Interleukin 1 (IL-1) is thought to mediate the inflammatory response present in inflammatory bowel disease (IBD). The recent discovery of a receptor antagonist specific for IL-1 (IL-1ra) has generated a great deal of interest in this process, especially because IL-1ra can suppress the pro-inflammatory activity of IL-1 found in experimental colitis in rabbits. The discovery of IL-1ra also suggests a potential strategy utilizing gene therapy for treatment of IBD: the targeted delivery of recombinant adenovirus encoding the IL-1ra to gut epithelium.In order to initiate gene transfer in the intestinal tract, an adenoviral vector should have sufficient stability to infect intestinal cells in vitro in a simulated luminal environment. Using an adenovirus containing the -galactosidase gene from E. coli that is specifically targeted for the nucleus (Ad.RSVntlacZ), we show that exposure of the adenovirus to gastrointestinal proteases and bile acids only slightly affected the infectivity of the virus over the testing period. Furthermore, variations of the luminal pH in the range of 5.5 to 7.4 did not lead to changes in infectivity.The human intestinal cell Caco-2 was used as an in vitro model for gene transfer to the gastrointestinal tract. Caco-2 monolayers develop tight junctions and form a significant diffusion barrier. They are morphologically polar and develop brush border membranes at their apical surface. By infecting at various times after seeding, we found that Ad.RSVntlacZ could indeed infect Caco-2 monolayers with a marked preference for nonconfluent and undifferentiated cells. Infection of Caco-2 cells grown on permeable supports with the adenoviral vector containing the IL-1ra gene (Ad.RSVIL-1ra) resulted in clearly detectable amounts of IL-1ra protein secreted into the apical as well as basolateral compartment. Secretion of IL-1ra protein has been maintained for at least 16 days after infection. Average secretion to the basolateral compartment, which would be the target side for IBD, was calculated to be about 10 ng/day. Additionally, the cell viability and integrity of the monolayers were not affected by infection with adenoviral vectors. In summary, we showed that adenoviral vectors were stable to a simulated luminal environment and were able to infect and express a transduced gene over the lifetime of the model Caco-2 monolayer. Furthermore, the IL-1ra protein was expressed at therapeutic levels. Thus, we feel that the Caco-2 cell line serves as an effective in vitro model system for developing adenoviral therapy in the treatment of IBD.