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Characterization of the Antigenic Determinant of Der f 7 Allergen Recognized by A Monoclonal Antibody HD12
Abstracts AB205
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2
802
804
803
805
Characterization of the Antigenic Determinant of Der f 7 Allergen Recognized by A Monoclonal Antibody HD12 H. Shen, H. Tai, H. Chou, Y. Chen; Taipei Veterans General Hospital, Taipei, TAIWAN. RATIONALE: The group 7 mite allergens Der p 7 and Der f 7 are implicated in atopic asthma in about 50% of mite-sensitive patients. However, little was known about their biological and structural features. The purpose of the present study is to characterize the antigenic determinant recognized by HD12, a Der f 7-specific monoclonal antibody. METHODS: The antigenic determinant of Der f 7 recognized by HD12 was analyzed by immunodot blot inhibition using synthetic peptides and site-directed mutagenesis. RESULTS: A linear epitope of five amino acids derived from Der f 7 (46GILDF50) can inhibit HD12 from binding to Der f 7. Der f 7 mutants with L48A or F50A substitution showed negative dot-blot immuno-reactivity against HD12. In addition, dot-blot inhibition results demonstrated that these mutants cannot inhibit HD12 from binding to Der f 7. With comparative protein modeling, a model was obtained for Der f 7. The critical core residues Leu48 and Phe50 of the HD12 antigenic determinant are located on a loop-like structure. CONCLUSIONS: A linear epitope [46GILDF50] with both Leu48 and Phe50 as critical core amino acid residues was mapped as the determinant of Der f 7 recognized by monoclonal antibody HD12. Based on comparative protein modeling, they were located on a loop structure of Der f 7. Results obtained provide a valuable basis for further understanding characteristics of the important group 7 mite allergens.
A Meta-analysis of Immune Epitope Data for Allergy and Asthma K. C. Vaughan; La Jolla Institute for Allergy and Immunology, La Jolla, CA. RATIONALE: The universe of allergens represents an incredible array of antigens from a broad spectrum of plants, animals and foods. Moreover, defining allergic sequelae is complex in the human host and in their respective animal models. It is therefore challenging to analyze the collective immunological data related to human allergy. The Immune Epitope Database (IEDB) provides scientists with a repository of immune epitope data. The IEDB captures epitope data from the scientific literature and contains antibody and T cell data for human and animal hosts for all disease categories. Analysis of epitope data provides an inventory of current immunological data and can highlight knowledge gaps and areas for future work. METHODS: A meta-analysis was performed on immune epitope data related to allergy and asthma as of August 2009. This report includes all epitope structures, their source (peptidic and non-peptidic) and associated immune reactivity, including assay details and disease-related data. RESULTS: More than 3,000 allergen-specific antibody and T cell epitopes have been captured. These data include human data and data from numerous animal models. Epitopes were defined in 8 different hosts and were derived from over 100 antigens, including plants, animals and foods. More than 1,700 epitopes were associated with human disease and nearly 70 epitopes were reported as having potential immunotherapeutic use. CONCLUSIONS: The IEDB contains a wealth of allergy-specific epitope data. This data is web accessible and freely available. Global analysis of the data provides, for the first time, an inventory of allergy data and serves to identify critical gaps in the knowledge.
Anti-inflammatory Effect of the Chinese Herbal FormulaFAHF-2 on PBMCs from Children with Crohn's Disease Y. Song1, D. Dunkin2, C. Ceballos2, K. Hoffstadter-Thal2, K. Benkov2, X. Li1; 1Pediatric Allergy & Immunology and Jaffe Food Allergy Institute, Mount Sinai School of Medicine, New York, NY, 2Pediatric Gastroenterology, Mount Sinai School of Medicine, New York, NY. RATIONALE: The prevalence of Crohn’s disease (CD) is increasing. Current therapies can have serious side effects. We previously developed an herbal formula, (FAHF-2), based on a classical traditional Chinese herbal formula that has long been used in China to treat colitis. This provided the basis for investigating the anti-inflammatory effects of FAHF-2 on peripheral blood mononuclear cells (PBMCs) from children with CD and RAW267.4 macrophages. METHODS: PBMCs isolated from 14 CD children (8-17 yrs old) were cultured for 24 hours with or without FAHF-2 in the presence or absence of LPS. Cytokine and chemokine production in the culture supernatants was measured by multiplex ELISA. The effect on TNF-a producing monocytes was determined by flow-cytometry. In addition, activation of NF-kB in LPS-stimulated RAW267.4 cells was determined by Western blot. RESULTS: FAHF-2 treatment in vitro significantly reduced LPS-induced TNF-a, IL-12, IFN-g, IP-10, MIG, and MIP-1b (p<0.05 to p<0.001) production, and increased GM-CSF production by PBMCs (p<0.05) (n514). FAHF-2 also significantly reduced the percent of TNF-a+CD14+ monocytes in LPS-stimulated PBMC samples (p<0.05) (n54). In addition, pre-incubation of RAW267.4 cells with FAHF-2 decreased IkB-a phosphorylation, IkB-a degradation and Akt phosphorylation following LPS stimulation. CONCLUSIONS: FAHF-2 inhibited pro-inflammatory cytokine production and number of TNF-a +CD14+ monocytes in PBMCs from CD children. FAHF-2 inhibition of LPS-stimulated TNF-a production may be due, at least partially, to blocking the NF-kB pathway. This study suggests that clinical investigation of FAHF-2 for treating CD is warranted.
