Researcher Database

Yasuhiko Suzuki
Research Center for Zoonosis Control Division of Bioresources

Researcher Profile and Settings


  • Research Center for Zoonosis Control Division of Bioresources

Job Title

  • Professor


  • Ph. D. (medicine)(Osaka University)

J-Global ID

Research Interests

  • 結核菌   PCR   遺伝子変異   リコンビナント蛋白質   分子疫学   多剤耐性   超多剤耐性   DNAチップ   DNAジャイレース   レクチン   補体   結核   tuberculosis   

Research Areas

  • Life sciences / Healthcare management, medical sociology
  • Life sciences / Hygiene and public health (non-laboratory)
  • Life sciences / Hygiene and public health (laboratory)
  • Life sciences / Hygiene and public health (non-laboratory)
  • Life sciences / Hygiene and public health (laboratory)
  • Life sciences / Healthcare management, medical sociology
  • Other / Other / Laboratory medicine
  • Life sciences / Bacteriology

Academic & Professional Experience

  • 2005 - Today Hokkaido University
  • 2003 - 2005 Tottori University Faculty of Medicine
  • 1997 - 2003 Osaka Prefectural Institute of Public Health
  • 1991 - 1997 Osaka Prefectural Institute of Public Health
  • 1988 - 1991 Osaka University Research Institute for Microbial Diseases


  • 1984 - 1988  大阪大学大学院
  •        - 1988  Osaka University  Graduate School, Division of Medical Science  Department of TuberculosisⅡ
  • 1983 - 1984  Osaka University  Research Institute for Microbial Diseases
  • 1981 - 1983  Shizuoka University  Graduate School of Science
  • 1977 - 1981  Shizuoka University  Faculty of Science
  •        - 1981  Shizuoka University  Faculty of Science  Department of Chemistry

Association Memberships

  • JAPANESE SOCIETY FOR TUBERCULOSIS   日本ハンセン病学会   日本細菌学会   Japanese Association of Bacteriology   

