Sequestration of T lymphocytes to body fluids in tuberculosis: reversal of anergy following chemotherapy.
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The specificity of CD4 T lymphocytes was investigated in 6 patients affected by tuberculosis who had negative tuberculin purified protein derivative (PPD) skin tests at diagnosis. Polyclonal CD4 T cell lines from the peripheral blood failed to proliferate to PPD and to the 16- or 38-kDa proteins of Mycobacterium tuberculosis, while CD4 cell lines from the disease site responded to PPD and to the 16- and 38-kDa proteins and derived epitopes in vitro. Four months after chemotherapy, the patients became responsive to PPD. The proliferative response to PPD and to the 16- or 38-kDa proteins and their derived peptides decreased in CD4 T cell lines from the disease site and increased in lines from the peripheral blood. These results indicate that CD4 T cells recognizing a vast array of M. tuberculosis epitopes are compartmentalized at the site of disease in anergic patients but appear in peripheral blood after chemotherapy. (+info)
Amplification of a 500-base-pair fragment from cultured isolates of Mycobacterium bovis.
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The presence of a 500-bp fragment which amplifies a region from the genome of Mycobacterium bovis (J. G. Rodriguez, G. A. Meija, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131-2138, 1995) was evaluated by carrying out PCR on 121 M. bovis isolates. The M. bovis strains, previously characterized by culture and biochemical tests, were isolated from cattle in different regions of Argentina, Mexico, and Colombia. Four additional strains isolated from sea lions that belong to the M. tuberculosis complex were also included in the study. All of the isolates tested were PCR positive, rendering the expected 500-bp band and giving a correlation of 100% with previous microbiological characterization. Southern blot analysis revealed a common band of 1, 800 bp and a polymorphic high-molecular-mass hybridization pattern. The results show that this assay may be useful for diagnosis and identification of M. bovis in cattle. (+info)
Inactivation of Mycobacterium tuberculosis for DNA typing analysis.
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DNA fingerprinting analysis of Mycobacterium tuberculosis is used for epidemiological studies and the control of laboratory cross-contamination. Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens. The technique ensures the inactivation of M. tuberculosis before DNA extraction without the loss of DNA integrity. The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months. (+info)
T-Cell hyporesponsiveness induced by activated macrophages through nitric oxide production in mice infected with Mycobacterium tuberculosis.
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In active tuberculosis, T-cell response to Mycobacterium tuberculosis is known to be reduced. In the course of Mycobacterium tuberculosis infection in mice, we observed that T-cell proliferation in response to M. tuberculosis purified protein derivative (PPD) reached the maximum level on day 7, then declined to the minimal level on day 14, and persisted at a low level through day 28 postinfection. The frequency of PPD-specific CD4 T cells in the spleen on day 28 decreased to one-sixth on day 7. To further investigate the mechanism of this T-cell hyporesponsiveness, we next analyzed the suppressive activity of spleen macrophages on T-cell function. The nonspecific proliferative response of naive T cells and the PPD-specific proliferative response of T cells were suppressed by day 28 macrophages, but not by day 7 macrophages or naive macrophages. This reduction of proliferative response was restored by addition of nitric oxide synthesis inhibitor, NG-monoethyl-L-arginine monoacetate, but not by monoclonal antibody against interleukin 10 or transforming growth factor beta. These data indicate that the macrophages from mice chronically infected with M. tuberculosis suppress T-cell response through production of nitric oxide, suggesting that nitric oxide-induced elimination mediated by activated macrophages may reduce the T-cell response and the number of mycobacterium-specific CD4 T cells in vivo. (+info)
Increase in gamma interferon-secreting CD8(+), as well as CD4(+), T cells in lungs following aerosol infection with Mycobacterium tuberculosis.
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Although it is well established that CD4(+) T cells are required for the protective immune response against tuberculosis (TB), there is some evidence that CD8(+) T cells are also involved in the host response to Mycobacterium tuberculosis. There is, however, a paucity of information on the pulmonary CD8(+) T-cell response during infection. We therefore have compared the changes in both CD8(+) and CD4(+) T cells following aerosol infection with M. tuberculosis. There was an observed delay between the peak of infection and the activated T-cell response in the lung. The kinetics of CD8(+) and CD4(+) T-cell responses in the lung were identical, both peaking at week 8, 4 weeks later than the peak of cellular response in draining lymph nodes. Similar changes in activation/memory phenotypes occurred on the pulmonary CD8(+) and CD4(+) T cells. Following in vitro restimulation, both subsets synthesized gamma interferon, a cytokine essential for controlling M. tuberculosis infection. Since lung CD8(+) T cells are actively expanded during aerosol M. tuberculosis infection, it is important that both CD8(+) and CD4(+) T cells be targeted in the design of future TB vaccines. (+info)
Role of CD40 ligand in Mycobacterium avium infection.
