- Email: [email protected]
The impact of oral food challenges on food-specific IgE antibody concentrations
S350
Abstracts
J ALLERGY CLIN IMMUNOL FEBRUARY 2003
1128 Cyc,osporinfor Chronic Idiopathic Urticaria in Children
1130 tinctFamUial D dCold loCi Autoinflammatory SUrticaria - f r Syndrome: o m AnytiE tn
D. R. Doshi l, M. M, Weinberger2; IUniversity of Iowa, Iowa City, IA, 2Pediatrics, University of Iowa, Iowa City, IA. RATIONALE: Chronic idiopathic urticaria (CIU) has been associated with functional autoantibodies to the high affinity IgE receptor. A controlled clinical trial with cyclosporin in adults (Greaves, e t a | , Br J Dermatol 2000) demonstrated a high degree of efficacy, safety, and induction of remission in adults. Out of a population of 33 children with CIU seen in our clinic during the last 2 years, 5 (ages 13-15) failed control with high doses of HI antihistamine therapy even with the addition of alternate morning prednisone. These patients had daily hives ranging 8-38 months prior to initiation of immunosuppressive therapy. METHODS: Low dose cyclosporin (Neoral brand), beginning with 3 mg/kg/day divided b.i.d, was used. Antihistamine therapy was continued during treatment. Cyclosporin blood levels, BUN, and creatinine were carefully monitored during treatment. RESULTS: Complete absence of urticaria was achieved in all five patients. Three patients are currently in remission and off cyclosporin therapy ranging from 2-15 months without symptoms. The remaining two patients are on cyclosporin without hives. Of these two patients, one has had recurrences while attempting to taper off cyclosporin. The second patient restarted cyclosporin following 16 months of remission with prompt control after restarting. CONCLUSIONS: All five patients tolerated cyclosporin with induction of remission or control of hives without clinical adverse effects. Our experience supports the efficacy and safety of cyclosporin for pediatric patients with CIU who fail to respond or do not tolerate traditional therapy with HI antihistamines with or without alternate morning prednisone.
R. L. Shpall 1, E. W. B. Jeffes 2,3, H. M. Hoffman4; ICollege of Medicine, University of California, Irvine, Irvine, CA, 2Division of Dermatology, VA Medical Center-Long Beach, Long Beach, CA, 3Department of Dermatology, University of California at Irvine, Orange, CA, 4Departments of Pediatrics and Medicine, University of California at San Diego, San Diego, CA. RATIONALE: Familial cold autoinflammatory syndrome (FCAS), commonly known as familial cold urticaria, is a rare autosomal dominant disorder characterized by a rash that develops in response to generalized cold exposure. Patients with this disease are often misdiagnosed with acquired cold urticaria (ACU), a mast cell mediated physical urticaria. METHODS/CASE: We describe a 66-year-old patient followed for several years with a diagnosis of cold urticaria and treated with antihistamines without relief. Since birth this patient has developed a pruritic rash 2-3 hours following prolonged generalized cold exposure. Episodes are often accompanied by arthralgia, fever, chills and sweating lasting several hours. Family history is notable for thirteen similarly affected members. Physical examination revealed a healthy male with discrete urticaria-like papules and plaques surrounded by macular erythema diffusely distributed across the patient's body. RESULTS: The ice cube test, performed by direct contact of ice to skin, was negative. Laboratory testing showed an elevation of white blood cell count, erythrocyte sedimentation rate, and C reactive protein. Skin biopsy demonstrated perivascular neutrophils and mononuclear cells without evidence of eosinophils or mast cells. Sequencing of the patient's DNA confirmed the presence of a mutation in CIAS1, the recently identified gene responsible for FCAS. CONCLUSION: FCAS can be differentiated from ACU by history, laboratory evaluation, and now by genetic testing. Appropriate diagnosis of FCAS can prevent ineffective treatments.
