Tubercle and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease最新文献

The Mycobacterium tuberculosis (MTB) SigF alternate sigma factor has been shown to have signficant homology to the Bacillus subtilis (BSU) stress-response sigma factor, SigB, as well as to the BSU developmental sigma factor, SigF. In this study we report that like both the BSU SigB and sigF genes, MTB sigF is preceded by an open reading frame (usfX) encoding a protein with significant homology to the previously described BSU anti-sigma factors, RsbW and SpollAB. Sequence analysis suggests that the usfX and sigF genes appear to be cotranscribed and translationally coupled. A second open reading frame called usfY precedes usfX, but has no significant homologues and may not be cotranscribed with the usfX and sigF. The sigF gene has been overexpressed in Escherichia coli, purified, and used to raise polyclonal antibodies. Immunoblotting demonstrates that MTB SigF is antigenically closer to BSU SigB than to BSU SigF. Fusion of the MTB sigF gene to the MTB hsp60 promoter has demonstrated that inappropriate overexpression of sigF is lethal for the slow-grower Mycobacterium bovis bacille Calmette-Guérin (BCG), but not for the rapid-grower Mycobacterium smegmatis which lacks a sigF homologue. Hence, sigF, encoding an MTB stress response, stationary phase transcription factor, is preceded by an antisigma factor homologue and is incompatible with growth when constitutively overexpressed in BCG.

There are several critical differences in the pulmonary granulomatous response to Mycobacterium tuberculosis between the mouse and other animal models such as the guinea pig or rabbit. One key difference is a conspicuous lack of central caseating necrosis in pulmonary lesions of immunologically intact mice. To determine whether normal mice could develop such pathology in response to highly virulent clinical isolates of M. tuberculosis, C57BL/6 mice were infected aerogenically with varying doses of three different strains, and the development of a granulomatous response was followed for as long as a year. Whereas such conditions failed to induce caseating necrosis in the lungs of these mice, all of the infections induced a granulomatous response which progressed similarly. We present here a descriptive report of the gross pathological progression of tuberculosis in the lungs of the mice. In each case, the disease progressed in five discrete stages, which were delineated on the basis of several criteria including the extent of granulomatous involvement, the cell types present, the degree of lymphocyte organization, and the presence of destructive sequelae such as airway epithelium erosion and airway debris. Quicker progression of disease along these five stages was induced by increasing the size of the inoculum or by the more virulent mycobacterial strains. The infections with the virulent strains were not resolved, and the later stages of the granulomatous response coincided with an increasing bacillary load and a loss of organized lymphocytes in the infected lungs which ultimately resulted in the death of the host. These results indicate that although C57BL/6 mice do not manifest a caseating form of pulmonary tuberculosis, they manifest an equally pathogenic granulomatous response which appears as a chronic interstitial fibrosing response that fails to contain the infection at a time that organized lymphocyte involvement wanes in the lung.

Setting: A study of multicase tuberculosis pedigrees from Northern Brazil.

Objective: To determine the model of inheritance for genetic susceptibility to tuberculosis, and to test the hypothesis that TNFA and NRAMP1 are candidate susceptibility genes.

Design: The study sample included 98 pedigrees, 704 individuals and 205 nuclear families. Segregation analyses were performed using the programs POINTER and COMDS. Combined segregation and linkage analysis was carried out within COMDS. Non-parametric linkage analyses were performed using BETA.

Results: A sporadic model for disease distribution in families was strongly rejected, as were polygenic and multifactorial models. A codominant single gene model provided the best fit (P< 0.001) to the data using POINTER. COMDS extended the analysis to compare single-gene and two-gene models. A general two-locus model for disease control was marginally favoured (0.01 < P< 0.05) over the codominant single-gene model. No evidence was found for linkage between susceptibility to disease per se and the TNF gene cluster. Weak linkage was observed using COMDS for genes (IL8RB, P = 0.039; D2S1471, P = 0.025) tightly linked (<150 kb) to NRAMP1, but not for NRAMP1 itself.

