting blood glucose, plasma insulin, HOMA-IR, alpha cell mass, beta cell mass, proliferation and apoptosis were measured. Comparable insulin resistance developed in the 2 db/db strains, which was well compensated for on both genetic backgrounds until 7 weeks of age. As expected, the BKS mice became hyperglycemic at 9 weeks. Beta cell proliferation was initially increased in both db/db strains but decreased rapidly in the BKS mice with advancing age. The rate of beta cell apoptosis was already higher in prediabetic BKS mice than in their B6 counterparts. Beta cell mass increased continuously in the B6 strain until 12 weeks of age, but declined from 7 weeks onwards in BKS. An age-dependent decline of beta cell proliferation and an increased rate of beta cell apoptosis already in the prediabetic state probably contribute to the diabetes susceptibility of db/db BKS mice. These factors could also play a role in the genetic predisposition for type 2 diabetes in humans.

An autologous cord blood infusion does not change the natural course of metabolic and immune parameters after disease onset. However, the content of CD34+ cells in the stored blood sample might offer potential for improvement of future cell therapies.

BackgroundThe transcription factor Pax6 functions in the specification and maintenance of the differentiated cell lineages in the endocrine pancreas. It has two DNA binding domains, the paired domain and the homeodomain, in addition to a C-terminal transactivation domain. The phenotype of Pax6-/- knockout mice suggests non-redundant functions of the transcription factor in the development of glucagon-expressing α-cells as this cell type is absent in the mutants. We ask the question of how the differentiation of pancreatic endocrine cells, in particular that of α-cells, is affected by selective inactivation of either one of the three major domains of Pax6.ResultsThe Pax6Aey18 mutant mouse line, in which the paired domain is inactivated, showed a phenotype similar to that of Pax6-/- knockout mice with a near complete absence of glucagon-positive α-cells (0-4 cells/section; ≤1% of wt), reduced β-cell area (74% of wt) and disorganized islets. The proportion of ghrelin-positive ε-cells was expanded. In Pax6Sey-Neu mutants, which lack the transactivation domain, α-and β-cells where reduced to 25 and 40% of wt, respectively. We also studied two mouse lines with mutations in the homeodomain, Pax64Neu and Pax6132-14Neu. Neighboring amino acids are affected in the two lines and both point mutations abolish DNA binding of the classical P3 homeodomain target sequence. The pancreatic phenotype of the two mutants however was divergent. While Pax64Neu homozygotes showed a reduction of α- and β-cells to 59 and 61%, respectively, pancreatic endocrine development was unaltered in the Pax6132-14Neu mutant strain.ConclusionsWe show that inactivation of the Pax6 paired domain leads to a more severe phenotype with regards to the differentiation of pancreatic α-cells than the loss of the transactivation domain. The analysis of two different homeodomain mutants suggests that the binding of Pax6 to P3 homeodomain consensus sequences is not required for α-cell development. It rather seems that the homeodomain has a modulating role in Pax6 function, possibly by facilitating a PH0-like binding confirmation on paired domain target genes like proglucagon. This function is differentially affected by the two homeodomain mutations analyzed in this study.

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IntroductionThe diabetes mellitus Incidence Cohort Registry (DiMelli) aims to characterize diabetes phenotypes by immunologic, metabolic, and genetic markers. We classified patients into three groups according to islet autoantibody status and examined whether patients with multiple diabetes-associated autoantibodies, one autoantibody, or without autoantibodies differed with respect to clinical, metabolic, and genetic parameters, including an insulin sensitivity (IS) score based on waist, HbA1c, and triglycerides. We also assessed whether metabolic markers predicted the immune status.Materials and MethodsAs of June 2012, 630 patients in Bavaria, Germany, aged <20 years diagnosed with any type of diabetes within the preceding 6 months were registered in DiMelli. We compared the clinical and laboratory parameters between islet autoantibody status defined patient groups. Parameters showing the strongest associations were included in principal component analysis. Receiver operating characteristic curves were used to assess the ability of the IS Score to predict islet autoantibody status.ResultsPatients with multiple islet autoantibodies, one autoantibody, or without autoantibodies were significantly different in terms of BMI percentile, weight loss before diagnosis, fasting C-peptide (all, P<0.001), and IS Score (P=0.034). However, principal component analysis revealed no distinct patterns according to autoantibody status. At the optimal IS Score cut-off for predicting islet autoantibody positivity (single compared to none), the specificity was 52.0% and the sensitivity was 86.8%. With respect to prediction of multiple autoantibodies (compared to none), specificity and sensitivity were slightly lower and in combination inferior to those obtained using the BMI percentile and fasting C-peptide.DiscussionThe DiMelli study indicated that patients with and without islet autoantibodies differed with respect to metabolic and genetic markers but there was considerable overlap of phenotypes, and autoantibody status could not be predicted by these parameters. Thus, our results suggest that refined diabetes classification may require both immune and metabolic phenotyping.