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Imported Fire Ant Venom Phospholipase, Sol i 1, is a Protein Allergen
S30 Abstracts
SATURDAY
115
Imported Fire Ant Venom Phospholipase, Sol i 1, is a Protein Allergen D. R. Hoffman1, C. M. Peroutka2, M. Schmidt3; 1Brody School Medicine East Carolina Univ, Greenville, NC, 2East Carolina Univ, Greenville, NC, 3 Dept Biology East Carolina Univ, Greenville, NC. RATIONALE: Natural Sol i 1 contains cross-reactive carbohydrate determinants, which account for at some of its IgE reactivity. Recombinant Sol i 1 has been prepared free of carbohydrate and tested for IgE binding activity. METHODS: DNA for Sol i 1 including the propeptide sequence, Nde1 and BamH1 restriction sites, and a C-terminal heptahistidine sequence was ligated into a pET-17b vector and transfected into Rosetta-gami chemically competent E. coli cells. This line forms intracytoplasmic disulfide bonds and has a codon usage similar to eukaryotes. The cell pellet was extracted with 6M guanidine and the recombinant protein isolated on Talon resin and extracted with 100 mM EDTA, pH 8.0. RESULTS: The identity of the recombinant protein was verified by SDSPAGE, western blotting and MALDI-TOF mass spectrometry of both the intact protein and tryptic digest. Peptides were identified corresponding to 66.5% of the sequence. IgE dot blots were positive for pooled and individual sera from fire ant venom allergic patients with both natural and recombinant Sol i 1. The recombinant protein did not have phospholipase enzyme activity. CONCLUSIONS: Since the recombinant carbohydrate free Sol i 1 has IgE binding activity, Sol i 1 is a protein allergen. Funding: Brody School of Medicine
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Fire Ant Immunotherapy Prescribing Patterns Among Practicing Allergists 1990-May2007 B. R. Haymore, R. L. McCoy, M. R. Nelson; Walter Reed Army Medical Center, Washington, DC. RATIONALE: To describe the prescribing patterns for imported fire ant (IFA) immunotherapy among practicing allergists in a large healthcare setting and compare them to published practice parameters. METHODS: Under an approved IRB protocol, data from the U.S. Army Centralized Allergen Extract Laboratory (USACAEL) was extracted and analyzed to determine immunotherapy prescribing patterns between Jan1990-May2007. This extract lab provides prescriptions for >320 Department of Defense, Veterans Administration, and Public Health Service clinics across the U.S. & military facilities overseas. RESULTS: There were 1092 patients given 1440 new or revised prescriptions for IFA immunotherapy during the specified time period. Prescriptions originated from 121 facilities in 28 states in the U.S. and 23 military facilities overseas. 87% of prescriptions came from states endemic for IFA. Monotherapy to Solenopsis invicta was prescribed in 146 (13.4%) individuals, monotherapy to Solenopsis richteri was given in one patient with the remainder of patients given both IFA antigens. The maintenance vial concentration most commonly prescribed was 1:200 in 30.9% of prescriptions. 29.4% of prescriptions had a maintenance vial concentration of 1:100, 32.6% were <1:100, 33.4% were between 1:100 - 1:10, and 4.6% were 1:10. The mean starting concentration was 4.4 10-fold dilutions below the maintenance concentration (mean of 5.4 vials per treatment set). CONCLUSIONS: The single most commonly prescribed maintenance vial concentration was 1:200. The majority (67.4%) of allergists prescribe maintenance concentrations recommended in the insect allergy practice parameters of 1:100 to 1:10. Both IFA antigens were used by the vast majority of clinicians.
117
Yellow Jacket Venom Hyaluronidase: Role of Peptid vs. Carbohydrate Epitopes and Specific Cross-Reactivity with Hyaluronidase from Honeybee Venom W. Hemmer1, C. Jin2, M. Focke3, R. Jarisch1, F. Altmann2; 1Floridsdorf Allergy Center, Vienna, AUSTRIA, 2Dept. of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, AUSTRIA, 3Center of Physiology and Pathophysiology, Medical University of Vienna, Vienna, AUSTRIA.
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
RATIONALE: Yellow jacket hyaluronidase (YJ-HYA) is considered a major allergen in YJ allergy and shows 50% sequence homology with hyaluronidase from honeybee venom (Api m 2). As IgE-binding to YJHYA and cross-reactivity with Api m 2 has been recently shown to be due to cross-reactive carbohydrate determinants, the significance of YJ-HYA as a true allergen needs to be re-evaluated. METHODS: Sera from 105 patients with an allergic reaction after a YJ sting (33 YJ CAP single-positive, 72 YJ-honeybee CAP double-positive) were selected and IgE-binding to electrophoretically separated Vespula venom allergens (mixture of V. vulgaris and germanica) was studied by Western blot. To discriminate between IgE-binding to YJ-HYA carbohydrate and peptide epitopes, Western blot inhibition was performed using MUXF-BSA (a synthetic neoglycoprotein consisting of isolated bromelain glycans attached to BSA) and purified Api m 2 as inhibitors. RESULTS: Among double-positive sera, 87% (63/72) reacted with YJHYA. IgE-binding was entirely due to YJ-HYA carbohydrate in 70% (39/ 56), 21% (12/56) showed a mixed antibody response to both peptide and carbohydrate epitopes, and 9% (5/56) reacted with the HYA peptide backbone alone. Nearly half of the sera reacting with YJ-HYA peptide epitopes (8/17) cross-reacted with Api m 2. Among YJ single-positive sera, only 9% (3/33) bound with YJ-HYA. CONCLUSIONS: YJ-HYA is only a minor allergen in YJ allergy and sensitization is primarily found in doube-positive patients. Cross-reactivity with honeybee venom HYA (Api m 2) can be observed in approximately 50% of cases.
