Establishment And Mechanisms Of In Vivo Digestibility And Food Allergy Model In WZS Minipigs

ObjectiveTo provide reference for the procedure and experimental method of the allergenicity potential assessment of GMO (genetically modified organisms). The Chinese native acclimated and cultivated inbred WZS minipigs were chosen as the model animal, (1) to establish the in vivo digestibility big animal model for the potential allergenicity assessment of GMO. After the stomach cannula and ileum cannula were surgically fit into the minipigs, gastric and intestine fluids were collected simultaneously at the selected time. Soybean and peanut were chosen as the positive controls, the in vivo digestibility of rice genetically modified with CpTI (Cowpea Trypsin Inhibitor) and milk genetically modified with LF (Lactoferrin) were compared and assessed. (2) to establish the big animal model for the potential allergenicity assessment of GMO. The oral sensitization WZS minipig model without use of adjuvant was tested with soybean glycinin andβ-conglycinin. The intraperitoneal sensitization WZS minipig model with cholera toxin was tested with soybeanβ-conglycinin.Methods1 Establishment and preliminary application of in vivo digestibility WZS minipig modelThe stomach cannula and ileum cannula were surgically fit into the adult male minipigs (22-30kg). The intestinal cannula was fit at the proximal end of the ileum, and the stomach one was fit at the arcus major ventriculi. Fourteen days after the surgery, the in vivo stability tests were performed. All of the minipigs freely ate the samples within 0.5-1 min to make no differences on the dwell times between different animals and experiments. The minipigs were fast 16h before the experiment, blank digestion fluids were collected before the sample giving. And then 250mL purified water mixed with 100g soybean, peanut powder, rice powder or 250mL milk were respectively given to the animals,1.5～2.0mL stomach digestion fluids at Oh,0.25h,0.5h,0.75h,1h, 2h,3h,4h,5h,6h were collected through the stomach cannula, and 0.5～1.5mL intestine digestion fluids at Oh,1h,2h,3h,4h,5h,6h were collected through the ileum cannula. The digestion fluids were centrifugated and the pH value of the supernatant were measured. After the protein of the digestion fluids supernatant quantitated with Coomassie assay and adjusted less than 10μg per well, the protein digestibility in the gastric or intestinal juice at different time after the soybean, peanut, rice and milk given were compared with SDS-PAGE. The monoclonal antibody against theβ-subunit of recombination soybean P-conglycinin was prepared, after three times cloning screening and affinity purification, and immunoblotted with the gastrointestinal digestion of Ji nf327 soybean to confirm the digestion and migration of theβ-subunit ofβ-conglycinin.2 Establishment and mechanisms of WZS minipig food allergy model2.1 Experimental study of anaphylactic reactions in WZS minipig orally induced by soybean proteinTwenty WZS minipigs from five litters were weaned at 45 days after birth. After 4 days of adaptation piglets were randomly allotted to five groups (negative control,4% 11S,8% 11S,4% 7S and 8% 7S) on day 0 of the experiment. Each treatment had four replicates. All experimental minipigs were weight every week. Physical activity and incidence of diarrhea were recorded daily throughout the entire experiment. For the 11S-and 7S-sensitized animals, during the sensitization phase (days 0-10 of the experiment) and excitation phase (days 16-18 and day 32 of the experiment),4% or 8% of daily diet 11S or 7S protein were orally given to pigs before feeding. The negative control animals were given soy-free diets. Blood was collected from the precaval vein on days 11,19, and 32. Cutaneous skin testing was conducted on day 25 of the experiment. On day 32 of the experiment, after the last excitation, all the piglets were slaughtered with anesthetic, jugular exsanguination and dissected. Serum IgG, IgE, histamine, TNF-α, IL-2, IFN-γ, IL-4, IL-10, and intestinal histamine ELISA kits were used for quantification. The expression of peripheral blood lymphocyte subsets CD3/CD4/CD8 were simultaneously measured with flow cytometer after the PBMC separated. The mast cells in the mucosa and submucosa were specifically stained with toluidine blue and quantified by randomly numbering mast cells in 10 areas of one segment histological slide with a Microcheck Grid.2.2 Experimental study of