Interaction Between the Epithelium and Mast Cells in Severe Asthma

描述（由申请人提供）：哮喘是一个重要的国家健康问题，影响美国人口的6%至8%，其中10%的哮喘患者患有严重疾病。哮喘的病理生理学是异质性的，涉及多种炎性细胞类型。肥大细胞，已知其在过敏和过敏反应中的作用，可能会导致对哮喘严重程度的更深入的了解，但对哮喘中上皮内和气道腔肥大细胞（MC）的水平或表型知之甚少，特别是在疾病严重程度方面。目的：确定与正常对照相比，不同哮喘严重程度的人类受试者气道室中存在的肥大细胞数量、表型和局部肥大细胞相互作用。具体目标：1）通过评估上皮、粘膜下组织和管腔（支气管肺泡灌洗细胞）区室中的肥大细胞标志物类胰蛋白酶、糜蛋白酶、羧肽酶3、造血前列腺素D2合酶和5-脂氧合酶，在mRNA和蛋白质水平上与轻度哮喘和正常对照相比，确定重度哮喘中气道肥大细胞的位置、表型和功能。2)测定灌洗液和痰液中肥大细胞活化标志物（包括类胰蛋白酶、组胺和前列腺素D2）的存在和相对浓度。3)与哈佛医学院/布里格姆妇女医院的博伊斯实验室合作，开发从人脐带血中体外收获肥大细胞群的必要技术技能。4)确定上皮产生的因子，包括TGF β 1/2和PGE 2，是否支持重度哮喘中上皮肥大细胞的生长和分化。研究设计/方法：将使用新鲜组织样本的定量真实的时间PCR、蛋白质印迹和免疫组织化学技术评估MC表型标志物。将使用EIA评估激活。还将评价源自人脐带血的体外肥大细胞的MC表型标志物。将培养的MC在体外暴露于TGFB 1/2、PGE 2和从哮喘和正常受试者生长的上皮细胞上清液，以进一步评估MC与上皮的相互作用。与公共卫生的相关性：哮喘是一个重要的国家健康问题，影响美国人口的6%至8%，其中10%的哮喘患者患有严重疾病。严重哮喘（SA）患者通常对标准哮喘治疗（如皮质类固醇）反应不佳。通过识别参与SA的不同细胞类型，如肥大细胞，新的靶向治疗可以改善患者的生活质量。

DESCRIPTION (provided by applicant): Asthma is an important national health problem, affecting 6 to 8% of the US population with 10% of all asthmatics having severe disease. The pathophysiology of asthma is heterogenous and involves multiple inflammatory cell types. Mast cells, known for their role in allergy and anaphylaxis, may lead to a deeper understanding of asthma severity but little is known regarding the levels or phenotype of intraepithelial and airway lumen mast cells (MCs) in asthma, particularly across disease severity. Objective: To determine the mast cell numbers, phenotype and local mast cell interactions present in the airway compartments of human subjects with a range of asthma severity compared to normal controls. Specific aims: 1) Determine the location, phenotype and function of airway mast cells in severe asthma, as compared to mild asthma and normal controls at the mRNA and protein level by evaluating the mast cell markers tryptase, chymase, carboxypeptidase 3, hematopoietic prostaglandin D2 synthase, and 5-lipoxygensae in epithelium, submucosal tissue and luminal (bronchoalveolar lavage cells) compartments. 2) Determine the presence and relative concentration of activation markers for mast cells including tryptase, histamine, and prostaglandin D2 in lavage fluid and sputum. 3) Develop the necessary technical skills to harvest mast cell populations in vitro from human cord blood in conjunction with the Boyce laboratory at Harvard Medical School/Brigham and Women's Hospital. 4) Determine whether factors produced by the epithelium, including TGFB1/2 and PGE2, support the growth and differentiation of epithelial mast cells in severe asthma. Research Design/Methods: MC phenotypic markers will be assessed using quantitative real time PCR, Western blot and immunohistochemistry techniques of fresh tissue samples. Activation will be assessed using EIA. In vitro mast cells derived from human cord blood will also be evaluated for MC phenotypic markers. Cultured MCs will be exposed in vitro to TGFB1/2, PGE2 and epithelial cell supernatants grown from asthmatic and normal subjects to further assess the interactions of the MC with the epithelium. Relevance to Public Health: Asthma is an important national health problem, affecting 6 to 8% of the US population with 10% of all asthmatics having severe disease. Patients with severe asthma (SA) often respond poorly to standard asthma treatment such corticosteroids. By identifying the distinct cell types involved in SA, such as the mast cell, new targeted therapies could improve patient quality of life.