Yih-Yuan Chen

Genetic diversity of the Mycobacterium tuberculosis Beijing family based on SNP and VNTR typing profiles in Asian countries.

The Mycobacterium tuberculosis (MTB) Beijing strain is highly virulent, drug resistant, and endemic over Asia. To explore the genetic diversity of this family in several different regions of eastern Asia, 338 Beijing strains collected in Taiwan (Republic of China) were analyzed by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and compared with published MIRU-VNTR profiles and by the Hunter-Gaston diversity index (HGDI) of Beijing strains from Japan and South Korea. The results revealed that VNTR2163b (HGDI>0.6) and five other loci (VNTR424, VNTR4052, VNTR1955, VNTR4156 and VNTR 2996; HGDI>0.3) could be used to discriminate the Beijing strains in a given geographic region. Analysis based on the number of VNTR repeats showed three VNTRs (VNTR424, 3192, and 1955) to be phylogenetically informative loci. In addition, to determine the geographic variation of sequence types in MTB populations, we also compared sequence type (ST) data of our strains with published ST profiles of Beijing strains from Japan and Thailand. ST10, ST22, and ST19 were found to be prevalent in Taiwan (82%) and Thailand (92%). Furthermore, classification of Beijing sublineages as ancient or modern in Taiwan was found to depend on the repeat number of VNTR424. Finally, phylogenetic relationships of MTB isolates in Taiwan, South Korea, and Japan were revealed by a minimum spanning tree based on MIRU-VNTR genotyping. In this topology, the MIRU-VNTR genotypes of the respective clusters were tightly correlated to other genotypic characters. These results are consistent with the hypothesis that clonal evolution of these MTB lineages has occurred.

The pattern of cytokine production in vitro induced by ancient and modern Beijing Mycobacterium tuberculosis strains.

It is unclear to what extent the host-responses elicited by Beijing versus non-Beijing strains of Mycobacterium tuberculosis (MTB) contribute to the predominance of modern Beijing strains in Taiwan and some other Asian countries. The purpose of this study was to compare the expression profiles of virulence-related genes in human monocyte-derived macrophages infected in vitro with Beijing (ancient and modern strains) and non-Beijing strains (EAI strains) of MTB that are epidemic in Taiwan. We found that modern Beijing strains induced lower levels of pro-inflammatory cytokines, whereas EAI strains induced higher levels. Notably, the most prevalent modern Beijing sub-lineage, possessing intact RD150 and RD142 chromosomal regions, induced very low levels of pro-inflammatory cytokines, especially interleukin-1β. Moreover, in an intracellular growth assay, the survival of the same modern Beijing strain in human monocyte-derived macrophages was significantly higher than that of an ancient Beijing strain and an EAI strain. Taken together, these results may explain why modern Beijing strains of MTB predominate in Taiwan.

Recombinant BCG coexpressing Ag85B, CFP10, and interleukin-12 induces multifunctional Th1 and memory T cells in mice

Lin C‐W, Su I‐J, Chang J‐R, Chen Y‐Y, Lu J‐J, Dou H‐Y. Recombinant BCG coexpressing Ag85B, CFP10, and interleukin‐12 induces multifunctional Th1 and memory T cells in mice. APMIS 2012; 120: 72–82.

Clonal Expansion of Both Modern and Ancient Genotypes of Mycobacterium tuberculosis in Southern Taiwan

