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Cross Reactivity in Mountain Cedar-Tomato Oral Allergy Syndrome
Abstracts S113
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1
ELISA and HPLC Comparison of Pediatric Amino Acid-based Nutritionals G. O. Duska-McEwen; Abbott Laboratories, Columbus, OH. RATIONALE: Amino acid-based nutritional products are often assumed to be free of intact protein based upon the product ingredient label. To test this assumption, we performed a comparative evaluation of the 6 pediatric amino acid-based nutritionals currently marketed in the US for the presence of casein and whey antigens and intact casein and whey protein. METHODS: Competitive inhibition ELISA methods were used to assess the immunologically active casein (IAC) and whey (IAW) antigen content of the nutritionals. Reverse phase HPLC was used to further characterize the protein components of the products. RESULTS: Pediatric E028Ò, which does not make a hypoallergenic claim, demonstrated consistently higher IAW content in all 4 lots tested. HPLC analysis of the same four lots detected the intact whey protein alpha-lactalbumin in each case, and at levels (ppm) consistent with the (independently determined) ELISA results. CONCLUSIONS: It is not valid or safe to assume that all amino acidbased nutritional products are protein-free based only upon the ingredient label. Only amino acid-based products with a documented hypoallergenic claim should be utilized when a protein-free nutritional is required. Funding: Abbott Laboratories
445
Peanut Allergic Child Increases Risk for Peanut Allergy in Siblings S. Huq1,2, J. Liem1,2, A. Kozyrskyj1,2, A. Becker1,2; 1University of Manitoba, Winnipeg, MB, CANADA, 2Manitoba Institute of Child Health, Winnipeg, MB, CANADA. RATIONALE: Parents are concerned about development of peanut allergy in siblings of peanut allergic children. We determined the risk of peanut allergy in these siblings. METHODS: Families of a cohort of children born in 1995 in Manitoba, Canada were given a questionnaire. Parents were asked whether their children (including the index child) had any food allergies. Skin prick tests 6 RASTs were performed for the index child. The likelihood of peanut allergy in siblings was then determined based on parental report of allergist assessment and dispensed EpiPen. RESULTS: We saw 514 index children. 32 (6.6%) children were peanut allergic (Skin prick test/RAST/oral challenge). Eight children were sensitized but not allergic. 26 children did not have siblings. There were 50 and 828 siblings of peanut allergic and non-peanut allergic children respectively. There were 15 siblings with peanut allergy (1.7% of total siblings). There were 4 (8%) and 11 (1.3%) with an index child being peanut allergic and not peanut allergic. The risk was increased for siblings of peanut allergic compared to sibling of non-peanut allergic. (OR56.46, 95%CI 1.9821.07). The OR for peanut allergy in the index child if older sibling was peanut allergic was 9.22 (95%CI 1.62-52.59). The risk of a younger sibling to the index child being peanut allergic was 4.80 (95%CI 0.93-24.69). CONCLUSIONS: Children are more likely to be allergic to peanut if they have a peanut allergic sibling. Allergists should consider testing of younger siblings before anticipated peanut ingestion. Funding: Manitoba Institute of Child Health
446
Study and Development of Food Allergy Herbal Formula (FAHF-2) Y. Chen, K. Srivastava, H. Sampson, X. Li; Mt. Sinai School of Medicine, New York, NY. RATIONALE: Food Allergy Herbal Formula (FAHF-2) can completely block peanut-induced anaphylaxis in a murine model. Comparable administration in man would require a large daily dose (24 g) of a very bitter tasting formulation, making it difficult for patients to ingest. Refining the current formulation of FAHF-2 is critical for patient acceptability. The purpose of this study was to investigate different extraction methods to reduce the required FAHF-2 dose while maintaining anti-anaphylaxis efficacy. METHODS: Butanol and 95% ethanol were used to extract FAHF-2. HPLC-PDA (high performance liquid chromatography-photo diode array) chromatography was used for quality control of extracts. Butanol and 95% ethanol extracts were then tested in a murine model of peanut allergy. RESULTS: Butanol extract greatly reduced the human daily dose equivalent to 3.14 g, and 95% ethanol reduced the daily dose to 8.47 g. HPLCPDA chromatography of the solvent extracts showed that they contained most of the peaks in the original water extracted FAHF-2 and had similar peak heights. Butanol extract contained fewer polar ingredients. Butanol and 95% ethanol extracts were tested for anti-peanut allergy efficacy. Both provided full protection against anaphylactic symptoms in peanutallergic mice. CONCLUSIONS: (1) Butanol extract of FAHF-2 maintained the full effectiveness of FAHF-2 while reducing the FAHF-2 daily dose to a level convenient for clinical use. (2) 95% ethanol extract was also effective, and although the required dose was higher than the butanol extract, it is still acceptable. 95% ethanol is much less expensive and more readily used in the botanical extract industry than butanol. Funding: NIH