anaphylactic reactions in WZS minipig peritoneally induced by soybean proteinEight WZS piglets from four litters were randomly allotted to adjuvant group (i.p.100μg cholera toxin) and 7S group (i.p.500μgβ-conglycinin and 100μg cholera toxin). The pregnant sows were gradually given soy-free diets before two weeks of the farrowing. The day of the birth was the day 0 of the experiment. The pigs were intraperitoneally sensitized on days 15,16,17 and boosted on day 25. On day 45, the piglets were weaned and firstly orally challenged two days later. On day 54, cutaneous skin testing was conducted and secondly challenged and dissected one week later. Blood was collected at five time point:(Ⅰ) day 15 before the first intraperitoneal sensitization (Ⅱ) day 25 after the fourth intraperitoneal sensitization (Ⅲ) day 35 after the fifth intraperitoneal sensitization (Ⅳ) day 49 after the first oral challenge (Ⅴ) day 61 after the second oral challenge. The animals were weight before the blood collection. Serum total IgG, IgE, histamine, intestinal histamine, peripheral blood lymphocyte subsets CD3/CD4/CD8 and mast cell were measured as in the oral allergy experiment. The level ofβ-conglycinin specific IgG in the serum was tested with indirect ELISA assay. The proportion of eosinophile and basophile granulocyte was analyzed on blood counting instrument. The transformation and proliferation function of peripheral blood lymphocyte was measured with MTT assay after the PBMC were isolated. The Th1/Th2 cytokines (IL-2, IFN-γ, IL-4) in the culture supernatant of PBMC were tested with ELISA kits.Results1 Establishment and preliminary application of in vivo digestibility WZS minipig modelCompared with the ileum cannula (several days to months), the stomach cannula could be kept in the minipigs longer (several months to fourteen months). After the minipigs were fasted for 16h, the pH value of blank gastric juice was low as 1.5～2, quickly increased when samples were given and descend at 4-6h. The time of pH value equal to 2 were different among different samples. It was 5h for peanut and soybean,0.25-0.5h for rice and 2～3h for milk.According to the comparison with the molecular marker, for the soybean, the protein fragments stable in the gastric juice for 0～1h was 40kD,0～2h were 150kD/80kD/71kD/67kD/20kD,0～3h was 15kD,0～6h were 50kD/34kD/17kD/13kD, the protein fragments stable in the intestinal juice for 4-6h were 50kD/34kD/20kD/17kD. Among them, the soybean protein subunits of 34kD and 20kD, which were gradually decreased in the gastric juice after 3h, appeared in the intestinal juice 4h later. The soybean protein subunits of 50kD, 20kD and 17kD were all expressed abundantly.For the peanut, the protein fragments stable in the gastric juice for 0～0.75h was 48.5kD,0～1 h were 58Kd/17.9Kd/15kD,0～2h were 63kD/39kD,0～3h was 23.5kD, and 0～6h was 12kD, the protein fragments stable in the intestinal juice for 2～6h was 23.5kD, which was disappeared in the stomach juice after 3h.The monoclonal antibody against theβ-subunit of recombination soybeanβ-conglycinin (McAb) not only could be identified byβ-subunit (50kD), but also by another soybean subunit of 34kD. After the McAb was immunoblotted with the in vivo soybean digestion, it was shown that the 50kD subunit was stable in the gastric juice for 6h, and the 34kD one was 2h. At the time of Oh, the strap of 28kD was evident in the stomach. However, in the intestine, not any strap was seen at 0～2h, the 50kD subunit was seen and distinctly seen during 4～6h. This immunoblotting results were similar to that from the SDS-PAGE.Not any fragment resistant to prolease was detected in the gastric juice 0.25h later after the rice was given. There weren't any significant differences between the parent strain rice (Minhui 86) and the genetically modified rice with CpTI on the protein digestion in the gastric or intestinal juice. The digestion reactions in the stomach of the milk were complex than that of the rice, and more protein straps were seen. But there weren't obvious differences on the LF molecular weight site between the control milk and genetically modified milk with LF.2 Establishment and mechanisms of WZS minipig food allergy model2.1 Experimental study of anaphylactic reactions in WZS minipig orally induced by soybean proteinTwo days after the pigs were orally sensitized with soybean protein, two of the 11S-sensitized animals and two of the 7S-sensitized animals showed diarrhea symptoms, which lasted for 2～4 