Furthermore, to ascertain if EMA and NFR belonged to distinct IgA subclasses, IgA1 and IgA2 EMA/NFR antibodies were searched in sera of the 11 patients in group 1 subjected to NFR characterization. Total IgA, IgA1 and IgA2 EMA/NFR antibodies were evaluated in sera diluted 1:5 by indirect immunofluorescence analysis (IFA) on cryostat sections of monkey oesophagus (Eurospital, Trieste, Italy). After sera incubation, the sections were stained by means

of fluorescein isothiocyanate (FITC)-conjugated anti-human IgA (Sigma, St Louis, MO, USA; diluted 1:100) and IgA1 (Sigma; diluted 1:20) monoclonal antibodies (mAbs), non-conjugated anti-human IgA2 mAb (ICN Biomedicals, Aurora, OH, USA; diluted 1:10) and its tetramethylrhodamine isothiocyanate

click here (TRITC)-conjugated detector (Sigma; diluted 1:20), all used according to the manufacturer’s instructions. Fluorescence for EMA (Fig. 1a) and NFR (Fig. 1b) was evaluated blindly Raf pathway by three trained observers, whose agreement rate was 99·6%. All FITC-conjugated and non-conjugated secondary mAbs, as well as the TRITC-conjugated anti-IgA2 mAb detector, were incubated further, alone or combined variously, on sections not exposed previously to serum antibodies. No fluorescence signal was observed after any of these control incubations, ensuring that there was no non-specific binding. To establish if EMA and NFR fluorescence patterns were related to distinct antibodies, and if the latter could be present simultaneously in the bloodstream, an indirect IFA-based double-staining assay was performed on monkey oesophagus sections (Eurospital) incubated first with sera of the 11 patients in group 1 subjected to NFR characterization. Because it was shown

during this study that EMA and NFR belong, respectively, to IgA1 and IgA2 isotypes (see below), the subsequent incubations with two different secondary mAbs (anti-human IgA1 and IgA2) detected by two different fluorochromes (FITC and TRITC, respectively) allowed the development, on every section, of a double-staining pattern. For interpretation, the appearance of two different and not overlapping fluorescence signals was considered indicative for the simultaneous presence of two distinct antibodies in CD patients’ sera. To investigate the possible contribution of anti-nuclear Acyl CoA dehydrogenase antibodies (ANA) in determining the NFR fluorescence pattern, classical ANA were searched in sera of all patients in group 1 using an indirect IFA-based commercial kit (Sigma) on both rat liver sections and human epithelial-2 (HEp-2) cell substrates. Results, evaluated blindly by three observers, were compared with positive controls presenting homogeneous (ANA-H), nucleolar (ANA-N) and speckled (ANA-S) antibody patterns. The occurrence of centromeric (ANA-C), peripheral (ANA-P) and cytoplasmic (Golgi apparatus, lysosomal, mitochondrial, ribosomal, speckled) HEp-2 antibody patterns, as well as nuclear subpatterns (e.g.