Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis.

Superoxide dismutase (SOD) is a ubiquitous metalloenzyme in aerobic organisms that catalyzes the conversion of superoxide anion to hydrogen peroxide. Mycobacterium tuberculosis is unusual in that it secretes large quantities of iron-cofactored SOD. To determine the role of SOD in pathogenesis, we constructed mutants of M. tuberculosis H37Rv with reduced SOD production. Compared with controls, SOD-diminished isolates were more susceptible to killing by hydrogen peroxide. The isolates were markedly attenuated, exhibiting nearly 100,000-fold fewer bacilli than virulent control strains in the lungs and spleens of C57BL/6 mice 4 wk after intravenous inoculation. In the lung, SOD-attenuated M. tuberculosis induced robust interstitial mononuclear cell infiltration within 24 h and many cells were apoptotic by TUNEL staining, whereas virulent H37Rv exhibited minimal early inflammatory response and only rare interstitial mononuclear cell apoptosis. During prolonged infections, C57BL/6 mice tolerated SOD-attenuated M. tuberculosis better than BCG, exhibiting 68% greater weight gain, quicker eradication of bacilli from the spleen, and less alveolar lung infiltration. These results establish the importance of SOD in the pathogenesis of tuberculosis. Its effect appears to be mediated in part by inhibiting innate host immune responses, including early mononuclear cell infiltration of infected tissues and apoptosis.

T cells enhance production of IL-18 by monocytes in response to an intracellular pathogen.

We studied the effect of T cells on IL-18 production by human monocytes in response to Mycobacterium tuberculosis. Addition of activated T cells markedly enhanced IL-18 production by monocytes exposed to M. tuberculosis. This effect was mediated by a soluble factor and did not require cell-to-cell contact. The effect of activated T cells was mimicked by recombinant IFN-gamma and was abrogated by neutralizing Abs to IFN-gamma. IFN-gamma also enhanced the capacity of alveolar macrophages to produce IL-18 in response to M. tuberculosis, suggesting that this mechanism also operates in the lung during mycobacterial infection. IFN-gamma increased IL-18 production by increasing cleavage of pro-IL-18 to mature IL-18, as it enhanced caspase-1 activity but did not increase IL-18 mRNA expression. These findings suggest that activated T cells can contribute to the initial immune response by augmenting IL-18 production by monocytes in response to an intracellular pathogen.

Naive human T cells develop into Th1 effectors after stimulation with Mycobacterium tuberculosis-infected macrophages or recombinant Ag85 proteins.

Most studies of human T-cell responses in tuberculosis have focused on persons with either active disease or latent infection. Although this work has been critical in defining T-cell correlates of successful versus failed host containment, little is known about the development of Mycobacterium-specific T-cell responses in uninfected persons. To explore this issue, naive T cells from uninfected donors were sensitized in vitro with avirulent Mycobacterium tuberculosis-infected autologous macrophages. T-cell lines primed in this manner proliferated and produced cytokines after challenge with mycobacterial antigens. Of 11 such lines, 8 were high Th1 responders, 2 were low Th1 responders, and 1 was a Th2 responder. Furthermore, similar patterns and magnitudes of proliferative and cytokine responses were seen when Mycobacterium infection-primed lines were challenged with recombinant antigen 85 (Ag85) proteins. The addition of interleukin 12 (IL-12) during the initial sensitization increased the magnitude of Th1 responses; however, antibody to IL-12 did not eliminate Th1 responses, suggesting that additional factors contributed to the differentiation of these cells. Finally, in the presence of IL-12, recombinant Ag85B was able to prime naive T cells for Th1 responses upon challenge with Mycobacterium-infected macrophages or Ag85B. Therefore, under the appropriate conditions, priming with whole bacteria or a subunit antigen can stimulate Mycobacterium-specific Th1 effector cell development. Further definition of the antigens and conditions required to drive naive human T cells to differentiate into Th1 effectors should facilitate the development of an improved tuberculosis vaccine.

Utility of bone marrow biopsy for rapid diagnosis of febrile illnesses in patients with human immunodeficiency virus infection.

BACKGROUND: Histochemical staining of bone marrow biopsy samples for microorganisms may provide a presumptive diagnosis weeks before culture. METHODS: To identify predictors of histochemical positivity, we reviewed 161 bone marrow biopsies from febrile patients with human immunodeficiency virus (HIV) infection. RESULTS: By multivariate analysis, both hematocrit value <30% and white blood cell count <4,000/mm3 predicted biopsy positivity by culture or staining, but only anemia predicted histochemical stain positivity. Of cases with serum lactate dehydrogenase (LDH) levels >600 U/L, histoplasmosis was diagnosed in 31.6% versus 7.8% with lower LDH levels. Among histoplasmosis cases, staining showed fungi in all, with LDH levels >600 U/L versus 44.4% with lower levels. CONCLUSIONS: Bone marrow biopsy will most likely provide a rapid diagnosis in patients with anemia. Markedly elevated LDH levels suggest stain positivity for Histoplasma capsulatum. Histopathologic patterns may also guide empiric therapy.

Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons.

A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins. To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E. coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase. Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E. coli 54-fold. The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes. Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, suggesting that the enhanced protein production was due primarily to enhanced efficiency of translation. Codon replacement in the genes encoding antigen 85A and superoxide dismutase yielded four- to sixfold increases in recombinant protein production, suggesting that this strategy may be generally applicable to overexpression of mycobacterial genes in E. coli.

Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis.

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics. beta-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses beta-lactam resistance. beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified. The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta-lactamase of M. tuberculosis is a class A beta-lactamase with predominant penicillinase activity. A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present.

Recombinant baculovirus influenza A hemagglutinin vaccines are well tolerated and immunogenic in healthy adults.

In a prospective double-blind trial, the reactogenicity and immunogenicity of recombinant baculovirus influenza A vaccines containing purified full-length hemagglutinin (HA) were compared with standard trivalent inactivated vaccine (TIV). The recombinant baculovirus influenza A vaccines (rHA0) were monovalent (containing 45 micrograms of A/Beijing/92[H3] and 15, 45, and 135 micrograms of A/Texas/91[H1]) and bivalent (containing 45 micrograms of both A/Beijing/92 and A/Texas/91). The bivalent rHA0 vaccine produced fewer local side effects than the TIV (50% vs. 88%, P = .003). The hemagglutinin inhibition (HAI) responses (defined as a > or = 4 increase in HAI) to A/Beijing rHA0 in the monovalent A/Beijing/92, the bivalent vaccine, and the TIV were 68%, 76%, and 46%, respectively (P = .086). Increasing doses of A/Texas rHA0 (15 micrograms [60%], 45 micrograms [69%], and 135 micrograms [76%]) and bivalent HA (76%) gave better immunologic responses to H1 than did TIV (31%; P = .003).