결핵균 30 kDa 단백항원 자극에 의하여 유도되는 IFN-Y 및 IL-4 mRNA 발현 양상과 세포독성능

백태현,조은경,박재하,박정규,김화중 충남대학교 생물공학연구소 1997 생물공학연구지 Vol.5 No.-

30 kDa protein antigen, a major secreted protein antigen of Mycobacterium tuberculosis, exhibits strong T cell stimulatory activity. In order to better understand the immunologic activities of 30 kDa antigen, lymphocyte proliferation by ^3H-thymidine incorporation, cytokine mRNA expression pattern using reverse transcription-polymerase chain reaction (RT-PCR), and cytotoxicity in response to in vitro stimulation of human peripheral blood mononuclear cells (PBMCs) with 30 kDa antigen were evaluated. Lymphoproliferative responses to 30 kDa and crude protein antigens in PBMCs of normal PPD-negative and positive donors were compared. As expected, PPD negative donors demonstrated no thymidine incorporation in response to 30 kDa and crude protein antigens, while PPD positive donors showed extensive proliferation to both antigens. Freshly isolated PBMCs were stimulated with 30 kDa antigen for 0, 6, 12, 24, 48 hr, 1 wk, and 2 wk and examined for the induction of IFN-γ and IL-4 mRNA using RT-PCR. The expression of IFN-γ mRNA was greatly augmented after 1 wk, and there was also early inductions at 6 and 24 hr, whereas IL-4 mRNA is consistently expressed through 0 to 48 hr and markedly decreased after 1 wk. In contrast, freshly isolated human tonsillar cells failed to express detectable level of IFN-γ mRNA but showed enhanced IL-4 mRNA expression after in vitro stimulation with 30 kDa antigen for 1 wk. Both natural killer (NK) and T cell cytotoxic activities induced by 30 kDa and crude antigens were also evaluated. PBMCs from PPD positive donors stimulated with 30 kDa antigen showed significantly higher NK and T cell cytotoxic activities than those in PPD negative donors. Crude antigen also induced similar level of cytotoxicity with that of 30 kDa antigen. These results suggest that 30 kDa antigen of M. tuberculosis may selectively activate Th1 cells and be a strong inducer of IFN-γ expression. In conclusion, 30 kDa antigen can be used as a standard T cell immunogen.

결핵성 수막염 환자에서 결핵균 30 kDa 및 32 kDa 단백항원에 대한 항체반응의 비교

백태현,김선영,이건수,김화중,김성호,조은경,박정규 충남대학교 의과대학 지역사회의학연구소 1996 충남의대잡지 Vol.23 No.2

The 30 and 32 kDa antigens from Mycobacterium tuberculosis H37Rv culture filtrates, identified as biologically important proteins in the immune responses against mycobacterial infection, were purified and used in enzyme linked ummunosorbent assay (ELISA) for the determination in specific IgG and IgM levels in cerebrospinal fluids of 15 patients with tuberculous meningitis and 17 controls with nontuberculous diseases. High reactivity to both antigens was observed in tuberculous meningitis. Mean IgG and IgM antibody levels differed significantly (P < 0.001) between patients and con trols. Mean IgG antibody levels were also higher than IgM levels in tuberculous meningitis. A comparison of the antibody levels against the 30 and 32 kDa antigen within the tuberculous meningitis patients showed higher IgG reactivity to the 30 kDa than to the 32 kDa antigen, suggesting that the antibody response of these patients is directed predominantly against the 30 kDa protein. However, the patterns of reactivity to 30/32 kDa, 30 kDa and 32 kDa antigen in individual subjects were similar. By the cut-off value adding 2 stsndard deviation to the mean absorbance of controls, the sensitivity and specificity of the IgG antibody to the 30 kDa antigen were 100% and 94.4%, respectively. These values were higher than those obtained by 30/ 32 kDa and 32 kDa antigen. From the above results, it is suggested that the 30 kDa antigen may be dominant antigen and more valuable in the diagnosis of tuberculous meningitis than 32 kDa antigen.

