Johanna Paronen

Dietary Intervention in Infancy and Later Signs of Beta-Cell Autoimmunity

BACKGROUND Early exposure to complex dietary proteins may increase the risk of beta-cell autoimmunity and type 1 diabetes in children with genetic susceptibility. We tested the hypothesis that supplementing breast milk with highly hydrolyzed milk formula would decrease the cumulative incidence of diabetes-associated autoantibodies in such children. METHODS In this double-blind, randomized trial, we assigned 230 infants with HLA-conferred susceptibility to type 1 diabetes and at least one family member with type 1 diabetes to receive either a casein hydrolysate formula or a conventional, cows-milk-based formula (control) whenever breast milk was not available during the first 6 to 8 months of life. Autoantibodies to insulin, glutamic acid decarboxylase (GAD), the insulinoma-associated 2 molecule (IA-2), and zinc transporter 8 were analyzed with the use of radiobinding assays, and islet-cell antibodies were analyzed with the use of immunofluorescence, during a median observation period of 10 years (mean, 7.5). The children were monitored for incident type 1 diabetes until they were 10 years of age. RESULTS The unadjusted hazard ratio for positivity for one or more autoantibodies in the casein hydrolysate group, as compared with the control group, was 0.54 (95% confidence interval [CI], 0.29 to 0.95), and the hazard ratio adjusted for an observed difference in the duration of exposure to the study formula was 0.51 (95% CI, 0.28 to 0.91). The unadjusted hazard ratio for positivity for two or more autoantibodies was 0.52 (95% CI, 0.21 to 1.17), and the adjusted hazard ratio was 0.47 (95% CI, 0.19 to 1.07). The rate of reported adverse events was similar in the two groups. CONCLUSIONS Dietary intervention during infancy appears to have a long-lasting effect on markers of beta-cell autoimmunity--markers that may reflect an autoimmune process leading to type 1 diabetes. (ClinicalTrials.gov number, NCT00570102.).

Evidence for a primary islet autoantigen (preproinsulin 1) for insulitis and diabetes in the nonobese diabetic mouse

It has been reported that an insulin 2 gene knockout, when bred onto nonobese diabetic (NOD) mice, accelerates diabetes. We produced insulin 1 gene knockout congenic NOD mice. In contrast to insulin 2, diabetes and insulitis were markedly reduced in insulin 1 knockout mice, with decreased and delayed diabetes in heterozygous females and no insulitis and diabetes in most homozygous female mice. Lack of insulitis was found for insulin 1 female homozygous knockout mice at 8, 12, and 37 weeks of age. Despite a lack of insulitis, insulin 1 homozygous knockout mice spontaneously expressed insulin autoantibodies. Administration of insulin peptide B:9-23 of both insulin 1 and 2 to NOD mice induced insulin autoantibodies. Insulin 1 is not the only lymphocytic target of NOD mice. Insulin 1 homozygous knockout islets, when transplanted into recently diabetic wild-type NOD mice, became infiltrated with lymphocytes and only transiently reversed diabetes. These observations indicate that loss of either insulin gene can influence progression to diabetes of NOD mice and suggest that the preproinsulin 1 gene is crucial for the spontaneous development of NOD insulitis and diabetes.

Dietary manipulation of beta cell autoimmunity in infants at increased risk of type 1 diabetes: a pilot study

Aims/hypothesisWe aimed to assess the feasibility of a dietary intervention trial with weaning to hydrolysed formula in infants at increased risk of type 1 diabetes and to study the effect of the intervention on the emergence of diabetes-associated autoantibodies in early childhood.MethodsWe studied 242 newborn infants who had a first-degree relative with type 1 diabetes and carried risk-associated HLA-DQB1 alleles. After exclusive breastfeeding, the infants underwent a double-blind, randomised pilot trial of either casein hydrolysate (Nutramigen; Mead Johnson) or conventional cow’s milk-based formula until the age of 6–8 months. During a mean observation period of 4.7 years, autoantibodies to insulin, anti-glutamic acid decarboxylase and insulinoma-associated antigen-2 were measured by radiobinding assays, and islet cell antibodies (ICA) by immunofluorescence.ResultsThe feasibility of screening and identifying a cohort of first-degree relatives with HLA-conferred disease susceptibility, enrolling them in a dietary intervention trial and following them for seroconversion to autoantibody positivity is established. The cumulative incidence of autoantibodies was somewhat smaller in the casein hydrolysate vs control formula group, suggesting the need for a larger well-powered study. After adjustment for duration of study formula feeding, life-table analysis showed a significant protection by the intervention from positivity for ICA (p=0.02) and at least one autoantibody (p=0.03).Conclusions/interpretationThe present study provides the first evidence ever in man, despite its limited power, that it may be possible to manipulate spontaneous beta cell autoimmunity by dietary intervention in infancy.

Glutamate Decarboxylase–Reactive Peripheral Blood Lymphocytes From Patients With IDDM Express Gut-Specific Homing Receptor α4β7-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 cows 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 α4β7-integrin, a homing receptor for MAdCAM-1, on GAD65-reactive lymphocytes. Using immunomagnetic cell sorting, we depleted the lymphocytes with high expression of α4β7-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 β-cells in IDDM.

