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Effects of Enzymatic Hydrolysis on Peanut Allergenicity
AB224 Abstracts
876
Epitope Mapping of the Main Four Shrimp Allergens and Comparison of IgE Recognition Between Children and Adults R. Ayuso1, S. Sanchez-Garcia2, J. Lin1, M. Iba´n˜ez3, C. Blanco4, T. Carrillo5, M. Goldis1, L. Bardina1, H. A. Sampson1; 1Mount Sinai Medical Center, New York, NY, 2Fundacion Jimenez-Diaz, Madrid, SPAIN, 3Hospital Universitario Nin˜o Jesus, Madrid, SPAIN, 4Hospital Universitario La Princesa, Madrid, SPAIN, 5Hospital Universitario Dr Negrin, Las Palmas de Gran Canaria, SPAIN. RATIONALE: Shellfish allergy is a long-lasting disorder typically persisting throughout life. We aimed to characterize the IgE-binding profiles of four shrimp allergens recognized by children and adults. METHODS: Sera from 34 children (P) and 19 adults (A), with immediate allergic reactions and elevated serum IgE to shrimp were tested by microarray analysis for IgE binding to overlapping synthetic peptides spanning the sequences of the shrimp allergens tropomyosin, myosin light chain (MLC), sarcoplasmic calcium-binding protein (SCP) and arginine kinase (AK). RESULTS: Median shrimp-specific IgE was 4 fold higher in children (47 versus 12.5 kUA/L). Frequency of allergen recognition was: tropomyosin 81% (94%P, 61%A), MLC 57% (70%P, 31%A), AK 51% (67%P, 21%A) and SCP 45% (59%P, 21%A). Identified epitopes in tropomyosin are: Epitope 1 (amino acids 1-39), Epitope 2 (40-63), Epitope 3 (61-81), Epitope 4 (82-105), Epitope 5a (115153), 5b (142-165), 5c (156-186), Epitope 6 (190-210), Epitope 7 (246284). In MLC: Epitope 1 (13-30), Epitope 2 (22-48), Epitope 3 (46-66), Epitope 4a (58-90), Epitope 4b (79-99), and Epitope 5 (118-141). In SCP: Epitope 1 (10-33), Epitope 2 (49-72) and Epitope 3 (130-147). And, in AK: Epitope 1 (1-18), Epitope 2a (61-87), 2b (79-96), Epitope 3 (121-141), Epitope 4 (160-192), Epitope 5 (232-249), Epitope 6 (319-342). CONCLUSIONS: Shrimp-allergic children have higher IgE levels, show more intense recognition of shrimp allergens and epitope diversity than adults, who almost only recognize tropomyosin. Our work is the first to show that sensitization to shrimp proteins is greater in children and that appears to decrease in adulthood.
877
TUESDAY
Differences In IgE Binding To Raw, Roasted And Denatured Ara h 1, A Major Peanut Allergen J. B. Nesbit, S. J. Maleki; US Dept. of Agriculture, New Orleans, LA. RATIONALE: Ara h 1 is a major peanut allergen known to bind higher IgE following the roasting process. Here, we will examine the contribution of the structural and chemical modifications to alterations in IgE binding properties of Ara h 1. METHODS: Ara h 1 purified from raw and roasted peanuts and, heat denatured raw or dithiotheratol treated Ara h 1 were subjected IgE binding with sera from peanut allergic and sensitized individuals using spot blot analysis. Circular dichroism (CD) spectroscopy was used to monitor or confirm changes in the secondary structure between raw, roasted and denatured Ara h 1. RESULTS: The structures of Ara h 1 purified from raw and roasted are very similar with an alpha plus beta secondary structure, but differ significantly in IgE binding. Treatment of raw Ara h 1 with heat resulted in significant loss of alpha helices, while treatment with the reducing agent while notable, was not as significant. The structure of the heat denatured Ara h 1 shifts strongly toward a random coil/undordered structure. Majority of IgE in allergic and non allergic patientsÕ sera bound from highest to lowest to roasted, raw and denatured Ara h 1 (roasted > raw> denatured), respectively. CONCLUSION: In raw Ara h 1, the structural epitopes, particularly the ones located in the alpha helical sections, are more important to IgE binding than the liner epitopes. Meanwhile, enhanced IgE binding to roasted Ara h 1 is more likely to be due to chemical modifications than structural changes.
