T-cell responses to enterovirus antigens in children with type 1 diabetes.

Enterovirus infections, implicated in the pathogenesis of type 1 diabetes in a number of studies, may precipitate the symptoms of clinical diabetes and play a role in the initiation of the beta-cell damaging process. The aim of this study was to evaluate whether cellular immune responses to enterovirus antigens are abnormal in children with type 1 diabetes. Lymphocyte proliferation responses to enterovirus antigens were analyzed in 41 children with new-onset type 1 diabetes, 23 children with type 1 diabetes for 4-72 months, and healthy control children in subgroups matched for HLA-DQB1 risk alleles, sex, and age. Children with diabetes for 4-72 months more often had T-cell responses to the Coxsackievirus B4-infected cell lysate antigen than children with new-onset diabetes (P < 0.01) or control children (P < 0.01). Responses to recombinant nonstructural protein 2C of Coxsackievirus B4 were also more frequent in children with type 1 diabetes for 4-72 months when compared with control subjects (P = 0.03), whereas the responses to purified Coxsackievirus B4 and recombinant VP0 protein, which did not contain nonstructural proteins, did not differ. These data suggest that T-cell responses to Coxsackievirus B4 proteins and particularly to the antigens containing the nonstructural proteins of the virus are increased in children with type 1 diabetes after the onset of the disease. However, in children with new-onset diabetes, responses were normal or even decreased. This phenomenon was specific for enteroviruses and could be caused by trapping of enterovirus-specific T-cells in the pancreas.

Induction of interferon-gamma and IL-4 production by mitogen and specific antigens in peripheral blood lymphocytes of Type 1 diabetes patients.

A functional imbalance in cytokine production resulting in dominance of Th1 over Th2 type response has been suggested to play a critical role in the pathogenesis of type 1 diabetes. In this study the cellular responses to pokeweed mitogen and a panel of specific antigens were analysed by measuring the production of IFN-gamma and IL-4 cytokines at the levels of mRNA expression (expression index=antigen/medium) and protein secretion in culture supernatants. Two enterovirus preparates were included due to the suggested significance of these viruses in the aetiology of type 1 diabetes. The study included 22 children with newly-diagnosed type 1 diabetes, 15 children with longer duration of disease and 20 healthy children. Comparisons were made between age- and sex-matched groups. Newly diagnosed diabetic patients had significantly higher IFN-gamma mRNA expression index (p<0.02) but also higher IL-4 mRNA expression index (p<0.05) in tetanus toxoid stimulated peripheral blood mononuclear cells compared to healthy controls. Also the diabetic patients studied 3-72 months after the diagnosis of type 1 diabetes showed a tendency to higher IFN-gamma mRNA expression index compared to controls (0.05

Enterovirus infections and enterovirus specific T-cell responses in infancy.

The development of enterovirus specific T-cell and antibody responses were examined in a cohort of 60 healthy infants at the ages of 3, 6, 9, and 12 months. By the age of 6 months, 68% of the infants had developed T-cell responses against enterovirus antigens by lymphocyte proliferation test, whereas only 30% had serological evidence of an enterovirus infection. By this age, only 7% of the infants had adenovirus specific T-cell responses and 3% had serologically verified adenovirus infection. Enterovirus specific T-cell responses correlated with the lack of enterovirus antibodies in cord blood and the number of sibs reflecting protection by maternal antibodies and the rate of exposures, respectively. T-cell responses cross-reacted between different enterovirus serotypes. The results show that enterovirus infections occur frequently in infancy and induce T-cell immunity. Cellular immunity may be a more sensitive indicator of neonatal enterovirus infections than antibodies.

Effect of coincident enterovirus infection and cows' milk exposure on immunisation to insulin in early infancy.

AIMS/HYPOTHESIS: Insulin autoantibodies appear often as the first autoantibody in children who develop islet-cell autoimmunity. Our recent studies indicate that primary immunisation to insulin is induced in early infancy by exposure to dietary bovine insulin present in cows' milk formulas. As gut-associated lymphoid tissue is also the primary replication site of enteroviruses, we tested whether enterovirus infections could modify the development of immune response to dietary insulin in early infancy. METHODS: We studied the development of IgG-antibodies to dietary bovine insulin by enzyme immunoassay in relation to enteroviral infections determined by T-cell proliferation response to the Coxsackie B4 virus and by serological tests for enterovirus antigens in 57 infants who carried the HLA DQB1(*)02/0302 diabetes risk genotype and participated in a Finnish population-based birth-cohort study (Diabetes Prediction and Prevention Project, DIPP, study). RESULTS: In the infants exposed to cows' milk formulas before the age of 3 months, those who had a T-cell proliferation response to enterovirus antigen at 3 months of age ( n = 12) had higher concentrations of IgG-antibodies to bovine insulin at the age of 6 and 9 months than those who did not have T-cell proliferation response to enterovirus antigen ( n = 25) (median OD were 0.742 vs 0.427, p = 0.04, and 0.477 vs 0.293, p = 0.02, respectively). CONCLUSION/INTERPRETATION: Our results suggest that two epidemiological risk factors of Type I (insulin-dependent) diabetes mellitus, enterovirus infections and exposure to cows' milk formulas, could modify the immunisation to insulin in early infancy.

Responses of coxsackievirus B4-specific T-cell lines to 2C protein-characterization of epitopes with special reference to the GAD65 homology region.

