Markers of Infection in Childhood Autoimmune Diabetes and Related Disorders

University dissertation from Department of Clinical Sciences, Lund University

Abstract: The main aim of this thesis was to contribute to the knowledge about mechanisms involved in the development of type 1 diabetes (T1D) and other autoimmune diseases in children. Immune markers in relation to genetic propensity in children with autoimmune disease, mainly T1D and a small group of juvenile idiopathic arthritis (JIA) and thyroiditis (THYR), as well as healthy controls have been studied. The possible influence by environmental factors as viral infections is supported by several studies. This thesis includes studies of antibodies to virus in blood samples from healthy children and from children at T1D onset and other organ specific autoimmune diseases in geographically different parts of Sweden. The virus analyses focus on antibodies to Ljungan virus (LV), a fairly recently defined parechovirus, reported to cause diabetes in voles that are found to carry LV, but LV is yet not shown to be pathogenic to humans. The last aim also included analyses of Ljungan virus antibodies (LVA) in voles as possible source of infection to humans. To test the specificity and possible cross-reactivity of the radiobinding assay (RBA) method for LVA analysis samples from children in the third study were analyzed for antibodies to several human parechoviruses (HPeV) and related viruses by a multiplex suspension array (SMIA). Islet autoantibodies and HLA-type of the children in study were analyzed for possible association to antibodies to LV, risk for infection and/ or autoimmune disease. The major objective was to study possible mechanisms of the etiology and pathogenesis of T1D at the time of clinical onset. The aim was also to compare features of T1D at onset with JIA and THYR which are less common but distinct autoimmune disorders.

Study I: Samples left at diagnosis by incident, 0-18y, T1D patients (n = 63), (October 2000 – February 2006) were studied along with age and sample time matched healthy controls (n = 126), all from Jämtland County in the northern part of Sweden. Results: The younger children (< 9 years) tended to have a higher T1D incidence rate during winter compared to summer months. The proportion of children with high level antibodies against LV were younger compared to the rest of the children and correlated with half yearly T1D IR.
Conclusion study I: High level LVA fluctuating with season and correlating with T1D IR indicates that past exposure to Ljungan virus may be associated with T1D.

Study II: Samples from children (n=676), 0-18y, from the Skåne study who left samples at T1D diagnoses from February 1996 - April 2005 were studied and compared to existing results from a control group of 309 healthy school children (11-13y) with sampling time January – March 1989. Results: High level LVA were found significantly more frequent in the youngest children. High level LVA in children <10 years of age correlated to HLA DQ2/8, 8/8 and 8/X. A significant correlation was found between high level LVA and IAA in children <10 years of age. Conclusions study II: LVA were common among the young and LVA levels were higher in the younger age groups; High level LVA correlated with IAA; There was correlation between high level LVA and high risk HLA as well as young age. The presence of LVA support the notion that humans are susceptible to LV infection and that LV may contribute to T1D.
Study III: Samples were obtained from a control group of healthy school children (n=295) from April 2009 – February 2011 to be compared with samples left for the Better Diabetes Diagnosis (BDD) study at diagnosis of T1D from 72 children during the same period of time, all in Jämtland County. A smaller number of children diagnosed with another autoimmune disease; 28 with juvenile idiopathic arthritis (JIA) and 16 with thyroiditis (THYR), contributed with blood samples during the same period. The sampling period coincided by chance with the InfluenzaA/H1N1 pandemic (Oct 2009 – May 2010) that took place in Sweden including Jämtland. Analyses of antibodies: LVA (RBA), GADA, IA-2A, IAA, ZnT8RA, WA and QA and HLA genotype were performed. Also, and specific for this study, testing the specificity and possible cross-reactivity of the radiobinding assay (RBA) method for LVA, analysis in a multiplex suspension array (SMIA) for antibodies to several human parechoviruses (HPeV) and related viruses for IgM and IgG were performed. Results: LVA was associated with young age at diagnosis and with IAA in young HLA-DQ8 subjects. Association was found between LVA and SMIA-IgG against LV-peptides of which LV-VP1_31-60-IgG also related to IAA and HLA-DQ8. LVA also associated to two of the HPeV3-peptides but with the difference that these HPeV3-peptides did not correlate to IAA or HLA-DQ8. No strong evidence was provided for that HPeV antibodies would solely cross-react with the LV translation product, instead more likely to be explained by that subjects may have been exposed to both LV and another parechovirus. The analyses for A/H1N1 antibody levels showed no relation to LVA or IgG-LV-VP1_31-60 suggesting specific immunoreactivity against the A/H1N1 that did not affect levels or presence of LV antibodies.
Conclusion study III: Both LVA and LV-VP1_31-60-IgG related to IAA and HLA-DQ8 suggesting possible role in T1D. It remains to be determined if cross-reactivity or concomitant exposure to LV and HPeV3 explains possible serological cross-reactions.

Study IV: Samples from bank voles trapped in Västerbotten County in northern Sweden were supplied by the Swedish University of Agricultural Sciences, SLU, Umeå, Sweden. The voles were captured during two weeks in early winter season in 1997 and early summer and early winter season in 1998. Results: LVA showed evidence for exposure to LV. The data indicate that these animals may be exposed to both LV and PUUV (analyses data provided by SLU) as older animals tended to be double positive. Conclusion study IV: The voles, as reservoirs of virus that infect humans, may play a role of LV in autoimmune diabetes.

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