Study On Potential Allergenicity Of Food Proteins And The Mechanism Of Food Allergy Using Cell Model

In recent years, food allergy appears to have increased in prevalence and affects approximately5%of young children and3-4%of adults in westernized countries. In order to prevent and control the occurrence of food allergy, the allergenicity assessment and the mechanism research of food allergy become hot spots in worldwide research. Currently, almost all of the approaches are made for investigating the potential allergenicity of novel proteins. However, there has not been a strategy for evaluating the allergenicity of food proteins. Furthermore, there are multiple signaling pathways in the activation phase of food allergy and multiple intersection points between the various pathways, which form an intricate network system for mast cell degranulation. However, some mechanisms remain to be further explored. In this study, we chosed different allergenic food proteins, including Glycinin, ovalbumin and potato acid phosphatase, to sensitize BALB/c mice for obtaining sensitized serum. Based on the analysis of the binding of rat basophilic leukemia (RBL) cells to mouse IgE and the functionality of RBL cells, we investigated a highly-reactive cell-based immunological assay, which further validated by methods recommended by the approaches used for assessing potential allergenicity of novel proteins, and then developed a novel strategy for evaluating the allergenicity of food proteins. It is critical for providing more accurate and comprehensive data to risk assessment and standardization of food allergens. Additionally, we explored immunological mechanisms for the activation phase of food allergy, and dissected the role of a key signaling protein Dok-1in regulating the FcεRI-mediated mast cell degranulation. It is of great significance for finding a potential therapeutic target in FcεRI-mediated inflammatory processes. The main results and conclusions are as follows:(1) We developed an RBL-2H3cell-based immunological assay. Based on its high sensitivity and specificity as well as its ability to differentiate different allergenic food proteins, an RBL-2H3cell-based immunological assay might be a useful tool for evaluating the allergenic activity of food proteins. Conversely, RBL-1cells, which cannot differentiate different allergenic food proteins, are unsuitable for evaluating the potential allergenicity of food proteins. Moreover, the results of the RBL-2H3cell-based immunological assay were found to be consistent with those of the bioinformatical analysis, pepsin digestion assay and BALB/c mice model assay.(2) We proposed a novel strategy for evaluating the allergenicity of food proteins. The bioinformatical analysis used can directly predict linear epitopes of food proteins in addition to the sequence homology comparison. Human serum studies used may validate the result of bioinformatical analysis based on the specific IgE levels and assess the clinical reactivity of food proteins based on the specific IgE/IgG4ratio. Furthermore, pepsin digestion assay is applied to determine the stability against pepsin digestion and the effect of heat treatment on the digestion of food proteins. When the result of bioinformatical analysis is inconsistent with that of pepsin digestion assay, an RBL-2H3cell-based immunological assay plays a "referee" or "verification" role for determining the allergenicity. The final step is to dissect mechanisms of allergic responses and potential changes due to modification of allergenic proteins by animal model assay. Thus comprehensive applications of the above-mentioned multiple methods will be a good choice for a complete evaluation of allergenic potential.(3) We explored the mechanism of Dok-1in negatively regulating the FcεRI-mediated mast cell degranulation and found a new signaling pathway. More specifically, following FcεRI activation, Dok-1interacts through its PH domain with PI(3,4,5)P3(the products of PI3K), which, in turn, is recruited to the plasma membrane, leading to its tyrosine phosphorylation by Src family kinases. Phosphorylated Dok-1inhibits FcεRI receptor-operated calcium influx via the TRPC1channel and then negatively regulates the FcεRI-mediated degranulation by recruiting Nck to the plasma membrane and sequentially inhibiting the disassembly of actin filament in a calcium-dependent manner.