• IMMUNE NETWORK
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• v.7 no.1
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• pp.26-30
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• 2007

Background: Mycobacterial antigens released as PIM, LM, LAM, lipoproteins and other cellular factors may contribute to macrophage and dendritic cell activation through pattern recognition receptors such as TLRs. In this study, we assessed cytokine production and ERK activation with stimulation of several major mycobacterial antigens. Methods: Purified mycobacterial antigens (10, 22, 30, 38kDa) and recombinant antigens (6, 16, 19, 38kDa, Ag85A antigen) were studied. The production of cytokines (TNF-${\alpha}$, IL-12, IL-6) was measured by ELISA. The ERK activation was detected by western blotting. The expression of TLR2 or TLR4 was measured by flow cytometry. Results: Among purified antigens only 30kDa antigen induced production of IL-6 or TNF-${\alpha}$ in THP-1 macrophage cells. When THP-1 macrophage cells were treated with 30kDa antigen, phosphorylation of ERK was detected. ERK activation also occurred in TLR2 transfectant HEK293 cells with 30kDa antigen stimulation. Conclusion: 30kDa antigen is one of the major mycobacterial antigens inducing cytokine production and MAP kinases phosphorylation in macrophages.

• Parasites, Hosts and Diseases
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• v.50 no.4
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• pp.281-286
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• 2012

Helminthic infections afflict over 1.5 billion people worldwide, while Mycobacterium tuberculosis infects one third of the world's population, resulting in 2 million deaths per year. Although tuberculosis and helminthic infections coexist in many parts of the world, and it has been demonstrated that the T-helper 2 and T-regulatory cell responses elicited by helminths can affect the ability of the host to control mycobacterial infection, it is still unclear whether helminth infections in fact affect tuberculosis disease. In this review article, current progress in the knowledge about the immunomodulation induced by helminths to diminish the protective immune responses to bacille Calmette-Guerin vaccination is reviewed, and the knowledge about the types of immune responses modulated by helminths and the consequences for tuberculosis are summarized. In addition, recent data supporting the significant reduction of both M. tuberculosis antigen-specific Toll-like receptor (TLR) 2 and TLR9 expression, and pro-inflammatory cytokine responses to TLR2 and TLR9 ligands in individuals with M. tuberculosis and helminth co-infection were discussed. This examination will allow to improve understanding of the immune responses to mycobacterial infection and also be of great relevance in combating human tuberculosis.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.119-123
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• 1993

The present study was intended to use the avidin-biotin-peroxidase-antiperoxidase complex (ABPAP) method for the identification of Mycobacterium bovis in the tissue sections of infected cattle. Antibodies and linksera for ABPAP procedure used in incubated order were rabbit anti-Mycobacterium polyvalent antibodies, goat anti-rabbit IgG, rabbit peroxidase-antiperoxidase complex, biotinyl-horse anti-rabbit IgG, and avidin-biotin-peroxidase complex. Where the bacterial antigen was localized by ABPAP, a dark brown deposit occurred in the cytoplasms of macrophages and Langerhans' giant cells of the granulomatous lesions. The method approved to be highly specific for the identification of the bacteria and allowed a precise localization of the bacterial antigen in infected cells.

• Tuberculosis and Respiratory Diseases
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• v.39 no.1
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• pp.1-6
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• 1992

The diagnosis of tuberculosis is usually established using staining and culturing techniques. Fluorescent stains have improved the sensitivity of direct microscopy. Improved culture media coupled with radiometric means of detecting early mycobacterial growth have shortened the time needed for cultural diagnosis. Rapid immunodiagnostic techniques based on the detection of mycobacterial antigen or of antibodies to theses antigens have not, however, come into widespread clinical use. The DNA or RNA hybridization tests with labeled specific probes which have been described so far are not sensitive enough to be used for clinical speicimens without prior culturing. The advent of the polymerase chain reaction (PCR) has opened new possibilities for diagnosis of microbial infections. This technique has already been applied to a number of microorganisms. In the field of mycobacteria the PCR has been used to identify and to detect DNAs extracted from various mycobacteria. However, despite the extraordinary enthusiasm surrounding this technique and the considerable investiment, PCR has not emerged from the developmental "trenches" in the passed several years. It may be a considerable lenth of time before clinical microbiology laboratories become PCR playgrounds because many details remain to be worked out.

