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Analysis of the upstream genetic structures of the ISEcp1-bla _CTX-M transposition units in Escherichia coli isolates carrying bla _CTX-M obtained from the Indonesian and Vietnamese communities.

Widyatama FS, Yagi N, Sarassari R, Shirakawa T, Le DT, Bui MHT, Kuntaman K, Hirai I

Microbiology and immunology     2021年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Comparison of species, virulence genes and clones of Aeromonas isolates from clinical specimens and well water in Okinawa Prefecture, Japan

Miyagi K.

Journal of Applied Microbiology ( Journal of Applied Microbiology )  131 ( 3 ) 1515 - 1530   2021年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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A high-throughput sequencing determination method for upstream genetic structure (UGS) of ISEcp1-bla_CTX-M transposition unit and application of the UGS to classification of bacterial isolates possessing bla_CTX-M.

Yagi N, Hamamoto K, Thi Bui KN, Ueda S, Tawata S, Le DT, Thi Bui MH, Hirai I

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy ( Journal of Infection and Chemotherapy )  27 ( 9 ) 1288 - 1294   2021年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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INTRODUCTION: Because blaCTX-M is responsible for resistance of bacteria to the third generation cephalosporins, location of blaCTX-M could be a good indicator for classifying bacterial isolates harboring blaCTX-M in molecular epidemiology. However, determination of blaCTX-M location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of blaCTX-M location. METHODS: Extracted DNA samples obtained from 168 Escherichia coli isolates possessing blaCTX-M were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer. RESULTS: Nucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures. CONCLUSIONS: Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-blaCTX-M transposition unit was useful for classification of bacterial isolates harboring blaCTX-M.

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Occurrence of Carriage of Multidrug Resistant Enterobacteriaceae among Pregnant Women in the Primary Health Center and Hospital Setting in Surabaya, Indonesia.

Oktaviani Sulikah SR, Hasanah M, Setyarini W, Parathon H, Kitagawa K, Nakanishi N, Nomoto R, Osawa K, Kinoshita S, Hirai I, Shirakawa T, Kuntaman K

Microbial drug resistance (Larchmont, N.Y.)     2021年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Objectives: The incidence of healthy individuals carrying multidrug resistant Enterobacteriaceae, including extended-spectrum β-lactamase producing Enterobacteriaceae (ESBL-E), especially extended-spectrum β-lactamase producing Escherichia coli (ESBL-EC) and extended-spectrum β-lactamase producing Klebsiella pneumoniae (ESBL-KP), is increasing worldwide. Although ESBL-E causes early or late onset of neonatal sepsis, the prevalence of ESBL-E carriage among pregnant women in Indonesia is not clear. In the present study, we compared the occurrence of carriage of ESBL-E among pregnant women in a primary health center (PHC) versus two hospitals. Materials and Methods: We collected rectal swab samples from 200 pregnant women who visited a PHC or were admitted to two hospitals in Surabaya, Indonesia from July to October 2018. The ESBL-E strains were isolated from the samples and phenotypically and genotypically analyzed. Results: ESBL-E strains were isolated from 25 (24.8%) pregnant women who visited the PHC and 49 (49.5%) pregnant women who were admitted to the hospitals. The rate of ESBL-E carriage of pregnant women in the hospitals was significantly higher than that in the PHC. Among the 74 isolated ESBL-E strains, ESBL-EC was most frequently isolated (62 strains), followed by ESBL-KP (12 strains). In addition, blaCTX-M-15 was the most frequent ESBL gene type of the isolated ESBL-E strains. Conclusions: Our results revealed the high occurrence of ESBL-E carriage in pregnant women, especially those who were admitted to the hospitals.

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Differential effects of chromosome and plasmid bla_CTX-M-15 genes on antibiotic susceptibilities in extended-spectrum beta-lactamase-producing Escherichia coli isolates from patients with urinary tract infection.

Yang YM, Osawa K, Kitagawa K, Hosoya S, Onishi R, Ishii A, Shirakawa T, Hirai I, Kuntaman K, Tanimoto H, Shigemura K, Fujisawa M

International journal of urology : official journal of the Japanese Urological Association ( International Journal of Urology )  28 ( 6 ) 623 - 628   2021年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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OBJECTIVES: To compare antibiotic susceptibilities between chromosomal and plasmid blaCTX-M-15 locations in urinary tract infection-causing extended-spectrum β-lactamases-producing Escherichia coli blaCTX-M-15 isolated in Indonesia. METHODS: A total of 84 strains identified as extended-spectrum β-lactamases-producing E. coli were isolated from patients with urinary tract infection in Indonesia in 2015. Antimicrobial susceptibility tests were performed on these strains using 18 antibiotics, and extended-spectrum β-lactamase bla genes were detected by polymerase chain reaction. Gene localization of blaCTX-M-15 -positive strains was confirmed by Southern blot hybridization, and epidemiological typing was conducted using multilocus sequence typing. RESULTS: Of 54 strains harboring the blaCTX-M-15 gene, 27 showed localization on chromosome, 20 on plasmid, and seven on chromosome and plasmid. Most multilocus sequence typing sequence types of the 27 strains with chromosomal blaCTX-M-15 were ST405 (25.9%) and ST131 (22.2%) strains, whereas the 20 strains with plasmid-blaCTX-M-15 were mostly ST410 (55.0%). CONCLUSIONS: Extended-spectrum β-lactamases-producing E. coli blaCTX-M-15 with plasmid genes show significantly higher resistant rates against piperacillin-tazobactam but lower resistant rates against chloramphenicol compared to chromosomal strains in Indonesian patients with urinary tract infection. Mechanistic investigations will be necessary to advance our knowledge of antimicrobial resistance in urinary tract infection.

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Observing inefficient action can induce infant preference and learning

Hirai M.

Developmental Science ( Developmental Science )    2021年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Characterization of CTX-M-type-extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolated from Indonesian undergraduate medical students of a university in Surabaya, Indonesia.

Rosantia S, Higa T, Yagi N, Tokunaga T, Higa S, Yakabi Y, Shirakawa T, Kuntaman K, Hirai I

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy ( Journal of Infection and Chemotherapy )  26 ( 6 ) 575 - 581   2020年06月 [ 査読有り ]

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High Prevalence of Colistin-Resistant Escherichia coli with Chromosomally Carried <i>mcr-1</i> in Healthy Residents in Vietnam.

Yamaguchi T, Kawahara R, Hamamoto K, Hirai I, Khong DT, Nguyen TN, Tran HT, Motooka D, Nakamura S, Yamamoto Y

mSphere   5 ( 2 )   2020年03月

掲載種別: 研究論文（学術雑誌）

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Characterization of bla_CTX-M-14 transposition from plasmid to chromosome in Escherichia coli experimental strain.

Hamamoto K, Tokunaga T, Yagi N, Hirai I

International journal of medical microbiology : IJMM ( International Journal of Medical Microbiology )  310 ( 2 ) 151395   2020年02月

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Characterisation of chromosomally-located bla_CTX-M and its surrounding sequence in CTX-M-type extended-spectrum β-lactamase-producing Escherichia coli isolates.

Hamamoto K, Hirai I

Journal of global antimicrobial resistance ( Journal of Global Antimicrobial Resistance )  17   53 - 57   2019年06月

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Characterization of CTX-M type ESBL-producing Enterobacteriaceae isolated from asymptomatic healthy individuals who live in a community of the Okinawa prefecture, Japan.

Higa S, Sarassari R, Hamamoto K, Yakabi Y, Higa K, Koja Y, Hirai I

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy ( Journal of Infection and Chemotherapy )  25 ( 4 ) 314 - 317   2019年04月

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A survey of extended-spectrum β-lactamase-producing Enterobacteriaceae in environmental water in Okinawa Prefecture of Japan and relationship with indicator organisms.

Miyagi K, Hirai I

Environmental science and pollution research international ( Environmental Science and Pollution Research )  26 ( 8 ) 7697 - 7710   2019年03月

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Wide dissemination of colistin-resistant Escherichia coli with the mobile resistance gene mcr in healthy residents in Vietnam.

Yamamoto Y, Kawahara R, Fujiya Y, Sasaki T, Hirai I, Khong DT, Nguyen TN, Nguyen BX

The Journal of antimicrobial chemotherapy ( Journal of Antimicrobial Chemotherapy )  74 ( 2 ) 523 - 524   2019年02月

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Potential transmission opportunity of CTX-M-producing Escherichia coli on a large-scale chicken farm in Vietnam.

Bui TKN, Bui TMH, Ueda S, Le DT, Yamamoto Y, Hirai I

Journal of global antimicrobial resistance ( Journal of Global Antimicrobial Resistance )  13   1 - 6   2017年10月

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Sanitary evaluation of domestic water supply facilities with storage tanks and detection of Aeromonas, enteric and related bacteria in domestic water facilities in Okinawa Prefecture of Japan.

Miyagi K, Sano K, Hirai I

Water research ( Water Research )  119   171 - 177   2017年08月

掲載種別: 研究論文（学術雑誌）

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Common findings of bla _CTX-M-55-encoding 104-139 kbp plasmids harbored by extended-spectrum β-lactamase-producing Escherichia coli in pork meat, wholesale market workers, and patients with urinary tract infection in Vietnam.

Hoang TAV, Nguyen TNH, Ueda S, Le QP, Tran TTN, Nguyen TND, Dao TVK, Tran MT, Le TTT, Le TL, Nakayama T, Hirai I, Do TH, Vien QM, Yamamoto Y

Current microbiology   74 ( 2 ) 203 - 211   2017年02月 [ 査読有り ]

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Distribution of Aeromonas species in environmental water used in daily life in Okinawa Prefecture, Japan.

