H37Rv Infection Research Articles

Role of mitogen-activated protein kinase pathways in the production of tumor necrosis factor-alpha, interleukin-10, and monocyte chemotactic protein-1 by Mycobacterium tuberculosis H37Rv-infected human monocytes.

The role of mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, IL-8, and monocyte chemotactic protein-1 (MCP-1) was investigated in human monocytes that were infected with Mycobacterium tuberculosis H37Rv. Analysis of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase showed rapid phosphorylation of both subfamilies in response to M. tuberculosis H37Rv. Using highly specific inhibitors of p38 (SB203580) and of MAPK kinase-1 (U0126 and PD98059), we found that both p38 and ERK were essential for M. tuberculosis H37Rv-induced TNF-alpha production, whereas activation of the p38 pathway, but not that of ERK, was essential for M. tuberculosis H37Rv-induced IL-10 production. Interestingly, the ERK pathway, but not that of p38, was critical for MCP-1 secretion from human monocytes that were infected with M. tuberculosis H37Rv. However, IL-8 secretion was not regulated by ERK1/2 or p38 MAPK. Collectively, these results suggest that induction of the MAPK pathway is required for the expression of TNF-alpha, IL-10, and MCP-1 by human monocytes during M. tuberculosis H37Rv infection.

Virulent but not avirulent Mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1-dependent, nitric oxide Synthase 2-independent defense in mice.

Control of infection with virulent Mycobacterium tuberculosis (Mtb) in mice is dependent on the generation of T helper (Th)1-mediated immunity that serves, via secretion of interferon (IFN)-γ and other cytokines, to upregulate the antimycobacterial function of macrophages of which the synthesis of inducible nitric oxide synthase (NOS)2 is an essential event. As a means to understanding the basis of Mtb virulence, the ability of gene-deleted mice incapable of making NOS2 (NOS2−/−), gp91Phox subunit of the respiratory burst NADPH-oxidase complex (Phox−/−), or either enzyme (NOS2/Phox−/−), to control airborne infection with the avirulent R1Rv and H37Ra strains of Mtb was compared with their ability control infection with the virulent H37Rv strain. NOS2−/−, Phox−/−, and NOS2/Phox−/− mice showed no deficiency in ability to control infection with either strain of avirulent Mtb. By contrast, NOS2−/− mice, but not Phox−/− mice, were incapable of controlling H37Rv infection and died early from neutrophil-dominated lung pathology. Control of infection with avirulent, as well as virulent Mtb, depended on the synthesis of IFN-γ, and was associated with a substantial increase in the synthesis in the lungs of mRNA for IFN-γ and NOS2, and with production of NOS2 by macrophages at sites of infection. The results indicate that virulent, but not avirulent, Mtb can overcome the growth inhibitory action of a Th1–dependent, NOS2-independent mechanism of defense.

Effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta and RANTES mRNA expression in guinea pig cells exposed to attenuated and virulent mycobacteria.

The effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta (IL-1 beta) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from naïve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1 beta or RANTES cDNA. Although IL-1 beta and RANTES mRNA could be detected in the spleen cells from naïve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1 beta expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1 beta mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1 beta and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.

Genetic control of susceptibility to infection with Mycobacterium tuberculosis in mice.

Genetic factors play a key role in host response, disease severity, and ultimate outcome of infection with Mycobacterium tuberculosis in humans. In the mouse, the DBA/2J strain is very susceptible to M. tuberculosis H37Rv infection, while the C57Bl/6J strain is resistant. In DBA/2J, a heavier bacterial burden causes a unique phenotype, that includes very severe and rapidly fatal pulmonary disease with extensive exudation of neutrophils and tissue necrosis, as opposed to slower progressive pulmonary disease characterized by the accumulation of epithelioid macrophages with protective immune and inflammatory responses in C57Bl/6J. To identify the genes responsible for differences in host response to M. tuberculosis in these two strains, 95 animals of an informative (C57Bl/6J x DBA/2J) F2 cross were infected intravenously with M. tuberculosis (1 x 10(5) CFU) and duration of survival was used as a quantitative phenotypic measure of susceptibility in a whole genome scan. Quantitative trait locus analysis (QTL) showed that the genetically controlled susceptibility was multigenic. QTL analysis identified two significant linkages on the distal portion of chromosome 1 (Trl-1, LOD, 4.80) and on the proximal portion of chromosome 7 (Trl-3, LOD, 4.66) that each account for approximately 21% of the phenotypic variance. A third suggestive linkage was identified on the proximal portion of chromosome 3 (Trl-2, LOD, 3.93; additional 18% of the variance). At each locus, homozygosity for the parental C57Bl/6J alleles was associated with increased resistance to infection. These novel mouse loci provide the basis for evaluating a possible association of the corresponding syntenic chromosomal regions in humans with susceptibility to tuberculosis.

