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58 Molecular cloning of kentucky bluegrass (KBG) pollen allergens
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THE EFFECTS OF DIETARY SUPPLEMENTATION WITH FISH OIL ON ASTHMATIC RESPONSES TO ANTIGEN. Jp Arm, CE Horton, NM Eiser, TJH Clark, TH Lee. London, U.K. Theeffects of dietary supplementation with fish-oil on neutrophil (PMN) biochemistry and immediate and late asthmatic function, and responses to antigen have been studied in 15 atopic asthmatic subjects. Nine subjects received 18 capsules a day containing 3.2 gm of acid (EPA) and 2.2 gm of eicosapentaenoic and six subjects received docosahexaenoic acid, identical placebo capsules containing olive-oil, for ten weeks in a double blind fashion. Following dietary supplementation with fishbut not following placebo, there was a oil, EPA greater than lo-fold increase in PMN content, and PMN chamotactic response to formyl - methionyl - leucyl - phenylalanine was markedly depressed. There were no significant extinction dose of changes in total serum IgE, antigen on skin prick testing (ED Ag), the dose of antigen causing a 35% fall in airways specific conductance (PD35), and the histamine PD35 following either placebo or fish-oil. The the antigen PD35 and the histamine PD35 ED Ag, were 0.034 gm %, 1.5 up and 0.48 umol (Geom means), respectively, before fish-oil and 0.014 1.8 ug and 0.42 umol, respectively, after gm %, fish-oil. The maximal fall in airways specific conductance during the late asthmatic response decreased by 36 + 6% (Mean + SEM, p
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DETECTION OF WHITE OAK POLLEN (Q.ALBA) ALLERGENS IMMUNOBLOTTING. R.C. Loria, M.D. and H.J. St. Louis, Missouri Wedner, M.D., Aqueous extracts of white oak pollen were separated by SDS-PAGE and transferred onto nitrocellulose (NC) membranes. The NC membranes were blocked with PBS/% nonfat dry milk, incubated with dilutions of atopic and control sera and probed with a radiolabeled antihuman IgE. The allergens were detected by auto23 allergenic bands were radiography. identified with molecular weights between 106-108 Kd (band 1) and 13.2-15.2 Kd (band 23). No allergen was recognized by every patient. Band 5 (MW 76.5 Kd) and band 21 (MW 16.6 Kd) were the most frequently recognized allergens. Multiple bands were recognized by 30%50% of the patients. All patients who were skin test positive to oak by prick testing and whose sera had been properly processed had positive Of 12 patients positive by inununoblots. intradermal skin testing only 4 were positive and 8 negative. The average number of allergens recognized by a single patient was 6.6. The maximum number of allergens to which any individual reacted was 18, the minimum number was one. The total number of allergenic bands (23) indicates that approximately 50% of the extractable proteins of white oak pollen, as seen on an SDS-polyacrylamide gel, are potentially allergenic. Several white oak allergens are recognized by the majority of oak allergic individuals although none of these Several allergens was universally recognized. minor allergens are present as well.
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MOLECULAR CLONING OF KENTUCKY BlJJEGRASS (KBGk POLLEN ALLERGENS. T.W. Hatton. R.D. Hill, A.K.M. Ekramoddoullah. F.T. Kisil and A.H. Sehon MRC Gray for Allergy Research, Depts. of Plant Science and Immunology, University of Manitoba, Winnipeg, MB., Canada.
