Browsing by Subject "Lipopolysaccharides"
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Item Constitutive Overexpression of Acyloxyacyl Hydrolase in Mus Musulus(2009-01-14) Ojogun, Noredia I.; Munford, Robert S.Acyloxyacyl hydrolase (AOAH) is a highly conserved host lipase that selectively removes the secondary acyl chains from lipid A, the bioactive center of Gram negative bacterial lipopolysaccharide (LPS). Deacylated LPS has a marked reduction in bioactivity and antagonizes the LPS signaling pathway. Thus, AOAH deacylation of LPS may represent a mechanism by which animals control responses to Gram-negative bacteria. Prior to the experiments described in this study, mice deficient in AOAH were found to be susceptible to the long-term effects of LPS. Aoah-/- mice developed long-lasting hepatomegaly, exaggerated antibody responses, and prolonged immunosuppression in response to small doses of LPS. In the studies described here, AOAH was overexpressed in mice by using CD68 promoter sequences which have been shown by others to drive transgene expression in macrophages. CD68p-AOAH transgenic mice had constitutive overexpression of AOAH in macrophages, dendritic cells and tissues rich in these cells (liver, spleen and lung). They also secreted the enzyme into blood and deacylated LPS at a faster rate both in vitro and in vivo. Importantly, constitutive overexpression of AOAH did not interfere with the initial pro-inflammatory responses to LPS, in keeping with prior observations that AOAH-mediated inactivation of LPS occurs over several hours and does not moderate acute reactions to LPS in vivo. The protective role of constitutive AOAH overexpression was determined by two test systems. First, after an intraperitoneal dose of LPS, CD68p-AOAH transgenic mice returned to their pre-challenge weights more rapidly than did the wildtype mice. Secondly, CD68p-AOAH transgenic mice were less susceptible to LPS and Gram-negative bacteria induced hepatosplenomegaly. These results suggest that overexpression of AOAH in macrophages could accelerate recovery from Gram-negative bacterial infections in animals, including humans.Item Identification of Acyloxyacyl Hydrolase, a Lipopolysaccharide Detoxifying Enzyme, in the Murine Urinary Tract(2003-10-8) Feulner, J. Amelia; Munford, Robert S.Acyloxyacyl hydrolase (AOAH) is a lipase that removes the secondary fatty acyl chains that are substituted to the hydroxyl groups of glucosamine-linked 3-hydroxyacyl residues in lipid A, the bioactive center of Gram-negative bacterial lipopolysaccharides (LPS). Such limited deacylation has been shown to attenuate cytokine and chemokine responses to LPS, suggesting a role for AOAH in modulating (downregulating) inflammatory responses to invading Gramnegative bacteria. Prior to the experiments described in this report, AOAH had only been found in myeloid lineage cells (monocyte-macrophages, neutrophils and dendritic cells). In the work presented here, AOAH was found in murine renal proximal tubule cells and in human renal v cortex. Proximal tubule cells are known targets for invading Gram-negative uropathogens and we hypothesize that possessing AOAH may help them degrade the LPS contained within these bacteria. I further found that AOAH is secreted from proximal tubules in vitro and that it can be detected in murine urine, where it is able to deacylate purified LPS. AOAH may also associate with downstream bladder epithelial cells (which do not express AOAH) and be processed by them to its more enzymatically active, mature form. Bladder cells that have taken up AOAH in vitro are able to deacylate LPS. To determine the in vivo role of AOAH, I induced ascending urinary tract infections (UTIs) in wild type and AOAH null mice. To my surprise, AOAH null mice were able to clear bacteria from their urine faster than did wild type mice. An analysis of the immune response by histological analysis of bladder tissue and enumeration of neutrophils in the urine did not show a significant difference between wild type and AOAH null mice at any of the time points examined. Although I do not yet understand the mechanism for such increased clearance in AOAH null animals, we hypothesize that, due to their inability to deacylate LPS, they might have a more effective immune response to invading Gram-negative bacteria. A more detailed analysis of such responses to invading Gram-negative uropathogens will be important for understanding the in vivo role of AOAH in the urinary tract.