Miyagi K, Hirai I, Sano K

Environmental health and preventive medicine ( Environmental Health and Preventive Medicine )  21 ( 5 ) 287 - 294   2016年09月

掲載種別: 研究論文（学術雑誌）

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Current status of extended spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan.

Nakama R, Shingaki A, Miyazato H, Higa R, Nagamoto C, Hamamoto K, Ueda S, Hachiman T, Touma Y, Miyagi K, Kawahara R, Toyosato T, Hirai I

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy ( Journal of Infection and Chemotherapy )  22 ( 5 ) 281 - 6   2016年05月

掲載種別: 研究論文（学術雑誌）

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High Prevalence of Chromosomal blaCTX-M-14 in Escherichia coli Isolates Possessing blaCTX-M-14.

Hamamoto K, Ueda S, Toyosato T, Yamamoto Y, Hirai I

Antimicrobial agents and chemotherapy   60 ( 4 ) 2582 - 4   2016年04月

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Antibody with an engineered Fc region as a therapeutic agent against dengue virus infection.

Ramadhany R, Hirai I, Sasaki T, Ono K, Ramasoota P, Ikuta K, Kurosu T

Antiviral research   124   61 - 8   2015年12月

掲載種別: 研究論文（学術雑誌）

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Carriage of Escherichia coli Producing CTX-M-Type Extended-Spectrum β-Lactamase in Healthy Vietnamese Individuals.

Bui TM, Hirai I, Ueda S, Bui TK, Hamamoto K, Toyosato T, Le DT, Yamamoto Y

Antimicrobial agents and chemotherapy ( Antimicrobial Agents and Chemotherapy )  59 ( 10 ) 6611 - 4   2015年10月

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Characteristics of Extended-Spectrum β-Lactamase-Producing Escherichia coli in Retail Meats and Shrimp at a Local Market in Vietnam.

Le QP, Ueda S, Nguyen TN, Dao TV, Van Hoang TA, Tran TT, Hirai I, Nakayama T, Kawahara R, Do TH, Vien QM, Yamamoto Y

Foodborne pathogens and disease   12 ( 8 ) 719 - 25   2015年08月

掲載種別: 研究論文（学術雑誌）

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Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds.

Hamamoto K, Ueda S, Yamamoto Y, Hirai I

Journal of clinical microbiology ( Journal of Clinical Microbiology )  53 ( 6 ) 1848 - 53   2015年06月

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Wide dissemination of extended-spectrum β-lactamase-producing Escherichia coli in community residents in the Indochinese peninsula.

Nakayama T, Ueda S, Huong BT, Tuyen le D, Komalamisra C, Kusolsuk T, Hirai I, Yamamoto Y

Infection and drug resistance ( Infection and Drug Resistance )  8   1 - 5   2015年

掲載種別: 研究論文（学術雑誌）

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Limited transmission of bla(CTX-M-9)-type-positive Escherichia coli between humans and poultry in Vietnam.

Ueda S, Ngan BT, Huong BT, Hirai I, Tuyen le D, Yamamoto Y

Antimicrobial agents and chemotherapy ( Antimicrobial Agents and Chemotherapy )  59 ( 6 ) 3574 - 7   2015年

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A highly conserved region between amino acids 221 and 266 of dengue virus non-structural protein 1 is a major epitope region in infected patients.

Omokoko MD, Pambudi S, Phanthanawiboon S, Masrinoul P, Setthapramote C, Sasaki T, Kuhara M, Ramasoota P, Yamashita A, Hirai I, Ikuta K, Kurosu T

The American journal of tropical medicine and hygiene ( AMER SOC TROP MED & HYGIENE )  91 ( 1 ) 146 - 55   2014年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The immune response to dengue virus (DENV) infection generates high levels of antibodies (Abs) against the DENV non-structural protein 1 (NS1), particularly in cases of secondary infection. Therefore, anti-NS1 Abs may play a role in severe dengue infections, possibly by interacting (directly or indirectly) with host factors or regulating virus production. If it does play a role, NS1 may contain epitopes that mimic those epitopes of host molecules. Previous attempts to map immunogenic regions within DENV-NS1 were undertaken using mouse monoclonal Abs (MAbs). The aim of this study was to characterize the epitope regions of nine anti-NS1 human monoclonal Abs (HuMAbs) derived from six patients secondarily infected with DENV-2. These anti-NS1 HuMAbs were cross-reactive with DENV-1, -2, and -3 but not DENV-4. All HuMAbs bound a common epitope region located between amino acids 221 and 266 of NS1. This study is the first report to map a DENV-NS1 epitope region using anti-DENV MAbs derived from patients.

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Detection of chromosomal blaCTX-M-15 in Escherichia coli O25b-B2-ST131 isolates from the Kinki region of Japan.

Hirai I, Fukui N, Taguchi M, Yamauchi K, Nakamura T, Okano S, Yamamoto Y

International journal of antimicrobial agents   42 ( 6 ) 500 - 6   2013年12月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Escherichia coli O25b-B2-ST131 isolates harbouring blaCTX-M-15 are distributed worldwide. The blaCTX-M-15 transposition unit has often been found on plasmids and the genetic contexts have been examined however, less is known about the frequency and contexts of the bla CTX-M-15 transposition unit on the chromosome. This study was performed to determine the chromosomal location of the blaCTX-M-15 transposition unit and to analyse the molecular structure of the region surrounding the blaCTX-M-15 transposition unit in E. coli O25b-B2-ST131 isolates. Twenty-two E. coli O25b-B2-ST131 strains harbouring blaCTX-M-15 that had been isolated from university hospital patients and nursing home residents in the Kinki region of Japan were examined. Inverse PCR (iPCR) targeting blaCTX-M-15 was performed to classify the molecular structure of the region surrounding the blaCTX-M-15 transposition unit. The isolates were classified into nine types (types A-I) considering the iPCR results type A was the most prevalent type (13/22 isolates). Sequences of the iPCR-amplified DNA fragments showed that the blaCTX-M-15 transposition unit consisted of ISEcp1, bla CTX-M-15 and orf477Δ. A homology search of the obtained sequences showed that the blaCTX-M-15 transposition unit was inserted into different chromosomal regions in eight of the nine classified types. Although 21 of the 22 E. coli isolates possessed chromosomally located bla CTX-M-15 transposition units, clonal spread was not evident on pulsed-field gel electrophoresis (PFGE) analysis. Taken together, these data indicate that certain E. coli O25b-B2-ST131 strains harbouring chromosomal blaCTX-M-15 have emerged and spread in the Kinki region of Japan. © 2013 Elsevier B.V. and the International Society of Chemotherapy.

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Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

Sasaki T, Setthapramote C, Kurosu T, Nishimura M, Asai A, Omokoko MD, Pipattanaboon C, Pitaksajjakul P, Limkittikul K, Subchareon A, Chaichana P, Okabayashi T, Hirai I, Leaungwutiwong P, Misaki R, Fujiyama K, Ono K, Okuno Y, Ramasoota P, Ikuta K

Antiviral research ( ELSEVIER SCIENCE BV )  98 ( 3 ) 423 - 31   2013年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')(2) fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection. (C) 2013 Elsevier B.V. All rights reserved.

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Jurkat cell proliferation is suppressed by Chlamydia (Chlamydophila) pneumoniae infection accompanied with attenuation of phosphorylation at Thr389 of host cellular p70S6K.

Hirai I, Ebara M, Nakanishi S, Yamamoto C, Sasaki T, Ikuta K, Yamamoto Y

Immunobiology   218 ( 4 ) 527 - 32   2013年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Chlamydia (Chlamydophila) pneumoniae infects T lymphocytes and multiplies within them. Our previous studies have indicated that C. pneumoniae infection suppresses proliferation of peripheral blood mononuclear cells stimulated with Staphylococcus-enterotoxin B however, the mechanism of suppression was unclear. In this study, we explored the molecular mechanism involved in C. pneumoniae infection by using human acute T cell leukemia cell line, Jurkat E6-1. Proliferation of Jurkat cells was suppressed in an m.o.i.-dependent manner by C. pneumoniae infection. The suppression by the infection was particularly evident during the initial 24. h of the infection, and down modulation of cyclin D3 protein levels were observed at the same time period by immunoblot analysis. The suppression of the Jurkat cell proliferation and the down modulation of cyclin D3 protein level were only induced by viable C. pneumoniae infection, not by exposure to UV-killed or heat-killed C. pneumoniae. Phosphorylations at Thr308 and Ser473 of AKT were induced by C. pneumoniae infection however, phosphorylation at Thr389 of the downstream kinase, p70S6K was inhibited by unidentified mechanism associated with C. pneumoniae infection. Taking into account that G1 arrest of the C. pneumoniae infected Jurkat cells were not observed and that p70S6K is one of the most important regulators of protein synthesis, it was suggested that the suppression of Jurkat cell proliferation by C. pneumoniae was at least in part mediated by down modulation of protein synthesis through attenuation of Thr389 phosphorylation of p70S6K. © 2012 Elsevier GmbH.

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Human monoclonal antibodies to neutralize all dengue virus serotypes using lymphocytes from patients at acute phase of the secondary infection (vol 423, pg 867, 2012)

Chayanee Setthapramote, Tadahiro Sasaki, Orapim Puiprom, Kriengsak Limkittikul, Pannamthip Pitaksajjakul, Chonlatip Pipattanaboon, Mikiko Sasayama, Pornsawan Leuangwutiwong, Weerapong Phumratanaprapin, Supat Chamnachanan, Teera Kusolsuk, Akanitt Jittmittraphap, Azusa Asai, Juan Fernando Arias, Itaru Hirai, Motoki Kuhara, Yoshinobu Okuno, Takeshi Kurosu, Pongrama Ramasoota, Kazuyoshi Ikuta

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  432 ( 1 ) 200 - 201   2013年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Chlamydophila pneumoniae in human immortal Jurkat cells and primary lymphocytes uncontrolled by interferon-γ.

