Early Secreted Antigenic Target of 6 kDa of Mycobacterium tuberculosis Stimulates Macrophage Chemoattractant Protein-1 Production by Macrophages and Its Regulation by p38 Mitogen-Activated Protein Kinases and Interleukin-4.

Early secreted antigenic target of 6 kDa (ESAT-6), the major virulence factor of Mycobacterium tuberculosis, affects host immunity and the formation of granulomas likely through inflammatory cytokines. To understand its role in this regard further, we investigated the effect of ESAT-6 on macrophages by determining the production of macrophage chemoattractant protein (MCP)-1, a major chemokine associated with tuberculosis pathogenesis, by murine bone marrow-derived macrophages (BMDMs) and its regulation by protein kinases and cytokines. The results revealed that ESAT-6, but not Ag85A and culture filtrate protein 10 kDa (CFP10), induced MCP-1 production by BMDMs dose and time dependently. Inhibition of p38 but not other mitogen-activated protein kinases (MAPK) and PI3K further enhanced ESAT-6-induced MCP-1 production by BMDMs. Inhibition of p38 MAPK enhanced ESAT-6-induced MCP-1 mRNA accumulation without affecting mRNA stability. ESAT-6 also induced TNF-α from BMDMs and MCP-1 from mouse lung epithelial cells, and these were suppressed by p38 MAPK inhibition, implying cytokine- and cell-specific effect of p38 MAPK inhibition on ESAT-6-induced MCP-1 by macrophages. Pretreatment of BMDMs with IL-4, but not other cytokines (IL-2, IL-10, TNF-α, IFN-γ and IL-1α) further elevated ESAT-6-stimulated MCP-1 production although IL-4 did not induce MCP-1 without ESAT-6. Both p38 MAPK inhibitor and IL-4 did not show additive effect on ESAT-6-induced MCP-1 protein level despite such effect on MCP-1 mRNA level was evident. In conclusion, these results indicate a specific role for both p38 MAPK and IL-4 in ESAT-6-induced MCP-1 production by macrophages and suggest a pathway with significance in tuberculosis pathogenesis.

Contribution of CD8(+) T cells to gamma interferon production in human tuberculosis.

The proportions of peripheral blood mononuclear cells (PBMC), CD4(+) T cells, and CD8(+) T cells that produce gamma interferon (IFN-gamma) in response to Mycobacterium tuberculosis were markedly reduced in tuberculosis patients, particularly in those with severe disease. Depletion of CD4(+) but not CD8(+) cells prior to stimulation of PBMC with M. tuberculosis abolished IFN-gamma production. These results show that (i) IFN-gamma production by CD8(+) and CD4(+) cells correlates with the clinical manifestations of M. tuberculosis infection and (ii) IFN-gamma production by CD8(+) cells depends on CD4(+) cells.

Cytokine profiles in immunocompetent persons infected with Mycobacterium avium complex.

To evaluate the immunologic factors that contribute to protection against Mycobacterium avium complex (MAC), cytokine production by peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus-negative persons with pulmonary MAC (MAC patients) and healthy control subjects with a delayed hypersensitivity skin test response to M. avium sensitin (MAS-positive control subjects) was measured. In MAC patients, mycobacterium-stimulated PBMC produced higher concentrations of interleukin (IL)-10 but lower concentrations of interferon (IFN)-gamma, IL-12, and tumor necrosis factor (TNF)-alpha, compared with PBMC from MAS-positive control subjects. Immunolabeling for intracellular IL-10 revealed that this cytokine was produced by both monocytes and T cells. Alveolar macrophages produced TNF-alpha and IL-10 in response to MAC, which suggests that these cytokines are produced in the lungs of patients with pulmonary disease caused by this pathogen. Our findings suggest that IFN-gamma, TNF-alpha, and IL-12 contribute to protection against MAC, whereas IL-10 is immunosuppressive.

Production of interleukin-18 in human tuberculosis.

To investigate the role of interleukin (IL)-18 in human tuberculosis, IL-18 production was evaluated in blood and at the site of disease in patients with tuberculosis. Mycobacterium tuberculosis-stimulated peripheral blood mononuclear cells (PBMC) from tuberculosis patients secreted less IL-18 and interferon-gamma (IFN-gamma) than did PBMC from healthy persons reactive to tuberculin. M. tuberculosis-induced IFN-gamma production was inhibited by anti-IL-18 and enhanced by recombinant IL-18. Alveolar macrophages secreted IL-18 in response to M. tuberculosis, and IL-18 and IFN-gamma concentrations were higher in pleural fluid of patients with tuberculosis than in pleural fluid of patients with nontuberculous diseases. These findings demonstrate that IL-18 production by PBMC correlates with IFN-gamma production and effective immunity to tuberculosis, suggesting that IL-18 contributes to a protective type 1 cytokine response in persons with mycobacterial infection.

Depressed CD40 ligand expression contributes to reduced gamma interferon production in human tuberculosis.

