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Determination of allergenicity of different food allergens in a rat model of food allergy
J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2
alpha-lactalbumin, beta-lactoglobulin and caseins. Exposure to cow milk protein should be avoided for the treatment of CMA until they are outgrown. In this study we evaluated the presence of major allergen in various dairy products, which is easily available, to see whether they are tolerated in CMA patients. METHODS: Commercially available cow milk and dairy products such as standard cow milk formula, cheese, yogurt, butter samples were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: SDS-PAGE of purified samples of cow milk and various dairy products showed similar pattern of 3 bands which corresponds to casein, alpha-lactalbumin, and beta-lactoglobulin. This pattern varies according to each product. CONCLUSIONS: Major allergens in cow milk protein are also included in dairy products, although the amount might be variable, suggesting that they should be restricted in children with CMA.
733
In Vitro Characterization of the Allergenicity and Protein Profiles of Two Kiwi Fruit Varieties
S. A. Lewis I, A. Pearce j, J. Lucas 2, J. O. Hourihanel; qnfection, Inflammation and Repair Division, University of Southampton, Southampton, UNITED KINGDOM, 2Child Health, Southampton University Hospital NHS Trust, Southampton, UNITED KINGDOM. RATIONALE: We investigated the in vitro allergenicity and protein profiles of two kiwi fruit varieties currently available in the UK: green and gold kiwi. METHODS: Protein extracts were prepared from Hayward (Zespri TM Green) and Zespri TM Gold varieties of kiwi fruit and separated by I and 2D SDS-PAGE. Western blots were performed using sera from patients with kiwi allergy. Protein were identified by mass spectrometry. RESULTS: 1 and 2-D SDS-PAGE revealed proteins common and unique to both varieties. A highly expressed protein in the Hayward variety was missing/expressed at levels too low to detect in the Zespri TM Gold variety using Coomassie stain. This protein had weight and pI features of Act c 1. Identification was confirmed by mass spectrometry. Western blotting showed proteins from both varieties bound IgE, again with bands common and unique to both varieties. A protein -30 kDa in the Hayward extract bound IgE (possibly Act c 1), but no corresponding band was seen with the Zespri TM Gold extract. CONCLUSIONS: This study has demonstrated differences in the in vitro allergenicity of two commonly available kiwi fruit varieties. The new variety of Zespri TM Gold may lack/express much lower levels of Act c l - - a major kiwi fruit allergen.
Funding: University of Southampton
734
Determination of Allergenicity of Different Food Allergens in a Rat Model of Food Allergy
B. Ahrens I, D. Quarcoo r, A. Meeuw j, J. Zagon 2, G. Reese3, S. Vieths3, E. Hamelmannl; 1Department of Pediatric Pneumology and Immunology, Charit6-Humboldt University, Berlin, GERMANY, 2Federal Institute for Health Protection of Consumers and Veterinary Medicine, BgVV, Berlin, GERMANY, 3Department of Allergology. Paul-Ehrlich Institute, Langen, GERMANY. Sensitization to food allergens often is the first detectable sign of atopy in early infancy, and may later lead to life-threatening disease. The underly-
Abstracts
$251
ing pathological mechanisms of food allergies are still not fully understood and require further investigation. Aim of the current study was to establish a rat model to compare the allergenicity of various food allergens and delineate systemic and local allergen-induced immune responses. Brown Norway rats were sensitized to protein extracts (soja, peanut, garden pea) or Ovalbumin (OVA) combined with pertussis and AI(OH)2 as adjuvans on day 1, 5, 10. On day 15 and 16 rats were locally challenged with allergen by gavage feeding. Allergen sensitization was determined by measurement of specific Ig serum levels and by proliferative responses of allergen-stimulated mononuclear spleen (SC) and mesenterical lymph node cells (MLN). Systemic and local immune responses were analyzed by flow cytometry of SC, MLN and intraepitbelial lymphocytes (IEL). Morphological changes of the intestinal tract after allergen challenges was assessed by histological staining. Allergen specific lg production and proliferative responses confirmed sensitization to different food proteins. However, strongest immune responses occurred following sensitization with peanut extract. Local histological changes after food challenges reflected allergen-mediated disease. The data confirmed that the degree of allergenicity of various food allergens is predictable, and that major immunological phenomena of food allergen-mediated disease are reflected utilizing an adequate animal model. This model thus may serve as a tool for further investigation of underlying mechanisms and potential therapies in the treatment and prevention of food allergies.