Conclusions: Tuberculosis susceptibility in this region of Brazil is under oligogenic control. Although a minor role for TNFA and NRAMP1 cannot be excluded, our data suggest that neither is a major gene involved in this oligogenic control.

Setting: Although nitric oxide (NO) is a major proximate mediator of microbicidal activity in murine macrophages against intracellular pathogens including mycobacteria, its production by and effector role in human macrophages is not clear.

Objective: To determine the capacity of Mycobacterium tuberculosis (MTB) to stimulate NO in human monocytes (MN) and alveolar macrophages (AM) and to assess the relationship between NO production and intracellular growth of MTB.

Design: NO production (measured as nitrite) by MTB (H37Ra)-infected macrophages and intracellular growth of MTB were measured in cells from 17 healthy subjects.

Results: MTB (5:1, MTB: cells) stimulated little to no NO by MN, but induced NO in AM at days 4 and 7 after infection. There was, however, variability in the response by AM to MTB: among seven subjects MTB-induced NO was low (4 ± 2 μM, mean ± SE); six subjects were moderate (56 ±11); four subjects were high (502 ± 167). NO synthase inhibitors inhibited the production of NO by AM but did not significantly affect the intracellular growth of MTB, although a trend towards increased intracellular growth was seen on day 4 of culture. Intracellular growth of MTB in AM from low NO producers was significantly higher than that in AM from moderate NO producers, P <- 0.05. Inducible NO synthase (iNOS) mRNA by RT-PCR was constitutively expressed by both MN and AM, but was further stimulated by MTB in AM > MN; MTB-induced iNOS protein was present in both MN and AM by Western blot analysis.

Conclusion: Thus, MTB-infected human AM are capable of producing NO and NO production correlates with intracellular growth inhibition of MTB in AM suggesting that NO may serve either directly or indirectly as a mycobactericidal mediator in human tissue macrophages.

Objective: Immunity to mycobacterial stress protein antigens was studied in response to vaccination and/or virulent infection.

Design: Guinea pigs, either vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), infected by the pulmonary route with virulent M. tuberculosis, or vaccinated then infected, were studied for the development of cellular and humoral immunity to two recombinant mycobacterial stress proteins, hsp 65 and hsp 70.

Results: Recombinant hsp 70 stimulated good proliferation in blood lymphocytes and, to a lesser extent, spleen and bronchotracheal lymph node lymphocytes from BCG-vaccinated guinea pigs. The proliferative responses to hsp 70 were diminished in both the spleen and lymph node cells following subsequent pulmonary challenge alone, but were boosted significantly by prior vaccination. Recombinant hsp 65 was much less active at inducing the proliferation of spleen and lymph node cells, with lowest responses observed in blood lymphocytes occurring in the cells from BCG-vaccinated, aerosol-challenged guinea pigs. Using a semi-quantitative dot blot procedure, serum antibodies to both hsp 65 and hsp 70 developed gradually following BCG vaccination, with all guinea pigs studied exhibiting significant seroreactivity after 15 weeks post-vaccination. In guinea pigs exposed to virulent M. tuberculosis by aerosol, serologic reactivity to hsp 70 was consistently stronger 6 weeks post-challenge in both vaccinated and non-vaccinated guinea pigs. In fact, 6 weeks following pulmonary exposure to M. tuberculosis in previously naive guinea pigs, 3 out of 6 animals had no detectable serum antibodies to hsp 65. Somewhat surprisingly, antibody levels to both hsp 65 and hsp 70 were only slightly increased by prior BCG vaccination in guinea pigs exposed to virulent M. tuberculosis by the respiratory route.

Conclusion: These results demonstrate that both hsp 65 and hsp 70 stimulate detectable humoral and cell-mediated immunity in guinea pigs vaccinated and/or infected under highly relevant conditions. There is little evidence that vaccination with BCG primes the guinea pig to make an anamnestic response to hsp 65 following virulent pulmonary challenge. The precise contribution of immunity to mycobacterial stress proteins to the pathogenesis of tuberculosis in this model remains to be elucidated.