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Mycobacterium avium is a common opportunistic pathogen in immunocompromised patients such as those infected with human immunodeficiency virus. Although M. avium is an intracellular organism replicating predominantly in macrophages, disseminated M. avium infection is seen in AIDS patients with CD4(+) cell counts of <50 cells/microliters, suggesting a possible involvement of a T cell-macrophage interaction for the elimination of M. avium. To determine whether CD40-CD40 ligand (CD40L) interactions play a role in M. avium infection, we studied the ability of CD40L to restrict M. avium replication in human monocyte-derived macrophages (MDM) in vitro. MDM were infected with M. avium and cocultured with CD40L-transfected 293 cells for 7 days. Intracellular growth of M. avium in these MDM was assessed by colony counting. CD40L-expressing cells inhibited growth of M. avium in MDM by 86.5% +/- 4.2% compared to MDM cultured with control cells. These findings were verified by assays using purified, soluble recombinant human CD40L (CD40LT). CD40LT (5 micrograms/ml) inhibited intracellular growth of M. avium by 76.9% +/- 18.0% compared to cells treated with medium alone. Inhibition by CD40LT was reduced by monoclonal antibodies (MAbs) against CD40 and CD40L. The inhibitory effect of CD40LT was not accompanied by enhancement of interleukin-12 (IL-12) production by M. avium-infected MDM, while CD40L-expressing cells stimulated IL-12 production by these cells. Treatment of M. avium-infected mice with MAb against murine CD40L resulted in recovery of larger numbers of organisms (0.8 to 1.0 log) from the spleens, livers, and lungs of these animals compared to infected mice which received normal immunoglobulin G. These results indicate that CD40-CD40L signaling may be an important step in host immune response against M. avium infection. (+info)
Fatal granuloma necrosis without exacerbated mycobacterial growth in tumor necrosis factor receptor p55 gene-deficient mice intravenously infected with Mycobacterium avium.
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The pathogenesis of mycobacterial infections is associated with the formation of granulomas in which both antibacterial protection and tissue damage take place concomitantly. We used murine Mycobacterium avium infection to compare the development of granulomatous lesions in intravenously infected tumor necrosis factor receptor p55 (TNFRp55) gene-deficient (p55(-/-)) mice to the development of granulomatous lesions in M. avium-infected syngeneic C57BL/6 (p55(+/+)) mice. Up to 5 weeks after infection with either the highly virulent M. avium strain TMC724 or the intermediately virulent M. avium strain SE01, bacterial counts in the liver, spleen, and lung of p55(-/-) mice were identical to or lower than those in infected p55(+/+) mice. However, the formation of mononuclear cell foci in the liver was delayed by approximately 2 to 3 weeks in p55(-/-) mice compared to the results obtained for infected p55(+/+) mice. Despite comparable bacterial loads, granulomas in p55(-/-) mice underwent progressive necrosis, causing damage to the surrounding liver tissue. The appearance of necrotizing granulomas was associated with the death of all infected p55(-/-) mice, regardless of the virulence of the M. avium strain used for infection. Granulomatous lesions in the liver contained three times as many CD3(+) cells in p55(-/-) mice yet appeared more diffuse than in p55(+/+) mice. Semiquantitative reverse transcription-PCR studies revealed that prior to mouse death, interleukin-12 (IL-12) and gamma interferon mRNA levels were up regulated in the livers of infected p55(-/-) mice, while mRNA levels for tumor necrosis factor, the inducible isoform of nitric-oxide synthase (iNOS), and IL-10 were similar to those found in infected p55(+/+) mice. In response to persistent mycobacterial infection, the absence of TNFRp55 causes the disregulation of T-cell-macrophage interactions and results in fatal granuloma necrosis even when adequate antibacterial functions are maintained. (+info)
Resistance of virulent Mycobacterium avium to gamma interferon-mediated antimicrobial activity suggests additional signals for induction of mycobacteriostasis.
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The cytokine gamma interferon (IFN-gamma) plays a major role in the control of Mycobacterium avium infections. We assessed whether the progressive growth of virulent strains of M. avium was associated with alterations in the production of this cytokine as evaluated by reverse transcription-PCR and detection of immunoreactive cytokine in the serum and in spleen homogenates. We found that IFN-gamma was induced during infection by a virulent strain of M. avium to similar or even higher extents than the levels found during infections by a less virulent strain whose growth was controlled. IFN-gamma produced during infection by both mycobacterial strains was partly derived from T cells and led to activation of macrophages, namely, those that were infected. Concomitant with the development of the infection with the virulent strain of M. avium there was an extensive depletion of lymphocytes in the spleen. Thymectomy alone promoted the proliferation of the virulent, but not of the less virulent, strain of M. avium. Our data indicate that virulent strains of M. avium resist the antimicrobial mechanisms of IFN-gamma-activated macrophages and raise the possibility that a second, T-cell-dependent signal is required for the effective control of mycobacterial replication inside macrophages. (+info)