RHINOVIRUS INFECTION OF HUMAN AIRWAY EPITHELIUM IN VIVO

University Of California San Francisco, San Francisco CA

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Abstract

In this project will infect cultures of human airway epithelium with rhinovirus, and measure changes in selected junctions deemed likely to contribute to asthma exacerbations. All experiments will use highly differentiated cultures of human tracheobronchial or nasal epithelium that are grown on porous supports, from tight junctions, and maintain epithelial polarity. Most experiments will be performed on intact cell sheets. However, because the proportion of cells infected may be small, we will attempt to separate infected from uninfected cells taking advantage of the ICAM-1 levels are dramatically increased by RV infection. Thus following viral exposure, cells will be dispersed, labeled with FITC-conjugated antibody to ICAM-1 and the most highly fluorescent cells will be separated from the others by FACS. Viral titers will confirm whether we have indeed separated infected from non-infected cells. If so, then gene expression measurements on the infected cells will indicate the functional assays to perform on the intact cell sheets. General functional integrity of cell sheets will be assessed from transepithelial electrical resistance. Short-circuit current (an index of salt and water transport) and mucus secretion will also be measured. Gross changes in cell morphology, such as deciliation, will be detected by scanning electron microscopy of the apical surface. Chemotactic activity of the bathing medium will be determined, and blocking antibodies used to indicate the agents responsible. The profile of inflammatory mediators, cytokines and chemokines secreted will be determined, as will levels of leukocyte-binding cell-to-cell adhesion molecules. We will further see whether viral infection alters the antigen presenting capacity of the epithelium as reflected in levels of MHC components and the ability to activate T-lymphocytes. In Aim #1m we will compare the effects of infection with different passages of RV-16. In Aim #2, we will study the effects of viruses from patients with colds with or without asthma exacerbations. Aim #3 will test the hypothesis that priming of the epithelium with Th-2 cytokines will increase the severity of viral infection. In Aim #4, we will grow cultures from nasal and bronchial brushings from healthy controls, and from patients with atopy and asthma, we will investigate the corresponding functional changes.