Final Report Summary - IGIFA (Impact of Gluten properties on Immune system and development of Food Allergy) Food allergies or food intolerances affect one out of three people. About 5% of children have clinically proven allergic reactions to foods. In teens and adults, food allergies occur in about 4% of the total population. A food allergy, or hypersensitivity, is an abnormal response to a food that is triggered by the immune system. It is extremely important for people who have true food allergies to identify them and prevent allergic reactions to food because these reactions can cause devastating illness and, in some cases, be fatal. It is well established that industrial food processing affects proteins. Indeed, the impact of these processes on food proteins features as well as their consequences on the development of immunity and physiopathology of allergy remain undetermined. Nevertheless, it is a key topic in order to reduce or stop the development of food allergies. Taken the wheat proteins as a model for food, we purified in a large scale wheat gliadin. The whole native gliadin extract (NG), was collected and purified by ion-exchange chromatography and reverse-phase HPLC. Then, on order to mimick industrial process, fraction of NG was used to obtain deamidated gliadin (DG) or hydrolysis gliadin (HG). Purified gliadin fractions chemically deamidated under acidic conditions displayed deamidation levels of 53%. The migration of native and deamidated gliadins in an acid-PAGE gel revealed differences, reflecting the increase in net charges of deamidated gliadins, and the broad heterogeneity of deamidated gliadins that results from chemical modification. HG were not detect within the experiment as fragment were too small to be detected confirming the efficacy of the hydrolysis. Regarding the immune properties of each fraction, IgE binding measured by F-ELISA from patients allergic to wheat proteins reacted against to wheat proteins according to their pathology. IgE from patients allergic to deamidated wheat proteins were much more reactive against deamidated gliadin fractions and inversely. Self-produced antibodies already existing in our laboratory has also been tested for their specificity to each fraction. Five of them were specific for deamidated samples. Three of them showed similar binding intensities for all deamidated fractions and bound weakly to the native gliadins. The two others displayed a more restricted profile of reactivity as it only bound strongly to deamidated fractions and no binding to the native gluten proteins. Biological properties of fractions have been measured by the percentage of degranulation of the RBL cell line reflected by the release of β-hexosaminidase or the expression of CD63 or CD203. We observed that the specific IgE from mice sensitized with DG and NG were able to induce RBL cell degranulation. Higher percentages of degranulation were observed when RBL cells were pre-incubated with IgE from DG sensitized mice and were then placed in contact with either DG or anti-IgE. To the opposite, lower percentages of degranulation were observed when RBL were pre-incubated with IgE from HG sensitized mice.Even if the allergic response is documented, the exact mechanisms of the immune response toward different form of the same protein are still unclear. Indeed, how the industrial processes modifying the wheat protein impact the immune mechanisms is unknown. Our work aimed to study the effect of three forms of gliadin on the sensitization phase and the elicitation phase on a murine model of food allergy. The immune response toward native gliadin, deamidated gliadin and enzymatic-hydrolyzed gliadin was studied on three different time points: after the first sensitization, after the second sensitization and after the oral challenge by gavage. To investigate if different industrial processes alter the immune response in a different way, we have studied the Ig response and the cell infiltration at the three different time points. These results will bring news data on the evolution of the immune mechanisms during the sensitization and the elicitation phase. Our results demonstrate that the sensitization with the deamidated and the native form of the gliadin induces a greater allergic response and bigger symptoms. These results suggest an important alteration of the intestinal epithelial layer due to the greater penetration by the allergen. Overall, these results tend to confirm that the deamidated form of the gliadin induces a greater systemic inflammation on mice.
