SHIRAKAWA Toshiro(Graduate School of Science, Technology and Innovation / Department of Science, Technology and Innovation)

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Affiliation

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Published Papers

SHIRAKAWA Toshiro

Graduate School of Science, Technology and Innovation / Department of Science, Technology and Innovation	Professor
School of Medicine / Faculty of Health Sciences
Graduate School of Medicine / Faculty of Medical Sciences
Graduate School of Health Sciences / Faculty of Health Sciences

Researcher Information

Research Keyword

Cancer Immunology

prostate

vaccine

urogenital cancer

oral vaccine

gene therapy

Research Areas

Life sciences / Tumor diagnostics and therapeutics

Research activity information

Award

Jun. 2017 日本ビフィズス菌センター/腸内細菌学会, 第21回腸内細菌学会最優秀発表賞, Anti-tumor immune responses induced by oral cancer vaccine using recombinant Bifidobacterium displaying Wilms’ tumor 1 protein
辰巳真帆, KITAGAWA KOICHI, 五ノ井玲菜, 斉藤大樹, 橋井佳子, 片山高嶺, SHIRAKAWA TOSHIRO
Japan society

Jun. 2008 日本遺伝子治療学会, 第13回JSGT学会賞, 学会発表演題
白川利朗

Aug. 2006 (財)クリタ水･環境科学振興財団, クリタ水・環境科学振興財団研究助成金, 命にかかわる下痢性感染症予防のための水質汚染モニタリング法の開発
SHIRAKAWA TOSHIRO

2004 前立腺研究財団, 前立腺研究財団優秀研究課題受賞, 腫瘍特異的増殖型アデノウイルスおよびキャリアー細胞を用いた前立腺癌に対する遺伝子治療法の開発
SHIRAKAWA Toshiro

Paper

Metagenome and Resistome Analysis of Beta-Lactam-Resistant Bacteria Isolated from River Waters in Surabaya, Indonesia.
Ryohei Nomoto, Kayo Osawa, Shohiro Kinoshita, Koichi Kitagawa, Noriko Nakanishi, Rosantia Sarassari, Dadik Raharjo, Masato Fujisawa, Kuntaman Kuntaman, Toshiro Shirakawa
Antimicrobial agents are administered to humans and livestock, and bacterial antimicrobial resistance (AMR) and antimicrobial agents are released into the environment. In this study, to investigate the trend of AMR in humans, livestock, and the environment, we performed a metagenomic analysis of multidrug-resistant bacteria with CHROMagar ESBL in environmental river water samples, which were collected using syringe filter units from waters near hospitals, downtown areas, residential areas, and water treatment plants in Surabaya, Indonesia. Our results showed that Acinetobacter, Pseudomonas, Aeromonas, Enterobacter, Escherichia, and Klebsiella grew in CHROMagar ESBL; they were most frequently detected in water samples from rivers surrounding hospitals contaminated with various AMR genes (ARGs) in high levels. These results identified bacteria as ARG reservoirs and revealed that hospitals could be sources for various ARGs disseminated into the environment. In conclusion, this study details a novel metagenomic analysis of collected bacteria in environmental water samples using a syringe filter unit for an AMR epidemiological study based on the One Health approach.
Jan. 2024, Microorganisms, 12(1) (1), English, International magazine
[Refereed]
Scientific journal

An oral cancer vaccine using Bifidobacterium vector augments combination of anti-PD-1 and anti-CTLA-4 antibodies in mouse renal cell carcinoma model.
Hideto Ueki, Koichi Kitagawa, Mako Kato, Shihoko Yanase, Yasuyoshi Okamura, Yukari Bando, Takuto Hara, Tomoaki Terakawa, Junya Furukawa, Yuzo Nakano, Masato Fujisawa, Toshiro Shirakawa
Recently, immune checkpoint inhibitor (ICI) based combination therapies, including anti-PD-1 antibody, nivolumab with anti-CTLA-4 antibody, and ipilimumab have become the primary treatment option for metastatic or unresectable renal cell carcinoma (RCC). However, despite the combination of two ICIs, 60-70% of patients are still resistant to first-line cancer immunotherapy. In the present study, undertook combination immunotherapy for RCC using an oral cancer vaccine (Bifidobacterium longum displaying WT1 tumor associated antigen (B. longum 420)) with anti-PD-1 and anti-CTLA-4 antibodies in a mouse syngeneic model of RCC to explore possible synergistic effects. We found that B. longum 420 significantly improved the survival of mice bearing RCC tumors treated by anti-PD-1 and anti-CTLA-4 antibodies compared to the mice treated by the antibodies alone. This result suggests that B. longum 420 oral cancer vaccine as an adjunct to ICIs could provide a novel treatment option for RCC patients. Our microbiome analysis revealed that the proportion of Lactobacilli was significantly increased by B. longum 420. Although the detailed mechanism of action is unknown, it is possible that microbiome alteration by B. longum 420 enhances the efficacy of the ICIs.
Jun. 2023, Scientific reports, 13(1) (1), 9994 - 9994, English, International magazine
Scientific journal
Link to Kobe Univ. Repository (Kernel)

Enhanced antitumor activity of a novel, oral, helper epitope-containing WT1 protein vaccine in a model of murine leukemia.
Hikaru Minagawa, Yoshiko Hashii, Hiroko Nakajima, Fumihiro Fujiki, Soyoko Morimoto, Jun Nakata, Toshiro Shirakawa, Takane Katayama, Akihiro Tsuboi, Keiichi Ozono
BACKGROUND: A Wilms' tumor 1 (WT1) oral vaccine, Bifidobacterium longum (B. longum) 420, in which the bacterium is used as a vector for WT1 protein, triggers immune responses through cellular immunity consisting of cytotoxic T lymphocytes (CTLs) and other immunocompetent cells (e.g., helper T cells). We developed a novel, oral, helper epitope-containing WT1 protein vaccine (B. longum 2656) to examine whether or not B. longum 420/2656 combination further accelerates the CD4+ T cell help-enhanced antitumor activity in a model of murine leukemia. METHODS: C1498-murine WT1-a genetically-engineered, murine leukemia cell line to express murine WT1-was used as tumor cell. Female C57BL/6 J mice were allocated to the B. longum 420, 2656, and 420/2656 combination groups. The day of subcutaneous inoculation of tumor cells was considered as day 0, and successful engraftment was verified on day 7. The oral administration of the vaccine by gavage was initiated on day 8. Tumor volume, the frequency and phenotypes of WT1-specific CTLs in CD8+ T cells in peripheral blood (PB) and tumor-infiltrating lymphocytes (TILs), as well as the proportion of interferon-gamma (INF-γ)-producing CD3+CD4+ T cells pulsed with WT135-52 peptide in splenocytes and TILs were determined. RESULTS: Tumor volume was significantly smaller (p < 0.01) in the B. longum 420/2656 combination group than in the B. longum 420 group on day 24. WT1-specific CTL frequency in CD8+ T cells in PB was significantly greater in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 (p < 0.05) and 6 (p < 0.01). The proportion of WT1-specific, effector memory CTLs in PB increased significantly in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 and 6 (p < 0.05 each). WT1-specific CTL frequency in intratumoral CD8+ T cells and the proportion of IFN-γ-producing CD3+CD4+ T cells in intratumoral CD4+ T cells increased significantly (p < 0.05 each) in the B. longum 420/2656 combination group than in the 420 group. CONCLUSIONS: B. longum 420/2656 combination further accelerated antitumor activity that relies on WT1-specific CTLs in the tumor compared with B. longum 420.
Feb. 2023, BMC cancer, 23(1) (1), 167 - 167, English, International magazine
Scientific journal

A recombinant adenovirus vector containing the synNotch receptor gene for the treatment of triple-negative breast cancer.
Ruhan A, Naoto Kunimura, Shoko Tominaga, Erika Hirata, Shunya Nishioka, Misato Uesugi, Rion Yamazaki, Hideto Ueki, Koichi Kitagawa, Masato Fujisawa, Toshiro Shirakawa
Triple-negative breast cancer (TNBC) is known as the most difficult molecular subtype of breast cancer to treat. Recent studies revealed that cancer stem cells (CSCs) play a critical role in TNBC recurrence and metastasis. In this study, we developed a recombinant replication-deficient adenoviral vector (Ad-CD44-N-HIF-3α4), which contains a gene encoding a synthetic Notch (synNotch) receptor composed of the extracellular domain of CD44 (CD44-ECD) and the hypoxia-inducible factor (HIF)-3α4 connected by the Notch core regulatory region. CD44 is a transmembrane glycoprotein and known as a CSC marker in breast cancer and other malignancies. HIF-3α4 is a dominant-negative regulator of HIF-1α and HIF-2α and inhibits hypoxia-inducing effect. Both CD44 and HIF signals contribute cancer stemness and maintaining CSCs in breast cancer. The CD44-ECD in the synNotch receptor acts as the CD44 decoy receptor, and after a ligand such as a hyaluronic acid binds to the CD44-ECD, HIF-3α4 is released from the Notch core domain. We performed an in vivo study using a mouse xenograft model of MDA-MB-231, a highly invasive TNBC cell, and confirmed the significant antitumor activity of the intratumoral injections of Ad-CD44-N-HIF3α4. Our findings in this study warrant the further development of Ad-CD44-N-HIF3α4 for the treatment of patients with TNBC.
2023, Frontiers in oncology, 13, 1147668 - 1147668, English, International magazine
Scientific journal
Link to Kobe Univ. Repository (Kernel)

Editorial: Targeting tumor vasculature to enhance cancer immunotherapy.
Jieying Yang, Li Fu, Toshiro Shirakawa, Tong Xiang
2023, Frontiers in oncology, 13, 1199811 - 1199811, English, International magazine

An oral WT1 protein vaccine composed of WT1-anchored, genetically engineered Bifidobacterium longum allows for intestinal immunity in mice with acute myeloid leukemia.
Natsuki Nakagawa, Yoshiko Hashii, Hisako Kayama, Ryu Okumura, Hiroko Nakajima, Hikaru Minagawa, Soyoko Morimoto, Fumihiro Fujiki, Jun Nakata, Toshiro Shirakawa, Takane Katayama, Kiyoshi Takeda, Akihiro Tsuboi, Keiichi Ozono
Wilms' tumor 1 (WT1) is a promising tumor-associated antigen for cancer immunotherapy. We developed an oral protein vaccine platform composed of WT1-anchored, genetically engineered Bifidobacterium longum (B. longum) and conducted an in vivo study in mice to examine its anticancer activity. Mice were orally treated with phosphate-buffered saline, wild-type B. longum105-A, B. longum 2012 displaying only galacto-N-biose/lacto-N-biose I-binding protein (GLBP), and WT1 protein- and GLBP-expressing B. longum 420. Tumor size reduced significantly in the B. longum 420 group than in the B. longum 105-A and 2012 groups (P < 0.00 l each), indicating B. longum 420's antitumor activity via WT1-specific immune responses. CD8+ T cells played a major role in the antitumor activity of B. longum 420. The proportion of CD103+CD11b+CD11c+ dendritic cells (DCs) increased in the Peyer's patches (PPs) from mice in the B. longum 420 group, indicating the definite activation of DCs. In the PPs, the number and proportion of CD8+ T cells capable of producing interferon-gamma were significantly greater in the B. longum 420 group than in the B. longum 2012 group (P < 0.05 or < 0.01). The production of WT1-specific IgG antibody was significantly higher in the B. longum 420 group than in the 2012 group (P < 0.05). The B. longum 420 group showed the most intense intratumoral infiltration of CD4+ and CD8+ T cells primed by activated DCs in the PPs of mice in the B. longum 420 group. Our findings provide insights into a novel, intestinal bacterium-based, cancer immunotherapy through intestinal immunity.
Jun. 2022, Cancer immunology, immunotherapy : CII, English, International magazine
Scientific journal

Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia.
Minori Takaichi, Kayo Osawa, Ryohei Nomoto, Noriko Nakanishi, Masanori Kameoka, Makiko Miura, Katsumi Shigemura, Shohiro Kinoshita, Koichi Kitagawa, Atsushi Uda, Takayuki Miyara, Ni Made Mertaniasih, Usman Hadi, Dadik Raharjo, Ratna Yulistiani, Masato Fujisawa, Kuntaman Kuntaman, Toshiro Shirakawa
The increase in antibiotic resistance in non-typhoidal Salmonella enterica (NTS) has been confirmed in Indonesia by this study. We confirmed the virulence genes and antimicrobial susceptibilities of clinical NTS (n = 50) isolated from chicken meat in Indonesia and also detected antimicrobial resistance genes. Of 50 strains, 30 (60%) were non-susceptible to nalidixic acid (NA) and all of them had amino acid mutations in gyrA. Among 27 tetracycline (TC) non-susceptible strains, 22 (81.5%) had tetA and/or tetB. The non-susceptibility rates to ampicillin, gentamicin or kanamycin were lower than that of NA or TC, but the prevalence of blaTEM or aadA was high. Non-susceptible strains showed a high prevalence of virulence genes compared with the susceptible strains (tcfA, p = 0.014; cdtB, p < 0.001; sfbA, p < 0.001; fimA, p = 0.002). S. Schwarzengrund was the most prevalent serotype (23 strains, 46%) and the most frequently detected as multi-antimicrobial resistant. The prevalence of virulence genes in S. Schwarzengrund was significantly higher than other serotypes in hlyE (p = 0.011) and phoP/Q (p = 0.011) in addition to the genes above. In conclusion, NTS strains isolated from Indonesian chicken had a high resistance to antibiotics and many virulence factors. In particular, S. Schwarzengrund strains were most frequently detected as multi-antimicrobial resistant and had a high prevalence of virulence genes.
May 2022, Pathogens (Basel, Switzerland), 11(5) (5), English, International magazine
Scientific journal

Expressions of PD-L1 and Nectin-4 in urothelial cancer patients treated with pembrolizumab.
H Ueki, N Hinata, K Kitagawa, T Hara, T Terakawa, J Furukawa, K Harada, Y Nakano, M Komatsu, M Fujisawa, T Shirakawa
OBJECTIVES: Recently, the standard of care for advanced urothelial cancer (UC) has been changed by developing immune-checkpoint inhibitors (ICIs). However, its response rate is limited to 20-30%. The identification of biomarkers to predict the therapeutic effects of ICIs is urgently needed. The present study explored the association between immunohistochemical biomarkers and clinical outcomes in UC patients treated with pembrolizumab. PATIENTS AND METHODS: A total of 85 patients with UC who received pembrolizumab after chemotherapy from January 2018 to May 2020 were retrospectively reviewed. Tumor tissues were obtained for immunohistochemical study from 47 out of 85 patients. The protein expressions of PD-L1, WT1, Nectin-4, CD4, CD8, Foxp3, and CD68 in tumor cells and/or tumor infiltrating lymphocytes were immunohistochemically examined. The associations between protein expressions and overall survival (OS), progression-free survival (PFS), and disease control rate (DCR) were statistically analyzed. RESULTS: Patients with positive PD-L1 in tumor cells showed significantly worse OS (Log-rank test: HR 5.146, p = 0.001, Cox regression analysis: HR 4.331, p = 0.014) and PFS (Log-rank test: HR 3.31. p = 0.022), along with significantly lower DCR (14.3%) compared to the PD-L1 negative patients (67.5%). In addition, patients with strong expression of Nectin-4 in tumor cells showed significantly higher DCR (100%) than the other patients (50%). CONCLUSION: PD-L1 expression in tumor cells was associated with poor prognosis (OS and PFS) and low DCR. Interestingly, the strong expression of Nectin-4 was correlated with high DCR. PD-L1 and Nectin-4 expression in tumor cells could be prognostic biomarkers useful for pembrolizumab in patients with advanced UC.
Oct. 2021, Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico, English, International magazine
Scientific journal

Analysis of the upstream genetic structures of the ISEcp1-blaCTX-M transposition units in Escherichia coli isolates carrying blaCTX-M obtained from the Indonesian and Vietnamese communities.
Fikri S Widyatama, Nobuyoshi Yagi, Rosantia Sarassari, Toshiro Shirakawa, Danh Tuyen Le, Mai Huong Thi Bui, Kuntaman Kuntaman, Itaru Hirai
Extended spectrum β-lactamase (ESBL)-producing Escherichia coli have been found in healthy individuals in Indonesia and Vietnam. The ISEcp1-blaCTX-M transposition unit of ESBL-producing bacterial isolates has been considered responsible for the production of CTX-M type ESBL and it is important for the dissemination of blaCTX-M . This study aimed to characterize the upstream genetic structure (UGS) of E. coli isolates possessing blaCTX-M-1 group and/or blaCTX-M-9 group genes obtained from healthy individuals in Indonesia and Vietnam. A total of 501 CTX-M type ESBL-producing E. coli isolates possessing blaCTX-M-1 group and/or blaCTX-M-9 group genes were obtained from healthy individuals of the two countries in 2018. The UGSs of the ISEcp1-blaCTX-M transposition unit of the 501 ESBL-producing E. coli isolates were amplified by barcode-adaptor-ligation-mediated PCR and analyzed using the Nanopore sequencer. The obtained sequence information was used to classify the UGSs of the ISEcp1-blaCTX-M transposition unit. From the 501 ESBL-producing E. coli isolates, 502 UGSs were obtained, which were classified into 85 UGS types based on the sequence. ISEcp1 of 359 (71.5%) of the 502 UGSs was disrupted by gene insertion, and ISEcp1-blaCTX-M transposition unit of most (87.1%) of the determined UGSs was confirmed as plasmidic. Only 6 (7.1%) of the 85 UGS types were common to both countries. Our results indicated that many different UGSs of ISEcp1-blaCTX-M transposition units were detected in Indonesia and Vietnam; hence, we suggest that structurally different kinds of plasmids harboring blaCTX-M were separately distributed in the two countries.
Sep. 2021, Microbiology and immunology, 65(12) (12), 542 - 550, English, International magazine
Scientific journal

An oral cancer vaccine using a Bifidobacterium vector suppresses tumor growth in a syngeneic mouse bladder cancer model.
Koichi Kitagawa, Maho Tatsumi, Mako Kato, Shota Komai, Hazuki Doi, Yoshiko Hashii, Takane Katayama, Masato Fujisawa, Toshiro Shirakawa
Cancer immunotherapy using immune-checkpoint inhibitors (ICIs) such as PD-1/PD-L1 inhibitors has been well established for various types of cancer. Monotherapy with ICIs, however, can achieve a durable response in only a subset of patients. There is a great unmet need for the ICI-resistant-tumors. Since patients who respond to ICIs should have preexisting antitumor T cell response, combining ICIs with cancer vaccines that forcibly induce an antitumor T cell response is a reasonable strategy. However, the preferred administration sequence of the combination of ICIs and cancer vaccines is unknown. In this study, we demonstrated that combining an oral WT1 cancer vaccine using a Bifidobacterium vector and following anti-PD-1 antibody treatment eliminated tumor growth in a syngeneic mouse model of bladder cancer. This vaccine induced T cell responses specific to multiple WT1 epitopes through the gut immune system. Moreover, in a tumor model poorly responsive to an initial anti-PD-1 antibody, this vaccine alone significantly inhibited the tumor growth, whereas combination with continuous anti-PD-1 antibody could not inhibit the tumor growth. These results suggest that this oral cancer vaccine alone or as an adjunct to anti-PD-1 antibody could provide a novel treatment option for patients with advanced urothelial cancer including bladder cancer.
Sep. 2021, Molecular therapy oncolytics, 22, 592 - 603, English, International magazine
Scientific journal
Link to Kobe Univ. Repository (Kernel)

Occurrence of Carriage of Multidrug Resistant Enterobacteriaceae among Pregnant Women in the Primary Health Center and Hospital Setting in Surabaya, Indonesia.
Siti Rochmanah Oktaviani Sulikah, Miratul Hasanah, Wahyu Setyarini, Hari Parathon, Koichi Kitagawa, Noriko Nakanishi, Ryohei Nomoto, Kayo Osawa, Shohiro Kinoshita, Itaru Hirai, Toshiro Shirakawa, Kuntaman Kuntaman
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.
Aug. 2021, Microbial drug resistance (Larchmont, N.Y.), 28(1) (1), 48 - 55, English, International magazine
Scientific journal

Pembrolizumab投与後7ヵ月目にirAEとして一型糖尿病、消化管壊死を来たした転移性膀胱癌の1例
松山直幹, 大西篤史, 原琢人, 千葉公嗣, 松下経, 古川順也, 原田健一, 石村武志, 重村克巳, 日向信之, 中野雄造, 白川利朗, 藤澤正人
泌尿器科紀要刊行会, Jun. 2021, 泌尿器科紀要, 67(6) (6), 259 - 259, Japanese

Differential effects of chromosome and plasmid blaCTX-M-15 genes on antibiotic susceptibilities in extended-spectrum beta-lactamase-producing Escherichia coli isolates from patients with urinary tract infection.
Young-Min Yang, Kayo Osawa, Koichi Kitagawa, Samiko Hosoya, Reo Onishi, Aya Ishii, Toshiro Shirakawa, Itaru Hirai, Kuntaman Kuntaman, Hiroshi Tanimoto, Katsumi Shigemura, Masato Fujisawa
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.
Jun. 2021, International journal of urology : official journal of the Japanese Urological Association, 28(6) (6), 623 - 628, English, International magazine
Scientific journal

