Interaction Between the Epithelium and Mast Cells in Severe Asthma

描述(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%到8%，所有哮喘患者中有10%患有严重疾病。哮喘的病理生理是异质性的，涉及多种炎性细胞类型。肥大细胞以其在过敏和过敏反应中的作用而闻名，它可能有助于更深入地了解哮喘的严重程度，但对哮喘上皮内和气道腔肥大细胞(MC)的水平或表型知之甚少，尤其是在不同疾病严重程度之间。目的：与正常对照组比较，研究哮喘严重程度不同的受试者呼吸道内肥大细胞的数量、表型和局部肥大细胞相互作用。目的：1)通过检测哮喘患者气道上皮、粘膜下组织和管腔(支气管肺泡灌洗细胞)中的肥大细胞标志物类胰蛋白酶、糜酶、羧基肽酶3、造血细胞前列腺素D2合成酶和5-脂氧合酶，从mRNA和蛋白水平确定重度哮喘患者气道肥大细胞的位置、表型和功能。2)测定灌洗液和痰中类胰蛋白酶、组胺和前列腺素D2等肥大细胞活化标志物的存在和相对浓度。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采集肥大细胞的必要技能。4)确定上皮细胞产生的因子，包括TGFB1/2和PGE2，是否支持重症哮喘上皮肥大细胞的生长和分化。研究设计/方法：采用实时定量聚合酶链式反应、免疫印迹和免疫组织化学技术对新鲜组织标本中的MC表型标志物进行评估。激活将使用EIA进行评估。在体外，来自人脐血的肥大细胞也将被评估MC表型标记。将体外培养的MC暴露于TGFB1/2、PGE2以及哮喘和正常人的上皮细胞培养上清液中，以进一步评价MC与上皮细胞的相互作用。与公共卫生相关：哮喘是一个重要的国家健康问题，影响着6%至8%的美国人口，10%的哮喘患者患有严重疾病。严重哮喘(SA)患者通常对标准的哮喘治疗反应不佳，如皮质类固醇。通过识别SA中涉及的不同细胞类型，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.