Metronidazole therapy in mice infected with tuberculosis.
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The capacity of metronidazole to inhibit the growth of Mycobacterium tuberculosis was tested in in vitro and in vivo mouse models. In vitro addition of metronidazole to cultures of infected bone marrow-derived macrophages had no effect, nor did it increase the reduction in bacterial load due to isoniazid. In vivo, metronidazole did not reduce bacterial numbers in the lungs of aerosol-infected mice during the active stage of the disease, during a phase of containment, or after prolonged isoniazid therapy (Cornell model). A small but significant reduction was seen if metronidazole therapy was given during an established chronic disease state 100 days after aerosol administration. These data indicate that under most conditions M. tuberculosis organisms are not in a metabolic state in which they are susceptible to the action of metronidazole and, hence, that this drug would be of limited clinical value. (+info)
An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic mycobacteria in axenic culture and in human monocytes: extracellular proteins as potential novel drug targets.
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Mycobacterium tuberculosis and other pathogenic mycobacteria export abundant quantities of proteins into their extracellular milieu when growing either axenically or within phagosomes of host cells. One major extracellular protein, the enzyme glutamine synthetase, is of particular interest because of its link to pathogenicity. Pathogenic mycobacteria, but not nonpathogenic mycobacteria, export large amounts of this protein. Interestingly, export of the enzyme is associated with the presence of a poly-L-glutamate/glutamine structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogenic and nonpathogenic mycobacteria and on the poly-L-glutamate/glutamine cell wall structure. The inhibitor L-methionine-S-sulfoximine rapidly inactivated purified M. tuberculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cultures of pathogenic mycobacteria, L-methionine- S-sulfoximine rapidly inhibited extracellular glutamine synthetase in a concentration-dependent manner but had only a minimal effect on cellular glutamine synthetase, a finding consistent with failure of the drug to cross the mycobacterial cell wall. Remarkably, the inhibitor selectively blocked the growth of pathogenic mycobacteria, all of which release glutamine synthetase extracellularly, but had no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms, none of which release glutamine synthetase extracellularly. The inhibitor was also bacteriostatic for M. tuberculosis in human mononuclear phagocytes (THP-1 cells), the pathogen's primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the amount of poly-L-glutamate/glutamine cell wall structure in M. tuberculosis. Although it is possible that glutamine synthetase inhibitors interact with additional extracellular proteins or structures, our findings support the concept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitute readily accessible targets of these relatively impermeable organisms, which are rapidly developing resistance to conventional antibiotics. (+info)
T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs.
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The three most abundant extracellular proteins of Mycobacterium tuberculosis, the 30-, 32-, and 16-kDa major extracellular proteins, are particularly promising vaccine candidates. We have mapped T-cell epitopes of these three proteins in outbred guinea pigs by immunizing the animals with each protein and assaying splenic lymphocyte proliferation against a series of overlapping synthetic peptides covering the entire length of the mature proteins. The 30-kDa protein contained nine immunodominant epitopes, the 32-kDa protein contained two immunodominant epitopes, and the 16-kDa protein contained a highly immunodominant region at its N terminus. The immunodominant epitopes of the 30- and 32-kDa proteins in outbred guinea pigs were frequently identified in healthy purified-protein-derivative-positive or BCG-vaccinated individuals in previous studies. The immunodominant epitopes of these major extracellular proteins have potential utility in an epitope-based vaccine against tuberculosis. (+info)
Mice deficient in CD4 T cells have only transiently diminished levels of IFN-gamma, yet succumb to tuberculosis.
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CD4 T cells are important in the protective immune response against tuberculosis. Two mouse models deficient in CD4 T cells were used to examine the mechanism by which these cells participate in protection against Mycobacterium tuberculosis challenge. Transgenic mice deficient in either MHC class II or CD4 molecules demonstrated increased susceptibility to M. tuberculosis, compared with wild-type mice. MHC class II-/- mice were more susceptible than CD4-/- mice, as measured by survival following M. tuberculosis challenge, but the relative resistance of CD4-/- mice did not appear to be due to increased numbers of CD4-8- (double-negative) T cells. Analysis of in vivo IFN-gamma production in the lungs of infected mice revealed that both mutant mouse strains were only transiently impaired in their ability to produce IFN-gamma following infection. At 2 wk postinfection, IFN-gamma production, assessed by RT-PCR and intracellular cytokine staining, in the mutant mice was reduced by >50% compared with that in wild-type mice. However, by 4 wk postinfection, both mutant and wild-type mice had similar levels of IFN-gamma mRNA and protein production. In CD4 T cell-deficient mice, IFN-gamma production was due to CD8 T cells. Thus, the importance of IFN-gamma production by CD4 T cells appears to be early in infection, lending support to the hypothesis that early events in M. tuberculosis infection are crucial determinants of the course of infection. (+info)
TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages.
