Number Of Bacteria In Spleen Research Articles

Insights into irr and rirA gene regulation on the virulence of Brucella melitensis M5-90.

Iron is a fundamental element required by most organisms, including Brucella. Several researchers have suggested that the iron response regulator (irr) and rhizobial iron regulator (rirA) genes regulate iron acquisition by Brucella abortus, influencing heme synthesis by and virulence of this pathogen. However, little is known about another Brucella species, Brucella melitensis. In this research, we successfully constructed two mutants: M5-90Δirr and M5-90ΔrirA. The adhesion, invasion, and intracellular survivability of these two mutants were evaluated in RAW264.7 cells infected with 1 × 106 CFU of M5-90Δirr, M5-90ΔrirA, or M5-90. We also tested the sensitivity of cells to hydrogen peroxide and their ability to grow. In addition, the virulence of these two mutants was evaluated in BALB/c mice. The results showed that the ability of these two mutants to invade and adhere inside the murine macrophages RAW264.7 was attenuated but their ability to replicate intracellularly was strengthened, enhancing the resistance to hydrogen peroxide. The M5-90Δirr mutant showed stronger growth ability than the parental strain under iron-limiting conditions. No differences were observed in the number of bacteria in spleen between M5-90 and M5-90Δirr at 7 or 15 days postinfection. However, the number of M5-90ΔrirA in spleen reduced significantly at 15 days postinfection. The splenic index of the M5-90Δirr group is evidently lower than that of M5-90. This is the first report that irr and rirA genes of B. melitensis are associated not only with virulence but also with growth ability. Together, our data suggest that M5-90Δirr is a promising Brucella vaccine candidate.

Role of PKCtheta in macrophage-mediated immune response to Salmonella typhimurium infection in mice.

BackgroundThe serine/threonine protein kinase C (PKC) theta has been firmly implicated in T cell-mediated immunity. Because its role in macrophages has remained undefined, we employed PKCtheta-deficient (PKCtheta−/−) mice in order to investigate if PKCtheta plays a role in macrophage-mediated immune responses during bacterial infections.ResultsOur results demonstrate that PKCtheta plays an important role in host defense against the Gram-negative, intracellular bacterium Salmonella typhimurium, as reflected both by markedly decreased survival and a significantly enhanced number of bacteria in spleen and liver of PKCtheta−/− mice, when compared to wild-type mice. Of note, albeit macrophages do not express detectable PKCtheta, PKCtheta mRNA expression was found to be profoundly upregulated during the first hours of lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-, but not IL-4-mediated cell polarization conditions in vitro. Mechanistically, despite expressing normal levels of classically activated macrophage (CAM) markers, PKCtheta-deficient CAMs expressed significantly higher levels of the anti-inflammatory cytokine IL-10 in vivo and in vitro when challenged with S. typhimurium or LPS/IFNgamma. Neutralization of IL-10 recovered immune control to S. typhimurium infection in PKCtheta-deficient macrophages.ConclusionsTaken together, our data provide genetic evidence that PKCtheta promotes a potent pro-inflammatory CAM phenotype that is instrumental to mounting protective anti-bacterial immunity. Mechanistically, PKCtheta exerts a host-protective role against S. typhimurium infection, and acts as an essential link between TLR4/IFNgammaR signaling and selective suppression of the anti-inflammatory cytokine IL-10 at the onset of CAM differentiation in the course of a bacterial infection.

Identification of (+)-Erythro-Mefloquine as an Active Enantiomer with Greater Efficacy than Mefloquine against Mycobacterium avium Infection in Mice

Infection caused by Mycobacterium avium is common in AIDS patients who do not receive treatment with highly active antiretroviral therapy (HAART) or who develop resistance to anti-HIV therapy. Mefloquine, a racemic mixture used for malaria prophylaxis and treatment, is bactericidal against M. avium in mice. MICs of (+)-erythro-, (-)-erythro-, (+)-threo-, and (-)-threo-mefloquine were 32 μg/ml, 32 μg/ml, 64 μg/ml, and 64 μg/ml, respectively. The postantibiotic effect for (+)-erythro-mefloquine was 36 h (MIC) and 41 h for a concentration of 4× MIC. The mefloquine postantibiotic effect was 25 h (MIC and 4× MIC). After baseline infection was established (7 days), the (+)- and (-)-isomers of the diastereomeric threo- and erythro-α-(2-piperidyl)-2,8-bis(trifluoromethyl)-4-quinolinemethanol were individually used to orally treat C57BL/6 bg(+)/bg(+) beige mice that were infected intravenously with M. avium. Mice were also treated with commercial mefloquine and diluent as controls. After 4 weeks of treatment, the mice were harvested, and the number of bacteria in spleen and liver was determined. Mice receiving (+)- or (-)-threo-mefloquine or (-)-erythro-mefloquine had numbers of bacterial load in tissues similar to those of untreated control mice at 4 weeks. Commercial mefloquine had a bactericidal effect. However, mice given the (+)-erythro-enantiomer for 4 weeks had a significantly greater reduction of bacterial load than those given mefloquine. Thus, (+)-erythro-mefloquine is the active enantiomer of mefloquine against M. avium and perhaps other mycobacteria.

