Bronchial Epithelial Cultures and Kawasaki Disease

支气管上皮培养与川崎病

• 批准号: 7599596
• 负责人: Susan C. Baker
• 金额: $ 19.22万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-03 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7599596
• 关键词: Acute; Aneurysm; Antibodies; Antigens; Arteries; Base Sequence; Blast Cell; CD8-Positive T-Lymphocytes; Cell Culture System; Cell Culture Techniques; Cells; Child; Childhood; Cilia; Ciliated Bronchial Epithelial Cell; Clinical; Collecting Cell; Coronary Aneurysm; Coronary artery; Cultured Cells; Cytoplasmic Inclusion; Developed Countries; Developing Countries; Development; Diagnosis; Diagnostic tests; Disease; Electron Microscopy; Epidemiologic Studies; Epithelial; Epithelial Cells; Etiology; Genes; Genetic; Genome; Goals; Heart Diseases; Human; Immune response; Immunoglobulin A; Immunoglobulin Variable Region; Immunohistochemistry; Immunologics; In Vitro; Inclusion Bodies; Incubated; Infectious Agent; Infiltration; Laboratories; Lung; Methods; Mucocutaneous Lymph Node Syndrome; Myocardial Infarction; Nucleic Acids; Nucleic acid sequencing; Pathogenesis; Patients; Pattern; Plasma Cells; Population; Prevention therapy; Reporting; Research Proposals; Respiratory System; Respiratory tract structure; Sampling; Sequence Analysis; Site; Staining method; Stains; Sudden Death; Supporting Cell; System; Techniques; Testing; Time; Tissues; Transmission Electron Microscopy; Virus Diseases; bronchial epithelium; disorder control; light transmission; macrophage; novel; pathogen; prevent; protein aggregate; public health relevance; rapid diagnosis; research study; respiratory; respiratory virus

DESCRIPTION (provided by applicant): The goal of this proposal is to develop an in vitro culture system to study the etiology and pathogenesis of Kawasaki Disease (KD). KD is the leading cause of acquired heart disease in children in developed nations, and can result in coronary artery aneurysms, myocardial infarction and sudden death. The etiologic agent of KD is unknown, although clinical and epidemiologic features point to a ubiquitous infectious agent that may cause severe disease in a genetically susceptible population. We found that KD patients harbor oligoclonal (antigen-driven) IgA plasma cells in the coronary arteries and other inflamed tissues. Using the variable regions from the IgA heavy chains, we made synthetic KD antibodies in vitro and used them in immunohistochemistry experiments on acute KD and control tissues. We found that KD synthetic antibodies detected antigen in acute KD but not control tissues. Interestingly, we detected antigen in the ciliated bronchial epithelium of KD patients, indicating that the respiratory tract is the likely portal of entry for the KD agent. Using light and transmission electron microscopy (TEM), we found that this antigen resides in cytoplasmic inclusion bodies consistent with aggregates of protein and associated nucleic acid. We hypothesize that bronchial epithelial cells are the initial site of replication of the KD agent, which then spreads to target tissues such as the coronary arteries. We propose to exploit human ciliated bronchial epithelial cells to cultivate the KD agent in vitro. Our specific aims are to 1) Determine if clinical respiratory samples from acute KD patients infect ciliated bronchial epithelial cell cultures in vitro and 2) Use sequence-independent amplification techniques to amplify, clone and sequence novel nucleic acid sequences from the cell-free supernatant of KD-infected cultures. These studies are exciting because for the first time, a cell likely to contain the KD agent, the ciliated bronchial epithelial cell, has been identified and can be subjected to careful study using electron microscopy and cell culture methods. Developing a cell culture system for the KD agent would allow for remarkable advances in diagnosis, therapy, and prevention of this increasingly recognized and potentially fatal childhood disease. PUBLIC HEALTH RELEVANCE. Kawasaki Disease (KD) is a childhood illness that can result in heart attacks and sudden death, but the cause of the disease is unknown, and therefore there is no specific test to allow for rapid diagnosis. We propose to culture cells from human airways and infect those cells with respiratory samples from KD patients to propagate the KD agent in the laboratory. Our goals are to identify the genetic information of the KD agent from these cells, and to develop a specific diagnostic test so that children with this disease can be diagnosed accurately and treated immediately to prevent fatalities.

描述（由申请人提供）：本提案的目标是开发一种体外培养系统来研究川崎病（KD）的病因和发病机制。 KD 是发达国家儿童获得性心脏病的主要原因，可导致冠状动脉瘤、心肌梗塞和猝死。川崎病的病因尚不清楚，但临床和流行病学特征表明存在一种普遍存在的传染原，可能在遗传易感人群中引起严重疾病。我们发现川崎病患者的冠状动脉和其他发炎组织中含有寡克隆（抗原驱动）IgA 浆细胞。利用 IgA 重链的可变区，我们在体外制备了合成的 KD 抗体，并将其用于急性 KD 和对照组织的免疫组织化学实验中。我们发现，KD 合成抗体可检测到急性 KD 中的抗原，但未检测到对照组织中的抗原。有趣的是，我们在川崎病患者的纤毛支气管上皮中检测到了抗原，表明呼吸道可能是川崎病药剂的进入门户。使用光学和透射电子显微镜（TEM），我们发现该抗原存在于与蛋白质和相关核酸的聚集体一致的细胞质包涵体中。我们假设支气管上皮细胞是 KD 剂复制的初始位点，然后扩散到冠状动脉等目标组织。我们建议利用人纤毛支气管上皮细胞在体外培养 KD 剂。我们的具体目标是 1) 确定来自急性川崎病患者的临床呼吸道样本是否在体外感染纤毛支气管上皮细胞培养物，以及 2) 使用序列无关的扩增技术从受川崎病感染的培养物的无细胞上清液中扩增、克隆和测序新的核酸序列。这些研究令人兴奋，因为首次鉴定出可能含有 KD 剂的细胞（纤毛支气管上皮细胞），并且可以使用电子显微镜和细胞培养方法对其进行仔细研究。为 KD 制剂开发细胞培养系统将使这种日益被认识的潜在致命儿童疾病的诊断、治疗和预防取得显着进展。公共卫生相关性。川崎病（KD）是一种儿童疾病，可导致心脏病发作和猝死，但该病的病因尚不清楚，因此没有特定的测试可以快速诊断。我们建议培养来自人类呼吸道的细胞，并用来自川崎病患者的呼吸道样本感染这些细胞，以在实验室中传播川崎病制剂。我们的目标是从这些细胞中识别 KD 因子的遗传信息，并开发一种特定的诊断测试，以便患有这种疾病的儿童能够得到准确的诊断和立即治疗，以防止死亡。

项目成果

Morphogenesis of Coronavirus HCoV-NL63 in Cell Culture: A Transmission Electron Microscopic Study.

• DOI: 10.2174/1874279300802010052
• 发表时间: 2009-01
• 期刊: The open infectious diseases journal
• 作者: J. Orenstein;Bridget S Banach;S. Baker