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The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival. (+info)
Genetically susceptible mice remain proportionally more susceptible to tuberculosis after vaccination.
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DBA/2 mice are much more susceptible to infection with Mycobacterium tuberculosis than major histocompatibility complex-compatible BALB/c mice. It is shown here that, although vaccination provided mice of both strains with a capacity to reduce the level of infection in their lungs, vaccinated DBA/2 mice remained much more susceptible in this organ than vaccinated BALB/c mice. Consequently, the former mice developed more lung pathology and died much earlier than the latter. On the other hand, colony-forming unit counts and histology suggest that vaccination provided mice of both strains with an increased and equal ability to express immunity in the liver and spleen, thereby indicating that they possessed equal systemic levels of vaccine-induced immunity at the time of M. tuberculosis challenge. The results indicate that inefficient expression of immunity in the lungs is likely to prove an obstacle to successful vaccination against tuberculosis in resistant and susceptible mouse strains, but more so in the latter strains. (+info)
Co-immunization with DNA vaccines expressing granulocyte-macrophage colony-stimulating factor and mycobacterial secreted proteins enhances T-cell immunity, but not protective efficacy against Mycobacterium tuberculosis.
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The development of more effective antituberculosis vaccines would assist in the control of the global problem of infection with Mycobacterium tuberculosis. One recent vaccination strategy is immunization with DNA plasmids encoding individual microbial genes. Using the genes for the M. tuberculosis-secreted proteins, MPT64 (23 000 MW) and Ag85B (30 000 MW) as candidate antigens, we previously prepared DNA vaccines and demonstrated their ability to stimulate T-cell responses and confer protection in a mouse model of aerosol tuberculosis (TB). The protective efficacy of the DNA vaccines was less than that promoted by the current vaccine Mycobacterium bovis bacille Calmette-Guerin (BCG). To improve the immunogenicity and protective efficacy of these mycobacterial vectors, co-immunization of a plasmid expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated. Intramuscular immunization with DNA expressing MPT64 or Ag85B and GM-CSF enhanced the antigen-specific cellular immune response, with increased proliferative response and production of interferon-gamma (IFN-gamma). The titre of antimycobacterial protein immunoglobulin G (IgG) antibodies was unchanged. Mice immunized with DNA vaccines showed reduced pulmonary bacterial load following an aerosol challenge of M. tuberculosis, but codelivery of the plasmid expressing GM-CSF did not increase the protective effect. Therefore, despite modifying the cellular immune response to DNA vaccines, GM-CSF does not improve their protective efficacy at the peak of infection after an aerosol challenge with 100 c.f.u. of M. tuberculosis. (+info)
Dominant recognition of a cross-reactive B-cell epitope in Mycobacterium leprae 10 K antigen by immunoglobulin G1 antibodies across the disease spectrum in leprosy.
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Mycobacterium leprae-specific immunoglobulin G1 (IgG1) antibodies in patients with leprosy show a direct correlation with bacterial load (rho=0.748; P<0002) suggesting that IgG1 B-cell responses may be surrogate markers of disease progression. To investigate if this upregulation was a general feature of IgG1 responses to all M. leprae (ML) antigens, we analysed responses to several recombinant purified ML heat-shock proteins (HSP). Three recombinant HSPs (ML10 K, ML 18 K and ML 65 K) were tested for their ability to induce various IgG subclasses in patients with either the lepromatous (LL/BL, n=26) or tuberculoid form (BT/TT, n=39) of the disease as well as in healthy households (HC, n=14) and endemic controls (EC=19). Our major findings were: (1) selective augmentation of IgG1 antibody responses to ML10 K; (2) recognition of a restricted number of epitopes across the disease spectrum and healthy controls by IgG1 antibodies; (3) dominant recognition of cross-reactive epitopes which were common to both ML and MT 10 K. This response was not related to contamination with endotoxin. Epitope mapping using 15-mer overlapping peptides spanning the ML 10 000 MW revealed an immunodominant IgG1 binding peptide (aa41-55) in patients as well as healthy controls. This peptide is a shared epitope with M. tuberculosis 10 K suggesting that postswitched IgG1 B cells recognizing this epitope rather than naive B cells are being expanded. (+info)