Ishida K, Kubo T, Saeki A, Yamane C, Matsuo J, Yimin, Nakamura S, Hayashi Y, Kunichika M, Yoshida M, Takahashi K, Hirai I, Yamamoto Y, Shibata K, Yamaguchi H

Microbes and infection ( ELSEVIER SCIENCE BV )  15 ( 3 ) 192 - 200   2013年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Lymphocytes are a potential host cell for Chlamydophila pneumoniae, although why the bacteria must hide in lymphocytes remains unknown. Meanwhile, interferon (IFN)-gamma is a crucial factor for eliminating chlamydiae from infected cells through indoleamine 2,3-dioxygenase (IDO) expression, resulting in depletion of tryptophan. We therefore assessed if lymphocytes could work as a shelter for the bacteria to escape IFN-gamma. C. pneumoniae grew normally in human lymphoid Jurkat cells, even in the presence of IFN-gamma or under stimulation with phorbol myristate acetate plus ionomycin. Although Jurkat cells expressed IFN-gamma receptor CD119, their lack of IDO expression was confirmed by RT-PCR and western blotting. Also, C. pneumoniae survived in enriched human peripheral blood lymphocytes, even in the presence of IFN-gamma. Furthermore, C. pneumoniae in spleen cells obtained from IFN-gamma knockout mice with C57BL16 background was maintained in a similar way to wild-type mice, supporting a minimal role of]FN-gamma-related response for eliminating C. pneumoniae from lymphocytes. Thus, we concluded that IFN-gamma did not remove C. pneumoniae from lymphocytes, possibly providing a shelter for C. pneumoniae to escape from the innate immune response, which has direct clinical significance. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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Fecal carriage of CTX-M β-lactamase-producing Enterobacteriaceae in nursing homes in the Kinki region of Japan.

Luvsansharav UO, Hirai I, Niki M, Nakata A, Yoshinaga A, Yamamoto A, Yamamoto M, Toyoshima H, Kawakami F, Matsuura N, Yamamoto Y

Infection and drug resistance   6   67 - 70   2013年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The detection rate of CTX-M-type β-lactamase-producing Enterobacteriaceae in Japan has significantly increased. Nursing homes may be a reservoir of antibiotic-resistant bacteria. Therefore, we determined the prevalence of, and risk factors associated with, fecal carriage of CTX-M-type β-lactamase-producing Enterobacteriaceae among nursing home residents. A total of 225 stool samples were collected for phenotypic and genotypic identification of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. Multivariate analysis was performed to identify the risk factors associated with fecal carriage of CTX-M producers. The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae, as confirmed by phenotypic and genotypic methods, was 19.6% (44 of 225 samples). Escherichia coli was the predominant CTX-M-type ESBL-producing bacterium among these isolates (41 of 44 isolates). Genotyping of blabla gene-positive isolates showed that 30 (68.2%), 13 (29.5%), and 1 (2.3%) of 44 samples belonged to groups CTX-M-9, CTX-M-1 and CTX-M-2, respectively. Among the CTX-M-type ESBL-producing Enterobacteriaceae found in nursing homes, 95.5% (42 of 44 isolates) were co-resistant to quinolone antibiotics. In multivariate logistic regression analysis, inability to turn over in bed, diabetes, and invasive procedures within the last 2 years were the only variables independently associated with fecal carriage of CTX-M-type ESBL producers. Nursing home residents in Japan exhibit a high prevalence of CTX-M-type ESBL-producing Enterobacteriaceae carriage, with a high level of co-resistance to quinolones. © 2013 Luvsansharav et al, publisher and licensee Dove Medical Press Ltd.

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Human monoclonal antibodies to neutralize all dengue virus serotypes using lymphocytes from patients at acute phase of the secondary infection.

Setthapramote C, Sasaki T, Puiprom O, Limkittikul K, Pitaksajjakul P, Pipattanaboon C, Sasayama M, Leuangwutiwong P, Phumratanaprapin W, Chamnachanan S, Kusolsuk T, Jittmittraphap A, Asai A, Arias JF, Hirai I, Kuhara M, Okuno Y, Kurosu T, Ramasoota P, Ikuta K

Biochemical and biophysical research communications ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  423 ( 4 ) 867 - 72   2012年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The global spread of the four dengue virus serotypes (DENV-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, we prepared hybridomas producing human monoclonal antibodies (HuMAbs) against DENV using peripheral blood mononuclear cells (PBMCs) from patients in the acute phase (around 1 week after the onset of illness) or the convalescent phase (around 2 weeks after the onset of illness) of secondary infection. Interestingly, a larger number of hybridoma clones was obtained from patients in the acute phase than from those in the convalescent phase. Most HuMAbs from acute-phase infections were cross-reactive with all four DENV serotypes and showed significant neutralization activity to all four DENV serotypes. Thus, secondary DENV infection plays a significant role in stimulating memory cells to transiently increase the number of antibody-secreting plasma cells in patients in the early phase after the secondary infection. These HuMAbs will enable us to better understand the protective and pathogenic effects of DENV infection, which could vary greatly among secondarily-infected individuals. (C) 2012 Elsevier Inc. All rights reserved.

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Chlamydia trachomatis serovar L2 infection model using human lymphoid Jurkat cells.

Kubo T, Ishida K, Matsuo J, Nakamura S, Hayashi Y, Sakai H, Yoshida M, Takahashi K, Hirai I, Yamamoto Y, Yamaguchi H

Microbial pathogenesis ( ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD )  53 ( 1 ) 1 - 11   2012年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Chlamydia trachomatis L2 invasively attacks lymphatic and subepithelial tissues of the genital tract during the formation of primary lesions. This subsequently results in lymphadenopathy, and suggests a greater propensity for systemic dissemination. However, whether lymphocytes are a potential vehicle cell for the dissemination of this infection remains unknown. We therefore assessed the growth properties of C trachomatis L2 in lymphoid Jurkat cells compared with those observed in epithelial HeLa cells. Both cells supported the growth of C trachomatis with a similar increase in infective progenies. Enriched human-blood lymphocytes also supported the C trachomatis growth as well as Jurkat cells. Bacteria infecting the Jurkat cells were more susceptible to antibiotics (doxycycline, azithromycin, ofloxacin) than those in HeLa cells. Of the sphingomyelin biosynthesis inhibitors tested, both myriocin and fumonisin B1 significantly inhibited bacterial growth in both cells types. A Jurkat cell mutant that impaired bacterial growth was established using ethylmethanesulfonate treatment. DNA microarray analysis with real-time reverse transcription-polymerase chain reaction revealed that the mutant cells over-expressed granzyme K gene. Immunofluorescence staining also indicated that granzyme K irregularly over-expressed among the mutant cells as compared with that of the wild cells, suggesting a possible mechanism refractory to C. trachomatis infection. Thus, we concluded that C. trachomatis 12 could infect Jurkat cells with lymphoid properties, providing a new tool for studying C. trachomatis dissemination to tissues via lymphocyte movement. (C) 2012 Elsevier Ltd. All rights reserved.

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Prevalence of and risk factors associated with faecal carriage of CTX-M β-lactamase-producing Enterobacteriaceae in rural Thai communities.

Luvsansharav UO, Hirai I, Nakata A, Imura K, Yamauchi K, Niki M, Komalamisra C, Kusolsuk T, Yamamoto Y

The Journal of antimicrobial chemotherapy ( OXFORD UNIV PRESS )  67 ( 7 ) 1769 - 74   2012年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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To determine the prevalence of CTX-M -lactamase-producing Enterobacteriaceae and to study the risk factors associated with faecal carriage in asymptomatic rural Thai people. In all, 417 stool samples were obtained from rural Thai people and screened for extended-spectrum -lactamases (ESBLs) using MacConkey agar supplemented with 2 mg/L cefotaxime. Results were confirmed using cefotaxime and ceftazidime with and without clavulanic acid. The bla(CTX-M) genes were identified and genotyped using PCR with bacterial DNA samples. Multivariate analysis was performed to investigate risk factors associated with the faecal carriage of CTX-M producers. The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae was 65.7. The CTX-M-9 group (60.6) was dominant, followed by the CTX-M-1 group (38.7). Most of the bacteria were Escherichia coli (85.4) and Klebsiella pneumoniae (4.7). Of a total of 234 E. coli strains, 48.7 belonged to phylogenetic group A, 28.6 to group B1, 15.8 to group D and 6.8 to group B2. Most CTX-M producers were susceptible to carbapenems and amikacin, but resistant to tetracycline and gentamicin. In a multivariate logistic regression model, better education status (OR 2.245; 95 CI 1.2973.884), history of hospitalization (OR 1.643; 95 CI 1.0362.603) and the use of antibiotics within the last 3 months (OR 1.883; 95 CI 1.2212.903) were independently associated with faecal carriage. Faecal carriage of CTX-M-type ESBL-producing Enterobacteriaceae among asymptomatic individuals in rural Thailand remains alarmingly high, and previous antibiotic use and a history of hospitalization may contribute to its dissemination.

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Epidemiology of Escherichia coli, Klebsiella species, and Proteus mirabilis strains producing extended-spectrum β-lactamases from clinical samples in the Kinki Region of Japan.