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Epidemiology Of Hymenoptera Sting Anaphylaxis In Irish School Children A. Jennings1, E. Duggan1, M. Graif2, I. Perry1, J. O. Hourihane1; 1University College Cork, Cork, IRELAND, 2Tel Aviv University, Tel Aviv, ISRAEL. RATIONALE: To generate the first epidemiological data regarding the prevalence of hymenoptera allergy among school children in Ireland. METHODS: Questionnaires including a set of 6 sting-specific questions (Graif, JACI 2006) were distributed to the parents of primary school children aged 6-8 years and 11-13 years, divided equally between rural and urban backgrounds. An anonymised review of E.R. attendance in the three Cork emergency hospitals from 2003-2007 was also carried out. RESULTS: 720 questionnaires were returned from 13 schools. 281 (40%) children had been stung. 199 (69%) were stung once, 65 (22.5%) twice and 22 (7.6%) more than twice. 15 (5.3%) of children stung had a large local reaction (LLR), and 8 (2.8%) had a mild (cutaneous) systemic reaction (MSR). 4 children (1.0%) experienced a moderate/severe systemic reaction (SSR) but none of these went to hospital. 4 children (2 LLR, 2 no adverse reaction) were taken to hospital as a result of the sting. From 2003 to 2007 21 children (4 LLR, 3 MSR, 14 no adverse reaction) presented to the E.R. departments in Cork city with insect stings. No child attending the ERs was referred to an allergist. CONCLUSIONS: Hymenoptera stings are common in Irish children but severe reactions are unusual, occurring in only 1% of those stung, which is a lower figure than those reported from Israel, using identical questions, and from rural Spain (Fernandez, 2005). There seems to be little awareness among the Irish general population or Irish Emergency physicians that insect stings can have a serious outcome. Funding: Health Research Board
SATURDAY
115
Imported Fire Ant Venom Phospholipase, Sol i 1, is a Protein Allergen D. R. Hoffman1, C. M. Peroutka2, M. Schmidt3; 1Brody School Medicine East Carolina Univ, Greenville, NC, 2East Carolina Univ, Greenville, NC, 3 Dept Biology East Carolina Univ, Greenville, NC. RATIONALE: Natural Sol i 1 contains cross-reactive carbohydrate determinants, which account for at some of its IgE reactivity. Recombinant Sol i 1 has been prepared free of carbohydrate and tested for IgE binding activity. METHODS: DNA for Sol i 1 including the propeptide sequence, Nde1 and BamH1 restriction sites, and a C-terminal heptahistidine sequence was ligated into a pET-17b vector and transfected into Rosetta-gami chemically competent E. coli cells. This line forms intracytoplasmic disulfide bonds and has a codon usage similar to eukaryotes. The cell pellet was extracted with 6M guanidine and the recombinant protein isolated on Talon resin and extracted with 100 mM EDTA, pH 8.0. RESULTS: The identity of the recombinant protein was verified by SDSPAGE, western blotting and MALDI-TOF mass spectrometry of both the intact protein and tryptic digest. Peptides were identified corresponding to 66.5% of the sequence. IgE dot blots were positive for pooled and individual sera from fire ant venom allergic patients with both natural and recombinant Sol i 1. The recombinant protein did not have phospholipase enzyme activity. CONCLUSIONS: Since the recombinant carbohydrate free Sol i 1 has IgE binding activity, Sol i 1 is a protein allergen. Funding: Brody School of Medicine
116
Fire Ant Immunotherapy Prescribing Patterns Among Practicing Allergists 1990-May2007 B. R. Haymore, R. L. McCoy, M. R. Nelson; Walter Reed Army Medical Center, Washington, DC. RATIONALE: To describe the prescribing patterns for imported fire ant (IFA) immunotherapy among practicing allergists in a large healthcare setting and compare them to published practice parameters. METHODS: Under an approved IRB protocol, data from the U.S. Army Centralized Allergen Extract Laboratory (USACAEL) was extracted and analyzed to determine immunotherapy prescribing patterns between Jan1990-May2007. This extract lab provides prescriptions for >320 Department of Defense, Veterans Administration, and Public Health Service clinics across the U.S. & military facilities overseas. RESULTS: There were 1092 patients given 1440 new or revised prescriptions for IFA immunotherapy during the specified time period. Prescriptions originated from 121 facilities in 28 states in the U.S. and 23 military facilities overseas. 87% of prescriptions came from states endemic for IFA. Monotherapy to Solenopsis invicta was prescribed in 146 (13.4%) individuals, monotherapy to Solenopsis richteri was given in one patient with the remainder of patients given both IFA antigens. The maintenance vial concentration most commonly prescribed was 1:200 in 30.9% of prescriptions. 29.4% of prescriptions had a maintenance vial concentration of 1:100, 32.6% were <1:100, 33.4% were between 1:100 - 1:10, and 4.6% were 1:10. The mean starting concentration was 4.4 10-fold dilutions below the maintenance concentration (mean of 5.4 vials per treatment set). CONCLUSIONS: The single most commonly prescribed maintenance vial concentration was 1:200. The majority (67.4%) of allergists prescribe maintenance concentrations recommended in the insect allergy practice parameters of 1:100 to 1:10. Both IFA antigens were used by the vast majority of clinicians.