Research Activities

Published Papers

  • Paudel S, Nakajima C, Mikota SK, Gairhe KP, Maharjan B, Subedi S, Poudel A, Sashika M, Shimozuru M, Suzuki Y, Tsubota T
    Emerging infectious diseases 25 (5) 1031 - 1032 1080-6040 2019/05 [Refereed][Not invited]
  • Usui M, Kajino A, Kon M, Fukuda A, Sato T, Shirakawa T, Kawanishi M, Harada K, Nakajima C, Suzuki Y, Tamura Y
    The Journal of veterinary medical science 0916-7250 2019/05 [Refereed][Not invited]
  • Yoshiki Hiyama, Satoshi Takahashi, Toyotaka Sato, Masaaki Shinagawa, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Naoya Masumori, Shin-Ichi Yokota
    Microbial drug resistance (Larchmont, N.Y.) 25 (3) 427 - 433 1076-6294 2019/04 [Refereed][Not invited]
    Neisseria gonorrhoeae is a principal pathogen for sexually transmitted infections, especially for male urethritis. Currently, the prevalence of multidrug resistance is increasing. Carbapenems are broad-spectrum antimicrobials that are widely used in the clinical setting, especially for multidrug-resistant Gram-negative bacteria. However, susceptibility to carbapenems has not been well evaluated for cephalosporin-resistant N. gonorrhoeae isolates. In this study, we determined the susceptibility to a series of carbapenems (meropenem, imipenem, doripenem, and biapenem) and faropenem against cephalosporin-resistant (resistant to cefixime, but susceptible to ceftriaxone) and cephalosporin-susceptible N. gonorrhoeae clinical isolates. The gene mutations associated with β-lactam resistance were evaluated. All cephalosporin-resistant N. gonorrhoeae isolates possessed mosaic mutation alleles in penA (NG-STAR penA-10.001, 27.001, or 108.001). They exhibited a low minimum inhibitory concentration (MIC) (≤0.125 mg/L) for meropenem and markedly high MICs (0.5-2 mg/L) for other carbapenems and faropenem. The strongest association was observed between the mosaic alleles in penA and decreased susceptibility to carbapenems and faropenem compared with mutations in mtrR, porB, and ponA. These results suggest that meropenem may serve as an alternative therapeutic agent for cephalosporin-resistant N. gonorrhoeae with a mosaic allele in penA, whereas other carbapenems and faropenem may be ineffective.
  • Usui M, Yokoo H, Tamura Y, Nakajima C, Suzuki Y, Ghigo JM, Beloin C
    Antimicrobial agents and chemotherapy 0066-4804 2019/04 [Refereed][Not invited]
  • Fujisawa S, Konnai S, Okagawa T, Maekawa N, Tanaka A, Suzuki Y, Murata S, Ohashi K
    BMC veterinary research 15 (1) 68  2019/02 [Refereed][Not invited]
  • Shah Y, Poudel A, Maharjan B, Thapa J, Yamaguchi T, Diab HM, Pandey BD, Solo E, Isoda N, Suzuki Y, Nakajima C
    Transactions of the Royal Society of Tropical Medicine and Hygiene 0035-9203 2019/01 [Refereed][Not invited]
  • Maharjan B, Nakajima C, Isoda N, Thapa J, Poudel A, Shah Y, Yamaguchi T, Shrestha B, Hoffmann H, Avsar K, Shrestha A, Gordon SV, Suzuki Y
    Scientific reports 8 (1) 16634  2018/11 [Refereed][Not invited]
  • Phetsuksiri B, Rudeeaneksin J, Srisungngam S, Bunchoo S, Klayut W, Sangkitporn S, Nakajima C, Hamada S, Suzuki Y
    Japanese journal of infectious diseases 1344-6304 2018/10 [Refereed][Not invited]
  • Hiroyuki Honda, Toyotaka Sato, Masaaki Shinagawa, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Tsukasa Shiraishi, Koji Kuronuma, Satoshi Takahashi, Hiroki Takahashi, Shin-Ichi Yokota
    Antimicrobial agents and chemotherapy 62 (9) 0066-4804 2018/09 [Refereed][Not invited]
    β-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of β-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were β-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.
  • Yasuo Ohkoshi, Toyotaka Sato, Takayuki Wada, Yukari Fukushima, Hiromi Murabayashi, Yasunari Takakuwa, Kaoru Nishiyama, Hiroyuki Honda, Tsukasa Shiraishi, Koji Kuronuma, Hiroki Takahashi, Chie Nakajima, Yasuhiko Suzuki, Shin-Ichi Yokota
    Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy 24 (8) 674 - 681 1341-321X 2018/08 [Refereed][Not invited]
    Multidrug-resistant Streptococcus pneumoniae strains were isolated from blood and sputum of a patient with disseminated intravascular coagulation in Sapporo city, Japan. These antibiograms were only susceptible to vancomycin, linezolid, daptomycin, some carbapenems, and some fluoroquinolones. Identical antibiograms, serotypes (19F), and sequence types (ST10017) suggested a shared origin of these isolates. Only one ST10017 strain has been isolated in the same city in Japan previously (2014), and the 2014 isolate is still susceptible to macrolides. The whole genome of the blood-derived isolate was sequenced. The strain harbored resistance mutations in parC, gyrA, pbp1a, pbp2a, pbp2b, and pbp2x, and harbored the resistance genes, ermB and tetM. The nucleotide sequences of parC and pbp2x genes of strain MDRSPN001 were clearly different from those of other S. pneumoniae strains and were similar to those of oral streptococci strains. These findings suggest that strain MDRSPN001 has been rapidly and drastically evolving multidrug resistance by gene replacement and accumulation of genes originating from other strains, such as oral streptococci, Streptococcus mitis.
  • Shiomi Yoshida, Tsubasa Araki, Tomohito Asai, Kazunari Tsuyuguchi, Kentaro Arikawa, Tomotada Iwamoto, Chie Nakajima, Yasuhiko Suzuki, Kenji Ohya, Tokuma Yanai, Takayuki Wada, Taro Yamamoto
    Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 62 122 - 129 1567-1348 2018/08 [Refereed][Not invited]
    Mycobacterium avium subspecies hominissuis (MAH) is an important cause of infection in human pulmonary and swine intestinal cases. Although MAH is isolated from environmental sources frequently, infections of other animals have rarely been analysed. Recently, we detected granulomatous inflammation in bovine lung as an abnormal postmortem inspection case. To ascertain its genetic profile, we conducted a variable numbers of tandem repeats (VNTR) analysis and genomic characterization using deep sequencing. The VNTR type was a unique profile that differed from reported genotypes, but it was assigned within a broad genotypic complex of isolates from human patients and bathrooms. Genomic comparison with 116 registered genome sequences of the subspecies revealed that the strain was separate from five major genetic population groups proposed previously. Although the infection source remains unclear, its isolation from various resources such as animal infection cases should be elucidated more extensively to reveal its genetic diversity and ecological context.
  • Thida Oo NA, San LL, Thapa J, Aye KS, Aung WW, Nakajima C, Suzuki Y
    Tuberculosis (Edinburgh, Scotland) 111 8 - 13 1472-9792 2018/07 [Refereed][Not invited]
  • Koide K, Kongsoi S, Ouchi Y, Yamaguchi T, Nakajima C, Suzuki Y
    Microbial drug resistance (Larchmont, N.Y.) 25 (1) 14 - 22 1076-6294 2018/07 [Refereed][Not invited]
  • Okagawa T, Konnai S, Nishimori A, Maekawa N, Goto S, Ikebuchi R, Kohara J, Suzuki Y, Yamada S, Kato Y, Murata S, Ohashi K
    Veterinary research 49 (1) 50  0928-4249 2018/06 [Refereed][Not invited]
  • San LL, Aye KS, Oo NAT, Shwe MM, Fukushima Y, Gordon SV, Suzuki Y, Nakajima C
    International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 76 109 - 119 1201-9712 2018/06 [Refereed][Not invited]
  • Sajiki Y, Konnai S, Okagawa T, Nishimori A, Maekawa N, Goto S, Ikebuchi R, Nagata R, Kawaji S, Kagawa Y, Yamada S, Kato Y, Nakajima C, Suzuki Y, Murata S, Mori Y, Ohashi K
    Infection and immunity 86 (5) 0019-9567 2018/05 [Refereed][Not invited]
  • Bwalya P, Yamaguchi T, Mulundu G, Nakajima C, Mbulo G, Solo ES, Fukushima Y, Kasakwa K, Suzuki Y
    Tuberculosis (Edinburgh, Scotland) 109 117 - 122 1472-9792 2018/03 [Refereed][Not invited]
  • Toyotaka Sato, Yasuo Ohkoshi, Takayuki Wada, Yukari Fukushima, Hiromi Murabayashi, Yasunari Takakuwa, Kaoru Nishiyama, Tsukasa Shiraishi, Chie Nakajima, Yasuhiko Suzuki, Shin-Ichi Yokota
    Genome announcements 5 (44) 2017/11/02 [Refereed][Not invited]
    Invasive infection of multidrug-resistant Streptococcus pneumoniae is a serious clinical concern. Here, we report the complete genome sequence of a multidrug-resistant S. pneumoniae serotype 19F strain isolated from a patient with an invasive infection in Sapporo, Japan.
  • Fuangfa Utrarachkij, Chie Nakajima, Ruchirada Changkwanyeun, Kanokrat Siripanichgon, Siriporn Kongsoi, Srirat Pornruangwong, Kanjana Changkaew, Risa Tsunoda, Yutaka Tamura, Orasa Suthienkul, Yasuhiko Suzuki
    MICROBIAL DRUG RESISTANCE 23 (7) 885 - 894 1076-6294 2017/10 [Refereed][Not invited]
    Salmonella Enteritidis has emerged as a global concern regarding quinolone resistance and invasive potential. Although quinolone-resistant S. Enteritidis has been observed with high frequency in Thailand, information on the mechanism of resistance acquisition is limited. To elucidate the mechanism, a total of 158 clinical isolates of nalidixic acid (NAL)-resistant S. Enteritidis were collected throughout Thailand, and the quinolone resistance determinants were investigated in the context of resistance levels to NAL, norfloxacin (NOR), and ciprofloxacin (CIP). The analysis of point mutations in type II topoisomerase genes and the detection of plasmid-mediated quinolone resistance genes showed that all but two harbored a gyrA mutation, the qnrS1 gene, or both. The most commonly affected codon in mutant gyrA was 87, followed by 83. Double codon mutation in gyrA was found in an isolate with high-level resistance to NAL, NOR, and CIP. A new mutation causing serine to isoleucine substitution at codon 83 was identified in eight isolates. In addition to eighteen qnrS1-carrying isolates showing nontypical quinolone resistance, one carrying both the qnrS1 gene and a gyrA mutation also showed a high level of resistance. Genotyping by multilocus variable number of tandem repeat analysis suggested a possible clonal expansion of NAL-resistant strains nationwide. Our data suggested that NAL-resistant isolates with single quinolone resistance determinant may potentially become fluoroquinolone resistant by acquiring secondary determinants. Restricted therapeutic and farming usage of quinolones is strongly recommended to prevent the emergence of fluoroquinolone-resistant isolates.
  • Yogendra Shah, Bhagwan Maharjan, Jeewan Thapa, Ajay Poudel, Hassan Mahmoud Diab, Basu Dev Pandey, Eddie S. Solo, Norikazu Isoda, Yasuhiko Suzuki, Chie Nakajima
    INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES 63 13 - 20 1201-9712 2017/10 [Refereed][Not invited]
    Objectives: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) poses a major public health problem in Nepal. Although it has been reported as one of the dominant genotypes of MTB in Nepal, little information on the Central Asian Strain (CAS) family is available, especially isolates related to multidrug resistance (MDR) cases. This study aimed to elucidate the genetic and epidemiological characteristics of MDR CAS isolates in Nepal. Methods: A total of 145 MDR CAS isolates collected in Nepal from 2008 to 2013 were characterized by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis, and drug resistance-associated gene sequencing. Results: Spoligotyping analysis showed CAS1_Delhi SIT26 as predominant (60/145, 41.4%). However, by combining spoligotyping and MIRU-VNTR typing, it was possible to successfully discriminate all 145 isolates into 116 different types including 18 clusters with 47 isolates (clustering rate 32.4%). About a half of these clustered isolates shared the same genetic and geographical characteristics with other isolates in each cluster, and some of them shared rare point mutations in rpoB that are thought to be associated with rifampicin resistance. Conclusions: Although the data obtained show little evidence that large outbreaks of MDR-TB caused by the CAS family have occurred in Nepal, they strongly suggest several MDR-MTB transmission cases. (C) 2017 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
  • Goto S, Konnai S, Okagawa T, Nishimori A, Maekawa N, Gondaira S, Higuchi H, Koiwa M, Tajima M, Kohara J, Ogasawara S, Kato Y, Suzuki Y, Murata S, Ohashi K
    Immunity, inflammation and disease 5 (3) 355 - 363 2017/09 [Refereed][Not invited]
  • Hirokazu Yano, Tomotada Lwamoto, Yukiko Nishiuchi, Chie Nakajima, Daria A. Starkova, Igor Mokrousov, Olga Narvskaya, Shiomi Yoshida, Kentaro Arikawa, Noriko Nakanishi, Ken Osaki, Ichiro Nakagawa, Manabu Ato, Yasuhiko Suzuki, Fumito Maruyama
    GENOME BIOLOGY AND EVOLUTION 9 (9) 2403 - 2417 1759-6653 2017/09 [Refereed][Not invited]
    Mycobacterium avium subsp. hominissuis (MAH) is one of the most common nontuberculous mycobacterial species responsible for chronic lung disease in humans. Despite increasing worldwide incidence, little is known about the genetic mechanisms behind the population evolution of MAH. To elucidate the local adaptation mechanisms of MAH, we assessed genetic population structure, the mutual homologous recombination, and gene content for 36 global MAH isolates, including 12 Japanese isolates sequenced in the present study. We identified five major MAH lineages and found that extensive mutual homologous recombination occurs among them. Two lineages (MahEastAsia1 and MahEastAsia2) were predominant in the Japanese isolates. We identified alleles unique to these two East Asian lineages in the loci responsible for trehalose biosynthesis (treS and mak) and in one mammalian cell entry operon, which presumably originated from as yet undiscovered mycobacterial lineages. Several genes and alleles unique to East Asian strains were located in the fragments introduced via recombination between East Asian lineages, suggesting implication of recombination in local adaptation. These patterns of MAH genomes are consistent with the signature of distribution conjugative transfer, a mode of sexual reproduction reported for other mycobacterial species.
  • Naoya Maekawa, Satoru Konnai, Satoshi Takagi, Yumiko Kagawa, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Tatsuya Deguchi, Chie Nakajima, Yukinari Kato, Keiichi Yamamoto, Hidetoshi Uemura, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    SCIENTIFIC REPORTS 7 (1) 8951  2045-2322 2017/08 [Refereed][Not invited]
    Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells. A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.
  • Xin-Ling Pan, Chun-Lei Zhang, Chie Nakajima, Jin Fu, Chang-Xia Shao, Li-Na Zhao, Jia-Yi Cui, Na Jiao, Chang-Long Fan, Yasuhiko Suzuki, Toshio Hattori, Di Li, Hong Ling
    EMERGING MICROBES & INFECTIONS 6 (7) e68  2222-1751 2017/07 [Refereed][Not invited]
    Although several optimal mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) loci have been suggested for genotyping homogenous Mycobacterium tuberculosis, including the Beijing genotype, a more efficient and convenient selection strategy for identifying optimal VNTR loci is needed. Here 281 M. tuberculosis isolates were analyzed. Beijing genotype and non-Beijing genotypes were identified, as well as Beijing sublineages, according to single nucleotide polymorphisms. A total of 22 MIRU-VNTR loci were used for genotyping. To efficiently select optimal MIRU-VNTR loci, we established accumulations of percentage differences (APDs) between the strains among the different genotypes. In addition, we constructed a minimum spanning tree for clustering analysis of the VNTR profiles. Our findings showed that eight MIRU-VNTR loci displayed disparities in h values of >= 0.2 between the Beijing genotype and non-Beijing genotype isolates. To efficiently discriminate Beijing and non-Beijing genotypes, an optimal VNTR set was established by adding loci with APDs ranging from 87.2% to 58.8%, resulting in the construction of a nine-locus set. We also found that QUB11a is a powerful locus for separating ST10s (including ST10, STF and STCH1) and ST22s (including ST22 and ST8) strains, whereas a combination of QUB11a, QUB4156, QUB18, Mtub21 and QUB26 could efficiently discriminate Beijing sublineages. Our findings suggested that two nine-locus sets were not only efficient for distinguishing the Beijing genotype from non-Beijing genotype strains, but were also suitable for sublineage genotyping with different discriminatory powers. These results indicate that APD represents a quantitative and efficient approach for selecting MIRU-VNTR loci to discriminate between divergent M. tuberculosis sublineages.
  • Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Chie Nakajima, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    FRONTIERS IN IMMUNOLOGY 8 650  1664-3224 2017/06 [Refereed][Not invited]
    Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1)/PD-ligand 1 (PD-L1), is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat-bovine chimeric monoclonal antibody 5D2 (Boch5D2) was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV). Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4(+) T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.
  • Jingge Zhao, Takashi Matsuba, Xiaoyan Zhang, Susan Leano, Chie Nakajima, Haorile Chagan-Yasutan, Elizabeth Freda Telan, Yasuhiko Suzuki, Toshio Hattori
    BMC INFECTIOUS DISEASES 17 (1) 344  1471-2334 2017/05 [Refereed][Not invited]
    Background: Strains of the Beijing genotype of Mycobacterium tuberculosis (MTB) are reportedly associated with the virulence of tuberculosis (TB) infection, unfavorable outcomes of anti-TB treatment, and the global TB pandemic. Rv0679c, a hypothetical membrane protein related to host cell invasion, has a Beijing genotype-specific mutation at residue 142 (Asn142Lys). Antigenicity differences between Rv0679c-Asn142 (N-type) and Rv0679c-Lys142 (K-type) have been previously observed in mice antigen-antibody responses. However, the immune response to Rv0679c in humans remains unknown. Therefore, we aimed to investigate the anti-Rv0679c immune response in TB patients from the endemic and non-endemic regions of the Beijing MTB genotype. Methods: We analyzed the Rv0679c-specific antibody responses in 84 subjects from the endemic region of the Beijing genotype MTB in China, including 45 pulmonary TB patients (C-PTB) and 39 healthy controls (C-HC), and 81 subjects from the Philippines (the endemic region of the non-Beijing genotype), including 51 pulmonary TB patients (P-PTB) and 30 healthy controls (P-HC). Anti-tuberculous-glycolipid (TBGL) antigen was used as the control antibody. Results: TBGL IgG titers were higher in both C-PTB and P-PTB than those in their corresponding HC (C-PTB median 4.2, P-PTB median 11.2; C-PTB vs. P-PTB, p > 0.05), suggesting immune response comparability in PTB from two different countries. C-PTB showed a higher response compared to C-HC for anti-K-type IgG (53.3%) than anti-N-type IgG (6.67%); this response was not observed in P-PTB (both N-type and K-type 9.80%). Conclusion: Dimorphic antigen Rv0679c was found to be associated with distinct immune response patterns, indicating the role of Beijing/non-Beijing genotype of MTB in stimulating specific responses in TB patients from the endemic region of Beijing MTB. Meanwhile, reactions to Rv0679c in patients and HC from non-endemic regions of the Beijing MTB may be caused by the response to the common epitope of Rv0679c N/K-type.
  • Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Yamato Sajiki, Yasuhiko Suzuki, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
    PLOS ONE 12 (4) e0174916  1932-6203 2017/04 [Refereed][Not invited]
    Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM(+) B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4(+) T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.
  • Masaru Usui, Mayuko Kawakura, Nobuki Yoshizawa, Lai Lai San, Chie Nakajima, Yasuhiko Suzuki, Yutaka Tamura
    ANAEROBE 43 15 - 20 1075-9964 2017/02 [Refereed][Not invited]
    Pigs, particularly piglets, have been identified as reservoir hosts of Clostridium difficile. To examine the survival ability of this pathogen in pig feces-based manure compost, C. difficile spores, which were prepared to contain as few vegetative cells as possible, were artificially inoculated into pig feces and incubated at different temperatures. While C. difficile survived in the feces incubated at temperatures below 37 C for over 30 days, cell numbers gradually decreased at thermophilic temperatures (over 55 C; p < 0.05). Next, to clarify the prevalence of C difficile in field manure compost, we isolated and characterized C difficile from the final products of manure compost products of 14 pig farms. A total of 11 C. difficile strains were isolated from 5 of 14 (36% positive rate) samples tested. Of these 11 strains, 82% were toxigenic, with ribotype 078 being the most prevalent. Thus, the application of composted manure to land therefore poses a possible risk of C. difficile transfer to the food chain. (C) 2016 Published by Elsevier Ltd.
  • Thanda Tun, Khin Saw Aye, Wint Wint Nyunt, John A. Crump, Chie Nakajima, Yasuhiko Suzuki, Kyi Kyi Thinn, Gregory M. Cook, Htin Lin Aung
    INFECTIOUS DISEASES 49 (3) 237 - 239 2374-4235 2017 [Refereed][Not invited]
  • Beata Shiratori, Jingge Zhao, Masao Okumura, Haorile Chagan-Yasutan, Hideki Yanai, Kazue Mizuno, Takashi Yoshiyama, Tadashi Idei, Yugo Ashino, Chie Nakajima, Yasuhiko Suzuki, Toshio Hattori
    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 18 (1) 1422-0067 2017/01 [Refereed][Not invited]
    Elevatedmatricellular proteins (MCPs), including osteopontin (OPN) and galectin-9 (Gal-9), were observed in the plasma of patients with Manila-type tuberculosis (TB) previously. Here, we quantified plasma OPN, Gal-9, and soluble CD44 (sCD44) by enzyme-linked immunosorbent assay (ELISA), and another 29 cytokines by Luminex assay in 36 patients with pulmonary TB, six subjects with latent tuberculosis (LTBI), and 19 healthy controls (HCs) from Japan for a better understanding of the roles of MCPs in TB. All TB subjects showed positive results of enzyme-linked immunospot assays (ELISPOTs). Spoligotyping showed that 20 out of 36 Mycobacterium tuberculosis (MTB) strains belong to the Beijing type. The levels of OPN, Gal-9, and sCD44 were higher in TB (positivity of 61.1%, 66.7%, and 63.9%, respectively) than in the HCs. Positive correlations between OPN and Gal-9, between OPN and sCD44, and negative correlation between OPN and ESAT-6-ELISPOT response, between chest X-ray severity score of cavitary TB and ESAT-6-ELISPOT response were observed. Instead of OPN, Gal-9, and sCD44, cytokines G-CSF, GM-CSF, IFN-alpha, IFN-gamma, IL-12p70, and IL-1RA levels were higher in Beijing MTB-infected patients. These findings suggest immunoregulatory, rather than inflammatory, effect of MCPs and can advance the understanding of the roles of MCPs in the context of TB pathology.
  • Insu Hwang, Kenichiro Mori, Katsuki Ohtani, Yasuyuki Matsuda, Nitai Roy, YounUck Kim, Yasuhiko Suzuki, Nobutaka Wakamiya
    JOURNAL OF INNATE IMMUNITY 9 (2) 217 - 228 1662-811X 2017 [Refereed][Not invited]
    Collectins are C-type lectins that are involved in innate immunity as pattern recognition molecules. Recently, collectin kidney 1 (CL-K1) has been discovered, and in vitro studies have shown that CL-K1 binds to microbes and activates the lectin complement pathway. However, in vivo functions of CL-K1 against microbes have not been elucidated. To investigate the biological functions of CL-K1, we generated CL-K1 knockout (CL-K1(-/-)) mice and then performed a Streptococcus pneumoniae infection analysis. First, we found that recombinant human CL-K1 bound to S. pneumoniae in a calcium-dependent manner, and induced complement activation. CL-K1(-/-) mice sera formed less C3 deposition on S. pneumoniae. Furthermore, immunofluorescence analysis in the wild-type (WT) mice demonstrated that CL-K1 and C3 were localized on S. pneumoniae in infected lungs. CL-K1(-/-) mice revealed decreased phagocytosis of S. pneumoniae. Consequently, less S. pneumoniae clearance was observed in their lungs. CL-K1(-/-) mice showed severe pulmonary inflammation and weight loss in comparison with WT mice. Finally, the decreased clearance and severe pulmonary inflammation caused by S. pneumoniae infection might cause higher CL-K1(-/-) mice lethality. Our results suggest that CL-K1 might play an important role in host protection against S. pneumoniae infection through the activation of the lectin complement pathway. (C) 2017 S. Karger AG, Basel
  • Jingge Zhao, Beata Shiratori, Masao Okumura, Hideki Yanai, Makoto Matsumoto, Chie Nakajima, Kazue Mizuno, Kenji Ono, Tetsuya Oda, Haorile Chagan-Yasutan, Yugo Ashino, Takashi Matsuba, Takashi Yoshiyama, Yasuhiko Suzuki, Toshio Hattori
    JOURNAL OF IMMUNOLOGY RESEARCH 2017 4797856  2314-8861 2017 [Refereed][Not invited]
    The Beijing genotype Mycobacterium tuberculosis (MTB), notorious for its virulence and predisposition to relapse, could be identified by spoligotyping based on genetic heterogeneity. The plasma samples from 20 cases of Beijing and 16 cases of non-Beijing MTB infected individuals and 24 healthy controls (HCs) were collected, and antibodies against 11 antigens (Rv0679c142Asn, Rv0679c142Lys, Ag85B, Ag85A, ARC, TDM-M, TDM-K, HBHA, MDP-1, LAM, and TBGL) were measured by ELISA. Compared to the HCs, the MTB infected subjects showed higher titers of anti-Ag85B IgG (positivity 58.2%) and anti-ACR IgG (positivity 48.2%). Of note, anti-ACR IgG showed higher titer in Beijing MTB infected tuberculosis (TB) patients than in HC (Kruskal-Wallis test, p < 0.05), while the levels of anti-Ag85B, anti-TBGL, anti-TDM-K, and anti-TDM-M IgG were higher in non-Beijing TB patients than in HC. Moreover, anti-Ag85B IgG showed higher response in non-Beijing TB patients than in Beijing TB patients (p < 0.05; sensitivity, 76.9% versus 44.4%). The sensitivity and specificity analysis showed that 78.8% Beijing infected individuals were negative in anti-TBGL-IgG or/and anti-Ag85B-IgG, while 75.0% of those were positive in anti-TBGL-IgA or/and anti-ACR-IgG tests. These results indicate the possibility of developing antibody-based test to identify Beijing MTB.
  • Tomoyuki Yamaguchi, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 (7) 1343 - 1347 0916-8451 2017 [Refereed][Not invited]
    Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.
  • Simon Peter Musinguzi, Keisuke Suganuma, Masahito Asada, Dusit Laohasinnarong, Thillaiampalam Sivakumar, Naoaki Yokoyama, Boniface Namangala, Chihiro Sugimoto, Yasuhiko Suzuki, Xuenan Xuan, Noboru Inoue
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (12) 1819 - 1824 0916-7250 2016/12 [Refereed][Not invited]
    We screened cattle and goats from the districts of Chama, Monze and Mumbwa in Zambia for animal African trypanosomes, Babesia bigemina and Theileria parva using PCRs; 38.1% of the samples tested positive for at least one of the parasite species. The most common parasite was Trypanosoma vivax (19.8%). Its incidence was significantly higher in goats than in cattle, (P<0.05). B. bigemina was found in samples from all the three areas, making it the most widespread of the parasites in Zambia. Among the tested samples, 12.0% of the positive samples were mixed infections. There were significant differences in the infection rates of T. vivax (Mumbwa had a significantly higher infection rate [39.6%, P<0.0001]), Th. parva (Monze had the only cases [P<0.0004]) and B. bigemina (Monze had a significantly higher infection rate [40.5%, P<0.0001]). According to the hematocrit values, the packed cell volume (%) among the cattle with mixed infections was significantly lower than that of the other cattle. The presence of multiple parasite species and mixed infections among the Zambian cattle and goat populations is of both clinical and economic importance to livestock farming. The absence of trypanosomosis among the samples from Monze can be attributed to tsetse eradication efforts that took place around Lake Kariba. This shows that the prevention and control of these parasitic diseases can have a significant impact on the disease status, which can translate directly into the improvement of the livestock sector in Zambia.
  • Marvin A. Villanueva, Claro N. Mingala, Michelle M. Balbin, Chie Nakajima, Norikazu Isoda, Yasuhiko Suzuki, Nobuo Koizumi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (11) 1649 - 1655 0916-7250 2016/11 [Refereed][Not invited]