단클론 항체를 이용하여 정제한 톡소포자충 30 kDa 항원의 면역학적 특성

이영화,노태진,신대환,Lee, Yeong-Hwa,No, Tae-Jin,Sin, Dae-Hwan 대한기생충학회 1997 The Korean Journal of Parasitology Vol.35 No.1

The molecular weight 30 kDa membrane protein of Toxoplusma Sondii (Toxoplasma 30 kDa) apparently conserved in most strains of T. gondii and sera of infected hosts. The present study aimed to elucidate Toxoplasmc 30 kDa as a useful diagnotic antigen for serodiagnisis of toxoplasmosis by ELISA and for induction of protective immunity. Murine spleen cells immunized with the membrane antigen of T. gondii were fused with mouse Sp2/0-Ag 14 myeloma cells. Out of 8 clones selected, five were IgG2b, the others belonged to IgG 1 and IgG2a. The 30 kDa antigen was distributed mainly on the surface membrane of tachyzoites by indirect fluorescence method. Murine peritoneal macrophages which were activated by 30 kDa antigen produced more amounts of NO2 compared with crude antigen-treated group, however there were no significant differences in toxoplamacidal activity between the two groups. Higher specificity of Toxoplosma 30 kDa antigen was recognized for serodiagnosis of toxoplasmosis than the crude antigen. From these results, ToxopLasmo 30 kDa antigen enhances the cytotoxic effect of macrophages as well as a more reliable means for the serodiagnosis of toxoplasmosis by ELISA. Key words: Toxoplosma gondii, 30 kDa antigen (p30), mouse, serodiagnosis, macrophage, cytotoxicity. 톡소포자충(ToxopLasmn gondii)은 다양한 항원을 가지고 있으며, 이들 항원의 분석은 세 포매개성 면역반응 및 톡소포자충증의 면역학적 진단방법의 연구에 매우 중요하다 본 연구는 톡소포자충의 여러 단백질중 대부분의 충주(strain)에 존재하는 분자량 30 kDa의 단백질을 단클론 항체를 이용하여 분리한 후. 30 kDa 항원의 면역학적 특성을 초음파 추출 조항원과 비교 평가하였다. 톡소포자충의 세포막 항원으로 면역한 마우스 비장세포와 마우스 Sp2/0-Agl4 골수종세포를 융합하여 8개의 단클론 항체를 Western blot으로 확인하였다 이들 단클론 항체는 높은 특이성을 보였으며, $IgG_{2b}가{\;}5개,{\;}IgG^{1}이{\;}2개,{\;}IgG_{2a}$가 1개였다. 간접형광항체법으로 충체내 위치를 관찰한 결과. 30 kDa 항원은 tachyzoite의 표면 세포막에 주로 분포하였다. 단클론 항체와 CNBr-activated Sepharose 4B를 coupling하여 만든 immunoafrnity chromatography를 이용 하여 30 kDa 항원을 분리하였다. 분리한 30 kDa 항원으로 자극시킨 마우스 복강대식세포의 $NO_2^{-}$ 생산량은 초음파 추출 조항원 사용군에 비해 유의하게 증가하였으나 대식세포의 탐식능은 유의한 차이가 없었다. 또한 ELISA로 톡소포자충증을 진단시, 톡소포자충 30 kDa 항원 사용군은 조항원 사용군에 비해 민감도의 변화는 없었으나 특이성은 증가하였다 이상으로 보아 톡소포자충 30 kDa 항원은 감염 방어 면역 효과가 있었으며 진단에 이용시 특이성을 더 높일 수 있었다.

결핵균 단백항원 자극에 의한 대식세포의 TNF-α 및 IL-6 생성과 ERK 활성화

안혜정(Ahn, Hae-Jeong),조상래(Cho, Sang-Nae),백태현(Paik, Tae-Hyun),이정림(Lee, Jung-Lim),최인홍(Choi, In-Hong) 대한면역학회 2007 Immune Network Vol.7 No.1

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-α, 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-α 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.

결핵균 단백항원 자극에 의한 대식세포의 TNF-${\alpha}$ 및 IL-6 생성과 ERK 활성화

안혜정,조상래,백태현,이정림,최인홍,Ahn, Hae-Jeong,Cho, Sang-Nae,Paik, Tae-Hyun,Lee, Jung-Lim,Choi, In-Hong 대한면역학회 2007 Immune Network Vol.7 No.1

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.