Funding: Universi~ of Iowa
1129 Cetirizine from Topical Rigid Liposomal Formulations: Evaluation of Peripheral Antihistaminic Activity and Systemic Absorption in a Rabbit Model K. J. Simons 1, A. A. W. Elzainy 1, X. Gu t, E. R. Simons2; Ipharmacy, University of Manitoba, Winnipeg, MB, CANADA, 2pediatrics, University of Manitoba, Winnipeg, MB, CANADA. RATIONALE: To assess cetirizine peripheral Hrantihistamine activity and extent of systemic absorption from cetirizine liposome formulations applied to the skin in a rabbit model. METHODS: Small unilamellar vesicles (SUV) and multi-lamellar vesicles (MLV) were prepared using L-~-hydrophosphatidylcholine. Glaxal Base (GB) was the control. In a randomized, crossover study, each formulation containing l0 mg of cetirizine was applied to the shaved backs of 6 female New Zealand rabbits (3.08_+0.05 kg). Intradermal tests with 0.05 ml histamine phosphate (l mg/ml), and blood sampling were performed before application, and at 0.5, l, 2, 3, 4, 5, 6, 8, 10, and 24 h. RESULTS: Compared to baseline, histamine-induced wheal formation was suppressed by cetirizine in MLV from 0.5-24 h, in SUV at 24 h and in GB from 0.5-10 h (p<0.05). Wheal suppression by cetirizine in MLV at 1 and 24 h (94_+2 and 76_+6%) and in SUV at 24 h (91_+5%), was greater than in GB (36_+7 to 61 +14%)(p<0.05); cetirizine in MLV at l, 2, 4, 5, and 8 h (92-+5 to 98__.1%) was superior to SUV (24_+6 to 44+12%) (p<0.05). Plasma cetirizine concentrations from SUV (6-+1 to 7_+0.1 ng/mL) were lower than MLV at 3,4 and l0 h (22-+4 to 28+6 ng/mL), and than GB from 0.5 to 3 h (35_+4 to 59-+5 ng/mL) (p<0.05). Cetirizine from MLV (27__.9 ng/mL) were lower than GB after 0.5-1 h. CONCLUSIONS: In this model, cetirizine from MLV has excellent topical Hi-antihistamine activity while systemic exposure was reduced.
Funding: Pfizer USA
Funding: NIH NIAID K08 AlO1508/Ludwig Institute of Cancer Research
1131 The Impact of Oral Food Challenges on Food-Specific IgE Antibody Concentrations E. J. Vukic, M. Mishoe, S. A. Noone, H. A. Sampson, S. H. Sicherer; Pediatrics, Mount Sinai School of Medicine, New York, NY. RATIONALE: It is unknown whether a food-allergic reaction consumes, primes or has no effect on specific lgE antibody concentration. METHODS: We compared food-specific IgE concentrations (Pharmacea CAP-Sytsem FEIA) to the challenge food and control allergen immediately before and approximately 1 hour following a positive double-blind, placebo-controlled food challenge (DBPCFC). In a subset, lgE was retested a year following challenge. RESULTS: Twenty-nine patients (mean age 7 yrs, 72% male, 86% atopic dermatitis) had positive DBPCFCs to: milk (10), peanut (7), egg (7), wheat (3), soy (1), and open challenge to mustard (1). Seventeen patients had an unchallenged food allergen for comparison. Symptom severity was 2.4 (2 organ systems involved) on a 1-5 scale. Pre- and post-challenge IgE concentrations declined a mean of 0.5 kIU/L (p=n.s.) and were highly correlated (r=0.995, p<0.001). Control food IgE declined a mean of 0.8 klU/L (r=0.998, p<0.001 ) which was of statistical (p=0.04), not clinical significance. There was no difference (p=0.84) in change of IgE concentration between challenge and control foods. IgE concentrations were obtained 8-16 months (mean, 12 months) following challenge (10 patients). One patient had an accidental ingestion and subsequent anaphylactic reaction to the challenge substance and an increase in lgE from 10.4 to >100 klU/L, the remainder had no further exposure. Excluding the patient with anaphylaxis, there was no difference compared to the immediate post-challenge IgE level (p=0.99). CONCLUSIONS: In children undergoing a positive food challenge with
J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2
mild-moderate symptoms and no subsequent consumption, no priming of IgE antibody was observed.