J ALLERGY CLIN IMMUNOL FEBRUARY 2010
878
Effects of Enzymatic Hydrolysis on Peanut Allergenicity B. Cabanillas1, M. M. Pedrosa2, C. Cuadrado2, C. Burbano2, M. Muzquiz2, J. Rodrı´guez1, J. F. Crespo1; 1Hospital Universitario 12 de Octubre, Madrid, SPAIN, 2Instituto Nacional de Investigaciones Agrarias, Madrid, SPAIN. RATIONALE: Enzymatic hydrolysis has been used to produce hypoallergenic dietary products derived from different protein sources. We sought to assess the effect of this procedure on the IgE-binding capacity of roasted peanut extracts. METHODS: Roasted peanut protein extract was hydrolyzed by individual and sequential action of an endoprotease (Alcalase) and an exoprotease (Flavourzyme). IgE binding was assessed by means of SDS-PAGE immunoblots and ELISA, using a pooled serum from 5 patients with clinical allergy to peanut confirmed on the basis of a positive doubled-blind, placebocontrolled, food challenge with peanut. RESULTS: Individual protein hydrolysis with Flavourzyme or Alcalase took place rapidly in the first 30 minutes; further, progression developed more slowly, reaching 24% and 17% degree of hydrolysis (DH) after 3h of processing with Flavourzyme or Alcalase respectively. The sequential treatment with Alcalase and Flavourzyme produced a 69% DH at the end of the hydrolytic process. SDS-PAGE immunoblots showed that roasted peanut extract appeared to be much more susceptible to the individual action of Alcalase and sequential action of Alcalase and Flavourzyme when compared with the native protein, indicating a reduction in its overall allergenicity. In contrast, roasted peanut extract showed little changes on the IgE-binding proteins after individual hydrolysis with Flavourzyme. In addition, roasted peanut extract hydrolyzed with Alcalase bound the lowest levels of IgE as determined by ELISA. CONCLUSIONS: Individual hydrolysis with Alcalase may result in a decrease of IgE immunoreactivity of roasted peanut allergens.
879
Inkt Cell Proliferation To Food-derived Sphingolipids In Food Allergic Vs Non-food Allergic Children S. Jyonouchi; Children’s Hospital of Philadelphia, Philadelphia, PA. RATIONALE: Invariant Natural Killer T (iNKT) cells are a subset of Tcells that modulate immune response through rapid release of Th1 and Th2 cytokines in response to sphingolipids. Thus, iNKT cells have the ability to affect disease processes by skewing T-cell cytokine profile. Our objective was to investigate the potential role of iNKT cells in food allergy (FA) by assessing frequency of peripheral blood (PB) frequency of iNKT cells and their responses to food derived sphingolipids in FA children and healthy controls. METHODS: This study includes 17 children with IgE mediated FA and 13 age-matched healthy controls. In addition to enumeration of PB iNKT cells, iNKT cells were expanded for 10 days in culture with stimuli of a-galactosylceramide (aGal), milk sphingomyelin, or egg ceramide. Expanded iNKT cells were enumerated and assessed for expression of activation marker (CD25) and intracellular cytokines. RESULTS: FA patients revealed lower numbers of iNKT cells than controls (p<0.05), but iNKT cell from FA children responded more vigorously in response to milk sphingomyelin and egg ceramide in culture (p<0.05). Moreover, FA iNKT cells revealed higher expression of CD25, IL-4, and IL-13 than controls (p<0.05). However, IFN-g expression did not differ between FA and Non-FA children. CONCLUSION: Our results indicated higher proliferative and Th2 skewed functional activation of FA iNKT cells in response to food allergen derived sphingolipids as compared to controls despite paucity of their numbers in PB. This, indicates a potential role of iNKT cells in FA by causing Th2 deviation of T cell responses.
