Evaluations of Immunogenetics, Viral infections, Humoral and cellular immune Responses in Children with Type 1 Diabetes Mellitus

This study was designed to investigate the role of HLA-class I and class II antigens in the etiology of type I diabetes mellitus (TIDM), the cellular changes of peripheral blood lymphocytes(PBL) including phenotyping of surface antigens(CD markers), the functional activities of PBL determined by measurement of  proliferative percentage in response to Con-A and in response to other viral antigens which proposed to be involved in the etiology of TIDM like, coxsackie virus type B(CVB5), poliovirus and adenovirus and assessment of other humoral mediators like cytokines, total immunoglobulins(Igs), Complement components, glutamic acid decarboxylase autoantibodies (GADA) and specific anti-viral IgG antibodies for (CVB₅, poliovirus and adenovirus) in order to define if there was previous exposure of TIDM patients to these viral infections.

Sixty TIDM patients who were newly onset of the disease (diagnosed less than five months) were selected from the National Diabetes Center at Al-Mustansiriya University. All the patients were treated with daily replacement doses of insulin.Their age ranged from 3-17 years. They  were divided into two groups according to their ages  as following: 36 patients equal or less than 10 years old and 24 patients more than 10 years old. Eighty apparently healthy control subjects, matched with age (4-17) years, sex and ethnic backgrounds   (Iraqi Arabs) underwent the HLA-typing examination. Finally 50 healthy individuals were selected randomly to undergo the same examination and tests of the study as TIDM patients. They were also divided into two groups according to their ages, 21 individuals equal or less than 10 years and 29 individuals more than 10 years. Another 50 healthy siblings of TIDM patients were available for investigation of HLA-typing, their ages range from 3 to 16 years.

The findings of this study are summarized in the following:

• A remarkable increase of glycosylated hemoglobin HbA1_C­% with a remarkable decrease of serum C-peptide levels in newly diagnosed TIDM patients in comparison to healthy controls. The age of children had no effect on the metabolic decomposition.

• At HLA-class I region, TIDM patients showed a significant increased frequency of antigen A9 (40.0 vs. 18.75%) and B8 (28.33 vs. 8.75%) as compared to control subjects, while at HLA-class II region, DR3 and DR4 were significantly increased in patients (53.33 vs. 26.25% and 50.0 vs. 12.5% respectively) as compared to controls and they might played an important role in the etiology of the disease. In addition to that, TIDM was significantly associated with DQ2 (33.33 vs. 15%) and DQ3 (40.0 vs. 20%) antigens as compared to controls, suggesting that these haplotypes had a role in disease susceptibility, while the frequency of DR2 and DQ1 antigens were significantly lowered in patients compared to controls ( 6.66 vs. 25%  and  6.66 vs. 22.5% respectively). These molecules might had protective effect. In siblings a significant increase frequency of DR4 antigen (34.0 vs. 12.5%) was observed in comparison to controls, suggesting that it might be much useful for predicting TIDM in affected families.
• TIDM patients showed a remarkable lowering in CD₃⁺, CD₄⁺ ,  CD₈⁺, CD₄₅RA⁺, and CD₅₆⁺ cells but the decrease in CD₄⁺ cells percentage was not significant in patients in comparison to healthy controls. In contrast, a significant elevation of activation markers includes (CD₄₅RO⁺, CD₃₈⁺ and HLA-DR⁺ cells were observed in TIDM patients in addition to a significant increase of CD₁₉⁺ cell percentage and CD₄⁺:CD₈⁺ ratio in the patients.
• No significant differences were shown in the PBL proliferative percentage in response to Con-A and tested viruses (CVB₅ and adenovirus) between TIDM and healthy controls, but PBL proliferative percentage of TIDM patients  showed a significant decline in response to poliovaccine. Strong T-cell proliferation in response to the tested viral antigens were observed to be related to HLA-DR4 and HLA-DQ3 antigens.
• By using ELISA technique, serum levels of a Th₁ cell cytokine, interferon gamma (IFN-γ), and Th₂ cell cytokines, interlukin-10(IL-10) and inflammatory cytokine (IL-6) were significantly higher in TIDM patients compared to healthy controls.
• Significant increase in total serum IgM, IgG and IgA levels were observed in patients with TIDM compared to healthy controls while the complement component C₃ and C₄ mean serum levels showed a significant decrease in TIDM patients.
• Anti-GAD autoantibodies were present in 50% of TIDM children especially in older ages and in females more than males. High proportion of GADA was found in the patients carrying HLA-DR3/DR4 heterozygous.
• High proportion of anti-CVB₅ IgG and anti-polio IgG were found in diabetic children compared to controls, while anti-adeno IgG were detected in diabetic patients only.