결핵균항원에 대한 폐결핵환자의 임파구 증식반응에 관한 연구

백태현,김준배,박정규,김화중,조은경,최대경 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

T lymphocytes are thought to play a central role in cell mediated immune response. To study the T lymphocyte proliferative response to purified 30-kDa antigen from Mycobacterium tuberculosis H37Rv, peripheral blood lymphocytes(PBL) isolated from healthy controls and tuberculosis patients were stimulated with the 30-kDa antigen, crude antigen and PHA. Healthy controls and tuberculosis patients were divided into PPD(+), PPD(-) groups and AFB(-), AFB(+) groups, respectively. PBL proliferation were determined by the ^3H-thymidine incorporation assay and MTT colorimetric assay. PBL proliferation with 30-kDa and crude protein antigens measured by both methods were almost identical(r=0.74, p<0.001). The lymphocyte proliferation to 30-kDa antigen and crude antigen were singnificantly increased in PPD(+) healthy controls and tuberculosis patients when compared to those in PPD(-) controls, but response to PHA was no significant difference. Analysis to T cell subsets of proliferated lymphocytes performed by the indirect immunoalkaline phosphatase techniques were no difference between not only healthy controls and patients groups, but also stimulated antigen or PHA, and especially, at all groups, the percentage of T8 cell was higher than that of T4 cell.

결핵균 PPD, 30-kDa 및 TSP 항원에 의한 치료전 폐결핵환자 말초혈액 단핵구의 IL-12 및 TNF-α 생성능

송창화,조은경,이지숙,김대수,임재현,김운옥,남현희,김화중,백태현,박정규,Song, Chang-Hwa,Jo, Eun-Kyeong,Lee, Ji-Suk,Kim, Dae-Su,Lim, Jae-Hyun,Kim, Un-Ok,Nam, Hyeon-Hui,Kim, Hwa-Jung,Paik, Tae-Hyun,Park, Jeong-Kyu 대한면역학회 2001 Immune Network Vol.1 No.3

To determine if initial infection with Mycobacterium tuberculosis changes the balance of cytokines between T cells and macrophages, we evaluated interferon (IFN)-${\gamma}$), interleukin-12 (IL)-12, and tumor necrosis factor (TNF)-${\alpha}$ productions by peripheral blood mononuclear cells (PBMC) from 15 untreated active pulmonary tuberculosis (TB) patients and 12 healthy tuberculin reactors (HTR). Freshly isolated PBMC were stimulated with Triton X-100 solubilized protein (TSP), 30-kDa or purified protein derivatives (PPD) antigen for 6, 18 and 96 hours. IL-12 p40 production by antigen-stimulated PBMC from TB patients was significantly decreased compared with that in HTR. In addition, IFN-${\gamma}$ production was significantly depressed in TB patients than that in HTR at a 96-hr stimulation. However, TNF-${\alpha}$ production was significantly higher in antigen-stimulated PBMC from TB than that of HTR. A pronounced increase in IFN-${\gamma}$ protein followed neutralization of IL-10 in early TB patients. However, neutralization of TNF-${\alpha}$ did not significantly alter IFN-${\gamma}$ induction in PBMC from TB patients. There were no significantly differences in the cytokine productions among three proteins, TSP, 30-kDa or PPD antigen. These results indicate that development of TB may be strongly associated with dysregulated productions of IL-12, IFN-${\gamma}$ and TNF-${\alpha}$, during the initial immune responses to M. tuberculosis. Further understanding of operative cytokine networks during human immune cell responses to protein antigens of M. tuberculosis may improve strategies for vaccine development.

Increased IL-12, but Depressed IL-18 Production after In Vitro Stimulation with a 30-kDa Mycobacterial Antigen in Tuberculous Pleural Mononuclear Cells

Song, Chang-Hwa,Jo, Eun-Kyeong,Kim, Seong-Ho,Kim, Hwa-Jung,Suhr, Ji-Won,Paik, Tae-Hyun,Nam, Hyun-Hee,Lim, Jae-Hyun,Kim, Un-Ok,Lee, Ji-Sook,Park, Jeong-Kyu The Korean Society for Microbiology 2001 Journal of Bacteriology and Virology Vol.31 No.3

In this study, we investigated interleukin (IL)-18 and IL-12 following in vitro stimulation with either the 30-kDa or purified protein derivative (PPD) antigens (Ag) of pleural mononuclear cells from 12 cases of tubercular pleurisy (TB-PMC) and 8 cases of malignant pleurisy (MG-PMC). Ag-stimulated TB-PMC produced significantly more IL-12 than did MG-PMC and the levels correlated with those of IFN-${\gamma}$. Although elevated IL-18 levels were found in freshly isolated pleural fluids, in vitro IL-18 production in response to either Ag was dramatically decreased in TB-PMC. Pro-IL-18 mRNA was detected before and after Ag stimulation in TB patients. Supernatants from the Ag-stimulated TB-PMC significantly suppressed IL-18 production in normal peripheral blood mononuclear cells (PBMC) and primary malignant cells over an 18 h incubation period. In addition, this suppressive activity was not inactivated by either heat or trypsin. Our findings imply that modulation of IL-12 and IL-18 levels may contribute to the Th1 elevation induced in human TB-PMC by the 30-kDa and PPD antigens.