    The extent of Leptospira infection in large ruminants resulting to economic problems in livestock industry in a leptospirosis-endemic country like the Philippines has not been extensively explored. Therefore, we determined the prevalence and carrier status of leptospirosis in large ruminants using molecular techniques and assessed the risk factors of acquiring leptospirosis in these animals. Water buffalo and cattle urine samples (n=831) collected from 21 farms during 2013-2015 were subjected to flaB-nested PCR to detect pathogenic Leptospira spp. Leptospiral flaB was detected in both species with a detection rate of 16.1%. Leptospiral DNA was detected only in samples from animals managed in communal farms. Sequence analysis of Leptospira flaB in large ruminants revealed the formation of three major clusters with L. borgpetersenii or L. kirschneri. One farm contained Leptospira flaB sequences from all clusters identified in this study, suggesting this farm was the main source of leptospires for other farms. This study suggested that these large ruminants are infected with various pathogenic Leptospira species causing possible major economic loss in the livestock industry as well as potential Leptospira reservoirs that can transmit infection to humans and other animals in the Philippines.
  • Siriporn Kongsoi, Ruchirada Changkwanyeun, Kazumasa Yokoyama, Chie Nakajima, Kanjana Changkaew, Orasa Suthienkul, Yasuhiko Suzuki
    DRUG TESTING AND ANALYSIS 8 (10) 1065 - 1070 1942-7603 2016/10 [Refereed][Not invited]
    The prevalence of quinolone-resistant Salmonella has become a public health concern. Amino acid substitutions have generally been found within the quinolone resistance-determining region in subunit A of DNA gyrase (GyrA) of Salmonella Typhimurium. However, direct evidence of the contribution of these substitutions to quinolone resistance remains to be shown. To investigate the significance of amino acid substitutions in S. Typhimurium GyrA to quinolone resistance, we expressed recombinant wild-type (WT) and five mutant DNA gyrases in Escherichia coli and characterized them in vitro. WT and mutant DNA gyrases were reconstituted in vitro by mixing recombinant subunits A and B of DNA gyrase. The correlation between the amino acid substitutions and resistance to quinolones ciprofloxacin, levofloxacin, nalidixic acid, and sitafloxacin was assessed by quinolone-inhibited supercoiling assays. All mutant DNA gyrases showed reduced susceptibility to all quinolones when compared with WT DNA gyrases. DNA gyrase with a double amino acid substitution in GyrA, serine to phenylalanine at codon 83 and aspartic acid to asparagine at 87 (GyrA-S83F-D87N), exhibited the lowest quinolone susceptibility amongst all mutant DNA gyrases. The effectiveness of sitafloxacin was shown by the low inhibitory concentration required for mutant DNA gyrases, including the DNA gyrase with GyrA-S83F-D87N. We suggest sitafloxacin as a candidate drug for the treatment of salmonellosis caused by ciprofloxacin-resistant S. Typhimurium. Copyright (C) 2015 John Wiley & Sons, Ltd.
  • Ruchirada Changkwanyeun, Tomoyuki Yamaguchi, Siriporn Kongsoi, Kanjana Changkaew, Kazumasa Yokoyama, Hyun Kim, Orasa Suthienkul, Masaru Usui, Yutaka Tamura, Chie Nakajima, Yasuhiko Suzuki
    DRUG TESTING AND ANALYSIS 8 (10) 1071 - 1076 1942-7603 2016/10 [Refereed][Not invited]
    Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance-determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild-type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni-NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP-dependent DNA supercoiling activity. A quinolone-inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone-resistant C. jejuni. An analysis of the structure-activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R-1 and a chloride substituent at R-8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin-resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP-resistant C. jejuni. Copyright (C) 2016 John Wiley & Sons, Ltd.
  • Haorile Chagan-Yasutan, Yue Chen, Talitha Lea Lacuesta, Prisca Susan A. Leano, Hiroko Iwasaki, Firmanto Hanan, Delsi Taurustiati, Yasukazu Ohmoto, Yugo Ashino, Hiroki Saitoh, Hideyasu Kiyomoto, Yasuhiko Suzuki, Freda O. Elizabeth Telan, Toshio Hattori
    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 17 (10) 1422-0067 2016/10 [Refereed][Not invited]
    Leptospirosis is a zoonotic disease whose severe forms are often accompanied by kidney dysfunction. In the present study, urinary markers were studied for potential prediction of disease severity. Urine samples from 135 patients with or without leptospirosis at San Lazaro Hospital, the Philippines, were analyzed. Urine levels of defensin alpha 1 (uDA1) were compared with those of neutrophil gelatinase-associated lipocalin (uNGAL) and N-acetyl-beta-D-glucosidase (uNAG). Serum creatinine (Cr) was used as a marker of kidney injury. The levels of uDA1/Cr, uNGAL/Cr, and uNAG/Cr were positive in 46%, 90%, and 80% of leptospirosis patients, and 69%, 70%, and 70% of non-leptospirosis patients, respectively. In leptospirosis patients, the correlation of uDA1/Cr, uNGAL/Cr and uNAG/Cr levels with serum Cr were r = 0.3 (p < 0.01), r = 0.29 (p < 0.01), and r = 0.02 (p = 0.81), respectively. uDA1/Cr levels were correlated with uNGAL/Cr levels (r = 0.49, p < 0.01) and uNAG/Cr levels (r = 0.47, p < 0.0001) in leptospirosis patients. These findings suggest that uDA1, uNGAL, and uNAG were elevated in leptospirosis patients and reflected various types of kidney damage. uDA1 and uNGAL can be used to track kidney injury in leptospirosis patients because of their correlation with the serum Cr level.
  • Yamaguchi T, Yokoyama K, Nakajima C, Suzuki Y
    PLoS neglected tropical diseases 10 (9) e0005013  1935-2727 2016/09 [Refereed][Not invited]
  • Sarad Paudel, Marvin A. Villanueva, Susan K. Mikota, Chie Nakajima, Kamal P. Gairhe, Suraj Subedi, Nabin Rayamajhi, Mariko Sashika, Michito Shimozuru, Takashi Matsuba, Yasuhiko Suzuki, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (7) 1117 - 1121 0916-7250 2016/07 [Refereed][Not invited]
    We developed an interferon-gamma release assay (IGRA) specific for Asian elephants (Elephas maximus). Whole blood collected from forty captive Asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (PHA), pokweed mitogen (PWM) and phorbol myristate aceteate/ionomycin (PMA/I). A sandwich ELISA that was able to recognize the recombinant elephant interferon-gamma (rEIFN-gamma) as well as native interferon-gamma from the Asian elephants was performed using anti-elephant IFN-gamma rabbit polyclonal antibodies as capture antibodies and biotinylated anti-elephant IFN-gamma rabbit polyclonal antibodies as detection antibodies. PMA/I was the best mitogen to use as a positive control for an Asian elephant IGRA. The development of an Asian elephant-specific IGRA that detects native IFN-gamma in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (TB) in Asian elephants.
  • Nitai Roy, Katsuki Ohtani, Yasuyuki Matsuda, Kenichiro Mori, Insu Hwang, Yasuhiko Suzuki, Norimitsu Inoue, Nobutaka Wakamiya
    Biochimica et biophysica acta 1860 (6) 1118 - 28 0006-3002 2016/06 [Refereed][Not invited]
    BACKGROUND: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown. METHODS: We prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1. RESULTS: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions. GENERAL SIGNIFICANCE: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction.
  • Naoya Maekawa, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
    PLOS ONE 11 (6) e0157176  1932-6203 2016/06 [Refereed][Not invited]
    Spontaneous cancers are common diseases in dogs. Among these, some malignant cancers such as oral melanoma, osteosarcoma, hemangiosarcoma, and mast cell tumor are often recognized as clinical problems because, despite their high frequencies, current treatments for these cancers may not always achieve satisfying outcomes. The absence of effective systemic therapies against these cancers leads researchers to investigate novel therapeutic modalities, including immunotherapy. Programmed death 1 (PD-1) is a costimulatory receptor with immunosuppressive function. When it binds its ligands, PD-ligand 1 (PD-L1) or PD-L2, PD-1 on T cells negatively regulates activating signals from the T cell receptor, resulting in the inhibition of the effector function of cytotoxic T lymphocytes. Aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancers. In clinical trials, anti-PD-1 or anti-PD-L1 antibodies induced tumor regression for several malignancies, including advanced melanoma, non-small cell lung carcinoma, and renal cell carcinoma. In this study, to assess the potential of the PD-1/PD-L1 axis as a novel therapeutic target for canine cancer immunotherapy, immunohistochemical analysis of PD-L1 expression in various malignant cancers of dogs was performed. Here, we show that dog oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumor, mammary adenocarcinoma, and prostate adenocarcinoma expressed PD-L1, whereas some other types of cancer did not. In addition, PD-1 was highly expressed on tumor-infiltrating lymphocytes obtained from oral melanoma, showing that lymphocytes in this cancer type might have been functionally exhausted. These results strongly encourage the clinical application of PD-1/PD-L1 inhibitors as novel therapeutic agents against these cancers in dogs.
  • Takashi Matsuba, Umme Ruman Siddiqi, Toshio Hattori, Chie Nakajima, Jun Fujii, Yasuhiko Suzuki
    FEMS MICROBIOLOGY LETTERS 363 (10) 0378-1097 2016/05 [Refereed][Not invited]
    The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains.
  • Hiroko Iwasaki, Haorile Chagan-Yasutan, Prisca Susan A. Leano, Nobuo Koizumi, Chie Nakajima, Delsi Taurustiati, Firmanto Hanan, Talitha Lea Lacuesta, Yugo Ashino, Yasuhiko Suzuki, Nina G. Gloriani, Elizabeth Freda O. Telan, Toshio Hattori
    DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE 84 (4) 287 - 291 0732-8893 2016/04 [Refereed][Not invited]
    Early diagnosis based on laboratory confirmation is essential for managing leptospirosis. This study investigated the effectiveness of a novel method of detecting leptospirosis that combines measurement of anti-Leptospira antibodies by the microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and immunochromatographic test (ICT) and leptospiral DNA by loop-mediated isothermal amplification (LAMP) and real-time PCR in plasma and 2 types of urine pellets. Of 113 suspected cases, 68.1%, 76.1%, and 60.2% were positive by MAT, ELISA, and ICT, respectively. Real-time PCR using DNA purified from urine pellets collected by low-speed centrifugation yielded positive signals for patients in late acute as well as early phase who were positive by LAMP using plasma DNA or urine pellets. Among antibody-negative patients, 9.5% were positive by DNA detection. These findings indicate that the leptospirosis detection rate is increased by combining antibody and DNA detection, providing a new tool for timely diagnosis of infection. (C) 2016 Elsevier Inc. All rights reserved.
  • Fuangfa Utrarachkij, Chie Nakajima, Kanokrat Siripanichgon, Kanjana Changkaew, Yuwanda Thongpanich, Srirat Pornraungwong, Orasa Suthienkul, Yasuhiko Suzuki
    JOURNAL OF INFECTION AND CHEMOTHERAPY 22 (4) 209 - 215 1341-321X 2016/04 [Refereed][Not invited]
    Objective: To trace the history of antimicrobial resistance in Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) circulating in Thailand, we characterised clinical isolates obtained during 2004-2007. Methods: Antimicrobial resistance profiles, multi-locus variable number tandem repeat analysis (MLVA) types and 3 representative virulence determinants (spvA, sodCI and sopE) were established from SE isolates (n = 192) collected from stool and blood of patients throughout Thailand during the period 2004-2007. Results: Resistance was found in SE against 10 out of 11 antimicrobials studied. The highest resistance ratios were observed for nalidixic acid (83.2%), ciprofloxacin (51.1%) and ampicillin (50.5%), and 25.5% were multidrug resistant. Based on five polymorphic tandem repeat loci analysis, MLVA identified 20 distinct types with three closely related predominant types. A significant increase of AMP resistance from 2004 to 2006 was strongly correlated with that of a MLVA type, 5-5-11-7-3. Conclusion: The usage of antimicrobials in human medicine or farm settings might act as selective pressures and cause the spread of resistant strains. Hence, a strict policy on antimicrobial usage needs to be implemented to achieve the control of resistant SE in Thailand. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
  • Toshio Hattori, Haorile Chagan-Yasutan, Beata Shiratori, Shinichi Egawa, Takako Izumi, Toru Kubo, Chie Nakajima, Yasuhiko Suzuki, Toshiro Niki, Bachti Alisjahbana, Elizabeth Telan
    TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE 238 (4) 287 - 293 0040-8727 2016/04 [Refereed][Not invited]
    After disaster, the victims lose their safe lives and are even exposed to nature where they could suffer from animal bites and vectors followed by suffering from zoonosis or vector-born diseases. Because of the urgent need for rapid and cheap diagnosis for infectious diseases after disaster, anonymous questionnaire clarified that leptospirosis, dengue, diarrhea, and cholera were recognized as common disaster-related infections in the Philippines, while diarrhea and pneumonia were more common in Indonesia. It should also be noted that infectious disease itself such as tuberculosis associated with acquired immune deficiency syndrome in South Africa is a disaster. Thus, the possible occurrence of similar situation in Asia should be prevented. We have conducted an international collaborative research in the Philippines and Indonesia on dengue virus, leptospira and mycobacterium tuberculosis (MTB) infectious diseases. Development of point-of-care testing for molecular diagnosis and disease severity was the principal purpose of the research. Loop-mediated isothermal amplification assay, which does not require a source of electricity, was developed for leptospirosis, dengue and MTB and has been proved to be useful where resource is limited. The plasma levels of matricellular proteins, including galectin-9 and osteopontin, were found to reflect the disease seventies in dengue virus and MTB infection, probably because matricellular proteins are one of the most functional extracellular proteins that are associated with inflammatory edema. The study on disaster-related infectious disease facilitates the international cooperation for development of point-of-care testing for tropical infectious diseases.
  • Khin Saw Aye, Chie Nakajima, Tomoyuki Yamaguchi, Min Min Win, Mu Mu Shwe, Aye Aye Win, Thandar Lwin, Wint Wint Nyunt, Ti Ti, Yasuhiko Suzuki
    JOURNAL OF INFECTION AND CHEMOTHERAPY 22 (3) 174 - 179 1341-321X 2016/03 [Refereed][Not invited]
    The number of multi-drug-resistant tuberculosis (MDR-TB) cases is rising worldwide. As a countermeasure against this situation, the implementation of rapid molecular tests to identify MDR-TB would be effective. To develop such tests, information on the frequency and distribution of mutations associating with phenotypic drug resistance in Mycobacterium tuberculosis is required in each country. During 2010, the common mutations in the rpoB, katG and inhA of 178 phenotypically MDR M. tuberculosis isolates collected by the National Tuberculosis Control Program (NTP) in Myanmar were investigated by DNA sequencing. Mutations affecting the 81-bp rifampicin (RIF) resistance-determining region (RRDR) of the rpoB were identified in 127 of 178 isolates (71.3%). Two of the most frequently affected codons were 531 and 526, with percentages of 48.3% and 14.0% respectively. For isoniazid (INH) resistance, 114 of 178 MDR-TB isolates (64.0%) had mutations in the katG in which a mutation-conferring amino acid substitution at codon 315 from Ser to Thr was the most common. Mutations in the inhA regulatory region were also detected in 20 (11.2%) isolates, with the majority at position -15. Distinct mutation rate and pattern from surrounding countries might suggest that MDR-TB has developed and spread domestically in Myanmar. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
  • Jeewan Thapa, Sarad Paudel, Amir Sadaula, Yogendra Shah, Bhagwan Maharjan, Gretchen E. Kaufman, Deborah McCauley, Kamal P. Gairhe, Toshio Tsubota, Yasuhiko Suzuki, Chie Nakajima
    EMERGING INFECTIOUS DISEASES 22 (3) 570 - 572 1080-6040 2016/03 [Refereed][Not invited]
  • Thomas J. Templeton, Masahito Asada, Montakan Jiratanh, Sohta A. Ishikawa, Sonthaya Tiawsirisup, Thillaiampalam Sivakumar, Boniface Namangala, Mika Takeda, Kingdao Mohkaew, Supawan Ngamjituea, Noboru Inoue, Chihiro Sugimoto, Yuji Inagaki, Yasuhiko Suzuki, Naoaki Yokoyama, Morakot Kaewthamasorn, Osamu Kaneko
    SCIENTIFIC REPORTS 6 23230  2045-2322 2016/03 [Refereed][Not invited]
    Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium.
  • Masato Nakaguro, Toyonori Tsuzuki, Satoko Shimada, Tetsuro Taki, Mari Tsuchiyama, Atsuko Kitamura, Yasuhiko Suzuki, Yojiro Nakano, Kenzo Ono
    PATHOLOGY INTERNATIONAL 66 (2) 108 - 113 1320-5463 2016/02 [Refereed][Not invited]
    Endocervicosis is a rare benign condition characterized by the presence of endocervical-type mucinous glands. Urinary bladder endocervicosis forms an elevated lesion in the posterior wall of the urinary bladder and is sometimes misdiagnosed as a malignant tumor clinically and pathologically. Herein we describe the first case of adenocarcinoma arising in urinary bladder endocervicosis. The patient, a 58-year-old woman, presented with asymptomatic hematuria. Cystoscopy revealed a nodular mass measuring 4cm in diameter in the posterior wall, and total cystectomy was performed. Histology revealed that the elevated lesion of the bladder wall was composed of haphazard proliferation of cystic glands lined by benign endocervical-type epithelium. An adenocarcinoma arose at the center of this endocervicosis. Mucin histochemistry revealed the presence of sulfomucin in both the endocervicosis and adenocarcinoma components. Immunohistochemically, the endocervicosis was positive for cytokeratin (CK) 7, AE1/AE3, CAM5.2, HBME1, CA19-9, and estrogen receptor (ER), and negative for CK20, CDX2, progesterone receptor (PR), MUC5AC, and -catenin. The adenocarcinoma showed similar immunohistochemical results, except for loss of ER expression and a slight increase in the ratio of Ki-67-positive cells. This case indicates that endocervicosis, known as a benign lesion, harbors the possibility of malignant transformation.
  • Marvin A. Villanueva, Claro N. Mingala, Nina G. Gloriani, Yasutake Yanagihara, Norikazu Isoda, Chie Nakajima, Yasuhiko Suzuki, Nobuo Koizumi
    JAPANESE JOURNAL OF VETERINARY RESEARCH 64 (1) 15 - 24 0047-1917 2016/02 [Refereed][Not invited]
    Water buffalo is an indispensable livestock in the Philippines. Leptospirosis is a serious zoonosis that can be fatal to humans and cause reproductive problems in livestock. Leptospirosis has been reported in some countries where water buffaloes are commercially raised, highlighting the Leptospira prevalence in this farming system, but information on leptospirosis in water buffalo farms in the Philippines is limited. In this study, we collected blood samples from rats (n = 21), and water buffaloes (n = 170) from different groups and locations in one intensive-type buffalo farm in the Philippines. Serum was analyzed by microscopic agglutination test (MAT). Anti-Leptospira antibodies reacting with serogroups Canicola, Icterohaemorrhagiae and Pomona were found in sera of 30% tested rats, and 48% of water buffalo sera tested positive for at least one Leptospira strain, in which serogroups Mini, Hebdomadis, Tarassovi and Pyrogenes were predominantly agglutinated. The number of seropositive young water buffaloes (<1 year-old) was lower than that of older seropositive ones. Furthermore, sera from younger water buffaloes were reactive with single serotypes with low MAT titers, but older animals were reactive with multiple Leptospira strains with variable MAT titers. In addition, antibodies against serogroups Icterohaemorrhagiae and Pomona were detected in both animals. Finally, Leptospira infection was found associated with age and animal grouping, highlighting the impact of management in the persistence of leptospirosis at intensive-type buffalo farm settings in the Philippines. Further investigation and appropriate control strategies are required to prevent leptospirosis from causing risks to public health and economic losses to the water buffalo farming industry.
  • Kaknokrat Chonsin, Shigeaki Matsuda, Chonchanok Theethakaew, Toshio Kodama, Jiraphan Junjhon, Yasuhiko Suzuki, Orasa Suthienkul, Tetsuya Iida
    FEMS MICROBIOLOGY LETTERS 363 (2) fnv222  0378-1097 2016/01 [Refereed][Not invited]
    Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms.
  • Hassan Mahmoud Diab, Chie Nakajima, Saber A. Kotb, Alaa Mokhtar, Nagwa F. M. Khder, Ahmed S. A. Abdelaal, Azza Hegazy, Ajay Poudel, Yogendra Shah, Yasuhiko Suzuki
    TUBERCULOSIS 96 13 - 20 1472-9792 2016/01 [Refereed][Not invited]
    The present study aimed to assess the population structure of Mycobacterium tuberculosis (MTB) isolates from Egypt. A total of 230 MTB isolates were analysed using spoligotyping, large sequence polymorphism (LSPs), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and multi-locus sequence typing (MLST). The majority of isolates (93.0%) belonged to lineage 4, including 44.3, 13.4 and 10.8% of the ill-defined T clade, LAM and Haarlem families, respectively, and lineage 3 was identified in 7.0% of the isolates. MIRU-VNTRs typing allowed efficient discrimination of the spoligotype-defined clusters, including spoligo-international types (SIT) 53, 34, and 4, into 56 patterns, including 13 clusters and 43 unique patterns. A new SNP at position 311614 was identified in all six isolates to form the biggest MIRU-VNTR cluster, which suggested a recent clonal expansion. This SNP could possibly be used as a genetic marker for robust discriminations of Egyptian MTB isolates belonging to SIT53. The combination of spoligotyping, 12 MIRU-VNTRs loci and MLST provided insight into the genetic diversity and transmission dynamics of the Egyptian MTB genotypes and could be a key to implementation of effective control measures by public health authorities. (C) 2015 Elsevier Ltd. All rights reserved.
  • Nobuo Koizumi, Hidemasa Izumiya, Jung-Jung Mu, Zbigniew Arent, Shou Okano, Chie Nakajima, Yasuhiko Suzuki, Maki Mizutani Muto, Tsutomu Tanikawa, Kyle R. Taylor, Noriyuki Komatsu, Kumiko Yoshimatsu, Hoang Thi Thu Ha, Makoto Ohnishi
    INFECTION GENETICS AND EVOLUTION 36 434 - 440 1567-1348 2015/12 [Refereed][Not invited]
    Leptospira spp. are the causative agents of a worldwide zoonosis, leptospirosis, maintained by various mammals. Each Leptospira serovar is frequently associated with a particular maintenance host, and recently, Leptospira genotype-host association has also been suggested to limit serovars to restricted areas. We investigated the molecular characteristics of L. interrogans and L. borgpetersenii which were isolated from small feral and wild animals in four East Asian states using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA using 11 loci was performed on 110 L. interrogans serogroups from Japan (79 strains of 5 serogroups from 3 animal species), Philippines (21; 3; 2), Taiwan (7; 2; 3), and Vietnam (3; 1; 1). A MLVA method using 4 loci for L. borgpetersenii was established and performed on 52 isolates from Japan (26; 3; 7), Philippines (13; 1; 2), and Taiwan (13; 1; 3). In L. interrogans, serogroups Autumnalis and Hebdomadis appeared more genetically diverse than serogroups Bataviae, Grippotyphosa, Icterohaemorrhagiae, Pomona, or Pyrogenes. The former serogroup strains with the exception of one Hebdomadis strain were isolated from Apodemus speciosus while all the latter serogroup strains with the exception of Grippotyphosa were isolated from Rattus norvegicus. L. borgpetersenii was isolated from at least 11 animal species while L. interrogans was isolated from five species, which might suggest a wider host range for L. borgpetersenii. Broad host preference in a single genotype was also observed, which colonized not only different species of the same genera but also multiple animal genera. This study demonstrates that there may be variability in the range of genetic diversity among different Leptospira serogroups, which may be attributed to maintenance host animals and environmental factors. (C) 2015 The Authors. Published by Elsevier B.V.
  • Tomoko Yoda, Yasuhiko Suzuki, Ikuko Aoyama, Kenji Yamazaki, Shuji Nakata, Kazuo Takahashi
    JOURNAL OF MEDICAL MICROBIOLOGY 64 (12) 1544 - 1547 0022-2615 2015/12 [Refereed][Not invited]
  • Muhammad Andrian Senoputra, Beata Shiratori, Fakhrial Mirwan Hasibuan, Raspati Cundarani Koesoemadinata, Lika Apriani, Yugo Ashino, Kenji Ono, Tetsuya Oda, Makoto Matsumoto, Yasuhiko Suzuki, Bachti Alisjahbana, Toshio Hattori
    DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE 83 (3) 278 - 285 0732-8893 2015/11 [Refereed][Not invited]
    We investigated the antibody responses to 10 prospective Mycobacterium tuberculosis (MTB) antigens and evaluated their ability to discriminate between latent (LTBI) and active pulmonary tuberculosis (TB). Our results indicate that plasma levels of anti-alpha-crystallin (ACR), antilipoarabinomannan, anti-trehalose 6,6'-dimycolate, and anti-tubercular-glycolipid antigen antibodies were higher in patients with active TB, compared to those in the LTBI and control subjects. No differences in the antibodies were observed between the control and LTBI subjects. Antibodies against the glycolipid antigens could not distinguish between Mycobacterium avium complex (MAC)-negative TB patients and MAC-infected LTBI individuals. The most useful serological marker was antibodies to ACR, with MAC-negative TB patients having higher titers than those observed in MAC-positive LTBI and control subjects. Our data indicate that antibody to ACR is a promising target for the serological diagnosis of patients with active TB patients. When dealing with antiglycolipid antibodies, MAC coinfection should always be considered in serological studies. (C) 2015 Elsevier Inc. All rights reserved.
  • Ryuji Kawahara, Kazuko Seto, Masumi Taguchi, Chie Nakajima, Yuko Kumeda, Yasuhiko Suzuki
    JOURNAL OF CLINICAL MICROBIOLOGY 53 (9) 3035 - 3038 0095-1137 2015/09 [Refereed][Not invited]
    We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored bla(CMY-2), a plasmid-mediated AmpC beta-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring bla(CMY-2).
  • Siriporn Kongsoi, Kazumasa Yokoyama, Apinun Suprasert, Fuangfa Utrarachkij, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
    DRUG TESTING AND ANALYSIS 7 (8) 714 - 720 1942-7603 2015/08 [Refereed][Not invited]
    Quinolones exhibit good antibacterial activity against Salmonella spp. isolates and are often the choice of treatment for life-threatening salmonellosis due to multi-drug resistant strains. To assess the properties of quinolones, we performed an in vitro assay to study the antibacterial activities of quinolones against recombinant DNA gyrase. We expressed the S. Typhimurium DNA gyrase A (GyrA) and B (GyrB) subunits in Escherichia coli. GyrA and GyrB were obtained at high purity (>95%) by nickel-nitrilotriacetic acid agarose resin column chromatography as His-tagged 97-kDa and 89-kDa proteins, respectively. Both subunits were shown to reconstitute an ATP-dependent DNA supercoiling activity. Drug concentrations that suppressed DNA supercoiling by 50% (IC(50)s) or generated DNA cleavage by 25% (CC(25)s) demonstrated that quinolones highly active against S. Typhimurium DNA gyrase share a fluorine atom at C-6. The relationships between the minimum inhibitory concentrations (MICs), IC(50)s and CC(25)s were assessed by estimating a linear regression between two components. MICs measured against S. Typhimurium NBRC 13245 correlated better with IC(50)s (R=0.9988) than CC(25)s (R=0.9685). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test to identify quinolones with promising activity against S. Typhimurium. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and N-1 cyclopropyl substituents are desirable structural features in targeting S. Typhimurium gyrase. Copyright (c) 2014 John Wiley & Sons, Ltd.