447
Cross Reactivity in Mountain Cedar-Tomato Oral Allergy Syndrome R. S. Bonds, R. Tiwari, B. Ning, E. W. Czerwinski, R. G. Goldblum, T. Midoro-Horiuti; University of Texas Medical Branch, Galveston, TX. RATIONALE: Oral allergy syndrome (OAS) is recognized in up to 50% of pollen-allergic adults. However, the mechanisms of cross-recognition are not well established. We describe here a mountain cedar-tomato OAS and propose a molecular basis for cross-reactivity. METHODS: Eleven patients with tomato OAS and cedar pollen hypersensitivity were identified by history and skin testing. A role for Jun a 1, the dominant allergen of mountain cedar, was evaluated by ImmunoCap inhibition. A panel of monoclonal antibodies (mAbs) to Jun a 1 was screened for reactivity with tomato crude extract (CE) by ELISA . Strongly reacting mAbs were tested by Western blotting and immunoprecipitation, followed by protein idenfification by mass spectrometry. RESULTS: Jun a 1 inhibited 38% of patient IgE binding to tomato ImmunoCaps. MAbs that reacted with partially heat sensitive (conformational) and heat resistant (linear) epitopes of Jun a 1 cross-reacted with tomato CE. Western blot analysis suggested that mAbs bound to several tomato proteins, including some known allergens. Immunoprecipitation and mass spectrometry indicated that the most striking interaction of several mAbs was with tomato pectin methylesterase. Computer overlays of the crystal structures of Jun a 1 and tomato pectin methylesterase demonstrated extensive structural homology, despite a very low degree of sequence identity. CONCLUSIONS: Our findings suggest extensive cross-reactivity between Jun a 1, the major allergen of mountain cedar, and tomato allergens, in particular pectin methylesterase. Careful analysis of the epitopes recognized by IgE in patients with cedar-tomato OAS may allow us to define patterns that predict local and systemic reactions to tomato. Funding: NIAID RO1, NIAID KO8, FDA
SUNDAY
444
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1
ELISA and HPLC Comparison of Pediatric Amino Acid-based Nutritionals G. O. Duska-McEwen; Abbott Laboratories, Columbus, OH. RATIONALE: Amino acid-based nutritional products are often assumed to be free of intact protein based upon the product ingredient label. To test this assumption, we performed a comparative evaluation of the 6 pediatric amino acid-based nutritionals currently marketed in the US for the presence of casein and whey antigens and intact casein and whey protein. METHODS: Competitive inhibition ELISA methods were used to assess the immunologically active casein (IAC) and whey (IAW) antigen content of the nutritionals. Reverse phase HPLC was used to further characterize the protein components of the products. RESULTS: Pediatric E028Ò, which does not make a hypoallergenic claim, demonstrated consistently higher IAW content in all 4 lots tested. HPLC analysis of the same four lots detected the intact whey protein alpha-lactalbumin in each case, and at levels (ppm) consistent with the (independently determined) ELISA results. CONCLUSIONS: It is not valid or safe to assume that all amino acidbased nutritional products are protein-free based only upon the ingredient label. Only amino acid-based products with a documented hypoallergenic claim should be utilized when a protein-free nutritional is required. Funding: Abbott Laboratories
445
Peanut Allergic Child Increases Risk for Peanut Allergy in Siblings S. Huq1,2, J. Liem1,2, A. Kozyrskyj1,2, A. Becker1,2; 1University of Manitoba, Winnipeg, MB, CANADA, 2Manitoba Institute of Child Health, Winnipeg, MB, CANADA. RATIONALE: Parents are concerned about development of peanut allergy in siblings of peanut allergic children. We determined the risk of peanut allergy in these siblings. METHODS: Families of a cohort of children born in 1995 in Manitoba, Canada were given a questionnaire. Parents were asked whether their children (including the index child) had any food allergies. Skin prick tests 6 RASTs were performed for the index child. The likelihood of peanut allergy in siblings was then determined based on parental report of allergist assessment and dispensed EpiPen. RESULTS: We saw 514 index children. 32 (6.6%) children were peanut allergic (Skin prick test/RAST/oral challenge). Eight children were sensitized but not allergic. 