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1.
J Immunol ; 173(9): 5872-9, 2004 Nov 01.
Article in English | MEDLINE | ID: mdl-15494541

ABSTRACT

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70-86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.


Subject(s)
Antimicrobial Cationic Peptides/immunology , Desensitization, Immunologic , Latex Hypersensitivity/immunology , Plant Lectins/immunology , T-Lymphocytes/immunology , Amino Acid Substitution/genetics , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Basophils/immunology , Basophils/metabolism , Binding, Competitive/genetics , Binding, Competitive/immunology , Cell Division/genetics , Cell Division/immunology , Cell Line , Desensitization, Immunologic/methods , Disulfides/chemistry , Down-Regulation , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Humans , Immunoglobulin E/biosynthesis , Immunoglobulin E/metabolism , Latex Hypersensitivity/diagnosis , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Peptide Fragments/genetics , Peptide Fragments/immunology , Peptide Fragments/metabolism , Plant Lectins/genetics , Plant Lectins/metabolism , Protein Binding/genetics , Protein Binding/immunology , Protein Structure, Tertiary/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , T-Lymphocytes/cytology
2.
Int Arch Allergy Immunol ; 127(3): 234-44, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11979049

ABSTRACT

BACKGROUND: Bermuda grass pollen (BGP) is an increasingly important seasonal aeroallergen in Australia and other subtropical and temperate regions. BGP shares minimal allergenic cross-reactivity with pollens of rye grass or other Pooideae grasses often used for desensitization regimens in grass pollen allergy. Current allergen immunotherapy is seldom used in asthmatic patients due to IgE-mediated side effects. Since clinically effective immunotherapy is linked with altered allergen-specific T cell response, characterisation of human T cell reactivity to Cyn d 1, the major B cell allergen of BGP, should permit the design of effective and safe immunotherapy for BGP allergy. METHODS: Short-term BGP-specific CD4+ T cell lines were established from peripheral blood of 14 BGP-sensitive patients before and after conventional 50% BGP and 50% 7-grass mix subcutaneous specific allergen immunotherapy (SIT). T cell diversity of antigen specificity and function was assessed by proliferation and cytokine production to BGP, Cyn d 1 and Cyn d 1 peptides. RESULTS: Three highly immunogenic regions of Cyn d 1 were identified in 13/14 patients pre-SIT: Cyn d 1 (109-128), (181-209) and (217-241). The SIT regimen was clinically efficacious. Following SIT, decreased proliferation to BGP, Cyn d 1 and Cyn d 1 peptides was observed with a marked decrease in the IL-5:IFN-gamma ratio. CONCLUSIONS: Cyn d 1 is a major T cell allergen of BGP. Decreased Cyn d 1-specific IL-5 dominant T cell responses were observed in association with clinically effective treatment with the 50% BGP and 50% 7-grass mix. Identified dominant T cell regions of Cyn d 1 should facilitate safer vaccine development for BGP-induced asthma in addition to rhinitis.


Subject(s)
Allergens , Desensitization, Immunologic , Plant Proteins/therapeutic use , Poaceae/immunology , Rhinitis, Allergic, Seasonal/therapy , T-Lymphocytes/immunology , Adult , Amino Acid Sequence , Antigens, Plant , Cell Line , Clone Cells , Cytokines/biosynthesis , Epitope Mapping , Epitopes, T-Lymphocyte/immunology , Female , Humans , Lymphocyte Activation , Male , Molecular Sequence Data , Peptides/immunology , Plant Proteins/chemistry , Plant Proteins/immunology , Pollen/chemistry , Pollen/immunology , Rhinitis, Allergic, Seasonal/diagnosis , Rhinitis, Allergic, Seasonal/immunology , Time Factors
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