• The Journal of the Korean Society for Microbiology
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• v.20 no.1
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• pp.79-89
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• 1985

It is important to discriminate between tuberculosis and tuberculosis-like disease by Mycobacteria other than tuberculosis in the serodiagnosis of tuberculosis. But because common antigens share among Mycobacteria, their antigenicities to human are similar. Therefore degree of cross-reactivity of antibody in the sera of patients with tuberculosis between M. tuberculosis and Mycobacteria other than tuberculosis should be checked to increase the specificity in the serodiagnosis of tuberculosis. The activity levels of IgG antibody in the sera of 106 patients confirmed as active pulmonary tuberculosis and 30 normal healthy control person to the pressate extract antigen (TE, BE, AE, and FE antigen) from M. tuberculosis, M. bovis, M. avium, and M. fortuitum were measured by enzyme-linked immunosorbent assay and the crossreactivity of IgG antibody with mycobacterial species was analysed. The results were as follows; 1. The activity level(O.D. at 492nm) of IgG to TE antigen in sera of patients with pulmonary tuberculosis was $0.228{\pm}0.167$ in minimal tuberculosis; moderately advanced, $0.556{\pm}0.616$; far advanced, $1.116{\pm}0.651$ and $0.315{\pm}0.245$ in miliary tuberculosis. 2. The activity level (O.D. at 492nm) of IgG to BE antigen in sera of patients with pulmonary tuberculosis was $0.190{\pm}0.162$ in minimal tuberculosis; moderately advanced, $0.337{\pm}0.361$; far advanced, $0.713[\pm}0.460$ and $0.204{\pm}0.162$ in miliary tuberculosis. 3. The activity level (O.D. at 492nm) of IgG to AE antigen in sera of patients with pulmonary tuberculosis was $0.165{\pm}0.114$ in minimal tuberculosis; moderately advanced, $0.392{\pm}0.494$; far advenced, $0.751{\pm}0.512$ and $0.233{\pm}0.191$ in miliary tuberculosis. 4. The activity level (O.D. at 492nm) of IgG to FE antigen in sera of patients with pulmonary tuberculosis was $0.280{\pm}0.227$ in minimal tuberculosis; moderately advanced, $0.460{\pm}0.564$ ; far advanced, $0.845{\pm}0.573$ and $0.257{\pm}0.103$ in miliary tuberculosis. 5. The activity level (O.D. at 492nm) of IgG in sera of healthy control person was $0.126{\pm}0.084$ to TE antigen. $0.105{\pm}0.041$ to BE antigen, $0.103{\pm}0.052$ to AE antigen, and $0.095{\pm}0.061$ to FE antigen. 6. Degree of correlation(r) in activity level of IgG between TE antigen and BE antigen was 0.905 ; between TE antigen and AE antigen, 0.760; between TE antigen and FE antigen, 0.790, and between AE antigen and FE antigen, 0.945. 7. As O.D. above 0.200 was determined positive for the serodiagnosis of pulmonary tuberculosis, the sensitivity and specificity in ELISA using TE antigen were 80% and 87% respectively, whereas in the case of using BE antigen, 66% and 100%; in the case of using AE antigen, 62% and 100%, and in the case of using FE antigen, 72% and 93%, respecitively.

• The Journal of the Korean Society for Microbiology
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• v.35 no.5_6
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• pp.358-358
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• 2000

• IMMUNE NETWORK
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• v.11 no.2
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• pp.123-133
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• 2011

Background: Mycobacterium tuberculosis (Mtb) heparin binding hemagglutinin (HBHA) is an Ag known to evoke effective host immune responses during tuberculosis infection. However, the molecular basis of the host immune response to HBHA has not been fully characterized. In this study, we examined the molecular mechanisms by which HBHA can induce the expression of proinflammatory cytokines in macrophages. Methods: HBHA-induced mRNA and protein levels of proinflammatory cytokines were determined in bone marrow-derived macrophages (BMDMs) using RT-PCR and ELISA analysis. The roles of intracellular signaling pathways for NF-${\kappa}B$, PI3-K/Akt, and MAPKs were investigated in macrophage proinflammatory responses after stimulation with HBHA. Results: HBHA robustly activated the expression of mRNA and protein of both TNF-${\alpha}$ and IL-6, and induced phosphorylation of NF-${\kappa}B$, Akt, and MAPKs in BMDMs. Both TNF-${\alpha}$ and IL-6 production by HBHA was regulated by the NF-${\kappa}B$, PI3-K, and MAPK pathways. Furthermore, PI3-K activity was required for the HBHA-induced activation of ERK1/2 and p38 MAPK, but not JNK, pathways. Conclusion: These data suggest that mycobacterial HBHA significantly induces proinflammatory responses through crosstalk between the PI3-K and MAPK pathways in macrophages.

• Korean Journal of Veterinary Research
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• v.52 no.2
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• pp.89-97
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• 2012

Mycobacterial cell-wall skeleton (CWS) is an immunoactive and biodegradable particulate adjuvant and has been tried to use for immunotherapy. The CWS of Mycobacterium bovis bacillus Calmette-Guerin (BCG-CWS) was studied as an universal vaccine vehicle for antigen conjugation, to develop potentially effective and safe vaccine. Although a variety of biological activities of BCG-CWS have been studied, the effects of BCG-CWS on spleen cells are not fully elucidated. Using MTT assay and trypan blue exclusion test, we found that BCG-CWS significantly enhanced the viability and proliferation of cells. Multiple clusters, indicating proliferation, were observed in BCG-CWS-treated spleen cells and surface marker staining assay revealed that BCG-CWS promoted the proliferation of $CD19^+$ B lymphocyte rather than $CD4^+$ or $CD8^+$ T lymphocyte. In addition, BCG-CWS up-regulated the expression of anti-apoptotic molecules such as bcl-2, bcl-xL. BCG-CWS increased the surface expression of CD25 and CD69 as well as IL-2 production of spleen cells, suggesting increased activation. Furthermore, BCG-CWS enhanced the antigen-specific cell proliferation and interferon-gamma production of spleen cells. Taken together, these results demonstrate the immunostimulatory effects of BCG-CWS on spleen cells via multiple mechanisms, providing valuable information to broaden the use of BCG-CWS in clinical and research settings.

