Bronchial Epithelial Cultures and Kawasaki Disease

描述（由申请人提供）：本申请的目标是建立一个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病因，可导致冠状动脉瘤、心肌梗死和猝死。KD的病因尚不清楚，但临床和流行病学特征表明，它是一种普遍存在的感染因子，可能在遗传易感人群中引起严重疾病。我们发现KD患者在冠状动脉和其他炎症组织中含有寡克隆（抗原驱动）IgA浆细胞。利用IgA重链的可变区，体外合成KD抗体，并应用于急性KD和对照组织的免疫组化实验。我们发现KD合成抗体在急性KD中检测抗原，而在对照组织中检测不到抗原。有趣的是，我们在KD患者的纤毛支气管上皮中检测到抗原，表明呼吸道可能是KD病原体的入口。利用光学和透射电子显微镜（TEM），我们发现这种抗原存在于与蛋白质和相关核酸聚集一致的细胞质包涵体中。我们假设支气管上皮细胞是KD试剂复制的初始位点，然后扩散到靶组织，如冠状动脉。我们拟利用人纤毛支气管上皮细胞体外培养KD因子。我们的具体目标是：1)确定急性KD患者的临床呼吸样本是否感染体外培养的纤毛支气管上皮细胞；2)使用序列无关扩增技术对KD感染培养物的无细胞上清进行扩增、克隆和测序新的核酸序列。这些研究是令人兴奋的，因为第一次发现了可能含有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进行仔细的研究。开发KD试剂的细胞培养系统将在诊断、治疗和预防这一日益被认识和潜在致命的儿童疾病方面取得显著进展。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，但疾病的原因尚不清楚，因此没有特定的检测方法可以快速诊断。我们建议培养人呼吸道细胞，用患者呼吸道样本感染这些细胞，在实验室繁殖KD因子。我们的目标是从这些细胞中确定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。

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.