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Essential Role of B-Cell-Intrinsic MyD88-Signaling in IgE Responses in Lungs
AB68 Abstracts
SATURDAY
217
Production of Naturally Occurring Human Allergen Specific IgE Monoclonal Antibodies (MAbs) Yasmin W. Khan, MD1, Scott A. Smith, MD, PhD2; 1Vanderbilt University, Nashville, TN, 2Infectious Diseases; Department of Medicine; Vanderbilt University School of Medicine, Nashville, TN. RATIONALE: Although food and environmental allergies are increasingly common in the developed world, our understanding of the properties and biology of the molecule mediating allergic disease, IgE, is incomplete. A better understanding of this molecule and its functional properties is needed and could lead to more targeted therapies. METHODS: Human memory B cells were cultured from the peripheral blood of allergic patients, transformed with Epstein-Barr Virus and then expanded in culture. Cultures were screened for IgE production via ELISA using murine anti-human IgE antibodies. The B cells from positively screened cultures were then fused with a myeloma cell line by electrical cytofusion to form human hybridomas, which secrete human allergen specific IgE mAb. RESULTS: Human memory B cells expressing IgE antibody were consistently isolated from peripheral blood, expanded in culture, then fused to make human hybridomas, which successfully produced large amounts of fully human IgE mAb. Panels of naturally occurring human IgE mAbs are being generated. To our knowledge these represent the first ever naturally occurring human IgE secreting human hybridomas. CONCLUSIONS: We have made human monoclonal IgE antibodies. Natural human derived monoclonal IgE will assist in investigation of the structural determinates of many clinically important antibody-antigen binding interactions. By improving our understanding of the IgE-antigen interaction, we hope to provide insights needed for the design of better allergen specific immunotherapies.
218
2-Methyl-1, 3, 6-Trihydroxy-9, 10-Anthraquinone Isolated from Rubia Cordifolia L Inhibits IgE Production Nayab Khan1, Changda Liu, PhD2, Janaki Patel1, Nan Yang, PhD2, Xiu-Min Li, MD, MS2; 1Icahn School of Medicine at Mount Sinai, New York, NY, 2Pediatric Allergy and Immunology, Icahn School of Medicine at Mount Sinai, New York, NY. RATIONALE: Medicinal herbs provide relief to a large percentage of the world population suffering from inflammatory diseases and a major resource for new drug development. The medicinal herb Rubia cordifolia L. is been widely used to treat inflammation. It also showed direct inhibition of IgE production in vitro and in vivo in our previous study. The aim of this study was to identify the bioactive compound in this herb that inhibits IgE production. METHODS: Liquid-liquid extraction, silica gel, and Sephadex LH20 column chromatographic methods were used for isolation and purification of compounds. Nnuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC-MS) techniques were used to identify the compound in Rubia cordifolia L. Rubia cordifolia L compound effects on suppression of IgE production by human B cells (U266 human myeloma cells) and peripheral blood mononuclear cells (PBMCs) from allergic patients was assessed. RESULTS: The compound that was isolated and purified (purity is>95%) from Rubia cordifolia was identified as L. 2-methyl-1, 3, 6-trihydroxy-9, 10-anthraquinone (MT- anthraquinone). This compound showed dosedependently inhibition of U266 cells IgE production with an IC50 of 3.2mg/mL (11.8mM) without any sign of cytotoxicity. MT- anthraquinone (10mg/mL) also non-toxically abolished IL-4 and anti-CD40 stimulated IgE production by PBMCs from food allergic patients. CONCLUSIONS: MT- anthraquinone inhibits IgE production in human B cell line and food allergic patient PBMCs. It may have potential for treatment of IgE associated inflammatory diseases, which requires further investigation.
J ALLERGY CLIN IMMUNOL FEBRUARY 2015
219
Essential Role of B-Cell-Intrinsic MyD88-Signaling in IgE Responses in Lungs Kazufumi Matsushita1, Tomohiro Yoshimoto1,2; 1Laboratory of Allergic Diseases, Institute for Advanced Medical Sciences, Hyogo College of Medicine, 2Department of Immunology and Medical Zoology, Hyogo College of Medicine. RATIONALE: Allergen-specific IgE is linked to asthma pathogenesis, but the underlying mechanisms of IgE production in response to allergen exposure are poorly understood. This study investigated the role of B-cellintrinsic myeloid differentiation factor 88 (MyD88) in IgE and IgG1 production evoked by ragweed pollen instillation into lungs. METHODS: Mice received ragweed pollen every 4 days, for a total of four times, and serum immunoglobulin levels were examined 24 h after final ragweed administration. B-cell-specific MyD88-deficient mice were generated by mixed bone marrow transfer system, in which lethally irradiated Rag2-deficient mice were administered mixed bone marrow cells comprising 80% B-cell-deficient mMT and 20% MyD88-deficient bone marrow. RESULTS: MyD88-deficient mice showed defective IgE/IgG1 production and germinal center responses to lung instillation of ragweed pollen. However, MyD88 was dispensable for dendritic cell activation and Th2 cell development. B-cell-specific deletion of MyD88 replicated the defective antibody production observed in MyD88-deficient mice. Although ragweed pollen contains Toll-like receptor (TLR) ligands, TLR2/4/9deficient mice developed normal allergic responses to ragweed pollen. However, anti-IL-1RI antibody-treated mice and IL-18-deficient mice showed decreased IgE/IgG1 production with normal Th2 development. CONCLUSIONS: Our data demonstrate that pollen instillation into lungs induces IL-1a/b and IL-18 production, which activates B-cell-intrinsic MyD88 signaling to promote germinal center responses and IgE/IgG1 production.
