To our knowledge, this test was replicated by another research group in a Norwegian cohort of adult CD patients [7,8]. In the present study we validated this method in a cohort of 14 young CD patients recruited in the south of Italy, and estimated the level of its reproducibility by exposing the same individual twice to gluten consumption. After the first
in-vivo challenge we observed a significant increase of IFN-γ-secreting cells in response to gliadin 6 days after the wheat intake, confirming the data reported in both Australian and Norwegian adult coeliac patients [4,7,8,23]. Similarly, the magnitude of the IFN-γ responses was comparable to the values IWR-1 chemical structure found in previous studies [4–7]. When we looked at individual responses we found that, upon wheat consumption, the frequency of IFN-γ-releasing cells to whole gliadin increased at least three times in eight of 14 (57%) subjects, barely within the average obtained in previous studies, that ranged from 40%  to 90%  of exposed coeliac patients. In agreement with these studies, the specific response to gluten elicited by the in-vivo challenge was mediated VEGFR inhibitor by CD4+ T cells and was DQ2-restricted. Furthermore, the IFN-γ-producing cells expressed
beta-7 integrin, indicating a phenotype of gut-homing cells. Short-term gluten consumption also induced a significant increase of T cells reacting to the immunodominant 33-mer peptide, although contrasting findings were reported on the
frequency of responder patients [2,3]. Anderson and co-workers reported that the great majority of coeliacs reacted to 33-mer (or to truncated peptide, α-gliadin (57–73) enough [5,6], while in a more recent study reactivity was observed in only six of 10 patients . Our results are in agreement with this latter finding, as we found an evident increase of IFN-γ responses induced by immunodominant gliadin peptide in 8 of 14 patients at first challenge. Unexpectedly, upon the second challenge the number of reacting subjects was far fewer (three of 13 subjects challenged). In this regard, we found that approximately 50% of intestinal T cell lines generated from south Italian CD patients who were assayed in vitro reacted to 33-mer, suggesting that only a subgroup of our coeliac donors seems to display a response to this epitope . These data are not surprising because, despite its strong immunogenicity, 33-mer is one of several gliadin-derived T cell epitopes active in coeliac patients [2,6], and this could explain the increased magnitude of IFN-γ-positive cells found in response to whole gliadin digest. In contrast to previous studies, in which the immune reactivity to gluten was very low, or totally absent, before wheat consumption at day 0, we also found substantial IFN-γ production instead.