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.
. 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.
| Orenstein, Jan Marc; Shulman, Stanford T; Fox, Linda M et al. (2012) Three linked vasculopathic processes characterize Kawasaki disease: a light and transmission electron microscopic study. PLoS One 7:e38998 |
| Rowley, Anne H; Baker, Susan C; Shulman, Stanford T et al. (2011) Ultrastructural, immunofluorescence, and RNA evidence support the hypothesis of a ""new"" virus associated with Kawasaki disease. J Infect Dis 203:1021-30 |
| S Banach, Bridget; Orenstein, Jan M; Fox, Linda M et al. (2009) Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model. J Virol Methods 156:19-26 |
| Orenstein, Jan M; Banach, Bridget; Baker, Susan C (2008) Morphogenesis of Coronavirus HCoV-NL63 in Cell Culture: A Transmission Electron Microscopic Study. Open Infect Dis J 2:52-58 |
