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Oral Immunotherapy In patients With Peanut Allergy And High Risk Of Anaphylactic Reactions
Abstracts S179
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Identification and Cloning of a Sarcoplasmic Calcium-binding Protein, a new Shrimp Allergen R. Ayuso1, G. Grishina1, M. D. Iba´n˜ez2, C. Blanco3, T. Carrillo4, H. A. Sampson1; 1Mount Sinai Medical Center, New York, NY, 2Hospital Nin˜o Jesus, Madrid, Spain, 3Hospital Universitario de la Princesa, Madrid, Spain, 4Hospital Universitario Dr. Negrı´n, Las Palmas de Gran Canaria, Spain. RATIONALE: Shellfish allergy is a long-lasting disorder usually persisting throughout life. Despite its high prevalence, there is limited knowledge about allergenic shrimp proteins. Recently a sarcoplasmic calcium-binding protein (SCP) was described as a shrimp allergen, but its allergenicity needs confirmation by recombinant methods. METHODS: Forty adult and pediatric patients were selected with immediate allergic reactions to shrimp and elevated shrimp-specific serum IgE. Shrimp proteins were separated by SDS-PAGE, followed by IgE-immunoblotting. Protein identification was done by MS/MS from in gel tryptic digests. cDNA encoding SCP was isolated from a shrimp cDNA library (Litopenaeus vannamei) by PCR with primers designed based on a published shrimp SCP sequence. Full length cDNA clones were isolated from the library, cloned in TOPO vector and sequenced. cDNA was then cloned in an expression vector. Cell lysates containing expressed recombinant SCP were tested with sera from shrimp-allergic subjects that recognized the 20 kDa band. RESULTS: Immunoblotting demonstrated IgE-binding to a 20 kDa shrimp protein by 24/40 sera (60%). Tryptic digestion of the protein followed by MS/MS analysis identified it as a sarcoplasmic calcium-binding protein. Amplified cDNA encoding for SCP was isolated and sequenced. Open reading frame translation provided the complete amino acid sequence of a new allergenic shrimp protein. Recombinant protein present in bacterial cell lysates was recognized by 13/19 subjects tested, but not by controls, confirming the allergenicity of shrimp SCP. CONCLUSIONS: We have identified and cloned a new shrimp allergen, a sarcoplasmic-calcium-binding protein (SCP) for which we suggest the name Lit v 4.
684
Oral Immunotherapy In patients With Peanut Allergy And High Risk Of Anaphylactic Reactions K. Blumchen, H. Ulbricht, U. Staden, K. Dobberstein, B. Niggemann, U. Wahn, K. Beyer; Dept of paediatric immunology/pneumology, Charite, Berlin, Germany. RATIONALE: As previously reported, rush oral immunotherapy seems not effective in reaching protective doses in peanut allergic children. However, conventional protocols over a prolonged period of time may be a more promising therapeutic strategy. METHODS: 22 patients finished rush-oral immunotherapy and went on a conventional protocol with further dose increments. We now report on five patients of this group with a median peanut-specific IgE >100 kU/l who finished the study protocol. Individual threshold levels and immunological parameters were determined at the beginning and end of the study. RESULTS: On first double-blind, placebo-controlled food challenge (DBPCFC) patients tolerated 0.125-1.0 g of peanut (median: 0.25 g). For a median of 88 days (range: 55-331 days) they received daily peanut doses with increases every other week up to 0.5-2.0 g of peanut (median: 1.0 g). In 52 of 1076 total daily doses mild to moderate side effects were observed. Patients had to be stable on this final dose for eight weeks followed by two weeks of a peanut-free diet and a final DBPCFC where the tolerated dose was raised to 0.25-2.0 g (median: 1.0 g). In 3/5 cases the symptoms observed at first and final DBPCFC involved different organ systems. No marked changes in peanut-specific IgE levels have been observed whereas peanut-specific IgG and IgG4 increased in all patients. IL-5 in supernatants of peanut-stimulated PBMCs decreased. CONCLUSIONS: Conventional oral immunotherapy appears to be safe and successful in reaching clinical relevant protective doses for peanut-allergic patients. Increase in threshold levels and changes of immunological parameters were observed.