Funding: NIH A1-44236 and reagents from Pharmacea Diagnostics
1132 Threshold Dose for Egg Allergy Determined by 0ral Challenge S. H e r e I, L. Christie 2, S. Sicherer 3, K. Althage 2, A. Burks 2, H. Sampson 3, S. Mofidi 3, S. Noone 3, L. Michaelis 4, S. Strobel 4, J. Hourihane 5, J. Nordlee t, S. Taylor l; ZUniversity of Nebraska, Food Allergy Research & Resource Program, Lincoln, NE, 2Department of Pediatrics, Arkansas Children's Hospital, University of Arkansas for Medical Sciences, Little Rock, AR, 3Department of Pediatrics, Mt. Sinai Hospital, New York, NY, 4Institute of Child Health, London, UNITED KINGDOM, 5Southampton University Hospital, Southampton, UNITED KINGDOM. RATIONALE: The amount of a food that can cause an allergic reaction in a sensitive individual is not known with certainty. An extremely small amount of data is available on determination of threshold levels for even commonly allergenic foods. It is important to obtain good threshold information to assist the food industry and regulatory agencies in designing approaches to protect food-allergic consumers. METHODS: Egg-allergic subjects were selected by the following criteria: good histories of allergy to egg and egg-specific immunoglobulin E (lgE) greater than the 95th percentile or a positive oral egg challenge in the last 4-6 months. Thirty-nine (39) egg-allergic subjects were selected and given increasing levels of spray-dried egg who egg (SDWE) in a DBPCFC. Oneounce servings of cherry-flavored gelatin were used as the vehicle for the doses of SDWE. The negative control was flavored gelatin without SDWE. RESULTS: Ten (10) challenges were positive. Four subjects reacted at a cumulative dose of 3.33 mg SDWE, and 6 reacted at 33.33 mg SDWE. Symptoms of reactions included pruritus, hives, vomiting, erythema, wheezing, abdominal pains, wheals on limbs, face and trunk, scratchy tongue, itchy mouth and throat, and nausea. Twenty-nine subjects did not react to a cumulative dose of 33.33 mg SDWE. CONCLUSIONS: The data presented here is a significant step toward determining the threshold level for egg. Threshold doses determined in statistically-based DBPCFC studies would be beneficial in assessing risk in the case of inadvertent ingestion of small amounts of undeclared egg.
Funding: Food Allergy Research & Resource Program
1133
Recognitionof Sequential and Conformational Structures of Ovomucoid Varies in Patients with Long-lasting and Transient Egg Allergy
K. M. Jarvinen, K. Beyer, L. Bardina, M. Mishoe, H. A. Sampson: Div. of Pediatric Allergy and Immunology and Jaffe Institute for Food Allergy, Mount Sinai School of Medicine, New York, NY. RATIONALE: The present study was performed to identify the immunodominant lgE-binding epitopes of ovomucoid, and to compare the pattern of recognition of linear and conformational structures by IgE antibodies of children with persistent and transient egg allergy. METHODS: Banked sera with elevated egg white-specific lgE antibodies from 46 children reactive to egg at that time, proven by DBPCFC, were identified. 23 of these sera were randomly chosen for individual labeling with overlapping decapeptides of ovomucoid synthesized on a cellulose-derivatized membrane. In addition, specific lgE antibodies against linear (reduced and alkylated) and conformational (native) ovomucoid were determined by CAP System FEIA (Pharmacia) using 18 of these children who in the follow-up retained their allergy beyond 9 years of age and 15 children who gained tolerance by then. RESULTS: Four major IgE-binding epitopes were identified. At the time of diagnosis, the patients with long-lasting egg allergy had a higher ratio of linear ovomucoid-specific IgE to egg white-specific IgE than the children who gained tolerance (p<0.01). An analysis of epitope-specific lgE antibodies showed that 7 out of the 8 patients with persistent egg allergy recognized the region corresponding to AA 1-10 and/or 11-20 whereas none of the 10 children who outgrew did. CONCLUSIONS: Patients with persistent egg allergy develop relatively
Abstracts
S351
more antibodies against linear structures of ovomucoid than the patients who will gain tolerance. The presence of serum lgE antibodies to AA 110 and 11-20 of ovomucoid may be used as a screening instrument to identify patients with persistent egg allergy.