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Role of Patient Education in the Management and Control of Asthma in the Adult Population Anil M. Patel, MD, Joseph S. Yusin, MD, FAAAAI; VA Greater Los Angeles Health Care System, Los Angeles, CA. RATIONALE: Despite advances in asthma medications and devices, studies showing durable improvements in asthma outcomes are lacking. To address this discrepancy, various asthma education programs have been developed. Data regarding the effect of these programs on asthma control and medication adherence are needed. Our study aims to measure the effectiveness of an asthma education session (AES) using established conventional asthma control measures and recently developed asthma medication adherence measures. METHODS: In this randomized, prospective study, patients(n511) with Asthma Control Test (ACT) scores <19 were randomized to an education intervention or a control group after consent. Intervention group patients(n57) received one 60 minute motivational interviewing AES by an allergist and were given an asthma action plan; patients in the control group (n54) did not receive either. Primary outcome measure was ACT score at 6 month follow-up. Secondary outcome measures included the Asthma Medication Ratio(AMR), Rescue Index(RI), and forced expiratory volume in 1 second(FEV1) by spirometry. RESULTS: At 6 months, ACT score improved in 50% of the control group and 43% of the intervention group. FEV1 scores improved on average by 7% in the control group vs 12% in the intervention group. No differences were noted in AMR or RI between the two groups. CONCLUSIONS: Although our education intervention did not demonstrate improvement in all asthma parameters measured, an improvement in FEV1 was observed. Future studies with larger sample size, longer followup periods, and stratification based on asthma severity are warranted. The role of education in improving asthma medication adherence measures remains to be determined.
SATURDAY
217
Production of Naturally Occurring Human Allergen Specific IgE Monoclonal Antibodies (MAbs) Yasmin W. Khan, MD1, Scott A. Smith, MD, PhD2; 1Vanderbilt University, Nashville, TN, 2Infectious Diseases; Department of Medicine; Vanderbilt University School of Medicine, Nashville, TN. RATIONALE: Although food and environmental allergies are increasingly common in the developed world, our understanding of the properties and biology of the molecule mediating allergic disease, IgE, is incomplete. A better understanding of this molecule and its functional properties is needed and could lead to more targeted therapies. METHODS: Human memory B cells were cultured from the peripheral blood of allergic patients, transformed with Epstein-Barr Virus and then expanded in culture. Cultures were screened for IgE production via ELISA using murine anti-human IgE antibodies. The B cells from positively screened cultures were then fused with a myeloma cell line by electrical cytofusion to form human hybridomas, which secrete human allergen specific IgE mAb. RESULTS: Human memory B cells expressing IgE antibody were consistently isolated from peripheral blood, expanded in culture, then fused to make human hybridomas, which successfully produced large amounts of fully human IgE mAb. Panels of naturally occurring human IgE mAbs are being generated. To our knowledge these represent the first ever naturally occurring human IgE secreting human hybridomas. CONCLUSIONS: We have made human monoclonal IgE antibodies. Natural human derived monoclonal IgE will assist in investigation of the structural determinates of many clinically important antibody-antigen binding interactions. By improving our understanding of the IgE-antigen interaction, we hope to provide insights needed for the design of better allergen specific immunotherapies.
