Mucosal biopsy immunophenotyping of lymphocytes: Multiparameter flow cytometric immunophenotyping of mucosal intraepithelial and lamina propria lymphocytes was performed. These lymphocytes were isolated from six duodenal biopsy specimens per time point through despite chemical and enzymatic dissociation[16]. The cells were stained with fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein and allophycocyanin-labelled monoclonal antibodies directed against CD3, CD4, CD8, CD16/56, CD19, CD45, CD45RA, HLA-DR, NKG2D, CD25 and TCR gamma-delta (all from BD Biosciences, San Jose, CA, United States), and appropriate isotype controls were included. Stained cells were analysed on a 4-colour flow cytometer (FACSCaliburTM, BD Biosciences) and the data were analysed using CellquestTM software (Becton Dickinson, San Jose, CA, United States).

Care was taken to analyse only viable cellular events based on light scatter properties. The mean fluorescence intensity index as compared to isotype controls was calculated for the markers included. Mucosal biopsy gluten-specific T-cell lines: Gut-resident, gluten-reactive T-cells are a hallmark of CD. To demonstrate that all patients possessed such cells, polyclonal T-cell lines were generated from small intestinal biopsies as described[17]. The resulting T-cell lines were tested for reactivity against a pepsin/trypsin digest of gluten and a pepsin/trypsin digest of gluten that had been treated with tissue transglutaminase in a T-cell proliferation assay as described[17]. In all patients gluten reactivity could be demonstrated (not shown).

Mucosal biopsy IgA-tTG deposits: Biopsies at the end of the randomisation study phase were stained for tTG-related extracellular IgA deposits and, in case of positivity, baseline biopsies were stained as well. Twelve unfixed, 5 ��m-thick frozen sections were examined per patient by double immunofluorescent labelling of IgA (green) and tTG (red) as previously described[18]. IgA is normally detected only inside plasma cells and at the luminal surface, whereas in active CD, subepithelial deposits composed of IgA-tTG are found along the surface and crypt basement membranes and around mucosal vessels, corresponding to the intestinal localisation of tTG. The CD-type IgA-tTG deposits were graded from 0 to 3 according to their intensity along the basement membranes in the villous-crypt area. As this study of the small intestinal IgA-tTG deposits is highly subjective, it was performed by an independent specialist in this field in a blind Drug_discovery manner to greatly increase its accuracy. Serum antibodies: Blood samples were collected by venipuncture to analyse CD-associated antibodies.