  • Ruchirada Changkwanyeun, Masaru Usui, Siriporn Kongsoi, Kazumasa Yokoyama, Hyun Kim, Orasa Suthienkul, Kanjana Changkaew, Chie Nakajima, Yutaka Tamura, Yasuhiko Suzuki
    JOURNAL OF INFECTION AND CHEMOTHERAPY 21 (8) 604 - 609 1341-321X 2015/08 [Refereed][Not invited]
    Quinolones have long been used as the first-line treatment for Campylobacter infections. However, an increased resistance to quinolones has raised public health concerns. The development of new quinolone-based antibiotics with high activity is critical for effective, as DNA gyrase, the target of quinolones, is an essential enzyme for bacterial growth in several mechanisms. The evaluation of antibiotic activity against Campylobacter jejuni largely relies on drug susceptibility tests, which require at least 2 days to produce results. Thus, an in vitro method for studying the activity of quinolones against the C. jejuni DNA gyrase is preferred. To identify potent quinolones, we investigated the interaction of C. jejuni DNA gyrase with a number of quinolones using recombinant subunits. The combination of purified subunits exhibited DNA supercoiling activity in an ATP dependent manner. Drug concentrations that inhibit DNA supercoiling by 50% (IC(50)s) of 10 different quinolones were estimated to range from 0.4 (sitafloxacin) to >100 mu g/mL (nalidixic acid). Sitafloxacin showed the highest inhibitory activity, and the analysis of the quinolone structure-activity relationship demonstrated that a fluorine atom at R-6 might play the important role in the inhibitory activity against C. jejuni gyrase. Measured quinolone IC50s correlated well with minimum inhibitory concentrations (R = 0.9943). These suggest that the in vitro supercoiling inhibition assay on purified recombinant C. jejuni DNA gyrase is a useful and predictive technique to monitor the antibacterial potency of quinolones. And furthermore, these data suggested that sitafloxacin might be a good candidate for clinical trials on campylobacteriosis. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
  • Kanjana Changkaew, Apiradee Intarapuk, Fuangfa Utrarachkij, Chie Nakajima, Orasa Suthienkul, Yasuhlko Suzuki
    JOURNAL OF FOOD PROTECTION 78 (8) 1442 - 1450 0362-028X 2015/08 [Refereed][Not invited]
    Administration of antimicrobials to food-producing animals increases the risk of higher antimicrobial resistance in the normal intestinal flora of these animals. The present cross-sectional study was conducted to investigate antimicrobial susceptibility and extended-spectrum beta-lactamase (ESBL) producing strains and to characterize class 1 integrons in Escherichia coil in healthy swine in Thailand. All 122 of the tested isolates had drug-resistant phenotypes. High resistance was found to ampicillin (98.4% of isolates), chloramphenicol (95.9%), gentamicin (78.7%), streptomycin (77.9%), tetracycline (74.6%), and cefotaxime (72.1%). Fifty-four (44.3%) of the E. coil isolates were confirmed as ESBL-producing strains. Among them, bla(CTX-M) (45 isolates) and bla(TEM) (41 isolates) were detected. Of the bla(CTX-M)-positive E. coli isolates, 37 carried the bla(CTX-M-1) cluster, 12 carried the bla(CTX-M-9) cluster, and 5 carried both clusters. Sequence analysis revealed bla(TEM-1), bla(TEM-135), and bla(TEM-175) in 38, 2, and 1 isolate, respectively. Eighty-seven (71%) of the 122isolates carried class 1 integrons, and eight distinct drug-resistance gene cassettes with seven different integron profiles were identified in 43 of these isolates. Gene cassettes were associated with resistance to aminoglycosides (aadAl, aadA2, aadA22, or aadA23), trimethoprim (dfrA5, dfrAl2, or dftA17), and lincosamide (linF). Genes encoding beta-lactamases were not found in class 1 integrons. This study is the first to report ESBL-producing E. coli with a class 1 integron carrying the linF gene cassette in swine in Thailand. Our findings confirm that swine can be a reservoir of ESBL-producing E. coli harboring class 1 integrons, which may become a potential health risk if these integrons are transmitted to humans. Intensive analyses of animal, human, and environmental isolates are needed to control the spread of ESBL-producing E. coil strains.
  • Jeewan Thapa, Chie Nakajima, Bhagwan Maharjan, Ajay Poudel, Yasuhiko Suzuki
    JAPANESE JOURNAL OF VETERINARY RESEARCH 63 (3) 151 - 158 0047-1917 2015/08 [Refereed][Not invited]
    Mycobacterium orygis, a new member of the Mycobacterium tuberculosis complex, was isolated from a captive spotted deer (Axis axis) and a blue bull (Boselaphus tragocamelus) in Nepal. Analyses by spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing, region of difference and single nucleotide polymorphism of genes gyrB, mmpL6, TbD1, PPE55 and Rv2042c confirmed the isolates as M. orygis. Moreover, analyses by spoligotyping (SIT587) as well as MIRU-VNTR showed that the isolates shared a similar pattern with many reported isolates. From previous and the present studies, it can be inferred that South Asia is one of the endemic regions for M. orygis. Further investigation including a larger sample size and different host interaction will help to understand the ecology and epidemiology of M. orygis in Nepal.
  • Usami O, Nakajima C, Endo S, Inomata S, Kanamori H, Hirakata Y, Uchiyama B, Kaku M, Suzuki Y, Hattori T
    Clinical case reports 3 (7) 622 - 625 2015/07 [Refereed][Not invited]
  • Hirohito Ogawa, Nobuo Koizumi, Aiko Ohnuma, Alisheke Mutemwa, Bernard M. Hang'ombe, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa
    INFECTION GENETICS AND EVOLUTION 32 143 - 147 1567-1348 2015/06 [Refereed][Not invited]
    The role played by bats as a potential source of transmission of Leptospira spp. to humans is poorly understood, despite various pathogenic Leptospira spp. being identified in these mammals. Here, we investigated the prevalence and diversity of pathogenic Leptospira spp. that infect the straw-colored fruit bat (Eidolon helvum). We captured this bat species, which is widely distributed in Africa, in Zambia during 2008-2013. We detected the flagellin B gene (flaB) from pathogenic Leptospira spp. in kidney samples from 79 of 529 E. helvum (14.9%) bats. Phylogenetic analysis of 70 flaB fragments amplified from E. helvum samples and previously reported sequences, revealed that 12 of the fragments grouped with Leptospira borgpetersenii and Leptospira kirschneri; however, the remaining 58 flaB fragments appeared not to be associated with any reported species. Additionally, the 16S ribosomal RNA gene (rrs) amplified from 27 randomly chosen flaB-positive samples was compared with previously reported sequences, including bat-derived Leptospira spp. All 27 rrs fragments clustered into a pathogenic group. Eight fragments were located in unique branches, the other 19 fragments were closely related to Leptospira spp. detected in bats. These results show that rrs sequences in bats are genetically related to each other without regional variation, suggesting that Leptospira are evolutionarily well-adapted to bats and have uniquely evolved in the bat population. Our study indicates that pathogenic Leptospira spp. in E. helvum in Zambia have unique genotypes. (C) 2015 The Authors. Published by Elsevier B.V.
  • Grace Mwikuma, Geoffry Kwenda, Bernard M. Hang'ombe, Edgar Simulundu, Trevor Kaile, Selestine Nzala, Seter Siziya, Yasuhiko Suzuki
    ANNALS OF CLINICAL MICROBIOLOGY AND ANTIMICROBIALS 14 1  1476-0711 2015/01 [Refereed][Not invited]
    Background: The emergence of Acquired Immunodeficiency Syndrome has highlighted the increased incidence and importance of the disease caused by Non-tuberculous Mycobacteria (NTM). While disease due to M. avium-intracellulare complex is apparently common throughout the world, other Non-tuberculous mycobacterial species have been isolated from both immunocompromised and immunocompetent individuals. The increasing number of infections caused by these organisms has made it clinically important to quickly identify mycobacterial species. The diagnosis of a pathogenic versus a non-pathogenic species not only has epidemiological implications but is also relevant to the demands of patient management. Since antibiotic treatment varies according to the species encountered, species identification would reduce the burden of some of these emerging opportunistic pathogens especially in immunocompromised patients and improve their quality of life. Findings: A total of 91 NTM suspected isolates from four regions of Zambia were included in the study. These isolates were identified using the sequence analysis of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Mycobacteria. Fifty-four of the 91 (59%) isolates were identified as NTM and these included M. intracellulare (27.8%), M. lentiflavum (16.7%), M. avium (14.8%), M. fortuitum (7.4%), M. gordonae (7.4%), M. kumamotonense (3.7%), M. indicus pranii (3.7%), M. peregrinum (3.7%), M. elephantis (1.85%), M. flavescens (1.85%), M. asiaticum (1.85%), M. bouchedurhonense (1.85%), M. chimaera (1.85%), M. europaeum (1.85%), M. neourum (1.85%), M. nonchromogenicum (1.5%). Conclusion: The study has shown that DNA sequencing of the ITS region may be useful in the preliminary identification of NTM species. All species identified in this study were potentially pathogenic.
  • Jingge Zhao, Zhaoqin Zhu, Xiaoyan Zhang, Yasuhiko Suzuki, Haorile Chagan-Yasutan, Haili Chen, Yanmin Wan, Jianqing Xu, Yugo Ashino, Toshio Hattori
    JOURNAL OF IMMUNOLOGY RESEARCH 2015 834749  2314-8861 2015 [Refereed][Not invited]
    Tuberculous glycolipid (TBGL) is a component of the Mycobacterium tuberculosis cell wall, and anti-TBGL antibodies are used for serodiagnosis of tuberculosis. Anti-TBGL IgG and IgA levels were measured in 45 pulmonary TB patients (PTB), 26 extrapulmonary TB patients (ETB), 16 AIDS-TB patients, and 58 healthy controls (HC) including 39 health care workers (HW) and 19 newly enrolled students (ST). Anti-TBGL IgG measurements yielded 68.9% and 46.2% sensitivity in PTB and ETB, respectively, and 81.0% specificity. However, anti-TBGL IgA measurements were significantly less sensitive in detecting ETB than PTB (15.4% versus 46.7% sensitivity) but showed up to 89.7% specificity. Samples from AIDS-TB patients exhibited low reaction of anti-TBGL IgG and IgA with 6.3% and 12.5% sensitivity, respectively. Unlike anti-lipoarabinomannan (LAM) IgG that was found to elevate in sputum smearpositive subjects, anti-TBGL IgG and IgA elevated in those with cavitation and bronchiectasis, respectively. Anti-TBGL IgG in cavitary TB yielded 78.2% sensitivity compared to 57.1% in those otherwise. Meanwhile, higher anti-TBGL IgA titers were observed in HW than in ST, and increasing anti-TBGL IgG titers were observed in HW on follow-up. Therefore, higher anti-TBGL antibody titers are present in patients presenting cavities and bronchiectasis and subjects under TB exposure risk.
  • Suzuki Y, Yamaguchi T, Kim H, Yokoyama K, Nakajima C
    Nihon Hansenbyo Gakkai zasshi = Japanese journal of leprosy : official organ of the Japanese Leprosy Association 83 (3) 21 - 27 1342-3681 2014/12 [Refereed][Not invited]
  • Kenichiro Mori, Katsuki Ohtani, SeongJae Jang, YounUck Kim, Insu Hwang, Nitai Roy, Yasuyuki Matsuda, Yasuhiko Suzuki, Nobutaka Wakamiya
    Biochimica et biophysica acta 1840 (12) 3345 - 56 0006-3002 2014/12 [Refereed][Not invited]
    BACKGROUND: Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD. METHODS: We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively. RESULTS: CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL. CONCLUSIONS: CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL. GENERAL SIGNIFICANCE: We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.
  • SeongJae Jang, Katsuki Ohtani, Atsushi Fukuoh, Kenichiro Mori, Takayuki Yoshizaki, Noritoshi Kitamoto, YounUck Kim, Yasuhiko Suzuki, Nobutaka Wakamiya
    Biochimica et biophysica acta 11 1840 (11) 3226 - 37 0006-3002 2014/11 [Refereed][Not invited]
    BACKGROUND: Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated. METHODS: We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in μ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out. RESULTS: We identified μ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2μ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules. CONCLUSIONS: Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2μ2. GENERAL SIGNIFICANCE: This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.
  • Di Li, Cai-Bo Dong, Jia-Yi Cui, Chie Nakajima, Chun-Lei Zhang, Xin-Ling Pan, Gao-Xiang Sun, En-Yu Dai, Yasuhiko Suzuki, Min Zhuang, Hong Ling
    INFECTION GENETICS AND EVOLUTION 27 294 - 299 1567-1348 2014/10 [Refereed][Not invited]
    Mycobacterium tuberculosis Beijing family includes a variety of sublineages. Knowledge of the distribution of a certain sublineage of the Beijing family may help to understand the mechanisms of its rapid spread and to establish an association between a certain genotype and the disease outcome. We have previously found that M. tuberculosis Beijing family clinical isolates represent approximately 90% of the clinical isolates from Heilongjiang Province, China. To clarify the distribution of M. tuberculosis Beijing family sublineages in Heilongjiang Province, China and to investigate the regularity rule for their evolution, we examined single nucleotide polymorphisms (SNPs) of 250 M. tuberculosis Beijing family clinical isolates using 10 SNP loci that have been identified as appropriate for defining Beijing sublineages. After determining the sequence type (ST) of each isolate, the sublineages of all M. tuberculosis Beijing family isolates were determined, and phylogenetic analysis was performed. We found that 9 out of the 10 SNP loci displayed polymorphisms, but locus 1548149 did not. In total, 92.8% of the isolates in Heilongjiang Province are modern sublineages. ST10 is the most prevalent sublineage (ST10 and ST22 accounted for 63.2% and 23.6% of all the Beijing family isolates, respectively). A new ST, accounting for 4% of the Beijing family isolates in this area, was found for the first time. Each new ST isolate showed a unique VNTR pattern, and none were clustered. The present findings suggest that controlling the spread of these modern sublineages is important in Heilongjiang Province and in China. (c) 2014 Published by Elsevier B.V.
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    IMMUNOLOGY 142 (4) 551 - 561 0019-2805 2014/08 [Refereed][Not invited]
    Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-gamma (IFN-gamma) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-gamma production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1.
  • Kanjana Changkaew, Fuangfa Utrarachkij, Kanokrat Siripanichgon, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
    JOURNAL OF FOOD PROTECTION 77 (8) 1394 - 1401 0362-028X 2014/08 [Refereed][Not invited]
    Antimicrobial resistance in bacteria associated with food and water is a global concern. To survey the risk, 312 Escherichia coli isolates from shrimp farms and markets in Thailand were examined for susceptibility to 10 antimicrobials. The results showed that 17.6% of isolates (55 of 312) were resistant to at least one of the tested drugs, and high resistance rates were observed to tetracycline (14.4%; 45 of 312), ampicillin (8.0%; 25 of 312), and trimethroprim (6.7%; 21 of 312); 29.1% (16 of 55) were multidrug resistant. PCR assay of the tet(A), tet(B), tet(C), tet(D), tet(E), and tet(G) genes detected one or more of these genes in 47 of the 55 resistant isolates. Among these genes, tet(A) (69.1%; 38 of 55) was the most common followed by tet(B) (56.4%; 31 of 55) and tet(C) (3.6%; 2 of 55). The resistant isolates were further investigated for class 1 integrons. Of the 55 resistant isolates, 16 carried class 1 integrons and 7 carried gene cassettes encoding trimethoprim resistance (dfrA12 or dfrA17) and aminoglycosides resistance (aadA2 or aadA5). Two class 1 integrons, In54 (dfrA17-aadA5) and In27 (dfrA12-orfF-aadA2), were found in four and three isolates, respectively. These results indicate a risk of drug-resistant E. coli contamination in shrimp farms and selling places. The occurrence of multidrug-resistant E. coli carrying tet genes and class 1 integrons indicates an urgent need to monitor the emergence of drug-resistant E. coli to control the dissemination of drug-resistant strains and the further spread of resistance genes to other pathogenic bacteria.
  • Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Chie Nakajima, Yasuhiko Suzuki, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi
    MICROBIOLOGY AND IMMUNOLOGY 58 (7) 388 - 397 0385-5600 2014/07 [Refereed][Not invited]
    Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)-1 receptor and its ligand, PD-L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD-L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD-L2 was analyzed in vitro. Recombinant PD-L2 (PD-L2-Ig), which comprises an extracellular domain of bovine PD-L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD-L2. Bovine PD-L2-Ig bound to bovine PD-1-expressing cells and addition of soluble bovine PD-1-Ig clearly inhibited the binding of PD-L2-Ig to membrane PD-1 in a dose-dependent manner. Cell proliferation and IFN-gamma production were significantly enhanced in the presence of PD-L2-Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD-L2-Ig significantly enhanced IFN-gamma production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-gamma production was further enhanced by treatment with anti-bovine PD-1 antibody. These data suggest potential applications of bovine PD-L2-Ig as a therapy for bovine diseases.
  • Mo Zhou, Keisuke Suganuma, Ngasaman Ruttayaporn, Thu-Thuy Nguyen, Shino Yamasaki, Ikuo Igarashi, Shin-ichiro Kawazu, Yasuhiko Suzuki, Noboru Inoue
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (6) 799 - 806 0916-7250 2014/06 [Refereed][Not invited]
    Trypanosoma congolense is a major livestock pathogen in Africa, causing large economic losses with serious effects on animal health. Reliable serodiagnostic tests are therefore urgently needed to control T congolense infection. In this study, we have identified one T congolense protein as a new candidate serodiagnostic antigen. The 46.4 kDa protein (TcP46, Gene ID: TcIL3000.0.25950) is expressed 5.36 times higher in metacyclic forms than epimastigote forms. The complete nucleotide sequences of TcP46 contained an open reading frame of 1,218 bp. Southern blot analysis indicated that at least two copies of the TcP46 gene were tandemly-arranged in the T congolense genome. The recombinant TcP46 (rTcP46) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Western blot analysis and confocal laser scanning microscopy revealed that the native TcP46 protein is expressed in the cytoplasm during all life-cycle stages of the parasite. Moreover, an enzyme-linked immunosorbent assay (ELISA) based on rTcP46 detected the specific antibodies as early as 8 days post-infection from mice experimentally infected with T congolense. No cross-reactivity was observed in the rTcP46-based ELISA against serum samples from cattle experimentally infected with Babesia bigemina, B. bovis and Anaplasma marginale. These results suggest that rTcP46 could be used as a serodiagnostic antigen for T congolense infection.
  • Naoya Maekawa, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Mami Adachi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    PLOS ONE 6 9 (6) e98415  1932-6203 2014/06 [Refereed][Not invited]
    Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the "exhausted'' status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1-expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-gamma production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed.
  • Yasushi Itoh, Reiko Yoshida, Shintaro Shichinohe, Megumi Higuchi, Hirohito Ishigaki, Misako Nakayama, Van Loi Pham, Hideaki Ishida, Mitsutaka Kitano, Masahiko Arikata, Naoko Kitagawa, Yachiyo Mitsuishi, Kazumasa Ogasawara, Hideaki Tsuchiya, Takahiro Hiono, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida, Mutsumi Ito, Le Quynh Mai, Yoshihiro Kawaoka, Hiroko Miyamoto, Mari Ishijima, Manabu Igarashi, Yasuhiko Suzuki, Ayato Takada
    PLOS PATHOGENS 10 (6) e1004192  1553-7366 2014/06 [Refereed][Not invited]
    Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.
  • Sarad Paudel, Susan K. Mikota, Chie Nakajima, Kamal P. Gairhe, Bhagwan Maharjan, Jeewan Thapa, Ajay Poudel, Michito Shimozuru, Yasuhiko Suzuki, Toshio Tsubota
    TUBERCULOSIS 3 94 (3) 287 - 292 1472-9792 2014/05 [Refereed][Not invited]
    Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts. (C) 2014 Elsevier Ltd. All rights reserved.
  • Thu-Thuy Nguyen, Mo Zhou, Ngasaman Ruttayaporn, Quoc Doanh Nguyen, Viet Khong Nguyen, Yasuyuki Goto, Yasuhiko Suzuki, Shin-ichiro Kawazu, Noboru Inoue
    VETERINARY PARASITOLOGY 201 (1-2) 18 - 23 0304-4017 2014/03 [Refereed][Not invited]
    Trypanosoma evansi infection, or surra, is currently affecting various species of animals, especially water buffaloes. Since diagnosis is an important aspect of surra control, development of novel diagnostic antigens is of interest to implement and improve the currently utilized methods. Our study evaluated the tandem repeat antigen TeGM6-4r in T. evansi antibody detection in water buffaloes. TeGM6-4r-based ELISA was performed with 20 positive and 8 negative controls and 484 field samples from water buffaloes in Northern Vietnam. To examine cross-reactivity, sera from Japanese cattle that had been experimentally infected with Theileria orientalis (n=10), Babesia bovis (n=3), Babesia bigemina (n = 7) and Trypanosoma theileri (n=59) were included in the study. The sensitivity of the test was 80%. TeGM6-4r did not react with Theileria or Babesia infected sera, however it showed cross reactivity with 11/59 T. theileri infected samples. The reference test, CATT/T. evansi also reacted with 3/59 T. theileri infected sera. The lysate antigen-based ELISA reacted with 4/59 T. theileri, 9/10 Theileria and 3/10 Babesia infected sera. In contrast, TeGM6-4r-based ELISA was 86.3% sensitive and 58.3% specific in the screening of field samples. The average seroprevalence of T. evansi infection among water buffaloes in Northern Vietnam was 27.1% by CATT/T. evansi and 53.7% by TeGM6-4r. Seroprevalence in the five surveyed provinces ranged from 17.4% to 39.8% in the reference test, and 47.3% to 67.3% in the recombinant antigen based test. The finding indicated that the disease is still widely endemic in the area and that surveillance programs need to be carried out regularly to better control surra. We proposed TeGM6-4r as a useful serodiagnostic antigen for the detection and epidemiological surveillance of T. evansi infection among water buffaloes. (C) 2014 Elsevier B.V. All rights reserved.
  • Beata Shiratori, Susan Leano, Chie Nakajima, Haorile Chagan-Yasutan, Toshiro Niki, Yugo Ashino, Yasuhiko Suzuki, Elisabeth Telan, Toshio Hattori
    MEDIATORS OF INFLAMMATION 2014 513263  0962-9351 2014 [Refereed][Not invited]
    Tuberculosis (TB) is the second most common cause of death from infectious diseases and results in high socioeconomic losses to many countries. Proper diagnosis is the first step in TB eradication. To develop a rapid, simple, and accurate diagnostic TB test and to characterize the prevalence of Mycobacterium tuberculosis (MTB) genotypes and immune profiles of TB patients, a total of 37 TB patients and 30 healthy control (HC) from Metro Manila were enrolled. Loop-mediated isothermal amplification (LAMP) reliably detected MTB infection. Manila genotype was identified by spoligotyping method in all TB patients. Osteopontin (OPN), interferon-gamma-induced protein 10 kDa (IP-10), and neutrophil counts were found to reflect the acute stage of MTB infection. The sensitivity and specificity were 94.6% and 93.3%, respectively, for both OPN and IP-10, and they were 83.8% and 78.6%, respectively, for neutrophils. The combination of OPN, IP-10, neutrophil count, IL-6, IL-8, TNF-alpha, MCP-1, platelets, galectin-9, and leukocyte count correctly identifies all the HC and 96.3% of TB patients. LAMP method may serve as a rapid, supportive method in addition to time-consuming culture methods. OPN, IP-10, and neutrophil counts are useful in detecting MTB infection and may have utility in monitoring the course of the disease.
  • Tomotada Iwamoto, Kentaro Arikawa, Chie Nakajima, Noriko Nakanishi, Yukiko Nishiuchi, Shiomi Yoshida, Aki Tamaru, Yutaka Tamura, Yoshihiko Hoshino, Heekyung Yoo, Young Kil Park, Hajime Saito, Yasuhiko Suzuki
    INFECTION GENETICS AND EVOLUTION 21 479 - 483 1567-1348 2014/01 [Refereed][Not invited]
    The PE (Pro-Glu) and PPE (Pro-Pro-Glu) multigene families are unique to mycobacteria, and are highly expanded in the pathogenic members of this genus. We determined the intra-subspecies genetic variability of the MACPPE12 gene, which is a specific PPE gene in Mycobacterium avium subsp. hominissuis (MAH), using 334 MAH isolates obtained from different isolation sources (222 human isolates, 145 Japanese and 77 Korean; 37 bathroom isolates; and 75 pig isolates). In total, 31 single-nucleotide polymorphisms (SNPs), which consisted of 16 synonymous SNPs and 15 nonsynonymous SNPs, were determined through comparison with the MACPPE12 gene sequence of MAH strain 104 as a reference. As the result, the 334 MAH isolates were classified into 19 and 13 different sequevars at the nucleic acid level (NA types) and amino acid level (AA types), respectively. Among the 13 AA types, only one type, the AA02 type, presented various NA types (7 different types) with synonymous SNPs, whereas all other AA types had a one-to-one correspondence with the NA types. This finding suggests that AA02 is a longer discernible lineage than the other AA types. Therefore, AA02 was classified as an ancestral type of the MACPPE12 gene, whereas the other AA types were classified as modern types. The ubiquitous presence of AA02 in all of the isolation sources and all different sequevars classified by the hsp65 genotype further supports this classification. In contrast to the ancestral type, the modern types showed remarkable differences in distribution between human isolates and pig isolates, and between Japanese isolates and Korean isolates. Divergence of the MACPPE12 gene may thus be a good indicator to characterize MAH strains in certain areas and/or hosts. (C) 2013 Elsevier B.V. All rights reserved.
  • Yukiko Nishiuchi, Aki Tamaru, Yasuhiko Suzuki, Seigo Kitada, Ryoji Maekura, Yoshitaka Tateishi, Mamiko Niki, Hisashi Ogura, Sohkichi Matsumoto
    JOURNAL OF WATER AND HEALTH 12 (2) 211 - 219 1477-8920 2014 [Refereed][Not invited]
    We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA (R) elute card,DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.
  • Mochabo KM, Zhou M, Suganuma K, Kawazu SI, Suzuki Y, Inoue N
    Parasitology research 0932-0113 2013/07 [Refereed][Not invited]
  • Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K
    Veterinary research 1 44 59  0928-4249 2013/07 [Refereed][Not invited]
  • Boniface Namangala, Elizabeth Oparaocha, Kiichi Kajino, Kyoko Hayashida, Ladslav Moonga, Noboru Inoue, Yasuhiko Suzuki, Chihiro Sugimoto
    AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE 89 (1) 116 - 118 0002-9637 2013/07 [Refereed][Not invited]
