Assessment of Allergenic Potential of Food

Specific Objective #1: 1) to determine the relative sensitization potencies of salt-soluble wheat protein fractions isolated from five different wheat genotypes in this model. Based on our own preliminary data and the information in the literature that wheat varieties may potentially differ in allergenicity, the hypothesis that the salt-soluble wheat protein fractions extracted from five distinct wheat genotypes (AA, DD, SS, AABB, AABBDD) differ intrinsically in their relative sensitization potencies will be tested using established methods. The salt-soluble proteins from five different wheat genotypes will be isolated and used in allergenicity testing. The in vivo sensitization potencies will be determined using our transdermal exposure adjuvant-free mouse model. Using the dose-response data, a quantitative comparative map of the sensitization potencies of five wheat genotypes will be established. Follow up studies using Western blot approach will identify specific IgE-binding protein allergens that are shared vs. unique to each wheat genotype.2) to determine the relative allergic disease elicitation potencies of salt-soluble wheat protein fractions isolated from five different wheat genotypes in this model. We postulate, again on the basis of our previous workand information in the literature that five distinct wheat genotypes will differ in their ability to elicit allergic disease upon systemic (intraperitoneal) as well as oral challenge in mice. Quantitative readouts of allergenicity will include hypothermia shock response and mucosal mast cell degranulation response as optimized by us before. A quantitative comparative map of the disease elicitation potencies of five wheat genotypes will be established. Follow up studies will be performed to elucidate molecular immune-activation signature patters by different wheat genotypes. For this tissues (spleen, mesenteric lymph nodes and skin) will be collected and protein extract will be used to measure cytokines and chemokines using a protein microarray-based biomarker mapping method we have used before. We will identify the differences in immune-activation patterns that correlate with differences in wheat allergenicity readouts among the five wheat genotypes.Specific Objective #3: To determine the effect of fermentation on wheat protein allergenicity in our mouse model--a pilot study. We propose to conduct a pilot study to evaluate the effect of sourdough fermentation on wheat protein allergenicity in this mouse model. For these studies we will test two genotypes (AABBDD and AABB) during this period. We plan to test other genotypes in the future. The wheat flour sourdough fermentation will be conducted using three selected lactic acid bacteria (based on literature) as described. Single fermentation reactions will be conducted with and without yeast as described earlier. Salt-soluble wheat protein fractions will be isolated using the methods established in the laboratory. The salt-soluble proteins isolated from fermented wheat flour vs. chemically acidified wheat flour vs. conventional wheat flour fermentation will be tested for in vivo allergenicity in our mouse model. We will conduct these studies in two parts: first we will evaluate the sensitization capacity of fermented wheat proteins. In the second part, we will evaluate the disease elicitation capacity of fermented wheat proteins.