Cross-Resistance and the Mechanisms of Cephalosporin-Resistant Bacteria in Urinary Tract Infections Isolated in Indonesia.
Aya Ishii, Katsumi Shigemura, Koichi Kitagawa, Mizuki Harada, Yuki Kan, Fuka Hayashi, Kayo Osawa, K Kuntaman, Toshiro Shirakawa, Masato Fujisawa
Urinary tract infection (UTI) by antibiotic-resistant strains has become increasingly problematic, with trends that differ from country to country. This study examined cross-resistance and the mechanisms of cephalosporin resistance in UTI-causative bacteria isolated in Indonesia. Antibiotic susceptibility tests based on Clinical Laboratory Standards Institute (CLSI) standards were done for UTI-causative strains (n = 50) isolated from patients in Indonesia in 2015-2016 and showed resistance against the third-generation cephalosporin. Mechanistic studies were carried out to confirm the presence of extended-spectrum β-lactamase (ESBL) genes, carbapenemase-related genes, the fosA3 gene related to fosfomycin resistance, and mutations of quinolone-resistance-related genes. Isolated UTI-causative bacteria included Escherichia coli (64.0%), Pseudomonas aeruginosa (16.0%), Klebsiella pneumoniae (10.0%), and others (10.0%). These strains showed 96.0% susceptibility to amikacin, 76.0% to fosfomycin, 90.0% to imipenem, 28.0% to levofloxacin, 92.0% to meropenem, and 74.0% to tazobactam/piperacillin. ESBL was produced by 68.0% of these strains. Mechanistic studies found no strains with carbapenemase genes but 6.0% of strains had the fosA3 gene. Seventy-two % of the strains had mutations in the gyrA gene and 74.0% in the parC gene. Most E. coli strains (87.5%) had Ser-83 → Leu and Asp-87 → Asn in gyrA and 93.8% of E. coli had Ser-80 → Ile in parC. There were significant correlations among mutations in gyrA and parC, and fosA3 gene detection (P < 0.05), respectively. To our knowledge, this is the first mechanistic study of antibiotic-cross-resistant UTI-causative bacteria in Indonesia. Further studies with a longer period of observation are necessary, especially for changes in carbapenem resistance without carbapenemase-related genes.
May 2021, Current microbiology, 78(5) (5), 1771 - 1777, English, International magazine
Scientific journal

インドネシアにおける鶏肉由来のキノロン耐性non-typhoidal Salmonella entericaの遺伝子解析
高市果希, 大澤佳代, 重村克巳, 北川孝一, 木下承晧, 亀岡正典, 楠木まり, 宮良高維, 藤沢正人, 白川利朗
(一社)日本感染症学会, Apr. 2021, 感染症学雑誌, 95(臨増) (臨増), 197 - 197, Japanese

インドネシアにおけるヒト及び環境由来のESBL産生Escherichia coliの分布調査
坂本夏那, 大澤佳代, 重村克巳, 北川孝一, 木下承晧, 亀岡正典, 楠木まり, 宮良高維, 藤沢正人, 白川利朗
(一社)日本感染症学会, Apr. 2021, 感染症学雑誌, 95(臨増) (臨増), 241 - 241, Japanese

Genetic analysis of ESBL-producing Klebsiella pneumoniae isolated from UTI patients in Indonesia.
Saya Yamasaki, Katsumi Shigemura, Kayo Osawa, Koichi Kitagawa, Aya Ishii, K Kuntaman, Toshiro Shirakawa, Takayuki Miyara, Masato Fujisawa
INTRODUCTION: Extended spectrum beta-lactamase (ESBL)-producing Klebsiellapneumoniae is a serious concern for nosocomial infection and the emergence rate in Indonesia is higher than that in developed countries. The purpose of this study was to investigate the genetic characteristics of ESBL-producing K. pneumoniae isolated from UTI patients in Indonesia. MATERIALS AND METHODS: We collected K. pneumoniae resistant to ceftazidime or cefotaxime isolated from UTI patients in Dr. Soetomo's Academic Hospital in Surabaya, Indonesia in 2015. Ninety-four strains were identified as ESBL-producing bacteria by confirmation tests. The isolates were investigated by antimicrobial susceptibility testing with 20 drugs and ESBL gene detection, plasmid replicon typing and virulence genes as hypermucoviscous (HMV) strains were tested by the string test. RESULTS: High rates of resistance to ciprofloxacin (86.2%), tetracycline (80.9%) and nalidixic acid (78.7%) were observed. CTX-M-15 was the most common ESBL gene (89.4%), 33 of which also carried SHV-type ESBL. IncF was the most prevalent plasmid replicon typing (47.6%). Sixteen (17.0%) strains were judged as HMV, all of which had rmpA and more than half of which had fimH, uge, and wab. IncL/M was the most common replicon plasmid in the HMV strains, and the difference in the positive rate was statistically significant (p = 0.0024). CONCLUSION: This study showed the high prevalence of multiple-drug resistant and predominately CTX-M-15-positive ESBL-producing K. pneumoniae in Indonesia. There was a correlation between IncL/M and the HMV phenotype in this study. As such hypervirulent strains continue to emerge, studying their dissemination with resistance determinants is an urgent priority.
Jan. 2021, Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 27(1) (1), 55 - 61, English, International magazine
Scientific journal

インドネシアの食肉から分離された薬剤耐性Salmonella entericaの遺伝子解析
高市果希, 大澤佳代, 重村克巳, 北川孝一, 木下承晧, 楠木まり, 宮良高維, Dadik Raharjo, Kuntaman Kuntaman, 亀岡正典, 藤澤正人, 白川利朗
(一社)日本臨床微生物学会, Dec. 2020, 日本臨床微生物学会雑誌, 31(Suppl.1) (Suppl.1), 201 - 201, Japanese

Combination of rAd-p53 in situ gene therapy and anti-PD-1 antibody immunotherapy induced anti-tumor activity in mouse syngeneic urogenital cancer models.
Naoto Kunimura, Koichi Kitagawa, Ryota Sako, Keita Narikiyo, Shoko Tominaga, Diosdado S Bautista, Wei Xu, Masato Fujisawa, Toshiro Shirakawa
In this study we undertook a novel combination therapy using rAd-p53 in situ gene therapy and immunotherapy with immune checkpoint inhibitor (ICI) anti-PD-1 antibody for urogenital cancers. Three mouse syngeneic tumor cell lines, TRAMP-C2 (prostate cancer derived from C57BL/6 mice), MBT-2 (bladder cancer derived from C3H mice) and Renca (kidney cancer derived from BALB/c mice) were used in this study. The highest coxsackie and adenovirus receptor (CAR) mRNA expression was observed in TRAMP-C2 cells, followed by Renca and then MBT-2 cells. Consistent with the CAR expressions, rAd-p53 at 160 multiplicity of infection (MOI) significantly inhibited the cell proliferation of TRAMP-C2 and Renca cells, but not MBT-2 cells. In in vivo experiments, the combination of intratumoral injections of rAd-p53 (1 × 109 plaque-forming units) every other day and intraperitoneal injections of anti-mouse PD-1 antibody (200 μg) twice a week suppressed tumor growth and prolonged survival compared to rAd-p53 or anti-PD-1 antibody monotherapy in both the TRAMP-C2 and Renca models. Our results encourage the clinical development of combination therapy comprised of in situ gene therapy with rAd-p53 and immunotherapy with an ICI anti-PD-1 antibody for urogenital cancers.
Oct. 2020, Scientific reports, 10(1) (1), 17464 - 17464, English, International magazine
Scientific journal
Link to Kobe Univ. Repository (Kernel)

インドネシア尿路感染症患者より検出されたCTX-M-15型ESBL産生E.coliの染色体性もしくはプラスミド性における薬剤感受性の比較
細谷砂美子, 重村克巳, 宮良高維, 北川孝一, 中西典子, 木下承晧, 大澤佳代, Kuntaman Kuntaman, 白川利朗, 藤澤正人
(公社)日本化学療法学会, Sep. 2020, 日本化学療法学会雑誌, 68(Suppl.A) (Suppl.A), 346 - 346, Japanese

Can ultrasound irradiation be a therapeutic option for prostate cancer?
Noriaki Maeshige, Koichi Kitagawa, Saya Yamasaki, Aya Ishii, Toshiro Shirakawa, Yong-Ming Yang, Shian-Ying Sung, Kuan-Chou Chen, Zhi-Min Yuan, Katsumi Shigemura, Masato Fujisawa
BACKGROUND: Focal therapies for prostate cancer (PC) can reduce adverse events and do not lead to androgen-independent progression. Ultrasound could be used for cancer treatments if the repetition frequency is fitted to the purpose. We investigated the possible therapeutic effect of ultrasound irradiation on PC cells. MATERIALS AND METHODS: We irradiated two PC cell lines, androgen-dependent LNCaP and -independent PC-3 with ultrasound (3.0 W/cm2 , 3 MHz, irradiation time rate: 20%) for 2 minutes for 1 day or 3 consecutive days at a repetition frequency of 1, 10, or 100 Hz in vitro. Cell proliferation and apoptosis were determined after irradiation. RESULTS: Cell proliferation of PC-3 was significantly inhibited after 1 day (P < .0001) and 3 days (P < .0001) of 10 Hz ultrasound irradiation, and that of LNCaP after 1 day (P < .0001) and 3 days (P < .0001) of irradiation. LNCaP was more sensitive to ultrasound at both lower and higher cell density but PC-3 was only sensitive at a lower cell density (P < .01). Irradiation with 10 Hz ultrasound-induced significantly more PC-3 apoptotic cells than control (1 day, P = .0137; 3 days, P = .0386) rather than irradiation with 1 Hz. Apoptosis via caspase-3 was induced at 10 Hz in 1-day (P < .05) irradiation in both cell lines. CONCLUSIONS: Ultrasound irradiation with even 1 day of 10 Hz significantly inhibited cell proliferation in both LNCaP and PC-3, especially by the remarkable induction of apoptosis in vitro. Our study indicated that ultrasound irradiation can be a therapeutic option for PC and further studies in vivo will be undertaken.
Sep. 2020, The Prostate, 80(12) (12), 986 - 992, English, International magazine
Scientific journal

Molecular detection of a new pathotype enteroaggregative haemorrhagic Escherichia coli (EAHEC) in Indonesia, 2015.
Wahyu Setyarini, Dadik Raharjo, Radita Yuniar Arizandy, Zakaria Pamoengkas, Subijanto Marto Sudarmo, Alpha Fardah Athiyyah, Toshiro Shirakawa
Enteroaggregative haemorrhagic Escherichia coli (E. Coli, EAHEC) has been identified as the agent responsible for one of the largest outbreaks of gastroenteritis and Haemolytic-uremic syndrome (HUS) that is transmitted through food in Germany in 2011. The hypervirulent pathotype has a unique combination of two pathogens namely enterohemorrhagic E.coli strain (EHEC) which produces shiga/verotoxin and enteroaggregative E.coli toxins (EAEC) which produces toxins similar to ST and hemolysin. The toxin produced by the EAHEC strain is a hybrid pathotype that combines the virulence potential of the EAEC and EHEC strains that will damage the microcirculation, cause vasculitis and other toxic effects. The purpose of this study was to determine the percentage of samples infected with enteroaggregative hemorrhagic E. coli bacteria (EAHEC) in pediatric diarrhea patients at DR. Soetomo Hospital, Surabaya, Indonesia, 2015. This study used PCR (Polymerase Chain Reaction) method to detect enteroaggregative E. coli strains (CVD432 and aaic genes) and enterohemorrhagic E.coli (eae gene).The results showed that 33 out of 40 (82,5%) stool samples examined were detected enteroaggregative E. coli (EAEC), 4 out of 40 (10%) enterohemorrhagic E. coli (EHEC) and 3 out of 40 (7,5%) enteroaggregative haemorrhagic E. coli bacteria (EAHEC), which caused diarrhea in pediatric diarrhea patients at Dr. Soetomo General Hospital. The unique combination of genomic features of the Surabaya outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype enteroaggregative haemorrhagic E. coli (EAHEC). It is expected that development of specific primer design and sequencing are needed to continue in this research.
Jul. 2020, Infectious disease reports, 12(Suppl 1) (Suppl 1), 8745 - 8745, English, International magazine
Scientific journal

Characterization of CTX-M-type-extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolated from Indonesian undergraduate medical students of a university in Surabaya, Indonesia.
Sarassari Rosantia, Takuya Higa, Nobuyoshi Yagi, Toshiro Tokunaga, Seina Higa, Yasuaki Yakabi, Toshiro Shirakawa, Kuntaman Kuntaman, Itaru Hirai
Enterobacteriaceae isolates producing CTX-M-type extended-spectrum β-lactamase (ESBL) has been found in hospitalized patients and healthy individuals in communities of the Southeast Asian countries. Medical students might have more risk of ESBL-producing Enterobacteriaceae contagion, because medical students who belong to communities have direct and indirect contacts with workers and patients in healthcare facilities. The aim of this study was to collect information for evaluation of the potential risk of ESBL-producing Enterobacteriaceae contagion in Indonesian undergraduate medical students by characterizing genotypic properties of Escherichia coli isolates-producing CTX-M-type ESBL. A total 141 fecal samples collected from 207 medical students of a university in Surabaya, Indonesia were subjected to PCR, XbaI and S1 nuclease-pulsed-field gel electrophoresis (PFGE), Southern blotting, and sequencing analysis. Eighty-two ESBL-producing Enterobacteriaceae, including 75 E. coli and 7 Klebsiella pneumoniae were isolated from 79 (56.0%) students. Among 75 ESBL-producing E. coli, blaCTX-M-15 was the most prevalent type (44.0%). Although XbaI-PFGE results showed genetic background of the E. coli isolates producing CTX-M-type ESBL were diverse, five clonal spread cases of certain E. coli producing CTX-M-type ESBL isolates were observed among the medical students. Our results suggested that ESBL-producing Enterobacteriaceae might be circulating among the medical students through contaminated environment such as in a university or communities they belonged.
Jun. 2020, Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 26(6) (6), 575 - 581, English, International magazine
[Refereed]
Scientific journal

Difference of Phenotype and Genotype Between Human and Environmental: Isolated Vibrio cholerae in Surabaya, Indonesia.
Kayo Osawa, Katsumi Shigemura, Koichi Kitagawa, K Kuntaman, Ni Made Mertaniasih, Wahyu Setyarini, Dita Arizandy, Dadik Rahadjo, Ro Osawa, Toshiro Shirakawa, Masato Fujisawa
Cholera due to Vibrio cholerae has been spreading worldwide, although the reports focusing on Indonesian V. cholerae are few. In this study, in order to investigate how V. cholerae transmitted to human from environment. We extended an epidemiological report that had investigated the genotype of V. cholerae isolated from human pediatric samples and environmental samples. We examined 44 strains of V. cholerae isolated from pediatric diarrhea patients and the environment such as shrimps or oysters collected in three adjacent towns in Surabaya, Indonesia. Susceptibilities were examined for 11 antibiotics. Serotype O1 or O139 genes and pathogenic genes including cholera toxin were detected. Multi-locus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC)-PCR were also performed to determine genetic diversity of those isolates. Serotype O1 was seen in 17 strains (38.6%) with all pathogenic genes among 44 isolates. Other isolates were non-O1/non-O139 V. cholerae. Regarding antibiotic susceptibilities, those isolates from environmental samples showed resistance to ampicillin (11.4%), streptomycin (9.1%) and nalidixic acid (2.3%) but those isolates from pediatric stools showed no resistance to those 3 kinds of antibiotics. MLST revealed sequence type (ST) 69 in 17 strains (38.6%), ST198 in 3 strains (6.8%) and non-types in 24 strains (54.5%). All the ST69 strains were classified to O1 type with more than 95% similarity by ERIC-PCR, including all 6 (13.6%) isolates from environmental samples with resistance to streptomycin. In conclusion, V. cholerae O1 ST69 strains has been clonally spreading in Surabaya, exhibiting pathogenic factors and antibiotic resistance to streptomycin, especially in the isolates from environment.
Jun. 2020, Indian journal of microbiology, 60(2) (2), 230 - 238, English, International magazine
[Refereed]
Scientific journal

Oncolytic effect of Midkine promoter-based conditionally replicating adenoviruses expressing EGFR siRNA in head and neck squamous cancer cell line T891
Natsumi Uehara, Naoki Otsuki, Mie Kubo, Junko Kitamoto, Yasutaka Kojima, Masanori Teshima, Hirotaka Shinomiya, Toshiro Shirakawa, Ken-ichi Nibu
Feb. 2020, Cancer Reports
[Refereed]
Scientific journal
Link to Kobe Univ. Repository (Kernel)

Overexpression of SOCS3 mediated by adenovirus vector in mouse and human castration-resistant prostate cancer cells increases the sensitivity to NK cells in vitro and in vivo.
Tomomi Yoneda, Naoto Kunimura, Koichi Kitagawa, Yuka Fukui, Hiroki Saito, Keita Narikiyo, Motoki Ishiko, Naoki Otsuki, Ken-Ichi Nibu, Masato Fujisawa, Satoshi Serada, Tetsuji Naka, Toshiro Shirakawa
Prostate cancer is one of the most common cancers in men. The overactivation of IL-6/JAK/STAT3 signaling and silencing of SOCS3 are frequently observed in prostate cancer. In the present study we undertook to develop Ad-SOCS3 gene therapy for the treatment of prostate cancer and also investigated whether Ad-SOCS3 increased sensitivity to NK cells. We demonstrated that Ad-SOCS3 could significantly inhibit growth of castration-resistant prostate cancer (CRPC) cell lines expressing pSTAT3, DU-145 (at 10, 20, and 40 MOI), and TRAMP-C2 (at 40 MOI), but not the PC-3 CRPC cell line with the STAT3 gene deleted. Ad-SOCS3 (40 MOI) could suppress IL-6 production in DU-145 cells and PD-L1 expression induced by IFN-γ in TRAMP-C2 cells, and increased the NK cell sensitivity of both TRAMP-C2 and DU-145 cells. In the DU-145 mouse xenograft tumor model, intratumoral injections (twice/week for 3 weeks) of 1 × 108 pfu of Ad-SOCS3 significantly inhibited tumor growth and combining the Ad-SOCS3 treatment with intratumoral injections (once/week for 2 weeks) of 1 × 107 human NK cells showed the highest tumor growth inhibitory effect. These results suggested that a combination of Ad-SOCS3 gene therapy and NK cell immunotherapy could be a powerful treatment option for advanced CRPC overexpressing pSTAT3.
Nov. 2019, Cancer gene therapy, 26(11-12) (11-12), 388 - 399, English, International magazine
[Refereed]
Scientific journal
Link to Kobe Univ. Repository (Kernel)

Prevalence of Quinolone Resistance of Extended-Spectrum β-Lactamase-Producing Escherichia coli with ST131-fimH30 in a City Hospital in Hyogo, Japan.
Masazumi Teramae, Kayo Osawa, Katsumi Shigemura, Koichi Kitagawa, Toshiro Shirakawa, Masato Fujisawa, Takayuki Miyara
Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates are known to tolerate superior quinolone antimicrobials compared with other antibacterial agents. Among the clones belonging to sequence type (ST) 131 by multilocus sequence typing, the involvement of the H30-Rx subclone has been reported worldwide with various fimH genes encoding type 1 pili. We investigated 83 isolates of ESBL-producing E. coli and performed antimicrobial susceptibility test, CH (fumC/fimH) ST131 by typing the specific PCR. Moreover, mutation analysis of genes involved in quinolone antibiotic resistance (gyrA and parC) and ESBL genotypes were determined. As a result, 54 of 83 isolates (65.1%) of CH40-30 clones corresponding to ST131-fimH30 were detected, and all were resistant to levofloxacin. Mutations associated with this resistance were common, and included S83L and D87N of gyrA and S80I and E84V of parC. Subclone analysis revealed a high proportion of fimH30-non-Rx (40 isolates, 74.1%). Each subclone was characterized by ESBL genotype, and the CTX-M-15 type was mainly seen for fimH30-Rx, with the CTX-M-14 type or CTX-M-27 type seen for fimH30-non-Rx. This study suggests that an increase in ESBL-producing quinolone-resistant E. coli in a city hospital in Hyogo, Japan, was caused by the spread of subclones belonging to fimH30-non-Rx of ST131.
Oct. 2019, International journal of molecular sciences, 20(20) (20), English, International magazine
[Refereed]
Scientific journal
Link to Kobe Univ. Repository (Kernel)