Expression of CCR5 Is Increased in Human Monocyte-Derived Macrophages and Alveolar Macrophages in the Course of in Vivo and in Vitro Mycobacterium tuberculosis Infection

Human immunodeficiency virus (HIV) replicates more efficiently in Mycobacterium tuberculosis (MTB)-infected macrophages than in uninfected controls. We investigated whether this may be partly explained by changes in expression of CCR5 in the course of mycobacterial infection, as this molecule has been shown to be a coreceptor for HIV entry. Since the lung is the preferential organ of HIV replication in the course of tuberculosis, we preliminarily analyzed beta-chemokine receptor expression in alveolar macrophages from patients with active tuberculosis, using flow cytometry based on an MIP-1alpha ligand-biotin/avidin-FITC detection system. Increased MIP-1alpha receptor (MIP-1alphaR) expression in alveolar macrophages from infected patients was observed whereas no detectable expression could be revealed in uninfected controls. Since MIP-la can also bind CCR1 and CCR4, the presence of CCR5 mRNA was investigated in bronchoalveolar lavage (BAL) cells and detected in alveolar macrophages from tuberculosis patients only. The study was then extended to in vitro MTB-infected macrophages. Monocyte-derived macrophages (MDMs) were left to differentiate for 7 days before MTB H37Rv infection, and CCR5 expression was monitored, by using a specific monoclonal antibody, on days 1, 6, and 11 after infection. Increased CCR5 expression in MTB-infected macrophages was observed, with a peak on day 6 (64% in MTB-infected versus 33% in control cultures) and a decrease by day 11 (25% in MTB infected versus 13% in control cultures). These results show that CCR5 expression is enhanced in the course of in vitro MTB infection and during active pulmonary tuberculosis.

Severity of tuberculosis in mice is linked to distal chromosome 3 and proximal chromosome 9.

Genetic factors play a role in host response to infection with Mycobacterium tuberculosis, the number one infectious killer worldwide. Mice of the inbred strains I/St and A/Sn show significant differences in disease severity after intravenous injection of a lethal dose of the virulent human isolate M. tuberculosis H37Rv. Following challenge with H37Rv, only I/St mice have rapid body weight loss and short survival times. A genome wide analysis for linkage with body weight after M. tuberculosis H37Rv infection was done in (A/SnxI/St)F1xI/St mice. Among females, quantitative trait loci (QTLs) on chromosomes 9 and 3 were significantly linked to postinfection body weight (logarithm of the odds ratio [LOD] scores of 6.68 and 3.92, respectively). Suggestive linkages were found for QTLs on chromosomes 8 and 17 (LOD scores of 3.01 and 2.95, respectively). For males, QTLs on chromosomes 5 and 10 showed suggestive linkages (LOD scores of 3.03 and 2.31, respectively). These linkages can be used to identify candidate regions for tuberculosis susceptibility loci in the human genome.

The 65 kDa protein of Mycobacterium habana and its putative role in immunity against experimental tuberculosis.

Mycobacteria including Mycobacterium tuberculosis and Mycobacterium leprae possess multiple antigens some of which inhibit other anti-mycobacterial immune responses. Whole cell vaccines are not free from these suppressive molecules and may adversely affect the immunogenic response(s). Purified protein components having only immunogenic properties should prove to be superior vaccine(s). Mycobacterium habana, a candidate vaccine for mycobacterial infections has been dissected for analysing its antigenic myriad. A 65 kDa protein of this mycobacterium has been isolated and characterized for its protective and cell mediated immune responses. The protein was isolated in pure form using an isotachophoresis (SDS-PAGE filtration) technique and identified with low molecular weight markers along with mAb using the immunoblot technique. Mab IIH9 has identified a 65 kDa protein in M. habana. This protein has been found to be immunoprotective in mice against M. tuberculosis H37Rv infection. It generates high levels of DTH responses in mice against M. tuberculosis and M. leprae antigens and inhibits migration of sensitized cells under the antigenic influence of homologous and heterologous origin. Possibilities of developing this protein as a subunit vaccine are discussed in this report.

Resistance to Mycobacterium tuberculosis H37Rv infection induced by Listeria and mycobacterial lipids.

The avirulent strain Listeria monocytogenes (Welshimer) induced upon a single injection 4 weeks prior to challenge with Mycobacterium tuberculosis H37Rv resistance in guinea-pigs, which was manifested by a significant decrease of spleen weight and Feldman's index in immunized animals. The degree of resistance was dependent on the immunizing dose and time of administration. Repeated high doses of Listeria yielded only a low or no effect. Further increase of resistance was obtained using the BCG lipids; however, stimulation of resistance with listeria lipids was not successful and, finally, Freund's incomplete adjuvant was significantly effective in the Feldman index only. The BCG lipids, extracted with ethanol, which contained nitrogen and water-soluble substances, induced a significant resistance against M. tuberculosis infection. The chloroform-methanol BCG lipids were also effective, however, significantly less than the ethanol-extracted material. The listeria factor Ei itself or together with Freund's incomplete adjuvant possessed only low effectiveness against mycobacterial infection. However, if injected together with ethanol-extracted BCG lipids, it produced a significant degree of resistance which was higher than that induced by lipids only. The degree of resistance was comparable with the effect of living BCG strain which served as a source of isolated lipids.

Antitubercular 2,8-bis(alkylaminomethyl)phenazines.

The preparation and antitubercular properties of a series of 2,8-bis(alkylaminomethyl)phenazines are described. These compounds all inhibited the growth of Mycobacterium smegmatis ATCC 607 in vitro. 2,8-Bis(dibutylaminomethyl)phenazine (5c) was also active against a lethal Mycobacterium tuberculosis H37Rv infection in mice.