Soluble adhesion molecules and oral antigen feeding in infants

Oral administration of foreign proteins, e.g. cows milk (CM) proteins, stimulates the immune system and induces humoral and cellular immune response against these antigens in infants. Up-regulation of adhesion molecules is known to be associated with activation of the immune system. The purpose of the study was to examine whether orally administered CM proteins induce elevation in soluble adhesion molecules, i.e. intercellular adhesion molecule-1 (ICAM-1) and L-selectin, in infants. In a double-blind trial, 10 infants received CM-based formula and 10 infants casein hydrolysate formula until the age of 9 mo. The infants of mothers with insulin-dependent diabetes mellitus (IDDM) were recruited into a pilot study of a trial for primary prevention of IDDM by elimination of CM proteins from the diet during early infancy. A cord blood sample and peripheral blood samples were taken at the ages of 3, 6, 9, and 12 mo of age. The levels of soluble ICAM-1 and L-selectin were measured by ELISA. The levels of soluble ICAM-1 were higher at the ages of 3, 6, 9, and 12 mo in infants who received CM-based formula than in infants who received hydrolyzed formula (p = 0.05). Instead, no difference was found in the the levels of soluble L-selectin. The levels of soluble ICAM-1 and L-selectin were higher in all infants when compared with the levels reported in adults or to the levels seen in cord blood. Orally fed CM proteins induce an elevation in soluble ICAM-1 in infants. This may reflect the generation of an immune response against these proteins, because ICAM-1 has an important costimulatory role in lymphocyte activation.

Nondepleting anti-CD4 monoclonal antibody prevents diabetes and blocks induction of insulin autoantibodies following insulin peptide B:9-23 immunization in the NOD mouse.

INTRODUCTION Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes that induces insulin autoantibodies and prevents diabetes in the NOD. However, immunization with peptide without adjuvant may be insufficient to reverse disease or induce long-term tolerance. Furthermore, recent experience has demonstrated the potential dangers of disease exacerbation or anaphylaxis with peptide immunotherapy. METHODS Combination therapy of B:9-23 with a nondepleting anti-CD4 monoclonal antibody (YTS 177.9) was studied in female NOD mice from 4 through 6 weeks of age. Injections of either B:9-23 in saline, YTS 177.9 antibody, or both peptide and antibody were given to mice. RESULTS By 52 weeks follow-up, 40% of B:9-23-treated, 100% of YTS177.9-treated, and 70% of B:9-23 and YTS177.9 combination-treated mice remained diabetes-free. IAA, both spontaneous and induced by B:9-23, was almost completely suppressed in mice receiving YTS 177.9. In addition to suppression of IAA expression, anti-B:9-23 peptide antibodies are also suppressed in mice receiving B:9-23 with YTS 177.9, compared to B:9-23 alone. CONCLUSION A brief course of the nondepleting anti-CD4 monoclonal antibody (YTS 177.9) in NOD mice confers long-term protection from diabetes and insulitis and profoundly blocks spontaneous and B:9-23 peptide-induced insulin autoantibodies.

Dietary insulin as an immunogen and tolerogen

We have shown that exposure to bovine insulin (BI) in cows milk (CM) formula induces an insulin‐specific immune response in infants. Here we studied the role of human insulin (HI) in breast milk as a modulator of the immune response to insulin. In a group of 128 children participating in the TRIGR pilot study, maternal breast milk samples were collected 3–7 days and/or 3 months after delivery. After exclusive breast‐feeding, the children received either CM formula or casein hydrolysate during the first 6–8 months of life. Insulin concentration in breast milk and immunoglobulin G (IgG) antibodies to BI in plasma samples were measured by EIA. The levels of insulin in breast milk samples were higher in mothers affected by type 1 diabetes than in non‐diabetic mothers (p = 0.007 and p < 0.001). The concentration of insulin in breast milk correlated inversely with the plasma levels of IgG antibodies to BI at 6 months of age in children who received CM formula (r = −0.39, p = 0.013), and at 12 months of age in all children (r = −0.25, p = 0.029). The levels of breast milk insulin were higher in the mothers of nine children who developed beta‐cell autoimmunity when compared with autoantibody‐negative children (p = 0.030); this holds true also when only children of diabetic mothers were included (p = 0.045). BI in CM induces higher levels of IgG to insulin in infants than does HI in breast‐fed children. Instead, HI in breast milk seems to be tolerogenic and may downregulate the IgG response to dietary BI. However, our results in infants who developed beta‐cell autoimmunity suggest that in this subgroup of children breast milk insulin does not promote tolerance.

Establishing Insulin 1 and Insulin 2 Knockout Congenic Strains on NOD Genetic Background

Abstract: As insulin is a major autoantigen in autoimmune diabetes and because the insulin gene region locus in humans has been linked to diabetes risk, we have bred insulin gene knockouts onto the NOD mouse. Mice differ from humans in terms that they express two nonallelic genes of insulin. Insulin 2 is the murine homologue of the human insulin gene and is located on mouse chromosome 7. Insulin 1 is thought to have evolved by a gene duplication event, lacks the second intron of the insulin 2 gene, and is located on mouse chromosome 19. The differential thymic expression of the insulin gene may be important for central tolerance induction. Here, we present the initial establishment of congenic knockouts and characterization of the congenic intervals corresponding to insulin 1 and insulin 2 knockout genes on mouse chromosome 19 and 7, respectively.

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.

Comparative study of oral versus subcutaneous B:9-23 insulin peptide in Balb/c mice as an experimental model for autoimmune diabetes.

Abstract: Insulin peptide B:9–23 (amino acids 9 to 23 of the B chain) can induce immune targeting of insulin and islets in normal Balb/c mice. The insulin autoantibodies induced react with insulin and not the immunizing peptide. Oral administration of insulin as well as subcutaneous insulin can sensitize to insulin.