Possible correlation of sonic hedgehog signaling with epithelial-mesenchymal transition in muscle-invasive bladder cancer progression.
Koichi Kitagawa, Katsumi Shigemura, Shian-Ying Sung, Kuan-Chou Chen, Chao-Ching Huang, Yi-Te Chiang, Ming-Che Liu, Tzu-Wen Huang, Fukashi Yamamichi, Toshiro Shirakawa, Masato Fujisawa
PURPOSE: To investigate the role of sonic hedgehog (Shh) signaling and epithelial-mesenchymal transition (EMT) in bladder cancer progression and invasion. METHODS: We cultured three bladder cancer cell lines, muscle-invasive T24 and 5637, and non-muscle-invasive KK47, in the presence of a recombinant-Shh (r-Shh) protein or cyclopamine, a Shh signaling inhibitor, to investigate proliferation and expression of EMT markers. Wound-healing assays and transwell assay were performed to evaluate cell invasion and migration. Mice were then inoculated with bladder cancer cells and treated with cyclopamine. Mouse tumor samples were stained for Shh signaling and EMT markers. RESULTS: R-Shh protein enhanced cell proliferation, whereas cyclopamine significantly suppressed cell proliferation, especially in invasive cancer (5637 and T24) (p < 0.05). R-Shh protein promoted EMT, suppressed E-cadherin and enhanced N-cadherin and vimentin and Gli1, an Shh downstream molecule, while cyclopamine blocked EMT, especially in 5637 and T24. Cyclopamine also inhibited cell invasion and migration in vitro. In the animal study, intraperitoneal injection of cyclopamine significantly suppressed tumor growth in 5637 and T24 in mice (p = 0.01 and p = 0.004, respectively) and slightly suppressing KK47 tumor growth (p = 0.298). Significant cyclopamine-induced suppression of Gli1 in 5637 and T24 mouse tumors (both p = 0.03) was seen, suggesting that muscle-invasive bladder cancer may be more dependent on Shh signaling than non-muscle-invasive bladder cancer. CONCLUSIONS: Shh signaling and EMT were especially enhanced in muscle-invasive bladder cancer progression and invasion, and suppressed by the inhibition of Shh signaling.
Sep. 2019, Journal of cancer research and clinical oncology, 145(9) (9), 2261 - 2271, English, International magazine
[Refereed]

Preclinical Development of a WT1 Oral Cancer Vaccine Using a Bacterial Vector to Treat Castration-Resistant Prostate Cancer.
Koichi Kitagawa, Reina Gonoi, Maho Tatsumi, Masahide Kadowaki, Takane Katayama, Yoshiko Hashii, Masato Fujisawa, Toshiro Shirakawa
Previously, we constructed a recombinant Bifidobacterium longum displaying a partial mouse Wilms' tumor 1 (WT1) protein (B. longum 420) as an oral cancer vaccine using a bacterial vector and demonstrated that oral administration of B. longum 420 significantly inhibited tumor growth compared with the Db126 WT1 peptide vaccine in the TRAMP-C2, mouse castration-resistant prostate cancer (CRPC) syngeneic tumor model. The present study demonstrated that oral administration of 1.0×109 colony-forming units of B. longum 420 induced significantly higher cytotoxicity against TRAMP-C2 cells than intraperitoneal injection of 100 μg of Db126, and the in vivo antitumor activity of B. longum 420 in the TRAMP-C2 tumor model could be augmented by intraperitoneal injections of 250 μg of anti-PD-1 antibody. For the clinical development, we produced the B440 pharmaceutical formulation, which is lyophilized powder of inactivated B. longum 440 displaying the partially modified human WT1 protein. We confirmed that B. longum 440 could induce cellular immunity specific to multiple WT1 epitopes. In a preclinical dosage study, B440 significantly inhibited growth of the TRAMP-C2 tumors compared with that of the control groups (PBS and B. longum not expressing WT1) at all dosages (1, 5, and 10 mg/body of B440). These mouse doses were considered to correspond with practical oral administration doses of 0.2, 1, and 2 g/body for humans. Taken together, these results suggest that the B440 WT1 oral cancer vaccine can be developed as a novel oral immuno-oncology drug to treat CRPC as a monotherapy or as an adjunct to immune checkpoint inhibitors.
May 2019, Molecular cancer therapeutics, 18(5) (5), 980 - 990, English, International magazine
[Refereed]

Stenotrophomonas maltophilia菌血症による死亡の危険因子
西本健人, 重村克巳, 大澤佳代, 北川孝一, 白川利朗, 宮良高維, 中野雄造
(一社)日本感染症学会, Mar. 2019, 感染症学雑誌, 93(臨増) (臨増), 343 - 343, Japanese

Bacteremia complicating urinary tract infection by Pseudomonas aeruginosa: Mortality risk factors.
Koichi Kitagawa, Katsumi Shigemura, Fukashi Yamamichi, Kayo Osawa, Atsushi Uda, Chihiro Koike, Issei Tokimatsu, Toshiro Shirakawa, Takayuki Miyara, Masato Fujisawa
OBJECTIVES: To examine the clinical risk factors for death within 30 days of diagnosis of Pseudomonas aeruginosa-causing bacteremia after a urinary tract infection. METHODS: A total of 62 patients with Pseudomonas aeruginosa isolated from both urine and blood at the same episode from January 2009 to December 2016 were enrolled in the present study. We retrospectively investigated clinical risk factors for death by comparison between surviving patients and those who died within 30 days after diagnosis of P. aeruginosa bacteremia. The comparison for risk factors for bacteremia-related death included 31 categories, such as age, laboratory data, underlying diseases, clinical history, history of surgery, care in the intensive care unit, P. aeruginosa susceptibility to the antibiotics used at the time of bacteremia diagnosis and consultation with urological department. RESULTS: The study included 48 men and 14 women aged 71.3 ± 10.4 years. Nine patients (14.5%) died of P. aeruginosa bacteremia. Statistical analysis showed that non-survivors had significantly lower albumin levels than survivors (2.07 ± 0.62 vs 2.62 ± 0.65; P = 0.023). The non-survivors had significantly higher rates of ventilator use, history of heart disease, septic shock and lower rates of consultation with urological departments after diagnosis (P < 0.05). CONCLUSIONS: Patients with bacteremia complicating urinary infection by P. aeruginosa have a low death rate. Earlier intervention by urologists might improve patients' outcome. Lower albumin levels, ventilator use, history of heart disease and septic shock are factors associated with higher mortality rate.
Mar. 2019, International journal of urology : official journal of the Japanese Urological Association, 26(3) (3), 358 - 362, English, International magazine
[Refereed]

Acquisition of antimicrobial-resistant variants in repeated infections caused by Pseudomonas aeruginosa revealed by whole genome sequencing.
Noriko Nakanishi, Ryohei Nomoto, Kanako Sato, Chihiro Koike, Mari Kusuki, Tatsuya Nakamura, Katsumi Shigemura, Toshiro Shirakawa, Masato Fujisawa, Issei Tokimatsu, Kayo Osawa
Pseudomonas aeruginosa, responsible for serious nosocomial-acquired infections, possesses intrinsic antibiotic resistance mechanisms and commonly exhibits multidrug resistance. Here, we report the evolving resistance profiles of strains isolated from the sputum of a patient being treated for repeated P. aeruginosa infections following cancer resection. Whole genome sequencing of six isolates obtained over a 2-month period revealed two key single nucleotide polymorphisms in the mexR and gyrB genes that affected efflux pump expression and antimicrobial resistance.
Feb. 2019, Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 25(2) (2), 154 - 156, English, International magazine
[Refereed]

Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study.
Alpha Fardah Athiyyah, Katsumi Shigemura, Koichi Kitagawa, Nazara Agustina, Andy Darma, Reza Ranuh, Dadik Raharjo, Toshiro Shirakawa, Masato Fujisawa, Subijanto Marto Sudarmo
Background: The objective of this study was to investigate the clinical manifestation of norovirus infection between norovirus genogroup and severity of acute diarrhea in pediatric patients at the Dr. Soetomo Hospital, Surabaya, Indonesia. Methods: This cross-sectional study involved 31 participants aged 1-60 months admitted to the hospital with acute diarrhea from April 2012 to March 2013. Norovirus genogroups (GI and II) were identified from patient stool using reverse transcription polymerase chain reaction (RT-PCR). Severity was measured using the Ruuska and Vesikari scoring system. Results: In total, 94 stool samples were obtained, of which 31 (19%) were norovirus positive. Norovirus GI was found in one sample with mild diarrhea. Norovirus GII was found in 30 samples (96.8%); one sample with mild diarrhea (3.3%), 20 samples with moderate diarrhea (66.7%), and nine samples with severe diarrhea (30%). Conclusion: Norovirus GII was the most prevalent cause of acute diarrhea and 30% of the cases manifested as severe diarrhea.
2019, F1000Research, 8, 2130 - 2130, English, International magazine
[Refereed]
Scientific journal
Link to Kobe Univ. Repository (Kernel)

Molecular characteristics of carbapenem-resistant Pseudomonas aeruginosa isolated from urine in Hyogo, Japan.
Kayo Osawa, Katsumi Shigemura, Koichi Kitagawa, Teruo Fukuda, Ayaka Takasaka, Sakie Wakabayashi, Kanako Sato, Fukashi Yamamichi, Toshiro Shirakawa, Masato Fujisawa
OBJECTIVES: To investigate the molecular characteristics and epidemiology of metallo-β-lactamase-producing Pseudomonas aeruginosa from urine of urinary tract infection patients in Hyogo Prefecture, Japan. METHODS: Carbapenem-resistant P. aeruginosa isolated from the urine of 21 urinary tract infection patients in three general hospitals in Hyogo Prefecture (Japan) were collected between 2007 and 2014. Their antibiotic susceptibilities, metallo-β-lactamase screening test, metallo-β-lactamase gene sequencing, multilocus sequence typing and repetitive-sequence-based polymerase chain reaction were determined for epidemiological analyses to investigate the genetic characteristics. RESULTS: Out of 21 isolates, 13 (61.9%) were positive for metallo-β-lactamase. There were 11 (52.4%) isolates with IMP-1 in them, one (4.5%) isolate with IMP-7 and one (4.5%) isolate with VIM-1. Metallo-β-lactamase-positive isolates were mainly identified as ST235, and metallo-β-lactamase-negative isolates were STs 357, 277, 234, 439 and 639. Repetitive-sequence-based polymerase chain reaction showed metallo-β-lactamase-positive isolates were grouped in eight clusters, and ST235 isolates with IMP-1 from three hospitals belonging to the identical group I, the other ST235 isolates with IMP-7 and VIM-1 were from two hospitals belonging to group II. CONCLUSIONS: Metallo-β-lactamase-positive P. aeruginosa of ST235 isolates with IPM-1 were mainly identified from the urine of urinary tract infection patients in Hyogo, Japan. A ST235 isolate with VIM-1 was found for the first time. Further investigation is necessary to follow the spread of metallo-β-lactamase-positive isolates.
Jan. 2019, International journal of urology : official journal of the Japanese Urological Association, 26(1) (1), 127 - 133, English, International magazine
[Refereed]

Clinical Risk Factors for Death Caused by Extended-Spectrum Beta-Lactamase: Producing Bacteria.
Yoshie Mita, Katsumi Shigemura, Kayo Osawa, Koichi Kitagawa, Tomohiro Kotaki, Toshiro Shirakawa, Takayuki Miyara, Masato Fujisawa
OBJECTIVES: Extended-spectrum beta-lactamase (ESBL)-producing bacteria often causes bacteremia, leading serious outcomes. In this study, we conducted a retrospective analysis to identify the risk factors associated with death by bacteremia of ESBL-producing bacteria. METHODS: Patients with bacteremia by ESBL-producing bacteria were retrospectively collected in Kobe University Hospital, Japan, between January 2011 and December 2015. Potential risk factors for death caused by ESBL-bacteremia were analyzed for patients' outcome (recovery or death) by univariate and multivariate analysis. RESULTS: A total of 101 patients (64 male and 37 female) were recruited. The most frequently detected ESBL-producing bacteria were Escherichia coli (91 cases; 90.1%), followed by Klebsiella pneumoniae (8 cases; 7.9%). Most frequently used antibiotics after the detection of bacteremia was meropenem (66.3%; 67/101) followed by cefmetazole (51.5%; 52/101). Univariate analysis showed a significantly positive correlation with mortality in ICU admission (p < 0.001), circulatory diseases (p = 0.022), shock (p = 0.044), and respirator requirement (p = 0.002). Multivariate analysis showed ICU admission remained significant risk factor for mortality (p = 0.0192). CONCLUSIONS: We showed ICU admission was significantly correlated with death from bacteremia by ESBL-producing bacteria. These factors should be monitored to estimate severity of ESBL causing-bacteremia for better patients' outcomes.
2019, Urologia internationalis, 102(2) (2), 205 - 211, English, International magazine
[Refereed]

Molecular characteristics of carbapenem-resistant Pseudomonas aeruginosa isolated from urine in Hyogo, Japan.
Kayo Osawa, Katsumi Shigemura, Koichi Kitagawa, Teruo Fukuda, Ayaka Takasaka, Sakie Wakabayashi, Kanako Sato, Fukashi Yamamichi, Toshiro Shirakawa, Masato Fujisawa
OBJECTIVES: To investigate the molecular characteristics and epidemiology of metallo-β-lactamase-producing Pseudomonas aeruginosa from urine of urinary tract infection patients in Hyogo Prefecture, Japan. METHODS: Carbapenem-resistant P. aeruginosa isolated from the urine of 21 urinary tract infection patients in three general hospitals in Hyogo Prefecture (Japan) were collected between 2007 and 2014. Their antibiotic susceptibilities, metallo-β-lactamase screening test, metallo-β-lactamase gene sequencing, multilocus sequence typing and repetitive-sequence-based polymerase chain reaction were determined for epidemiological analyses to investigate the genetic characteristics. RESULTS: Out of 21 isolates, 13 (61.9%) were positive for metallo-β-lactamase. There were 11 (52.4%) isolates with IMP-1 in them, one (4.5%) isolate with IMP-7 and one (4.5%) isolate with VIM-1. Metallo-β-lactamase-positive isolates were mainly identified as ST235, and metallo-β-lactamase-negative isolates were STs 357, 277, 234, 439 and 639. Repetitive-sequence-based polymerase chain reaction showed metallo-β-lactamase-positive isolates were grouped in eight clusters, and ST235 isolates with IMP-1 from three hospitals belonging to the identical group I, the other ST235 isolates with IMP-7 and VIM-1 were from two hospitals belonging to group II. CONCLUSIONS: Metallo-β-lactamase-positive P. aeruginosa of ST235 isolates with IPM-1 were mainly identified from the urine of urinary tract infection patients in Hyogo, Japan. A ST235 isolate with VIM-1 was found for the first time. Further investigation is necessary to follow the spread of metallo-β-lactamase-positive isolates.
Jan. 2019, International journal of urology : official journal of the Japanese Urological Association, 26(1) (1), 127 - 133, English, International magazine
[Refereed]
Scientific journal

Occurrence and characterization of carbapenem-resistant Gram-negative bacilli: A collaborative study of antibiotic-resistant bacteria between Indonesia and Japan.
Kuntaman Kuntaman, Katsumi Shigemura, Kayo Osawa, Koichi Kitagawa, Koharu Sato, Naoki Yamada, Kento Nishimoto, Fukashi Yamamichi, Dadik Rahardjo, Usman Hadi, Ni Made Mertaniasih, Shohiro Kinoshita, Masato Fujisawa, Toshiro Shirakawa
OBJECTIVES: To explore the occurrence and characterization of carbapenemase-producing pathogens among carbapenem-resistant Gram-negative bacilli isolated from hospitalized patients with urinary tract infection in Indonesia. METHODS: This was a study promoted by the Japanese-Indonesian collaborative research program in the Japan Initiative for Global Research Network on Infectious Diseases. Bacterial pathogens were prospectively isolated from urine specimens of hospitalized urinary tract infection patients at Dr. Soetomo Hospital (Surabaya, Indonesia). All Gram-negative bacteria resistant to third-generation cephalosporin or carbapenem were included in this study. Carbapenemase genes were investigated for phenotype and genotype. RESULTS: In total, 1082 Gram-negative bacilli were isolated, of which 116 strains were resistant to imipenem or meropenem (carbapenem-resistant Gram-negative bacilli), and 22 strains were carbapenemase-producing Gram-negative bacilli. Carbapenemase-producing Gram-negative bacilli consisted of Acinetobacter baumannii (n = 4), Pseudomonas aeruginosa (n = 4), Klebsiella pneumoniae (n = 5), Providencia rettgeri (n = 4) and five others. The carbapenemase-producing Gram-negative bacilli included NDM-1 (n = 18, 81.8%, in Enterobacteriaceae and Acinetobacter spp.) and IMP-7 (n = 4, 18.2%, all in P. aeruginosa). Among carbapenem-resistant Gram-negative bacilli, all four P. aeruginosa were sensitive to colistin, and all six Acinetobacter spp. were sensitive to minocycline, colistin and tigecycline. Of those patients harboring carbapenemase-producing Gram-negative bacilli, 12 (54.5%) were seriously ill at the time of admission, with longer hospital stays and three deaths (13.6% mortality rate). CONCLUSIONS: Urinary tract infection-causing carbapenem-resistant Gram-negative bacilli are widely disseminated in Indonesia. The NDM-1 phenotype seems to be dominant, and it can be treated with colistin and tigecycline in most cases. Most patients harboring carbapenemase-producing Gram-negative bacilli are seriously ill, have a bad prognosis, with a longer hospital stay and a significant mortality rate.
Nov. 2018, International journal of urology : official journal of the Japanese Urological Association, 25(11) (11), 966 - 972, English, International magazine
[Refereed]
Scientific journal

Adenovirus-mediated transfer of HPV 16 E6/E7 antisense RNA combined with cisplatin inhibits cellular growth and induces apoptosis in HPV-positive head and neck cancer cells.
Yasutaka Kojima, Naoki Otsuki, Mie Kubo, Junko Kitamoto, Eri Takata, Hiroki Saito, Kyoko Kosaka, Naoya Morishita, Natsumi Uehara, Toshiro Shirakawa, Ken-Ich Nibu
Human papillomavirus (HPV) infection has been identified as an etiologic factor of head and neck cancers (HNCs). We explored the potential use of antisense HPV RNA transcripts for gene therapy and its effect in combination with cisplatin (CDDP) for HPV-positive HNCs. We introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into UM-SCC-47 cells harboring HPV 16 and YCU-T892 cells that were HPV-negative using a recombinant adenoviral vector, Ad-E6/E7-AS. We then analyzed the effects of the introduction of Ad-E7-AS on cell and tumor growth and the synergistic effect with CDDP in vitro and in vivo. After infection of Ad-E6/E7-AS, the cellular growth of UM-SCC-47 cells were suppressed, but not that of YCU-T892 cells. E7 protein expression was suppressed, and p53 and pRb protein expression increased after infection of Ad-E7-AS. Cell growth and tumorigenicity were greatly suppressed in combination with CDDP compared with Ad-E7-AS or CDDP treatment alone in vitro. Ad-E7-AS combined with CDDP treatment significantly reduced the volumes of established subcutaneous tumors. Transfection with HPV 16 E7 antisense RNA combined with CDDP treatment might be a potentially useful approach to the therapy of HPV 16-positive HNC.
Oct. 2018, Cancer gene therapy, 25(9-10) (9-10), 274 - 283, English, International magazine
[Refereed]

Determination of the antimicrobial susceptibility and molecular profile of clarithromycin resistance in the Mycobacterium abscessus complex in Japan by variable number tandem repeat analysis.
Mari Kusuki, Kayo Osawa, Kentaro Arikawa, Miho Tamura, Katsumi Shigemura, Toshiro Shirakawa, Tatsuya Nakamura, Yuji Nakamachi, Masato Fujisawa, Jun Saegusa, Issei Tokimatsu
Mycobacterium abscessus complex, including three subspecies-M. abscessus, M. massiliense, and M. bolletii-is resistant to a variety of antibiotics so limited treatment options are available. The susceptibility of these subspecies to antimicrobial agents depends in particular on the erm(41) sequevar and rrl mutations in the 23S rRNA, which are potentially related to clarithromycin (CLR) resistance. The purpose of this study was to carry out identification and molecular characterization of these subspecies based on variable number of tandem repeats (VNTR) analysis. Twenty-four M. abscessus complex strains were identified as M. abscessus and M. massiliense and these subspecies could be discriminated between based on their resistance to CLR, as determined by truncation or mutation of erm(41) or mutation of rrl, as illustrated by their VNTR patterns. In conclusion, we confirmed that the CLR susceptibility profiles could be differentiated according to the subspecies of M. abscessus complex strains by their VNTR patterns.
Jul. 2018, Diagnostic microbiology and infectious disease, 91(3) (3), 256 - 259, English, International magazine
[Refereed]
Scientific journal