Funding: N1H, NIAID, Bunning Family Fund
1134
Mutational Analysis of IgE-Binding Epitopes of the Major Cow's Milk Allergen 13-CaseinShowed a HeterogenousPattern of Critical Amino Acids between Individual Patients and Pooled Sera
R. R. Cocco, K. M. Jarvinen, N. Han, P. Chatchatee, H. A. Sampson, K. Beyer; Pediatric Allergy and Immunology, Mount Sinai School of Medicine, New York, NY. RATIONALE: In order to alter a cDNA to encode !3-casein with reduced lgE-binding capacity for vaccine development, critical amino acids (AA) necessary for IgE-binding B-cell epitopes were identified. METHODS: Eleven peptides, 10 to 14 AA in length, were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with pooled sera from 15 cow's milk allergic patients and individual sera from six of the patients. IgE antibody binding was detected utilizing an immunoenzymatic method. The ODs of the peptide spots were quantitated on x-ray film. In case no decrease in binding was gained with single substitutions, peptides with double AA substitutions were generated and labeled. RESULTS: Using pooled patient sera, single AA substitution led to a total abrogation of IgE binding in seven of eleven peptides. One to six critical AA were identified per epitope. For the remaining peptides double substitution led to a clear reduction in lgE binding. However, in four of the eleven peptides, modification of the critical AA identified with pooled sera did not result in abrogation of binding with at least one individual patient serum. For these patients AAs other than those implicated by the patient pool were identified. indicating a more heterogeneous pattern in IgE recognition than expected. C O N C L U S I O N S : For future immunotherapeutic interventions with mutated peptides, critical AA should be determined with individual patient sera to account tbr the B-cell epitope heterogeneity between patients.
Funding: Grant Monies
1135 Soybeans Comparison of Genetically Modified Soybeans and Wild in ImmunologicAspects H. Yum; Pediatrics, Pochon Cha University, Seoul, REPUBLIC OF KOREA. RATIONALE: To assess the allergenic potential of new proteins in genetically engineered crops, the immunological and physicochemical characterization is needed. METHODS: We made crude extract from GMO and wild soybeans, curd and soy milk, then performed SDS-PAGE. To evaluate the changes of protein composition, the samples were heated or added with pepsin. We tried PCR to detect GMOs. Sera from patients sensitive to soybean antigen were used for immunoblot of GMO and wild soybeans. RESULTS: SDS-PAGE showed definite protein bands at 68kD in GMO, 50kD in wild soybean. After heating, that difference disappeared. The same protein bands of 68, 37, and 20kD were observed in globulin fraction after acidification. After adding of pepsin 20kD and 68kD bands were preserved in GMO and wild soybeans. The PCR procedures with primers specific to GMO soybeans showed that GMO soybeans and some curd samples included GMO component. Skin test results of 49 patients showed 13 positive results to wild soybeans and 8 positive results to GMO soybean. One patient showed positive skin test result to GMO soybean only. GMO soybean showed unique strong lgE protein band at 25kD in some patients and wild soybean showed strong IgE band at 30-36kD. CONCLUSIONS: Protein band distribution between GMO and wild soybean were similar in heat and pepsin treatment. Immunoblot results shows 25kD protein of GMO soybean reacting with IgE of some patients. To assess the allergenicity of GMO food. the more researches including in vitro and in vivo immunoassay are needed.
