Autoantibodies and high-risk HLA susceptibility markers in first-degree relatives of Brazilian patients with type 1 diabetes mellitus: a progression to disease based study.

PURPOSE: The objective of this study was to determine the frequencies of autoantibodies to heterogeneous islet-cell cytoplasmic antigens (ICA), glutamic acid decarboxylase(65) (GAD(65)A), insulinoma-associated antigen-2 (IA-2A) and insulin (IAA)-and human leukocyte antigen (HLA) class II markers (HLA-DR and -DQ) in first degree relatives of heterogeneous Brazilian patients with type I diabetes (T1DM). A major focus of this study was to determine the influence of age, gender, proband characteristics and ancestry on the prevalence of autoantibodies and HLA-DR and -DQ alleles on disease progression and genetic predisposition to T1DM among the first-degree relatives. METHODS: IAA, ICA, GAD(65)A, IA-2A and HLA- class II alleles were determined in 546 first-degree-relatives, 244 siblings, 55 offspring and 233 parents of 178 Brazilian patients with T1DM. RESULTS: Overall, 8.9% of the relatives were positive for one or more autoantibodies. IAA was the only antibody detected in parents. GAD(65) was the most prevalent antibody in offspring and siblings as compared to parents and it was the sole antibody detected in offspring. Five siblings were positive for the IA-2 antibody. A significant number (62.1%) of siblings had 1 or 2 high risk HLA haplotypes. During a 4-year follow-up study, 5 siblings (expressing HLA-DR3 or -DR4 alleles) and 1 offspring positive for GAD(65)A progressed to diabetes. CONCLUSIONS: The data indicated that the GAD(65) and IA-2 antibodies were the strongest predictors of T1DM in our study population. The high risk HLA haplotypes alone were not predictive of progression to overt diabetes.

Family-based association of HLA class II alleles and haplotypes with type I diabetes in Brazilians reveals some characteristics of a highly diversified population.

The association of HLA class II haplotypes with type I diabetes was analyzed in 56 Southeastern Brazilian families using affected family-based controls (AFBAC) method. DRB1-DQA1-DQB1 alleles were determined by polymerase chain reaction/sequence-specific primer genotyping. This study first revealed the great haplotype diversity of Brazilians (65 different haplotypes even with incomplete DRB1 subtyping), probably due to the admixture of Africans genes with European and Amerindian genes in this population. The results revealed increased frequencies of the DRB1*03-DQA1*0501-DQB1*02 and DRB1*0401-DQA1*03-DQB1*0302 haplotypes in the patient group The highest risk for type I diabetes was associated with the heterozygote DRB1*03/*04 genotype as largely reported, and DRB1*03/X and DRB1*04/Y genotypes conferred a significant, but much lower disease risk. Protection from type I diabetes revealed some peculiarities in Southeastern Brazilians: a lack of significant protecting effect of the DRB1*1501-DQA1*0102-DQB1*0602 haplotype, and an apparent protection conferred by the DRB1*13-DQB1*0301, DRB1*11-DQB1*0301, and DRB1*01-DQB1*0501 two-locus haplotypes. The risk to type I diabetes in the highly diversified Southeastern Brazilians evidenced specific information to the prediction of the disease in this region of the country.

Distribution of HLA-DRB1 alleles in a mixed population with insulin-dependent diabetes mellitus from the southeast of Brazil.

HLA class II genes are strongly associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). The present study reports the HLA-DRB1 genotyping of 41 IDDM patients and 99 healthy subjects from the Southeast of Brazil (Campinas region). Both groups consisted of an ethnic mixture of Caucasian, African Negro and Amerindian origin. HLA-DRB1*03 and *04 alleles were found at significantly higher frequencies among IDDM patients compared to the controls (DRB1*03: 48.8% vs 18.2%, P < 0.005, RR = 4.27; DRB1*04: 43.9% vs 15.1%, P < 0.008, RR = 4.37) and were associated with a susceptibility to the disease. DRB1*03/*04 heterozygosity conferred a strong IDDM risk (RR = 5.44). In contrast, the HLA-DRB1*11 allele frequency was lower among IDDM patients (7.3% vs 26.3% in controls), but the difference was not significant. These data agree with those described for other populations and allow genetic characterization of IDDM in Brazil.

Insulin secretion by pancreas of athymic mice injected with peripheral mononuclear cells from insulin-dependent diabetic patients.