Improved bacterial identification directly from urine samples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Koichi Kitagawa, Katsumi Shigemura, Ken-Ichiro Onuma, Masako Nishida, Mayu Fujiwara, Saori Kobayashi, Mika Yamasaki, Tatsuya Nakamura, Fukashi Yamamichi, Toshiro Shirakawa, Issei Tokimatsu, Masato Fujisawa
Background: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) contributes to rapid identification of pathogens in the clinic but has not yet performed especially well for Gram-positive cocci (GPC) causing complicated urinary tract infection (UTI). The goal of this study was to investigate the possible clinical use of MALDI-TOF MS as a rapid method for bacterial identification directly from urine in complicated UTI. Methods: MALDI-TOF MS was applied to urine samples gathered from 142 suspected complicated UTI patients in 2015-2017. We modified the standard procedure (Method 1) for sample preparation by adding an initial 10 minutes of ultrasonication followed by centrifugation at 500 g for 1 minutes to remove debris such as epithelial cells and leukocytes from the urine (Method 2). Results: In 133 urine culture-positive bacteria, the rate of corresponded with urine culture in GPC by MALDI-TOF MS in urine with standard sample preparation (Method 1) was 16.7%, but the modified sample preparation (Method 2) significantly improved that rate to 52.2% (P=.045). Method 2 also improved the identification accuracy for Gram-negative rods (GNR) from 77.1% to 94.2% (P=.022). The modified Method 2 significantly improved the average MALDI score from 1.408±0.153 to 2.166±0.045 (P=.000) for GPC and slightly improved the score from 2.107±0.061 to 2.164±0.037 for GNR. Conclusion: The modified sample preparation for MALDI-TOF MS can improve identification accuracy for complicated UTI causative bacteria. This simple modification offers a rapid and accurate routine diagnosis for UTI, and may possibly be a substitute for urine cultures.
John Wiley and Sons Inc., Mar. 2018, Journal of Clinical Laboratory Analysis, 32(3) (3), English
[Refereed]
Scientific journal

Pseudomonas aeruginosaの連続感染における抗菌耐性機序の分析(Analysis of antimicrobial resistance mechanism in successive infections of Pseudomonas aeruginosa)
中西典子, 野本竜平, 佐藤加奈子, 小池千裕, 楠木まり, 中村竜也, 重村克巳, 白川利朗, 時松一成, 大澤佳代
日本細菌学会, Feb. 2018, 日本細菌学雑誌, 73(1) (1), 141 - 141, English

Antitumor effect of oral cancer vaccine with Bifidobacterium delivering WT1 protein to gut immune system is superior to WT1 peptide vaccine
Toshiro Shirakawa, Koichi Kitagawa
Despite the revolutionary progress of immune checkpoint inhibitors (CPIs) for cancer immunotherapy, CPIs are effective only in a subset of patients. Combining CPIs and cancer vaccines to achieve better clinical outcomes is a reasonable approach since CPI enhances cancer vaccine-induced tumor-associated antigen (TAA) specific CTL. Among the various TAAs so far identified, WT1 protein is one of the most promising TAAs as a cancer vaccine target. Until now clinical trials of WT1 vaccine have demonstrated only modest clinical efficacy. These WT1 vaccines were based on peptides or dendritic cells (DCs), and there was no oral cancer vaccine. Recently, we developed a WT1 oral cancer vaccine using a recombinant Bifidobacterium displaying WT1 protein, which can efficiently deliver WT1 protein to the gut immune system, and we demonstrated that this oral cancer vaccine had a significant anti-tumor effect in a C1498-WT1 murine leukemia syngeneic tumor model. The WT1 protein displayed in this vaccine consists of about 70% of the WT1 amino acid sequence including multiple known CD4 and CD8 T-cell epitopes of WT1. In this commentary, we introduce our recent data indicating the superior anti-tumor effect of a WT1 oral cancer vaccine delivering WT1 protein to the gut immune system compared to a peptide vaccine.
Taylor and Francis Inc., Jan. 2018, Human Vaccines and Immunotherapeutics, 14(1) (1), 159 - 162, English
[Refereed]
Scientific journal

International comparison of causative bacteria and antimicrobial susceptibilities of urinary tract infections between Kobe, Japan, and Surabaya, Indonesia
Koichi Kitagawa, Katsumi Shigemura, Fukashi Yamamichi, Lindawati Alimsardjono, Dadik Rahardjo, Kuntaman Kuntaman, Toshiro Shirakawa, Masato Fujisawa
Variation by country in urinary tract infection (UTI)-causative bacteria is partly due to the differences in the use of antibiotics. We compared their frequencies and antibiotic susceptibilities in the treatment of patients with UTI from 2 cities, Kobe, Japan, and Surabaya, Indonesia. We retrospectively analyzed 1,804 urine samples collected from patients with UTI in 2014 (1,251 collected in 11 months at Kobe University Hospital in Kobe and 544 collected in 2 months at Dr. Soetomo Hospital in Surabaya). Surabaya data were divided into adult and pediatric patients because a substantial number of specimens from pediatric-patients had been collected. The results indicated that Escherichia coli was the most common uropathogen (24.1% in Kobe and 39.3% in Surabaya) and was significantly resistant to ampicillin and substantially to first- and third-generation cephalosporins in Surabaya adults but not in Kobe adults (p < 0.01). Enterococcus faecalis was often isolated in Kobe (14.0%), but not in Surabaya (5.3%). Klebsiella spp. were isolated at a higher rate in Surabaya pediatric patients (20.3%) than in Surabaya adults (13.6%) and Kobe adults (6.6%). The antibiotic susceptibilities of the isolates form Surabaya isolates tended to be lower than the ones from Kobe. Extended-spectrum β-lactamaseproducing Gram-negative bacteria were detected at a significantly higher rate in Surabaya than in Kobe (p < 0.001). These results showed that the antimicrobial resistance patterns of UTI-causative bacteria are highly variable among 2 countries, and the continuous surveillance of trends in antibiotic resistance patterns of uropathogens is necessary for the future revision of antibiotic use.
National Institute of Health, 2018, Japanese Journal of Infectious Diseases, 71(1) (1), 8 - 13, English
[Refereed]
Scientific journal

Wilms'tumor 1抗原発現ビフィズス菌を用いた経口癌ワクチンのマウス前立腺癌に対する抗腫瘍効果の検討
五ノ井玲菜, 北川孝一, 古田拓也, 辰巳真帆, 橋井佳子, 片山高嶺, 白川利朗
生命科学系学会合同年次大会運営事務局, Dec. 2017, 生命科学系学会合同年次大会, 2017年度, [2P - 0893], Japanese

penA, ponA, porB1, and mtrR Mutations and Molecular Epidemiological Typing of Neisseria gonorrhoeae with Decreased Susceptibility to Cephalosporins
Kayo Osawa, Katsumi Shigemura, Yukie Nukata, Koichi Kitagawa, Fukashi Yamamichi, Hiroyuki Yoshida, Toshiro Shirakawa, Soichi Arakawa, Masato Fujisawa
AMER SOC MICROBIOLOGY, Aug. 2017, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 61(8) (8), pii: e01174 - 17., English
[Refereed]
Scientific journal

Combination of p53-DC vaccine and rAd-p53 gene therapy induced CTLs cytotoxic against p53-deleted human prostate cancer cells in vitro
H. Saito, K. Kitagawa, T. Yoneda, Y. Fukui, M. Fujsawa, D. Bautista, T. Shirakawa
Recently, the US FDA approved sipuleucel-T, which is composed of autologous DCs stimulated with a recombinant fusion protein of prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF), as the first immunotherapeutic agent for metastatic castration resistant prostate cancer (mCRPC). However, sipuleucel-T demonstrated only modest efficacy in mCPRC patients. Researchers are now investigating the potential of p53 protein as a tumor-associated antigen (TAA) loaded in DC-based cancer vaccine. Approximately half of all tumors overexpress p53, and up to 20% of prostate cancer cells overexpresses p53. In this study, we evaluated the feasibility of combining p53-DC vaccine and rAd-p53 gene therapy, using the p53-overexpressing and non-expressing prostate cancer cells in vitro. We successfully generated the p53-DC vaccine by culturing autologous DCs infected with rAd-p53. This p53-DC vaccine can differentiate CTLs specifically cytotoxic to p53-overexpressing prostate cancer cells. In addition, rAd-p53 infection can induce overexpression of p53 and thus the cytotoxicity of CTLs differentiated by the p53-DC vaccine in p53 non-expressing prostate cancer cells. These findings suggest that this combination therapy using p53-DC vaccine and rAd-p53 gene therapy together may represent a new paradigm for the treatment of mCRPC.
NATURE PUBLISHING GROUP, Jul. 2017, CANCER GENE THERAPY, 24(7) (7), 289 - 296, English
[Refereed]
Scientific journal

Antibiotic Susceptibilities and Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Stools of Pediatric Diarrhea Patients in Surabaya, Indonesia
Eddy Bagus Wasitou, Katsumi Shigemura, Kayo Osawa, Alpha Fardah, Akiho Kanaida, Dadik Raharjo, K. Kuntaman, Usman Hadi, Sugeng Harijono, Subijanto Marto Sudarmo, Tatsuya Nakamura, Keigo Shibayama, Masato Fujisawa, Toshiro Shirakawa
The purpose of this study was to investigate extended-spectrum beta-lactamase (ES-BL)-producing Escherichia coli isolates from pediatric (aged 0 to 3 years) diarrhea patients in Surabaya, Indonesia, where this kind of survey is rare; our study included assessment of their antibiotic susceptibilides, as well as ESBL typing, multilocus sequence typing (MLST), and diarrheagenic E. coli (DEC)-typing. ESBL-producing E. coli were detected in 18.8% of all the samples. Many ESBL-producing E. coli had significantly lower susceptibility to gentamicin (p < 0.0001) and the quinolones nalidixic acid (p = 0.004) and ciprofloxacin (p < 0.0001) than non-producers. In ESBL-producing E. coli, 84.0% of strains expressed CTX-M-15 alone or in combination with other ESBL types. MLST revealed that 24.0% of ESBL-producers had sequence type 617, all of which expressed the CTX-M-15 gene; we also detected expression of 3 DEC-related genes: 2 enteroaggregative E. coli genes and 1 enteropathogenic E. coli gene. In conclusion, CTX-M-15-type ESBL-producing E. coli ST617 appear to have spread to Indonesia.
NATL INST INFECTIOUS DISEASES, Jul. 2017, JAPANESE JOURNAL OF INFECTIOUS DISEASES, 70(4) (4), 378 - 382, English
[Refereed]
Scientific journal

Development of oral cancer vaccine using recombinant Bifidobacterium displaying Wilms' tumor 1 protein
Koichi Kitagawa, Tsugumi Oda, Hiroki Saito, Ayame Araki, Reina Gonoi, Katsumi Shigemura, Yoshiko Hashii, Takane Katayama, Masato Fujisawa, Toshiro Shirakawa
Several types of vaccine-delivering tumor-associated antigens (TAAs) have been developed in basic and clinical research. Wilms' tumor 1 (WT1), identified as a gene responsible for pediatric renal neoplasm, is one of the most promising TAA for cancer immunotherapy. Peptide and dendritic cell-based WT1 cancer vaccines showed some therapeutic efficacy in clinical and pre-clinical studies but as yet no oral WT1 vaccine can be administrated in a simple and easy way. In the present study, we constructed a novel oral cancer vaccine using a recombinant Bifidobacterium longum displaying WT1 protein. B. longum 420 was orally administered into mice inoculated with WT1-expressing tumor cells for 4 weeks to examine anti-tumor effects. To analyze the WT1-specific cellular immune responses to oral B. longum 420, mice splenocytes were isolated and cytokine production and cytotoxic activities were determined. Oral administrations of B. longum 420 significantly inhibited WT1-expressing tumor growth and prolonged survival in mice. Immunohistochemical study and immunological assays revealed that B. longum 420 substantially induced tumor infiltration of CD4(+)T and CD8(+)T cells, systemic WT1-specific cytokine production, and cytotoxic activity mediated by WT1-epitope specific cytotoxic T lymphocytes, with no apparent adverse effects. Our novel oral cancer vaccine safely induced WT1-specific cellular immunity via activation of the gut mucosal immune system and achieved therapeutic efficacy with several practical advantages over existing non-oral vaccines.
SPRINGER, Jun. 2017, CANCER IMMUNOLOGY IMMUNOTHERAPY, 66(6) (6), 787 - 798, English
[Refereed]
Scientific journal

Prevalence of Antibiotic-resistance Enterobacteriaceae strains Isolated from Chicken Meat at Traditional Markets in Surabaya, Indonesia
R. Yulistiani, D. Praseptiangga, Supyani, Sudibya, D. Raharjo, T. Shirakawa
Antibiotic resistance in bacteria from the family Enterobacteriaceae is an important indicator of the emergence of resistant bacterial strains in the community. This study investigated the prevalence of antibiotic-resistant Enterobacteriaceae isolated from chicken meat sold at traditional markets in Surabaya Indonesia. In all, 203 isolates (43 Salmonella spp., 53 Escherichia coli, 16 Shigella spp., 22 Citrobacter spp., 13 Klebsiella spp, 24 Proteus spp., 15 Yersinia spp., 7 Enterobacter spp., 6 Serratia spp., 3 Edwardsiella spp. were resistant to tetracycline (69.95 %), nalidixid acid (54.19 %), sulfamethoxazole/sulfamethizole (42.36 %), chloramphenicol (12.81%), cefoxitin (6.40 %), gentamicin (5.91 %). Tetracycline was the antimicrobial that showed the highest frequency of resistance among Salmonella, E. coli, Citrobacter, Proteus and Erdwardsiella isolates, and nalidixid acid was second frequency of resistance. Overall, 124 (61.08 %) out of 203 isolates demonstrated multidrug resistance to at least two unrelated antimicrobial agents. The high rate of antimicrobial resistance in bacterial isolates from chicken meat may have major implications for human and animal health with adverse economic implications.
Institute of Physics Publishing, May 2017, IOP Conference Series: Materials Science and Engineering, 193(1) (1), English
[Refereed]
International conference proceedings

Wilms' tumor 1タンパク質発現ビフィズス菌を用いた経口癌ワクチンの抗腫瘍免疫誘導効果に関する検討
北川孝一, 辰巳真帆, 五ノ井玲菜, 斉藤大樹, 橋井佳子, 片山高嶺, 白川利朗
(公財)腸内細菌学会, Apr. 2017, 腸内細菌学雑誌, 31(2) (2), 112 - 112, Japanese

Oral Combination Vaccine, Comprising Bifidobacterium Displaying Hepatitis C Virus Nonstructural Protein 3 and Interferon-alpha, Induces Strong Cellular Immunity Specific to Nonstructural Protein 3 in Mice
Koichi Kitagawa, Chika Omoto, Tsugumi Oda, Ayame Araki, Hiroki Saito, Katsumi Shigemura, Takane Katayama, Hak Hotta, Toshiro Shirakawa
We previously generated an oral hepatitis C virus (HCV) vaccine using Bifidobacterium displaying the HCV nonstructural protein 3 (NS3) polypeptide. NS3-specific cellular immunity is important for viral clearance and recovery from HCV infection. In this study, we enhanced the cellular immune responses induced by our oral HCV vaccine, Bifidobacterium longum 2165 (B. longum 2165), by combining interferon-alpha (IFN-alpha) as an adjuvant with the vaccine in a mouse experimental model. IFN-alpha is a widely used cytokine meeting the standard of care (SOC) for HCV infection and plays various immunoregulatory roles. We treated C57BL/6N mice with B. longum 2165 every other day and/or IFN-alpha twice a week for a month and then analyzed the immune responses using spleen cells. We determined the induction of NS3-specific cellular immunity by cytokine quantification, intracellular cytokine staining, and a cytotoxic T lymphocyte (CTL) assay targeting EL4 tumor cells expressing NS3/4A protein (EL4-NS3/4A). We also treated mice bearing EL4-NS3/4A tumor with the combination therapy in vivo. The results confirmed that the combination therapy of B. longum 2165 and IFN-a induced significantly higher IFN-gamma secretion, higher population of CD4(+)T and CD8(+)T cells secreting IFN-gamma, and higher CTL activity against EL4-NS3/4A cells compared with the control groups of phosphate-buffered saline, B. longum 2165 alone, and IFN-alpha alone (p < 0.05). We also confirmed that the combination therapy strongly enhanced tumor growth inhibitory effects in vivo with no serious adverse effects (p < 0.05). These results suggest that the combination of B. longum 2165 and IFN-alpha could induce a strong cellular immunity specific to NS3 protein as a combination therapy augmenting the current SOC immunotherapy against chronic HCV infection.
MARY ANN LIEBERT, INC, Apr. 2017, VIRAL IMMUNOLOGY, 30(3) (3), 196 - 203, English
[Refereed]
Scientific journal

High Prevalence of Diarrheagenic Escherichia coli among Children with Diarrhea in Kenya
Yoshio Iijimal, Joseph O. Oundo, Takumi Hibino, Suleiman M. Saidi, Atsushi Hinenoya, Kayo Osawa, Toshiro Shirakawa, Ro Osawa, Shinji Yamasaki
Diarrheagenic Escherichia coli (DEC) is an important agent of endemic and epidemic diarrhea worldwide, particularly in developing countries. DEC cannot be differentiated from commensal E. coli on selective media, although there are a few exceptions. Most studies use the colony isolation method, which cannot detect low numbers of DEC, and therefore, these studies might underestimate the incidence of DEC. In the present study, we employed a colony sweep method with real-time PCR targeting virulence genes of 5 categories of DEC; this technique can detect very low numbers of DEC among hundreds of commensal E. coli. DEC was detected in 171 (55.9%) of 306 children with diarrhea in Kenya. The prevalence of DEC in Kenya was notably higher than that (30 in 143, 21.0%) in Indonesia. Occurrences of multiple DEC infection in Kenya were frequent (69 in 306, 23.2%), suggesting that the source of DEC infection may be related to grossly contaminated food and water. In contrast, only 9 (6.0%) of 150 healthy adults in Kenya carried DEC. Considering that healthy adults naturally harbor non-DEC, it is interesting how children exclude DEC but not non-DEC as they grow up. Several mechanisms, such as mucosal immunity and intestinal microbiota, might be involved in the exclusion of DEC.
NATL INST INFECTIOUS DISEASES, Jan. 2017, JAPANESE JOURNAL OF INFECTIOUS DISEASES, 70(1) (1), 80 - 83, English
[Refereed]
Scientific journal

セフェム系抗菌薬低感受性淋菌の遺伝学的解析
額田雪絵, 大澤佳代, Shigemura Katsumi, 吉田弘之, 藤澤正人, 荒川創一, 白川利朗
May 2016, 日本化学療法学会雑誌, 64(Suppl.A) (Suppl.A), 164, Japanese
Research society

カルバペネム耐性腸内細菌科細菌の分離状況
朝比奈桃花, 大澤佳代, Shigemura Katsumi, 吉田弘之, 高羽桂, 時松一成, 藤澤正人, 荒川創一, 白川利朗
May 2016, 日本化学療法学会雑誌, 64(Suppl.A) (Suppl.A), 178, Japanese
Research society

兵庫県内で分離されたメタロβラクタマーゼ産生緑膿菌の解析
高坂綾香, 大澤佳代, Shigemura Katsumi, 山道深, 吉田弘之, 中村竜也, 藤澤正人, 荒川創一, 白川利朗
Mar. 2016, 感染症学雑誌, 90(2号) (2号), 183 - 184, Japanese
Research society

MRSA疫学手法としてもPOT法とrep-PCR法の比較
大澤佳代, Shigemura Katsumi, 吉田弘之, 白川利朗, 藤澤正人, 荒川創一
Mar. 2016, 感染症学雑誌, 90(臨増) (臨増), 345, Japanese
Research society

Association of overexpression of efflux pump genes with antibiotic resistance in Pseudomonas aeruginosa strains clinically isolated from urinary tract infection patients
Katsumi Shigemura, Kayo Osawa, Ayaka Kato, Issei Tokimatsu, Soichi Arakawa, Toshiro Shirakawa, Masato Fujisawa
There are several mechanisms for antibiotic-resistant Pseudomonas aeruginosa. The purpose of this study is to investigate the association between the expression of efflux pump-coding genes and antibiotic resistance in P. aeruginosa causing urinary tract infections (UTIs). We extracted the RNA from 105 clinical strains of P. aeruginosa isolated from UTI patients with full data on antibiotic MICs and assayed real-time quantitative reverse-transcription PCR. We investigated the gene expressions of four resistance nodulation cell division-type multi-drug efflux pump systems (MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY (-OprA)) and the correlation of the MICs of nine antibiotics, risk factors and antibiotic resistance-related genes with expressions of mexB, mexC, mexE and mexY. Multivariate statistical data demonstrated a significant relationship between increased expression of mexB or mexC and complicated UTI (Odds ratio=8.03, P<0.001 and Odds ratio=8.86, P=0.032, respectively). We also found a significant association between the increased expression of mexC and resistance to levofloxacin (LVFX) (Odds ratio=4.48, P=0.035). In conclusion, increased expression of mexC leads to LVFX resistance in P. aeruginosa causing UTI. These results contribute to our knowledge of the efflux pump system and antibiotic resistance.
JAPAN ANTIBIOTICS RESEARCH ASSOC, Sep. 2015, JOURNAL OF ANTIBIOTICS, 68(9) (9), 568 - 572, English
[Refereed]
Scientific journal