Nakamura T, Komatsu M, Yamasaki K, Fukuda S, Miyamoto Y, Higuchi T, Ono T, Nishio H, Sueyoshi N, Kida K, Satoh K, Toda H, Toyokawa M, Nishi I, Sakamoto M, Akagi M, Nakai I, Kofuku T, Orita T, Wada Y, Zikimoto T, Koike C, Kinoshita S, Hirai I, Takahashi H, Matsuura N, Yamamoto Y

American journal of clinical pathology ( AMER SOC CLINICAL PATHOLOGY )  137 ( 4 ) 620 - 6   2012年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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In the present study, nonduplicate, clinical isolates of extended-spectrum beta-lactamase (ESBL)-producing Escherichia colt, Klebsiella spp, and Proteus mirabilis were collected during a 10-year period from 2000 to 2009 at several hospitals in the Kinki region, Japan. The detection rate of E coli markedly increased from 0.24% to 7.25%. The detection rate of Klebsiella pneumoniae increased from 0% to 2.44% and that of P mirabilis from 6.97% to 12.85%. The most frequently detected genotypes were the CTX-M9 group for E coli, the CTX-M2 group for K pneumoniae, and the CTX-M2 group for P mirabilis. E coli clone O25:H4-ST131 producing CTX-M-15, which is spreading worldwide, was first detected in 2007. The most common replicon type of E coli was the IncF type, particularly FIB, detected in 466 strains (69.7%). Of the K pneumoniae strains, 47 (55.3%) were of the IncN type; 77 P mirabilis strains (96.3%) were of the IncT type. In the future, the surveillance of various resistant bacteria, mainly ESBL-producing Enterobacteriaceae, should be expanded to prevent their spread.

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Extended-spectrum β-lactamase-producing Escherichia coli strains in the feces of carriers contribute substantially to urinary tract infections in these patients.

Niki M, Hirai I, Yoshinaga A, Ulzii-Orshikh L, Nakata A, Yamamoto A, Yamamoto M, Yamamoto Y

Infection ( SPRINGER HEIDELBERG )  39 ( 5 ) 467 - 471   2011年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Introduction The current increase in the incidence of urinary tract infections (UTIs) worldwide caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli may be due to the high number of ESBL-producing Enterobacteriaceae carriers in the community. However, whether ESBL-producing bacteria can cause UTIs in carriers remains uncertain. Materials and methods In this study, 21 fecal carriers of ESBL-producing Enterobacteriaceae were assessed for UTIs caused by ESBL-producing E. coli. Bacterial isolates obtained from patients&apos; urine and stool specimens were phenotypically and genotypically examined. Clonal similarities of isolates were assessed by multilocus sequence typing (MLST) and random amplified polymorphic DNA (RAPD) fingerprinting. Results The study revealed that 9 of 21 carriers developed UTIs, and genetic analysis showed that 44% of the UTIs developed were caused by the same ESBL-producing E. coli as that found in the feces of the patients. Conclusions The ESBL-producing E. coli in carriers can cause UTIs.

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Prevalence of fecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae among healthy adult people in Japan.

Luvsansharav UO, Hirai I, Niki M, Nakata A, Yoshinaga A, Moriyama T, Yamamoto Y

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy ( SPRINGER TOKYO )  17 ( 5 ) 722 - 5   2011年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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In Japan, the prevalence of extended-spectrum beta-lactamase (ESBL) producers in hospital settings has shown an increasing trend. In the present study, we investigated the prevalence of the fecal carriage of ESBL-producing Enterobacteriaceae among healthy adults. Stool samples were collected for the phenotypic and genotypic identification of ESBL-producing Enterobacteriaceae. We found the prevalence of ESBL producers to be 6.4% by phenotypic identification, and 92.9% of them possessed the bla(CTX-M) gene. Among the CTX-M ESBL-producing Enterobacteriaceae, we identified 11 Escherichia coli and 2 Klebsiella pneumonia strains. The findings suggest that the fecal carriage of CTX-M-type ESBL producers by healthy people is rapidly increasing in Japan. This may be one of the causes of the increased spread of ESBL-producing bacteria in hospitals.

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Chlamydophila pneumoniae attachment and infection in low proteoglycan expressing human lymphoid Jurkat cells.

Kobayashi M, Ishida K, Matsuo J, Nakamura S, Nagasawa A, Motohashi K, Yao T, Hirai I, Yamamoto Y, Suzuki H, Shimizu C, Matsuno K, Yamaguchi H

Microbial pathogenesis ( ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD )  51 ( 3 ) 209 - 16   2011年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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This study investigated the proteoglycan (PG)-dependent mechanism of Chlamydophila pneumoniae attachment to lymphocytic cells. Lymphoid Jurkat cells and epithelial HEp-2 cells were statically infected with C. pneumoniae (TW183). Transmission electron microscopy and assessment of inclusion-forming units indicated that the bacteria grew normally in Jurkat cells and were capable of producing secondary infection; however, they grew at a slower rate than in HEp-2 cells. RT-PCR analysis indicated that HEp-2 cells strongly expressed PG-core protein encoding genes, thereby sustaining glycosaminoglycans (GAGs), such as heparin, on the cellular surface. Similar gene expression levels were not observed in Jurkat cells, with the exception of glypican-1. Immunofluorescence analysis also supported strong heparin expression in HEp-2 cells and minimal expression in Jurkat cells, although heparan sulfate pretreatment significantly inhibited bacterial attachment to both cell types. Immunofluorescent co-staining with antibodies against chlamydial LPS and heparin did not identify bacterial and heparin co-localization on Jurkat cells. We also confirmed that when C. pneumoniae was statically infected to human CD4(+) peripheral blood lymphocytes known not expressing detectable level of heparin, the bacteria attached to and formed inclusion bodies in the cells. Thus, the attachment mechanism of C pneumoniae to Jurkat cells with low PG expression is unique when compared with HEp-2 cells and potentially independent of GAGs such as heparin. (C) 2011 Elsevier Ltd. All rights reserved.

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Analysis of risk factors for a high prevalence of extended-spectrum {beta}-lactamase-producing Enterobacteriaceae in asymptomatic individuals in rural Thailand.

Luvsansharav UO, Hirai I, Niki M, Sasaki T, Makimoto K, Komalamisra C, Maipanich W, Kusolsuk T, Sa-Nguankiat S, Pubampen S, Yamamoto Y

Journal of medical microbiology ( SOC GENERAL MICROBIOLOGY )  60 ( Pt 5 ) 619 - 624   2011年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The prevalence of and risk factors associated with extended-spectrum beta-lactamase (ESBL)-producing micro-organisms have not been well studied in healthy individuals. The aim of this study was to determine this in healthy individuals in Thailand. Stool samples and questionnaires obtained from 445 participants from three provinces in Thailand were analysed. The antimicrobial susceptibility of the isolates was assessed using phenotypic and genotypic methods. PCR analysis was performed to detect and group the bla(CTX-M) genes. The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae in the three provinces was as follows: 29.3% in Nan (43/147), 29.9% in Nakhon Si Thammarat (43/144) and 50.6% in Kanchanaburi (78/154) (P&lt;0.001). Of the 445 samples, 33 (7.4%), 1 (0.2%) and 127 (28.5%) isolates belonged to the bla(CTX-M) gene groups I, Ill and IV, respectively. Escherichia coli was the predominant member of the Enterobacteriaceae producing CTX-M-type ESBLs (40/43, 39/43 and 70/78 isolates in Nan, Nakhon Si Thammarat and Kanchanaburi, respectively). No statistically significant association was observed between the presence of ESBL-producing bacteria and gender, age, education, food habits or antibiotic usage. However, the provinces that had the highest prevalence of ESBL-producing Enterobacteriaceae also had the highest prevalence of use and purchase of antibiotics without a prescription. Thus, this study revealed that faecal carriage of ESBL-producing Enterobacteriaceae is very high in asymptomatic individuals in Thailand, with some variations among the provinces. This high prevalence may be linked to antibiotic abuse.

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Sequence analysis of East Asian cagA of Helicobacter pylori isolated from asymptomatic healthy Japanese and Thai individuals.

Hirai I, Yoshinaga A, Kimoto A, Sasaki T, Yamamoto Y

Current microbiology ( SPRINGER )  62 ( 3 ) 855 - 60   2011年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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CagA, especially East Asian type, is one of the most important virulence factors of Helicobacter pylori, which is believed to contribute to the gastric cancer development. There is extreme sequence heterogeneity on 3&apos; region of cagA gene, demonstrated by the sequence analysis of cagA of H. pylori strains isolated from gastric disease patients. However, whether such heterogeneity of the cagA gene sequence is related to the pathogenicity of H. pylori in the gastric cancer development is not certain. Therefore, in this study, the 3&apos; region of cagA sequences isolated from asymptomatic healthy individuals in Japan and Thailand, which show high and low gastric cancer prevalence, respectively, were analyzed and compared with those from patients with gastric cancer. The CagA sequences analysis in 21 and 12 H. pylori DNA samples obtained from Japanese and Thai individuals, respectively, by the molecular phylogenetic method showed that the sequences were more conserved in the Thai individuals (concordance rates among Thai sequences, 93.9-100%) than in the Japanese individuals (concordance rates among Japanese sequences, 82.8-100%) as shown by unrooted neighbor-joining (N-J) consensus trees constructed with the sequence between Asn869 and Ala967 in CagA. CagA sequences in gastric cancer patients were obtained from published data; analysis of these sequences revealed that CagA sequences from almost all Thai individuals were concentrated in one branch. In contrast, CagA sequences from Japanese individuals were uniformly distributed throughout the N-J consensus tree. These results suggest that the difference in the CagA sequences between asymptomatic healthy Japanese and Thai individuals may be linked to the incidence of gastric cancer in Japan and Thailand.

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Molecular typing of Bartonella henselae DNA extracted from human clinical specimens and cat isolates in Japan.