876
Epitope Mapping of the Main Four Shrimp Allergens and Comparison of IgE Recognition Between Children and Adults R. Ayuso1, S. Sanchez-Garcia2, J. Lin1, M. Iba´n˜ez3, C. Blanco4, T. Carrillo5, M. Goldis1, L. Bardina1, H. A. Sampson1; 1Mount Sinai Medical Center, New York, NY, 2Fundacion Jimenez-Diaz, Madrid, SPAIN, 3Hospital Universitario Nin˜o Jesus, Madrid, SPAIN, 4Hospital Universitario La Princesa, Madrid, SPAIN, 5Hospital Universitario Dr Negrin, Las Palmas de Gran Canaria, SPAIN. RATIONALE: Shellfish allergy is a long-lasting disorder typically persisting throughout life. We aimed to characterize the IgE-binding profiles of four shrimp allergens recognized by children and adults. METHODS: Sera from 34 children (P) and 19 adults (A), with immediate allergic reactions and elevated serum IgE to shrimp were tested by microarray analysis for IgE binding to overlapping synthetic peptides spanning the sequences of the shrimp allergens tropomyosin, myosin light chain (MLC), sarcoplasmic calcium-binding protein (SCP) and arginine kinase (AK). RESULTS: Median shrimp-specific IgE was 4 fold higher in children (47 versus 12.5 kUA/L). Frequency of allergen recognition was: tropomyosin 81% (94%P, 61%A), MLC 57% (70%P, 31%A), AK 51% (67%P, 21%A) and SCP 45% (59%P, 21%A). Identified epitopes in tropomyosin are: Epitope 1 (amino acids 1-39), Epitope 2 (40-63), Epitope 3 (61-81), Epitope 4 (82-105), Epitope 5a (115153), 5b (142-165), 5c (156-186), Epitope 6 (190-210), Epitope 7 (246284). In MLC: Epitope 1 (13-30), Epitope 2 (22-48), Epitope 3 (46-66), Epitope 4a (58-90), Epitope 4b (79-99), and Epitope 5 (118-141). In SCP: Epitope 1 (10-33), Epitope 2 (49-72) and Epitope 3 (130-147). And, in AK: Epitope 1 (1-18), Epitope 2a (61-87), 2b (79-96), Epitope 3 (121-141), Epitope 4 (160-192), Epitope 5 (232-249), Epitope 6 (319-342). CONCLUSIONS: Shrimp-allergic children have higher IgE levels, show more intense recognition of shrimp allergens and epitope diversity than adults, who almost only recognize tropomyosin. Our work is the first to show that sensitization to shrimp proteins is greater in children and that appears to decrease in adulthood.
877
TUESDAY
Differences In IgE Binding To Raw, Roasted And Denatured Ara h 1, A Major Peanut Allergen J. B. Nesbit, S. J. Maleki; US Dept. of Agriculture, New Orleans, LA. RATIONALE: Ara h 1 is a major peanut allergen known to bind higher IgE following the roasting process. Here, we will examine the contribution of the structural and chemical modifications to alterations in IgE binding properties of Ara h 1. METHODS: Ara h 1 purified from raw and roasted peanuts and, heat denatured raw or dithiotheratol treated Ara h 1 were subjected IgE binding with sera from peanut allergic and sensitized individuals using spot blot analysis. Circular dichroism (CD) spectroscopy was used to monitor or confirm changes in the secondary structure between raw, roasted and denatured Ara h 1. RESULTS: The structures of Ara h 1 purified from raw and roasted are very similar with an alpha plus beta secondary structure, but differ significantly in IgE binding. Treatment of raw Ara h 1 with heat resulted in significant loss of alpha helices, while treatment with the reducing agent while notable, was not as significant. The structure of the heat denatured Ara h 1 shifts strongly toward a random coil/undordered structure. Majority of IgE in allergic and non allergic patientsÕ sera bound from highest to lowest to roasted, raw and denatured Ara h 1 (roasted > raw> denatured), respectively. CONCLUSION: In raw Ara h 1, the structural epitopes, particularly the ones located in the alpha helical sections, are more important to IgE binding than the liner epitopes. Meanwhile, enhanced IgE binding to roasted Ara h 1 is more likely to be due to chemical modifications than structural changes.