TUESDAY
Pathologic Characteristics and Major Histocompatibility Complex Class II Expression in Pediatric Patients with Eosinophilic Esophagitis D. J. Mulder, D. J. Hurlbut, C. J. Justinich; Queen’s University, Kingston, ON, CANADA. RATIONALE: Eosinophilic esophagitis (EoE) is a clinicopathologic disorder with an unknown etiology. EoE is associated with atopic disorders, a Th2 cytokine profile and can be treated by eliminating dietary antigens. Thus, EoE may result from presentation of antigen in the esophageal mucosa through expression of proteins such as HLA-DR. Hypothesis: Presentation of antigen by HLA-DR results in the initiation of EoE. METHODS: Patients with EoE (n540) were identified and characterized. From these EoE patients, mucosal biopsies obtained and processed by standard histology methods. Additional patients with normal esophagus (n512) and gastroesophageal reflux disease (GERD, n514) were used as controls. Slides from each biopsy were stained with hematoxylin-phloxine-saffron and anti-HLA-DR antibody. RESULTS: The average age at EoE diagnosis was 10.5 years and 73% of the patients were male. Symptoms occurred in an age specific manner. All but two patients had concurrent atopic disorders and 27.5% had two or more. Endoscopic features included furrowing, white papules, friability, concentric rings and polyps. Intraepithelial eosinophil number, basal zone thickness, vascular papillae length and inflammatory cell HLA-DR expression were all significantly increased in EoE biopsies compared with normal and GERD biopsies. HLA-DR expression occurred on epithelial cells in a subset (42.1%) of EoE patients only. CONCLUSIONS: HLA-DR expression is increased in the esophageal mucosae of patients with EoE and may correspond to antigen presentation initiating a Th2 inflammatory response.
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2
802
804
803
805
Characterization of the Antigenic Determinant of Der f 7 Allergen Recognized by A Monoclonal Antibody HD12 H. Shen, H. Tai, H. Chou, Y. Chen; Taipei Veterans General Hospital, Taipei, TAIWAN. RATIONALE: The group 7 mite allergens Der p 7 and Der f 7 are implicated in atopic asthma in about 50% of mite-sensitive patients. However, little was known about their biological and structural features. The purpose of the present study is to characterize the antigenic determinant recognized by HD12, a Der f 7-specific monoclonal antibody. METHODS: The antigenic determinant of Der f 7 recognized by HD12 was analyzed by immunodot blot inhibition using synthetic peptides and site-directed mutagenesis. RESULTS: A linear epitope of five amino acids derived from Der f 7 (46GILDF50) can inhibit HD12 from binding to Der f 7. Der f 7 mutants with L48A or F50A substitution showed negative dot-blot immuno-reactivity against HD12. In addition, dot-blot inhibition results demonstrated that these mutants cannot inhibit HD12 from binding to Der f 7. With comparative protein modeling, a model was obtained for Der f 7. The critical core residues Leu48 and Phe50 of the HD12 antigenic determinant are located on a loop-like structure. CONCLUSIONS: A linear epitope [46GILDF50] with both Leu48 and Phe50 as critical core amino acid residues was mapped as the determinant of Der f 7 recognized by monoclonal antibody HD12. Based on comparative protein modeling, they were located on a loop structure of Der f 7. Results obtained provide a valuable basis for further understanding characteristics of the important group 7 mite allergens.
