Khandaker Ahtesham Ahmed

Cytoprotective function of heme oxygenase 1 induced by a nitrated cyclic nucleotide formed during murine salmonellosis.

Signaling mechanisms of NO-mediated host defense are yet to be elucidated. In this study, we report a unique signal pathway for cytoprotection during Salmonella infection that involves heme oxygenase 1 (HO-1) induced by a nitrated cyclic nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP). Wild-type C57BL/6 mice and C57BL/6 mice lacking inducible NO synthase (iNOS) were infected with Salmonella enterica serovar Typhimurium LT2. HO-1 was markedly up-regulated during the infection, the level being significantly higher in wild-type mice than in iNOS-deficient mice. HO-1 up-regulation was associated with 8-nitro-cGMP formation detected immunohistochemically in Salmonella-infected mouse liver and peritoneal macrophages. 8-Nitro-cGMP either exogenously added or formed endogenously induced HO-1 in cultured macrophages infected with Salmonella. HO-1 inhibition by polyethylene glycol-conjugated zinc-protoporphyrin IX impaired intracellular killing of bacteria in mouse liver and in both RAW 264 cells and peritoneal macrophages. Infection-associated apoptosis was also markedly increased in polyethylene glycol-conjugated zinc-protoporphyrin IX-treated mouse liver cells and cultured macrophages. This effect of HO-1 inhibition was further confirmed by using HO-1 short interfering RNA in peritoneal macrophages. Our results suggest that HO-1 induced by NO-mediated 8-nitro-cGMP formation contributes, via its potent cytoprotective function, to host defense during murine salmonellosis.

Nitric oxide produced in Peyer's patches exhibits antiapoptotic activity contributing to an antimicrobial effect in murine salmonellosis

Salmonella species normally infect hosts via the oral‐fecal route. We previously reported that NO had potent host defense functions in murine salmonellosis, not only via a direct antibacterial effect but also because it was cytoprotective for infected host cells. Here, we used an oral route to infect iNOS‐deficient mice infected with S. enterica serovar Typhimurium to further investigate the cytoprotective role of NO in preventing damage caused by Salmonella organisms in PP. Oral bacterial challenge (2 × 105 CFU, or >100 LD50) produced a more severe infection and greater lethality in iNOS‐deficient mice than in iNOS‐competent mice. We used specific antibodies to S. enterica Typhimurium, neutrophils, iNOS, nitrotyrosine, and dendritic cells (CD11c‐positive) in histochemical and immunohistochemical studies to examine infected PP tissues. S. enterica Typhimurium colonization in PP from iNOS‐deficient mice was significantly higher than that in wild‐type mice. Histochemical assays showed extensive cellular damage in PP. We then examined PP tissues for apoptosis by means of in situ TUNEL analysis and by measuring caspase‐3 specific activity in tissue homogenates. Increased numbers of TUNEL‐positive cells and severe granulomatous inflammation with increased infiltration of neutrophils and macrophages were observed during infection in iNOS‐deficient mice compared with wild‐type mice. iNOS‐deficient mice had increased numbers of dendritic cells and significantly higher caspase‐3‐specific activity in PP. These data confirm that NO exerts its protective function not only through direct antibacterial action, but also by preventing apoptosis and thereby contributing to antimicrobial defense during salmonellosis.

Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses

Helicobacter cinaedi is the most common enterohepatic Helicobacter species that causes bacteremia in humans, but its pathogenicity is unclear. Here, we investigated the possible association of H. cinaedi with atherosclerosis in vivo and in vitro. We found that H. cinaedi infection significantly enhanced atherosclerosis in hyperlipidaemic mice. Aortic root lesions in infected mice showed increased accumulation of neutrophils and F4/80+ foam cells, which was due, at least partly, to bacteria-mediated increased expression of proinflammatory genes. Although infection was asymptomatic, detection of cytolethal distending toxin RNA of H. cinaedi indicated aorta infection. H. cinaedi infection altered expression of cholesterol receptors and transporters in cultured macrophages and caused foam cell formation. Also, infection induced differentiation of THP-1 monocytes. These data provide the first evidence of a pathogenic role of H. cinaedi in atherosclerosis in experimental models, thereby justifying additional investigations of the possible role of enterohepatic Helicobacter spp. in atherosclerosis and cardiovascular disease.

Vascular responses to 8-nitro-cyclic GMP in non-diabetic and diabetic mice

BACKGROUND AND PURPOSE 8‐Nitroguanosine 3′,5′‐cyclic monophosphate (8‐nitro‐cGMP), formed nitric oxide (NO)‐dependently, is a physiological second messenger, yet little is known about its role in the pathophysiology of vascular diseases. To study the pharmacological activity of 8‐nitro‐cGMP in diabetic mice, we compared its effects on vascular reactivity of aortas from non‐diabetic and diabetic mice.

Measuring nitrate reductase activity from human and rodent tongues

Reduction of salivary nitrate to nitrite by oral microbes expressing nitrate-reductase has emerged as a crucial pathway in systemic NO homeostasis in humans and other mammals. Selective depletion of oral microbes prevents dietary nitrate-dependent lowering of blood pressure, inhibition of platelet aggregation and ischemic injury. To date, most studies interrogate enterosalivary nitrate reduction by following changes in saliva or plasma nitrite and NO-signaling (functional) end points. Little is known about whether, and if so how, nitrate-reductase enzymatic activity per se (i.e. independent of nitrate levels) is a variable and may account for any individual to individual variation. Here, we describe a minimally invasive protocol that allows for NR activity determination from human, rat and mouse tongue scrapes/swabs. We validate this method using selective application of antiseptic agents to the distal tongue surface which decreased NR activity by >80% and show that bacterial number is a significant variable in measured NR activities between males and females. Also, we show that NR activity is >80% lower in smokers (humans) and after bromine gas exposure (mice), suggesting that exposure to inhaled reactive substances inhibit NR activity identifying a potentially new mechanism by which environmental toxicants promote dysfunction in NO-bioavailability. The described method will facilitate studies testing whether NR specific activity is a variable in different pathophysiologic settings, and in turn how this activity modulates enterosalivary nitrate-reduction.