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IDENTIFICATION AND CHARACTERIZATION OF A CLINICALLY RELEVANT COCKROACH ALLERGEN(S). Richard P. Stankus, M.D., Ph.D., New Orleans, Louisiana. Several studies have supported the clinical importance of cockroach allergens in the induction/exacerbation of allergic respiratory disease. This report describes a fractionation procedure for the isolation of a clinically relevant cockroach allergen(s). Crude whole body extracts of American cockroach (Periplaneta americana) were fractionated by Sephadex G-75 column chromatography followed by chromatofocusing on polybuffer exchanger. Protein fractions eluting in the molecular weight range of 10,000 to 75,000 daltons and in the pH range of 4.0 to 4.5 were identified as containing an important cockroach allergen(s) as determined by the direct radioallergosorbent test (RAST), RAST inhibition and prick skin testing. A similar p1 (4.5) for this major allergen(s) was demonstrated by isoelectrofocusing and immunoprinting. Twenty atopic individuals with immediate skin test reactivity to crude cockroach extract, demonstrated both skin test and RAST reactivity to this important cockroach allergen(s). Employing identical fractionation procedures, a major allergen(s) was isolated from German (Blatella germanica) cockroach whole body extracts. This allergen(s) also eluted in the molecular weight range of 10,000 to 75,000 daltons and in the pH range of 4.0 to 4.5. Collectively, these studies outline an effective procedure for the isolation of an important cockroach allergen(s) and demonstrate the presence of similar allergen(s) in both American and German cockroach whole body extracts.
A group of proteins in the range of lo-58 kDa allergens were recently characterized as potent of KBG (Poa pratensis) pollen. in extracts Complementary DNA clones are ideally suited for the determination of protein structures and for large scale production of these proteins. Hence, with a view to producing pure KBG allergens for use in immunotherapy, in this study a cDNA library was constructed for KBG pollen proteins. By passage of total RNA of RBG pollen over oligo-dT cellulose or a poly U-Sepharose the mRNA was isolated, converted to the column, complementary DNA, which in turn was incorporated into lambda gt 11. A library of 300,000 phages with 85% recombinant clones was thus generated and screened by a pool of sera of grass pollen sensitive patients and anti-KBG murine monoclonal antibodies (Mabs). Several positive clones were detected with the pool of human sera and ten clones with the anti-Poa p I Mab #60. Because of the known crossreactivity of Poa p I and Lo1 p I allergens, a synthetic oligonucleotide was prepared which was based on the available partial amino acid sequence of the latter allergen. Strong hybridization of the DNA prepared from Clone 15.1 with a 32 P-labelled 24-mer probe was revealed by Southern blots. We, therefore, believe that clone 15.1 contains the cDNA for Poa p I.
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THE EFFECTS OF DIETARY SUPPLEMENTATION WITH FISH OIL ON ASTHMATIC RESPONSES TO ANTIGEN. Jp Arm, CE Horton, NM Eiser, TJH Clark, TH Lee. London, U.K. Theeffects of dietary supplementation with fish-oil on neutrophil (PMN) biochemistry and immediate and late asthmatic function, and responses to antigen have been studied in 15 atopic asthmatic subjects. Nine subjects received 18 capsules a day containing 3.2 gm of acid (EPA) and 2.2 gm of eicosapentaenoic and six subjects received docosahexaenoic acid, identical placebo capsules containing olive-oil, for ten weeks in a double blind fashion. Following dietary supplementation with fishbut not following placebo, there was a oil, EPA greater than lo-fold increase in PMN content, and PMN chamotactic response to formyl - methionyl - leucyl - phenylalanine was markedly depressed. There were no significant extinction dose of changes in total serum IgE, antigen on skin prick testing (ED Ag), the dose of antigen causing a 35% fall in airways specific conductance (PD35), and the histamine PD35 following either placebo or fish-oil. The the antigen PD35 and the histamine PD35 ED Ag, were 0.034 gm %, 1.5 up and 0.48 umol (Geom means), respectively, before fish-oil and 0.014 1.8 ug and 0.42 umol, respectively, after gm %, fish-oil. The maximal fall in airways specific conductance during the late asthmatic response decreased by 36 + 6% (Mean + SEM, p
59
DETECTION OF WHITE OAK POLLEN (Q.ALBA) ALLERGENS IMMUNOBLOTTING. R.C. Loria, M.D. and H.J. St. Louis, Missouri Wedner, M.D., Aqueous extracts of white oak pollen were separated by SDS-PAGE and transferred onto nitrocellulose (NC) membranes. The NC membranes were blocked with PBS/% nonfat dry milk, incubated with dilutions of atopic and control sera and probed with a radiolabeled antihuman IgE. The allergens were detected by auto23 allergenic bands were radiography. identified with molecular weights between 106-108 Kd (band 1) and 13.2-15.2 Kd (band 23). No allergen was recognized by every patient. Band 5 (MW 76.5 Kd) and band 21 (MW 16.6 Kd) were the most frequently recognized allergens. Multiple bands were recognized by 30%50% of the patients. All patients who were skin test positive to oak by prick testing and whose sera had been properly processed had positive Of 12 patients positive by inununoblots. intradermal skin testing only 4 were positive and 8 negative. The average number of allergens recognized by a single patient was 6.6. The maximum number of allergens to which any individual reacted was 18, the minimum number was one. The total number of allergenic bands (23) indicates that approximately 50% of the extractable proteins of white oak pollen, as seen on an SDS-polyacrylamide gel, are potentially allergenic. Several white oak allergens are recognized by the majority of oak allergic individuals although none of these Several allergens was universally recognized. minor allergens are present as well.