BACTERIAL IDENTIFICATION USING ssRA ENCODING TRANSFER-MESSENGER RNA
Kayo Osawa, Katsumi Shigemura, Hiroki Shirai, Ayaka Kato, Yuma Okuya, Takumi Jikimoto, Soichi Arakawa, Masato Fujisawa, Toshiro Shirakawa
Ribosomal DNA (rDNA) sequences are widely used for phylogenetic and bacterial identification. However, rDNA of different species often reveals similar or identical same sequences. This study employed the bacterial stable small RNA (ssrA) gene encoding transfer-messenger RNA (tmRNA) as a tool for identification of Staphylococcus aureus, Enterococcus spp, Pseudomonas spp and Enterobacteriaceae from clinical isolates as representative groups using PCR and species specific primers. The method correctly identified 11 standard strains and 99 clinical isolates. Quantitative PCR revealed a limit of detection of 10(-5)mu g of DNA for S. aureus and Enterococcus spp, and 10(-6)mu g for Pseudomonas spp and Enterobacteriaceae. Further studies with a greater number of bacteria especially from clinical samples will need to be undertaken before this bacterial molecular marker can be applied in a clinical setting.
SOUTHEAST ASIAN MINISTERS EDUC ORGANIZATION, Jul. 2015, SOUTHEAST ASIAN JOURNAL OF TROPICAL MEDICINE AND PUBLIC HEALTH, 46(4) (4), 720 - 727, English
[Refereed]
Scientific journal

A clinical protocol to inhibit the HGF/c-Met pathway for malignant mesothelioma with an intrapleural injection of adenoviruses expressing the NK4 gene
Yuji Tada, Kenzo Hiroshima, Hideaki Shimada, Naoya Morishita, Toshiro Shirakawa, Kunio Matsumoto, Masato Shingyoji, Ikuo Sekine, Koichiro Tatsumi, Masatoshi Tagawa
Background: The hepatocyte growth factor (HGF)/c-Met signal pathway is up-regulated in human mesothelioma and suppression of the HGF/c-Met signaling with a competitive inhibitor, NK4 homologous to HGF in the structure, produced anti-tumor effects to mesothelioma in a preclinical study. Mesothelioma is highly resistant to a number of chemotherapeutic agents but distant metastasis to extra-thoracic organs is relatively infrequent until the late stage. Methods/design: We planned to conduct a clinical study of gene therapy with adenoviruses expressing the NK4 gene (Ad-NK4) to control the local tumor growth. The study is designed to inject Ad-NK4 into the intrapleural cavity with a dose escalation manner from 1010 to 1012 virus particles per patient and to examine safety and possible clinical benefits. The clinical investigation is a first-in-human trial to use the NK4 gene and to block the HGF/c-Met pathway with gene medicine. We conducted in vivo animal experiments to examine the safety level as one of the preclinical studies, and showed that Ad DNA administered in the pleural cavity was detected in many parenchymal organs. Biochemical and pathological analyses showed that liver damages were the major adverse effects with little toxicity to other organs. These studies firstly demonstrated biodistribution and transgene expression after an intrapleural injection of Ad vectors in an animal study, which contrasts with an intravenous injection showing relatively rapid clearance of Ad-NK4. Discussion: The clinical study can also provide information regarding production of NK4 protein and antibody against NK4, and inhibition levels of the HGF/c-Met pathway by detecting dephosphorylation of c-Met in mesothelioma cells. These data will be crucial to judge whether local production of NK4 molecules can be an anti-cancer strategy.
SPRINGER INTERNATIONAL PUBLISHING AG, Jul. 2015, SPRINGERPLUS, 4, 358, English
[Refereed]
Scientific journal

Azithromycin Resistance and Its Mechanism in Neisseria gonorrhoeae Strains in Hyogo, Japan
Katsumi Shigemura, Kayo Osawa, Makiko Miura, Kazushi Tanaka, Soichi Arakawa, Toshiro Shirakawa, Masato Fujisawa
Therapeutic options are limited for Neisseria gonorrhoeae infection, especially for oral drugs. The purpose of this study was to investigate the susceptibility of N. gonorrhoeae to oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains of N. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59 N. gonorrhoeae strains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 mu g/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 mu g/ml or less) (4/22; 18.2%) (P < 0.0001). However, a mutation of codon 40 (Ala -> Asp) in the mtrR gene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P < 0.0001). In N. gonorrhoeae multiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or - intermediate strains, as previously described. In conclusion, a deletion mutation in the mtrR promoter region may be a significant indicator for higher MIC (0.5 mu g/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance in N. gonorrhoeae infection.
AMER SOC MICROBIOLOGY, May 2015, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 59(5) (5), 2695 - 2699, English
[Refereed]
Scientific journal

Molecular Characteristics of Extended-Spectrum beta-Lactamase-Producing Escherichia coli in a University Teaching Hospital
Kayo Osawa, Katsumi Shigemura, Rika Shimizu, Ayaka Kato, Mari Kusuki, Takumi Jikimoto, Tatsuya Nakamura, Hiroyuki Yoshida, Soichi Arakawa, Masato Fujisawa, Toshiro Shirakawa
The prevalence of extended-spectrum beta-lactamases (ESBLs) has been increasing worldwide. Recently, a pandemic clone of Escherichia coli O25:H4, sequence type 131 (ST131), producing ESBL-type CTX-M-15 has been reported as a major problem. In this study, we investigated the molecular characteristics of 72 ESBL-producing E. coli isolates. We detected the ESBL bla(CTX-M) gene and nine virulence factor genes (papC, papEF, fimH, hlyA, iutA, sfa, eaeA, bfpA, and aggR) by PCR and DNA sequencing, plasmid replicon typing, phylogenetic grouping, repetitive-sequence-based PCR (rep-PCR), and multilocus sequence typing. All strains were positive for bla(CTX-M). Twenty-two (30.6%) strains in CTX-M-1 group included 9 (12.5%) of CTX-M-15, 3 (4.2%) in CTX-M-2 group, and 47 (65.3%) strains in CTX-M-9 group. The CTX-M-15-producing E. coli O25:H4 ST131 was derived from phylogenetic group B2 and rep-PCR pattern d. The most prevalent virulence factor was fimH (72 strains; 100%) and the most common replicon type was the IncF type (65 strains; 90.3%). The CTX-M-9 group was significantly associated with the presence of papC and papEF [OR (95% CI)=9.22 (1.32-64.7), p=0.025] or hlyA [OR (95% CI)=5.57 (1.17-26.4), p=0.031]. In conclusion, we confirmed that CTX-M-15-producing E. coli O25:H4 ST131 has emerged in Japan and found significant virulence factors with CTX-M-9 group.
MARY ANN LIEBERT, INC, Apr. 2015, MICROBIAL DRUG RESISTANCE, 21(2) (2), 130 - 139, English
[Refereed]
Scientific journal

Genotyping and clinical factors in pediatric diarrhea caused by rotaviruses: one-year surveillance in Surabaya, Indonesia
Subijanto Marto Sudarmo, Katsumi Shigemura, Alpha Fardah Athiyyah, Kayo Osawa, Oktavian Prasetia Wardana, Andy Darma, Reza Ranuh, Dadik Raharjo, Soichi Arakawa, Masato Fujisawa, Toshiro Shirakawa
Background: Rotavirus infections are a major cause of diarrhea in children in both developed and developing countries. Rotavirus genetics, patient immunity, and environmental factors are thought to be related to the severity of acute diarrhea due to rotavirus in infants and young children. The objective of this study was to provide a correlation between rotavirus genotypes, clinical factors and degree of severity of acute diarrhea in children under 5 years old in Surabaya, Indonesia. Methods: A cross-sectional study was conducted in children aged 1-60 months with acute diarrhea hospitalized in Soetomo Hospital, Surabaya, Indonesia from April to December 2013. Rotavirus in stool specimens was identified by ELISA and genotyping (G-type and P-type) using multiplex reverse transcription PCR. Severity was measured using the Ruuska and Vesikari scoring system. The clinical factors were investigated included patient's age (months), hydration, antibiotic administration, nutritional state, co-bacterial infection and co-viral infection. Results: A total of 88 children met the criteria; 80.7% were aged 6-24 months, watery diarrhea was the most common type (77.3%) and 73.6% of the subjects were co-infected with bacteria, of which pathogenic Escherichia coli was the most common (42.5%). The predominant VP7 genotyping (G-type) was G2 (31.8%) and that of VP4 genotyping (P-type) was P[4] (31.8%). The predominant rotavirus genotype was G2P[4] (19.3%); G1P[4] and G9P[4] were uncommon with a prevalence of 4.5%. There were significant differences between the common genotype and uncommon genotype with respect to the total severity score of diarrhea (p < 0.05). G3, G4 and G9 were significantly correlated with severe diarrhea (p = 0.009) in multivariate analyses and with frequency of diarrhea (> 10 times a day) (p = 0.045) in univariate analyses, but there was no significant correlation between P typing and severity of diarrhea. For combination genotyping of G and P, G2P[4] was significantly correlated with severe diarrhea in multivariate analyses (p = 0.029). Conclusions: There is a correlation between rotavirus genotype and severity of acute diarrhea in children. Genotype G2P[4] has the highest prevalence. G3, G4, G9 and G2P[4] combination genotype were found to be associated with severe diarrhea.
BIOMED CENTRAL LTD, Feb. 2015, GUT PATHOGENS, 7, 3, English
[Refereed]
Scientific journal

[Development of the novel oral vaccine against hepatitis C virus utilizing bifidobacteria]
Shirakawa Toshiro
日本臨床社, Feb. 2015, Nihon Rinsho, 73(2) (2), 239 - 242, English
Scientific journal

Beta-3 adrenergic receptors could be significant factors for overactive bladder-related symptoms
Fukashi Yamamichi, Katsumi Shigemura, Hosny M. Behnsawy, Masuo Yamashita, Toshiro Shirakawa, Masato Fujisawa
The treatment failure often happens in overactive bladder (OAB) partly owing to its unknown pathogenesis. The purpose of this study is to find significant receptors or biological markers for OAB-related symptoms for establishment of potential order-made therapeutic strategies. The overactive bladder symptom scores (OABSS) and international prostate symptom scores (IPSS)/quality of life (QOL) were questioned in all the 18 patients with OAB diagnosis. Their bladder mucosal tissues were taken from the random biopsy of bladder cancer suspected patients without any finding such as inflammation or carcinoma in situ. They were investigated quantitatively by immunohistochemical (IHC) stainings for inflammatory or immune-system (Interleukin (IL)-6 and cyclooxygenase-2 (Cox-2)), Caspase-3 apoptosis markers, angiogenesis (CD-31), epithelial-mesenchymal transition (E-cadherin) and muscarinic receptor (Muscarine-2 (M)-2), adrenergic receptors (ARs) (alpha 1-d (alpha 1-d) and beta-3 (beta-3)). The statistical correlation between the expressions of these 5 markers and 3 receptors and these symptom scores were examined under the comparison between OAB patients and control patients who had urgency score with less than 2 in OABSS. The OABSS and IPSS/QOL was 7.39 +/- 2.69 and 21.2 +/- 6.59/4.33 +/- 1.33, respectively but those of control patients were 2.00 +/- 1.41 and 10.1 +/- 9.52/2.14 +/- 1.46, respectively (P<0.05). Regarding the correlation of those markers' expressions and symptom scores, in OAB patients, OABSS total significantly correlated with beta-3 AR expressions (P=0.0457). IPSS post-voiding significantly correlated with beta-3 AR expressions (P=0.0308) but no significant relationship in control patients (P>0.05). In conclusion, this study demonstrated that beta-3 AR in our tested 8 markers or receptors was correlated strongly with OAB-related symptoms. These data may help elucidate the pathophysiology of OAB and offer possible strategy for its order-made therapies.
E-CENTURY PUBLISHING CORP, 2015, INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL PATHOLOGY, 8(9) (9), 11863 - 11870, English
[Refereed]
Scientific journal

Sonic hedgehog and androgen signaling in tumor and stromal compartments drives epithelial-mesenchymal transition in prostate cancer
Fukashi Yamamichi, Katsumi Shigemura, Hosny M. Behnsawy, Fatma Y. Meligy, Wen-Chin Huang, Xiangyan Li, Kunito Yamanaka, Keisuke Hanioka, Hideaki Miyake, Kazushi Tanaka, Masato Kawabata, Toshiro Shirakawa, Masato Fujisawa
Objective. Sonic hedgehog (Shh) signaling, androgens and epithelial-mesenchymal transition (EMT) are related to prostate cancer (PCa) progression. The aim of this study was to investigate how Shh and androgen [dihydrotestosterone (DHT)] signaling act in prostate epithelial and stromal compartments and whether this signaling pathway drives EMT and promotes PCa progression. Material and methods. LNCaP, normal prostate fibroblast (NPF) and cancer-associated prostate fibroblast (CPF) cells were studied with DHT and/or the Shh signaling inhibitor cyclopamine. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the expressions of a potential Shh target gene, osteonectin (ON) and EMT-associated markers (E-cadherin, N-cadherin and vimentin). Immunohistochemical studies using PCa prostatectomy samples were performed to assess the expression levels of ON, Gli-1, androgen receptor, Shh, E-cadherin, N-cadherin and vimentin. Results. While DHT enhanced cell proliferation in CPF more than LNCaP or NPF, cyclopamine inhibited cell proliferation enhanced by DHT in CPF. Real-time RT-PCR showed whereas both Shh and DHT induced N-cadherin and vimentin, DHT also induced the expression of osteonectin in LNCaP and cyclopamine blocked these expressions in osteonectin, N-cadherin and vimentin (p = 0.0084, 0.0002 and 0.0373, respectively). Immunohistochemistry showed that high expression of stromal, but, not epithelial, ON was significantly correlated with serum prostate-specific antigen (PSA) (p = 0.031), and high expression of Gli-1 and low expression of stromal ON with PSA recurrence (p = 0.0114 and p = 0.0005, respectively). Conclusions. Shh and androgen signaling in prostate tumor and stromal compartments drives EMT, and thus may play some role in PCa progression. Cyclopamine may be one therapeutic strategy for PCa.
INFORMA HEALTHCARE, Dec. 2014, SCANDINAVIAN JOURNAL OF UROLOGY, 48(6) (6), 523 - 532, English
[Refereed]
Scientific journal

兵庫県におけるアジスロマイシン(AZM)耐性淋菌の分子遺伝学的解析
三浦真希子, Shigemura Katsumi, 大澤佳代, 吉田弘之, 藤原美樹, 澤村暢, 奈須聖子, 荒川創一, 藤澤正人, 白川利朗
(一社)日本性感染症学会, Oct. 2014, 日本性感染症学会誌, 25(2号) (2号), 78 - 78, Japanese
Research society

セフェム系薬剤感受性低下Neisseria gonorrhoeaeの遺伝解析
大澤佳代, Shigemura Katsumi, 額田雪絵, 吉田弘之, 藤原美樹, 奈須聖子, 白川利朗, 藤澤正人, 荒川創一
Oct. 2014, 日本性感染症学会誌, 25(2号) (2号), 78, Japanese
Research society

Comparison between phage-open-reading frame typing and automated repetitive-sequence-based PCR for typing MRSA isolates
Kayo Osawa, Katsumi Shigemura, Takumi Jikimoto, Toshiro Shirakawa, Masato Fujisawa, Soichi Arakawa
The methods for typing and epidemiological study for especially antibiotic-resistant bacteria has been issued but there are the debates regarding which method is best for this purpose. The purpose of this study is to investigate and apply a comparatively new technology, phage-open-reading frame typing (POT) and repetitive-sequence-based PCR (rep-PCR) using DiversiLab system and compare for the discrimination of major methicillin-resistant Staphylococcus aureus (MRSA) lineages in epidemiological surveillance. We analyzed 47 representative MRSA stains isolated in Kobe University Hospital between January and December 2009. We performed MRSA typing using the POT kit and rep-PCR using the DiversiLab system. POT method classified all the MRSA strains into 35 clusters, whereas rep-PCR method typed all the MRSA strains in 10 kinds of clusters with a definition of 95% similarity. The discriminatory power and congruence between the methods were compared using the Simpson's index of diversity, adjusted Rand's and Wallace's coefficients. Our statistical analyses showed that the POT (POT 1-2-3 and POT 2-3) revealed a higher discriminatory power in the Simpson's index of diversity (SID; 0.969, range 0.939-4.000 and 0.967, range 0.935-0.998, respectively) for MRSA isolates than the rep-PCR (0.821 (0.767-0.876)). The adjusted Rand's and Wallace's coefficients did not show higher concordance among the methods. In conclusion, we demonstrated that the POT can perform accurate and reliable epidemiological surveillance studies for analyzing the genetic relatedness of MRSA strains.
JAPAN ANTIBIOTICS RESEARCH ASSOC, Aug. 2014, JOURNAL OF ANTIBIOTICS, 67(8) (8), 565 - 569, English
[Refereed]
Scientific journal

A Combined Lymphokine-activated Killer (LAK) Cell Immunotherapy and Adenovirus-p53 Gene Therapy for Head and Neck Squamous Cell Carcinoma
Hiroki Saito, Satoshi Ando, Naoya Morishita, Kyung-Mi Lee, Dante Dator, David Dy, Katsumi Shigemura, Zainal Adhim, Ken-Ichi Nibu, Masato Fujisawa, Toshiro Shirakawa
Background: The antitumor activity of lymphokine activated killer (LAK) cells immunotherapy is not always effective in all patients, especially when used alone. In this study, we investigated the in vitro antitumor activities of a combination of LAK immunotherapy and gene therapy employing an adenovirus carrying the p53 gene (Ad-p53) in human head and neck squamous cell carcinoma. Materials and Methods: The in vitro cytotoxicity of LAK cells was tested in H891 cells infected with or without Ad-p53, and the mRNA expression levels of natural killer group 2D ligands (UL16 binding protein (ULBP) 1 to 5) and tumor necrosis factor (TNF-alpha) in these cells were measured by real-time reverse transcription polymerase chain reaction. Results: Ad-p53 infection increased the cytotoxicity of LAK cells against H891 cells, and also increased the mRNA expression levels of the ULBPs in H891 cells and TNF-alpha in the LAK cells. Conclusion: The antitumor activities of LAK cells in H891 cells were enhanced by Ad-p53. Conclusion: The combinational therapy of LAK immunotherapy and Ad-p53 gene therapy may represent a new paradigm for the treatment of head and neck cancer.
INT INST ANTICANCER RESEARCH, Jul. 2014, ANTICANCER RESEARCH, 34(7) (7), 3365 - 3370, English
[Refereed]
Scientific journal

兵庫県下で分離されたメタロβラクタマーゼ産生腸内細菌の解析
大澤佳代, Shigemura Katsumi, 吉田弘之, 藤原美樹, 荒川創一, 藤澤正人, 白川利朗
May 2014, 日本化学療法学会雑誌, 62(Suppl.A) (Suppl.A), 402, Japanese
Research society

当院で分離されたESBLs産生Escherichia coliの遺伝子解析
大澤佳代, 吉田弘之, Shigemura Katsumi, 楠木まり, 直本拓己, 中村竜也, 荒川創一, 藤澤正人, 白川利朗
May 2014, 日本化学療法学会雑誌, 62(Suppl.A) (Suppl.A), 400, Japanese
Research society

Oral administration of genetically modified Bifidobacterium displaying HCV-NS3 multi-epitope fusion protein could induce an HCV-NS3-specific systemic immune response in mice
Saki Takei, Chika Omoto, Koichi Kitagawa, Naoya Morishita, Takane Katayama, Katsumi Shigemura, Masato Fujisawa, Masato Kawabata, Hak Hotta, Toshiro Shirakawa
More than 170 million people worldwide are chronic HCV (Hepatitis C virus) carriers, and about 30% of them will develop progressive liver disease, such as cirrhosis and hepatocellular carcinoma. A combination of pegylated interferon-alpha with ribavirin, the standard treatment for HCV infection, has been effective in fewer than 50% of patients infected with HCV genotype 1. A strong T cell response against the nonstructural protein 3 (NS3) is important for recovery from acute HCV infection, and an early multi-specific CD4+ helper and CD8+ cytotoxic T cell response is critical for HCV clearance. In the present study, we successfully constructed a genetically modified Bifidobacterium longum (B. longum) displaying recombinant HCV-NS3 peptides containing some CD4 and CD8 epitopes located in the HCV-NS3 region as an oral vaccine against chronic HCV infection. The oral administration of this vaccine could induce NS3-specific immune responses in mice through intestinal mucosal immunity. Our findings suggest that this novel oral vaccine has great potential as a novel oral vaccine against chronic HCV infection. (C) 2014 Elsevier Ltd. All rights reserved.
ELSEVIER SCI LTD, May 2014, VACCINE, 32(25) (25), 3066 - 3074, English
[Refereed]
Scientific journal

