In order to study the effects of cytokine-dependent tissue damage in response to lung infections in pigs, we have developed adenoviral vectors which contain anti-inflammatory cytokines for use in gene transfer experiments. The purpose of this study was to determine whether porcine cells could be infected by non-replicating adenovirus-5 constructs encoding for /3-galactosidase (AdS/ja-gal) or human interleukin-1 receptor antagonist (Ad-5/hIL-lra). METHODS: PK(15) cells were infected with Ad-5 constructs and incubated for 48 hr. Extracts from Ad-5//3-gal-infected cells were analyzed for /3-galactosidase activity using o-nitrophenol-/?-D-galactoside (ONPG) as a substrate. Supernatants from Ad-5/hIL-lra-infected cells were analyzed for hlL-lra by ELISA. Pigs were endotracheally inoculated with 10U pfu of Ad-5/hIL-lra, sacrificed up to 10 days post-infection, and lung lavages analyzed for IL-Ira by ELISA. RESULTS: Cells infected in vitro with Ad-5//3-gal expressed /3-galactosidase over 48 hr. The activity increased from 388 pmol ONPG hydrolyzed/min/mg protein at 24 hr to 1030 pmol ONPG hydrolyzed/min/mg protein at 48 hr. Similarly, hlL-lra was detected at a concentration of 54.9 pg/ml in culture supernatants taken from Ad-5/hIL-Ira-infected cells 48 hr after infection. In vivo, expression of hlL-lra was detected 48 hr post-infection (38.5 pg/ml) and reached maximal levels at day 7 (667 pg/ml). CONCLUSION: Porcine cells are able to express hlL-lra both in vitro and in vivo from genes delivered in recombinant adenoviruses.
|Original language||English (US)|
|State||Published - Dec 1 1996|