58
MOLECULAR CLONING OF KENTUCKY BlJJEGRASS (KBGk POLLEN ALLERGENS. T.W. Hatton. R.D. Hill, A.K.M. Ekramoddoullah. F.T. Kisil and A.H. Sehon MRC Gray for Allergy Research, Depts. of Plant Science and Immunology, University of Manitoba, Winnipeg, MB., Canada.
60
IDENTIFICATION AND CHARACTERIZATION OF A CLINICALLY RELEVANT COCKROACH ALLERGEN(S). Richard P. Stankus, M.D., Ph.D., New Orleans, Louisiana. Several studies have supported the clinical importance of cockroach allergens in the induction/exacerbation of allergic respiratory disease. This report describes a fractionation procedure for the isolation of a clinically relevant cockroach allergen(s). Crude whole body extracts of American cockroach (Periplaneta americana) were fractionated by Sephadex G-75 column chromatography followed by chromatofocusing on polybuffer exchanger. Protein fractions eluting in the molecular weight range of 10,000 to 75,000 daltons and in the pH range of 4.0 to 4.5 were identified as containing an important cockroach allergen(s) as determined by the direct radioallergosorbent test (RAST), RAST inhibition and prick skin testing. A similar p1 (4.5) for this major allergen(s) was demonstrated by isoelectrofocusing and immunoprinting. Twenty atopic individuals with immediate skin test reactivity to crude cockroach extract, demonstrated both skin test and RAST reactivity to this important cockroach allergen(s). Employing identical fractionation procedures, a major allergen(s) was isolated from German (Blatella germanica) cockroach whole body extracts. This allergen(s) also eluted in the molecular weight range of 10,000 to 75,000 daltons and in the pH range of 4.0 to 4.5. Collectively, these studies outline an effective procedure for the isolation of an important cockroach allergen(s) and demonstrate the presence of similar allergen(s) in both American and German cockroach whole body extracts.
A group of proteins in the range of lo-58 kDa allergens were recently characterized as potent of KBG (Poa pratensis) pollen. in extracts Complementary DNA clones are ideally suited for the determination of protein structures and for large scale production of these proteins. Hence, with a view to producing pure KBG allergens for use in immunotherapy, in this study a cDNA library was constructed for KBG pollen proteins. By passage of total RNA of RBG pollen over oligo-dT cellulose or a poly U-Sepharose the mRNA was isolated, converted to the column, complementary DNA, which in turn was incorporated into lambda gt 11. A library of 300,000 phages with 85% recombinant clones was thus generated and screened by a pool of sera of grass pollen sensitive patients and anti-KBG murine monoclonal antibodies (Mabs). Several positive clones were detected with the pool of human sera and ten clones with the anti-Poa p I Mab #60. Because of the known crossreactivity of Poa p I and Lo1 p I allergens, a synthetic oligonucleotide was prepared which was based on the available partial amino acid sequence of the latter allergen. Strong hybridization of the DNA prepared from Clone 15.1 with a 32 P-labelled 24-mer probe was revealed by Southern blots. We, therefore, believe that clone 15.1 contains the cDNA for Poa p I.
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