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955 IgE antibodies to arthropods
Abstracts
J ALLERGY CLIN IMMUNOL VOLUME 105, NUMBER 1, PART 2
955
956
IgE Antibodies to Arthropods J-J Tsai*. I Holmquisrf, L Ymanf *Section of Allergy and Clinical Immunology, Cathay General Hospital, Taipei. Taiwan tPhannacia & Upjohn Diagnostics AB, Uppsala. Sweden The prevalence of IgE antibodies to cockroach in allergic subjects varies with climate and housing conditions, from 0% in dry, high altitude regions to about 70% in asthmatic urban populations. Several cockroach species are widely distributed and still expanding by accidental introduction into new areas. Quantitative IgE-antibody measurements (UniCAP@) to cockroaches of the genera Blattellu. Blotta. and Peripluneta (P. ameriC(IR~, E filiginosa) in allergic patients from Taiwan with asthma and/or rhinitis (age 3-58 y, n=88) confirmed that common genera share allergens, but also contain genus- and possibly species-specific allergenic epitopes. Prevalences of IgE antibodies were 50%. 58%. and 66%, respectively. High prevalence of IgE antibodies was found also to a related insect, Forjcula (earwig) 50%. and to a landliving crustacean, Porcellio (woodlouse or sowbug) 75%. Dermatophagoides-specific IgE antibodies were found in 89%. A subgroup of the patients showed strong correlation between co&roach. common earwig, and sowbug indicating allergens shared by arthropods. No allergen relationship was observed between cockroach and house dust mite. The relative importance of cockroach as sensitizer can be expected to vary between regions depending not only on the exposure to cockroaches, but also on the exposure to other arthropod allergens. Further regional studies on potential constituents of inhaled dusts and their role in allergic disease would contribute to improvement of diagnosis and management of indoor allergy. Expression of Cockroach Tropomyosin in Pichia pastoris and Purification of the Natural Allergen From Periplaneta americana Extract Ana Beatriz R. Santos+, Wginia P L. Ferriani*, Rob Aalbersef,
Lisa
D.
Vailesf,
MD
Chapmanf.
L. Karla
Arruda*
*School of Medicine of Ribeirlo Preto-USP, Brazil ICLB. Amsterdam, The Netherlands tuniversity of Virginia, Charlottesville, VA, USA We have previously reported the identification of tropomyosin as an important allergen from Periplaneta americana (Santos et al. JACl 1999;104:329-337). Cockroach tropomyosin shows 80%-82% sequence identity to tropomyosin from mites (D. pteronyssinus and D. farinae) and shrimp. A monoclonal antibody directed to D. pteronyssinus tropomyosin (mAb IA6). which also reacted with shrimp tropomyosin. showed strong binding to cockroach striated muscle on immunofluorescence. Control experiments with unrelated antibody (anti-Der p 2) showed no reactivity. Cockroach tropomyosin was designated as Per a 7, according to the WHOfiUIS allergen nomenclature. Recombinant Per a 7 was expressed in the yeast Pichia pastoris. PCR-amplified Per a 7 DNA was subcloned into the SnaB I and Not I sites of the pPIC9 vector, which contains the S. cerevisae a-factor signal sequence, to target protein expression to the secretory pathway. pPIC9 containing Per a 7 DNA was linearized with Bgl II, to generate the His+MutS GS 1 I5 phenotype
S325
following transformation. In addition, Sal I linearized pPIC9 was used to produce either His+Mut+ or His+MutS GSI I5 or KM71 phenotype transformants. respectively. Twenty four colonies were selected by screening for Mut+ or MutS in minimal medium with or without Methanol, and 4/16 selected colonies had the correct Per a 7 insert, as analyzed by PCR. Three of these clones secreted Per a 7 in the supematant, following culture in YPD medium strictly at 3OoC. A 32 kDa band was detected on silver-stained SDS-PAGE gel under reducing conditions, after 24 hours of culture. Time course experiments revealed that Per a 7 production peaks at 96 hours for all three transformants. Recombinant Per a 7 was purified from culture supernatants by affinity chromatography using mAb IA6, and the amino acid sequence of the purified protein was confirmed by mass spectrophotometry. The final yield was 7 mg purified protein/L of culture. Natural