• 대한임상검사과학회지
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• 제50권2호
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• pp.118-125
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• 2018

결핵은 본질적으로 MTB에 의해 발생하는 감염성 질환이지만 발병의 과정에는 숙주의 면역계와 연관성 있는 하는 유전자가 관여한다. ITGB2 유전자는 인테그린 beta 2 chain인 CD18 단백질을 암호화 하고 있는 유전자로 염색체 21번에 존재하고 있다. 인테그린 beat 2 chain은 백혈구에서 발현하는 인테그린으로 백혈구의 성숙 및 부착에 매우 중요한 역할을 수행하는 단백질이다. ITGB2는 결핵 발병에서 MTB의 탐식과 백혈구의 집합에도 중요한 역할을 수행한다고 알려졌다. 따라서 이번 연구는 한국인의 유전체 데이터를 활용하여 결핵 발생 환자들과 정상 대조군 사이에서 ITGB2의 유전적 다형성의 빈도에 통계적으로 유의한 차이가 존재하는지를 알아보고자 하였다. 그 결과 10개의 SNP에서 유의한 상관관계를 확인할 수 있었다. 가장 유의성 있는 SNP는 rs113421921 였다 (OR=0.69, CI: 0.53~0.90, $P=5.8{\times}10^{-3}$). 또한 rs173098의 경우는 전사 보조인자인 p300이 결합할 가능성이 있는 염기서열이 존재하여 유전적 다형성에 따라 ITGB2 유전자 발현에 영향을 미칠 수 있음을 확인할 수 있었다. 이러한 결과는 결핵의 발병 기전이 백혈구 집합이나 부착과 같은 숙주의 면역 기능과 관련된 다양한 유전적 요인에 의해 영향을 받을 수 있음을 시사한다. 이 연구결과는 결핵의 발병에 숙주 면역계의 유전자들이 영향을 줄 수 있다고 볼 수 있다. 이러한 결과들을 통해 MTB 감염에 대해 각 사람들 별로 감염의 진행과정과 결과에 차이를 가져다 주는 유전적 배경에 대한 이해에 기반을 제공할 것으로 기대한다.

• 대한의생명과학회지
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• 제16권2호
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• pp.119-122
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• 2010

Interleukin-1${\beta}$ $(IL-1{\beta})$ is one of the key proinflammatory cytokines and it plays an important role for the antimycobacterial host defense mechanisms. In this study, we examined Mycobacterium tuberculosis (MTB)-stimulated induction of IL-1${\beta}$ and evaluated the associated signal transduction pathways. In PMA-differentiated THP-1 cells, MTB infection increased mRNA expression of IL-$1{\beta}$ in a dose-dependent manner. The expression of IL-1${\beta}$ mRNA began to be induced at 1.5 h after infection, and induced expression of IL-1${\beta}$ was retained for 48 h after MTB infection. The increase in expression of IL-1${\beta}$ caused by MTB was reduced in cells treated with Ro-31-8425 (an inhibitor of PK$C{\alpha}$, ${\beta}I$, ${\beta}II$, ${\gamma}$, ${\varepsilon}$) or PD98059 (an inhibitor of MEK1), meanwhile, pre-treatment with $G\ddot{o}6976$ (an inhibitor of $Ca^{2+}$ dependent PK$C{\alpha}$ and PK$C{\beta}I$) or Rottlerin (an inhibitor of PK$C{\delta}$) has no effect on MTB-induced expression of $IL-1{\beta}$ mRNA. These results show that the expression of $IL-1{\beta}$ mRNA caused by MTB may be mediated via MEK1 and PKC isoforms including PK$C{\beta}II$, $PKC{\gamma}$, or $PKC{\varepsilon}$. Further studies are required to determine whether other PKC isoforms $(PKC {\eta},\;{\theta},\;{\varepsilon},\;and\;{\lambda}/{\iota})$, except $PKC{\delta}$, $PKC{\alpha}$, and $PKC{\beta}I$, are also involved in $IL-1{\beta}$ mRNA expression after mycobacterial infection.

• 대한의생명과학회지
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• 제25권4호
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• pp.426-430
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• 2019

Currently, molecular diagnostic assays based on nucleic acid amplification tests have been shown to effectively detect mycobacterial infections in various types of specimen, however, variable sensitivity was shown in FFPE samples according to the kind of commercial kit used. The present study therefore used automated PCR-reverse blot hybridization assay (REBA) system, REBA Myco-ID HybREAD 480^®, for the rapid identification of Mycobacterium species in various types of human tissue and compared the conventional one-tube nested-PCR assay for detecting Mycobacterium tuberculosis (MTB). In conventional nested-PCR tests, 25 samples (48%) were MTB positive and 27 samples (52%) were negative. In contrast, when conducted PCR-REBA assay, 11 samples (21%) were MTB positive, 20 samples (39%) were NTM positive, 8 samples (15%) were MTB-NTM double positive, and 13 samples (25%) were negative. To determine the accuracy and reliability of the two molecular diagnostic tests, the one-tube nested-PCR and PCR-REBA assays, were compared with histopathological diagnosis in discordant samples. When conducted nested-PCR assay, 10 samples (59%) were MTB positive and seven samples (41%) were negative. In contrast, when conducted PCR-REBA test, three samples (17%) were MTB positive, 10 samples (59%) were NTM positive and four samples (24%) were negative. In conclusion, the automated PCR-REBA system proved useful to identify Mycobacterium species more rapidly and with higher sensitivity and specificity than the conventional molecular assay, one-tube nested-PCR; it might therefore be the most suitable tool for identifying Mycobacterium species in various types of human tissue for precise and accurate diagnosis of mycobacterial infection.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.338-346
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• 2018

