The proposal concerns the development of the G.A.T.CD4 (Gliadin-activated CD4+ T cells) method which allows, in peripheral blood, the identification of CD4+ T lymphocytes reactive to toxic peptides of gliadin, the main gluten protein of cereals. Through the use of specific markers of CD4+ T lymphocytes activation, the G.A.T.CD4 method allows the frequency and reactivity of antigen-specific T helper lymphocytes in a blood sample, following antigen stimulation and subsequent analysis with multiparametric flow cytometry. The test is based on the preparation of the leukocyte fraction (PBMC) from a venous sampling, cells culture in plates in the presence of the antigenic gliadin peptides pool or the entire deamidated gliadin protein. After 48 hours of incubation, only the CD4 T lymphocytes specific for the gliadin antigen will be activated and proliferate and can be stained with a cocktail of 4 monoclonal antibodies and analyzed by flow cytometry. The test will be positive only in patients suffering from celiac disease acute, and will be negative for those patients who are in remission of the disease, on a gluten-free diet.
The diagnosis of celiac disease is carried out by determining the titre of anti-transgutaminase and anti-endomysial antibodies in the blood. In controversial cases, for which it is not possible to make a diagnosis on the basis of antibodies and clinical symptoms, gastroscopy with biopsy is performed. The method G.A.T.CD4 aims to the identification of CD4+ T lymphocytes reactive towards gluten, a widely consumed food protein and responsible for one of the most common forms of food intolerance globally. The applications of the G.A.T.CD4 method are:
1. Diagnosis of acute celiac disease without histopathological analysis of intestinal mucosal tissue (EGDS examination or esophagogastroduodenoscopy).
2. Support for the diagnosis of active celiac disease in doubtful cases of EGDS.
3. Monitoring of disease remission following the gluten-free diet.
4. Support for research in order to analyze the phenotype and functions of subpopulations of pathogenic CD4+ T lymphocytes, reactive to gliadin.
Italy