tropomyosin was purified from P. americana whole body extract using mAb IA6 affinity column. A 32 kDa and a 64 kDa (dimer) bands were present on SDS-PAGE gel after elution with 5mM Glycine pH 2.8, and the sequence was also confirmed by mass spectrophotometry. Preliminary results showed that both recombinant and natural Per a 7 induced positive skin prick tests in 2 selected cockroach allergic asthmatics, whereas no reaction was found in 2 non-allergic subjects. Our results suggest that the yeast rPer a 7 will make possible further studies on the strucmre of tropomyosin and on the immunological responses to this cockroach allergen in patients with asthma. 957
Long-Term
Reduction Strategies Z G Stamenkovic Municipal Institute for Pulmonary Diseases, Beograd, Yugoslavia Asthma severity among mite-sensitized individuals appears to be related to the degree of mite allergen exposure. The objective of this study was to assess the long-term effectiveness of mite avoidance measures in the homes of asthmatics. The concentration of house dust mite allergen (Der p 1) was measured on the mattress surfaces and bedroom floors in I5 homes on IO occasions over a I2 month period. After the first three visits, all mattresses were covered with a special encasement, and carpeted bedroom floors were randomly allocated to regular applications of a placebo or an “anti-mite” shampoo. The concentration of Der p I recovered was initially high in the carpeted bedrooms and mattresses. During the initial observation period the concentration of mite allergen fell in the treatment or placebo groups. During the seven treatment periods, no differences were seen between the Der p I concentrations in the groups using the “anti-mite” shampoo or placebo shampoo. In the contrast, the concentration of Der p I in samples from uncarpeted floors and mattress encasements was low. Soon we had not enough dust for analysis from these sites. Conclusions: There was no additional benefit from the use of an “anti-mite” shampoo. The absence of carpets and the use of mattress encasements was a useful long-term strategy for mite allergen avoidance.
Plavsic.
Efficacy
L Sagic.
of House
D Poppvac,
Dust
Mite
D Knezevic,
J ALLERGY CLIN IMMUNOL VOLUME 105, NUMBER 1, PART 2
955
956
IgE Antibodies to Arthropods J-J Tsai*. I Holmquisrf, L Ymanf *Section of Allergy and Clinical Immunology, Cathay General Hospital, Taipei. Taiwan tPhannacia & Upjohn Diagnostics AB, Uppsala. Sweden The prevalence of IgE antibodies to cockroach in allergic subjects varies with climate and housing conditions, from 0% in dry, high altitude regions to about 70% in asthmatic urban populations. Several cockroach species are widely distributed and still expanding by accidental introduction into new areas. Quantitative IgE-antibody measurements (UniCAP@) to cockroaches of the genera Blattellu. Blotta. and Peripluneta (P. ameriC(IR~, E filiginosa) in allergic patients from Taiwan with asthma and/or rhinitis (age 3-58 y, n=88) confirmed that common genera share allergens, but also contain genus- and possibly species-specific allergenic epitopes. Prevalences of IgE antibodies were 50%. 58%. and 66%, respectively. High prevalence of IgE antibodies was found also to a related insect, Forjcula (earwig) 50%. and to a landliving crustacean, Porcellio (woodlouse or sowbug) 75%. Dermatophagoides-specific IgE antibodies were found in 89%. A subgroup of the patients showed strong correlation between co&roach. common earwig, and sowbug indicating allergens shared by arthropods. No allergen relationship was observed between cockroach and house dust mite. The relative importance of cockroach as sensitizer can be expected to vary between regions depending not only on the exposure to cockroaches, but also on the exposure to other arthropod allergens. Further regional studies on potential constituents of inhaled dusts and their role in allergic disease would contribute to improvement of diagnosis and management of indoor allergy. Expression of Cockroach Tropomyosin in Pichia pastoris and Purification of the Natural Allergen From Periplaneta americana Extract Ana Beatriz R. Santos+, Wginia P L. Ferriani*, Rob Aalbersef,
Lisa
D.
Vailesf,
MD
Chapmanf.