    Canine African trypanosomosis (CAT) is rarely reported in the literature. In this preliminary study, we evaluated the performance of loop-mediated isothermal amplification (LAMP) against microscopy to detect CAT in six exotic dog breeds naturally infected with trypanosomes from Zambia's South Luangwa National Park and Chiawa Game Management Area. To our knowledge, this is the first report of CAT in Zambia. The patients exhibited a variety of aspecific clinical signs. The LAMP did not only confirm all six parasitologically positive CAT cases detected passively between April 2010 and January 2012, but was also critical in trypanosome speciation. According to LAMP, the majority of the dogs had monolytic infections with either Trypanosoma congolense or Trypanosoma brucei rhodesiense. The LAMP is thus a potential simple and cost-effective tool for trypanosome diagnosis in endemic regions. The rare report of zoonotic trypanosomes in dogs in Zambia has public health implications and justifies further investigations of CAT.
  • Nakajima C, Tamaru A, Rahim Z, Poudel A, Maharjan B, Aye KS, Ling H, Hattori T, Iwamoto T, Fukushima Y, Suzuki H, Suzuki Y, Matsuba T
    J. Clin. Microbiol. 51 (7) 2025 - 2032 2013/07 [Refereed][Not invited]
  • Phetsuksiri B, Rudeeaneksin J, Srisungngam S, Bunchoo S, Roienthong D, Mukai T, Nakajima C, Hamada S, Suzuki Y
    Jpn. J. Infect. Dis. 66 (3) 249 - 251 1344-6304 2013/05 [Refereed][Not invited]
  • Ajay Poudel, Bhagwan Maharjan, Chie Nakajima, Yukari Fukushima, Basu D. Pandey, Antje Beneke, Yasuhiko Suzuki
    TUBERCULOSIS 93 (1) 84 - 88 1472-9792 2013/01 [Refereed][Not invited]
    The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global control of TB. Although molecular characterization of drug resistance-associated mutations in multidrug-resistant isolates in Nepal has been made, mutations in XDR isolates and their genotypes have not been reported previously. In this study, we identified and characterized 13 XDR Mycobacterium tuberculosis isolates from clinical isolates in Nepal. The most prevalent mutations involved in rifampicin, isoniazid, ofloxacin, and kanamycin/capreomycin resistance were Ser531Leu in rpoB gene (92.3%), Ser315Thr in katG gene (92.3%), Asp94Gly in gyrA gene (53.9%) and A1400G in rrs gene (61.5%), respectively. Spoligotyping and multilocus sequence typing revealed that 69% belonged to Beijing family, especially modern types. Further typing with 26-loci variable number of tandem repeats suggested the current spread of XDR M. tuberculosis. Our result highlights the need to reinforce the TB policy in Nepal with regard to control and detection strategies. (C) 2012 Elsevier Ltd. All rights reserved.
  • Zeaur Rahim, Chie Nakajima, Rubhana Raqib, Khalequ Zaman, Hubert P. Endtz, Adri G. M. van der Zanden, Yasuhiko Suzuki
    TUBERCULOSIS 92 (6) 529 - 534 1472-9792 2012/11 [Refereed][Not invited]
    Despite having 100% coverage of directly observed treatment short-course, multi drug-resistant (MDR) tuberculosis (TB) is still increasing in Bangladesh. Early detection of MDR-TB by rapid molecular test and early initiation of treatment will effectively stop this trend. To develop rapid diagnostic tools, molecular characterization of genes conferring Mycobacterium tuberculosis resistance to rifampicin (RIF) and isoniazid (INH) will be required. Hence, this study elucidated the molecular mechanism RIF and INH resistance in 218 MDR strains from hospitalized (n = 161) and non-hospitalized (n = 57) TB patients in Bangladesh. Mutations in rpoB gene were detected in 207 (95.0%) with majority at codon 531 (52.3%). Mutations in katG or inhA or both were detected in 206 (94.5%) with majority at codon 315 of katG (83.9%). It was noteworthy that a novel C to T mutation at position 34 and G to A mutations at position -47 in inhA regulatory region were found, respectively, in combination with mutation at codon 315 of katG. This is the first comprehensive molecular analysis of rpoB and katG genes and inhA regulatory regions of MDR isolates from Bangladesh. This study provides basic data for the construction of low cost tailor-made molecular system for rapid diagnosis of MDR-TB in Bangladesh. (C) 2012 Elsevier Ltd. All rights reserved.
  • Mudenda B. Hang'ombe, Musso Munyeme, Chie Nakajima, Yukari Fukushima, Haruka Suzuki, Wigganson Matandiko, Akihiro Ishii, Aaron S. Mweene, Yasuhiko Suzuki
    BMC VETERINARY RESEARCH 8 221  1746-6148 2012/11 [Refereed][Not invited]
    Background: In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important. Results: A PCR identified 15 and 37 M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia. Conclusions: These results provide strong evidence of M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host.
  • Peter S. Lee, Reiko Yoshida, Damian C. Ekiert, Naoki Sakai, Yasuhiko Suzuki, Ayato Takada, Ian A. Wilson
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 (42) 17040 - 17045 0027-8424 2012/10 [Refereed][Not invited]
    Continual and rapid mutation of seasonal influenza viruses by antigenic drift necessitates the almost annual reformulation of flu vaccines, which may offer little protection if the match to the dominant circulating strain is poor. S139/1 is a cross-reactive antibody that neutralizes multiple HA strains and subtypes, including those from H1N1 and H3N2 viruses that currently infect humans. The crystal structure of the S139/1 Fab in complex with the HA from the A/Victoria/3/1975 (H3N2) virus reveals that the antibody targets highly conserved residues in the receptor binding site and contacts antigenic sites A, B, and D. Binding and plaque reduction assays show that the monovalent Fab alone can protect against H3 strains, but the enhanced avidity from binding of bivalent IgG increases the breadth of neutralization to additional strains from the H1, H2, H13, and H16 subtypes. Thus, antibodies making relatively low affinity Fab interactions with the receptor binding site can have significant antiviral activity when enhanced by avidity through bivalent interactions of the IgG, thereby extending the breadth of binding and neutralization to highly divergent influenza virus strains and subtypes.
  • Zeaur Rahim, Mst. Sabiha Banu Momi, Sajal K. Saha, K. Zaman, Khwaja Nazim Uddin, S. N. A. Ashraf Jamil, Nazmun Nahar, A. K. Azad Khan, E. A. W. D. Cooreman, Motiuddin Ahmed, A. G. M. van der Zanden, Chie Nakajima, Yasuhiko Suzuki
    INTERNATIONAL JOURNAL OF TUBERCULOSIS AND LUNG DISEASE 16 (8) 1132 - 1133 1027-3719 2012/08 [Refereed][Not invited]
  • Aki Tamaru, Chie Nakajima, Takayuki Wada, Yajun Wang, Manabu Inoue, Ryuji Kawahara, Ryoji Maekura, Yuriko Ozeki, Hisashi Ogura, Kazuo Kobayashi, Yasuhiko Suzuki, Sohkichi Matsumoto
    PLOS ONE 7 (8) e42505  1932-6203 2012/08 [Refereed][Not invited]
    Infection and transmission of multidrug-resistant Mycobacterium tuberculosis (MDR-Mtb) and extensively drug-resistant M. tuberculosis (XDR-Mtb) is a serious health problem. We analyzed a total of 1,110 Mtb isolates in Osaka Prefecture and neighboring areas from April 2000 to March 2009. A total of 89 MDR-Mtb were identified, 36 (48.5%) of which were determined to be XDR-Mtb. Among the 89 MDR-Mtb isolates, 24 (27.0%) phylogenetically distributed into six clusters based on mycobacterial interspersed repetitive units-various number of tandem repeats (MIRU-VNTR) typing. Among these six clusters, the MIRU-VNTR patterns of four (OM-V02, OM-V03, OM-V04, and OM-V06) were only found for MDR-Mtb. Further analysis revealed that all isolates belonging to OM-V02 and OM-V03, and two isolates from OM-V04 were clonal. Importantly such genotypes were not observed for drug-sensitive isolates. These suggest that few but transmissible clones can transmit after acquiring multidrug resistance and colonize even in a country with a developed, well-organized healthcare system.
  • Janisara Rudeeaneksin, Supranee Bunchoo, Sopa Srisungngam, Pathom Sawanpanyalert, Sawet Chamnangrom, Atipa Kamolwat, Porntip Thanasripakdeekul, Tooru Taniguchi, Chie Nakajima, Yasuhiko Suzuki, Benjawan Phetsuksiri
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 65 (4) 306 - 311 1344-6304 2012/07 [Refereed][Not invited]
    Definitive diagnosis of tuberculosis (TB) by conventional culture, followed by bacterial identification based on biochemical tests is time-consuming and tedious. Simple loop-mediated isothermal amplification (LAMP) specific for Mycobacterium tuberculosis complex, targeting the M. tuberculosis 16S ribosomal RNA gene, termed TB-LAMP, was evaluated as an alternative for rapid culture confirmation. TB-LAMP was assessed for its ability to detect M. tuberculosis complex in BACTEC MGIT 960-positive cultures. Of the 103 cultures evaluated, 100 were identified to contain M. tuberculosis complex by TB-LAMP and had concordant results with standard biochemical tests of niacin accumulation, nitrate reductase, lack of heat-stable catalase, and susceptibility to para-nitrobenzoic acid. These results indicate that TB-LAMP in combination with BACTEC MOLT 960 is a specific, reliable, and technically feasible method for rapid and accurate identification of M. tuberculosis complex.
  • Tomotada Iwamoto, Chie Nakajima, Yukiko Nishiuchi, Tomoko Kato, Shiomi Yoshida, Noriko Nakanishi, Aki Tamaru, Yutaka Tamura, Yasuhiko Suzuki, Masao Nasu
    INFECTION GENETICS AND EVOLUTION 12 (4) 846 - 852 1567-1348 2012/06 [Refereed][Not invited]
    Mycobacterium avium subsp. hominissuis (MAH) strains are genetically diverse and cause infections in pigs and humans. To elucidate the geographical and host-dependent variations in the genetic diversity of MAH, we performed variable numbers of tandem repeat (VNTR) analysis targeting 19 loci for MAH samples from humans (n = 146), bathroom environments (n = 37), and pigs (n = 75) in Japan; these data were then compared with previously reported VNTR data from other countries. The minimum spanning tree (MST) and the multi-dimensional scaling (MDS) analyses based on the VNTR data indicated a high degree of genetic relatedness between isolates from humans and bathrooms in Japan, but a low degree of similarity with the isolates from France and Finland. Moreover, the comparison showed a higher similarity of isolates from Japanese pigs with those from French humans and pigs and Finnish humans and pigs than with other isolates from humans and bathrooms in Japan. The singularity of the Japanese MAH was characterized as the prevalence of hsp65 sequevar code 15 and ISMav6 for the human and bathroom isolates; however, none of the isolates obtained from the pigs belonged to the code 15 or possessed ISMav6. The genetic diversity of MAH and its regional variations imply a possible regional or local specific source of infection and route of transmission of MAH for humans. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.
  • Nobuo Koizumi, Chie Nakajima, Tsunehito Harunari, Tsutomu Tanikawa, Toshihiro Tokiwa, Eriko Uchimura, Tokujiro Furuya, Claro Niegos Mingala, Marvin Ardeza Villanueva, Makoto Ohnishi, Yasuhiko Suzuki
    JOURNAL OF CLINICAL MICROBIOLOGY 50 (6) 2072 - 2074 0095-1137 2012/06 [Refereed][Not invited]
    We developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction mixture following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB nested PCR.
  • Beata Shiratori, Jing Zhang, Osamu Usami, Haorile Chagan-Yasutan, Yasuhiko Suzuki, Chie Nakajima, Toshimitsu Uede, Toshio Hattori
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 56 (6) 2868 - 2872 0066-4804 2012/06 [Refereed][Not invited]
    Quinolones, in addition to their antibacterial activities, act as immunomodulators. Osteopontin (OPN), a member of the extracellular matrix proteins, was found to play a role in the immune and inflammatory response. We found that quinolones significantly enhanced OPN secretion, namely, garenoxacin (220%), moxifloxacin (62%), gatifloxacin (82%), sparfloxacin, (79%), and sitafloxacin (60%). Enhancement of OPN secretion was shown to be due to the effect of quinolones on the OPN gene promoter activity. We also examined the role of quinolones on apoptosis and found that sparfloxacin decreased the late apoptosis of A549 cells, but garenoxacin did not show the antiapoptotic effect. The antiapoptotic effects of quinolones do not appear to be associated with OPN elevation.
  • Ajay Poudel, Chie Nakajima, Yukari Fukushima, Haruka Suzuki, Basu Dev Pandey, Bhagwan Maharjan, Yasuhiko Suzuki
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 56 (6) 2831 - 2836 0066-4804 2012/06 [Refereed][Not invited]
    Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIP-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.
  • Yasuhiko Suzuki, Chie Nakajima, Aki Tamaru, Hyun Kim, Takashi Matsuba, Hajime Saito
    INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS 39 (5) 435 - 439 0924-8579 2012/05 [Refereed][Not invited]
    Minimum inhibitory concentrations of sitafloxacin, gatifloxacin, moxifloxacin, sparfloxacin, levofloxacin and ciprofloxacin against 59 ciprofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Japan were determined. The isolates were most susceptible to sitafloxacin and gatifloxacin. To understand better the basis for drug resistance, nucleotide sequences encoding the gyrA and gyrB quinolone resistance-determining region were determined. Predicted amino acid sequences revealed distinct mutational patterns likely to be responsible for fluoroquinolone resistance. Double gyrA mutations as well as mutations in both gyrA and gyrB correlated with increased resistance to all fluoroquinolones. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
  • Aixiao Bi, Chie Nakajima, Yukari Fukushima, Aki Tamaru, Isamu Sugawara, Akio Kimura, Ryuji Kawahara, Zhongyi Hu, Yasuhiko Suzuki
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 65 (3) 247 - 251 1344-6304 2012/05 [Refereed][Not invited]
    A rapid, simple, and low-cost diagnostic tool for tuberculosis (TB) detection is urgently needed in countries with a high TB burden. Here, we report a novel loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene for the rapid detection of Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti. The specificity of this assay was evaluated using 4 reference strains of Mycobacterium tuberculosis complex (MTC), 22 species of non-tuberculous mycobacteria (NTM), 7 non-mycobacterial species, and 50 clinical M. tuberculosis isolates. All the reference MTC strains and M. tuberculosis clinical isolates were successfully detected by this method, and there were no false-positive results with NTM or non-mycobacterial species, which demonstrates the high specificity of this assay for MTC. The detection limit was 10 copies of MTC genome within 27 min, and the detection speed of this assay was higher than that of any other isothermal methods reported so far. Because of its speed, simplicity, sensitivity, specificity, and inexpensiveness, the TB hspX LAMP assay is a potential gene diagnostic method for TB detection in developing countries with a high TB burden.
  • Umme Ruman Siddiqi, Haorile Chagan-Yasutan, Chie Nakajima, Hiroki Saitoh, Yugo Ashino, Osamu Usami, Beata Shiratori, Motoki Usuzawa, Yasuhiko Suzuki, Toshio Hattori
    TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE 226 (4) 313 - 319 0040-8727 2012/04 [Refereed][Not invited]
    Nontuberculous mycobacteria (NTM) diseases are in the face of a progressive increase even in immune-competent subjects, and the clinical features of NTM diseases are heterogenous. The decision to institute treatment of the patients should be made after a period of follow up, because therapy is often prolonged, and frequently ineffective. The reasons why some patients develop severe NTM diseases are not clear. Here we observed the involvement of latent tuberculosis infection (LTBI) in clinical and laboratory features of NTM diseases. We evaluated various tuberculosis-related inflammatory markers including osteopontin (OPN), pentraxin-3 (PTX-3), and soluble IL-2 receptor (sIL-2R) in NTM infected patients with or without LTBI. Eight NTM and 5 tuberculosis (TB) patients, and 5 healthy subjects were enrolled. Polymerase Chain Reaction (PCR) analysis confirmed the absence of tuberculosis specific gene (RD1 region), among clinical isolates from NTM patients. Interferon-gamma (IFN-gamma) release assay (IGRA) using Early Secreted Antigenic Target-6 (ESAT-6) and CFP-10, the RD1-encoded protein, was employed for determining LTBI. IGRA was positive in 4/8 NTM (NTM with LTBI, 50%) and 5/5 TB patients. Only 2 of 4 NTM with LTBI were under chemotherapy among all NTM patients, and others were followed up. The plasma levels of OPN, PTX3 and sIL-2R were significantly higher in NTM patients with LTBI than in those without LTBI (P < 0.05). The two patients under therapy showed the highest OPN levels that persisted after treatment. The increased inflammatory levels in NTM patients with LTBI indicate enhanced inflammatory reaction. Extensive therapy may be necessary in such patients.
  • Andrea Marzi, Reiko Yoshida, Hiroko Miyamoto, Mari Ishijima, Yasuhiko Suzuki, Megumi Higuchi, Yukie Matsuyama, Manabu Igarashi, Eri Nakayama, Makoto Kuroda, Masayuki Saijo, Friederike Feldmann, Douglas Brining, Heinz Feldmann, Ayato Takada
    PLOS ONE 7 (4) e36192  1932-6203 2012/04 [Refereed][Not invited]
    Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.
  • Fuangfa Utrarachkij, Srirat Pornraungwong, Kanokrat Siripanichgon, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul
    INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY 154 (1-2) 73 - 78 0168-1605 2012/03 [Refereed][Not invited]
    Salmonella contamination of eggshells, egg contents, reusable egg trays, and various environmental samples was assessed. Although the overall Salmonella contamination rate from egg farms was low (3.2%), over a quarter (26.7%) of egg trays from farms and more than one third (36.7%) of trays from the market were contaminated. Salmonella strains isolated from reusable egg trays were analyzed by serotyping, antimicrobial susceptibility test and Xbal pulsed-field gel electrophoresis (PFGE) typing. Five serovars (S. Braenderup, S. Emek, S. Weltevreden, S. Stanley, and S. Derby) were isolated, and half of the strains assessed were found to be resistant to one or more of the six antimicrobial agents examined. The overall resistance rates to nalidixic acid, trimethoprim-sulfamethoxazole, tetracycline, and ampicillin were 40.7%, 36.0%, 26.7% and 3.5%, respectively. The PFGE types were matched against sample location and drug resistance. S. Braenderup PFGE type A2 (susceptible to all tested drugs) was isolated from all sample sites; PFGE type A2 (resistant to nalidixic acid) was isolated from Farm C and the market. S. Braenderup PFGE type A1 (resistant to four drugs) was isolated from Farms A and C. S. Weltevreden PFGE type C3 (susceptible to all tested drugs) was isolated from Farms A and B and type C4 (susceptible to all tested drugs) was isolated from Farm A and the market. The distribution of the related genotypes and resistance patterns of Salmonella in egg farms and the market indicate drug-resistant strains of Salmonella may be spread on reusable egg trays. (C) 2011 Elsevier B.V. All rights reserved.
  • Kazumasa Yokoyama, Hyun Kim, Tetsu Mukai, Masanori Matsuoka, Chie Nakajima, Yasuhiko Suzuki
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 56 (2) 697 - 702 0066-4804 2012/02 [Refereed][Not invited]
    Amino acid substitutions at position 89 or 91 in GyrA of fluoroquinolone-resistant Mycobacterium leprae clinical isolates have been reported. In contrast, those at position 94 in M. tuberculosis, equivalent to position 95 in M. leprae, have been identified most frequently. To verify the possible contribution of amino acid substitutions at position 95 in M. leprae to fluoroquinolone resistance, we conducted an in vitro assay using wild-type and mutant recombinant DNA gyrases. Fluoroquinolone-mediated supercoiling activity inhibition assay and DNA cleavage assay revealed the potent contribution of an amino acid substitution of Asp to Gly or Asn at position 95 to fluoroquinolone resistance. These results suggested the possible future emergence of quinolone-resistant M. leprae isolates with these amino acid substitutions and the usefulness of detecting these mutations for the rapid identification of fluoroquinolone resistance in leprosy.
  • Takayuki Yoshizaki, Katsuki Ohtani, Wataru Motomura, Seong-Jae Jang, Ken-ichiro Mori, Noritoshi Kitamoto, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
    JOURNAL OF BIOCHEMISTRY 151 (1) 57 - 64 0021-924X 2012/01 [Refereed][Not invited]
    Mannan-binding lectin (MBL) was first discovered as a collectin in animal blood, and was shown to have such unique characteristics as a collage-like domain and a carbohydrate recognition domain. We recently identified human collectin kidney 1 (CL-K1, COLEC11) from a human kidney cDNA library. To quantitate the CL-K1 concentration in blood, we developed several polyclonal and monoclonal antibodies using recombinant human CL-K1 in CHO cells and the CL-K1 fragment in Escherichia coli. Using these antibodies, we established a sandwich enzyme-linked immunosorbent assay (ELISA) system. The concentration of CL-K1 in human plasma was 0.34 +/- 0.13 mu g/ml and that in MBL was 1.72 +/- 1.51 mu g/ml. Concentrations of MBL are often low due to its single nucleotide polymorphisms (SNPs) which seem to be related to an opsonic defect. However, no low concentrations of CL-K1 were observed on testing over two hundred blood samples. We also found that the blood concentration of CL-K1 was not dependent on gender or age and did not correlate completely with that of MBL. The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of CL-K1 in humans.
  • Influence of Lineage-Specific Amino Acid Dimorphisms in GyrA on Mycobacterium tuberculosis Resistance to Fluoroquinolones
    Hyun Kim, Chie Nakajima, Youn Uck Kim, Kazumasa Yokoyama, Yasuhiko Suzuki
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 65 (1) 72 - 74 1344-6304 2012/01 [Refereed][Not invited]
    We conducted in vitro DNA supercoiling assays, utilizing recombinant DNA gyrases, to elucidate the influence of the lineage-specific serine or threonine residue at position 95 of GyrA on fluoroquinolone resistance in Mycobacterium tuberculosis. There was little effect of the GyrA-Ala74Ser amino acid substitution on activity of the GyrA-Ser95 gyrase, while activity of the GyrA-Asp94Gly-Ser95 gyrase was reduced. These findings were in striking contrast to previous reports analyzing GyrA with Thr95 and suggest an important impact of the amino acid in the development of fluoroquinolone resistance.
  • Umme Ruman Siddiqi, Prisca Susan A. Leano, Haorile Chagan-Yasutan, Beata Shiratori, Hiroki Saitoh, Yugo Ashino, Yasuhiko Suzuki, Toshio Hattori, Elizabeth Freda O. Telan
    CLINICAL & DEVELOPMENTAL IMMUNOLOGY 2012 610707  1740-2522 2012 [Refereed][Not invited]
    Anti-tubercular-glycolipid-IgG (TBGL-IgG) and -IgA (TBGL-IgA) antibodies, and the QuantiFERON-TB Gold test (QFT) were compared in healthcare workers (HCWs, n = 31) and asymptomatic human immunodeficiency virus-carriers (HIV-AC, n = 56) in Manila. In HCWs, 48%, 51%, and 19% were positive in QFT, TBGL-IgG, and -IgA, respectively. The TBGL-IgG positivity was significantly higher (P = 0.02) in QFT-positive than QFT-negative HCWs. Both TBGL-IgG- and -IgA-positive cases were only found in QFT-positive HCWs (27%). The plasma IFN-gamma levels positively correlated with TBGL-IgA titers (r = 0.74, P = 0.005), but not TBGL-IgG titers in this group, indicating that mucosal immunity is involved in LTBI in immunocompetent individuals. The QFT positivity in HIV-AC was 31% in those with CD4+ cell counts >350/mu L and 12.5% in low CD4 group (<350/mu L). 59 % and 29% were positive for TBGL-IgG and -IgA, respectively, in HIV-AC, but no association was found between QFT and TBGL assays. TBGL-IgG-positive rates in QFT-positive and QFT-negative HIV-AC were 61% and 58%, and those of TBGL-IgA were 23% and 30%, respectively. The titers of TBGL-IgA were associated with serum IgA (P = 0.02) in HIV-AC. Elevations of TBGL-IgG and -IgA were related to latent tuberculosis infection in HCWs, but careful interpretation is necessary in HIV-AC.
  • Katsuki Ohtani, Yasuhiko Suzuki, Nobutaka Wakamiya
    JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY 2012 493945  1110-7243 2012 [Refereed][Not invited]
    Collectins are characterized by a collagen-like sequence and a carbohydrate recognition domain and are members of the vertebrate C-type lectin superfamily. Recently, "novel collectins", different from "classical collectins" consisting of mannan-binding lectin (MBL) and surfactant proteins A and D (SP-A and SP-D), have been found by reverse genetics. These "novel collectins" consist of collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1), and collectin placenta 1 (CL-P1) and are encoded by three separate genes. Experimental findings on human and animal collectins have shown that both novel collectins and classical collectins play an important role in innate immunity. Based on our recent results and those of others, in this paper, we summarize the new biological functions of these novel collectins in embryonic morphogenesis and development.
  • Yokoyama K, Kim H, Mukai T, Matsuoka M, Nakajima C, Suzuki Y
    PLoS neglected tropical diseases 10 6 e1838  1935-2727 2012 [Refereed][Not invited]
  • Mitsuko Fukuda, Katsuki Ohtani, Seong-Jae Jang, Takayuki Yoshizaki, Ken-Ichiro Mori, Wataru Motomura, Itsuro Yoshida, Yasuhiko Suzuki, Yutaka Kohgo, Nobutaka Wakamiya
    Biochimica et biophysica acta 1810 (12) 1150 - 9 0006-3002 2011/12 [Refereed][Not invited]
    BACKGROUND: Scavenger receptors are generally expressed in macrophages and vascular endothelial cells and some scavenger receptors are thought to contribute to the development of atherosclerosis. METHODS: We cloned the cDNA of a zebrafish CL-P1 (collectin placenta 1) and performed a knockdown study using its antisense morpholino oligonucleotides (MO). RESULTS: Zebrafish CL-P1 (zCL-P1) is 51% identical to human CL-P1 in its amino acid sequence. Microbes and OxLDL bound to zCL-P1 cDNA transfected cells. zCL-P1 mRNA expression gradually increased after 6hours post-fertilization (hpf), reached its highest level at 24hpf, and then decreased, which is similar to the gene expression pattern of Tie-2. The knockdown of zCL-P1 led to an increase in the number of zebrafish embryos with severe morphological abnormalities such as short body lengths and defects in the dorsal aorta at 48hpf. Simultaneous injection of both MO and synthetic zCL-P1 or zVEGF mRNA rescued the abnormal phenotype. CONCLUSIONS: In vivo knockdown study shows that zCL-P1 is implicated in vasculogenesis and those of our in vitro study support its role as a scavenger receptor. GENERAL SIGNIFICANCE: These results suggest that zCL-P1 might be essential for vasculogenesis during the early embryonic phase in bone fish.
  • Satoshi Koyama, Katsuki Ohtani, Jun Fukuzawa, Naoyuki Yao, Mitsuko Fukuda, Seong-Jae Jang, Naoyuki Hasebe, Kenjiro Kikuchi, Hiroyuki Itabe, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
    Biochimica et biophysica acta 1810 (9) 836 - 42 0006-3002 2011/09 [Refereed][Not invited]
    BACKGROUND: Oxidative stress-induced endothelial dysfunction and oxidized low-density lipoprotein (LDL) might play a key role in the pathogenesis of atherosclerosis. We recently identified a vascular endothelial scavenger receptor, collectin placenta 1 (CL-P1), which acts as a receptor for oxidized LDL as well as for microbes. METHODS: We demonstrate how hypoxic and oxidative stress induced CL-P1 expression and compared their effects with the expression of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), an endothelial scavenger receptor expressed by oxidative stress. RESULTS: Hypoxia/reoxygenation induced CL-P1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs). The expression of LOX-1 mRNA in these cells peaked slightly at 24 h, while the expression of CL-P1 had an onset at 72 h and was sustained for 120 h after reoxygenation. Furthermore, the exposure of rat carotid artery endothelium to ischemia/reperfusion increased the maximal CL-P1 mRNA expression at 72 h and expression of its protein peaked at 7 days after this treatment. We demonstrate that CL-P1 up-regulation is induced in vitro and in vivo by oxidative stress. GENERAL SIGNIFICANCE: The inducible expression of CL-P1 by oxidative stress might play a crucial role in endothelial dysfunction or chronic activation leading to the pathogenesis of atherosclerosis.