We studied the effect of peripheral blood mononuclear cells (PBMNC) from insulin-dependent diabetic (IDDM) children on the insulin secretion pattern of the pancreas from recipient athymic mice. PBMNC from healthy controls or IDDM patients in different stages of disease were injected into athymic mice. PBMNC from newly diagnosed IDDM children elicited basal nonfasting hyperglycemia and in vitro inhibition of the first and second phases of glucose-stimulated insulin secretion in recipient mice. Animals injected with cells from chronically IDDM children showed normoglycemia, abnormal tolerance to glucose, and inhibition of first-phase insulin secretion. Mitomycin C treatment of MNC from IDDM patients abolished insulin secretion inhibition in recipient mice. PBMNC from newly diagnosed and chronically IDDM patients showed positive anti-beta-cell cellular immune aggression. Mice injected with cells from patients during the remission period showed normoglycemia and no alteration of insulin secretion patterns. When relapsed to their former clinical stage, injection of the cells significantly inhibited first-phase glucose-induced insulin secretion in recipients. PBMNC from newly diagnosed IDDM patients were found to migrate to the pancreas of recipient mice preferably as compared with cells from controls. Cells from chronically IDDM patients cultured with concanavalin A (Con A) increased insulin secretion inhibition; despite this, cells from children during the remission period cultured with Con A failed to modify insulin secretion in recipients. These results show that injection of PBMNC from diabetic patients leads to insulin secretion impairment in recipient mice pancreas, and provide a basis for the study of mechanisms involved in the onset and modulation of anti-beta-cell cellular immune aggression induced by human PBMNC.

Beta-cell function in mice injected with mononuclear splenocytes from multiple-dose streptozotocin diabetic mice.

Multiple low doses of streptozotocin (mid sz 40 mg/kg/day, five consecutive days) induce autoimmune diabetes in mice. The aim of the present work was to study beta-cell function in mice injected with splenocytes from mid-sz diabetic mice. Mononuclear splenocytes (MS) from control or diabetic donors were injected into syngeneic C57BL/6J healthy mice (5 x 10(7) MS, ip). MS from diabetic donors did not produce basal hyperglycemia, but they induced abnormal ip glucose tolerance in recipient mice. These "diabetic" MS were also preferentially trapped by the recipient's pancreas. Perifused pancreas from mice injected with MS from mid sz-diabetic donors showed a diminished first and second phase of glucose-induced insulin secretion after 15 days of the cell injection. At this time, pancreatic insulin content among MS recipients did not differ from that found in controls or diabetic donors. Diabetic MS treated with Mitomycin C prior to transfer did not inhibit insulin secretion in recipient mice. Injection of MS from mice made diabetic by a single high sz dose (200 mg/kg) did not induce any alterations of the insulin secretion in recipients. There is enough evidence when using athymic and euthymic (BALB/c nu/nu and +/nu) mice to suggest that proliferation of the injected splenocytes enhanced the progression to the diabetic state, and that both donor and recipient T lymphocytes played an important part in this progression. The results suggest that injection of MS from mid sz-diabetic mice interfere with glucose-stimulated insulin secretion in recipient mice and provide a basis for the study of the mechanisms involved in the onset and modulation of autoimmune pancreatic aggression.

Aberrant distribution of CD4 and CD8 lymphocyte subsets in recent-onset IDDM patients. Effect of cyclosporin.

The monoclonal antibodies 2H4 and 4B4 are specifically directed against CD45RA and CD29 antigens, respectively, which are expressed on both CD4+ and CD8+ lymphocytes. We used them to monitor the distribution of these T-cell subsets in 19 recent-onset IDDM patients (26 +/- 7 yr of age [mean +/- SD throughout]) receiving cyclosporin, and who had been treated with insulin for < 1 mo. Blood samples were examined before starting cyclosporin and after 3 and 9-12 mo of therapy. CD45RA and CD29 are expressed on distinct subsets of CD4+ T-cells, whereas in both healthy subjects and IDDM patients, 2H4 and 4B4 labeling does not always differentiate two distinct CD8+ populations. Various proportions of double-labeled CD8+ CD45RA+ CD29+ cells were detected in 59% of IDDM patients and 70% of healthy control subjects. Before cyclosporin treatment, recent-onset IDDM patients had a significantly lower proportion of CD4+CD29+ cells than the healthy control subjects (42 +/- 11 vs. 52 +/- 9%, P < 0.004). This imbalance corresponded to a significant increase in the CD4+ CD45RA+/CD4+ CD29+ ratio in the IDDM patients (1.37 +/- 0.93 vs. 0.78 +/- 0.36, P < 0.014). Independent of the presence of double-labeled cells, the proportion of CD8+ lymphocytes expressing CD45RA was significantly higher in the patients than in control subjects (72 +/- 15 vs. 57 +/- 15%, P < 0.004). The mean density of the CD45RA antigen (but not that of CD29) on both CD4+ and CD8+ cells was significantly higher in the IDDM patients before treatment than in the healthy control subjects. Cyclosporin induced complete or partial remission of the disease in 12 of 19 patients at 4-6 mo of treatment, but did not correct the imbalance between the regulatory T-cell subsets, regardless of clinical effectiveness. Cyclosporin use was associated with a significant decrease in CD45RA antigen density on both CD4+ and CD8+ T-cells.