Abstract
Refractory coeliac disease (RCD) is a very rare and dangerous form of CD, in which gluten-free diet loses its therapeutic effect and the damage of intestinal mucosa persists. Because of the adherence to the diet, serological markers of CD [immunoglobulin A (IgA) antibodies against gliadin, tissue transglutaminase (tTG) and endomysium] are often missing in RCD patients. We found substantially elevated levels of IgA anti-calreticulin (CRT) antibodies in the sera of almost all RCD patients tested. These sera were negative for IgA antibodies to gliadin and tTG and only some of them showed IgA antibodies to enterocytes. Analysis of patients' IgA reactivity to CRT fragments (quarters and halves) by Western blotting revealed differences in the specificity of IgA antibodies between RCD and CD patients. We therefore used the Pepscan technique with synthetic overlapping decapeptides of CRT to characterize antigenic epitopes recognized by serum IgA antibodies of RCD patients. Employing this method we demonstrated several dominant antigenic epitopes recognized by IgA antibodies of RCD patients on the CRT molecule. Epitope GVTKAAEKQMKD was recognized predominantly by serum IgA of RCD patients. Our results suggest that testing for serum IgA antibodies against CRT and its selected peptide could be a very useful tool in RCD differential diagnosis.