Probiotics Protect Mice Against Experimental Infections

Our group has concerned itself with the study of the effect of probiotics on the resistance to infections using experimental models. Here, we will focus on evidence that the UFV-H2b20 strain of Lactobacillus delbrueckii var. bulgaricus may be considered a probiotic and has protective effects on mice against a variety of bacterial infections. Germ-free, monoassociated, and conventional mice were used. Mice were treated with probiotics and challenged with Escherichia coli, Salmonella enterica serovar Typhimurium, or Listeria monocytogenes, and the outcome of infection was measured as mortality, quantification of bacteria in target organs, and systemic of local cytokine production. L. delbrueckii increased clearance of E. coli and production of systemic inflammatory cytokines. This strain also protected monoassociated and conventional mice against infection with S. enterica serovar Typhimurium. Finally, monoassociated mice were more resistant to L. monocytogenes as measured by mortality and the number of bacteria in spleen and liver. In addition, monoassociated mice challenged with L. monocytogenes showed increased production of inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) and nitric oxide. Interestingly, interleukin-10 levels were not altered by monoassociation or infection. L. delbrueckii UFV-H2b20 protects mice against infection, apparently by eliciting the up-regulation of production of inflammatory cytokines.

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca(2+)-activated K(+) (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK(-/-)) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O(2)(-) and H(2)O(2) production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn(2+), which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca(2+) and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca(2+) by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK(-/-) and BK(+/+) mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity.

Alcohol consumption by C57BL/6 mice is associated with depletion of lymphoid cells from the gut-associated lymphoid tissues and altered resistance to oral infections with Salmonella typhimurium.

Studies were done to test whether ethanol (ETOH) consumption alters resistance to mucosal and systemic infections by Salmonella typhimurium. S. typhimurium-immune and -nonimmune mice were fed 1 of 3 diets (an ETOH-containing liquid diet, an isocaloric liquid diet equal in volume to that of the ETOH-treated group, or laboratory chow) in a pair-feeding design and were infected orally or intravenously with S. typhimurium. The number of bacteria in spleen and liver and the effect of ETOH feeding and infection on the number of lymphoid cells in the gut-associated lymphoid tissues (GALT) were determined. ETOH feeding resulted in profound loss of GALT lymphoid cells and an increased number of Salmonella organisms in the intestines, liver, and spleen of infected nonimmune, but not of immune, mice. These data show that ETOH consumption in this model impairs host defense mechanisms that control mucosal infections and inhibits the mechanisms that control levels of bacteria in the central organs.

Clarithromycin significantly improves interleukin-12-mediated anti-Mycobacterium avium activity and abolishes toxicity in mice.

Treatment of experimental murine Mycobacterium avium (MAC) infection with interleukin-12 (IL-12) significantly decreased MAC organisms in tissue but resulted in toxicity. Because IL-12-related toxicity was seen only in infected mice, IL-12 was combined with clarithromycin in an attempt to decrease bacterial burden. Clarithromycin (200 mg/kg/day) was administered alone to M. avium-infected mice for 1 week, and from week 2, IL-12 (20 microg/kg twice per week) was added to the regimen for 4 weeks. Treatment with IL-12 resulted in 60% mortality, compared with 40% mortality in untreated control mice and 20% when IL-12 was given with clarithromycin (P < .05). Clarithromycin plus IL-12 resulted in increased activity compared with either clarithromycin or IL-12 alone in reducing the number of bacteria in spleen and blood. Although potentially toxic, IL-12 is an effective immunotherapy for MAC infection, and combination with clarithromycin reduces IL-12 toxicity.

Toxoplasma gondii: Decreased resistance to intracellular bacteria in mice

The effect of sublethal inocula of Toxoplasma gondii on the course of listeriosis and salmonellosis in mice was investigated. Intravenous injection of T. gondii 24 hr after inoculation of Listeria monocytogenes increased mortality from 16% ( L. monocytogenes alone) to 68% ( L. monocytogenes + T. gondii) ( P < 0.001). Multiplication of L. monocytogenes in spleens also was increased significantly in mice given T. gondii. By 3 days after infection, mice that had received T. gondii and L. monocytogenes had approximately 10 times the number of L. monocytogenes per spleen compared to mice receiving L. monocytogenes alone. Similarly, mortality and the number of bacteria in spleens were increased in mice injected with Salmonella typhimurium and then inoculated with T. gondii. An in vitro assay of macrophage listeriacidal activity was used to investigate the mechanism of this decreased resistance. Peritoneal macrophages from mice injected with T. gondii were less bactericidal than macrophages from uninfected mice. Delayed hypersensitivity responses to L. monocytogenes antigen were markedly suppressed in mice injected with T. gondii. T. gondii infection appears to suppress both macrophage and T-lymphocyte function and may result in decreased resistance to infections caused by intracellular bacteria.