685
Prognostic Factors Associated to Persistent IgE-mediated Cow's Milk Allergy (CMA) ˆ . B.F. Fomin, L. A. Watanabe, V. Z. C. M. A. Jacob, A. C. Pastorino, A Fucci, A. K. F. Gushken, A. Branda˜o, U. Doria, C. A. Silva, A. B. M. Castro; DEPTO. OF PEDIATRICS, Sa˜o Paulo, Brazil. RATIONALE: Cow’s milk allergy (CMA) is the most common food allergy in infants affecting 2.5% of the general population. Most patients developing tolerance by age of 3, however, the prognostic factors for tolerance are not well defined. The aim of this study is to determine the prognostic factors at diagnosis associated to the persistent CMA at age of 5. METHODS: 89 patients (5 years) with IgE mediated CMA were evaluated and the following criteria adopted for this diagnosis were: anaphylaxis triggered by cow’s milk (CM) or immediate clinical reaction to DBPC test. Tolerance was defined as the absence of response to the DBPC test or during the accidental exposure to CM. RESULTS: At the age of five, 40 patients became tolerant and 49 persisted with CMA, without difference regarding gender (p 5 0,321), age of the onset symptoms (p 5 0, 5470) and the age at the first CM contact (p 5 0,052). In the persistent group, anaphylaxis (p 5 0,001), sensitization to other foods (p 5 0,018), the serum concentrations of total IgE and specific IgE for milk and its fractions were significantly more frequent. It was possible to establish, using the laboratorial data at diagnosis, the cut-off points of specific IgE for CM (4.88 kU/L, sensitivity and specificity of 89.7%) and casein (3.5kU/L, sensitivity of 79.4% and specificity of 89.4%) associated to persistent CMA. CONCLUSIONS: In this study was possible to determine clinical and laboratorial data associated to the persistent CMA that could be useful to improve the approach to CMA patients.
686
Over-reliance on Serum Testing for IgE to Food Allergens Results in Inappropriate Food Elimination Diets G. C. Spears1, N. K. Miyazawa1, M. C. Gleason1, E. A. Gyorkos1, C. G. Wilson1, J. R. Murphy1, J. M. Um1, D. Atkins1, A. Bock2, D. Y. Leung1, D. M. Fleischer1; 1National Jewish Health, Denver, CO, 2Boulder Valley Asthma and Allergy Clinic, Boulder, CO. RATIONALE: Recently, we have observed that many children, especially those with eczema, are placed on food elimination diets based on positive radioallergosorbent test (RAST)/ImmunoCap results. To evaluate this observation, we reviewed data from the past two years of children evaluated for food allergy at National Jewish Health (NJH) and a local allergy practice that underwent oral food challenges (OFCs) to the foods eliminated based on these tests. METHODS: A retrospective chart review of 115 children aged 1-19 years (median 3 years) undergoing OFCs between 2007-2008 was performed. RAST/ImmunoCap and OFC results to 5 of the major allergenic foods (egg, milk, peanut, soy, wheat) were collected. RESULTS: Of the 142 OFCs performed for foods eliminated based on positive RAST/ImmunoCAP, 109 were negative (77%). Comparing results of the initial RAST/ImmunoCap tests and the NJH ImmunoCap tests obtained at the time of OFC (Paired T-Test) showed no significant difference. Ninety-three subjects (81%) had eczema (52 [56%] with >50% body involvement), and 59 subjects had total IgE levels: range 3-66,520 IU/L, median 1,147). CONCLUSIONS: Only 23% of the OFCs in this cohort were positive. Our data indicate that, in the absence of anaphylaxis, OFCs should be performed to confirm food allergy status as most of these children were placed on restricted diets unnecessarily. Given the commercial availability of RAST/ImmunoCAP testing, referral to an allergy center capable of serum testing review and allergist-supervised OFCs is warranted in patients with a history of adverse food reactions and positive serum testing before they are subjected to long-term elimination diets.