We present the first comprehensive analysis of Mycobacterium tuberculosis isolates circulating in the Kaohsiung region of southern Taiwan. The major spoligotypes found in the 224 isolates studied were Beijing lineages (n = 97; 43.3%), EAI lineages (n = 72; 32.1%) and Haarlem lineages (n = 18; 8.0%). By 24 MIRU-VNTR typing, 174 patterns were identified, including 24 clusters of 74 isolates and 150 unique patterns. The combination of spoligotyping and 12-MIRU-VNTR revealed that 129 (57.6%) of the 224 isolates were clustered in 18 genotypes. Moreover, 63.6% (7/11) of infected persons younger than 30 years had a Beijing strain, which could suggest recent spread among younger persons by this family of TB strains in Kaohsiung. Among the 94 Beijing family (SIT1, SIT250 and SIT1674) isolates further analyzed for SNPs by mass spectrometry, the most frequent strain found was ST10 (n = 49; 52%), followed by ST22 (n = 17; 18%) and ST19 (n = 11; 12%). Among the EAI-Manila family isolates analyzed by region deletion-based subtyping, the most frequent strain found was RD type 1 (n = 63; 87.5%), followed by RD type 2 (n = 9; 12.5%). In our previous study, the proportion of modern Beijing strains (52.5%) in northern Taiwan was significantly higher than the proportion of EAI strains (11%). In contrast, in the present study, EAI strains comprised up to 32% of Beijing strains in southern Taiwan. In conclusion, both ‘modern’ (Beijing) and ‘ancient’ (EAI) M. tuberculosis strains are prevalent in the Kaohsiung region, perhaps suggesting that both strains are somehow more adapted to southern Taiwan. It will be interesting to investigate the dynamics of the lineage composition by different selection pressures.

Molecular epidemiology of Mycobacterium tuberculosis in aboriginal peoples of Taiwan, 2006–2011

Previous research revealed a 6-fold higher incidence of tuberculosis (TB) amongst aborigines compared to Han Chinese in Taiwan. To investigate the reasons for this disparity, we genotyped Mycobacterium tuberculosis (MTB) strains obtained from members of different aboriginal tribes in different geographical regions of Taiwan by using molecular methods. In total, 177 isolates of MTB collected from patients at four hospitals in Taiwan from January 2006 to December 2011 were analysed by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem-repeat (MIRU-VNTR) typing. The most prevalent strains in the eastern and central regions of Taiwan were Beijing (45.7% in eastern) and Haarlem (39.1% in eastern, 37.1% in central) lineages, whereas in southern regions the most prevalent strains were EAI (47.7%) and Haarlem (20.5%) lineages. The high prevalence of EAI in southern Taiwan aborigines may be closely associated with Austronesian culture. This study provides a first overview of the M. tuberculosis strains circulating in aboriginal populations in Taiwan. The high prevalences of certain MTB lineages within aboriginal sub-populations suggest that transmission of MTB may have been restricted to close contacts.

Prevalence of Mycobacterium tuberculosis strain genotypes in Taiwan reveals a close link to ethnic and population migration

Taiwan is a relatively isolated island, serving as a mixing vessel for colonization by different waves of ethnic and migratory groups over the past 4 centuries. The potential transmission pattern of Mycobacterium tuberculosis in different ethnic and migratory populations remains to be elucidated. By using mycobacterial tandem repeat sequences as genetic markers, the prevalence of M. tuberculosis strains in Taiwan revealed a close link to the historical migration. Interestingly, the M. tuberculosis strain in the aborigines of Eastern and Central Taiwan had a dominance of the Haarlem (Dutch) strain while those in Southern Taiwan had a dominance of the East-African Indian (EAI) strain. The prevalence of different M. tuberculosis strains in specific ethnic populations suggests that M. tuberculosis transmission is limited and restricted to close contact. The prevalence of the Beijing modern strain in the young population causes a concern for M. tuberculosis control, because of high virulence and drug resistance. Furthermore, our data using molecular genotyping should provide valuable information on the historical study of the origin and migration of aborigines in Taiwan.

Molecular epidemiology of tuberculosis in Kaohsiung City located at southern Taiwan, 2000-2008.

Background We present the first comprehensive analysis of Mycobacterium tuberculosis (MTB) isolates circulating in southern Taiwan. In this 9-year population-based study, the TB situation in the Kaohsiung region was characterized by genotypic analysis of 421 MTB isolates. Methods All 421 isolates of MTB were analyzed by spoligotyping and MIRU-VNTR typing. Drug-resistance patterns were also analyzed. Results The percentage of EAI (East African-Indian) strains increased across sampling years (2000–2008) in southern Taiwan, whereas the proportion of Beijing lineages remained unchanged. Clustering was more frequent with EAI genotype infections (odds ratio = 3.6, p<0.0001) when compared to Beijing genotypes. Notably, MTB resistance to streptomycin (STR) had significantly increased over time, but resistance to other antibiotics, including multidrug resistance, had not. Three major genes (gidB, rpsL and rrs) implicated in STR resistance were sequenced and specific mutations identified. Conclusions This study revealed that EAI strains were highly transmissible and that STR resistance has increased between 2000 and 2008 in Kaohsiung, Taiwan.