Funding: Ministry of Health and Welfare of the Republic of Korea
Abstracts
J ALLERGY CLIN IMMUNOL FEBRUARY 2003
1128 Cyc,osporinfor Chronic Idiopathic Urticaria in Children
1130 tinctFamUial D dCold loCi Autoinflammatory SUrticaria - f r Syndrome: o m AnytiE tn
D. R. Doshi l, M. M, Weinberger2; IUniversity of Iowa, Iowa City, IA, 2Pediatrics, University of Iowa, Iowa City, IA. RATIONALE: Chronic idiopathic urticaria (CIU) has been associated with functional autoantibodies to the high affinity IgE receptor. A controlled clinical trial with cyclosporin in adults (Greaves, e t a | , Br J Dermatol 2000) demonstrated a high degree of efficacy, safety, and induction of remission in adults. Out of a population of 33 children with CIU seen in our clinic during the last 2 years, 5 (ages 13-15) failed control with high doses of HI antihistamine therapy even with the addition of alternate morning prednisone. These patients had daily hives ranging 8-38 months prior to initiation of immunosuppressive therapy. METHODS: Low dose cyclosporin (Neoral brand), beginning with 3 mg/kg/day divided b.i.d, was used. Antihistamine therapy was continued during treatment. Cyclosporin blood levels, BUN, and creatinine were carefully monitored during treatment. RESULTS: Complete absence of urticaria was achieved in all five patients. Three patients are currently in remission and off cyclosporin therapy ranging from 2-15 months without symptoms. The remaining two patients are on cyclosporin without hives. Of these two patients, one has had recurrences while attempting to taper off cyclosporin. The second patient restarted cyclosporin following 16 months of remission with prompt control after restarting. CONCLUSIONS: All five patients tolerated cyclosporin with induction of remission or control of hives without clinical adverse effects. Our experience supports the efficacy and safety of cyclosporin for pediatric patients with CIU who fail to respond or do not tolerate traditional therapy with HI antihistamines with or without alternate morning prednisone.
R. L. Shpall 1, E. W. B. Jeffes 2,3, H. M. Hoffman4; ICollege of Medicine, University of California, Irvine, Irvine, CA, 2Division of Dermatology, VA Medical Center-Long Beach, Long Beach, CA, 3Department of Dermatology, University of California at Irvine, Orange, CA, 4Departments of Pediatrics and Medicine, University of California at San Diego, San Diego, CA. RATIONALE: Familial cold autoinflammatory syndrome (FCAS), commonly known as familial cold urticaria, is a rare autosomal dominant disorder characterized by a rash that develops in response to generalized cold exposure. Patients with this disease are often misdiagnosed with acquired cold urticaria (ACU), a mast cell mediated physical urticaria. METHODS/CASE: We describe a 66-year-old patient followed for several years with a diagnosis of cold urticaria and treated with antihistamines without relief. Since birth this patient has developed a pruritic rash 2-3 hours following prolonged generalized cold exposure. Episodes are often accompanied by arthralgia, fever, chills and sweating lasting several hours. Family history is notable for thirteen similarly affected members. Physical examination revealed a healthy male with discrete urticaria-like papules and plaques surrounded by macular erythema diffusely distributed across the patient's body. RESULTS: The ice cube test, performed by direct contact of ice to skin, was negative. Laboratory testing showed an elevation of white blood cell count, erythrocyte sedimentation rate, and C reactive protein. Skin biopsy demonstrated perivascular neutrophils and mononuclear cells without evidence of eosinophils or mast cells. Sequencing of the patient's DNA confirmed the presence of a mutation in CIAS1, the recently identified gene responsible for FCAS. CONCLUSION: FCAS can be differentiated from ACU by history, laboratory evaluation, and now by genetic testing. Appropriate diagnosis of FCAS can prevent ineffective treatments.