  • Hyun Kim, Chie Nakajima, Kazumasa Yokoyama, Zeaur Rahim, Youn Uck Kim, Hiroki Oguri, Yasuhiko Suzuki
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 55 (8) 3661 - 3667 0066-4804 2011/08 [Refereed][Not invited]
    Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.
  • Youn Uck Kim, Katsuki Ohtani, Kenichiro Mori, Seong Jae Jang, Yasuhiko Suzuki, Nobutaka Wakamiya
    GENES & GENOMICS 33 (3) 273 - 281 1976-9571 2011/06 [Refereed][Not invited]
    Scavenger receptors including collection-placenta 1 (CL-P1) are cell surface glycoproteins capable of binding to oxidized low density lipoprotein (oxLDL), which are expressed on endothelial cells, macrophages, and smooth muscle cells. We cloned and characterized a 750 base-pair fragment containing the 5'-flanking region of the human CL-P1 gene using a human genomic library. The fragment obtained from the translational start site, which contained three specificity protein-1 (Sp1) sites upstream of transcriptional start site and putative binding sites for granulocyte chemotactic factor and early growth response factor-1, was ligated to the pGL4.10-basic vector, and promoter activity was confirmed by transfection studies. Luciferase and electrophoretic gel motility shift assays revealed that three Sp1 binding sites showed specific DNA-protein binding. Deletion and mutation analyses showed that the region comprising the three Sp1 sites was required for CL-P1 proximal promoter activity in human umbilical vein endothelial cells, Hs683 cells, and SK-LMS cells. Furthermore, the third Sp1 (-28/-20) binding site was regulated through some factor, not the Sp1 protein, by hydrogen peroxide stimulation.
  • Juan Wang, Yan Liu, Chun-Lei Zhang, Bin-Ying Ji, Liu-Zhuo Zhang, Yong-Zhen Shao, Shui-Lian Jiang, Yasuhiko Suzuki, Chie Nakajima, Chang-Long Fan, Yuan-Ping Ma, Geng-Wen Tian, Toshio Hattori, Hong Ling
    JOURNAL OF CLINICAL MICROBIOLOGY 49 (4) 1354 - 1362 0095-1137 2011/04 [Refereed][Not invited]
    For the last decade China has occupied second place, after India, among the top five countries with high burdens of tuberculosis (TB). Heilongjiang Province is located in northeastern China. The prevalence of drug-resistant TB in Heilongjiang Province is higher than the average level in China. To determine the transmission characteristics of Mycobacterium tuberculosis strains isolated in this area and their genetic relationships, especially among the Beijing family strains, we investigated their genotypes. From May 2007 to October 2008, 200 M. tuberculosis isolates from patients presenting pulmonary TB were analyzed by molecular typing using PCR-based methods: spacer-oligonucleotide typing (spoligotyping), Beijing family-specific PCR (detection of the deletion of region of difference 105 [RD105]), and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Different combinations of MIRU-VNTR loci were evaluated to define the genotypes and clustering characteristics of the local strains. We found that Beijing family strains represented 89.5% of the isolates studied. However, the rates of multidrug-resistant (MDR) M. tuberculosis among Beijing and non-Beijing family strains were not statistically different. The 15-locus set is considered the optimal MIRU-VNTR locus combination for analyzing the M. tuberculosis strains epidemic in this area, while the 10-locus set is an ideal set for first-line molecular typing. We found that the clustering rate of all the M. tuberculosis isolates analyzed was 10.0% using the 15-locus set typing. We conclude that the Beijing family genotype is predominant and that highly epidemic TB and MDR TB are less likely associated with the active transmission of M. tuberculosis in the study area.
  • Rapid detection of Mycobacterium tuberculosis complex in Cattle and Lechwe (Kobus leche kafuensis) at the slaughter house.
    Hang’ombe M B, Nakajima C, Ishii A, Fukushima Y, Munyeme M, Matandiko W, Mweene A S, Suzuki Y
    Vet. Sci. Dev. 1 e5  2011 [Refereed][Not invited]
  • Possible Mode of Emergence for Drug-Resistant Leprosy Is Revealed by an Analysis of Samples from Mexico
    Masanori Matsuoka, Yasuhiko Suzuki, Iris Estrada Garcia, Mary Fafutis-Morris, Alberto Vargas-Gonzalez, Cristina Carreno-Martinez, Yukari Fukushima, Clue Nakajima
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 63 (6) 412 - 416 1344-6304 2010/11 [Refereed][Not invited]
    Mexico is a country with sporadic leprosy cases, and the reemergence of drug resistance is a concern In this study, molecular analysis of Mycobacterium leprae was employed to clarify the spread of drug-resistant leprosy Thus, drug resistance-determining regions in the folP1, rpoB, and gyrA genes, which are associated with resistance to dapsone, rifampicin, and ofloxacin, respectively, were analyzed by direct sequencing of the PCR product No mutations in the folP1 gene were observed in any of the 72 slit skin samples obtained from 38 patients, although two samples carrying a mutation at codon 425 in the rpoB gene, which confers resistance to rifampicin, a key component of multidrug therapy, were identified In addition, a mutation at codon 91 in the gyrA gene, which correlates with ofloxacin resistance, was found in one sample These results demonstrate the existence of rifampicin- and ofloxacin-resistant leprosy Interestingly, wild-type and mutant sequences in the gyrA gene were found to coexist in one clinical sample In addition, all three drug resistance-related mutations were found in only one of the two earlobes of the patients concerned, suggesting a possible pathway for the spread of drug-resistant M leprae
  • Kanako Ishihara, Natsumi Shimokubo, Akie Sakagami, Hiroshi Ueno, Yasukazu Muramatsu, Tsuyoshi Kadosawa, Chie Yanagisawa, Hideaki Hanaki, Chie Nakajima, Yasuhiko Suzuki, Yutaka Tamura
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 76 (15) 5165 - 5174 0099-2240 2010/08 [Refereed][Not invited]
    Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.
  • A Novel Method for the Purification of DNA by Capturing Nucleic Acid and Magnesium Complexes on Non-Woven Fabric Filters under Alkaline Conditions for the Gene Diagnosis of Tuberculosis by Loop-Mediated Isothermal Amplification (LAMP)
    Tadashi Fukasawa, Naozumi Oda, Yasunao Wada, Aki Tamaru, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 63 (4) 246 - 250 1344-6304 2010/07 [Refereed][Not invited]
    A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg2+) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg2+ complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg2+ complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg2+ was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.
  • Chie Nakajima, Zeaur Rahim, Yukari Fukushima, Isamu Sugawara, Adri G. M. van der Zanden, Aki Tamaru, Yasuhiko Suzuki
    BMC INFECTIOUS DISEASES 10 118  1471-2334 2010/05 [Refereed][Not invited]
    Background: Species identification of isolates belonging to the Mycobacterium tuberculosis complex (MTC) seems to be important for the appropriate treatment of patients, since M. bovis is naturally resistant to a first line anti-tuberculosis (TB) drug, pyrazinamide, while most of the other MTC members are susceptible to this antimicrobial agent. A simple and low-cost differentiation method was needed in higher TB burden countries, such as Bangladesh, where the prevalence of M. bovis among people or cattle has not been investigated. Methods: Genetic regions cfp32, RD9 and RD12 were chosen as targets for a species distinguishable multiplex PCR and the system was evaluated with twenty reference strains of mycobacterial species including non-tubercular mycobacteria (NTM). A total of 350 clinical MTC isolates obtained in Bangladesh were then analyzed with this multiplex PCR. Results: All of the MTC reference strains gave expected banding patterns and no non-specific amplifications were observed in the NTM strains. Out of 350 clinical isolates examined by this method, 347 (99.1%) were positive for all of the cfp32, RD9 and RD12 and determined as M. tuberculosis. Two isolates lacked cfp32 PCR product and one lacked RD12, however, those three samples were further examined and identified as M. tuberculosis by the sequence analyses of hsp65 and gyrB. Conclusions: The MTC-discrimination multiplex PCR (MTCD-MPCR) developed in this study showed high specificity and was thought to be very useful as a routine test because of its simplicity. In the current survey, all the 350 MTC isolates obtained from Bangladesh TB patients were determined as M. tuberculosis and no other MTC were detected. This result suggested the general TB treatment regimen including pyrazinamide to be the first choice in Bangladesh.
  • Kazuhisa Okada, Siriporn Chantaroj, Tooru Taniguchi, Yasuhiko Suzuki, Amonrattana Roobthaisong, Orapim Puiprom, Takeshi Honda, Pathom Sawanpanyalert
    DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE 66 (2) 135 - 139 0732-8893 2010/02 [Refereed][Not invited]
    Loop-mediated isothermal amplification (LAMP) method was designed for clinical diagnosis of Vibrio cholerae carrying the call gene. The detection limits of the method were 5 fg of purified genomic DNA/reaction and 0.54 CFU/reaction. The method was applied to rectal swab samples from cholera patients and healthy volunteers (19 subjects each) and yielded the same results as the "gold standard" culture method, while the polymerase chain reaction-based method failed to detect V cholerae in 8 of the positive samples. Direct application of this LAMP method without precultivation enabled the rapid detection of 5 asymptomatic carriers from rectal swabs of 21 household contacts of cholera patients. This LAMP method could be a sensitive, specific, inexpensive, and rapid detection tool for V cholerae carrying the ctxA gene in the clinical laboratory and in the field. (C) 2010 Elsevier Inc. All rights reserved.
  • Susumu Uchiyama, Yasuhiko Suzuki, Kentaro Otake, Masami Yokoyama, Mitsuo Ohta, Shuichi Aikawa, Midori Komatsu, Tetsuji Sawada, Yoshitoyo Kagami, Yasuo Morishima, Kiichi Fukui
    CANCER SCIENCE 101 (1) 201 - 209 1347-9032 2010/01 [Refereed][Not invited]
    We describe novel humanized anti-CD20 monoclonal antibodies (mAbs) developed for therapeutic use on the basis of their physicochemical properties and cellular cytotoxicity. A distinct correlation between apparent dissociation constants (K(d)) and apoptotic activity for eight murine anti-CD20 mAbs (OUBM1-OUBM8) and previously-developed murine anti-CD20 mAbs enabled us to categorize anti-CD20 mAbs into two groups. Group A mAbs had lower K(d) values and did not induce definite apoptosis, while Group B mAbs had greater K(d) values and did induce definite apoptosis. A murine version mAb of rituximab, 2B8, belongs to Group B. An epitope analysis showed that the epitope of two murine mAbs, OUBM3 and OUBM6, differed from that of 2B8 or 2F2 (ofatumumab). Two mAbs, OUBM3 from Group A and OUBM6 from Group B, were selected and humanized. As expected, the humanized OUBM3 with the lower K(d) did not induce apoptosis, while the humanized OUBM6 (hOUBM6) with the greater K(d) did. Both hOUBM3 and hOUBM6 induced highly-effective, complement-dependent cytotoxicity and antibody-dependent, cell-mediated cytotoxicity against Burkitt's and follicular lymphomas. Importantly, hOUBM6 exhibited cellular cytotoxicity against diffuse, large B cells that are less effectively depleted by rituximab and also exhibited effective cytotoxicity against tumor cells from human CD20(+) leukemia and lymphoma patients. These results suggest the potential impact of the further development of our anti-CD20 mAbs. Our study shows that the selection of mAbs based on their physicochemical parameters, followed by the biological activity assessment for the selected mAbs, is a rational and efficient approach for pharmaceutical mAb development. (Cancer Sci 2009).
  • Tomoko Yoda, Yasuhiko Suzuki, Kenji Yamazaki, Naomi Sakon, Masashi Kanki, Tetsuo Kase, Kazuo Takahashi, Kiyoshi Inoue
    JOURNAL OF MEDICAL VIROLOGY 81 (12) 2072 - 2078 0146-6615 2009/12 [Refereed][Not invited]
    Norovirus is a major etiologic agent in worldwide outbreaks of gastroenteritis associated with food as well as person-to-person transmission. The ubiquitous nature of Norovirus necessitates simple and rapid detection methods with high accuracy and sensitivity. To this end, several investigators have evaluated the usefulness of commercial reverse-transcription loop-mediated isothermal amplification (RT-LAMP) kits for detecting Norovirus genogroups I (GI) and II (GII). In previous studies, the conventional Loopamp kit for Norovirus GII showed a relatively high detection rate, while that for Norovirus GI showed a relatively low detection rate. In the present study, clinical Norovirus specimens were used to compare the detection rate of a modified Loopamp kit for Norovirus GI with the rates of the conventional Loopamp kit for Norovirus GI and an "in-house" RT-LAMP GI primer set, methods which had a high detection rate. Results from the present study showed that the modified Loopamp kit for Norovirus GI had a higher detection rate for two viral genotypes (GI.3, GI.11). On comparison with an "in-house" GII primer set using genotype GII.4 viruses circulating recently, the detection rate by the Loopamp kit for Norovirus GII was found to be higher, with a 98% detection rate. These results indicate the applicability of the modified LAMP kit for GI and the conventional LAMP kit for GII for detection of Noroviruses in clinical samples. J. Med. Virol. 81:2072-2078, 2009. (C) 2009 Wiley-Liss, Inc.
  • Loop-Mediated Isothermal Amplification (LAMP) for the Direct Detection of Human Pulmonary Infections with Environmental (Nontuberculosis) Mycobacteria
    Bal Ram Adhikari, Basu Dev Pandey, Prakash Ghimire, Bhawana Shrestha, Manoj Khadka, Tomoko Yoda, Yasuhiko Suzuki
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 62 (3) 212 - 214 1344-6304 2009/05 [Refereed][Not invited]
    Most first-line anti-tuberculosis drugs have less in vitro activity against atypical mycobacteria. Loop-mediated isothermal amplification (LAMP) was used for the rapid diagnosis of mycobacterial species. The sensitivity of LAMP was 96.1% (49/51) in smear-positive and culture-positive sputum samples and 85.0% (17/20) in smear-negative and culture-positive samples. Of the 77 total LAMP-positive samples, 75 (97.4%) were identified as Mycobacterium tuberculosis and 2 (2.6%) as M. intracellulare. One of the M. intracellulare-infected cases was identified in a patient with suspected mycobacteriosis and another was found in a follow-up patient.
  • SeongJae Jang, Katsuki Ohtani, Atsushi Fukuoh, Takayuki Yoshizaki, Mitsuko Fukuda, Wataru Motomura, Kenichiro Mori, Jun Fukuzawa, Noritoshi Kitamoto, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 (6) 3956 - 3965 0021-9258 2009/02 [Refereed][Not invited]
    Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs, although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.
  • Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum.
    Poudel A, Pandey BD, Lekhak B, Rijal B, Sapkota BR, Suzuki Y
    Kathmandu Univ. Med. J 7 109 - 114 2009 [Refereed][Not invited]
  • A. Edagawa, A. Kimura, H. Doi, H. Tanaka, K. Tomioka, K. Sakabe, C. Nakajima, Y. Suzuki
    JOURNAL OF APPLIED MICROBIOLOGY 105 (6) 2104 - 2114 1364-5072 2008/12 [Not refereed][Not invited]
    To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20.0%) water samples from 17 (42.5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real-time PCR (from 1.7 x 10(5) to 2.6 x 10(11) cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0.05). Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.
  • Masanori Matsuoka, Khin Saw Aye, Kyaw Kyaw, Esterlina Virtudes Tan, Ma Victoria Balagon, Paul Saunderson, Robert Gelber, Masanao Makino, Chie Nakajima, Yasuhiko Suzuki
    JOURNAL OF MEDICAL MICROBIOLOGY 57 (10) 1213 - 1219 0022-2615 2008/10 [Refereed][Not invited]
    A simple method to detect mutations in the genome of Mycobacterium leprae that confer resistance to key drugs for leprosy was exploited on the basis of a reverse hybridization system. A series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB and gyrA genes for dapsone, rifampicin and ofloxacin resistance, respectively, were selected and fixed on a glass slide as capture probes, to develop a DNA microarray termed the leprosy drug susceptibility-DNA microarray (LDS-DA). Mutations in clinical isolates of M. leprae were successfully identified by the LDS-DA. Feasibility studies were conducted to evaluate the performance of the LDS-DA in two developing countries, Myanmar and the Philippines. The high concordance of the results obtained by this method with the results of nucleotide sequencing strongly supports the applicability of the LDS-DA as a drug susceptibility test in place of sequencing, a time-consuming and costly procedure. This is a rapid and simple method for the simultaneous susceptibility testing of three front-line drugs for leprosy, and solves the problems of previously reported methods.
  • Basu Dev Pandey, Ajay Poudel, Tomoko Yoda, Aki Tamaru, Naozumi Oda, Yukari Fukushima, Binod Lekhak, Basista Risal, Bishnu Acharya, Bishwa Sapkota, Chie Nakajima, Tooru Taniguchi, Benjawan Phetsuksiri, Yasuhiko Suzuki
    JOURNAL OF MEDICAL MICROBIOLOGY 57 (4) 439 - 443 0022-2615 2008/04 [Refereed][Not invited]
    A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 % respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.
  • Wataru Motomura, Takayuki Yoshizaki, Katsuki Ohtani, Toshikatsu Okumura, Mituko Fukuda, Jun Fukuzawa, Kenichiro Mori, Seong-Jae Jang, Naoki Nomura, Itsuro Yoshida, Yasuhiko Suzuki, Yutaka Kohgo, Nobutaka Wakamiya
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 56 (3) 243 - 252 0022-1554 2008/03 [Refereed][Not invited]
    We have recently identified a novel collectin, CL-K1, that may play a role in innate immunity as a member of the collectin family. In this study using mice, we investigated the tissue distribution of CL-K1 for better understanding of its pathophysiological relevance. Real-time PCR analyses demonstrated that CL-K1 mRNA was expressed in all tissues tested. Immunohistochemical analyses demonstrated that CL-K1 was expressed in proximal tubules of kidney, in mucosa of the gastrointestinal tract, and in bronchial glands of bronchioles similar to the localization of SP-A and SP-D in these pulmonary structures. Immunohistochemistry also showed that CL-K1 was highly expressed in hepatocytes around the central which suggests that murine CL-K1 may be mainly produced in the liver and veins in liver, secreted into the blood stream as is human CL-K1. CL-K1 was especially detected in vascular smooth muscle in several types of tissues. In addition, it was also expressed in intestinal Paneth cells, in mesangial cells of kidney, in pancreatic islet D cells, and in neurons of the brain. It is of interest that this profile of CL-K1 expression is unique among the collectins. Together these histological findings may be useful for understanding the biological function of this novel collectin.
  • Megumi Higuchi, Aya Matsuo, Masashi Shingai, Kyoko Shida, Akihiro Ishii, Kenji Funami, Yasuhiko Suzuki, Hiroyuki Oshiumi, Misako Matsumoto, Tsukasa Seya
    DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 32 (2) 147 - 155 0145-305X 2008 [Refereed][Not invited]
    Human Toll-like receptor 2 (TLR2) subfamily recognizes bacterial lipoproteins (BLP) and peptidoglycan (PGN). According to the genome information, chicken has structural orthologs of TLRs1 and 2, in addition to TLRs3, 4, 5 and 7. Chicken has two additional TLRs, TLR15 and TLR21, whose orthologs human lacks. The chicken (ch)TLR1 and 2 genes are individually duplicated to encode for four different proteins, chTLR1-1, 1-2, 2-1 and 2-2, of the TLR2 subfamily. Here we investigated the functional profile of these TLR2 subfamily proteins of chicken. By NF-kappa B reporter assay using HEK293 cells, we found that chTLR2-1 and chTLR1-2 cooperatively signal the presence of PGN. A combination of chTLR2-1 and chTLR1-2 also most efficiently recognized diacylated BLP, macrophage-activating lipopeptide 2 kDa (Malp-2), while the combination of chTLR2-1 and chTLR1-1 failed to recognize Malp-2. All combinations, however, recognized triacylated BLP, Pamm3. Consistent with these results, human TLR2-stimulating mycobacteria preparations, BCG-cell wall and cell lysate of Mycobacterium avium, induced activation of NF-kappa B in cells expressing chTLR2-1 and 1-2 and to lesser extents, cells with chTLR2-2 and either of chTLR1. Strikingly, expression of either of these alone did not activate the reporter for NF-kappa B. These chTLRs are likely to have the combination functional feature as in the human TLR2 subfamily. Confocal and immunoprecipitation analyses of human cell transfectants showed that they cluster on the cell surface by a physical molecular association, causing all of them to merge and coprecipitate. These results suggest that chTLR2 subfamily members discriminate between their ligands by combinational events. (C) 2007 Elsevier Ltd. All rights reserved.
  • Ikuo Shiratori, Yasuhiko Suzuki, Hiroyuki Oshiumi, Nasim A. Begum, Takashi Ebihara, Misako Matsumoto, Kaoru Hazeki, Ken Kodama, Yasuo Kashiwazaki, Tsukasa Seya
    CANCER SCIENCE 98 (12) 1936 - 1942 1347-9032 2007/12 [Refereed][Not invited]
    Interleukin (IL)-12 and IL-18 are secreted by myeloid cells activated with adjuvants such as Bacillus Calmette-Guerin (BCG) cell wall. They induce T-helper 1 polarization in the host immune system and upregulate production of lymphocyte interferon-gamma, which leads to the induction of an antitumor gene program. It has been reported that humans have an immune system that more closely resembles that of the guinea pig in adjuvant-response features rather than the mouse system, which prevents the mouse results being extrapolated to human immunotherapy. Here we have constructed a tumor-implant system in guinea pigs to evaluate the antitumor potential of guinea pig IL-12 (gpIL-12) and guinea pig IL-18 (gpIL-18). Purified recombinant gpIL-12 and gpIL-18 were prepared and applied intraperitoneally to tumor-bearing (line 10 hepatoma) guinea pigs as the basis of the adjuvant immunotherapy. Intraperitoneal administration of gpIL-12 and gpIL-18 led to retardation of primary tumor growth and suppression of lymph-node metastasis in tumor-bearing guinea pigs. The permissible range of IL-12 appeared wider in guinea pigs than in mice. Even at an IL-12 dose higher than that in mice, there was no evidence of side-effects until day 26, when the guinea pigs were killed. gpIL-18 augmented the antitumor effect of gpIL-12 but exerted less ability to suppress lymph-node metastasis. The effects of gpIL-12 and gpIL-18 on the tumors implanted in guinea pigs will encourage us to use IL-12- and IL-18-inducible adjuvants for immunotherapy in human patients with solid cancer.
  • Yukihiro Tambe, Atsuko Yoshioka-Yamashita, Ken-ichi Mukaisho, Seiki Haraguchi, Tokuhiro Chano, Takahiro Isono, Takao Kawai, Yasuhiko Suzuki, Ryoji Kushima, Takanori Hattori, Motohito Goto, Shuichi Yamada, Makoto Kiso, Yumiko Saga, Hirokazu Inoue
    CARCINOGENESIS 28 (4) 777 - 784 0143-3334 2007/04 [Not refereed][Not invited]
    The drs gene was originally isolated as a suppressor of v-src transformation. Expression of drs mRNA is markedly downregulated in a variety of human cancer cell lines and tissues, suggesting the potential role of this gene as a tumor suppressor. Previously, we found that Drs protein associates with ASY/Nogo-B/RTN-x(S), an apoptosis-inducing protein in the endoplasmic reticulum, and sequentially activates caspases to induce apoptosis in human cancer cells without involvement of the mitochondria. In this study, we investigated the tumor suppressor function of drs and the correlation between Drs-mediated apoptosis and tumor suppression by generating a gene-knockout (KO) mouse. Between 7 and 12 months after birth, malignant tumors including lymphomas, lung adenocarcinomas and hepatomas were generated in about 30% of the drs KO mice, whereas no tumors were found in any of the wild-type mice during the same period of time. drs KO embryonic fibroblasts also showed enhanced sensitivity to transformation by v-src oncogene. Reintroduction of drs into a tumor cell line derived from the tumor of a drs KO mouse led to the suppression of tumor formation in nude mice, which was accompanied by enhanced apoptosis and the activation of caspase-9 and -3. Furthermore, introduction of drs into this cell line enhanced sensitivity to apoptosis mediated by caspase-3, -9 and -12 under low serum culture conditions. The present results thus indicate that drs contributes to the suppression of malignant tumor formation, and this suppression is closely correlated with drs-mediated apoptosis.
  • Takashi Matsuba, Yasuhiko Suzuki, Yoshinori Tanaka
    ARCHIVES OF MICROBIOLOGY 187 (4) 297 - 311 0302-8933 2007/04 [Refereed][Not invited]
    The Rv0679c gene in Mycobacterium tuberculosis H37Rv encodes a protein with a predicted molecular mass of 16,586 Da consisting of 165 amino acids which contains a putative N-terminal signal sequence and a consensus lipoprotein-processing motif. Globomycin treatment, Triton X-114 separation and mass spectrometry analyses clarified a property of the Rv0679c protein as a lipoprotein. In addition, trifluoromethanesulphonic acid treatment of the lysate revealed an association of the recombinant Rv0679c protein with carbohydrates. The Rv0679c protein homolog of Mycobacterium bovis BCG was also expressed as the protein associated with lipids and carbohydrates. In Western blot analysis, each of the protein homolog and Lipoarabinomannan (LAM) was detected as a similar pattern by anti-Rv0679c and anti-LAM antibodies, respectively. Interestingly, the Rv0679c protein was detected in commercially available LAM purified from M. tuberculosis. Inhibition assay of LAM synthesis in M. bovis BCG by ethambutol showed an altered migration pattern of the Rv0679c protein to low molecular mass similar to that of LAM. The results suggest that the Rv0679c protein exists as a tight complex with LAM in M. tuberculosis/M. bovis BCG.
  • Tomoko Yoda, Yasuhiko Suzuki, Kenji Yamazaki, Naomi Sakon, Masashi Kanki, Ikuko Aoyama, Teizo Tsukamoto
    JOURNAL OF MEDICAL VIROLOGY 79 (3) 326 - 334 0146-6615 2007/03 [Refereed][Not invited]
    A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT-LAMP assay and compared with the results of routine RT-PCR. The results of the RT-LAMP assay corresponded well to that of RT-PCR. These findings suggest the practical application of the RT-LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT-LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT-LAMP system is able to detect NVs in clinical specimens within a wide range.
  • Susumu Okamoto, Aki Tamaru, Chie Nakajima, Kenji Nishimura, Yukinori Tanaka, Shinji Tokuyama, Yasuhiko Suzuki, Kozo Ochi
    MOLECULAR MICROBIOLOGY 63 (4) 1096 - 1106 0950-382X 2007/02 [Refereed][Not invited]
    Streptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycin-resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low-level streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m(7)G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so-called 530 loop. Thus, we have identified gidB as a new streptomycin-resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m(7)G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10(-6) and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.
  • Kawai T, Kase T, Suzuki Y, Eda S, Sakamoto T, Ohtani K, Wakamiya N
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 2 69 (2) 221 - 224 0916-7250 2007/02 [Refereed][Not invited]
  • Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction
    Rungrat Nintasen, Fuangfa Utrarachkij, Kanokrat Siripanichgon, Adisak Bhumiratana, Yasuhiko Suzuki, Orasa Suthienkul
    MICROBIOLOGY AND IMMUNOLOGY 51 (8) 777 - 785 0385-5600 2007 [Refereed][Not invited]