DEVELOPMENT OF MULTIPLEX PCR FOR RAPID IDENTIFICATION OF FOUR SALMONELLA SEROVARS MOST COMMONLY ISOLATED IN JAPAN
Rika Shimizu, Kayo Osawa, Katsumi Shigemura, Hiroyuki Yoshida, Miki Fujiwara, Yoshio Iijima, Masato Fujisawa, Toshiro Shirakawa
More than 2,500 serovars of Salmonella species have been reported to date. A multiplex-PCR method was developed and evaluated for discriminating the four Salmonella enterica subsp enterica serovars, namely, S. Enteritidis, S. Typhimurium, S. Thompson and S. Infantis, most commonly isolated in Japan. Twenty-two serovars of 84 Salmonella strains and 7 species of non-Salmonella strains were evaluated using primer pairs specific for the detection of Salmonella spp. Multiplex PCR generated, with 100% specificity, the expected amplicon of 333, 413,551 and 658 bp of S. Enteritidis, S. Inf antis, S. Typhimurium, and S. Thompson, respectively, while an additional non-specific amplicon (about 1,000 bp) was observed for S. Infantis, but it had no practical impact in the bacterial detection. This multiplex PCR assay can be applied to identify and discriminate clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological examination.
SOUTHEAST ASIAN MINISTERS EDUC ORGANIZATION, May 2014, SOUTHEAST ASIAN JOURNAL OF TROPICAL MEDICINE AND PUBLIC HEALTH, 45(3) (3), 654 - 661, English
[Refereed]
Scientific journal

前立腺癌増悪において上皮ならびに間質でのShhとandrogenシグナル伝達が上皮間葉移行を作動させる
山道深, Shigemura Katsumi, ホスニー・ベンソイ, ファトマ・メリジ, ウェンチン・ファン, シャン・ギアン・リ, リーランド・チャン, 川端眞人, 後藤章暢, 三宅秀明, 田中一志, 白川利朗, 藤澤正人
Apr. 2014, 日本泌尿器科学会総会 102回, 415, Japanese
Research society

Biosafety studies of carrier cells infected with a replication-competent adenovirus introduced by IAI.3B promoter
Katsuyuki Hamada, Toshiro Shirakawa, Shuji Terao, Akinobu Gotoh, Kenzaburo Tani, Wenlin Huang
The use of carrier cells infected with oncolytic viruses in cancer gene therapy is an attractive method because it can overcome viral immunogenicity and induce tumor immunity and significant antitumor activity. To enable human clinical trials of this treatment, acute and chronic toxicity tests must first be performed to ensure safety. IAI.3B promoter, oncolytic adenovirus AdE3-IAI.3B introduced by IAI.3B promoter, and A549 carrier cells infected with AdE3-IAI.3B were highly active in cancer cells but not in normal cells. Freeze-thawing increased the antitumor effect of A549 carrier cells by promoting the translocation of oncolytic adenovirus particles from the nucleus to the cytoplasm following the rupture of the nuclear membranes. No deaths or abnormal blood test data resulted from acute toxicity tests conducted in nude mice after a single dose. In chronic toxicity tests in rabbits, there were no serious side effects after eight doses of 1.25 x 10(7) cells/kg or less for 4 weeks; a significant immune response is known to elicit increased numbers of antiadenovirus antibodies and enlarge the spleen. From these results, it could be concluded that cancer gene therapy of recurrent solid tumors using carrier cells can be safely trialed in humans.
CELL PRESS, 2014, MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT, 1, 14019, English
[Refereed]
Scientific journal

Antimicrobial Resistance in Salmonella Strains Clinically Isolated in Hyogo, Japan (2009-2012)
Kayo Osawa, Katsumi Shigemura, Rika Shimizu, Ayaka Kato, Mayuha Kimura, Yuki Katayama, Yuma Okuya, Shunichiro Yutaka, Akiko Nishimoto, Akane Kishi, Miki Fujiwara, Hiroyuki Yoshida, Yoshio Iijima, Masato Fujisawa, Toshiro Shirakawa
The purpose of this study was to examine the in vitro susceptibilities to antimicrobial agents and genetic diversity of 195 clinical strains of Salmonella spp., which were isolated and examined for the extended-spectrum beta-lactamase (ESBL) bla(CTX-M) gene and the presence of gyrA, gyrB, parC, and parE genes mutations in Hyogo, Japan, from 2009 to 2012. Forty-three of the 195 strains were antimicrobial resistant. Two Salmonella enterica subsp. enterica strains, 1 serovar Schwarzengrund, and 1 serovar Enteritidis were identified as ESBL-producing strains possessing bla(CTX-M-15) and bla(CTX-M-2), respectively. Among 8 nalidixic acid-resistant strains, 7 had mutations in gyrA alone or in gyrA and parC. In conclusion, we identified CTX-M ESBL-producing Salmonella clinical strains with multidrug resistance. Further studies are needed to monitor these serious drug-resistant Salmonella strains in Japan.
NATL INST INFECTIOUS DISEASES, Jan. 2014, JAPANESE JOURNAL OF INFECTIOUS DISEASES, 67(1) (1), 54 - 57, English
[Refereed]
Scientific journal

BPH/LUTSに対するデュタステリドの有用性に関する検討
Tanaka Kazushi
Sep. 2013, 日本排尿機能学会誌, 24(1号) (1号), 282, Japanese
[Refereed]
Research society

Silodosin versus naftopidil in Japanese patients with lower urinary tract symptoms associated with benign prostatic hyperplasia: A randomized multicenter study
Toshiro Shirakawa, Takahiro Haraguchi, Katsumi Shigemura, Shinichi Morishita, Kohji Minayoshi, Jiro Miyazaki, Yuji Yamada, Hideaki Miyake, Kazushi Tanaka, Masato Fujisawa
Objectives: Silodosin is a novel drug that is highly selective to subtype alpha 1A and, since 2006, has been used in Japan for treating benign prostatic hyperplasia. This study aimed to compare the clinical effects of the alpha-adrenoceptor antagonist, silodosin, with those of naftopidil in patients presenting lower urinary tract symptoms associated with benign prostatic hyperplasia. Methods: This was a randomized, open-label, controlled multicenter study carried out in Japan. Overall, 121 participants with lower urinary tract symptoms associated with benign prostatic hyperplasia were randomized to receive silodosin (4mg twice daily) or naftopidil (50mg once daily) for 4 or 8 weeks. Patients were divided into four groups: the alpha-blocker-naive groups received silodosin (35 patients) or naftopidil (33 patients) and the drug-switching groups changed from tamsulosin to silodosin (26 patients) or naftopidil (27 patients). The outcomes parameters were the International Prostate Symptom Score, quality of life, maximum urinary flow rate and post-void residual urine volume. P<0.05 was considered statistically significant by using the Wilcoxon signed-rank and rank-sum tests, and analysis of covariance. Results: In all the groups, silodosin and naftopidil significantly improved the total International Prostate Symptom Score and quality of life. However, silodosin obtained significantly better improvement in total International Prostate Symptom Score in the alpha-blocker-naive patients at 4 and 8 weeks. The maximum urinary flow rate and residual urine did not change significantly in all the treatment groups. Conclusions: The present study confirms the clinical usefulness of silodosin in the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia.
WILEY-BLACKWELL, Sep. 2013, INTERNATIONAL JOURNAL OF UROLOGY, 20(9) (9), 903 - 910, English
[Refereed]
Scientific journal

Modulation of O-antigen chain length by the wzz gene in Escherichia coli O157 influences its sensitivities to serum complement
Kayo Osawa, Katsumi Shigemura, Atsushi Iguchi, Hiroki Shirai, Toru Imayama, Kazuko Seto, Dadik Raharjo, Masato Fujisawa, Ro Osawa, Toshiro Shirakawa
Escherichia coli O157 strains belonging to a distinct lineage and expressing different O-antigen (Oag) lengths were isolated. Although the function of wzz in E. coli has not been adequately investigated, this gene is known to be associated with regulation of Oag length. Using E. coli O157:H7 ATCC43888 (wild-type), several wzz mutants of E. coli O157, including a wzz deletion mutant, were generated and the relationship between the length of Oag modulated by the wzz gene and sensitivities to serum complement investigated. SDS-PAGE, immunoblot analyses and sensitivity tests to human serum complement were performed on these strains. The lengths of the O157-antigen could be modulated by the wzz gene mutations and were classified into long, intermediate and short groups. The short chain mutant was more serum sensitive than the wild-type strain and the other wzz mutants (P<0.001). In conclusion, Oag chain length modulated by the wzz gene in E. coli O157 influences its sensitivities to serum complement. The present findings suggest that E. coli O157 strains with intermediate or long length Oag chains might show greater resistance to serum complement than those with short chains.
WILEY-BLACKWELL, Sep. 2013, MICROBIOLOGY AND IMMUNOLOGY, 57(9) (9), 616 - 623, English
[Refereed]
Scientific journal

Frequency of Diarrheagenic Escherichia coli among Children in Surabaya, Indonesia
Kayo Osawa, Dadik Raharjo, Eddy Bagus Wasito, Sugeng Harijono, Katsumi Shigemura, Ro Osawa, Subijanto Marto Sudarmo, Yoshio Iijima, Toshiro Shirakawa
Diarrheagenic Escherichia coli (DEC) is a major etiologic agent of childhood diarrhea in developing countries. We investigated the frequency of DEC in stool samples from 125 diarrheal children (age, 1-10 years) and 92 non-diarrheal children in Surabaya, Indonesia. The non-diarrheal children served as healthy controls. DEC was detected in 23 of 125 (18.4%) and 47 of 92 (51.1%) samples in the diarrheal and non-diarrheal children, respectively. Enteropathogenic E. coli was the most prevalent in the non-diarrheal children (25.0%), and its prevalence was significantly higher than that in the diarrheal children (0.8%) (P < 0.0001). Interestingly, Shiga toxin-producing E. coli (4.3%) was detected only in the non-diarrheal children (P = 0.031). This is the first study comparing between diarrheal children with non-diarrheal or healthy children to investigate the role of DEC in pediatric diarrheal diseases in Indonesia.
NATL INST INFECTIOUS DISEASES, Sep. 2013, JAPANESE JOURNAL OF INFECTIOUS DISEASES, 66(5) (5), 446 - 448, English
[Refereed]
Scientific journal

Gene silencing with siRNA targeting E6/E7 as a therapeutic intervention against head and neck cancer-containing HPV16 cell lines
Zainal Adhim, Naoki Otsuki, Junko Kitamoto, Naoya Morishita, Masato Kawabata, Toshiro Shirakawa, Ken-Ichi Nibu
Conclusion: Our results indicate that siRNA E6 and/or E7 may have potential as a gene-specific therapy for human papillomavirus (HPV) type 16 (HPV16)-related squamous cell carcinoma of the head and neck (HNSCC). Objectives: To evaluate the effectiveness of siRNA targeting E6 and/or E7 on the in vitro and in vivo growth suppression of HPV16-related HNSCC. Methods: HPV16-related HNSCC (UM-SCC47) cell lines were used for the present study. Expression of HPV viral oncogenes E6 and/or E7 and their cellular targets, p53 and pRb, was evaluated by real-time PCR, Western blotting, and immunofluorescence staining. To study the effect of siRNA on tumor growth in vivo, we developed animal models. Representative tumors harvested from each group were processed for apoptosis analyses (TUNEL assay) and immunofluorescence staining for p53 and pRb. Results: E6 and E7 oncogenes of HPV16 were down-regulated by E6 and/or E7 targeting siRNAs, respectively. The expression of p53 and pRb proteins in both the E6 siRNA group and E7 siRNA group was up-regulated compared with those of control groups. The cellular proliferation and apoptosis indexes of E6 and/or E7 siRNA groups were higher than those of controls. In vivo studies showed significant inhibitory effect of E6 and/or E7 siRNA compared with those of control groups, which was consistent with in vitro studies. © 2013 Informa Healthcare.
Jul. 2013, Acta Oto-Laryngologica, 133(7) (7), 761 - 771, English
[Refereed]
Scientific journal

Possible role of sonic hedgehog and epithelial-mesenchymal transition in renal cell cancer progression
Hosny M. Behnsawy, Katsumi Shigemura, Fatma Y. Meligy, Fukashi Yamamichi, Masuo Yamashita, Wen-Chin Haung, Xiangyan Li, Hideaki Miyake, Kazushi Tanaka, Masato Kawabata, Toshiro Shirakawa, Masato Fujisawa
Purpose: Sonic hedgehog (Shh) signaling and epithelial-mesenchymal transition (EMT) are both known to relate to cancer progression. The purpose of this study was to investigate the role of Shh signaling and EMT in renal cell carcinoma (RCC). Materials andMethods: Cell proliferation was assayed in RCC cell lines in the presence or absence of a Shh signaling stimulator, recombinant Shh (r-Shh) protein, or a Shh signaling inhibitor, cyclopamine. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to study the expression of EMT markers (E-cadherin, N-cadherin, and vimentin) and osteonectin. The expression of Ki-67, Gli-1, osteonectin, and EMT markers in nephrectomy specimens from RCC patients was also measured by immunohistochemical (IHC) staining. Results: RCC cells showed enhanced cell proliferation by r-Shh protein, whereas cell proliferation was suppressed by the addition of cyclopamine in RenCa cells. Real-time RT-PCR showed that r-Shh suppressed the expression of E-cadherin and that this suppression was partly blocked by cyclopamine alone in RenCa cells. In the IHC results, osteonectin significantly correlated with vein sinus invasion (p=0.0218), and the expression of vimentin significantly correlated with lymphatic invasion (p=0.0392). Conclusions: Shh signaling and EMT play roles in RCC progression, and the Shh signaling inhibitor cyclopamine might be a possible molecular targeted therapeutic strategy for RCC. © The Korean Urological Association, 2013.
Jun. 2013, Korean Journal of Urology, 54(8) (8), 547 - 554, English
[Refereed]
Scientific journal

尿路感染症患者より分離したPseudomonas aeruginosa薬剤耐性株におけるefflux pump遺伝子の発現についての検討
加藤綾香, 大澤佳代, Shigemura Katsumi, 田中一志, 荒川創一, 藤澤正人, 白川利朗
May 2013, 感染症学雑誌, 87(臨増) (臨増), 297, Japanese
Research society

Does mutation in gyrA and/or parC or efflux pump expression play the main role in fluoroquinolone resistance in Escherichia coli urinary tract infections?: A statistical analysis study
Katsumi Shigemura, Kazushi Tanaka, Fukashi Yamamichi, Toshiro Shirakawa, Hideaki Miyake, Masato Fujisawa
There are several mechanisms of fluoroquinolone (FQ) resistance, such as mutations in the quinolone resistance-determining regions (QRDRs) of target genes and efflux pump expression. The purpose of this study was to investigate which factor plays the main role in FQ resistance in Escherichia coli causing urinary tract infections (UTIs) from a statistical analysis of our two previous works. DNA sequencing of the QRDRs of the FQ resistance-related genes gyrA and parC as well as real-time reverse transcriptase (RT)-PCR for the expression of efflux pump genes such as marA or yhiU were performed and the correlations of mutations or efflux pump gene expression with FQ minimal inhibitory concentrations (MICs) were investigated. Significant factors for high MICs of sitafloxacin (STFX), ciprofloxacin (CPFX) and levofloxacin were the mutations S83L and D87N in gyrA and S80I and E84V in parC as well as the expression of marA. Mutations in the QRDRs of gyrA or parC had a greater effect on FQ MICs than efflux pump gene expression. Based on the regression coefficient, these mutations correlated with MICs to CPFX most, and STFX had the lowest effects from these mutations among the three tested FQs. In conclusion, in E. coli causing UTIs, mutations in the QRDRs of gyrA or parC had a greater effect on FQ resistance, especially to CPFX, than efflux pump gene expression from a statistical analysis study of our two previous works. Further research into the molecular basis for FQ resistance could lead to new therapeutic strategies against FQ-resistant E. coli. (c) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
ELSEVIER SCIENCE BV, Dec. 2012, INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS, 40(6) (6), 516 - 520, English
[Refereed]
Scientific journal

Mutations in the gyrA and parC genes and in vitro activities of fluoroquinolones in 114 clinical isolates of Pseudomonas aeruginosa derived from urinary tract infections and their rapid detection by denaturing high-performance liquid chromatography
Minori Matsumoto, Katsumi Shigemura, Toshiro Shirakawa, Yuzo Nakano, Hideaki Miyake, Kazushi Tanaka, Shohiro Kinoshita, Soichi Arakawa, Masato Kawabata, Masato Fujisawa
Fluoroquinolone (FQ) resistance in Pseudomonas aeruginosa has spread. The purpose of this study was to investigate the correlation between representative FQ, i.e. levofloxacin (LVX), resistance and mutations in the gyrA and parC genes of P. aeruginosa clinical isolates from the urine of urinary tract infection patients and their rapid detection by denaturing high-performance liquid chromatography (DHPLC). The susceptibility to LVX of 114 clinical isolates was measured and the quinolone resistance-determining regions (QRDRs) in the gyrA and parC genes of these isolates were sequenced. DHPLC was undertaken to correlate the distinctive chromatograms with their DNA mutation patterns. Among 114 isolates tested, 22 isolates (19.3%) were resistant to LVX. Six amino acid mutations were detected (Thr83Ile, Asp87Tyr and Asp87Asn in gyrA and Ser87Leu, Ser87Trp and Glu91Arg in parC), existing alone or in combination. There were 10 kinds of mutation patterns. The presence of two or more kinds of mutation significantly correlated with LVX resistance compared with the wild-type or a single mutation (P < 0.0001). DHPLC data identified the number of amino acid mutations with reproducibility distinguishable by peak number and profile of the DHPLC chromatogram. In conclusion, two or more mutations in gyrA and parC were significantly related to LVX resistance in P. aeruginosa. DHPLC facilitated the detection of resistant alleles, providing a rapid (5 min per sample), economical (96 samples per run) and reliable technique for characterising LVX resistance in P. aeruginosa. This rapid detection system could forecast LVX resistance by the DHPLC profile. (C) 2012 Published by Elsevier B.V.
ELSEVIER SCIENCE BV, Nov. 2012, INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS, 40(5) (5), 440 - 444, English
[Refereed]
Scientific journal

Augmentation of natural cytotoxicity by chronic low-dose ionizing radiation in murine natural killer cells primed by IL-2
Chung Hee Sonn, Jong Rip Choi, Tae-Jin Kim, Young-Bin Yu, Kwanghee Kim, Suk Chul Shin, Gil-Hong Park, Toshiro Shirakawa, Hee Sun Kim, Kyung-Mi Lee
The possible beneficial effects of chronic low-dose irradiation (LDR) and its mechanism of action in a variety of pathophysiological processes such as cancer are a subject of intense investigation. While animal studies involving long-term exposure to LDR have yielded encouraging results, the influence of LDR at the cellular level has been less well defined. We reasoned that since natural killer (NK) cells constitute an early responder to exogenous stress, NK cells may reveal sentinel alterations in function upon exposure to LDR. When purified NK cells received LDR at 4.2 mGy/h for a total of 0.2 Gy in vitro, no significant difference in cell viability was observed. Likewise, no functional changes were detected in LDR-exposed NK cells, demonstrating that LDR alone was insufficient to generate changes at the cellular level. Nonetheless, significant augmentation of cytotoxic, but not proliferative, function was detected when NK cells were stimulated with low-dose IL-2 prior to irradiation. This enhancement of NK cytotoxicity was not due to alterations in NK-activating receptors, NK1.1, NKG2D, CD69 and 2B4, or changes in the rate of early or late apoptosis. Therefore, LDR, in the presence of suboptimal cytokine levels, can facilitate anti-tumor cytotoxicity of NK cells without influencing cellular proliferation or apoptosis. Whether these results translate to in vivo consequences remains to be seen; however, our data provide initial evidence that exposure to LDR can lead to subtle immune-enhancing effects on NK cells and may explain, in part, the functional basis underlying, diverse beneficial effects seen in the animals chronically exposed to LDR.
OXFORD UNIV PRESS, Nov. 2012, JOURNAL OF RADIATION RESEARCH, 53(6) (6), 823 - 829, English
[Refereed]
Scientific journal