J ALLERGY CLIN IMMUNOL FEBRUARY 2010
878
Effects of Enzymatic Hydrolysis on Peanut Allergenicity B. Cabanillas1, M. M. Pedrosa2, C. Cuadrado2, C. Burbano2, M. Muzquiz2, J. Rodrı´guez1, J. F. Crespo1; 1Hospital Universitario 12 de Octubre, Madrid, SPAIN, 2Instituto Nacional de Investigaciones Agrarias, Madrid, SPAIN. RATIONALE: Enzymatic hydrolysis has been used to produce hypoallergenic dietary products derived from different protein sources. We sought to assess the effect of this procedure on the IgE-binding capacity of roasted peanut extracts. METHODS: Roasted peanut protein extract was hydrolyzed by individual and sequential action of an endoprotease (Alcalase) and an exoprotease (Flavourzyme). IgE binding was assessed by means of SDS-PAGE immunoblots and ELISA, using a pooled serum from 5 patients with clinical allergy to peanut confirmed on the basis of a positive doubled-blind, placebocontrolled, food challenge with peanut. RESULTS: Individual protein hydrolysis with Flavourzyme or Alcalase took place rapidly in the first 30 minutes; further, progression developed more slowly, reaching 24% and 17% degree of hydrolysis (DH) after 3h of processing with Flavourzyme or Alcalase respectively. The sequential treatment with Alcalase and Flavourzyme produced a 69% DH at the end of the hydrolytic process. SDS-PAGE immunoblots showed that roasted peanut extract appeared to be much more susceptible to the individual action of Alcalase and sequential action of Alcalase and Flavourzyme when compared with the native protein, indicating a reduction in its overall allergenicity. In contrast, roasted peanut extract showed little changes on the IgE-binding proteins after individual hydrolysis with Flavourzyme. In addition, roasted peanut extract hydrolyzed with Alcalase bound the lowest levels of IgE as determined by ELISA. CONCLUSIONS: Individual hydrolysis with Alcalase may result in a decrease of IgE immunoreactivity of roasted peanut allergens.
879
Inkt Cell Proliferation To Food-derived Sphingolipids In Food Allergic Vs Non-food Allergic Children S. Jyonouchi; Children’s Hospital of Philadelphia, Philadelphia, PA. RATIONALE: Invariant Natural Killer T (iNKT) cells are a subset of Tcells that modulate immune response through rapid release of Th1 and Th2 cytokines in response to sphingolipids. Thus, iNKT cells have the ability to affect disease processes by skewing T-cell cytokine profile. Our objective was to investigate the potential role of iNKT cells in food allergy (FA) by assessing frequency of peripheral blood (PB) frequency of iNKT cells and their responses to food derived sphingolipids in FA children and healthy controls. METHODS: This study includes 17 children with IgE mediated FA and 13 age-matched healthy controls. In addition to enumeration of PB iNKT cells, iNKT cells were expanded for 10 days in culture with stimuli of a-galactosylceramide (aGal), milk sphingomyelin, or egg ceramide. Expanded iNKT cells were enumerated and assessed for expression of activation marker (CD25) and intracellular cytokines. RESULTS: FA patients revealed lower numbers of iNKT cells than controls (p<0.05), but iNKT cell from FA children responded more vigorously in response to milk sphingomyelin and egg ceramide in culture (p<0.05). Moreover, FA iNKT cells revealed higher expression of CD25, IL-4, and IL-13 than controls (p<0.05). However, IFN-g expression did not differ between FA and Non-FA children. CONCLUSION: Our results indicated higher proliferative and Th2 skewed functional activation of FA iNKT cells in response to food allergen derived sphingolipids as compared to controls despite paucity of their numbers in PB. This, indicates a potential role of iNKT cells in FA by causing Th2 deviation of T cell responses.