Coxsackie B viruses (CBV) have been indicated as environmental triggers initiating autoimmune destruction of insulin-producing pancreatic beta-cells, and molecular mimicry might be the mechanism. A prime candidate for inducing cross-reactive immune responses is a homology sequence, PEVKEK, found both in CBV4 2C protein and in GAD65. To characterize the CBV4-specific T-cell epitopes, overlapping peptides covering the 2C protein were synthesized and CBV4-specific T-cell lines were established from healthy and diabetic subjects. The T-cell epitopes were dependent on the HLA-DR genotype of the T-cell donor, but no difference between diabetic and healthy subjects could be detected. Peptide p4, which included the PEVKEK sequence, contained an HLA-DR1-restricted T-cell epitope. Three randomly selected CBV4-specific T-cell lines, which responded to peptide p4, failed to recognize GAD65 protein or GAD65 peptides containing the PEVKEK sequence. We conclude that the CBV4 2C protein is strongly immunogenic for T-cells and PEVKEK is included in a T-cell epitope. However, presentation of this epitope in the context of neutral HLA-DR1 allele does not support its role in pathogenesis of type 1 diabetes.

Comparison of enterovirus-specific cellular immunity in two populations of young children vaccinated with inactivated or live poliovirus vaccines.

Enterovirus-specific cellular immunity was studied in Estonian and in Finnish children at the age of 9 months. The aim was to evaluate the level of responsiveness in two neighbouring countries with different poliovirus immunization practices and striking differences in the incidence of insulin-dependent diabetes mellitus (IDDM), a disease in which early enterovirus infections are an aetiological risk factor. The Estonian children immunized with live attenuated polio vaccine had stronger T cell responses to coxsackievirus B4 and poliovirus type 1 when compared with Finnish children immunized with inactivated polio vaccine (median stimulation indices 10.4 and 6.3 in Estonian children and 1.9 and 2.9 in Finnish children, respectively; P < 0.05). Lymphocytes stimulated by poliovirus type 1 antigen expressed interferon-gamma (IFN-gamma) mRNAs, which strongly correlated with the level of proliferation responses. Lymphocytes of Estonian children had a tendency towards stronger expression of IFN-gamma upon poliovirus challenge when compared with Finnish children. The number of children who had experienced coxsackievirus B infections, as determined by the presence of neutralizing antibodies, did not differ between Estonian and Finnish children. The results show that Finnish children have weaker cellular immunity against enteroviruses at the age of 9 months compared with Estonian children at the same age. This is most probably due to the difference in polio vaccination schedules; in Estonia live poliovirus vaccine is used and given at earlier ages than the inactivated vaccines in Finland. This leads to stronger T cell immunity which cross-reacts with other enterovirus serotypes. This may explain the lower incidence of IDDM in Estonia by providing effective protection against diabetogenic enterovirus strains in Estonian children.

T cell responses to enterovirus antigens and to beta-cell autoantigens in unaffected children positive for IDDM-associated autoantibodies.

Enterovirus infections have been implicated in the pathogenesis of IDDM in a number of studies. The aim of the present study was to evaluate whether the cellular immune response to enterovirus antigens is abnormal in children who test positive for IDDM-associated autoantibodies. Lymphocyte proliferation responses were analysed to enterovirus antigens and to a panel of beta-cell autoantigen preparations in 31 non-diabetic ICA and/or GAD65 antibody-positive children and in 19 ICA/GAD65-negative control children. The responses to highly purified enteroviruses did not differ between autoantibody (AA)-positive and -negative subjects. However, proliferation responses to coxsackievirus-infected cell lysate, which also included non-structural proteins of the virus, were higher in AA-positive than in AA-negative subjects (P<0.05). This difference was most marked in children carrying the HLA-DQB1*02 allele (P=0.01). AA-positive subjects also had higher responses to one of the three GAD65 antigen preparations compared to AA-negative subjects (P<0.05). Proliferation responses to the adenovirus hexon protein did not differ between the groups. These results show that the increased responses to virus infected cell lysates are associated with early phases of beta-cell autoimmunity.

Rotavirus infections and development of diabetes-associated autoantibodies during the first 2 years of life.

Rotavirus, the most common cause of childhood gastroenteritis, has been implicated as one of the viral triggers of diabetes-associated autoimmunity. To study the possible association between rotavirus infections and the development of diabetes-associated autoantibodies, we measured the prevalence of rotavirus antibodies in serum samples collected at 3-6-month intervals up to the age of 2 years from 177 children selected from consecutive newborns because they carried HLA-DQB1 alleles associated with increased risk for type 1 diabetes. Twenty-nine of the children developed at least two of four diabetes-associated autoantibodies (ICA, IAA, GADA or IA-2A) during the first 2 years of life (the cases), whereas 148 children remained autoantibody-negative matched with the cases for date of birth, gender, living region and HLA-DQB1 alleles. The temporal association between the development of the first-appearing diabetes-associated autoantibody and rotavirus infections was studied by analysing whether the cases had a diagnostic increase in rotavirus antibody titre more often during the 6-month period that preceded seroconversion to autoantibody positivity than the controls. By the age of 12 months one of the 13 case children (7%), who had a serum sample drawn at that age and who had developed at least one type of diabetes-associated autoantibodies, had experienced a rotavirus infection, while 12 of the 61 (20%) autoantibody-negative control children had had a rotavirus infection. By 18 months, four of the 22 autoantibody-positive cases (18%) and 18 of the 89 controls (20%) had rotavirus antibodies, and by the age of 24 months the respective numbers were five of the 27 cases (19%) and 32 of the 113 (28%) controls. A rotavirus infection occurred during the 6 months preceding the sample which was positive for an autoantibody in four of the 25 periods (16%) for which both necessary samples were available, while the controls had a rotavirus infection during 55 of the 370-such periods (15%). Accordingly, our data suggest that rotavirus infections are unlikely triggers of beta-cell autoimmunity in young children with genetic susceptibility to type 1 diabetes.