Funding: Universi~ of Iowa
1129 Cetirizine from Topical Rigid Liposomal Formulations: Evaluation of Peripheral Antihistaminic Activity and Systemic Absorption in a Rabbit Model K. J. Simons 1, A. A. W. Elzainy 1, X. Gu t, E. R. Simons2; Ipharmacy, University of Manitoba, Winnipeg, MB, CANADA, 2pediatrics, University of Manitoba, Winnipeg, MB, CANADA. RATIONALE: To assess cetirizine peripheral Hrantihistamine activity and extent of systemic absorption from cetirizine liposome formulations applied to the skin in a rabbit model. METHODS: Small unilamellar vesicles (SUV) and multi-lamellar vesicles (MLV) were prepared using L-~-hydrophosphatidylcholine. Glaxal Base (GB) was the control. In a randomized, crossover study, each formulation containing l0 mg of cetirizine was applied to the shaved backs of 6 female New Zealand rabbits (3.08_+0.05 kg). Intradermal tests with 0.05 ml histamine phosphate (l mg/ml), and blood sampling were performed before application, and at 0.5, l, 2, 3, 4, 5, 6, 8, 10, and 24 h. RESULTS: Compared to baseline, histamine-induced wheal formation was suppressed by cetirizine in MLV from 0.5-24 h, in SUV at 24 h and in GB from 0.5-10 h (p<0.05). Wheal suppression by cetirizine in MLV at 1 and 24 h (94_+2 and 76_+6%) and in SUV at 24 h (91_+5%), was greater than in GB (36_+7 to 61 +14%)(p<0.05); cetirizine in MLV at l, 2, 4, 5, and 8 h (92-+5 to 98__.1%) was superior to SUV (24_+6 to 44+12%) (p<0.05). Plasma cetirizine concentrations from SUV (6-+1 to 7_+0.1 ng/mL) were lower than MLV at 3,4 and l0 h (22-+4 to 28+6 ng/mL), and than GB from 0.5 to 3 h (35_+4 to 59-+5 ng/mL) (p<0.05). Cetirizine from MLV (27__.9 ng/mL) were lower than GB after 0.5-1 h. CONCLUSIONS: In this model, cetirizine from MLV has excellent topical Hi-antihistamine activity while systemic exposure was reduced.
Funding: Pfizer USA
Funding: NIH NIAID K08 AlO1508/Ludwig Institute of Cancer Research
1131 The Impact of Oral Food Challenges on Food-Specific IgE Antibody Concentrations E. J. Vukic, M. Mishoe, S. A. Noone, H. A. Sampson, S. H. Sicherer; Pediatrics, Mount Sinai School of Medicine, New York, NY. RATIONALE: It is unknown whether a food-allergic reaction consumes, primes or has no effect on specific lgE antibody concentration. METHODS: We compared food-specific IgE concentrations (Pharmacea CAP-Sytsem FEIA) to the challenge food and control allergen immediately before and approximately 1 hour following a positive double-blind, placebo-controlled food challenge (DBPCFC). In a subset, lgE was retested a year following challenge. RESULTS: Twenty-nine patients (mean age 7 yrs, 72% male, 86% atopic dermatitis) had positive DBPCFCs to: milk (10), peanut (7), egg (7), wheat (3), soy (1), and open challenge to mustard (1). Seventeen patients had an unchallenged food allergen for comparison. Symptom severity was 2.4 (2 organ systems involved) on a 1-5 scale. Pre- and post-challenge IgE concentrations declined a mean of 0.5 kIU/L (p=n.s.) and were highly correlated (r=0.995, p<0.001). Control food IgE declined a mean of 0.8 klU/L (r=0.998, p<0.001 ) which was of statistical (p=0.04), not clinical significance. There was no difference (p=0.84) in change of IgE concentration between challenge and control foods. IgE concentrations were obtained 8-16 months (mean, 12 months) following challenge (10 patients). One patient had an accidental ingestion and subsequent anaphylactic reaction to the challenge substance and an increase in lgE from 10.4 to >100 klU/L, the remainder had no further exposure. Excluding the patient with anaphylaxis, there was no difference compared to the immediate post-challenge IgE level (p=0.99). CONCLUSIONS: In children undergoing a positive food challenge with
J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2
mild-moderate symptoms and no subsequent consumption, no priming of IgE antibody was observed.