    The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (inip) gene-specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100-treated water samples, or by using silica-gel membrane spin column-eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3'proximity of the mip-coding gene of L. pneumophila deposited in genome databases. EcoRI- or KpnI-digested PCR fragments with expected sizes were also confirmed in all 52 PCR-positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR-positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments.
  • Wataru Sugiura, Tomoko Yoda, Takashi Matsuba, Yoshinori Tanaka, Yasuhiko Suzuki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 70 (7) 1655 - 1665 0916-8451 2006/07 [Refereed][Not invited]
    Azoreductases have been characterized as enzymes that can decolorize azo dyes by reducing azo groups. In this study, genes encoding proteins having homology with theazoreductase gene of Bacillus sp. OY1-2 were obtained from Bacillus subtilis ATCC6633, B. subtilis ISW1214, and Geobacillus stearotherophilus IF013737 by polymerase chain reaction. All three genes encoded proteins with 174 amino acids. The deduced amino acid sequences of azoreductase homologs from B. subtilis ISW1214,B-.. subtilis ATCC6633, and G. stearotherophilus IF013737 showed similarity of 53.3, 53.9, and 53.3 % respectively to that of Bacillus sp. OY1-2. All three genes were expressed in Escherichia coli, and were characterized as having the decolorizing activity of azo dyes in a beta-NADPH dependent manner. The transformation of several azo dyes into colorless compounds by recombinant enzymes was demonstrated to have distinct substrate specificity from that of azoreductase from Bacillus sp. OY1-2.
  • Identification and characterization of a novel human collectin CL-K1
    Hiroyuki Keshi, Takashi Sakamoto, Takao Kawai, Katsuki Ohtani, Tsuyoshi Katoh, Seong-Jae Jang, Wataru Motomura, Takayuki Yoshizaki, Mitsuko Fukuda, Satoshi Koyama, Jun Fukuzawa, Atsushi Fukuoh, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
    MICROBIOLOGY AND IMMUNOLOGY 50 (12) 1001 - 1013 0385-5600 2006 [Refereed][Not invited]
    Collectins are a family of C-type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host-defense. Here we report the cloning and characterization of a novel collectin CL-K1. RT-PCR analyses showed CL-K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL-K1 cDNA expressing CHO cells revealed that CL-K1 is expressed as a secreted protein. CL-K1 is found in blood by immunoblotting and partial amino acid analyses. CL-K1 showed Ca2+-dependent sugar binding activity of fucose and weakly mannose but not N-acetyl-galactosamine, N-acetyl-glucosamine, or maltose, though mannose-binding lectin (MBL) containing similar amino acid motif. CL-K1 can recognize specially several bacterial saccharides due to specific sugar-binding, character. Elucidation of the role of two ancestor collectins of CL-K1 and CL-L1 could lead to see the biological function of collectin family.
  • H Matsumoto, S Adachi, Y Suzuki
    ARCHIVES OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY 48 (4) 459 - 466 0090-4341 2005/05 [Not refereed][Not invited]
    To survey the estrogenic activity of the organic extracts from particulate matter of urban ambient outdoor air, samples were collected on glass fiber filters using a high-volume air sampler on the rooftop of our institute for 6 months (six filters/month). After extracting the organic materials and separating them into three fractions, i.e., acidic, neutral, and basic, we applied a cell-growth assay using MCF-7 human breast cancer cells to the original extract and the extracts of the fractions. Only the extract in the acidic fraction showed cell proliferation activity in a dose-response manner. To survey the chemical(s) responsible for the activity, a gas chromatography/ mass spectrometry (GC/MS) analysis was conducted after silylating the extract. The presence of bisphenol A (BPA) was confirmed, because the retention times and the MS fragment patterns between the silylated derivative of a component in the sample and that of BPA itself were the same. By using a GC/ MS-SIM (selective ion monitoring) technique, the average value was found to be 0.51 ng/m(3) of air (range: 0.02 similar to 1.92 ng/m(3) of air). The trend of the residual levels in air particulates showed seasonal variation, increasing from autumn to winter and decreasing from winter to spring. The only exception was that the value in January was lower than those in December and February. Considering the content of BPA in the extract of the acidic fraction and the strength of the activities with the extract and BPA itself, the estrogenic activity due to BPA in the fraction seemed to decrease. In spite of this decline, the possibility remains that the estrogenic activity mainly originated from BPA.
  • Y Kumai, Y Suzuki, Y Tanaka, K Shima, RK Bhadra, S Yamasaki, K Kuroda, G Endo
    EPIDEMIOLOGY AND INFECTION 133 (1) 59 - 70 0950-2688 2005/02 [Refereed][Not invited]
    A total of 455 highly tetracycline-resistant Escherichia coli strains were isolated from 84 healthy swine from abattoirs and it was found that 56.9, 43.1, 22.2, 15.4, 2.6 and 1.5 % of strains were resistant to chloramphenicol, ampicillin, kanamycin, trimethoprim-sulphamethoxazole, ofloxacin and gentamicin respectively. Interestingly, E. coli strains isolated from certain finisher hog groups exhibited resistance against 2-7 antimicrobials, but strains isolated from multiparous sow groups in each herd were resistant to only 2-4 antimicrobial agents. When randomly selected 108 tetracycline-resistant isolates were tested for the presence of resistance genes, the following genes tet(A) (n = 6), tet(B) (n = 95), tet(D) (n = 1) or both tet(A) and tet(B) (n = 6) were found to be distributed among them. Furthermore, 52 isolates carried the integrase 1 gene and 24 strains gave live different PCR amplicon profiles using primers from the variable region of integron. Extensive nucleotide sequence analyses of these amplicons revealed the presence of dhfrI, dhfrXII, dfr17, aadA, aadA2, aadA5, aadA21, aacA4 and catB3 genes which code for different antibacterial resistance proteins.
  • 足立伸一, 山本康次, 織田肇, 小野芳朗, 鈴木定彦
    水環境学会誌 28 (7) 451 - 456 0916-8958 2005 [Not refereed][Not invited]
  • 松本比佐志, 足立伸一, 鈴木定彦
    薬学雑誌 125 (8) 643 - 652 0031-6903 2005 [Not refereed][Not invited]
  • Kimura A, Suzuki Y, Matsui T
    J Vet Med Sci 66 (7) 879 - 881 0916-7250 2004 [Not refereed][Not invited]
  • K Suzuki, Y Suzuki, S Kitamura
    JOURNAL OF EXPERIMENTAL BOTANY 54 (389) 1997 - 1999 0022-0957 2003/08 [Refereed][Not invited]
    A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-D-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-D-glucuronic acid to UDP-D-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots.
  • T Yoda, Y Suzuki, Y Terano, K Yamazaki, N Sakon, T Kuzuguchi, H Oda, T Tsukamoto
    JOURNAL OF CLINICAL MICROBIOLOGY 41 (6) 2367 - 2371 0095-1137 2003/06 [Refereed][Not invited]
    We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.
  • DNAマイクロアレイを用いた臨床分離結核菌の薬剤耐性判定
    吉川陽子, 市原竜生, 鈴木定
    臨床微生物迅速診断研究会誌 14 45 - 50 2003 [Not refereed][Not invited]
  • Characterization of clinical isolates of Mycobacterium tuberculosis resistant to drugs and detection of RpoB mutation in multidrug-resistant tuberculosis in the Philippines
    Int J Tuberc Lung Dis (7) 1104 - 8 2003 [Not refereed][Not invited]
  • H Ohmori, Y Makita, M Funamizu, S Chiba, K Ohtani, Y Suzuki, N Wakamiya, A Hata
    JOURNAL OF HUMAN GENETICS 48 (2) 82 - 85 1434-5161 2003 [Refereed][Not invited]
    Collectins are a family of C-type lectins found in vertebrates. These proteins have four regions, a relatively short N-terminal region, a collagen-like region, an alphahelical coiled coil, and a carbohydrate recognition domain. Collectins are involved in host defense through their ability to bind carbohydrate antigens on microorganisms. Type A scavenger receptors are classical-type scavenger receptors that also have collagen-like domains. We previously described a new scavenger receptor, collectin from placenta [collectin placenta 1 (CL-P1)]. CL-P1 is a type II membrane protein with all four regions. We found that CL-P1 can bind and phagocytize both bacteria and yeast. In addition to that, it reacts with oxidized low-density lipoprotein (LDL) but not with acetylated LDL. These results suggest that CL-P1 might play important roles in host defenses and/or atherosclerosis formation. One rational strategy to study the role of CL-P1 in these pathological conditions would be to perform a haplotype association study using human samples. As a first step for this strategy, we analyzed the haplotype structure of the CL-P1 gene. By sequencing the CL-P1 gene in ten Japanese volunteers, we identified five single-nucleotide polymorphisms (SNPs) with a minor allele frequency of at least 29%. To obtain SNPs in the 5'-upstream region of the gene, we screened a total of 20 SNPs described in the database and finally picked up one SNP for the present study. Thus, a total of six SNPs, one in the 5'-upstream region, two in intron 2, one in exon 5, and two in exon 6, were used to analyze the haplotype structure of the gene, with DNAs derived from 54 individuals (108 alleles). The analysis revealed that only two of six SNPs showed significant linkage disequilibrium (r(2) > 0.5) with each other. This haplotype information may be useful in disease-association studies in which a contribution of the CL-P1 gene has been suspected, especially in immunological disturbance or atherosclerosis. Two SNPs in exon 6, both leading to amino acid substitutions, could be candidates for influencing disease susceptibility.
  • T Kawai, Y Suzuki, S Eda, T Kase, K Ohtani, Y Sakai, H Keshi, A Fukuoh, T Sakamoto, M Nozaki, NG Copeland, NA Jenkins, N Wakamiya
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 (10) 2134 - 2145 0916-8451 2002/10 [Refereed][Not invited]
    Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense. We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1). In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88% identical with human CL-L1. Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos. In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age. In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac. The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q. CL-L1 was a highly conserved protein in mammals, birds, and fish.
  • T Kawai, Y Suzuki, A Yamashita, H Inoue
    CANCER LETTERS 183 (1) 79 - 86 0304-3835 2002/09 [Refereed][Not invited]
    The drs gene was isolated as a transformation suppressor against the v-src oncogene. Drs protein has a transmembrane domain and three consensus repeats (CRs) called Sushi motifs in the extracellular domain. The dry gene also has the ability to suppress anchorage-independent growth of human cancer cell lines. In this paper, we report the isolation of a novel variant cDNA of mouse drs (mDRS-2) containing two CRs, in addition to a Mouse homolog of drs (mDRS-1) containing three CRs. We investigated the Suppressor function of these mDRS cDNAs in human cancer cells and found that the lack of one CR is critical for suppression of anchorage-independent growth by drs. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.
  • Y Suzuki, A Suzuki, A Tamaru, C Katsukawa, H Oda
    JOURNAL OF CLINICAL MICROBIOLOGY 40 (2) 501 - 507 0095-1137 2002/02 [Refereed][Not invited]
    Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterialpyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5' ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis.
  • Y Hakozaki, M Yoshiba, K Sekiyama, E Seike, J Iwamoto, K Mitani, M Mine, T Morizane, K Ohtani, Y Suzuki, N Wakamiya
    LIVER 22 (1) 29 - 34 0106-9543 2002/02 [Refereed][Not invited]
    Background/Aims: The mannose-binding lectin (MBL) gene was reported to play an important role in determining the clinical outcome of persistent hepatitis B virus (HBV) infection. We investigated serum MBL concentrations and MBL gene mutations to determine whether they were related to the prognosis of patients with fulminant hepatic failure (FHF) caused by HBV infection. Methods: We investigated serum MBL concentrations and MBL gene mutations in 43 HBV-infected Japanese patients with FHF and 260 HBsAg-negative healthy controls. Serum MBL concentrations were measured by an enzyme-linked immunosorbent assay, and mutations in the MBL gene were analysed by nested PCR and direct DNA sequencing. Results: Only a mutation in codon 54 of the MBL gene was found. The frequency of this mutation in nonsurvivors (40%, 8/20) was higher than in survivors (13%, 3/23), and the difference was slightly significant (p = 0.043). The H allele frequency in survivors (70.5%, 31/44) was higher than in nonsurvivors (39.5%, 15/38) (p = 0.0048). Because of these factors the mean serum MBL concentration in survivors, 1.61 mug/ml (range 0.3-3.86), was significantly higher than in nonsurvivors, 0.79 mug/ml (range 0.04-1.51) (p < 0.0001). The likelihood ratio for nonsurvival was 0 for over 2.0 mug/ml, 0.67 for 1.0-2.0 mug/ml, and 2.24 for 0-1.0 mug/ml. Conclusions: The mutation in codon 54 of the MBL gene tended to be higher in nonsurvivors than in survivors. The H allele frequency (high producing allele in H/Y) in survivors was higher than that in nonsurvivors. High levels of serum MBL correlated with the survival of patients with FHF due to HBV infection. Serum MBL may be useful as a predictive factor for the survival of patients with FHF caused by HBV.
  • Hui Zhao, Nobutaka Wakamiya, Yasuhiko Suzuki, Matthew T Hamonko, Gregory L Stahl
    Hybridoma and hybridomics 21 (1) 25 - 36 1536-8599 2002/02 [Refereed][Not invited]
    Mannose binding lectin (MBL) binding initiates activation of the lectin complement pathway. Recent studies from our laboratory have demonstrated that MBL-dependent complement activation mediates cellular injury following oxidative stress in vivo and in vitro. A panel of novel inhibitory monoclonal antibodies (MAbs) against MBL (e.g., MAb 3F8, 2A9, and hMBL1.2) has been developed that inhibit MBL binding and lectin pathway activation. Here, we further characterized the interactions of these MAbs and their Fab fragments to MBL. Whole MAbs or their Fab fragments bound to MBL with relatively high affinity. Fab fragments of 3F8 were functionally effective in inhibiting MBL-dependent complement activation, however, steric hindrance of MAb 2A9 was essential for inhibition of MBL-dependent complement activation. We identified the hinge region, and residues EDCVLLL within the carbohydrate recognition domain of MBL as the recognition sites for MAb 3F8 and 2A9, respectively. The interaction of MAbs (e.g., 3F8 and 2A9) to MBL was dependent on the conformation of their recognition sites. These findings demonstrate that MBL binding can be inhibited by at least two separate and independent mechanisms.
  • Arylhdrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphnls in genetically engineered C57BL/6J mice
    Carcinogenesis (23) 1199 - 1207 2002 [Not refereed][Not invited]
  • Mannose-binding lectin gene: polymorphisms in Japanese patients with systemic lupus erythematosus, rheumatoid arthritis and Sjogren's syndrome
    A Tsutsumi, K Sasaki, N Wakamiya, K Ichikawa, T Atsumi, K Ohtani, Y Suzuki, T Koike, T Sumida
    GENES AND IMMUNITY 2 (2) 99 - 104 1466-4879 2001/04 [Not refereed][Not invited]
    Mannose-binding lectin (MBL) is a key element of the innate immunity, with a structure similar to complement C1q. Serum MBL levels are greatly affected by the polymorphisms of the MBL gene. In particular, codon 54 mutation of the MBL gene results in a significant reduction of serum MEL. To determine whether polymorphism of the MBL gene is associated with occurrence of systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjogren's syndrome in the Japanese population, we analyzed the MBL gene polymophisms of these patients and controls, by polymerase chain reaction-restriction fragment length polymorphism methods. We found that patients studied had a significantly higher frequency of having homozygous codon 54 mutation compared to controls. In particular patients with SLE or Sjogren's syndrome showed higher probabilities of being homozygous for this mutation. Among subjects with the same genotype, SLE patients tended to have higher serum MBL concentration than controls. Analysis of the promotor region suggested that SLE patients heterozygous for the codon 54 mutation have a higher probability of having a low producing haplotype for the gene without the codon 54 mutation. We conclude that persons homozygous for codon 54 mutation of the MBL gene may be prone to occurrence of autoimmune disorders including SLE, in the Japanese. MBL may have protective effects on occurrence and progression of SLE.
    J Biol Chem 276 (47) 44222 - 44228 0021-9258 2001 [Not refereed][Not invited]
  • Characterization of Norwalk virus GI specific monoclonal antibodies generated against Escherichia coil expressed capsid protein and reactivity of two broadly reactive monoclonal antibodies generated against GII capsid toward GI recombinant fragments
    BMC Micobiol (1) 24  2001 [Not refereed][Not invited]
    Int Immunopharmacol 2001 1 (4) 677 - 687 1567-5769 2001 [Not refereed][Not invited]
    J. Biol. Chem. 276 (26) 23456 - 23463 0021-9258 2001 [Not refereed][Not invited]
    J. Biol. Chem 276 (12) 9059 - 9065 0021-9258 2001 [Not refereed][Not invited]
  • アミン ルフル, 鈴木 定彦, 高鳥毛 敏雄, 多田羅 浩三, 白倉 良太
    Kekkaku 76 (1) 9 - 18 0022-9776 2001 [Not refereed][Not invited]
  • 日本人におけるMBL(mannan-binding lectin)遺伝子変異と血中濃度
    芥子宏行, 大谷克城, 坂本隆志, 岸雄一郎, 荒木宏昌, 鈴木定彦, 若宮伸隆
    医学のあゆみ 194 957 - 968 2000 [Refereed][Not invited]
  • Mannose-binding lectin polymorphisms in patients with hepatitis C virus infection
    Scand J Gastroenterol (35) 960 - 5 2000 [Not refereed][Not invited]
    J Interferon Cytokine Res 20 (2) 179 - 185 1079-9907 2000 [Not refereed][Not invited]
  • Expression of recombinant Norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection
    J. Med Virol. (60) 475 - 481 2000 [Not refereed][Not invited]
  • T Kase, Y Suzuki, T Kawai, T Sakamoto, K Ohtani, S Eda, A Maeda, Y Okuno, T Kurimura, N Wakamiya
    IMMUNOLOGY 97 (3) 385 - 392 0019-2805 1999/07 [Not refereed][Not invited]
    Mannan-binding lectin (MBL) is a C-type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagenlike sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro-organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV-A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti-human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non-damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.
  • 田丸亜貴, 鈴木定彦
    Kekkaku 74 (7) 555 - 561 0022-9776 1999 [Refereed][Not invited]
  • Preparation of recombinant alpha2 antigen of M. leprae in E. coli and the application for sero-diagnosis of leprosy.
    Yin Y, Suzuki Y, Makino M, Wu Q, Hou W
    Chin. Med. Sci. J. 14 106  1999 [Refereed][Not invited]
  • Gene cloning and expression of Mycobacterium leprae alpha 2 antigen.
    Yin Y, Suzuki Y, Makino M, Wu Q
    Zhongguo Yi Xue Ke Xue Yuan Xue Bao 21 62 - 68 1999 [Refereed][Not invited]
    Eur J Immunol 29 (11) 3603 - 3608 0014-2980 1999 [Not refereed][Not invited]
    J. Biol. Chem. 274 (19) 13681 - 13689 0021-9258 1999 [Not refereed][Not invited]
    J. Immunol. Methods 222 (1/2) 135 - 144 0022-1759 1999 [Not refereed][Not invited]
  • S Eda, Y Suzuki, T Kawai, K Ohtani, T Kase, T Sakamoto, N Wakamiya
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 62 (7) 1326 - 1331 0916-8451 1998/07 [Not refereed][Not invited]
    Mannan-binding protein (MBP) is a calcium-dependent mammalian serum lectin important in first-line host defense. MBP belongs to the collectin family, which is characterized by an NH2-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain (CRD). We have expressed a recombinant human MBP, consisting of the short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain, and the CRD, in Escherichia coli. The truncated MBP was capable of forming trimers by association of the neck domain and could bind sugar with a specificity similar to that of the native form. Results of hemagglutination inhibition (HI) assay of influenza A virus showed that the truncated MBP inhibited hemagglutination less strongly, although the native MBP induced the HI phenomenon. These results suggest that an oligomeric structure is an advantage for MBP to have full biological activity against influenza A virus.
  • T Hara, Y Suzuki, T Nakazawa, H Nishimura, S Nagasawa, M Nishiguchi, M Matsumoto, M Hatanaka, M Kitamura, T Seya
    IMMUNOLOGY 93 (4) 546 - 555 0019-2805 1998/04 [Not refereed][Not invited]
    We obtained a unique CD46 cDNA, STc/CY4, from the human testis, the predicted amino acid sequence of which suggested the presence of a novel isoform of CD46. This message was present predominantly in the testis, and the predicted isoform possessed a short (11 amino acids) transmembrane section (TM) and an unidentified cytoplasmic tail (CT). When expressed in Chinese hamster ovary (CHO) cells, this CD46 isoform underwent no O-glycosylation and was mostly retained in the endoplasmic reticulum, This unusual behaviour of the new isoform was due in part to the short TM and the unusual sequences of the CY. The molecular mass of this isoform was 42 000, approximately 20 000 smaller than conventional CD46. These properties of the STc/CY4 isoform were similar to those of sperm CD46. The only difference between sperm CD46 and the STc/CY4 isoform expressed on CHO cells was that only the latter possessed N-linked sugars of high mannose types, Since the STc/CY4 isoform may behave like sperm CD46 in cellular localization and post-translational modification, studies of sperm-egg interassociation were performed using hamster eggs and CHO cell clones expressing various isoforms including the STc/CY4. Rosette formation was seen most effectively between hamster eggs and STc/CY4-expressing CHO cells. These results infer that O-gycosylation perturbs CD46-mediated sperm-binding to eggs and thus sperm CD46 lacking O-linked sugars can serve as an adhesion molecule, The possible role of CD46 in fertilization and the structural differences between sperm and conventional CD46 are discussed.
  • T Kawai, Y Suzuki, S Eda, K Ohtani, T Kase, T Sakamoto, H Uemura, N Wakamiya
    GLYCOBIOLOGY 8 (3) 237 - 244 0959-6658 1998/03 [Not refereed][Not invited]
    Mannan-binding protein (MBP) is a member of the collectin family of protein, There are two types of MBP, MBP-A and MBP-C, which were found in rodent (rats and mice), rhesus monkey, and cynomolgus monkey, while chimpanzee and human have only one MBP, It was considered that the loss of one MBP gene occurred during hominoid evolution, In this article two rabbit MBP, a liver and serum MBP, were characterized biologically and genetically, Analyses by SDS-PAGE under reduced condition and their amino acid sequences of both MBPs showed that they have a same molecular weight of 32 kDa and their amino acid sequences were identical, A serum MBP has a higher ability to activate complement than does a liver MBP; however, a liver MBP inhibits hemagglutination by influenza virus as strongly as a serum MBP does, cDNA clones encoding the rabbit MBP were isolated from a rabbit cDNA liver library using whole cDNA of mouse MBP-C as a probe. The cDNA carried an insert of 744 bp coding for a protein of 247 acid residues with a signal peptide of 22 residues. The deduced amino acid sequence of the cDNA was identical to that of amino acid sequences of the 32 kDa proteins determined here, Northern blot analysis showed that mRNA transcripts of about 0.9 and 3.0 kb were expressed only in the liver. The analysis of the phylogenetic tree of rabbit and bovine MBPs and other collectins indicates that the loss of MBP gene occurred not only during hominoid evolution but also at some points after the separation of birds and mammals.
  • Regulation of promoter and intron enhancer activity in immunoglobulin heavy chain genes through late stage of B cell development
    Microbiol . Immunol (42) 399 - 405 1998 [Not refereed][Not invited]
    Biochem. Pharmacol. 56 (2) 243 - 251 0006-2952 1998 [Not refereed][Not invited]
  • Detection of kanamycin resistant Mycobacterium tuberculosis by searching mutations on the 16S ribosomal RNA gene
    J. Clin. Microbiol (36) 1220 - 1225 1998 [Not refereed][Not invited]
  • T Kawai, Y Suzuki, S Eda, K Ohtani, T Kase, Y Fujinaga, T Sakamoto, T Kurimura, N Wakamiya
    GENE 186 (2) 161 - 165 0378-1119 1997/02 [Not refereed][Not invited]
    To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE, The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.
    Biochem Biophys. Res. Commun. 238 (3) 856 - 860 0006-291X 1997 [Not refereed][Not invited]
    Proc. Natl. Acad. Sci. USA 94 (11) 5967 - 5972 0027-8424 1997 [Not refereed][Not invited]
  • Recombinant human surfactant protein D, lacking the N-terminal and collagenouse domains, has less bacterial agglutinating activity but is able to inhibit haemagglutination by influenze A virus
    Biochem. J. (323) 393 - 399 1997 [Not refereed][Not invited]
    J. Appl. Microbiol 83 (5) 634 - 640 1364-5072 1997 [Not refereed][Not invited]
  • Recombinant bovine conglutinin, lacking the N-terminal and collagenouse domains, has less conglutination activity but is able to inhibit haemagglutination by influenze A Virus
    Biochem. J. (316) 43 - 48 1996 [Not refereed][Not invited]
  • High expression of membrane cofactor protein of complement (CD46) in human leukaemia cell lines
    Scand. J. Immunol. (42) 581 - 590 1995 [Not refereed][Not invited]
  • Mutations in rpoB gene of rifampicin resistant clinical isolates of Mycobacterium tuberculosis in Japan
    J. Jpn. Assoc. Infect. Dis. (69) 413 - 41 1995 [Not refereed][Not invited]
    J. Human Genet 39 (3) 337 - 343 0916-8478 1994 [Not refereed][Not invited]
    J. Exp. Med. 179 (2) 395 - 403 0022-1007 1994 [Not refereed][Not invited]
    Biochem Biophys. Res. Commun 191 (2) 335 - 342 0006-291X 1993 [Not refereed][Not invited]
  • Inducibility of Protein-reactive anibodies by Peptide Immunization:Comparizon of Three Peptide of Hen Egg-White Lysozyme
    J. Biochem (111) 259 - 264 1992 [Not refereed][Not invited]
    Proc. Natl. Acad. Sci. USA 88 (9) 4020 - 4024 0027-8424 1991 [Not refereed][Not invited]
  • Analysis of the promoter region in the rRNA operon from Mycobacterium bovis BCG
    Antonie van Leeuwenhoek (60) 7 - 11 1991 [Not refereed][Not invited]
  • Activation of ideotype-specific CD4+ T-cell line: Cellular processing of exogenous self-immunoglobulin
    Immunology (71) 153 - 157 1990 [Not refereed][Not invited]
  • Primary Structure of Rat Mrain Prostaglandin D Synthetase Deduced from cDNA Sequence
    J. Biol Che. (264) 1041 - 1045 1989 [Not refereed][Not invited]
  • 鈴木定彦, 山田毅
    Kekkaku 63 (1) 1 - 3 0022-9776 1988 [Refereed][Not invited]
  • Complete Nucleotide Sequence of the 16S rRNA Gene of Mycobacterium bovis BCG
    J. Bacteriol (170) 2886 - 2889 1988 [Not refereed][Not invited]
  • Molecular Cloning and Characterization of an r RNA Operon in Streptomyces lividans TK21
    J. Bacteriol (170) 1631 - 1636 1988 [Not refereed][Not invited]
  • Study on rRNA Genes in Mycobacterium smegmatis
    Microbiol Immunol (32) 1259 - 1262 1988 [Not refereed][Not invited]
    Nucleic Acids Research 16 (1) 370  0305-1048 1988 [Not refereed][Not invited]
  • Chlorella chloroplast DNA seguence containing a gene for the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase and a part of a possible gene for the beta’s subunit of RNA polymerase
    Plant Mol. Biol (10) 245 - 250 1988 [Not refereed][Not invited]
    JOURNAL OF BACTERIOLOGY 169 (2) 839 - 843 0021-9193 1987/02 [Not refereed][Not invited]
  • The number of rihosomal RNA genes in Mycobacterium lepraemurium
    FEMS Microbiology Letters (44) 73 - 76 1987 [Not refereed][Not invited]
  • Antimicrob. Agents and Chemother (27) 921 - 924 1985 [Not refereed][Not invited]
  • Agric Biol Chem 47 (4) 919 - 920 0002-1369 1983 [Not refereed][Not invited]

