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，影响了美国6 - 8%的人口，其中10%的哮喘患者患有严重疾病。哮喘的病理生理是异质性的，涉及多种炎症细胞类型。肥大细胞因其在过敏和过敏反应中的作用而闻名，可能导致对哮喘严重程度的更深入了解，但对哮喘中上皮内和气道管腔肥大细胞（MCs）的水平或表型知之甚少，特别是在疾病严重程度上。目的：与正常对照相比，确定不同哮喘严重程度人类受试者气道间室中存在的肥大细胞数量、表型和局部肥大细胞相互作用。具体目的：1)通过评估肥大细胞标志物胰蛋白酶、淀粉酶、羧肽酶3、造血前列腺素D2合成酶和5-脂氧酶在上皮、粘膜下组织和管腔（支气管肺泡灌洗细胞）室中的表达，确定重度哮喘患者气道肥大细胞的位置、表型和功能，并与轻度哮喘和正常对照进行mRNA和蛋白水平的比较。2)测定灌洗液和痰液中肥大细胞激活标志物（包括胰蛋白酶、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Boyce实验室合作，发展必要的技术技能，从人脐带血中体外收获肥大细胞群。4)确定上皮产生的因子，包括TGFB1/2和PGE2，是否支持严重哮喘上皮肥大细胞的生长和分化。研究设计/方法：使用实时定量PCR、Western blot和新鲜组织样本免疫组织化学技术评估MC表型标记。活化工程将采用环评方法进行评估。来自人脐带血的体外肥大细胞也将被评估为MC表型标记。体外培养的MCs将暴露于TGFB1/2、PGE2和来自哮喘和正常受试者的上皮细胞上清液中，以进一步评估MCs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，影响到美国人口的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。通过识别与SA相关的不同细胞类型，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.