Funding: NIH A1-44236 and reagents from Pharmacea Diagnostics
1132 Threshold Dose for Egg Allergy Determined by 0ral Challenge S. H e r e I, L. Christie 2, S. Sicherer 3, K. Althage 2, A. Burks 2, H. Sampson 3, S. Mofidi 3, S. Noone 3, L. Michaelis 4, S. Strobel 4, J. Hourihane 5, J. Nordlee t, S. Taylor l; ZUniversity of Nebraska, Food Allergy Research & Resource Program, Lincoln, NE, 2Department of Pediatrics, Arkansas Children's Hospital, University of Arkansas for Medical Sciences, Little Rock, AR, 3Department of Pediatrics, Mt. Sinai Hospital, New York, NY, 4Institute of Child Health, London, UNITED KINGDOM, 5Southampton University Hospital, Southampton, UNITED KINGDOM. RATIONALE: The amount of a food that can cause an allergic reaction in a sensitive individual is not known with certainty. An extremely small amount of data is available on determination of threshold levels for even commonly allergenic foods. It is important to obtain good threshold information to assist the food industry and regulatory agencies in designing approaches to protect food-allergic consumers. METHODS: Egg-allergic subjects were selected by the following criteria: good histories of allergy to egg and egg-specific immunoglobulin E (lgE) greater than the 95th percentile or a positive oral egg challenge in the last 4-6 months. Thirty-nine (39) egg-allergic subjects were selected and given increasing levels of spray-dried egg who egg (SDWE) in a DBPCFC. Oneounce servings of cherry-flavored gelatin were used as the vehicle for the doses of SDWE. The negative control was flavored gelatin without SDWE. RESULTS: Ten (10) challenges were positive. Four subjects reacted at a cumulative dose of 3.33 mg SDWE, and 6 reacted at 33.33 mg SDWE. Symptoms of reactions included pruritus, hives, vomiting, erythema, wheezing, abdominal pains, wheals on limbs, face and trunk, scratchy tongue, itchy mouth and throat, and nausea. Twenty-nine subjects did not react to a cumulative dose of 33.33 mg SDWE. CONCLUSIONS: The data presented here is a significant step toward determining the threshold level for egg. Threshold doses determined in statistically-based DBPCFC studies would be beneficial in assessing risk in the case of inadvertent ingestion of small amounts of undeclared egg.
Funding: Food Allergy Research & Resource Program
1133
Recognitionof Sequential and Conformational Structures of Ovomucoid Varies in Patients with Long-lasting and Transient Egg Allergy
K. M. Jarvinen, K. Beyer, L. Bardina, M. Mishoe, H. A. Sampson: Div. of Pediatric Allergy and Immunology and Jaffe Institute for Food Allergy, Mount Sinai School of Medicine, New York, NY. RATIONALE: The present study was performed to identify the immunodominant lgE-binding epitopes of ovomucoid, and to compare the pattern of recognition of linear and conformational structures by IgE antibodies of children with persistent and transient egg allergy. METHODS: Banked sera with elevated egg white-specific lgE antibodies from 46 children reactive to egg at that time, proven by DBPCFC, were identified. 23 of these sera were randomly chosen for individual labeling with overlapping decapeptides of ovomucoid synthesized on a cellulose-derivatized membrane. In addition, specific lgE antibodies against linear (reduced and alkylated) and conformational (native) ovomucoid were determined by CAP System FEIA (Pharmacia) using 18 of these children who in the follow-up retained their allergy beyond 9 years of age and 15 children who gained tolerance by then. RESULTS: Four major IgE-binding epitopes were identified. At the time of diagnosis, the patients with long-lasting egg allergy had a higher ratio of linear ovomucoid-specific IgE to egg white-specific IgE than the children who gained tolerance (p<0.01). An analysis of epitope-specific lgE antibodies showed that 7 out of the 8 patients with persistent egg allergy recognized the region corresponding to AA 1-10 and/or 11-20 whereas none of the 10 children who outgrew did. CONCLUSIONS: Patients with persistent egg allergy develop relatively
Abstracts
S351
more antibodies against linear structures of ovomucoid than the patients who will gain tolerance. The presence of serum lgE antibodies to AA 110 and 11-20 of ovomucoid may be used as a screening instrument to identify patients with persistent egg allergy.
