Tissue transglutaminase autoantibodies in childhood celiac disease

Sammanfattning: Celiac disease is a prevalent small bowel disease in children caused by permanent intolerance against gliadin, which is the alcohol-soluble fraction of the cereal protein gluten in wheat, rye and barley. Celiac disease is characterized by an inflammatory infiltrate of CD4+ T lymphocytes in the intestinal mucosa leading to damage of villous structure and loss of absorptive surface. As a consequence, gastrointestinal symptoms and signs of malnutrition often occur in children. The exclusion of gluten from the diet results in healing of the intestinal mucosa. Celiac disease is also strongly associated with tissue transglutaminase (tTG) autoantibodies that are considered to be markers for the disease. Moreover, levels of tTG autoantibodies decline after gluten withdrawal, which enables objective evaluation of how patients respond to treatment with gluten-free diet. The use of serological markers for diagnostic purposes in celiac disease will most likely increase as screenings of large populations are performed. It is therefore important to test the current methods for autoantibody detection. The aim of the present thesis was to evaluate radioimmunoassays for the assessment of tTG autoantibodies in children with celiac disease. In Study I, the diagnostic sensitivity and specificity were determined for autoantibodies against human recombinant tTG with liquid phase radioligand binding immunoassays. It was shown that the diagnostic sensitivities for IgA-tTG and IgG-tTG were 85% and 84%, respectively, and the specificity for both isotypes was 94%. The sensitivities were lower compared with previous studies based on similar radioimmunoassays and mainly explained by the inclusion of children diagnosed at two years of age or younger. In Study II, it was examined how human recombinant tTG affected the binding of tTG autoantibodies in liquid phase compared with solid phase immunoassays. We hypothesized that epitopes may be hidden for autoantibodies in the solid phase, but results showed high concordance for IgA-tTG in children with untreated celiac disease. However, the specific binding of IgG-tTG was significantly reduced compared with the two IgA-assays. In contrast, using protein A for binding IgG-tTG in the radioligand binding assays nearly displayed as high diagnostic accuracy as IgA-tTG. In Study III, the effect of gluten-free diet on tTG autoantibody levels and subsets of peripheral blood lymphocytes by flow cytometry were investigated in children with newly diagnosed celiac disease. It was observed that the decline in tTG autoantibody levels subsequent to gluten-free diet was associated with concomitant changes in B cells and a subset of CD4+ regulatory T cells expressing the markers CD25 and CCR4, indicating that flow cytometry might be a useful complement to tTG autoantibodies in monitoring of the disease activity in celiac disease. In Study IV, tTG autoantibodies and endomysial autoantibodies were measured in a screening for celiac disease performed on normal 3-year old children. The outcome of the study diagnosed 0.5% of children with previously unrecognized celiac disease. Although the study revealed a high number of children with transient autoantibodies, the combination of IgA-tTG and IgG-tTG seemed to better predict persistent autoantibodies and a pathological biopsy than any of the autoantibodies alone. In conlusion, this thesis adds new aspects in terms of how tTG autoantibodies can be used as serological markers in children investigated for celiac disease. It is proposed that both IgA-tTG and IgG-tTG are analyzed with radioligand binding assays in children screened for celiac disease with the primary goal to develop a standardized immunoassay in near future.