26 children did not have siblings. There were 50 and 828 siblings of peanut allergic and non-peanut allergic children respectively. There were 15 siblings with peanut allergy (1.7% of total siblings). There were 4 (8%) and 11 (1.3%) with an index child being peanut allergic and not peanut allergic. The risk was increased for siblings of peanut allergic compared to sibling of non-peanut allergic. (OR56.46, 95%CI 1.9821.07). The OR for peanut allergy in the index child if older sibling was peanut allergic was 9.22 (95%CI 1.62-52.59). The risk of a younger sibling to the index child being peanut allergic was 4.80 (95%CI 0.93-24.69). CONCLUSIONS: Children are more likely to be allergic to peanut if they have a peanut allergic sibling. Allergists should consider testing of younger siblings before anticipated peanut ingestion. Funding: Manitoba Institute of Child Health
446
Study and Development of Food Allergy Herbal Formula (FAHF-2) Y. Chen, K. Srivastava, H. Sampson, X. Li; Mt. Sinai School of Medicine, New York, NY. RATIONALE: Food Allergy Herbal Formula (FAHF-2) can completely block peanut-induced anaphylaxis in a murine model. Comparable administration in man would require a large daily dose (24 g) of a very bitter tasting formulation, making it difficult for patients to ingest. Refining the current formulation of FAHF-2 is critical for patient acceptability. The purpose of this study was to investigate different extraction methods to reduce the required FAHF-2 dose while maintaining anti-anaphylaxis efficacy. METHODS: Butanol and 95% ethanol were used to extract FAHF-2. HPLC-PDA (high performance liquid chromatography-photo diode array) chromatography was used for quality control of extracts. Butanol and 95% ethanol extracts were then tested in a murine model of peanut allergy. RESULTS: Butanol extract greatly reduced the human daily dose equivalent to 3.14 g, and 95% ethanol reduced the daily dose to 8.47 g. HPLCPDA chromatography of the solvent extracts showed that they contained most of the peaks in the original water extracted FAHF-2 and had similar peak heights. Butanol extract contained fewer polar ingredients. Butanol and 95% ethanol extracts were tested for anti-peanut allergy efficacy. Both provided full protection against anaphylactic symptoms in peanutallergic mice. CONCLUSIONS: (1) Butanol extract of FAHF-2 maintained the full effectiveness of FAHF-2 while reducing the FAHF-2 daily dose to a level convenient for clinical use. (2) 95% ethanol extract was also effective, and although the required dose was higher than the butanol extract, it is still acceptable. 95% ethanol is much less expensive and more readily used in the botanical extract industry than butanol. Funding: NIH
447
Cross Reactivity in Mountain Cedar-Tomato Oral Allergy Syndrome R. S. Bonds, R. Tiwari, B. Ning, E. W. Czerwinski, R. G. Goldblum, T. Midoro-Horiuti; University of Texas Medical Branch, Galveston, TX. RATIONALE: Oral allergy syndrome (OAS) is recognized in up to 50% of pollen-allergic adults. However, the mechanisms of cross-recognition are not well established. We describe here a mountain cedar-tomato OAS and propose a molecular basis for cross-reactivity. METHODS: Eleven patients with tomato OAS and cedar pollen hypersensitivity were identified by history and skin testing. A role for Jun a 1, the dominant allergen of mountain cedar, was evaluated by ImmunoCap inhibition. A panel of monoclonal antibodies (mAbs) to Jun a 1 was screened for reactivity with tomato crude extract (CE) by ELISA . Strongly reacting mAbs were tested by Western blotting and immunoprecipitation, followed by protein idenfification by mass spectrometry. RESULTS: Jun a 1 inhibited 38% of patient IgE binding to tomato ImmunoCaps. MAbs that reacted with partially heat sensitive (conformational) and heat resistant (linear) epitopes of Jun a 1 cross-reacted with tomato CE. Western blot analysis suggested that mAbs bound to several tomato proteins, including some known allergens. Immunoprecipitation and mass spectrometry indicated that the most striking interaction of several mAbs was with tomato pectin methylesterase. Computer overlays of the crystal structures of Jun a 1 and tomato pectin methylesterase demonstrated extensive structural homology, despite a very low degree of sequence identity. CONCLUSIONS: Our findings suggest extensive cross-reactivity between Jun a 1, the major allergen of mountain cedar, and tomato allergens, in particular pectin methylesterase. Careful analysis of the epitopes recognized by IgE in patients with cedar-tomato OAS may allow us to define patterns that predict local and systemic reactions to tomato. Funding: NIAID RO1, NIAID KO8, FDA
SUNDAY
444