Two molecular epidemiologic methods, IS6110 restriction fragment length polymorphism (IS6110-RFLP) and 24-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR), are used worldwide in studies of Mycobacterium tuberculosis (MTB). Conversely, because of its poor resolution, pulsed-field gel electrophoresis (PFGE) is not widely used for MTB. In this study, we improved the 24-locus MIRU-VNTR and PFGE protocols and compared the effectiveness of these approaches for the molecular typing of MTB using 75 clinical isolates obtained from a cohort investigation of high-risk populations infected with MTB. The 24-locus MIRU-VNTR method demonstrated superior discriminatory ability, followed by PFGE and IS6110-RFLP. Next, we analyzed six isolates with clear epidemiologic connections; that is, isolates from patients who attended the same school. IS6110-RFLP and PFGE identified these samples as the same type. By contrast, according to MIRU-VNTR, two isolates differed from four other isolates at one locus each; one isolate was identified as Mtub29 and the other as QUB-26. In summary, the 24-locus MIRU-VNTR assay was the most useful molecular typing method among the three methods investigated due to its discriminatory power, short time required, and availability as an epidemiologic investigation tool. PFGE was the second-best method. Compared with the other loci assessed in the 24-locus MIRU-VNTR assay, the Mtub29 and QUB-26 loci appeared to exhibit greater variability during transmission.

• Journal of Microbiology and Biotechnology
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• 제24권7호
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• pp.1004-1007
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• 2014

Culture is the gold standard for diagnosis of tuberculosis, but it takes 6 to 8 weeks to confirm the result. This issue is complemented by the detection method using polymerase chain reaction, which is now widely used in a routine microbiology laboratory. In this study, we evaluated the performance of the Seegene Anyplex TB PCR to assess its diagnostic sensitivity and specificity, and compared its results with the Roche Cobas TaqMan MTB PCR, one of the most widely used assays in the world. Five university hospitals located in the Chungcheong area in South Korea participated in the study. A total of 1,167 respiratory specimens ordered for acid-fast bacilli staining and culture were collected for four months, analyzed via the Seegene Anyplex TB PCR, and its results were compared with the Roche Cobas TaqMan MTB PCR. For detection of Mycobacterium tuberculosis, the diagnostic sensitivity and specificity of the Anyplex TB PCR were 87.5% and 98.2% respectively, whereas those of the Cobas TaqMan were 92.0% and 98.0% respectively (p value > 0.05). For smear-positive specimens, the sensitivity of the Anyplex TB PCR was 95.2%, which was exactly the same as that of the Cobas TaqMan. For smear-negative specimens, the sensitivity of the Anyplex TB PCR was 69.2%, whereas that of the Cobas TaqMan TB PCR was 84.6%. For detection of MTB, the Seegene Anyplex TB PCR showed excellent diagnostic performance, and high sensitivity and specificity, which were comparable to the Roche Cobas TaqMan MTB PCR. In conclusion, the Anyplex TB PCR can be a useful diagnostic tool for the early detection of tuberculosis in clinical laboratories.

• Genomics & Informatics
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• 제21권3호
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• pp.42.1-42.23
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• 2023

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Tolllike receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

• IMMUNE NETWORK
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• 제23권2호
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• pp.16.1-16.19
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• 2023

Bacillus Calmette-Guerin (BCG) vaccine is the only licensed vaccine for tuberculosis (TB) prevention. Previously, our group demonstrated the vaccine potential of Rv0351 and Rv3628 against Mycobacterium tuberculosis (Mtb) infection by directing Th1-biased CD4⁺ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the lungs. Here, we assessed immunogenicity and vaccine potential of the combined Ags (Rv0351/Rv3628) formulated in different adjuvants as subunit booster in BCG-primed mice against hypervirulent clinical Mtb strain K (Mtb K). Compared to BCG-only or subunit-only vaccine, BCG prime and subunit boost regimen exhibited significantly enhanced Th1 response. Next, we evaluated the immunogenicity to the combined Ags when formulated with four different types of monophosphoryl lipid A (MPL)-based adjuvants: 1) dimethyldioctadecylammonium bromide (DDA), MPL, and trehalose dicorynomycolate (TDM) in liposome form (DMT), 2) MPL and Poly I:C in liposome form (MP), 3) MPL, Poly I:C, and QS21 in liposome form (MPQ), and 4) MPL and Poly I:C in squalene emulsion form (MPS). MPQ and MPS displayed greater adjuvancity in Th1 induction than DMT or MP did. Especially, BCG prime and subunit-MPS boost regimen significantly reduced the bacterial loads and pulmonary inflammation against Mtb K infection when compared to BCG-only vaccine at a chronic stage of TB disease. Collectively, our findings highlighted the importance of adjuvant components and formulation to induce the enhanced protection with an optimal Th1 response.