L. Karla
Arruda*
*School of Medicine of Ribeirlo Preto-USP, Brazil ICLB. Amsterdam, The Netherlands tuniversity of Virginia, Charlottesville, VA, USA We have previously reported the identification of tropomyosin as an important allergen from Periplaneta americana (Santos et al. JACl 1999;104:329-337). Cockroach tropomyosin shows 80%-82% sequence identity to tropomyosin from mites (D. pteronyssinus and D. farinae) and shrimp. A monoclonal antibody directed to D. pteronyssinus tropomyosin (mAb IA6). which also reacted with shrimp tropomyosin. showed strong binding to cockroach striated muscle on immunofluorescence. Control experiments with unrelated antibody (anti-Der p 2) showed no reactivity. Cockroach tropomyosin was designated as Per a 7, according to the WHOfiUIS allergen nomenclature. Recombinant Per a 7 was expressed in the yeast Pichia pastoris. PCR-amplified Per a 7 DNA was subcloned into the SnaB I and Not I sites of the pPIC9 vector, which contains the S. cerevisae a-factor signal sequence, to target protein expression to the secretory pathway. pPIC9 containing Per a 7 DNA was linearized with Bgl II, to generate the His+MutS GS 1 I5 phenotype
S325
following transformation. In addition, Sal I linearized pPIC9 was used to produce either His+Mut+ or His+MutS GSI I5 or KM71 phenotype transformants. respectively. Twenty four colonies were selected by screening for Mut+ or MutS in minimal medium with or without Methanol, and 4/16 selected colonies had the correct Per a 7 insert, as analyzed by PCR. Three of these clones secreted Per a 7 in the supematant, following culture in YPD medium strictly at 3OoC. A 32 kDa band was detected on silver-stained SDS-PAGE gel under reducing conditions, after 24 hours of culture. Time course experiments revealed that Per a 7 production peaks at 96 hours for all three transformants. Recombinant Per a 7 was purified from culture supernatants by affinity chromatography using mAb IA6, and the amino acid sequence of the purified protein was confirmed by mass spectrophotometry. The final yield was 7 mg purified protein/L of culture. Natural tropomyosin was purified from P. americana whole body extract using mAb IA6 affinity column. A 32 kDa and a 64 kDa (dimer) bands were present on SDS-PAGE gel after elution with 5mM Glycine pH 2.8, and the sequence was also confirmed by mass spectrophotometry. Preliminary results showed that both recombinant and natural Per a 7 induced positive skin prick tests in 2 selected cockroach allergic asthmatics, whereas no reaction was found in 2 non-allergic subjects. Our results suggest that the yeast rPer a 7 will make possible further studies on the strucmre of tropomyosin and on the immunological responses to this cockroach allergen in patients with asthma. 957
Long-Term
Reduction Strategies Z G Stamenkovic Municipal Institute for Pulmonary Diseases, Beograd, Yugoslavia Asthma severity among mite-sensitized individuals appears to be related to the degree of mite allergen exposure. The objective of this study was to assess the long-term effectiveness of mite avoidance measures in the homes of asthmatics. The concentration of house dust mite allergen (Der p 1) was measured on the mattress surfaces and bedroom floors in I5 homes on IO occasions over a I2 month period. After the first three visits, all mattresses were covered with a special encasement, and carpeted bedroom floors were randomly allocated to regular applications of a placebo or an “anti-mite” shampoo. The concentration of Der p I recovered was initially high in the carpeted bedrooms and mattresses. During the initial observation period the concentration of mite allergen fell in the treatment or placebo groups. During the seven treatment periods, no differences were seen between the Der p I concentrations in the groups using the “anti-mite” shampoo or placebo shampoo. In the contrast, the concentration of Der p I in samples from uncarpeted floors and mattress encasements was low. Soon we had not enough dust for analysis from these sites. Conclusions: There was no additional benefit from the use of an “anti-mite” shampoo. The absence of carpets and the use of mattress encasements was a useful long-term strategy for mite allergen avoidance.
Plavsic.
Efficacy
L Sagic.
of House
D Poppvac,
Dust
Mite
D Knezevic,