The main function of the Vector, Protein & Mechanistic Core is to support mechanistic studies being conducted in the three proposed projects of this Consortium. One of the hypotheses guiding this Consortium application is that bacterially expressed modified allergen (peanut allergens Ara hi, 2, and 3) delivered via the rectal route encapsulated within heat-killed E. coli (EMP-123) provides a combination of signals resulting in the activation of regulatory T cells. These cells then suppress the Th2 biased response in a peanut sensitized individual. The goals of this core are to provide reagents for the murine mechanistic studies and to acquire subject samples from the clinical sites of Consortium members and perform the assays required to address this hypothesis and determine the mechanism of action of EMP-123. This core will also serve as the acquisition and distribution center for standardized reagents and materials required for local processing of blood samples, i.e., for the initial activation of T cells in fresh PBMCs, and subsequent analyses for both the interventional [Project 1] and observational [Project 2] studies. These goals will be accomplished by the following aims: 1) Generation of E. coli expressing recombinant modified Ara hi, 2, & 3 and purification of recombinant proteins for use in Project 3. To track bacteria and protein, these reagents will be labeled with either fluorescent or radioactive labels. 2) Providing mechanistic insights into the effects of EMP-123 in the interventional and observational studies (Project 1 and 2). This will be accomplished by analysis of the activation of Tregs assessed by FoxPS expression (mRNA and flow), IL10 production (mRNA and protein by ELISA), TGF-b production (active form by ELISA) and either Th2 to Th1 conversion or the development of anergy (real time PCR for GATA-3, T-bet, IL4, IL10, IL5, IL13, IFN-g mRNA as well as protein measurements (by ELISA) in supernatants from stimulated (CPE, Ara H1, PMA/PHA) patient and control T cells. The use of common laboratory supplies and reagents, highly specific protocols for handling of PBMCs at local sites, and centralized training and certification of all lab personnel will minimize laboratory variability and allow meaningful analyses of data generated. The results of these studies should help us to gain insights into mechanisms of action of the modified peanut protein delivery system, allow us to define pathways whereby novel interventions can be approached, and provide insights into mechanisms and biomarkers associated with the natural development and loss of food.
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