• Tuberculosis and Respiratory Diseases
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• v.47 no.6
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• pp.757-767
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• 1999

Background: Diagnosis by smear and/or cultures of the Mycobacterium tuberculosis from body fluid or biopsy specimen is "Gold standard". However the sensitivity of the direct microscopy is relatively low and culture of mycobacteria is time consuming. Despite an explosion in the techniques of rapid identification of mycobacteria by molecular genetic means, it is laborious and expensive and then rapid, inexpensive serodiagnosis is interested in diagnosis of tuberculosis. But sensitivity and specificity of known serologic antigen is not full sufficient level and then new antigen develop and combination cocktails of new developed antigens by ELISA are needed. Method: To compare the efficacy of different mycobacterial specific antigen and to assess the applicability of the combination of several different antigens in the diagnosis of tuberculosis, five ELISA tests derived 14KDa, 16KDa, 19KDa, 23KDa, 38KDa were evaluated in 57 active pulmonary patient and 24 inactive post-therapy follow up patient and 48 normal control. Results: The optical densities of ELISA test with 14KDa, 16KDa, 19KDa, 23KDa, 38KDa were significantly higher in active tuberculosis cases than in normal control(P<0.001, P<0.001, P<0.027, P<0.001, P<0.001) and those with 16KDa, 38KDa were significant higher in active tuberculosis cases than in inactive post-therapy follow up cases(P<0.01. P<0.001) and those of 14KDa, 16KDa, 23KDa, 38KDa were significant higher in inactive post-therapy follow up cases than in normal control(P<0.008. P<0.01. P<0.006. P<0.001). The sensitivity of 14KDa, 16KDa, 19KDa, 23KDa, 38KDa in active pulmonary patient cases was 42.1%, 43.9%, 15.8%, 28.0%, 70.2%, respectively and the specificity of 14KDa, 16KDa, 19KDa, 23KDa, 38KDa in active pulmonary patient cases was 95.8%, 95.8%, 91.7%, 89.6%, 93.8%, respectively. The sensitivity and specificity of combination 38KDa with 16KDa was 87% and 93.7%. Conclusion: The sensitivity and specificity of new antigens for serodiagnosis of the tuberculosis still remains limited at around 70%, which makes its a poor diagnostic tool for disease confirmation. A combination of cocktail antigens provided by cut-off value adjustment for serodiagnosis of tuberculosis some improved diagnostic yield than single antigen serologic test.

• Tuberculosis and Respiratory Diseases
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• v.47 no.1
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• pp.13-25
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• 1999

Background: Ineffective cell-mediated immune response in human tuberculosis is associated with a depressed Thl cytokine response and reduced production of IFN-$\gamma$. Most persons infected with Mycobacterium tuberculosis are healthy tuberculin reactors with protective immunity, but a minority with ineffective immunity develop extensive pulmonary tuberculosis. The cell-mediated immune response is an important aspect of host resistance to mycobacterial infection and is believed to be tightly regulated by a balance between Th1 cytokines including IFN-$\gamma$, IL-12, IL-18, regulated on activation, normal T cell expressed and secreted (RANTES) and Th2 counterparts such as IL-4, monocyte chemoattractant protein-l (MCP-l). Methods: Proliferation and mRNA expression of IFN-$\gamma$, RANTES and MCP-l by RT-PCR in peripheral blood mononuclear cells (PBMCs) in response to in vitro stimulation with mycobacterial antigens were compared in pulmonary tuberculosis patients with cured and treatment failure and in tuberculin-positive and tuberculin-negative healthy subjects. Results: Defective proliferative responsiveness to aqueous TSP antigen was involved with treatment failure tuberculosis patients. Aqueous TSP antigen-induced IFN-$\gamma$ and RANTES mRNA expression was decreased in treatment failure tuberculosis patients compared with healthy tuberculin reactors and cured tuberculosis patients (23.1 % versus 90.0% for IFN-$\gamma$ and 46.2% versus 70.0% versus 46.2% for RANTES). The frequency of MCP-l mRNA expression to aqueous TSP antigen in treatment failure tuberculosis patients was greater than in healthy tuberculin reactors and cured tuberculosis patients (76.9% versus 40.0%). Conclusion: The increasing expression of MCP-1 mRNA in response to aqueous TSP antigen might be predicted to favor Th1 responses and restricted Th1 responses in treatment failure of pulmonary tuberculosis.