days. Erythematous responses in the corresponding protein sites were positive at different degrees in both 11 S-sensitized and 7S-sensitized animals, but both no more severe than that in the histamine site. Control animals fed soybean-free diets failed to show any symptoms.The serum IgG, IgE and histamine levels (logarithmic transforming values) all reached a maximum after animals were orally challenged with soybean protein at 19d. In contrast to the control, serum IgG, IgE and histamine levels at 19d and IgE levels at 32d of 4% 11S and 4% 7S sensitized groups, and serum IgG and histamine levels of 4% 11S at 32d were all significantly increased. The serum IgG, IgE, and histamine of the 20 pigs at days 11,19, and 32 were positively correlated. The jejunum histamine level was positively correlated with that of the duodenum and ileum, but negatively correlated with the mast cell numbers in the jejunum mucosa and sub-mucosa. Compared with the control, the jejunum histamine level of 4% 7S sensitized animals was increased and the mast cell number in the jejunum sub-mucosa of 4% 11S sensitized animals was decreased. The serum IFN-y content of 4% 7S group at 19d was significantly decreased, while the CD4+/CD8+ ratio of 8% 11S group was significantly increased.2.2 Experimental study of anaphylactic reactions in WZS minipig peritoneally induced by soybean proteinAfter the fourth intraperitoneal sensitization, two of the 7S-sensitized animals showed diarrhea symptoms and lasted for 3 days.Erythematous responses in the 7S injection site of 4 cholera toxin control animals were negative, while that of the two 7S-sensitized animals were positive. Compared with the cholera toxin control, the level ofβ-conglycinin specific IgG in the serum was higher. After the first oral challenge, the ratio of CD4+/CD8+ and the proliferation function of peripheral blood lymphocyte of the 7S-sensitized animals were increased in contrast to the control.Conclusions1 Through the surgery of stomach and ileum cannula fit in the WZS minipig, we originally established the in vivo minipig model for the simultaneous analysis of gastrointestinal digestion juice to assess the allergenicity of GMO. The in vivo digestion stability of different food allergen (peanut, soybean) were analyzed and initial evaluated with SDS-PAGE.2 The soybean digestion protein was confirmed after immunoblotted with the originally prepared monoclonal antibody against theβ-subunit of recombination soybeanβ-conglycinin. The validation assay for digestion protein of in vivo digestibility WZS minipig animal was set up.3 We originally applyed the in vivo digestibility WZS minipig model on the allergenicity assessment of rice genetically modified with CpTI and milk genetically modified with LF. It was preliminaryly proved that the in vivo digestability of rice genetically modified with CpTI and parent Ming 86 rice, milk genetically modified with LF and control milk were substantial equivalent.4 It is proved that both of oral and intraperitoneal sensitization routes for WZS minipig allergy model are eligible for the food potential allergenicity assessment. Diarrhea, cutaneous skin testing, serum total and specific IgG or IgE antibody, serum histamine, mast cell number, peripheral blood lymphocyte subsets and proliferation function are available different endpoints and testing index for the model evaluation. Appropriate routes of exposure should considering the characteristics and available amount of target protein.5 The WZS minipig allergy models orally induced by soybean glycinin andβ-conglycinin represent type-Ⅰhypersensitivity reactions mediated by IgE, and the possible mechanisms may related to the disturbance of CD4+/CD8+ proportion and increase of histamine release caused by mast cell degranulation after the intestinal mast cell proliferation and activation. The food hypersensitivity response induced by soybean antigen protein is not absolutely correlated with the dose.6 With the help of adjuvant, the WZS minipig allergy model peritoneally induced by soybeanβ-conglycinin represents type-Ⅰhypersensitivity reactions mediated by IgE, and the possible mechanisms may related to the disturbance of CD4+/CD8+ proportion and T lymphocytes hyperproliferation. Compared with the serum total IgG and IgE, serum specific IgG will be more sensitive for the evaluation of allergencity peritoneally induced byβ-conglycinin, and it is cumulative and peaks after multiple peritoneal boosts and oral challenges.