Funding: BMBF
735
E.ecI of Suplatast Tosilate, a Th2 Cytokine Inhibitor, on Murine Food Allergy Model on the Small Intestine and Liver Damages
Y. Ilkura, S. Yofu, T. Takahashi, K. Ueno; Showa University School of Medicine, Tokyo, JAPAN. OBJECTIVE: We previously reported that liver damage may occur in individuals with food allergy and have conducted various studies on methods for treating this. The aim of the present study was to investigate the inhibitory effect of the antiallergic drug suplatast tosilate (IPD) on the reaction caused by food allergy. METHODS: Ovalbumin (OVA)-sensitized mice (Nc/Nga) were challenged by administering OVA via the gastrointestinal tract. Three hours later, the livers and small intestines were resected, and hematoxylin-andeosin-stained specimens were prepared. The various parameters were expressed numerically using diagnostic imaging software, and the results were then analyzed for significant differences using statistical analysis software. Serum AST and ALT levels were also compared. Group H was given IPD at a dose of 100 mg/kg/day and group L was given a dose of 10 mg/kg/day. A non-OVA-sensitized group and a group given an inactive placebo instead of IPD were used as the controls. RESULTS: In the IPD-treated groups, the hepatocyte degeneration and necrosis, inflammatory cell infiltration in the liver, elevation of AST and ALT levels, and edematous degeneration in the villus lumen in the small intestine seen in the OVA-sensitized group had resolved to the extent that the tissue was virtually the same as the normal tissue in the non-sensitized group, and the liver function values also normalized. C O N C L U S I O N S : The results indicate that IPD is useful for treating pathological changes associated with damage to the small intestine and liver caused by tbod allergy and suggest that the drug exhibits its effect by inhibiting inflammatory cell infiltration, preventing tissue degeneration. and facilitating tissue repair.
alpha-lactalbumin, beta-lactoglobulin and caseins. Exposure to cow milk protein should be avoided for the treatment of CMA until they are outgrown. In this study we evaluated the presence of major allergen in various dairy products, which is easily available, to see whether they are tolerated in CMA patients. METHODS: Commercially available cow milk and dairy products such as standard cow milk formula, cheese, yogurt, butter samples were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: SDS-PAGE of purified samples of cow milk and various dairy products showed similar pattern of 3 bands which corresponds to casein, alpha-lactalbumin, and beta-lactoglobulin. This pattern varies according to each product. CONCLUSIONS: Major allergens in cow milk protein are also included in dairy products, although the amount might be variable, suggesting that they should be restricted in children with CMA.
733
In Vitro Characterization of the Allergenicity and Protein Profiles of Two Kiwi Fruit Varieties
S. A. Lewis I, A. Pearce j, J. Lucas 2, J. O. Hourihanel; qnfection, Inflammation and Repair Division, University of Southampton, Southampton, UNITED KINGDOM, 2Child Health, Southampton University Hospital NHS Trust, Southampton, UNITED KINGDOM. RATIONALE: We investigated the in vitro allergenicity and protein profiles of two kiwi fruit varieties currently available in the UK: green and gold kiwi. METHODS: Protein extracts were prepared from Hayward (Zespri TM Green) and Zespri TM Gold varieties of kiwi fruit and separated by I and 2D SDS-PAGE. Western blots were performed using sera from patients with kiwi allergy. Protein were identified by mass spectrometry. RESULTS: 1 and 2-D SDS-PAGE revealed proteins common and unique to both varieties. A highly expressed protein in the Hayward variety was missing/expressed at levels too low to detect in the Zespri TM Gold variety using Coomassie stain. This protein had weight and pI features of Act c 1. Identification was confirmed by mass spectrometry. Western blotting showed proteins from both varieties bound IgE, again with bands common and unique to both varieties. A protein -30 kDa in the Hayward extract bound IgE (possibly Act c 1), but no corresponding band was seen with the Zespri TM Gold extract. CONCLUSIONS: This study has demonstrated differences in the in vitro allergenicity of two commonly available kiwi fruit varieties. The new variety of Zespri TM Gold may lack/express much lower levels of Act c l - - a major kiwi fruit allergen.