MONDAY
683
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Identification and Cloning of a Sarcoplasmic Calcium-binding Protein, a new Shrimp Allergen R. Ayuso1, G. Grishina1, M. D. Iba´n˜ez2, C. Blanco3, T. Carrillo4, H. A. Sampson1; 1Mount Sinai Medical Center, New York, NY, 2Hospital Nin˜o Jesus, Madrid, Spain, 3Hospital Universitario de la Princesa, Madrid, Spain, 4Hospital Universitario Dr. Negrı´n, Las Palmas de Gran Canaria, Spain. RATIONALE: Shellfish allergy is a long-lasting disorder usually persisting throughout life. Despite its high prevalence, there is limited knowledge about allergenic shrimp proteins. Recently a sarcoplasmic calcium-binding protein (SCP) was described as a shrimp allergen, but its allergenicity needs confirmation by recombinant methods. METHODS: Forty adult and pediatric patients were selected with immediate allergic reactions to shrimp and elevated shrimp-specific serum IgE. Shrimp proteins were separated by SDS-PAGE, followed by IgE-immunoblotting. Protein identification was done by MS/MS from in gel tryptic digests. cDNA encoding SCP was isolated from a shrimp cDNA library (Litopenaeus vannamei) by PCR with primers designed based on a published shrimp SCP sequence. Full length cDNA clones were isolated from the library, cloned in TOPO vector and sequenced. cDNA was then cloned in an expression vector. Cell lysates containing expressed recombinant SCP were tested with sera from shrimp-allergic subjects that recognized the 20 kDa band. RESULTS: Immunoblotting demonstrated IgE-binding to a 20 kDa shrimp protein by 24/40 sera (60%). Tryptic digestion of the protein followed by MS/MS analysis identified it as a sarcoplasmic calcium-binding protein. Amplified cDNA encoding for SCP was isolated and sequenced. Open reading frame translation provided the complete amino acid sequence of a new allergenic shrimp protein. Recombinant protein present in bacterial cell lysates was recognized by 13/19 subjects tested, but not by controls, confirming the allergenicity of shrimp SCP. CONCLUSIONS: We have identified and cloned a new shrimp allergen, a sarcoplasmic-calcium-binding protein (SCP) for which we suggest the name Lit v 4.
684
Oral Immunotherapy In patients With Peanut Allergy And High Risk Of Anaphylactic Reactions K. Blumchen, H. Ulbricht, U. Staden, K. Dobberstein, B. Niggemann, U. Wahn, K. Beyer; Dept of paediatric immunology/pneumology, Charite, Berlin, Germany. RATIONALE: As previously reported, rush oral immunotherapy seems not effective in reaching protective doses in peanut allergic children. However, conventional protocols over a prolonged period of time may be a more promising therapeutic strategy. METHODS: 22 patients finished rush-oral immunotherapy and went on a conventional protocol with further dose increments. We now report on five patients of this group with a median peanut-specific IgE >100 kU/l who finished the study protocol. Individual threshold levels and immunological parameters were determined at the beginning and end of the study. RESULTS: On first double-blind, placebo-controlled food challenge (DBPCFC) patients tolerated 0.125-1.0 g of peanut (median: 0.25 g). For a median of 88 days (range: 55-331 days) they received daily peanut doses with increases every other week up to 0.5-2.0 g of peanut (median: 1.0 g). In 52 of 1076 total daily doses mild to moderate side effects were observed. Patients had to be stable on this final dose for eight weeks followed by two weeks of a peanut-free diet and a final DBPCFC where the tolerated dose was raised to 0.25-2.0 g (median: 1.0 g). In 3/5 cases the symptoms observed at first and final DBPCFC involved different organ systems. No marked changes in peanut-specific IgE levels have been observed whereas peanut-specific IgG and IgG4 increased in all patients. IL-5 in supernatants of peanut-stimulated PBMCs decreased. CONCLUSIONS: Conventional oral immunotherapy appears to be safe and successful in reaching clinical relevant protective doses for peanut-allergic patients. Increase in threshold levels and changes of immunological parameters were observed.
