Research On Mechanism Of Allergic Reactions Caused By Soybean Glycinin And β-conglycinin In Balb/c Mice

Food allergy is becoming a medical, economical and social problem. Soybean is one of the major sources of protein in human and animal nutrition, and has also been well characterized as a major allergenic source. Food allergy caused by the ingestion of soybean products has been documented in many reports about clinical symptoms, which range from mild gastrointestinal distress to suspected anaphylaxis. Soybean glycinin andβ-conglycinin represent up to one third of protein in the soybean and were identified as binding IgE of subjects with soy allergy, and many results showed that the antibody response generated against soy protein was mainly reactive towards the storage protein glycinin and P-conglycinin.The purpose of this study was to investigate allergy syndrome and anaphylactic reactions induced by soybean glycinin orβ-conglycinin with an intragastric feeding protocol without using the adjuvant as hypersensitivity reaction in Balb/c mice.1. Extract, separate and purify soybean glycinin andβ-conglycinin from defatted soybeanThe objective of this study was to extract, separate and purify soybean glycinin andβ-conglycinin from defatted soybean. Glycinin and P-conglycinin contents in soybean seeds and in protein fractions were immunologically assayed. Treatment with 10%Nacl and ammonium sulfate fractionation improved the glycinin plus P-conglycinin content. Each fraction of the ammonium sulfate fractionated was finally purified on a Sepharose CL-6B column and purified 7S and 11S globulins achieved by these operations were designated glycinin and P-conglycinin.2. Establishement of murine model orally sensitized by soybean glycinin and P-conglycininTo investigate oral allergy syndrome and anaphylactic reactions in Balb/c mice caused by soybean glycinin and P-conglycinin with an intragastric feeding protocol without using an adjuvant. Balb/c mice were sensitized by gavages with glycinin and P-conglycinin, and allergen-specific IgE and IgG1 responses were studied by a passive cutaneous anaphylaxis assay. Intestinal histamine release and blood pressure were measured according to other methods. Epithelium and mast cell dye used the method of light microscopy. Sensitization with soybean allergens induced high levels of antigen-specific IgE and IgGl and increased intestinal histamine in Balb/c mice. Percentiles of intact mast cell of small intestine in mice sensitized with glycinin and P-conglyinin significantly decreased for 28 days. Degranulation of mast cells and damage of the epithelium in the small intestine of mice sensitized with globulins were observed. The level of blood pressure in sensitized mice reached a minimum at 3 h. The results suggested that glycinin and P-conglyinin induced allergic reaction in mice and Balb/c is a well anmimal for evaluating food allergy.3. The study of immediated IgE-mediated hypersensitivity induced by soybean glycinin andβ-conglyininTo develop and characterize a murine model of IgE-mediated soybean glycinin andβ-conglyinin sensitization induced by intragastric immunization. Sensitization with both 0.1mg/ml and lmg/ml glycinin andβ-conglyinin induced high levels of antigen-specific IgE and IgG and IgG1 and low levels of specific IgG2a and a concomitant increase of IL-4, IL-5 and IFN-y (P<0.01) production in spleocyte supernatants. Proliferative response both in mice sensitized with 1mg/ml glycinin andβ-conglyinin was detectable (P<0.01). Relative expression of cytokines IL-4, and IFN-y mRNA in spleen cells sensitized with lmg/ml glycinin andβ-conglyinin has increased significantly and IL-10 mRNA expression reduced (P＜0.01).4. Mechanism of immune-mediated gut hypersensitivity induced by soybean glycinin and P-conglyininHypersensitivity against soybean allergens can lead inflammatory disorders like food allergy and current models reflect a variety of causes but do not reveal the detailed modulation of gut immunity in response to food antigens after breakdown in mucosal tolerance. Balb/c mice were orally sensitized and challenged with soybean glycinin andβ-conglycinin and gut segments were collected for subsequent analyses. Results showed that T cell counts included CD3+ and CD4+ LPL increased (P<0.01) significantly in mice orally sensitized with soybean glycinin and increased CD3+ and CD8+ LPL (P<0.01) and CD4+ LPL cell counts sensitized with P-conglycinin andα4β7 LPL had also a significant increase (P＜0.01) in two experimental group. T cell counts of CD3+ and CD4+ IEL (P＜0.05) andα4β7 IEL (P<0.01) were higher in mice sensitized with soybean glycinin than counts of contror group. And the counts of CD3+, CD4+, CD8+ andα4β7 IEL (P＜0.01) and the ratio of CD4+/CD8+(P<0.05) in mice sensitized with soybeanβ-conglycinin have also increased compared with control. Modulation of mucosal immunity was mediated by increased expression of IL-4 and IFN-y mRNA in contrast to low IL-10 and TGFβ1 in the gut. The alteration of IgA+ plasma cells demonstrated that soybean allergens could lead gut inflammatory response.