Expression of CD40 ligand (CD40L) correlated directly with Mycobacterium tuberculosis-stimulated gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) from tuberculosis patients and healthy tuberculin reactors. The CD40L agonist increased M. tuberculosis-induced IFN-gamma production by PBMC, and anti-CD40 or anti-CD40L antibodies reduced IFN-gamma production. CD40L expression on PBMC was reduced by exposure to B cells and to soluble factors from M. tuberculosis-infected monocytes. These findings suggest that CD40L dysregulation contributes to reduced IFN-gamma production in human tuberculosis.

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.

Cytokine production in children with tuberculous infection and disease.

To determine if the manifestations of initial infection with Mycobacterium tuberculosis reflect changes in the balance of T cell cytokines, we evaluated cytokine production by M. tuberculosis-stimulated peripheral blood mononuclear cells (PBMCs) from 24 children with tuberculosis and 22 children who were healthy tuberculin reactors. PBMCs from patients with tuberculosis had lower production and mRNA expression of interferon gamma (IFN-gamma) than did PBMCs from healthy tuberculin reactors. IFN-gamma production was most severely depressed in patients with moderately advanced and far-advanced pulmonary disease and in malnourished patients. Production of IL-12, IL-4, and IL-10 was similar in tuberculosis patients and healthy tuberculin reactors. These results indicate that, during the initial immune response to M. tuberculosis, development of tuberculosis is associated with diminished IFN-gamma production, which is not due to reduced production of IL-12 or enhanced production of IL-4 or IL-10.

Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages.

To determine whether the extent of spread of Mycobacterium tuberculosis strains in the community correlated with their capacity to replicate in human macrophages, intracellular growth rates of M. tuberculosis patient isolates were measured. Strain 210 caused disease in 43 patients in central Los Angeles, 3 "small-cluster" strains caused disease in 8-23 patients, and 5 "unique" strains each caused disease in only 1 patient who was positive by sputum acid-fast smear and spent substantial amounts of time at homeless shelters that were tuberculosis transmission sites. Strain 210 isolates grew significantly more rapidly than small-cluster and unique strains in macrophages. All strains elicited production of similar amounts of tumor necrosis factor-alpha, interleukin (IL)-6, IL-10, and IL-12 and were equally susceptible to reactive nitrogen intermediates. It was concluded that the extensive spread of an M. tuberculosis strain correlated with its capacity to replicate rapidly in human macrophages, which may be a marker of virulence.

[Separation of human bladder cancer cells from bone marrow with a kind of immunomagnetic microspheres].

By binding a monoclonal antibody BDI-1 to polystyrene magnetic micospheres using physical and chemical methods together, a kind of immunomagnetic microspheres (IMMS), which can specifically bind target cells and give it magnetic response, was sucessfully constituted. The results of indirect immunofluorescence and cell binding suggest that IMMS can effectively bind target cells while do not have non-specific binding activity. The influence of antibody added and the ratio between IMMS and cells were also studied. After purging cancer cells from bone marrow of mice with IMMS, the cancer cells were effectively removed while most of the bone marrow cells were recovered.

[Labeling of antihuman bladder carcinoma monoclonal antibody with a technetium-99m and radioimmunoimaging of human bladder carcinoma xenograft in nude mice].

Anti-human bladder carcinoma cell line BIU-87 monoclonal antibody-BDI-1 was labeled with 99mTc by direct mercaptoethanol reducing method. Quality control tests showed that the labeling yield was 69.9%; the radiochemical purity was higher than 90%; the immunoreactive fraction of 99mTc-BDI-1 was 81% and the association constant was 1.22 x 10(-9) M-1. The radioimmunoimaging of human bladder carcinoma xenograft in nude mice and the biodistribution of 99mTc-BDI-1 were studied. After having been scintigraphed at three time intervals (at 4, 16 and 22 hour), the animals were sacrificed for biodistribution of 99mTc-BDI-1. Tumor can be seen clearly at 22 hour after intravenous injection of 99mTc-BDI-1; %ID/g (percentage of the injected dose per gram of tissue) of tumor was 20.70, the average T/NT was 10.52, the minimum T/NT was 2.90 (tumor/kidney) and the maximum T/NT was 20.70 (tumor/small intestine or muscule). The results above indicated that 99mTc-BDI-1 can be used for radioimmunodetection of human bladder carcinoma in vivo and BDI-1 may be suitable as a targeting device in patients.

[Study on the specific killing activity of albumin nanoparticles containing adriamycin targeted by monoclonal antibody BDI-1 to human bladder cancer cells].

A highly specific immuno-nanoparticle (ADR-NP-Ab) has been constituted by chemically coupling a monoclonal antibody BDI-1 to albumin nanoparticle containing adriamycin (ADR-NP). Different molecular ratios of antibody to ADR-NP were tried to determine the optimal condition for preparing the immuno-nanoparticle. The results of immunoflurecence and microphotographic analysis showed that the activity of ADR-NP-Ab was well preserved. The result of the cytotoxicity of ADR-NP-Ab in vitro assay showed strong killing activity of ADR-NP-Ab to bladder cancer cells (EJ), while no apparent cytotoxic activity to non-targeted human colon carcinoma cells (Lovo) was observed.