Books etc

  • 非結核性抗酸菌の基礎と臨床
    医薬ジャーナル社 2010
  • Leprosy
    Tokai University Press 2010
  • 最新獣医公衆衛生学
    緑書房 2009
  • 床検査項辞典
    医歯薬出版 2008
    Research Spotlight 2007
  • 総説現代ハンセン病医学
    東海大学出版会 2007
  • DNAチップの開発
    シーエムシー出版 2005
  • ハンセン病医学
    東海大学出版会 1997
  • 新内科学
    南江堂 1996
  • 免疫学辞典
    東京化学同人 1993

Conference Activities & Talks


Industrial Property Rights

Awards & Honors

  • 2010 日本ハンセン病学会賞

Research Grants & Projects

  • ハンセン病の予防法及び診断・治療法の開発・普及に関する研究
    Date (from‐to) : 2012 -2015 
    Author : 向井 徹
  • 文部科学省:科学研究費補助金(基盤研究(B))
    Date (from‐to) : 2012 -2014 
    Author : 鈴木 定彦
  • アフリカにおける研究ネットワーク構築に関する研究
    Date (from‐to) : 2011 -2013 
    Author : 服部俊夫
  • LTBI 検出のための新規血清学的アッセイの開発とLTBI 根治薬もしくは結核再燃防止薬の開発
    Date (from‐to) : 2010 -2013 
    Author : 松本 真
  • 結核及びトリパノソーマ症の新規診断法・治療法の開発
    Date (from‐to) : 2009 -2013 
    Author : 鈴木 定彦
  • インフルエンザウイルスライブラリーを活用した抗体作出・創薬応用基盤研究
    Date (from‐to) : 2009 -2011 
    Author : 喜田 宏
  • 文部科学省:科学研究費補助金(基盤研究(B))
    Date (from‐to) : 2009 -2011 
    Author : 鈴木 定彦, 中島 千絵
  • 蛋白質大量発現細胞株の確立と産生蛋白質(バイオジェネリック医薬品等)の有効性評価
    Date (from‐to) : 2008 -2010 
    Author : 鈴木 定彦
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2007 -2009 
    Author : Nobutaka WAKAMIYA, 福澤 純, Ysuhiko SUZUKI, Itsuro YOHIDA, Wataru MOTOMURA, Katsuki OHTANI, Seong-jae JANG
  • 文部科学省:科学研究費補助金(萌芽研究)
    Date (from‐to) : 2007 -2008 
    Author : 吉田 玲子, 高田 礼人, 鈴木 定彦
    ウイルス感染症やがんの有効な遺伝子治療法を開発するために、モノクローナル抗体を利用した標的細胞に効率よく目的遺伝子を導入できるターゲティングウイルス(標的指向性ウイルス)の作製を目指した。本研究ではインフルエンザ感染に対するターゲティングウイルスを作製することを目的に、インフルエンザウイルスHAに対するモノクローナル抗体のFab領域を発現するプラスミド、および膜融合タンパク質としてセンダイウイルスFタンパク質を発現するプラスミドを構築した。293T細胞にプラスミドを導入したところ、タンパク質の単独および共発現が確認できた。さらに、抗体と融合タンパク質を細胞に発現させ、G遺伝子をGFPに置換したVSVを感染させて、増殖したウイルスを回収した。作出したウイルス粒子の構造を電子顕微鏡で観察するとともに、抗体とFタンパク質のウイルス粒子内への取り込みを、SDS-PAGEとWestern blotで解析したところ、抗体タンパク質のウイルス粒子への取り込み効率が極めて低いことが判明した。また、ターゲティングウイルスとしての活性も認められなかった。そこで、抗体タンパク質のウイルス取り込み効率を上昇させるために、VSV-Gタンパク質およびFタンパク質が三量体であることから、抗体タンパク質を三量体で発現させることを試みた。抗体遺伝子の3'側にVSV-Gタンパク質またはFタンパク質の細胞外領域4...
  • 結核感染経路解明のためのDNAマイクロアレイの開発
    Date (from‐to) : 2007 -2007 
    Author : 鈴木 定彦
  • ニューキノロン剤耐性結核菌迅速検出法の開発
    Date (from‐to) : 2007 -2007 
    Author : 鈴木 定彦
  • 抗酸菌の遺伝子型のデジタル化とデータベース構築
    Date (from‐to) : 2000 -2007
  • Digitalization of the geno type of Mycobacteria and construction
    Special Coordination Funds for Promoting Science and Technology
    Date (from‐to) : 2000 -2007
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2004 -2006 
    Author : Nobutaka WAKAMIYA, 福澤 純, 鈴木 定彦, 吉田 逸朗, 小笠原 正洋, 大谷 克城
    CL-P1 is a new collectin which was found by Ohtani at 2001. It was expressed in vascular endothelial cells in murine tissues and also found in vascular endothelial cell lines. Here we established the antibody against above CL-P1 to investigate their biological functions. We have immunized mice with recombinant CL-P1 protein and made hybridoma for monoclonal antibodies. Several monoclonal antibodies were established and were divided to two or three groups. We tried to make the analysis method to detect CL-P1 using above monoclonal antibodies. We made several prototype assay systems but we co...
  • 結核迅速診断法の開発
    Date (from‐to) : 2005 
  • Development of rapid diagnostic method for tuberculosis
    Date (from‐to) : 2005
  • 文部科学省:科学研究費補助金(萌芽研究)
    Date (from‐to) : 2002 -2003 
    Author : 若宮 伸隆, 鈴木 定彦
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2001 -2003 
    Author : Nobutaka WAKAMIYA, 福澤 純, 小笠原 正洋, 吉田 逸朗, 鈴木 定彦
    Collectins are a family of C type lectins which have collagen-like sequences and carbohydrate recognition domains (CRD). The scavenger receptors type A and MARCO are classical type scavenger receptors which have internal collagen-like domains. Here, we found a new scavenger receptor which is a membrane type collectin from placenta (collectin placenta 1=CL-P1) that has a typical collectin collagen-like domain and a CRD. To elucidate the functions in CL-P1 in animal body is our purpose. In 3 years, we finished seven projects below,1.Cloning of CL-P1 cDNA in human, mouse, rat, zebra fish, xeno...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2000 -2003 
    Author : Yashihiko SUZUKI, 織田 肇, 田丸 亜貴, 松葉 隆司
    We have investigated followings1)A new method for the rapid typing of clinically isolated Mycobacterium tuberculosis strains was constructed and termed as self-ligation mediated polymerase chain reaction (SL-PCR)2)Extended spacer oligonucleotide DNA micro array was established and the performance was compared with conventional spoligotyping method. Extended spacer oligonucleotide DNA micro array could discriminate the strains that can not be discriminated by spoligotyping method.3)The IS6110 preferential locus (ipl) of clinically isolated M.tuberculosis strains in Japan were analyzed to lac...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 1999 -2001 
    Author : Chihiro KATSUKAWA, 鈴木 定彦
    Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidaseactivity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this study, we developed a new method for t...
  • 文部科学省:科学研究費補助金(特定領域研究(A))
    Date (from‐to) : 2000 -2000 
    Author : 若宮 伸隆, 鈴木 定彦
    O-GlcNAc糖鎖は、蛋白のセリンやスレオニンのアミノ酸残基(Ser/Thr)に結合する糖鎖であり、UDP-GlcNAc-β-N-acetylglucosaminyltransferase(O-GlcNAc transferase)によって糖付加が行われると考えられている。この糖鎖の付加は細胞内蛋白(核蛋白、細胞質存在蛋白)のみに認められ、生命現象に重要な役目を担っていることが推測される。我々は、コレクチンの糖結合活性が上記の糖鎖とオーバーラップすることから、コレクチンのモチーフを利用して新規コレクチンのクローニングを企画し、細胞内糖鎖の機能を探ろうと考えた。1)我々は、膜タイプの新規コレクチンを想定し、その遺伝子クローニングを試み、本年度候補遺伝子の一つCL-P1遺伝子のクローニングに成功した。さらにCL-P1遺伝子を大腸菌で発現させ、抗体作成を行い、本レクチンが膜型のレクチンであることを証明した(論文投稿中)。その後本レクチンのマウスホモローグをクローニングした。本レクチン遺伝子は非常に遺伝子保存率が高く、生体にとって重要な働きをしていることが示唆された。2)レクチン遺伝子の機能を探るには、生体での役割探索が必須であり、いろんな大学、病院との共同研究をおこなっている。始めに大阪某市における健康診断において、ボランテアでの日本人正常値の検討を行った。約500人におけるMB...
  • 文部科学省:科学研究費補助金(特定領域研究(A))
    Date (from‐to) : 1999 -1999 
    Author : 若宮 伸隆, 鈴木 定彦
    O-GlcNAc糖鎖は、蛋白のセリンやスレオニンのアミノ酸残基(Ser/Thr)に結合する糖鎖であり、UDP-GlcNAc-β-N-acetylglucosaminyltransferase(O-GlcNAc transferase)によって糖付加が行われると考えられている。この糖鎖の付加は細胞内蛋白(核蛋白、細胞質存在蛋白)のみに認められ、生命現象に重要な役目を担っていることが推測される。我々は、コレクチンの糖結合活性が上記の糖鎖とオーバーラップすることから、コレクチンのモチーフを利用して新規コレクチンのクローニングを企画し、細胞内糖鎖の機能を探ろうと考えた。1)我々は、細胞質存在タイプの新規コレクチンを想定し、その遺伝子クローニングを試み、本年度候補遺伝子の一つCL-L1遺伝子のクローニングに成功した。さらにCL-L1遺伝子を大腸菌で発現させ、抗体作成を行い、本レクチンが細胞質存在型のレクチンであることを証明した(J.Biol.Chem.1999)。その後本レクチンのマウスホモローグをクローニングした。本レクチン遺伝子は非常に遺伝子保存率が高く、生体にとって重要な働きをしていることが示唆された。2)レクチン遺伝子の機能を探るには、真核細胞での発現系が必須であり、新しいベクターを作成して、その発現系を確立し、その生物学的活性を比較検討した(J.lmmunol.Methods...
  • 文部科学省:科学研究費補助金(特定領域研究(A))
    Date (from‐to) : 1998 -1998 
    Author : 若宮 伸隆, 鈴木 定彦
    O-GlcNAc糖鎖は、蛋白のセリンやスレオニンのアミノ酸残基(Ser/Thr)に結合する糖鎖であり、UDP-GlcNAc-β-N-acetylglucosaminyltransferase(O-GlcNAc transferase)によって糖付加が行われると考えられている。この糖鎖の付加は細胞内蛋白(核蛋白、細胞質存在蛋白)のみに認められ、生命現象に重要な役目を担っていることが推測される。我々は、コレクチンの糖結合活性が上記の糖鎖とオーバーラップすることから、コレクチンのモチーフを利用して新規コレクチンのクローニングを企画し、細胞内糖鎖の機能を探ろうと考えた。1) 我々は、O-GlcNacの糖鎖に結合するものとして、細胞質存在タイプの新規コレクチンを想定し、その遺伝子クローニングを試み、本年度候補遺伝子の一つCL-L1遺伝子のクローニングに成功した。さらにCL-L1遺伝子を大腸菌で発現させ、抗体作成を行い、本レクチンが細胞質存在型のレクチンであることを証明した(J.Biol.Chem.ln press)。現在、このレクチンにって詳細な解析をおこなっている。2) レクチン遺伝子の機能を探るには、真核細胞での発現系が必須であり、新しいベクターを作成して、その発現系を確立し、その生物学的活性を比較検討した(Eda,Biosci.Biotech.Bioch.,1988.,Ohtan...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 1997 -1998 
    Author : Nobutaka WAKAMIYA, 鈴木 定彦
    Mannan-binding lectin (MBL) is a C-type serum lectin which is believed to play an important role in innate immunity.It is one of the collectin family which are characterized by having a collagen-like sequence and a carbohydrate recognition domain.Collectin can bind to sugar determinants of several microorganisms, neutralize them and inhibit infection by the of complement activation through the lectin pathway and opsonization by collectin receptors.First we tried to isolate collectin genes in several animal and establish the production system of collectin in E.coli and CHO cells.Next to reve...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    Date (from‐to) : 1993 -1993 
    Author : 鈴木 定彦
  • 文部科学省:科学研究費補助金(一般研究(B))
    Date (from‐to) : 1992 -1993 
    Author : 牧野 正直, 東 逸男, 鈴木 定彦, 勝川 千尋
    学校や塾における集団発生や海外からの移入等は、今日結核感染において注目されている問題の一つである。しかし、原因菌に対する疫学的調査の方法はこれまでに確たるものは存在していない。そこで我々は結核菌の疫学的同定法の確立を目指して研究を進めている。16SリボゾームRNA(rRNA)遺伝子と23S rRNA遺伝子の間の非コード領域の構造の多様化に着目し、この部分の塩基配列を決定、比較することにより結核菌の疫学的同定が可能かどうかについて検討を行なった。1)プライマーの選択プライマーは16S rRNA遺伝子の3′末端付近と23S rRNA遺伝子5′末端付近で、様々な菌種の間で保存性に基づいて選択した。結核菌H37Rv株より抽出したDNAを鋳型としてプライマーのペアおよび反応条件の検討を行なった。2)塩基配列の決定および比較結核菌標準株4株、結核菌以外の抗酸菌20株及び臨床分離結核菌40株よりPCRにより16S-23S非コード領域を増幅し、大腸菌ベクターpUC18にサブクローニングした。得られたプラスミドDNAを鋳型としてA.L.F.DNAシーケンサにより塩基配列を決定後、それぞれの比較を行なった。その結果、結核菌とその他の抗酸菌の間にはこの領域の塩基配列に明らかな違いが見られ、結核菌の他の抗酸菌よりの鑑別には適していることが判明した。結核菌標準株どうしには1から4個の塩基置換が見られた...

Educational Activities

Teaching Experience

  • 感染症学特別研究Ⅰ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Microbiology
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 医学部
    キーワード : 細菌、ウイルス、グラム陽性菌、グラム陰性菌、抗酸性菌、感染症、ワクチン、プリオン、寄生虫、原虫、マイコプラズマ、クラミジア、スピロヘータ、真菌
  • 感染症学特別演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Zoonotic Science
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 人獣共通感染症、新興・再興感染症、疫学、自然宿主、媒介動物、感染環、ウイルス、細菌、原虫、寄生虫 
  • 研究倫理演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Environment and People
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : インフルエンザ、エイズ、結核、エボラ出血熱、免疫
  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 感染症学特別研究ⅡA
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 感染症学特別研究ⅡB
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 海外インターンシップA
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 海外インターンシップB
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学・感染症学基礎科目 微生物学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 国際感染症学コア科目Ⅰ 人獣共通感染症制御学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学特別研究
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学特論演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 動物実験倫理特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 獣医微生物学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目A 人獣共通感染症制御学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 研究倫理演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • インターンシップ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学基礎科目 微生物学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院

Campus Position History

  • 2016年4月1日 
  • 2016年4月1日 
  • 2018年4月1日 
  • 2018年4月1日 

Position History

  • 2016年4月1日 
  • 2016年4月1日 
  • 2018年4月1日 
  • 2018年4月1日 

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