Alcoholism increases patient susceptibility to pneumonia and tuberculosis by an unknown mechanism. Ethanol may inhibit pulmonary host defense by down-regulating tumor necrosis factor(alpha) (TNFalpha) and NO synthesis in alveolar macrophage (AM) and recruited neutrophils (PMN). Moreover, ETOH increases phagocyte intracellular iron (Fei) which can decrease bacterial killing by free No. To test this hypothesis we will use virus free SPF rats (n-5/gp) on a non-alcoholic, isocaloric diet (NAI), NAI given a bolus dose of ETOH (AR), NAI on a standard alcohol diet for 6 wk (Chronic) and Chronic given a bolus dose of (Chronic AR). We stipulate four Specific Aims: (a). Determine whether ETOH-mediated down regulation of NOS II in PM and AM attenuates pulmonary host defense. Planned steps include: give rats K. pneumonia or endotoxin (LPS) directly into the lung. Obtain PMN and AM by lavage and circulating PMN from blood at fixed histology in relation to changes in PMN and AM release of free NO measured with microelectrodes at the surface of the PMN and AM. Measure NOS II activity by conversion of 14 C-L-arginine to citrulline and NO. Measure mRNA for NOS II in PMN and AM using competitive reverse transcriptase polymerase chain reaction. Determine if down-regulation of NO is 1) associated with decreased bacterial killing, 2) mediated by ETOH or acetaldehyde or 3) results from changes in cytokine-mediated cell signalling. (b) Determine whether ETOH-mediated suppression of TNF and increases of corticosterone down regulates NOS II in PMN and AM. We ill up and down regulated TNF and corticosterone assessing their effects on ETOH-mediated alterations in NOS II, bacterial killing and the lung's ability to clear an infection (see a). (c) Determine whether ETOH- mediated down regulation of NOS II results from a direct effect on gene expression for NOS II or NOS II activity. We will up and down regulated adenosine, cAMP and Ca2+ assessing their effect on ETOH or acetaldehyde- mediated inhibition of superoxide (ferricytochrome-c-reductase assay), NOS II and bacterial killing (see a). (d) Determine whether ETOH- mediated increases in phagocyte Fei or decreases of biotin attenuate the bactericidal activity of NO. We will up and down regulate Fei and biotin, measure their concentrations in PMN and AM and assess their effects on bacterial killing, No and NOS II and the lung's ability to clear an infection (see a). This project will engender new approaches for preventing/treating opportunistic infections in alcoholics.
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