117
Yellow Jacket Venom Hyaluronidase: Role of Peptid vs. Carbohydrate Epitopes and Specific Cross-Reactivity with Hyaluronidase from Honeybee Venom W. Hemmer1, C. Jin2, M. Focke3, R. Jarisch1, F. Altmann2; 1Floridsdorf Allergy Center, Vienna, AUSTRIA, 2Dept. of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, AUSTRIA, 3Center of Physiology and Pathophysiology, Medical University of Vienna, Vienna, AUSTRIA.
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
RATIONALE: Yellow jacket hyaluronidase (YJ-HYA) is considered a major allergen in YJ allergy and shows 50% sequence homology with hyaluronidase from honeybee venom (Api m 2). As IgE-binding to YJHYA and cross-reactivity with Api m 2 has been recently shown to be due to cross-reactive carbohydrate determinants, the significance of YJ-HYA as a true allergen needs to be re-evaluated. METHODS: Sera from 105 patients with an allergic reaction after a YJ sting (33 YJ CAP single-positive, 72 YJ-honeybee CAP double-positive) were selected and IgE-binding to electrophoretically separated Vespula venom allergens (mixture of V. vulgaris and germanica) was studied by Western blot. To discriminate between IgE-binding to YJ-HYA carbohydrate and peptide epitopes, Western blot inhibition was performed using MUXF-BSA (a synthetic neoglycoprotein consisting of isolated bromelain glycans attached to BSA) and purified Api m 2 as inhibitors. RESULTS: Among double-positive sera, 87% (63/72) reacted with YJHYA. IgE-binding was entirely due to YJ-HYA carbohydrate in 70% (39/ 56), 21% (12/56) showed a mixed antibody response to both peptide and carbohydrate epitopes, and 9% (5/56) reacted with the HYA peptide backbone alone. Nearly half of the sera reacting with YJ-HYA peptide epitopes (8/17) cross-reacted with Api m 2. Among YJ single-positive sera, only 9% (3/33) bound with YJ-HYA. CONCLUSIONS: YJ-HYA is only a minor allergen in YJ allergy and sensitization is primarily found in doube-positive patients. Cross-reactivity with honeybee venom HYA (Api m 2) can be observed in approximately 50% of cases.
118
Epidemiology Of Hymenoptera Sting Anaphylaxis In Irish School Children A. Jennings1, E. Duggan1, M. Graif2, I. Perry1, J. O. Hourihane1; 1University College Cork, Cork, IRELAND, 2Tel Aviv University, Tel Aviv, ISRAEL. RATIONALE: To generate the first epidemiological data regarding the prevalence of hymenoptera allergy among school children in Ireland. METHODS: Questionnaires including a set of 6 sting-specific questions (Graif, JACI 2006) were distributed to the parents of primary school children aged 6-8 years and 11-13 years, divided equally between rural and urban backgrounds. An anonymised review of E.R. attendance in the three Cork emergency hospitals from 2003-2007 was also carried out. RESULTS: 720 questionnaires were returned from 13 schools. 281 (40%) children had been stung. 199 (69%) were stung once, 65 (22.5%) twice and 22 (7.6%) more than twice. 15 (5.3%) of children stung had a large local reaction (LLR), and 8 (2.8%) had a mild (cutaneous) systemic reaction (MSR). 4 children (1.0%) experienced a moderate/severe systemic reaction (SSR) but none of these went to hospital. 4 children (2 LLR, 2 no adverse reaction) were taken to hospital as a result of the sting. From 2003 to 2007 21 children (4 LLR, 3 MSR, 14 no adverse reaction) presented to the E.R. departments in Cork city with insect stings. No child attending the ERs was referred to an allergist. CONCLUSIONS: Hymenoptera stings are common in Irish children but severe reactions are unusual, occurring in only 1% of those stung, which is a lower figure than those reported from Israel, using identical questions, and from rural Spain (Fernandez, 2005). There seems to be little awareness among the Irish general population or Irish Emergency physicians that insect stings can have a serious outcome. Funding: Health Research Board