Reversine Induced Multinucleated Cells, Cell Apoptosis and Autophagy in Human Non-Small Cell Lung Cancer Cells.

Reversine, an A3 adenosine receptor antagonist, has been shown to induce differentiated myogenic-lineage committed cells to become multipotent mesenchymal progenitor cells. We and others have reported that reversine has an effect on human tumor suppression. This study revealed anti-tumor effects of reversine on proliferation, apoptosis and autophagy induction in human non-small cell lung cancer cells. Treatment of these cells with reversine suppressed cell growth in a time- and dosage-dependent manner. Moreover, polyploidy occurred after reversine treatment. In addition, caspase-dependent apoptosis and activation of autophagy by reversine in a dosage-dependent manner were also observed. We demonstrated in this study that reversine contributes to growth inhibition, apoptosis and autophagy induction in human lung cancer cells. Therefore, reversine used as a potential therapeutic agent for human lung cancer is worthy of further investigation.

Pros and cons of the tuberculosis drugome approach--an empirical analysis.

Drug-resistant Mycobacterium tuberculosis (MTB), the causative pathogen of tuberculosis (TB), has become a serious threat to global public health. Yet the development of novel drugs against MTB has been lagging. One potentially powerful approach to drug development is computation-aided repositioning of current drugs. However, the effectiveness of this approach has rarely been examined. Here we select the “TB drugome” approach – a protein structure-based method for drug repositioning for tuberculosis treatment – to (1) experimentally validate the efficacy of the identified drug candidates for inhibiting MTB growth, and (2) computationally examine how consistently drug candidates are prioritized, considering changes in input data. Twenty three drugs in the TB drugome were tested. Of them, only two drugs (tamoxifen and 4-hydroxytamoxifen) effectively suppressed MTB growth at relatively high concentrations. Both drugs significantly enhanced the inhibitory effects of three first-line anti-TB drugs (rifampin, isoniazid, and ethambutol). However, tamoxifen is not a top-listed drug in the TB drugome, and 4-hydroxytamoxifen is not approved for use in humans. Computational re-examination of the TB drugome indicated that the rankings were subject to technical and data-related biases. Thus, although our results support the effectiveness of the TB drugome approach for identifying drugs that can potentially be repositioned for stand-alone applications or for combination treatments for TB, the approach requires further refinements via incorporation of additional biological information. Our findings can also be extended to other structure-based drug repositioning methods.

Recombinant bacille Calmette–Guerin coexpressing Ag85b, CFP10, and interleukin-12 elicits effective protection against Mycobacterium tuberculosis

BACKGROUND The tuberculosis (TB) pandemic remains a leading cause of human morbidity and mortality, despite widespread use of the only licensed anti-TB vaccine, bacille Calmette-Guerin (BCG). The protective efficacy of BCG in preventing pulmonary TB is highly variable; therefore, an effective new vaccine is urgently required. METHODS In the present study, we assessed the ability of novel recombinant BCG vaccine (rBCG) against Mycobacterium tuberculosis by using modern immunological methods. RESULTS Enzyme-linked immunospot assays demonstrated that the rBCG vaccine, which coexpresses two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 (rBCG2) elicits greater interferon-γ (IFN-γ) release in the mouse lung and spleen, compared to the parental BCG. In addition, rBCG2 triggers a Th1-polarized response. Our results also showed that rBCG2 vaccination significantly limits M. tuberculosis H37Rv multiplication in macrophages. The rBCG2 vaccine surprisingly induces significantly higher tumor necrosis factor-α (TNF-α) production by peripheral blood mononuclear cells that were exposed to a nonmycobacterial stimulus, compared to the parental BCG. CONCLUSION In this study, we demonstrated that the novel rBCG2 vaccine may be a promising candidate vaccine against M. tuberculosis infection.