Yanagihara M, Tsuneoka H, Hoshide S, Ishido E, Umeda A, Tsukahara M, Nojima J, Ichihara K, Hino K, Hirai I, Yamamoto Y

FEMS immunology and medical microbiology ( WILEY-BLACKWELL )  60 ( 1 ) 44 - 8   2010年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Bartonella henselae is the causative agent of cat scratch disease (CSD). To clarify the population structure and relationship between human and cat strains of B. henselae, 55 specimens isolated in Japan, including 24 B. henselae DNA-positive clinical samples from CSD patients and 31 B. henselae isolates from domestic cats, were characterized by multilocus sequence typing (MLST) and the 16S-23S tRNA-Ala/tRNA-Ile intergenic spacer (S1) sequence, which were used previously for strain typing of B. henselae. Three different sequence types (STs) were identified by MLST, one of which was novel. Fifty-two strains (94.5%), including all strains detected in CSD patients, were assigned to ST-1. Eight S1 genotypes were observed, three of which were novel. The 52 ST-1 strains were classified into seven S1 genotypes, two of which were predominant in both human and cat strains. In addition, 5.5% of the strains (3/55) contained two different intergenic spacer S1 copies. These results indicate that the predominant B. henselae MLST ST-1 in Japan is a significantly genetically diverse population on the basis of the sequence diversity of intergenic spacer S1, and that highly prevalent S1 genotypes among cats are often involved in human infections.

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Laboratory surveillance for prospective plasmid-mediated AmpC beta-lactamases in the Kinki region of Japan.

Yamasaki K, Komatsu M, Abe N, Fukuda S, Miyamoto Y, Higuchi T, Ono T, Nishio H, Sueyoshi N, Kida K, Satoh K, Toyokawa M, Nishi I, Sakamoto M, Akagi M, Nakai I, Kofuku T, Orita T, Wada Y, Jikimoto T, Kinoshita S, Miyamoto K, Hirai I, Yamamoto Y

Journal of clinical microbiology ( AMER SOC MICROBIOLOGY )  48 ( 9 ) 3267 - 73   2010年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Extended-spectrum beta-lactamases, plasmid-mediated AmpC beta-lactamases (PABLs), and plasmid-mediated metallo-beta-lactamases confer resistance to many beta-lactams. In Japan, although several reports exist on the prevalence of extended-spectrum beta-lactamases and metallo-beta-lactamases, the prevalence and characteristics of PABLs remain unknown. To investigate the production of PABLs, a total of 22,869 strains of 4 enterobacterial species, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis, were collected during six 6-month periods from 17 clinical laboratories in the Kinki region of Japan. PABLs were detected in 29 (0.13%) of 22,869 isolates by the 3-dimensional test, PCR analysis, and DNA sequencing analysis. PABL-positive isolates were detected among isolates from 13 laboratories. Seventeen of 13,995 (0.12%) E. coli isolates, 8 of 5,970 (0.13%) K. pneumoniae isolates, 3 of 1,722 (0.17%) K. oxytoca isolates, and 1 of 1,182 (0.08%) P. mirabilis isolates were positive for PABLs. Of these 29 PABL-positive strains, 20 (69.0%), 6 (20.7%), 2 (6.9%), and 1 (3.4%) carried the genes for CMY-2, DHA-1, CMY-8, and MOX-1 PABLs, respectively. Pattern analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoretic analysis revealed that the prevalence of CMY-2-producing E. coli strains was not due to epidemic strains and that 3 DHA-1-producing K. pneumoniae strains were identical, suggesting their clonal relatedness. In conclusion, the DHA-1 PABLs were predominantly present in K. pneumoniae strains, but CMY-2 PABLs were predominantly present in E. coli strains. The present findings will provide significant information to assist in preventing the emergence and further spread of PABL-producing bacteria.

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Characterisation of anti-Staphylococcus aureus activity of quercetin

Itaru Hirai, Mana Okuno, Reika Katsuma, Namie Arita, Masahiro Tachibana, Yoshimasa Yamamoto

INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY ( WILEY-BLACKWELL )  45 ( 6 ) 1250 - 1254   2010年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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P&gt;Although many antibiotics are available for the treatment of bacterial infections, the emergence and global spread of antibiotic-resistant bacteria is a community-wide problem. To overcome this problem, we must explore alternative antimicrobials. This study investigated the antibacterial properties of quercetin, a flavonoid present in vegetables and fruits. Quercetin was tested against gram-positive and gram-negative bacteria and was found to exert selective antibacterial activity against Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), and Staphylococcus epidermidis. Some clinical MRSA strains showed remarkable susceptibility to quercetin. In combination with antibiotics, such as oxacillin, ampicillin, vancomycin, gentamicin, and erythromycin, quercetin showed markedly enhanced antibacterial activity against MRSA. We also report quercetin-induced aggregation of S. aureus cells; the morphological changes in these cells, as assessed by electron microscopy; and the colony-spreading ability of quercetin-sensitive MRSA, all of which revealed the unique antibacterial properties of quercetin against S. aureus.

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Chlamydia pneumoniae infection suppresses Staphylococcus enterotoxin B-induced proliferation associated with down-expression of CD25 in lymphocytes

Itaru Hirai, Maki Utsumi, Hiroyuki Yamaguchi, Yoshimasa Yamamoto

CANADIAN JOURNAL OF MICROBIOLOGY ( NATL RESEARCH COUNCIL CANADA-N R C RESEARCH PRESS )  56 ( 4 ) 289 - 294   2010年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Chlamydia pneumoniae (Chlamydophila pneumoniae) infects lymphocytes and modulates their immune functions; this is critical in the development of chronic inflammatory diseases associated with this pathogen. Therefore, to clarify this immune modulation due to C. pneumoniae infection, the effect of this infection on the proliferation of human peripheral blood lymphocytes was examined. Lymphocytes were proliferated by stimulation with Staphylococcus aureus enterotoxin B, and the cell number was increased up to 3 times the unstimulated lymphocyte number. Further, induction of CD25 expression was observed in 55.8% of lymphocytes. Infection with C. pneumoniae suppressed the proliferation of almost half the lymphocytes induced by stimulation with S. aureus enterotoxin B, and CD25 induction was inhibited in 64.7% of lymphocytes. Inhibition of CD25 expression was observed in both infected and uninfected lymphocytes in culture. However, the expression of VLA4 was not affected by C. pneumoniae infection. Furthermore, inhibition was observed only by infection with viable C. pneumoniae and not by the heat-killed bacteria. These results suggest that C. pneumoniae affects lymphocyte function by inhibiting proliferation and CD25 expression in response to immunological stimulation, possibly via humoral mediator(s).

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High prevalence of CTX-M beta-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in Thailand.

Sasaki T, Hirai I, Niki M, Nakamura T, Komalamisra C, Maipanich W, Kusolsuk T, Sa-Nguankiat S, Pubampen S, Yamamoto Y

The Journal of antimicrobial chemotherapy ( OXFORD UNIV PRESS )  65 ( 4 ) 666 - 8   2010年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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To determine the prevalence of CTX-M beta-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in a rural area of Thailand. Bacteria in stool specimens were screened for extended-spectrum beta-lactamase (ESBL) production on McConkey agar with cefotaxime and confirmed by the double-disc synergy test. Genetic detection and genotyping of CTX-M-type ESBL was performed by PCR with bacterial DNA extracted from isolates. A markedly high number (82 of 141, 58.2%) of the specimens showed the presence of CTX-M beta-lactamase-producing Enterobacteriaceae, as confirmed by both phenotypic and genetic examinations. The majority of the CTX-M beta-lactamase-producing bacteria were Escherichia coli (85.1%). The study revealed the wide dissemination of CTX-M beta-lactamase-producing Enterobacteriaceae in the healthy population.

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Infection of less virulent Helicobacter pylori strains in asymptomatic healthy individuals in Thailand as a potential contributing factor to the Asian enigma.

Hirai I, Sasaki T, Kimoto A, Yamamoto Y, Azuma T, Mahachai V, Hansomburana P, Lertkupinit C, Luangjaru S, Noophan P, Chanatrirattanapan R, Piyanirandr V, Sappajit T, Suthivarakom K, Sangsuk L, Wangroongsarb P

Microbes and infection ( ELSEVIER SCIENCE BV )  12 ( 3 ) 227 - 30   2010年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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In Thailand, gastric cancer incidence is considerably low despite the high prevalence of Helicobacter pylori infection. We investigated the prevalence of H. pylori infection and the genotypes of cagA by using 179 stool specimens obtained from asymptomatic Thai individuals. In this study, the prevalence of H. pylori infection was 43.6%, and the detection rate of cagA-positive strains was 43.5%. In addition, the proportion of the highly virulent East-Asian type of cagA was 7.2%. These results indicate that the low prevalence of cagA-positive H. pylori strain as well as the low prevalence of East-Asian genotype cagA-positive strains may contribute to the low gastric cancer incidence. (C) 2009 Elsevier Masson SAS. All rights reserved.

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エクアドルおよびパナマ在住の健常人におけるHelicobacter pylori感染について―寄生虫共感染およびH.pylori病原因子の検討―

佐々木正大, 平井到, 山本容正

Clin Parasitol   20 ( 1 ) 105 - 107   2010年02月

掲載種別: 研究論文（学術雑誌）

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  J-GLOBAL

Establishment of shared antigen reactive cytotoxic T lymphocyte using co-stimulatory molecule introduced autologous cancer cells.

Hirohashi Y, Torigoe T, Hirai I, Tamura Y, Nakatsugawa M, Inoue Y, Kanaseki T, Kamiguchi K, Ikeda H, Sasaki A, Yamanaka N, Sato N

Experimental and molecular pathology ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  88 ( 1 ) 128 - 32   2010年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs. (C) 2009 Elsevier Inc. All rights reserved.