• Tuberculosis and Respiratory Diseases
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• 제63권4호
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• pp.331-336
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• 2007

연구배경: 결핵은 호흡기를 통한 전염성 질환이지만 말초혈액에서 결핵균 PCR이 양성이거나 결핵균이 배양된 보고가 일부 있었다. 이는 결핵의 진단에 있어서의 유용성과 헌혈을 통한 결핵의 전염 가능성의 두 가지 문제를 제기하게 되었고, 저자들은 상기 문제들에 대한 향후 연구방향의 기초자료로 사용하고자 연구를 시행하여 보았다. 방법: 속립성 결핵이 아닌 폐결핵, 비결핵항산균 폐질환, 폐암 및 폐렴 환자 69명을 대상으로 말초혈액에서 결핵균 PCR을 시행하였고, 결핵균이 많을 것으로 추정되는 10명의 폐결핵 환자의 말초혈액을 대상으로 결핵균 배양을 시행하였다. 결핵균 PCR은 nested PCR을 이용한 TB-taq (M&D, Korea)을 이용하였고 혈액배양에는 BACTEC Myco/F Lytic 혈액배양병(Becton Dickinson, Sparks, Md)을 사용하였다. 결과: 결핵균 PCR을 시행한 환자는 69명으로 각각 폐결핵 35명, 비결핵항산균 폐질환 6명, 폐암 20명, 폐렴 8명이었다. 폐렴이나 폐암 환자 28명 모두에서 PCR은 음성이었고, 비결핵항산균 폐질환 6명 중 1명(16.7%), 폐결핵 35명 중 8명(22.8%)에서 양성이었다. 혈액 배양은 폐결핵 10명 모두에서 음성이었다. 결론: 미만성 결핵이 동반되지 않은 폐결핵 환자의 말초혈액에서 결핵균의 DNA가 검출됨을 확인할 수 있었으나 균배양이 되지 않는 것으로 보아 살아있는 균이 존재하기 보다는 균의 일부 성분만 존재할 가능성을 제시하였다.

• 동의생리병리학회지
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• 제18권3호
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• pp.783-788
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• 2004

Cheonmabanhwa- Tang (CBT) has been used in the Oriental Medicine for many centuries as a therapeutic agent for dizziness due to Poong-Dam, We reported that CBT had effects on the cerebral hemodynamics [regional cerebral blood flow (rCBF), pial arterial diameter (PAD), and mean arterial blood pressure (MABP)] in normal and cerebral ischemic rats. Therefor we designed to determine the mechanism of action of CBT. The changes of rCBF and MABP were determinated by laser-Doppler flowmetry(LDF), and the change of PAD was determinated by video-microscopy. The results were as follows: The CBT-induced increase in rCBF was significnatly inhibited by pretreatment with indomethacin (IDN, 1 ㎎/㎏, i.p.), an inhibitor of cyclooxygenase, or methylene blue (MTB, 10 ㎍/㎏, i.p.), an inhibitor of guanylate cyclase. The CBT-induced increase in PAD was also significantly inhibited by pretreatment with IDN or MTB. The CBT-induced increase in MABP was also significantly inhibited by pretreatment with IDN or MTB. In conclusion, it is suggested that CBT causes a diverse effect on cerebral hemodynamics via mediation of cyclooxygenase and guanylate cyclase.

• Tuberculosis and Respiratory Diseases
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• 제60권5호
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• pp.497-509
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• 2006

Until recently, the tuberculin skin test (TST) has been the only tool available for diagnosing a latent TB infection. However, the development of new diagnostic tools, using the Mycobacterium tuberculosis (MTB)-specific early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) antigens, should improve the control of tuberculosis (TB) by allowing a more accurate identification of a latent TB infection (LTBI). Antigen-specific interferon-gamma ($IFN-{\gamma}$) assays have greater specificity in BCG-vaccinated individuals, and as less biased by nontuberculous mycobacterial infections. Many comparative studies have suggested that those assays have a higher specificity than the TST, and the sensitivity of these assays are expected to remarkably improved if more MTB-specific antigens can become available. Nevertheless, the major obstacle to the widespread use of these tests is the limited financial resources. Similar to other diagnostic tests, the predictive value of $IFN-{\gamma}$ assays depends on the prevalence of a MTB infection in the population being tested. Therefore, prospective studies will be meeded to establish the applicability of these new assays at multiple geographic locations among patients of different ethnicities, and to determine if the $IFN-{\gamma}$ responses can indicate those with a high risk of progressing to active TB.