Funding: University of Southampton
734
Determination of Allergenicity of Different Food Allergens in a Rat Model of Food Allergy
B. Ahrens I, D. Quarcoo r, A. Meeuw j, J. Zagon 2, G. Reese3, S. Vieths3, E. Hamelmannl; 1Department of Pediatric Pneumology and Immunology, Charit6-Humboldt University, Berlin, GERMANY, 2Federal Institute for Health Protection of Consumers and Veterinary Medicine, BgVV, Berlin, GERMANY, 3Department of Allergology. Paul-Ehrlich Institute, Langen, GERMANY. Sensitization to food allergens often is the first detectable sign of atopy in early infancy, and may later lead to life-threatening disease. The underly-
Abstracts
$251
ing pathological mechanisms of food allergies are still not fully understood and require further investigation. Aim of the current study was to establish a rat model to compare the allergenicity of various food allergens and delineate systemic and local allergen-induced immune responses. Brown Norway rats were sensitized to protein extracts (soja, peanut, garden pea) or Ovalbumin (OVA) combined with pertussis and AI(OH)2 as adjuvans on day 1, 5, 10. On day 15 and 16 rats were locally challenged with allergen by gavage feeding. Allergen sensitization was determined by measurement of specific Ig serum levels and by proliferative responses of allergen-stimulated mononuclear spleen (SC) and mesenterical lymph node cells (MLN). Systemic and local immune responses were analyzed by flow cytometry of SC, MLN and intraepitbelial lymphocytes (IEL). Morphological changes of the intestinal tract after allergen challenges was assessed by histological staining. Allergen specific lg production and proliferative responses confirmed sensitization to different food proteins. However, strongest immune responses occurred following sensitization with peanut extract. Local histological changes after food challenges reflected allergen-mediated disease. The data confirmed that the degree of allergenicity of various food allergens is predictable, and that major immunological phenomena of food allergen-mediated disease are reflected utilizing an adequate animal model. This model thus may serve as a tool for further investigation of underlying mechanisms and potential therapies in the treatment and prevention of food allergies.
Funding: BMBF
735
E.ecI of Suplatast Tosilate, a Th2 Cytokine Inhibitor, on Murine Food Allergy Model on the Small Intestine and Liver Damages
Y. Ilkura, S. Yofu, T. Takahashi, K. Ueno; Showa University School of Medicine, Tokyo, JAPAN. OBJECTIVE: We previously reported that liver damage may occur in individuals with food allergy and have conducted various studies on methods for treating this. The aim of the present study was to investigate the inhibitory effect of the antiallergic drug suplatast tosilate (IPD) on the reaction caused by food allergy. METHODS: Ovalbumin (OVA)-sensitized mice (Nc/Nga) were challenged by administering OVA via the gastrointestinal tract. Three hours later, the livers and small intestines were resected, and hematoxylin-andeosin-stained specimens were prepared. The various parameters were expressed numerically using diagnostic imaging software, and the results were then analyzed for significant differences using statistical analysis software. Serum AST and ALT levels were also compared. Group H was given IPD at a dose of 100 mg/kg/day and group L was given a dose of 10 mg/kg/day. A non-OVA-sensitized group and a group given an inactive placebo instead of IPD were used as the controls. RESULTS: In the IPD-treated groups, the hepatocyte degeneration and necrosis, inflammatory cell infiltration in the liver, elevation of AST and ALT levels, and edematous degeneration in the villus lumen in the small intestine seen in the OVA-sensitized group had resolved to the extent that the tissue was virtually the same as the normal tissue in the non-sensitized group, and the liver function values also normalized. C O N C L U S I O N S : The results indicate that IPD is useful for treating pathological changes associated with damage to the small intestine and liver caused by tbod allergy and suggest that the drug exhibits its effect by inhibiting inflammatory cell infiltration, preventing tissue degeneration. and facilitating tissue repair.