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Stability of Chlamydophila pneumoniae in a harsh environment without a requirement for acanthamoebae.

Matsuo J, Kobayashi M, Nakamura S, Mizutani Y, Yao T, Hirai I, Yamamoto Y, Yamaguchi H

Microbiology and immunology ( WILEY-BLACKWELL PUBLISHING, INC )  54 ( 2 ) 63 - 73   2010年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The effect of actual interactions between Chlamydophila pneumoniae and amoebae (Acanthamoeba) on the survival of C. pneumoniae was investigated. C. pneumoniae and amoebae were detected in 75 soil samples by IFU assay and PCR. Although C. pneumoniae could not be cultured, the DNA prevalence of C. pneumoniae and amoebae in natural soil was 20% and 92% (no correlation between the prevalence of DNA was observed). The viability of C. pneumoniae spiked in autoclaved soil was assessed by IFU assay and RT-PCR. Although the number of infective progeny decreased for three days, transcripts of C. pneumoniae were detected for up to 98 days independently of amoebae. The stability of C. pneumoniae in liquid medium was also assessed by IFU assay and transmission electron microscopy. The bacteria could survive at 15 degrees C for 14 days independently of amoebae. Bacteria cultured without amoebae were confirmed to have normal structures. Thus, the presence of amoebae has no effect on C. pneumoniae survival, and the bacteria can survive in the absence of host cells for an extended period of time.

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Assessment of East Asian-type cagA-positive Helicobacter pylori using stool specimens from asymptomatic healthy Japanese individuals.

Hirai I, Sasaki T, Kimoto A, Fujimoto S, Moriyama T, Yamamoto Y

Journal of medical microbiology ( SOC GENERAL MICROBIOLOGY )  58 ( Pt 9 ) 1149 - 1153   2009年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Recent investigations have suggested that CagA, a virulence factor of Helicobacter pylori and known to have multiple genotypes, plays a critical role in the development of stomach cancer. However, the prevalence of cagA-positive H. pylori strains and the cagA genotypes have not been well studied in healthy individuals because of the difficulty in collecting gastric specimens. In the present study, we assessed the prevalence of infection with H. pylori, particularly the strains with the East Asian cagA genotype (which is more potent in causing gastric diseases), among healthy asymptomatic Japanese individuals by a noninvasive method using stool specimens. The H. pylori antigen was detected in 40.3 % of healthy asymptomatic adult individuals (n=186) enrolled in the study. For the detection and genotyping of the cagA gene, DNA was extracted from the stool specimens of these individuals and analysed by PCR. We detected the East Asian cagA genotype in the DNA samples of a significantly high number (63.1 %) of healthy asymptomatic Japanese individuals. These results indicate that a significant number of asymptomatic healthy Japanese individuals were infected with highly virulent H. pylori.

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Analysis of Helicobacter pylori Genotype in Stool Specimens of Asymptomatic People

Tadahiro Sasaki, Itaru Hirai, Ricardo Izurieta, Boo Hoe Kwa, Edmundo Estevez, Azael Saldana, Jose Calzada, Saori Fujimoto, Yoshimasa Yamamoto

LABMEDICINE ( AMER SOC CLINICAL PATHOLOGY )  40 ( 7 ) 412 - 414   2009年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Background. Genotyping of Helicobacter pylori virulence factor is important in human health care. However, the genotyping of H. pylori infecting asymptomatic people is not well studied due to the difficulty in collecting gastric specimens. Methods: We analyzed 164 asymptomatic participants in Ecuador and Panama by utilizing stool specimens. H, pylori was detected using immunological methods. H. pylori virulence factor cagA was genotyped by PCR. Results: Helicobacter pylori antigen was found in 72.2% of participants from Ecuador and 54.1% from Panama. Among H. pylori DNA-positive samples, cagA was detected in 45.9% from Ecuador and 20.0% from Panama. Over 80% of the detected cagA from both countries was East-Asian genotype. Conclusions: The results suggest that our method is valuable for assessing the H. pylori infection status of asymptomatic people and that the prevalence of H. pylori infection and/or the occurrence of cagA in H. pylori may be related to the risk of stomach cancer.

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A method for assessment of Helicobacter pylori genotype using stool specimens.

Hirai I, Sasaki T, Fujimoto S, Moriyama T, Azuma T, Yamamoto Y

FEMS immunology and medical microbiology ( WILEY-BLACKWELL PUBLISHING, INC )  56 ( 1 ) 63 - 6   2009年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Helicobacter pylori infection has been regarded as a major factor associated with the development of gastric diseases. The characterization of infected H. pylori in asymptomatic individuals is important for the prediction of the onset of such diseases. However, because of the difficulty in obtaining gastric biopsy samples, H. pylori in healthy subjects have not been studied sufficiently. Therefore, we tested a noninvasive method for the characterization of H. pylori using stool specimens. This method involved H. pylori antigen detection in stool specimens by immuno-chromatography; confirmation of H. pylori DNA by real-time PCR that involved the detection of its 16S rRNA gene in the DNA extracted from stool specimens; and nested PCR with genotype-specific primer pairs. A total of 80 samples obtained from asymptomatic subjects were assessed using this method. The results showed that the prevalence of H. pylori in asymptomatic Japanese individuals was 37.5%. The detection rate of the virulence factor gene cagA was 18.8%. Furthermore, all the detected cagA belonged to the highly virulent East-Asian type. These data suggest that the method used in this study is valuable for studying the molecular epidemiology of H. pylori infection in asymptomatic people.

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Immunosuppressive effect on T cell activation by interleukin-16- and interleukin-10-cDNA-double-transfected human squamous cell line.

Matsumoto Y, Fujita T, Hirai I, Sahara H, Torigoe T, Ezoe K, Saito T, Cruikshank WW, Yotsuyanagi T, Sato N

Burns : journal of the International Society for Burn Injuries ( ELSEVIER SCI LTD )  35 ( 3 ) 383 - 9   2009年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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It is well known that induction of immunotolerance with allogeneic skin transplantation is generally difficult. This study attempted to find an immunosuppressive protocol for skin allograft rejection involving interleukin-16 (IL-16) and interleukin-10 (IL-10), because both are known to inhibit mixed lymphocyte reaction (MLR). The data indicated that IL-16 enhanced the immunosuppressive effect of IL-10. IL-16-cDNA- and IL-10-cDNA-double-transfected squamous cell carcinoma cell line were used as an in vitro model and they produced more than 20 ng/ml of IL-16 and 100 pg/ml of IL-10 in the supernatant, which significantly inhibited MLR and also the activation of allogeneic lymphocytes, which were stimulated directly by allogeneic double-cDNA-transfectant cells. Thus allogeneic skin graft producing IL-16 and IL-10 might have a local immunosuppressive action that could prolong graft survival. (c) 2008 Elsevier Ltd and ISBI. All rights reserved.

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[Analysis of Helicobacter pylori infection in a healthy Panamanian population].

Sasaki T, Hirai I, Yamamoto Y

Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases   83 ( 2 ) 127 - 32   2009年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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健常人保菌Helicobacter pyloriの検出と病原因子遺伝子解析

佐々木正大, 平井到, IZURIETA Richard, IZURIETA Richard, KWA Boo, 山本容正

臨床と微生物   35 ( 5 ) 475 - 480   2008年09月

掲載種別: 研究論文（学術雑誌）

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Disruption of the association of 73 kDa heat shock cognate protein with transporters associated with antigen processing (TAP) decreases TAP-dependent translocation of antigenic peptides into the endoplasmic reticulum.

Kamiguchi K, Torigoe T, Fujiwara O, Ohshima S, Hirohashi Y, Sahara H, Hirai I, Kohgo Y, Sato N

Microbiology and immunology ( BLACKWELL PUBLISHING )  52 ( 2 ) 94 - 106   2008年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Major histocompatibility complex class I-bound antigenic peptides generated in the cytosol are translocated into the ER by TAP. In the present study, the physical association of HSC73 with TAP in human lymphoblastoid T I cells was demonstrated. The dissociation was induced in the presence of 10mM ATP, indicating that the ADP-binding form of HSC73 might be associated with TAP. We found that HSC73-binding immunosuppressant, MeDSG disrupted the HSC73-TAP association, whereas it did not affect the binding of HSC73 to a substrate protein. MHC class I expression on the cell surface was also downregulated. Then, the effect of MeDSG on the TAP-mediated ER translocation was examined using two homologous model peptides, NGT-Bw4 and NGT-Bw6, which had distinct binding affinity to HSC73. Although high-affinity peptide NGT-Bw4 was translocated by TAP, low-affinity peptide NGT-Bw6 was not. The TAP-dependent translocation of NGT-Bw4 was abolished in the presence of MeDSG. Decreased presentation on the cell surface was shown for the human leukocyte antigen (HLA) -A31-restricted natural antigenic peptide F4.2, which had high affinity to HSC73, in the presence of MeDSG. It was indicated that disruption of the HSC73-TAP association resulted in inhibition of TAP-dependent translocation of HSC73-bound peptides. Our findings highlighted an important role of HSC73 for feeding antigenic peptides to TAP, and suggested a possibility that a synthetic polyamine might inhibit the function of HSC73, thereby suppressing MHC class I-restricted presentation of HSC73-bound antigenic peptides.

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Loss of RPA1 induces Chk2 phosphorylation through a caffeine-sensitive pathway.