685
Prognostic Factors Associated to Persistent IgE-mediated Cow's Milk Allergy (CMA) ˆ . B.F. Fomin, L. A. Watanabe, V. Z. C. M. A. Jacob, A. C. Pastorino, A Fucci, A. K. F. Gushken, A. Branda˜o, U. Doria, C. A. Silva, A. B. M. Castro; DEPTO. OF PEDIATRICS, Sa˜o Paulo, Brazil. RATIONALE: Cow’s milk allergy (CMA) is the most common food allergy in infants affecting 2.5% of the general population. Most patients developing tolerance by age of 3, however, the prognostic factors for tolerance are not well defined. The aim of this study is to determine the prognostic factors at diagnosis associated to the persistent CMA at age of 5. METHODS: 89 patients (5 years) with IgE mediated CMA were evaluated and the following criteria adopted for this diagnosis were: anaphylaxis triggered by cow’s milk (CM) or immediate clinical reaction to DBPC test. Tolerance was defined as the absence of response to the DBPC test or during the accidental exposure to CM. RESULTS: At the age of five, 40 patients became tolerant and 49 persisted with CMA, without difference regarding gender (p 5 0,321), age of the onset symptoms (p 5 0, 5470) and the age at the first CM contact (p 5 0,052). In the persistent group, anaphylaxis (p 5 0,001), sensitization to other foods (p 5 0,018), the serum concentrations of total IgE and specific IgE for milk and its fractions were significantly more frequent. It was possible to establish, using the laboratorial data at diagnosis, the cut-off points of specific IgE for CM (4.88 kU/L, sensitivity and specificity of 89.7%) and casein (3.5kU/L, sensitivity of 79.4% and specificity of 89.4%) associated to persistent CMA. CONCLUSIONS: In this study was possible to determine clinical and laboratorial data associated to the persistent CMA that could be useful to improve the approach to CMA patients.
686
Over-reliance on Serum Testing for IgE to Food Allergens Results in Inappropriate Food Elimination Diets G. C. Spears1, N. K. Miyazawa1, M. C. Gleason1, E. A. Gyorkos1, C. G. Wilson1, J. R. Murphy1, J. M. Um1, D. Atkins1, A. Bock2, D. Y. Leung1, D. M. Fleischer1; 1National Jewish Health, Denver, CO, 2Boulder Valley Asthma and Allergy Clinic, Boulder, CO. RATIONALE: Recently, we have observed that many children, especially those with eczema, are placed on food elimination diets based on positive radioallergosorbent test (RAST)/ImmunoCap results. To evaluate this observation, we reviewed data from the past two years of children evaluated for food allergy at National Jewish Health (NJH) and a local allergy practice that underwent oral food challenges (OFCs) to the foods eliminated based on these tests. METHODS: A retrospective chart review of 115 children aged 1-19 years (median 3 years) undergoing OFCs between 2007-2008 was performed. RAST/ImmunoCap and OFC results to 5 of the major allergenic foods (egg, milk, peanut, soy, wheat) were collected. RESULTS: Of the 142 OFCs performed for foods eliminated based on positive RAST/ImmunoCAP, 109 were negative (77%). Comparing results of the initial RAST/ImmunoCap tests and the NJH ImmunoCap tests obtained at the time of OFC (Paired T-Test) showed no significant difference. Ninety-three subjects (81%) had eczema (52 [56%] with >50% body involvement), and 59 subjects had total IgE levels: range 3-66,520 IU/L, median 1,147). CONCLUSIONS: Only 23% of the OFCs in this cohort were positive. Our data indicate that, in the absence of anaphylaxis, OFCs should be performed to confirm food allergy status as most of these children were placed on restricted diets unnecessarily. Given the commercial availability of RAST/ImmunoCAP testing, referral to an allergy center capable of serum testing review and allergist-supervised OFCs is warranted in patients with a history of adverse food reactions and positive serum testing before they are subjected to long-term elimination diets.
MONDAY
683