218
2-Methyl-1, 3, 6-Trihydroxy-9, 10-Anthraquinone Isolated from Rubia Cordifolia L Inhibits IgE Production Nayab Khan1, Changda Liu, PhD2, Janaki Patel1, Nan Yang, PhD2, Xiu-Min Li, MD, MS2; 1Icahn School of Medicine at Mount Sinai, New York, NY, 2Pediatric Allergy and Immunology, Icahn School of Medicine at Mount Sinai, New York, NY. RATIONALE: Medicinal herbs provide relief to a large percentage of the world population suffering from inflammatory diseases and a major resource for new drug development. The medicinal herb Rubia cordifolia L. is been widely used to treat inflammation. It also showed direct inhibition of IgE production in vitro and in vivo in our previous study. The aim of this study was to identify the bioactive compound in this herb that inhibits IgE production. METHODS: Liquid-liquid extraction, silica gel, and Sephadex LH20 column chromatographic methods were used for isolation and purification of compounds. Nnuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC-MS) techniques were used to identify the compound in Rubia cordifolia L. Rubia cordifolia L compound effects on suppression of IgE production by human B cells (U266 human myeloma cells) and peripheral blood mononuclear cells (PBMCs) from allergic patients was assessed. RESULTS: The compound that was isolated and purified (purity is>95%) from Rubia cordifolia was identified as L. 2-methyl-1, 3, 6-trihydroxy-9, 10-anthraquinone (MT- anthraquinone). This compound showed dosedependently inhibition of U266 cells IgE production with an IC50 of 3.2mg/mL (11.8mM) without any sign of cytotoxicity. MT- anthraquinone (10mg/mL) also non-toxically abolished IL-4 and anti-CD40 stimulated IgE production by PBMCs from food allergic patients. CONCLUSIONS: MT- anthraquinone inhibits IgE production in human B cell line and food allergic patient PBMCs. It may have potential for treatment of IgE associated inflammatory diseases, which requires further investigation.
J ALLERGY CLIN IMMUNOL FEBRUARY 2015
219
Essential Role of B-Cell-Intrinsic MyD88-Signaling in IgE Responses in Lungs Kazufumi Matsushita1, Tomohiro Yoshimoto1,2; 1Laboratory of Allergic Diseases, Institute for Advanced Medical Sciences, Hyogo College of Medicine, 2Department of Immunology and Medical Zoology, Hyogo College of Medicine. RATIONALE: Allergen-specific IgE is linked to asthma pathogenesis, but the underlying mechanisms of IgE production in response to allergen exposure are poorly understood. This study investigated the role of B-cellintrinsic myeloid differentiation factor 88 (MyD88) in IgE and IgG1 production evoked by ragweed pollen instillation into lungs. METHODS: Mice received ragweed pollen every 4 days, for a total of four times, and serum immunoglobulin levels were examined 24 h after final ragweed administration. B-cell-specific MyD88-deficient mice were generated by mixed bone marrow transfer system, in which lethally irradiated Rag2-deficient mice were administered mixed bone marrow cells comprising 80% B-cell-deficient mMT and 20% MyD88-deficient bone marrow. RESULTS: MyD88-deficient mice showed defective IgE/IgG1 production and germinal center responses to lung instillation of ragweed pollen. However, MyD88 was dispensable for dendritic cell activation and Th2 cell development. B-cell-specific deletion of MyD88 replicated the defective antibody production observed in MyD88-deficient mice. Although ragweed pollen contains Toll-like receptor (TLR) ligands, TLR2/4/9deficient mice developed normal allergic responses to ragweed pollen. However, anti-IL-1RI antibody-treated mice and IL-18-deficient mice showed decreased IgE/IgG1 production with normal Th2 development. CONCLUSIONS: Our data demonstrate that pollen instillation into lungs induces IL-1a/b and IL-18 production, which activates B-cell-intrinsic MyD88 signaling to promote germinal center responses and IgE/IgG1 production.
220
Role of Patient Education in the Management and Control of Asthma in the Adult Population Anil M. Patel, MD, Joseph S. Yusin, MD, FAAAAI; VA Greater Los Angeles Health Care System, Los Angeles, CA. RATIONALE: Despite advances in asthma medications and devices, studies showing durable improvements in asthma outcomes are lacking. To address this discrepancy, various asthma education programs have been developed. Data regarding the effect of these programs on asthma control and medication adherence are needed. Our study aims to measure the effectiveness of an asthma education session (AES) using established conventional asthma control measures and recently developed asthma medication adherence measures. METHODS: In this randomized, prospective study, patients(n511) with Asthma Control Test (ACT) scores <19 were randomized to an education intervention or a control group after consent. Intervention group patients(n57) received one 60 minute motivational interviewing AES by an allergist and were given an asthma action plan; patients in the control group (n54) did not receive either. Primary outcome measure was ACT score at 6 month follow-up. Secondary outcome measures included the Asthma Medication Ratio(AMR), Rescue Index(RI), and forced expiratory volume in 1 second(FEV1) by spirometry. RESULTS: At 6 months, ACT score improved in 50% of the control group and 43% of the intervention group. FEV1 scores improved on average by 7% in the control group vs 12% in the intervention group. No differences were noted in AMR or RI between the two groups. CONCLUSIONS: Although our education intervention did not demonstrate improvement in all asthma parameters measured, an improvement in FEV1 was observed. Future studies with larger sample size, longer followup periods, and stratification based on asthma severity are warranted. The role of education in improving asthma medication adherence measures remains to be determined.