Araya R, Hirai I, Meyerkord CL, Wang HG

FEBS letters ( ELSEVIER SCIENCE BV )  579 ( 1 ) 157 - 61   2005年01月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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RPA is an important component of DNA replication, repair and recombination, but its involvement in the signaling of cell-cycle checkpoints is not well understood. In this study, we show that knockdown of RPAI by siRNA duplexes induces ATM (Ser1981) and Chk2 (Thr68), but not Chk1 (Ser345) phosphorylation and results in p21 upregulation in HeLa cells. However, the induction of Chk2 (Thr68) phosphorylation and p21 expression by RPA1 siRNA transfection can be completely blocked by the ATM inhibitor caffeine. Moreover, transfection of siRNAs targeting ATM dramatically reduces Chk2 (Thr68) phosphorylation in RPA1 knockdown cells. Taken together, these results suggest that loss of RPA1 activates the Chk2 signaling pathway in an ATM-dependent manner. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Human hRad1 but not hRad9 protects hHus1 from ubiquitin-proteasomal degradation.

Hirai I, Sasaki T, Wang HG

Oncogene ( NATURE PUBLISHING GROUP )  23 ( 30 ) 5124 - 30   2004年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Three of the Rad family proteins, Rad9, Rad1, and Hus1, can interact with each other and form a heterotrimeric complex that is thought to play a role in the sensing step of the DNA integrity checkpoint pathways, but the nature of the Rad9 - Rad1 - Hus1 complex assembly remains enigmatic. Here, we demonstrate that the human hRad1 protein plays a significant role as molecular chaperone in the process of the hRad9 - hRad1 - hHus1 heterotrimeric complex formation. In contrast to hRad1, hHus1 is an unstable protein that is actively degraded via the ubiquitin - proteasome pathway. We show that treating cells with proteasome-specific inhibitors stabilizes hHus1 expression. Moreover, hRad1 can associate with hHus1 in the absence of hRad9 and protect hHus1 from ubiquitination and degradation in the cytoplasm. Importantly, genotoxic stress induces hRad1 expression and stabilizes the hHus1 protein. Taken together, these findings suggest a novel role of hRad1 as a potential intrinsic chaperone in the stabilization of hHus1 for the hRad9 - hRad1 - hHus1 checkpoint complex formation.

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Tumor-derived heat shock protein 70-pulsed dendritic cells elicit tumor-specific cytotoxic T lymphocytes (CTLs) and tumor immunity.

Ueda G, Tamura Y, Hirai I, Kamiguchi K, Ichimiya S, Torigoe T, Hiratsuka H, Sunakawa H, Sato N

Cancer science ( JAPANESE CANCER ASSOC )  95 ( 3 ) 248 - 53   2004年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Vaccination with autologous tumor-derived heat shock proteins (Hsp), such as Hsp70, Hsp90 and gp96, has been demonstrated to elicit specific immune responses against the tumor from which the Hsps were isolated. The effect of Hsp immunization is wholly dependent on the presence of functional antigen-presenting cells (APCs) in the immunized host, and Hsp receptors on APCs have recently been identified. Here we show that bone marrow-derived dendritic cells (DCs) are able to internalize HSP-peptide complex and that peptides are re-presented by DCs via the major histocompatibility complex (MHC) class I presentation pathway. In addition, immunization with tumor-derived HSP-pulsed IDCs induces strong cytotoxic T cell (CTL) responses against multiple antigenic peptides in a transporter-associated antigen processing (TAP)-dependent manner. The results of the present study provide strong evidence of an efficient cross-priming activity of Hsp70, which could be exploited in the development of new and more effective immunotherapeutic strategies for cancer patients.

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Caspase-3-mediated cleavage of Rad9 during apoptosis.

Lee MW, Hirai I, Wang HG

Oncogene ( NATURE PUBLISHING GROUP )  22 ( 41 ) 6340 - 6   2003年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The activation of caspases is a critical event for the execution phase of programmed cell death. Caspases are highly specific in their ability to activate or inhibit many crucial proteins in the cell via cleavage. In this study, we report the identification of several caspase-3-like cleavage sites in the cell-cycle checkpoint protein Rad9. We demonstrate that human Rad9 can be specifically cleaved in cells induced to enter apoptosis by both DNA damage and staurosporine treatment. Indeed, we show that human Rad9 can be effectively cleaved both in vitro and in vivo, which can be inhibited by either a pan-caspase inhibitor or a caspase-3-specific inhibitor. Additionally, no cleavage of Rad9 can be seen in the caspase-3-deficient cell line MCF-7. Site-directed mutagenesis of three of the most conserved cleavage sites dramatically abrogates cleavage of Rad9 by caspase-3 in vitro, and in intact cells after DNA damage. Expression of the cleavage-resistant mutant Rad9 DDD/AAA appears to protect the cell from DNA damage-induced apoptosis. Immunofluorescence studies of Rad9 localization before and after induction of apoptosis show a translocation of Rad9 from the nucleus to the cytosol, concomitant to the appearance of apoptotic morphology. Furthermore, analysis of a truncated Rad9 mutant that corresponds to a putative N-terminal cleavage fragment shows that the N-terminal portion of Rad9 localizes in the cytosol, binds to Bcl-XL, and induces apoptosis. These results support a dual role for cleavage of Rad9: ( 1) the liberation and translocation of the BH3 domain-containing N-terminus of Rad9 to the cytosol, as a means of promoting apoptosis via antagonism of Bcl-XL, and ( 2) the disruption of the Rad9-Rad1-Hus1 DNA damage checkpoint complex.

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Analysis of a shared pancreatic cancer antigen recognized by an HLA-A*2601-restricted cytotoxic T-lymphocyte clone.

Kashiwagi K, Ikeda H, Hirohashi Y, Yamamoto M, Idenoue S, Hirai I, Kamiguchi K, Tamura Y, Torigoe T, Wada Y, Hirata K, Sato N

Pancreas   26 ( 4 ) e81 - 8   2003年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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IgG and IgA antibody titers against human heat-shock protein (hsp60) in sera of rheumatoid arthritis and osteoarthritis patients.

Watanabe S, Takubo N, Hirai I, Hitsumoto Y

Modern rheumatology   13 ( 1 ) 22 - 26   2003年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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T-cell receptor variable gamma chain gene expression in the interaction between rat gammadelta-type T cells and heat-shock protein 70-like molecule.

Ichinohe T, Ichimiya S, Kishi A, Tamura Y, Kondo N, Ueda G, Torigoe T, Yamaguchi A, Hiratsuka H, Hirai I, Kohama GI, Sato N

Microbiology and immunology   47 ( 5 ) 351 - 7   2003年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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A role of the C-terminal region of human Rad9 (hRad9) in nuclear transport of the hRad9 checkpoint complex.

Hirai I, Wang HG

The Journal of biological chemistry ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC )  277 ( 28 ) 25722 - 7   2002年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Rad9, Rad1, and Hus1 are members of the Rad family of checkpoint proteins that are required for both DNA replication and DNA damage checkpoints and are thought to function as sensors in the DNA integrity checkpoint control. These proteins can interact with each other and form a stable proliferating cell nuclear antigen-related Rad9-Rad1-Hus1 heterotrimeric complex that might encircle DNA at or near the damaged sites. In this study, we demonstrate that the human Rad9 (hRad9) protein contains a predicted nuclear localization sequence (NLS) near its C terminus, which plays an essential role in the hRad9-mediated G2 checkpoint. Deletion experiments indicate that the NLS-containing region of hRad9 is critical for the nuclear transport of not only hRad9 but also human Rad1 (hRad1) and human Hus1 (hHus1), although this region is not required for hRad9-hRad1-hHus1 complex formation. In support of the role that hRad9 NI'S plays in the nuclear targeting of the hRad9-hRad1-hHus1 complex, overexpression of a deletion mutant of hRad9 lacking the NLS-containing C-terminal region can bypass the G(2) checkpoint and result in cell death after ionizing radiation or hydroxyurea treatment. Moreover, knockdown of hRad9 expression by small interfering RNA (siRNA) results in hRad1 accumulation in the cytoplasm and significantly abrogates the G, checkpoint in the presence of damaged DNA or incomplete DNA replication. Thus, the C-terminal region of human Rad9 protein is important for G(2) checkpoint control by operating the transport of the hRad9-hRad1-hHus1 checkpoint complex into the nucleus.

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Identification of germline mutation of PTEN gene and analysis of apoptosis resistance of the lymphocytes in a patient with Cowden disease.

Kanaseki T, Torigoe T, Hirohashi Y, Hirai I, Himi T, Sato N

Pathobiology : journal of immunopathology, molecular and cellular biology ( KARGER )  70 ( 1 ) 34 - 9   2002年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Mutations of the tumor suppressor gene PTEN have been reported in patients with Cowden disease (CD) and in several malignant tumors. We analyzed a germline mutation of the PTEN gene in a patient with CD and identified a 4-bp deletion in exon 8 of the PTEN gene. The same germline mutation was detected in 3 members of her family. The mutated gene was predicted to encode a C-terminal truncated PTEN protein. Immunoblotting analysis revealed that the expression level of the wildtype PTEN protein in the patient's lymphocytes was reduced to almost half the level of the control lymphocytes, and the predicted truncated mutant PTEN could not be detected. Since PTEN is known to function as a negative regulator of the phosphatidylinositol-3-kinase signal pathway that promotes cell survival, the patient's lymphocytes were tested for the resistance against the apoptotic stimulus. It was shown that the patient's lymphocytes were more resistant to apoptosis induced by calcium ionophore than the healthy control lymphocytes. These results indicate that the germline mutation of the PTEN gene and the consequent loss of heterozygous expression may lead to an increase in the survival potential of cells, thereby elucidating a role of PTEN in the pathogenesis of tumor generation and hyperplasia of lymphoid tissue in CID. Copyright (C) 2002 S. Karger AG, Basel.