Funding: N1H, NIAID, Bunning Family Fund
1134
Mutational Analysis of IgE-Binding Epitopes of the Major Cow's Milk Allergen 13-CaseinShowed a HeterogenousPattern of Critical Amino Acids between Individual Patients and Pooled Sera
R. R. Cocco, K. M. Jarvinen, N. Han, P. Chatchatee, H. A. Sampson, K. Beyer; Pediatric Allergy and Immunology, Mount Sinai School of Medicine, New York, NY. RATIONALE: In order to alter a cDNA to encode !3-casein with reduced lgE-binding capacity for vaccine development, critical amino acids (AA) necessary for IgE-binding B-cell epitopes were identified. METHODS: Eleven peptides, 10 to 14 AA in length, were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with pooled sera from 15 cow's milk allergic patients and individual sera from six of the patients. IgE antibody binding was detected utilizing an immunoenzymatic method. The ODs of the peptide spots were quantitated on x-ray film. In case no decrease in binding was gained with single substitutions, peptides with double AA substitutions were generated and labeled. RESULTS: Using pooled patient sera, single AA substitution led to a total abrogation of IgE binding in seven of eleven peptides. One to six critical AA were identified per epitope. For the remaining peptides double substitution led to a clear reduction in lgE binding. However, in four of the eleven peptides, modification of the critical AA identified with pooled sera did not result in abrogation of binding with at least one individual patient serum. For these patients AAs other than those implicated by the patient pool were identified. indicating a more heterogeneous pattern in IgE recognition than expected. C O N C L U S I O N S : For future immunotherapeutic interventions with mutated peptides, critical AA should be determined with individual patient sera to account tbr the B-cell epitope heterogeneity between patients.
Funding: Grant Monies
1135 Soybeans Comparison of Genetically Modified Soybeans and Wild in ImmunologicAspects H. Yum; Pediatrics, Pochon Cha University, Seoul, REPUBLIC OF KOREA. RATIONALE: To assess the allergenic potential of new proteins in genetically engineered crops, the immunological and physicochemical characterization is needed. METHODS: We made crude extract from GMO and wild soybeans, curd and soy milk, then performed SDS-PAGE. To evaluate the changes of protein composition, the samples were heated or added with pepsin. We tried PCR to detect GMOs. Sera from patients sensitive to soybean antigen were used for immunoblot of GMO and wild soybeans. RESULTS: SDS-PAGE showed definite protein bands at 68kD in GMO, 50kD in wild soybean. After heating, that difference disappeared. The same protein bands of 68, 37, and 20kD were observed in globulin fraction after acidification. After adding of pepsin 20kD and 68kD bands were preserved in GMO and wild soybeans. The PCR procedures with primers specific to GMO soybeans showed that GMO soybeans and some curd samples included GMO component. Skin test results of 49 patients showed 13 positive results to wild soybeans and 8 positive results to GMO soybean. One patient showed positive skin test result to GMO soybean only. GMO soybean showed unique strong lgE protein band at 25kD in some patients and wild soybean showed strong IgE band at 30-36kD. CONCLUSIONS: Protein band distribution between GMO and wild soybean were similar in heat and pepsin treatment. Immunoblot results shows 25kD protein of GMO soybean reacting with IgE of some patients. To assess the allergenicity of GMO food. the more researches including in vitro and in vivo immunoassay are needed.
Funding: Ministry of Health and Welfare of the Republic of Korea