Celiac disease (CD) is a malabsorption disorder characterized by a small intestinal enteropathy that reverts to normal on removal of dietary gluten. The exact gluten peptide(s) that exacerbate CD are unknown, although toxicity is known to reside in wheat gliadin. The condition affects 130,000 individuals in the USA. CD is strongly associated with the class II human leucocyte antigens (HLA) DR17(3), and DQ2. The pathogenesis is poorly understood, although it is thought to be mediated by HLA restricted gliadin-sensitive T-cells in the small intestine. Investigation of a gliadin-specific T cell clone isolated from the peripheral blood of a celiac patient has permitted the identification of a peptide corresponding to amino acid residues 31-49 of A-gliadin. Subsequent in vivo challenge experiments in 4 treated CD patient volunteers with this and 2 control A-gliadin peptides has confirmed that this peptide exacerbates CD while the control peptides do not. We now propose to extend these studies by producing additional gliadin- specific T cell clones from both the peripheral blood and small intestine of CD patients. The specificity of these cells will be investigated using a panel of amino acid sequenced gliadin peptides. The HLA-class II restriction of the gliadin-specific T cells will be analyzed using HLA homozygous antigen presenting cells and mouse fibroblasts (L cells) that express transfected HLA-class II genes in lymphocyte proliferation assays. To undertake these studies we will produce additional gliadin peptides. These will include a set of peptides corresponding to the l9 amino acid residue peptide that we have shown exacerbates celiac disease, but in which single amino acid residues will be substituted with alanine. Binding of these wheat gliadin peptides to HLA class II molecules associated with celiac disease will be investigated, focusing primarily on DR17(3) and DQ2. For these studies, the gliadin peptides will be labeled with long chain biotin. Their binding to HLA molecules expressed on B cell lines from patients with celiac disease, homozygous B cell lines, controls and transfected L cells will be determined. These studies will provide a better understanding of the pathogenesis of CD at the molecular level and identify the precise amino acid structure of the cereal peptide(s) that exacerbate CD. Dr. Ciclitira's clinic of more than 500 patients with CD combined with Dr. Rosen-Bronson's expertise with transfected cell lines and peptide binding assays offers a unique opportunity to investigate celiac disease at the molecular level.
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