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Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-X-L but not Bcl-2

Hirai, I, HG Wang

BIOCHEMICAL JOURNAL ( PORTLAND PRESS )  359 ( Pt 2 ) 345 - 352   2001年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The pro-apoptotic Bcl-2-family protein Bad heterodimerizes with Bcl-2 and Bel-x(L) in the outer mitochondrial membranes, nullifying their anti-apoptotic activities and promoting cell death. We report that interleukin-3 (IL-3) stimulation induces Bad phosphorylation and triggers its translocation from mitochondria to cytoplasm in cells expressing Bcl-x(L) but not Bcl-2. Over-expression of Bad sensitized Bcl-x(L)-expressing FL5.12 cells to apoptosis induced by IL-3 deprivation, but had no effect on the viability of cells expressing Bcl-2. IL-3 stimulation induced Bad phosphorylation at Ser-112, impairing its binding to Bcl-x(L) and resulting in its association with 14-3-3 proteins in the cytosol. However, Ser-112 phosphorylation could not trigger Bad dissociation from mitochondria in FL5.12 cells expressing Bcl-2. In 293T cells expressing Bcl-x(L), Bad was phosphorylated at three serines, 112, 136 and 155, and was largely localized in the cytosolic fraction. In contrast, overexpression of Bcl-2 prevented phosphorylation of Bad at Ser-136 and Ser-155, sequestering this protein in the mitochondrial membranes. When the N-terminal regions of Bcl-2 and Bel-x(L) were swapped with each other, the Bcl-x(L)(N)-Bcl-2 chimaeric protein (containing the N-terminal region of Bel-x,) failed to prevent Bad phosphorylation in cells and was unable to block the cytosolic distribution of this proapoptotic protein. Additional experiments with the Bcl-2(N)Bcl-x(L) chimaeric protein (containing the N-terminal region of Bcl-2) indicated that, although the N-terminal region of Bcl-2 is necessary, it is not sufficient for sequestering Bad in the mitochondrial membranes. These observations suggest that growth-factor-mediated phosphorylation of Bad contributes to the cytoprotective function of Bcl-x(L) but not Bcl-2.

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The cell surface-expressed HSC70-like molecule preferentially reacts with the rat T-cell receptor V delta 6 family

A Kishi, T Ichinohe, Hirai, I, K Kamiguchi, Y Tamura, M Kinebuchi, T Torigoe, S Ichimiya, N Kondo, K Ishitani, T Yoshikawa, M Kondo, A Matsuura, N Sato

IMMUNOGENETICS ( SPRINGER-VERLAG )  53 ( 5 ) 401 - 409   2001年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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We previously showed that the cell surface-expressed M-r 70,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3(+), CD4(-), CD8(-), T-cell receptor (TCR)alpha beta (-), natural killer recetor-Pl(-) T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the V delta6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR gamma delta chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR V delta6 family are likely to contribute to host resistance to tumor cells.

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The expression of a novel natural killer inhibitory molecule, Cho-1, on the chorionic cytotrophoblast cells of successful pregnancy, but not of spontaneous abortion

M Nagata, T Akazawa, Y Tamura, K Kamiguchi, Hirai, I, S Ohtani, S Sagae, R Kudo, T Torigoe, N Sato

PATHOLOGY INTERNATIONAL ( BLACKWELL SCIENCE ASIA )  50 ( 10 ) 824 - 831   2000年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The regulatory mechanism of the recognition and cytotoxicity by natural killer (NK) cells in placental tissue remains unclarified. Previous reports indicated that monoclonal antibody Cho-1-defined molecule (Cho-1 molecule) may act as the negative regulator in the cytotoxicity by human NK cells. The Cho-1 molecule is composed of non-covalently asssociated cell surface molecules of approximately 200 kDa and 40 kDa. In the present study we analyzed the expression of this novel molecule in extravillous cytotrophoblast cells, which are presumed to be exposed to the cytotoxic action by maternal NK cells, from clinical cases of successful pregnancy and spontaneous abortion. By using monoclonal antibody Cho-1, our immunohistochemical data indicated that the Cho-1 molecule is clearly expressed in the cytotrophoblast cells of the early phase of successful pregnancy, but only weakly expressed in those from spontaneous abortion. The cytotrophoblast cells in the late phase (9-10 months) of pregnancy also expressed this molecule. Fluorescence-activated cell sorter analysis also showed that it is expressed on the cytotrophoblast cell surface of successful pregnancy but not on that of spontaneous abortion, suggesting that Cho-1 antigen may act as a negative regulator of the cytotoxicity by NK cells in successful pregnancy of the fetus.

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Human CD8 and CD4 T cell epitopes of epithelial cancer antigens

N Sato, Y Nabeta, H Kondo, H Sahara, Y Hirohashi, K Kashiwagi, T Kanaseki, Y Sato, S Rong, Hirai, I, K Kamiguchi, Y Tamura, A Matsuura, S Takahashi, T Torigoe, H Ikeda

CANCER CHEMOTHERAPY AND PHARMACOLOGY ( SPRINGER-VERLAG )  46   S86 - S90   2000年06月

掲載種別: 研究論文（学術雑誌）

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Recent human tumor immunology research has identified several genes coding immunogenic peptides recognized by CD8 cytotoxic T lymphocytes (CTLs) in melanoma tumors. Very recently, CD4 T cell antigenic epitopes were also determined in certain melanoma tumors. The use of these peptides in conjunction with human immunotherapy could prove to be of great benefit. However, such peptides in clinically common tumors of epithelial cell origin, such as of the stomach, colon, lung, etc., have not yet been determined extensively. We describe for the first time an HLA-A31 (A*31012)-restricted natural antigenic peptide recognized by the CD8 CTL TcHST-2 of gastric signet ring cell carcinoma cell line HST-2. We also identified the HLA-DRB1*08032-restricted peptide recognized by the CD4 T cell line TcOSC-20 of squamous cell carcinoma OSC-20 derived from the oral cavity. The antigenic peptide of HST-2, designated F4.2, is composed of 10 amino acid residues with two anchor motif residues necessary for binding to HLA-A31 molecules. The synthetic F4.2 peptide enhanced the reactivity of TcHST-2 against HST-2 cells. Furthermore, introduction of an expression minigene coding F4.2 peptide to HLA-A31(+) cells conferred cytotoxic susceptibility to TcHST-2 on the cells. Some stomach cancer lines into which the HLA-A31 gene had been introduced, such as MKN28-A31-2, were lysed by TcHST-2, suggesting the presence of F4.2 peptide in at least some HLA-A31(+) stomach cancers. Furthermore, F4.2 peptide induced an F4.2 peptide-specific CTL response in at least 30-40% of HLA-A31(+) peripheral blood lymphocytes from gastric cancer patients, suggesting that F4.2 peptide could be used as a cancer vaccine for gastric tumors. The natural antigenic peptide of OSC-20 was also determined using acid extraction and biochemical separation and by mass spectrometry. Consequently, OSC-20 peptide was designated as the 6-1-5 peptide, an HLA-DRB1*08032-restricted 16-mer peptide with two possible anchor motifs. It has an amino acid sequence identical to that of human alpha-enolase, suggesting that it was derived from the processed parental alpha-enolase protein. We are presently attempting to determine the genes that code tumor rejection antigens recognized by HLA-A24- and A26-restricted T cells, including those of pulmonary and pancreatic carcinomas. The search for these antigenic peptides may lead to the identification of immunogenic peptide antigens that would be suitable for clinical use in commonly occurring epithelial cancers.

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  PubMed

Immunosuppressive effect on T cell activation by interleukin-16-cDNA-transfected human squamous cell line

T Fujita, Y Matsumoto, Hirai, I, K Ezoe, T Saito, A Yagihashi, T Torigoe, K Homma, S Takahashi, WW Cruikshank, K Jimbow, N Sato

CELLULAR IMMUNOLOGY ( ACADEMIC PRESS INC )  202 ( 1 ) 54 - 60   2000年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

It is well known that it is difficult to induce an immunotolerance with allogeneic skin transplantation. We attempted to find the immunosuppressive protocol for prolonging skin allograft rejection by using interleukin-16 because IL-16 is considered one of the natural ligands to CD4 molecules. First we examined whether synergistic immunosuppressive effects of recombinant IL-16 plus anti-CD4 mAbs are induced in mixed lymphocyte reaction (MLR). Next we used IL-16-cDNA-transfected OSC-20 (human oral squamous cell carcinoma cell line) as an in vitro model of the epidermal keratinocyte equivalent and examined whether this transfectant could inhibit the activation of allogeneic T cells. Our data indicated that IL-16 clearly inhibited human MLR and that IL-16 increased synergistically the immunosuppressive effect of anti-CD4 mAb. We also used IL-16 transfectant and this produced more than 50 ng/ml of IL-16 in the supernatant by which human MLR was significantly inhibited. Furthermore, this transfectant also inhibited the activation of allogeneic lymphocytes stimulated directly with transfectant cells. These results indicated that the IL-16-producing allogeneic skin graft might have a local immunosuppressive action that would prolong graft survival. (C) 2000 Academic Press.

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  DOI
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  PubMed

Molecular cloning of rat NK target structure - The possibility of CD44 involvement in NK cell-mediated lysis

K Kanki, T Torigoe, Hirai, I, H Sahara, K Kamiguchi, Y Tamura, A Yagihashi, N Sato

MICROBIOLOGY AND IMMUNOLOGY ( CENTER ACADEMIC PUBL JAPAN )  44 ( 12 ) 1051 - 1061   2000年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

The nature of target molecules of natural killer (NK) cell-mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor-associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H-ras oncogene, 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell-mediated lysis against W31 and W14, Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44, Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti-rat CD44 mAb OX-50 recognize the same protein of W31 cell lysates with an 86 kDa molecular size. CD44 was suggested to be a target structure of NK cell-mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up-regulation of target cell susceptibility to NK cell-mediated lysis, Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.

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