Senger Stefania, Maurano Francesco, Mazzeo Maria F, Gaita Marcello, Fierro Olga, David Chella S, Troncone Riccardo, Auricchio Salvatore, Siciliano Rosa A, Rossi Mauro
Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche, Avellino, Italy.
J Immunol. 2005 Dec 15;175(12):8087-95. doi: 10.4049/jimmunol.175.12.8087.
Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant alpha-gliadin (r-alpha-gliadin) showing the most conserved sequence among the fraction of alpha-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-alpha-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-alpha-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120-139) and p23 (aa 220-239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-alpha-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-gamma secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.
乳糜泻由小麦醇溶蛋白以及来自大麦和黑麦的相关醇溶谷蛋白引发，其特征是与HLA - DQ2和HLA - DQ8基因密切相关。醇溶蛋白是多种蛋白质的混合物，这使得主要免疫显性表位的鉴定变得困难。为了解决这个问题，我们在大肠杆菌中表达了一种重组α - 醇溶蛋白（r - α - 醇溶蛋白），它在α - 醇溶蛋白部分中显示出最保守的序列。食用无麸质饮食的HLA - DQ8小鼠，用r - α - 醇溶蛋白的胰凝乳蛋白酶消化物与霍乱毒素作为佐剂进行胃内免疫。使用一组涵盖r - α - 醇溶蛋白整个序列的合成肽，对脾和肠系膜淋巴结T细胞反应进行体外增殖测定分析。鉴定出了与肽p13（氨基酸120 - 139）和p23（氨基酸220 - 239）相对应的两个免疫显性表位。该反应限于DQ且由CD4 + T细胞介导。两种肽的体外组织转谷氨酰胺酶脱酰胺作用并未增强反应；此外，组织转谷氨酰胺酶在体外沿整个r - α - 醇溶蛋白分子催化广泛的脱酰胺作用，但未能引发新的免疫原性决定簇。令人惊讶的是，细胞因子谱分析表明，尽管使用了通常诱导对旁观者抗原产生Th2反应的黏膜佐剂霍乱毒素，但脱酰胺肽和天然肽均优先诱导IFN - γ分泌。综上所述，这些数据表明，在这种麸质超敏反应模型中，脱酰胺不是引发麸质反应的先决条件。