Elevated Levels of Interferon-inducible Protein-10 (IP)-10/CXCL10, but not of $Interferon-{\gamma}$, in Patients with Pulmonary Tuberculosis

Lee, Ji-Sook,Lee, Ji-Yeon,Choi, Hong-Hee,Son, Ji-Woong,Kim, Ki-Hye,Paik, Tae-Hyun,Jo, Eun-Kyeong The Korean Society for Microbiology 2007 Journal of Bacteriology and Virology Vol.37 No.3

Mycobacterial strains are potent inducers of cytokines/chemokines by mononuclear phagocytes, which constitute an important cellular component of the first line of defense in the innate immune system. Interferon $(IFN)-{\gamma}-inducible$ protein (IP-10 or CXCL10) is a potent chemoattractant; however, little is known about the IP-10 profiles attributable to the Th1 regulation associated with active tuberculosis (TB). In this study, we investigated the production of IP-10, interleukin (IL)-12 p40, and $IFN-{\gamma}$ by the peripheral blood mononuclear cells (PBMCs) of patients with active pulmonary TB in response to in vitro stimulation with Triton X-100 soluble proteins (TSPs) or the 30-kDa antigen. The TSP antigens used in the present study were isolated and purified from Mycobacterium tuberculosis H37Rv (virulent strain), M. tuberculosis H37Ra (avirulent strain), and Mycobacterium bovis BCG. The results were compared with those obtained for healthy tuberculin reactors (HTRs). Concordant with earlier studies, $IFN-{\gamma}$ production was significantly depressed in the PBMCs from TB patients compared with those in the HTR group. However, the IP-10 levels in the PBMCs from TB patients were significantly elevated 18 h after stimulation compared to those in the PBMCs from HTRs. IP-10 release was correlated in a significant manner with the release of $IFN-{\gamma}$in the HTRs, but this was not the case for the TB patients. Collectively, these data suggest that TB patients show altered regulation of Th1-driving cytokine and chemokine production in response to a variety of mycobacterial antigens.

Depressed CCL5 Expression in Human Pulmonary Tuberculosis

Lee, Ji-Sook,Kim, Ki-Hye,Lee, Da-Youn,Choi, Hong-Hee,Lee, Hye-Mi,Son, Ji-Woong,Paik, Tae-Hyun,Jo, Eun-Kyeong The Korean Society for Microbiology 2008 Journal of Bacteriology and Virology Vol.38 No.3

CCL5/regulated on activation, normal T expressed and secreted production (RANTES) is a principal CC chemokine, and can activate macrophages and Th1 lymphocytes, however, little is known about the CCL5 profiles associated with active tuberculosis (TB). In this study, we investigated the production of CCL5 by the peripheral blood mononuclear cells (PBMCs) of patients with active pulmonary TB after stimulation with Triton X-100 soluble proteins (TSP) or the 30-kDa antigen. The profiles of cytokines/chemokines [CXCL8/interleukin (IL)-8, IL-12 p40, and interferon (IFN)-$\gamma$] were also examined by PBMCs from TB patients, and compared with those obtained from healthy tuberculin reactors (HTR). Concordant with earlier studies, IFN-$\gamma$ production was significantly depressed in the PBMCs from TB patients compared with those from HTR. In addition, the CCL5, but not CXCL8, levels in the PBMCs from TB patients were significantly depressed after stimulation for 18 hr compared to those in the PBMCs from HTRs. The CCL5 release was not significantly correlated with the release of IFN-$\gamma$ in the cells from TB patients and HTRs. Further, inhibitor studies show that the 30-kDa- or TSP-induced CCL5 mRNA expression is sensitive to inhibitors of mitogen-activated protein kinase kinase (MEK) 1/2 and Janus kinase (JAK) 2, but not p38, pathway activation, suggesting a MEK1/2- or JAK2-based mechanism is responsible for modulating of the CCL5 expression in human PBMCs. Collectively, these data suggest that TB patients show depressed production of CCL5 secretion, which can be modulated by MEK- and JAK2-based transcriptional regulatory mechanisms, in response to the mycobacterial antigens.