Potential establishment of lung metastatic xenograft model of androgen receptor-positive and androgen-independent prostate cancer (C4-2B).
Fukashi Yamamichi, Takayuki Matsuoka, Katsumi Shigemura, Masato Kawabata, Toshiro Shirakawa, Masato Fujisawa
OBJECTIVE: To establish a mouse xenograft model of metastatic prostate cancer (PCa) and investigate the relationship between metastasis and circulating tumor cells. METHODS: Flow cytometry (FACS) was used to detect suitable PCa cells and markers for detecting circulating tumor cells in vivo. We orthotopically injected androgen receptor-positive and androgen-independent C4-2B PCa cells into 12 severe combined immunodeficiency (SCID) mouse prostates, including 1 vehicle control. We measured the serum prostate-specific antigen levels biweekly after tumor inoculation. Circulating tumor cells (CTCs) were measured qualitatively by fluorescent microscopy immediately after the mice were sacrificed. The mouse prostates and lungs were examined for tumor formation using immunohistochemistry because we found no apparent metastasis, except in the lung. RESULTS: FACS analyses in vitro identified the marker, prostate-specific membrane antigen, and C4-2B cells to be appropriate for additional in vivo study. We confirmed that the serum prostate-specific antigen increase was dependent on time and prostate tumor weight in mice. Of the 11 mice, 6 could be used as the mouse PCa xenograft model. Fluorescent microscopy detected CTCs in the peripheral blood in 5 of the 6 mice constituting the PCa model. Human prostate-specific antigen expression was detected by immunohistochemistry in the prostates of all the mice and in the lung of 2 of the 6 mice, suggesting 2 mice with lung metastasis. CONCLUSION: We have shown the potential establishment of a mouse lung metastatic xenograft model of androgen receptor-positive and androgen-independent C4-2B PCa tumor. However, the present model requires improvement to be a more reproducible, accurate and complete experimental model. Additional study is necessary to verify the relationship between metastasis and CTCs.
Oct. 2012, Urology, 80(4) (4), 951.e1-7 - 7, English, International magazine
[Refereed]
Scientific journal

排尿障害患者におけるデュタステリド内服療法の性機能および血中テストステロン値への影響についての検討
Chiba Koji, Nakano Yuzo, Haraguchi Takahiro, Shirakawa Toshiro, Tanaka Kazushi, Arakawa Soichi, Fujisawa Masato
(一社)日本性機能学会, Sep. 2012, 日本性機能学会雑誌, 27(2号) (2号), 175 - 175, Japanese
Research society

BPH/LUTSに対するデュタステリドの有用性に関する検討
Haraguchi Takahiro, Miyake Hideaki, Shirakawa Toshiro, Tanaka Kazushi, Fujisawa Masato
Aug. 2012, 日本排尿機能学会誌, 23(1号) (1号), 194, Japanese
Research society

Detection of tumor markers in prostate cancer and comparison of sensitivity between real time and nested PCR.
Takayuki Matsuoka, Katsumi Shigemura, Fukashi Yamamichi, Masato Fujisawa, Masato Kawabata, Toshiro Shirakawa
The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivity by real time PCR and nested PCR. In real time PCR, there was a significant correlation between cell number and the RNA concentration obtained (R(2)=0.9944) for PSA, PSMA, and AR. We found it possible to detect these markers from a single LNCaP cell in both real time and nested PCR. By comparison, nested PCR reached a linear curve in fewer PCR cycles than real time PCR, suggesting that nested PCR may offer PCR results more quickly than real time PCR. In conclusion, nested PCR may offer tumor maker detection in PCa cells more quickly (with fewer PCR cycles) with the same high sensitivity as real time PCR. Further study is necessary to establish and evaluate the best tool for PCa tumor marker detection.
Jun. 2012, The Kobe journal of medical sciences, 58(2) (2), E51-9 - 9, English, Domestic magazine
[Refereed]
Scientific journal
Link to Kobe Univ. Repository (Kernel)

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue, bone marrow, and skeletal muscle tissue
Fatma Y. Meligy, Katsumi Shigemura, Hosny M. Behnsawy, Masato Fujisawa, Masato Kawabata, Toshiro Shirakawa
The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.
SPRINGER, Apr. 2012, IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL, 48(4) (4), 203 - 215, English
[Refereed]
Scientific journal

Efficient antitumor effects of carrier cells loaded with a fiber-substituted conditionally replicating adenovirus on CAR-negative tumor cells
K. Iguchi, F. Sakurai, K. Tomita, K. Katayama, T. Yamaguchi, K. Kawabata, M. Tagawa, M. Kawabata, T. Shirakawa, H. Mizuguchi
Carrier cells delivering a conditionally replicating adenovirus (CRAd), which selectively replicates in tumor cells and induces tumor cell lysis, have promising potential for treatment of cancer because CRAd-loaded carrier cells evade inhibition by neutralizing anti-adenovirus (Ad) antibodies and because the carrier cells are locally retained at the injection point after local injection. A previous study by Hamada et al. demonstrated that carrier cells (CRAd-containing cell fragments derived from the carrier cells) are engulfed into the target cells, probably through a pathway independent of the primary receptor for Ad, the coxsackievirus and Ad receptor (CAR) (Mol Ther, 15: 1121-1128; 2007); however, it remains to be elucidated whether carrier cells infected with a conventional CRAd, which is composed of subgroup-C Ad serotype-5 (Ad5), mediate antitumor effects on CAR-negative cells. In order to examine whether carrier cells delivering a conventional CRAd (Carrier-F5) induce lysis of CAR-negative tumor cells, CAR-positive and CAR-negative tumor cells were incubated with Carrier-F5. Carrier-F5 mediated efficient killing of CAR-positive tumor cells; however, CAR-negative tumor cells were almost refractory to Carrier-F5. On the other hand, carrier cells loaded with a fiber-substituted CRAd containing fiber proteins of Ad serotype-35 (Ad35) (CRAd-F35), which binds to human CD46 for infection, showed efficient killing of both CAR-positive and CAR-negative tumor cells. Intra-tumoral injection of carrier cells loaded with CRAd-F35 (Carrier-F35) also resulted in efficient regression of both CAR-positive and CAR-negative tumors. These results demonstrated that the expression levels of receptors for Ad are an important factor for CRAd-loaded carrier cell-mediated cancer therapy, and that Carrier-F35 would have potential as a cancer treatment for not only CAR-positive tumors but also CAR-negative tumors. Cancer Gene Therapy (2012) 19, 118-125; doi: 10.1038/cgt.2011.74; published online 11 November 2011
NATURE PUBLISHING GROUP, Feb. 2012, CANCER GENE THERAPY, 19(2) (2), 118 - 125, English
[Refereed]
Scientific journal

E10A, an adenovirus-carrying endostatin gene, dramatically increased the tumor drug concentration of metronomic chemotherapy with low-dose cisplatin in a xenograft mouse model for head and neck squamous-cell carcinoma
Z. Adhim, X. Lin, W. Huang, N. Morishita, T. Nakamura, H. Yasui, N. Otsuki, K. Shigemura, M. Fujisawa, K. Nibu, T. Shirakawa
Most cancer chemotherapeutic agents are administered at the maximum-tolerated dose (MTD) in short cycles with treatment breaks. However, MTD-based chemotherapies are often associated with significant toxicity and treatment breaks allow the opportunity for tumor regrowth and acquisition of chemoresistance. To minimize these drawbacks, a metronomic strategy, in which chemotherapeutics are administered at doses significantly below the MTD without treatment breaks, has been suggested by many investigators. The antitumor effect of metronomic chemotherapy may be partially due to inhibition of tumor angiogenesis, and it could be enhanced by a combination therapy, including antiangiogenic agents. In this study, we evaluated the synergistic effect of E10A, an adenovirus carrying the endostatin gene, the most potent inhibitors of tumor angiogenesis, in combination with weekly low-dose cisplatin in a xenograft mouse model for head and neck squamous-cell carcinoma. The E10A induced mRNA and protein expressions of endostatin in H891 cells in vitro. E10A significantly enhanced the in vivo tumor growth inhibitory effect of cisplatin. Immunohistochemical analysis with a TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay and anti-CD31 antibodies revealed that the combination of E10A and cisplatin induced high levels of cell apoptosis and inhibited tumor angiogenesis. Importantly, E10A increased the platinum concentrations in tumors to fivefold higher than that induced by cisplatin alone. Cancer Gene Therapy (2012) 19, 144-152; doi: 10.1038/cgt.2011.79; published online 25 November 2011
NATURE PUBLISHING GROUP, Feb. 2012, CANCER GENE THERAPY, 19(2) (2), 144 - 152, English
Scientific journal

Mechanisms of and Risk Factors for Fluoroquinolone Resistance in Clinical Enterococcus faecalis Isolates from Patients with Urinary Tract Infections
Tomihiko Yasufuku, Katsumi Shigemura, Toshiro Shirakawa, Minori Matsumoto, Yuzo Nakano, Kazushi Tanaka, Soichi Arakawa, Masato Kawabata, Masato Fujisawa
We examined Enterococcus faecalis strains clinically isolated from 100 patients with urinary tract infections (UTIs) for their susceptibility to levofloxacin (LVX) by measuring the MIC and investigated amino acid mutations by direct DNA sequencing, which were then correlated with LVX resistance. Next, we studied risk factors for LVX resistance, such as age, gender, and previous fluoroquinolone use, and investigated the statistical correlation of these risk factors with each amino acid mutation and LVX resistance. Of the 100 isolates tested, 14 isolates showed LVX resistance and all of these isolates had amino acid mutations. We demonstrated that 2 out of 4 mutations (Ser83-to-Ile in gyrA and Ser80-to-Ile in parC) had a significant correlation with LVX resistance. There was a significant relationship between isolates with 2 or 3 amino acid mutations and LVX resistance. In addition, we found a significant correlation between the previous use of fluoroquinolones and LVX resistance or the presence of mutations and also demonstrated that previous use of other types of antibiotics was significantly related to the presence of mutations by multivariate analysis. In conclusion, we found significant correlation between amino acid mutations in E. faecalis, LVX resistance, and risk factors such as previous use of fluoroquinolones.
AMER SOC MICROBIOLOGY, Nov. 2011, JOURNAL OF CLINICAL MICROBIOLOGY, 49(11) (11), 3912 - 3916, English
[Refereed]
Scientific journal

In vitro and in vivo inhibitory effect of three Cox-2 inhibitors and epithelial-to-mesenchymal transition in human bladder cancer cell lines
Z. Adhim, T. Matsuoka, T. Bito, K. Shigemura, K-M Lee, M. Kawabata, M. Fujisawa, K. Nibu, T. Shirakawa
BACKGROUND: Although the anti-tumour effect of cyclooxygenase-2 (Cox-2) inhibitors in invasive bladder cancer has been confirmed, its mechanisms of action are unclear. Recently, the concept of an epithelial-to-mesenchymal transition (EMT) promoting carcinoma progression has been suggested, and a key feature of the EMT is the downregulation of E-cadherin. In this study, we investigated the effect of Cox-2 inhibitors on reversal EMT and tumour growth inhibition in bladder cancer cells. METHODS: We used three Cox-2 inhibitors, etodolac, celecoxib and NS-398 and three human bladder cancer cell lines, T24, 5637 and KK47, in this study. T24 xenograft tumour mouse model was used in the in vivo study. RESULTS: Within the clinical drug concentrations, only etodolac showed the in vitro growth inhibition in T24 not in the other cell lines. Etodolac reduced SNAIL mRNA and vimentin cell surface expression, and induced E-cadherin mRNA and E-cadherin cell surface expression, in T24. Etodolac also most strongly inhibited the cell migration of T24 in vitro and showed the highest tumour growth inhibition in T24 tumour in vivo. CONCLUSION: Etodolac at clinical doses exhibited induced in vitro and in vivo anti-tumour effects and reversal effect of EMT in T24. These results suggest that etodolac is a good candidate for an anti-tumour or chemopreventive reagent for high-grade bladder cancer. British Journal of Cancer (2011) 105, 393-402. doi:10.1038/bjc.2011.262 www.bjcancer.com Published online 12 July 2011 (C) 2011 Cancer Research UK
NATURE PUBLISHING GROUP, Jul. 2011, BRITISH JOURNAL OF CANCER, 105(3) (3), 393 - 402, English
[Refereed]
Scientific journal

Intravenous injection of irradiated tumor cell vaccine carrying oncolytic adenovirus suppressed the growth of multiple lung tumors in a mouse squamous cell carcinoma model
Aya Saito, Naoya Morishita, Chihomi Mitsuoka, Shunichi Kitajima, Katsuyuki Hamada, Kyung-Mi Lee, Masato Kawabata, Masato Fujisawa, Toshiro Shirakawa
Background Although cancer therapy using replication-selective oncolytic adenoviruses has been available for many years, its anti-tumor efficacy is suboptimal as a result of low and nonspecific infectivity that depends on coxsackie adenovirus receptor expression of the target cancer and normal cells, and generation of an anti-adenovirus neutralizing antibody. In addition, concerns of triggering a severe innate immune response against the adenovirus limit the systemic administration. We developed the carrier cell-based oncolytic virus system (CBOVS) using irradiated tumor cells as carrier cells and concealing the adenovirus (Ad-IAI.3B) inside to improve the specific infectivity. We investigated the anti-tumor effect of CBOVS in a multiple lung tumor mouse model. Methods The ability of CBOVS to infect Ad-IAI.3B to the target cancer cells was examined in vitro in the presence of anti-adenovirus antibodies. To evaluate the systemic effect of CBOVS, we intravenously injected CBOVS into mice with lung tumors (KLN205 cell lines). Results CBOVS enhanced the infectivity of Ad-IAI.3B to tumor cells in the presence of anti-adenovirus antibodies in vitro. Intravenous injections of CBOVS produced an accumulation of the adenovirus in the lung-bearing tumors and produced a strong anti-tumor effect in vivo. Furthermore, lymphocytes collected from the CBOVS-treated mice induced an increase in cytokines related to the Th1 response (interferon-gamma, interleukin-12) by pulsing with KLN205. Conclusions These findings suggest that CBOVS could protect adenoviruses from neutralizing antibodies and systemically deliver them to lung tumors. Furthermore, CBOVS appears to have potential as a tumor cell vaccine that activates cytotoxic immunity against cancer cells. Copyright (C) 2011 John Wiley & Sons, Ltd.
WILEY-BLACKWELL, Jun. 2011, JOURNAL OF GENE MEDICINE, 13(6) (6), 353 - 361, English
Scientific journal

Risk factors and mechanisms of fluoroquinolone resistance in 156 Escherichia coli strains clinically isolated from urinary tract infections
Tomihiko Yasufuku, Katsumi Shigemura, Toshiro Shirakawa, Yuzo Nakano, Kazushi Tanaka, Soichi Arakawa, Shouhiro Kinoshita, Kunihiro Nishimura, Masato Kawabata, Masato Fujisawa
As fluoroquinolone-resistant strains of Escherichia coli emerge, several risk factors for fluoroquinolone resistance have become evident, such as amino acid mutations in the quinolone resistance determining regions (QRDR) of gyrA and parC and previous use of fluoroquinolone. This study investigated risk factors for fluoroquinolone resistance and amino acid mutation in the QRDR in E. coli. We investigated the statistical correlation between each amino acid mutation and resistance to levofloxacin. We examined the minimum inhibitory concentration (MIC) of levofloxacin and the amino acid mutations of gyrA and parC by direct DNA sequence in E. coli clinically isolated from urinary tract infection (UTI) patients. We investigated risk factors for levofloxacin resistance, such as age, sex, and previous use of fluoroquinolone. We found a significant correlation between the number of mutations and resistance to levofloxacin (p < 0.001) and between the presence of underlying urinary tract disease and the presence of mutations (p = 0.004) by multivariate analyses. Three mutations in QRDR were demonstrated to be significantly correlated with levofloxacin resistance. In conclusion, these findings contribute to our understanding of the molecular mechanisms and risk factors for fluoroquinolone resistance.
INFORMA HEALTHCARE, Feb. 2011, SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES, 43(2) (2), 83 - 88, English
[Refereed]
Scientific journal

PP-344 尿路感染症由来緑膿菌臨床株における高速液体クロマトグラフィー法によるフルオロキノロン系抗菌薬耐性の迅速診断法確立への検討(一般演題ポスター発表・討論,一般演題ポスター,第99回日本泌尿器科学会総会)
松本穣, 重村克巳, 白川利朗, 安福富彦, 中野雄造, 田中一志, 木下承皓, 川端眞人, 荒川創一, 藤澤正人
一般社団法人日本泌尿器科学会, 2011, 日本泌尿器科学会雑誌, 102(2) (2), 461 - 461, Japanese

OP-106 腸球菌の尿路感染症臨床株におけるgyrA,parC遺伝子変異とフルオロキノロン系抗菌薬耐性の相関についての検討(感染症1,一般演題口演,第99回日本泌尿器科学会総会)
安福富彦, 重村克巳, 白川利朗, 松本穰, 中野雄造, 田中一志, 木下承皓, 川端眞人, 荒川創一, 藤澤正人
一般社団法人日本泌尿器科学会, 2011, 日本泌尿器科学会雑誌, 102(2) (2), 358 - 358, Japanese

Phenotypic and Genotypic Characterization of Vibrio cholerae Clinically Isolated in Surabaya, Indonesia
Tomoyuki Nishibori, Garry Cores de Vries, Dadik Rahardjo, Eddy Bagus Wasito, Ismoedijanto De, Shouhiro Kinoshita, Yoshitake Hayashi, Hak Hotta, Masato Kawabata, Toshiro Shirakawa, Yoshio Iijima, Ro Osawa
The phenotypic and genotypic characteristics of 6 clinical strains of Vibrio cholerae isolated in Surabaya, Indonesia in 2009 were examined. The DNA fingerprints obtained suggested that these isolates were not from a single clone. Furthermore, all isolates produced cholera toxin and possessed the classical type of toxin B subunit gene, thus meaning that this is the first report of the occurrence of El Tor variants of V. cholerae in Indonesia. Although all isolates were sensitive to almost all antibiotics tested, including ampicillin, chloramphenicol, ciprofloxacin, gentamicin, levofloxacin, kanamycin, nalidixic acid, norfloxacin, streptomycin, trimethoprim-sulfamethoxazole, and tetracycline, and had no mutation in the gyrA and parC genes, they nevertheless possessed the class 1 integron that is a molecular vehicle for the acquisition of antibiotic resistance genes, suggesting that they have the potential to acquire the genetic element for drug resistance.
NATL INST INFECTIOUS DISEASES, Jan. 2011, JAPANESE JOURNAL OF INFECTIOUS DISEASES, 64(1) (1), 7 - 12, English
[Refereed]
Scientific journal

Correlation of Overexpression of Efflux Pump Genes with Antibiotic Resistance in Escherichia coli Strains Clinically Isolated from Urinary Tract Infection Patients
Tomihiko Yasufuku, Katsumi Shigemura, Toshiro Shirakawa, Minori Matsumoto, Yuzo Nakano, Kazushi Tanaka, Soichi Arakawa, Shouhiro Kinoshita, Masato Kawabata, Masato Fujisawa
Escherichia coli is one of the most common pathogens in urinary tract infections (UTIs), and antibiotic resistance in E. coli is becoming a serious problem in treating UTI. Efflux system overexpression is reported to contribute to E. coli resistance to several antibiotics. This study investigated the correlation of antibiotic susceptibilities with the overexpression of the efflux pump genes such as marA, yhiU, yhiV, and mdfA and with risk factors for antibiotic resistance in E. coli isolated from UTI patients. We examined the expression level of efflux pump genes using quantitative real-time reverse transcription-PCR (qRT-PCR). We also tested the in vitro susceptibilities to 12 kinds of antibiotics in 64 clinical strains of E. coli isolated from UTI patients. By multivariate analyses we revealed significant relationships between the overexpression of (i) marA and MICs of cefepime (FEP) and nalidixic acid (NAL), (ii) yhiV and MICs of minocycline (MIN), and (iii) mdfA and MICs of sitafloxacin (STX). In our investigation of the efflux pump genes, risk factors such as gender and the previous use of fluoroquinolones correlated with the overexpression of marA, and indwelling catheter use correlated with the overexpression of mdfA. In conclusion, we demonstrated that the increased expression of efflux pump genes such as marA and mdfA can lead to fluoroquinolone resistance in E. coli. These results contribute to our knowledge of the efflux system and raise the possibility of developing new agents, such as efflux pump inhibitors (EPIs), to antibiotic-resistant E. coli.
AMER SOC MICROBIOLOGY, Jan. 2011, JOURNAL OF CLINICAL MICROBIOLOGY, 49(1) (1), 189 - 194, English
[Refereed]
Scientific journal

Genetically modified Bifidobacterium displaying Salmonella-antigen protects mice from lethal challenge of Salmonella Typhimurium in a murine typhoid fever model
Sakura Yamamoto, Jun Wada, Takane Katayama, Takumi Jikimoto, Masakuni Nakamura, Shohiro Kinoshita, Kyung-Mi Lee, Masato Kawabata, Toshiro Shirakawa
We developed a novel vaccine platform utilizing Bifidobacterium as an antigen delivery vehicle for mucosal immunization. Genetically modified Bifidobacterium longum displaying Salmonella-flagellin on the cell surface was constructed for the oral typhoid vaccine. The efficiency of this vaccine was evaluated in a murine model of typhoid fever. We then orally administered 2.5 x 10(7) CFU of the recombinant Bifidobacterium longum (vaccine) or parental Bifidobacterium longum, or PBS to BALB/C mice every other day for 2 weeks. After the administration, a total of 42 mice (14 mice in each group) were challenged with Salmonella Typhimurium (1.0 x 10(7) CFU/mouse). While 12 mice in the PBS group, and 9 in the parental Bifidobacterium longum group died (median survival: 14 and 25 days), only two in the vaccine group died. These data support that our genetically modified Bifidobacterium antigen delivery system offers a promising vaccine platform for inducing efficient mucosal immunity. (C) 2010 Elsevier Ltd. All rights reserved.
ELSEVIER SCI LTD, Sep. 2010, VACCINE, 28(41) (41), 6684 - 6691, English
[Refereed]
Scientific journal

