Bacterial pneumonia is a frequent cause of morbidity and mortality in persons abusing alcohol. One proposed mechanism of the susceptibility of the host to pneumonia is a diminished release of cytokines, specifically Tumor Necrosis Factor in the lung of alcoholics in response to a bacterial challenge. Our previous work has demonstrated that TNF is suppressed in vivo in acutely alcoholic animals and that increasing TNF in vivo by pre- treatment of alcoholic animals with interferon-gamma augments pulmonary host defense. The goals of this project are two-fold: 1) to investigate the molecular mechanisms of ethanol's effect on TNF gene expression and 2) to utilize gene therapy to modulate TNF responses in the alcoholic host to augment the immune response to intrapulmonary pathogens. It is our hypothesis that acute ethanol downregulates TNF through a posttrancriptional mechanism with a resultant impairment of pulmonary host defense. Moreover, we hypothesize that overcoming this post- transcriptional block by gene therapy will improve pulmonary host defense to intrapulmonary pathogens. This proposal will test this hypothesis using a rat model and has three specific aims: 1) to characterize the effect of ethanol on TNF gene regulation and protein processing in vivo, 2) To assess the ability of recombinant adenoviral vectors encoding TNF or Interferon-gamma to upregulate TNF in vitro and in vivo, and 3) to utilize gene therapy to upregulate intrapulmonary TNF in vivo to augment pulmonary host defense in alcohol suppressed animals challenged with intrapulmonary pathogens. To accomplish these aims we will: a) investigate the in vivo effect of ethanol on TNF mRNA levels and protein processing of this cytokine, b) to generate recombinant adenoviruses encoding human TNF and murine interferon-gamma, c) study the effect of ethanol on the expression of these two vectors in vitro and in vivo, d) investigate whether these vectors can augment recruitment of polymorphonuclear lymphocytes into the lung of alcoholic animals challenged with endotoxin and e) whether these vectors can augment bacterial killing in alcoholic animals. The identification of the precise block of TNF production will yield new insights into ethanol's effect on gene expression and the use of cytokine gene therapy will further elucidate the role of these cytokines in the alcoholic host as well as lead to potential new treatment strategies.
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