A Meta-analysis of Immune Epitope Data for Allergy and Asthma K. C. Vaughan; La Jolla Institute for Allergy and Immunology, La Jolla, CA. RATIONALE: The universe of allergens represents an incredible array of antigens from a broad spectrum of plants, animals and foods. Moreover, defining allergic sequelae is complex in the human host and in their respective animal models. It is therefore challenging to analyze the collective immunological data related to human allergy. The Immune Epitope Database (IEDB) provides scientists with a repository of immune epitope data. The IEDB captures epitope data from the scientific literature and contains antibody and T cell data for human and animal hosts for all disease categories. Analysis of epitope data provides an inventory of current immunological data and can highlight knowledge gaps and areas for future work. METHODS: A meta-analysis was performed on immune epitope data related to allergy and asthma as of August 2009. This report includes all epitope structures, their source (peptidic and non-peptidic) and associated immune reactivity, including assay details and disease-related data. RESULTS: More than 3,000 allergen-specific antibody and T cell epitopes have been captured. These data include human data and data from numerous animal models. Epitopes were defined in 8 different hosts and were derived from over 100 antigens, including plants, animals and foods. More than 1,700 epitopes were associated with human disease and nearly 70 epitopes were reported as having potential immunotherapeutic use. CONCLUSIONS: The IEDB contains a wealth of allergy-specific epitope data. This data is web accessible and freely available. Global analysis of the data provides, for the first time, an inventory of allergy data and serves to identify critical gaps in the knowledge.
Anti-inflammatory Effect of the Chinese Herbal FormulaFAHF-2 on PBMCs from Children with Crohn's Disease Y. Song1, D. Dunkin2, C. Ceballos2, K. Hoffstadter-Thal2, K. Benkov2, X. Li1; 1Pediatric Allergy & Immunology and Jaffe Food Allergy Institute, Mount Sinai School of Medicine, New York, NY, 2Pediatric Gastroenterology, Mount Sinai School of Medicine, New York, NY. RATIONALE: The prevalence of Crohn’s disease (CD) is increasing. Current therapies can have serious side effects. We previously developed an herbal formula, (FAHF-2), based on a classical traditional Chinese herbal formula that has long been used in China to treat colitis. This provided the basis for investigating the anti-inflammatory effects of FAHF-2 on peripheral blood mononuclear cells (PBMCs) from children with CD and RAW267.4 macrophages. METHODS: PBMCs isolated from 14 CD children (8-17 yrs old) were cultured for 24 hours with or without FAHF-2 in the presence or absence of LPS. Cytokine and chemokine production in the culture supernatants was measured by multiplex ELISA. The effect on TNF-a producing monocytes was determined by flow-cytometry. In addition, activation of NF-kB in LPS-stimulated RAW267.4 cells was determined by Western blot. RESULTS: FAHF-2 treatment in vitro significantly reduced LPS-induced TNF-a, IL-12, IFN-g, IP-10, MIG, and MIP-1b (p<0.05 to p<0.001) production, and increased GM-CSF production by PBMCs (p<0.05) (n514). FAHF-2 also significantly reduced the percent of TNF-a+CD14+ monocytes in LPS-stimulated PBMC samples (p<0.05) (n54). In addition, pre-incubation of RAW267.4 cells with FAHF-2 decreased IkB-a phosphorylation, IkB-a degradation and Akt phosphorylation following LPS stimulation. CONCLUSIONS: FAHF-2 inhibited pro-inflammatory cytokine production and number of TNF-a +CD14+ monocytes in PBMCs from CD children. FAHF-2 inhibition of LPS-stimulated TNF-a production may be due, at least partially, to blocking the NF-kB pathway. This study suggests that clinical investigation of FAHF-2 for treating CD is warranted.
TUESDAY
Pathologic Characteristics and Major Histocompatibility Complex Class II Expression in Pediatric Patients with Eosinophilic Esophagitis D. J. Mulder, D. J. Hurlbut, C. J. Justinich; Queen’s University, Kingston, ON, CANADA. RATIONALE: Eosinophilic esophagitis (EoE) is a clinicopathologic disorder with an unknown etiology. EoE is associated with atopic disorders, a Th2 cytokine profile and can be treated by eliminating dietary antigens. Thus, EoE may result from presentation of antigen in the esophageal mucosa through expression of proteins such as HLA-DR. Hypothesis: Presentation of antigen by HLA-DR results in the initiation of EoE. METHODS: Patients with EoE (n540) were identified and characterized. From these EoE patients, mucosal biopsies obtained and processed by standard histology methods. Additional patients with normal esophagus (n512) and gastroesophageal reflux disease (GERD, n514) were used as controls. Slides from each biopsy were stained with hematoxylin-phloxine-saffron and anti-HLA-DR antibody. RESULTS: The average age at EoE diagnosis was 10.5 years and 73% of the patients were male. Symptoms occurred in an age specific manner. All but two patients had concurrent atopic disorders and 27.5% had two or more. Endoscopic features included furrowing, white papules, friability, concentric rings and polyps. Intraepithelial eosinophil number, basal zone thickness, vascular papillae length and inflammatory cell HLA-DR expression were all significantly increased in EoE biopsies compared with normal and GERD biopsies. HLA-DR expression occurred on epithelial cells in a subset (42.1%) of EoE patients only. CONCLUSIONS: HLA-DR expression is increased in the esophageal mucosae of patients with EoE and may correspond to antigen presentation initiating a Th2 inflammatory response.