Ultraviolet light induces Stat3 activation in human keratinocytes and fibroblasts through reactive oxygen species and DNA damage
Toshinori Bito, Naoko Sumita, Taro Masaki, Toshiro Shirakawa, Masato Ueda, Ryutaro Yoshiki, Yoshiki Tokura, Chikako Nishigori
Stat3 is activated by the outer stressors, such as ultraviolet (UV) exposure. In this study, we investigated the Stat3 response to UV irradiation in human epidermal keratinocytes and dermal fibroblasts. Results indicated that UVB and UVC differentially activate Stat3 in these cells. The UV-induced Stat3 activation was mediated by both reactive oxygen species (ROS) and DNA damage, and the dominancy of ROS and DNA damage to activate Stat3 depended on the wavelength of UV. By using fibroblasts from a patient with xeroderma pigmentosum A (XP-A) and those transfected with human XPA gene, we found that UVB activates Stat3 via both ROS and DNA damage, while UVC does so mainly via DNA damage. The present data suggest that Stat3 activation in UV-exposed human skin is one of the initial events where DNA damage and ROS are involved.
WILEY-BLACKWELL, Jul. 2010, EXPERIMENTAL DERMATOLOGY, 19(7) (7), 654 - 660, English
[Refereed]
Scientific journal

NRAMP1/SLC11A1 GENE POLYMORPHISMS AND HOST SUSCEPTIBILITY TO MYCOBACTERIUM TUBERCULOSIS AND M. LEPRAE IN SOUTH SULAWESI, INDONESIA
Mochammad Hatta, Ratnawati, Motoko Tanaka, Jun Ito, Toshiro Shirakawa, Masato Kawabata
Genetic host factor may play an important role in controlling mycobacterial infections such as tuberculosis and leprosy Natural resistance associated macrophage protein 1 (Nramp1, alias Slc11a1) gene has been suggested to an associated gene of the host susceptibility to mycobacterium infection. To determine the association of Nramp1/Slc11a1 with tuberculosis and leprosy, we analyzed using polymerase chain reaction restriction fragment length polymorphisms threevariants (D543N, 3'UTR and INT4) of Nramp1/Slc11a1 gene in 58 tuberculosis patients (mean age, 34 0 +/- 13 1), 42 leprosy patients (mean age, 35 0 +/- 14 3) and 198 healthy controls (mean age, 32 0 +/- 12 9) from South Sulawesi, Indonesia We observed an association of INT4 polymorphism with paucibacillary type of leprosy (p=0.032, 1df, OR=2 975, CI=1.057-8.373), but not to multibacillary type (p=0 173, 1df, OR=2 248, CI=0.682-7.404). No significant association was found in the three variants with tuberculosis in this population.
SOUTHEAST ASIAN MINISTERS EDUC ORGANIZATION, Mar. 2010, SOUTHEAST ASIAN JOURNAL OF TROPICAL MEDICINE AND PUBLIC HEALTH, 41(2) (2), 386 - 394, English
[Refereed]
Scientific journal

OP-008 尿路感染症由来緑膿菌臨床株におけるgyrA、parC遺伝子変異とキノロン系抗菌薬耐性の関連についての検討(感染症1,一般演題口演,第98回日本泌尿器科学会総会)
松本穣, 重村克巳, 白川利朗, 安福富彦, 中野雄造, 田中一志, 武中篤, 荒川創一, 水下承皓, 川端眞人, 藤澤正人
一般社団法人日本泌尿器科学会, 2010, 日本泌尿器科学会雑誌, 101(2) (2), 234 - 234, Japanese

APP-001 尿路感染症由来大腸菌の臨床株における薬剤排出ポンプ遺伝子の発現と各種抗菌薬耐性との関連の解析(発表・討論,総会賞応募ポスター,第98回日本泌尿器科学総会)
安福富彦, 重村克巳, 白川利朗, 中野雄造, 田中一志, 武中篤, 荒川創一, 木下承皓, 川端眞人, 藤澤正人
一般社団法人日本泌尿器科学会, 2010, 日本泌尿器科学会雑誌, 101(2) (2), 181 - 181, Japanese

Emergence of fluoroquinolone-resistant strains of Salmonella enterica in Surabaya, Indonesia
Daisuke Yanagi, Garry Cores de Vries, Dadik Rahardjo, Lindawati Alimsardjono, Eddy Bagus Wasito, Ismoedijanto De, Shouhiro Kinoshita, Yoshitake Hayashi, Hak Hotta, Ro Osawa, Masato Kawabata, Toshiro Shirakawa
Typhoid fever remains a major health problem in developing countries. Fluoroquinolones such as ciprofloxacin emerged as the 1st-choice treatment of enteric fever, including typhoid, in the 1990s. Recently, Salmonella typhi strains with resistance to ciprofloxacin have been increasingly reported in several countries, although the fluoroquinolone-resistant clinical strain has not been reported in Indonesia. In the present study, we examined the drug susceptibility and the presence of gyrA mutations in 17 clinical strains of S. typhi isolated from Surabaya, Indonesia, in 2006 (9 strains) and 2008 (8 strains). Although all 9 isolates from 2006 were sensitive to all tested antibiotics and had no mutation in the gvrA gene, all 8 isolates from 2008 were resistant to nalidixic acid and ampicillin and had a gyrA mutation at codon 87. In addition, 3 of 8 strains from 2008 showed multiple drug resistance, including resistance to chloramphenicol, trimethoprim-sulfamethoxazole, and ciprofloxacin. Therefore, newer drugs, such as ceftriaxone, cefixime, and azithromycin, might be effective in this situation. This is the 1st report of the emergence of fluoroquinolone-resistant clinical strains of S. typhi with gyrA mutation, and it reveals a health risk due to multidrug-resistant strains in Indonesia. (C) 2009 Elsevier Inc. All rights reserved.
ELSEVIER SCIENCE INC, Aug. 2009, DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 64(4) (4), 422 - 426, English
[Refereed]
Scientific journal

A Pilot Study of Quality of Life of Patients with Hormone-refractory Prostate Cancer after Gene Therapy
Shuji Terao, Toshiro Shirakawa, Bishnu Acharya, Masahiro Miyata, Nobuyuki Hinata, Kazushi Tanaka, Atsushi Takenaka, Isao Hara, Michio Naoe, Kohzo Fuji, Takatsugu Okegawa, Eiji Higashihara, Sadao Kamidono, Masato Fujisawa, Akinobu Gotoh
Background: The effects on quality of life (QOL) after a Phase I/II clinical trial of a combination of osteocalcin promoter-driven herpes simplex virus thymidine kinase (Ad-OC-TK) gene therapy and valacyclovir (VAL) were investigated for patients with hormone-refractory prostate cancer (HRPC). Patients and Methods: The QOL of six patients was prospectively assessed after gene therapy on days 0, 14, and 28. A modified questionnaire was created based on the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire's prostate cancer-specific module (EORTC QLQ-PR25). Results: The scores of all items significantly improved along with the total score. Further, bodily pain was significantly reduced on day 28. Moreover, the rate of change in the serum prostate-specific antigen levels from day 0 to day 28 was significantly correlated with the rate of change in bodily pain. Conclusion: In this clinical trial, Ad-OC-TK plus VAL treatment significantly improved the short-term QOL and bodily pain of patients with localized recurrence or bone metastases of HRPC.
INT INST ANTICANCER RESEARCH, May 2009, ANTICANCER RESEARCH, 29(5) (5), 1533 - 1537, English
[Refereed]
Scientific journal

Differential expression of osteocalcin during the metastatic progression of prostate cancer
Thomas A. Gardner, Sang-Jin Lee, Sang-Don Lee, Xiong Li, Toshihiro Shirakawa, Dong Deuk Kwon, Ra Young Park, Kyu Youn Ahn, Chaeyong Jung
Osteocalcin expression is restricted to osteoblasts and serum osteocalcin level is elevated in metastatic bone tumors including prostate tumours, which predominantly metastasizes to the bone and causes typical osteoblastic lesions. Previously, we have reported that osteocalcin RNA is widely expressed but incompletely spliced in the prostate including prostate tumors. Considering that many studies using osteocalcin-driven gene therapy have been conducted to treat hormone refractory metastatic tumors, detailed mechanisms controlling osteocalcin expression needs to be clarified. We aim to learn how osteocalcin expression is regulated during the metastatic process of prostate cancer. We applied assays of immunohistochemistry and RNA in situ hybridization in prostate tumors acquired from prostate (15) and metastatic sites, 13 from lymph node and 14 from bone. RT-PCR analysis in various cultured prostate cells was also performed. As predicted, osteocalcin RNA was highly expressed in most prostate epithelial cells of tumors, regardless of metastatic status of the tumor. However, osteocalcin protein was undetectable in tumors acquired from the primary site or lymph nodes whereas protein was highly expressed in the majority of bone-metastasized prostate tumors. RT-PCR analysis demonstrated that there was more completely spliced form of osteocalcin RNA present in bone-derived prostate cancer cells. Our data suggest that osteocalcin RNA was expressed but not completely spliced in non-bone environment, ultimately resulting in improper production of osteocalcin protein. This study explains why serum osteocalcin level is increased in patients with bone-metastasized prostate cancers. Yet, it remains to be clarified what regulates bone-specific osteocalcin RNA splicing in prostate tumors.
SPANDIDOS PUBL LTD, Apr. 2009, ONCOLOGY REPORTS, 21(4) (4), 903 - 908, English
[Refereed]
Scientific journal

Cyclooxygenase 2 Promoter-Based Replication-Selective Adenoviral Vector for Hypopharyngeal Cancer
Takahiro Nakagawa, Hironori Tanaka, Toshiro Shirakawa, Akinobu Gotoh, Yoshitake Hayashi, Katsuyuki Hamada, Mamoru Tsukuda, Ken-ichi Nibu
Objective: To explore the potential clinical application of the oncolytic activity of cyclooxygenase 2 (COX-2) promoter-based, conditional, replication-selective adenovirus vector for hypopharyngeal squamous cell carcinoma. Design: In vivo study and retrospective study. Setting: Kobe University Hospital, Kobe, Japan. Subjects: Expression of COX-2 in hypopharyngeal cancers treated at Kobe University Hospital was immunohistochemically investigated. In addition, nude mice bearing human hypopharyngeal cancer cells (H891) were used to analyze oncolytic activity of a conditional replication-selective adenovirus vector in which the expression of E1a, required for viral replication, is controlled by the COX-2 promoter Ad-COX2-E1a. Results: In vivo assays showed significant growth suppression in the murine hypopharyngeal model. Cyclooxygenase 2 expression was observed in 75.3% of hypopharyngeal cancers, especially in differentiated tumor cells (P =. 001; r = 0.433). Conclusion: In this study, we demonstrated the potential of oncolytic therapy using the COX-2-promoter based, conditional, replication-selective adenovirus for COX-2-expressing hypopharyngeal squamous cell carcinomas.
AMER MEDICAL ASSOC, Mar. 2009, ARCHIVES OF OTOLARYNGOLOGY-HEAD & NECK SURGERY, 135(3) (3), 282 - 286, English
[Refereed]
Scientific journal

OP-021 大腸菌の臨床株におけるgyrA,parC遺伝子変異とフルオロキノロン系抗菌薬耐性の相関についての検討(尿路性器感染症,一般演題口演,第97回日本泌尿器科学会総会)
安福富彦, 重村克巳, 白川利朗, 中野雄造, 田中一志, 武中篤, 荒川創一, 木下承皓, 川端眞人, 藤澤正人
一般社団法人日本泌尿器科学会, 2009, 日本泌尿器科学会雑誌, 100(2) (2), 194 - 194, Japanese

OP-078 前立腺肥大症患者におけるシロドシンおよびナフトピジルの臨床効果に関する比較検討(BPH/Male LUTS2,一般演題口演,第97回日本泌尿器科学会総会)
白川利朗, 原口貴裕, 松本穣, 竹田雅, 森下真一, 山道深, 源吉顕治, 原田健一, 田中一志, 武中篤, 藤澤正人
一般社団法人日本泌尿器科学会, 2009, 日本泌尿器科学会雑誌, 100(2) (2), 208 - 208, Japanese

Quantitative proteomic analysis to discover potential diagnostic markers and therapeutic targets in human renal cell carcinoma
Noboru Okamura, Taro Masuda, Akinobu Gotoh, Toshiro Shirakawa, Shuji Terao, Naoki Kaneko, Kazuki Suganuma, Makoto Watanabe, Toshiya Matsubara, Ryota Seto, Jun Matsumoto, Megumi Kawakami, Motohiro Yamamori, Tsutomu Nakamura, Tatsurou Yagami, Toshiyuki Sakaeda, Masato Fujisawa, Osamu Nishimura, Katsuhiko Okumura
Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Recent advances in drug development are providing novel agents for the treatment of RCC, but the effects are still minimal. In addition, there is an urgent need to identify diagnostic markers for RCC. In this report, to discover potential diagnostic markers and therapeutic targets, we subjected RCC samples to a quantitative proteomic analysis utilizing 2-nitrobenzenesulfenyl (NBS) reagent. Proteins were extracted from RCC and adjacent normal tissue, obtained surgically from patients, and labeled with NBS reagent containing six C-12 or C-13. This was followed by trypsin digestion and the enrichment of labeled peptides. Samples were then subjected to analysis by MALDI-TOF MS. NBS-labeled peptides with a 6 Da difference were identified by MS/MS. Thirty-four proteins were upregulated in more than 60% of the patients of which some were previously known, and some were novel. The identity of a few proteins was confirmed by Western blotting and quantitative real time RT-PCR. The results suggest that NBS-based quantitative proteomic analysis is useful for discovering diagnostic markers and therapeutic targets for RCC.
WILEY-V C H VERLAG GMBH, Aug. 2008, PROTEOMICS, 8(15) (15), 3194 - 3203, English
[Refereed]
Scientific journal

Clinical study results of a phase I/II study of AD-OC-TK/VAL gene therapy for the patients with metastatic or local recurrent prostate cancer
Toshiro Shirakawa, Akinobu Gotoh, Shuji Terao, Nobuyuki Hinata, Kazumasa Goda, Kazushi Tanka, Atsushi Takenaka, Isao Hara, Sadao Kamidono, Masato Fujisawa
JOHN WILEY & SONS LTD, Apr. 2008, JOURNAL OF GENE MEDICINE, 10(4) (4), 433 - 434, English
[Refereed]

Impact and perspective on chronic kidney disease in an Asian developing country: A large-scale survey in north Vietnam
Jun Ito, Dinh Thi Kim Dung, Mai Tuyet Vuong, Do Gia Tuyen, Le Danh Vinh, Nguyen Thi Huong, Tran Bich Ngoc, Nguyen Thi Bich Ngoc, Mai Thi Hien, Dang Duc Hao, Lam Thi Kim Oanh, Do Thi Lieu, Masato Fujisawa, Masato Kawabata, Toshiro Shirakawa
Background: The prevalence of chronic kidney disease (CKD) in Asia is expected to increase along with increases of hypertension and diabetes. Most cases are not treated and progress to end-stage renal disease (ESRD) with an increased risk for cardiovascular complications. Renal replacement therapies are so expensive that most ESRD patients die without treatment. Thus, countermeasures against early stages of CKD are urgently needed. Nevertheless, basic information for CKD has not been reported in Vietnam. Methods: We conducted a survey of CKD in 8,505 inhabitants aged 1 40 years in Vietnam. Subjects with abnormal urinary findings were further examined, including serum creatinine levels. In this study, CKD was defined as <60 ml/min/1.73 m(2) of estimated creatinine clearance by the Cockcroft-Gault method. Results: We identified 3.1% of subjects as CKD (stages 3-5) with positive findings in urine test. We also found that elderly hypertension and malnutrition were independent risk factors for CKD in this population. Conclusion: We found a significant number of CKD patients in Vietnam. To avoid a CKD pandemic in Asia including Vietnam, we strongly suggest further analyses of risk factors and prognosis of CKD in these populations, and the development of efficient management systems suitable for Asia. Copyright (C) 2008 S. Karger AG, Basel.
KARGER, 2008, NEPHRON CLINICAL PRACTICE, 109(1) (1), C25 - C32, English
[Refereed]
Scientific journal

Impact and perspective on chronic kidney disease in an Asian developing country: a large-scale survey in North Vietnam
Fujisawa Masato, Shirakawa Toshiro
2008, Nephron Clinical practice, Vol. 109, No. 1, pp. c25-32(1) (1), c25 - 32, English
[Refereed]
Scientific journal

Etodolac, a selective cyclooxygenase-2 inhibitor, induces Upregulation of e-cadherin and has antitumor effect on human bladder cancer cells in vitro and in vivo
Asako Okamoto, Toshiro Shirakawa, Toshinori Bito, Katsumi Shigemura, Katsuyuki Hamada, Akinobu Gotoh, Masato Fujisawa, Masato Kawabata
OBJECTIVES Cyclooxygenase-2 (COX-2) is highly expressed in several human cancers, including bladder cancer. Thus, a selective COX-2 inhibitor could be useful as an antitumor agent for a range of cancers. In the present study, we investigated the antitumor effect and E-cadherin induction of etodolac, a highly selective COX-2 inhibitor, on human bladder cancer cells in vitro and in vivo. METHODS We examined the cytotoxicity of etodolac against three human bladder cancer cell lines, T24, 5637, and KK47, and performed quantitative reverse transcriptase-polymerase chain reaction to measure the mRNA expression of COX-2, and E-cadherin. RESULTS Etodolac showed significant cytotoxicity only to T24 cells, which expressed the greatest level of COX-2 mRNA and the lowest level of E-cadherin mRNA among the three cell lines. Etodolac also increased the E-cadherin. mRNA expression in T24 cells in vitro. We also found that etodolac suppressed tumor growth and induced E-cadherin expression and cell apoptosis in a T24 tumor xenograft mouse model. CONCLUSIONS Etodolac exhibited antitumor activity and induced E-cadherin expression in bladder cancer cells and might be useful for the clinical treatment and prevention of bladder cancer, especially in poorly differentiated bladder cancer with high COX-2 and low E-cadherin expression.
ELSEVIER SCIENCE INC, Jan. 2008, UROLOGY, 71(1) (1), 156 - 160, English
[Refereed]
Scientific journal

Long-term outcome of phase I/II clinical trial of Ad-OC-TK/VAL gene therapy for hormone-refractory metastatic prostate cancer
Toshiro Shirakawa, Shuji Terao, Nobuyuki Hinata, Kazushi Tanaka, Atsushi Takenaka, Isao Hara, Kazuro Sugimura, Masafumi Matsuo, Katsuyuki Hamada, Kohzo Fuji, Takatsugu Okegawa, Eiji Higashihara, Thomas A. Gardner, Chinghai Kao, Leland W. K. Chung, Sadao Kamidono, Masato Fujisawa, Akinobu Gotoh
We evaluated the long-term safety and efficacy of Ad-OC-TK (recombinant adenoviral vector carrying an osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) gene therapy for hormone-refractory prostate cancer. Ad-OC-TK/VAL therapy is the first in vivo adenovirus-mediated gene therapy to be used to treat metastatic prostate cancer, including bone metastasis. Six patients were enrolled in this trial, and two doses of Ad-OC-TK (2.5 x 10(9) or 2.5 x 10(10) plaque-forming units) were injected into locally recurrent tumor or bone metastasis on day 1 and day 8. Patients were also given VAL (3 g/day) for 21 days. Safety and efficacy were evaluated for at least 8 months in each patient. All patients tolerated this therapy with no serious adverse events. One prostate-specific antigen (PSA) response (from 318.3 to 4.9 ng/ml) was observed with a time to PSA progression (TTP) of 12 months. Docetaxel (30 mg/m(2) per week) and estramustine (560 mg/day) combination chemotherapy (DE) was given to three docetaxel-naive patients on PSA failure after gene therapy. All three patients had a PSA response to DE therapy with 21, 7, and 4 months of TTP. These results suggest that additional trials are warranted.
MARY ANN LIEBERT INC, Dec. 2007, HUMAN GENE THERAPY, 18(12) (12), 1225 - 1232, English
[Refereed]
Scientific journal

Midkine promoter-based conditionally replicative adenovirus for targeting midkine-expressing human bladder cancer model
Shuji Terao, Toshiro Shirakawa, Shuji Kubo, Acharya Bishunu, Sang-Jin Lee, Kazumasa Goda, Mamoru Tsukuda, Katsuyuk Hamada, Masatoshi Tagawa, Atsushi Takenaka, Masato Fujisawa, Akinobu Gotoh
OBJECTIVES To develop a novel therapeutic strategy against human bladder cancer using Ad-MK-Ela-a midkine (MK) promoter-regulated, conditionally replicating, adenovirus. METHODS We tested several human cancer cell lines in vitro, including those of bladder cancer (KK47, 5637, and T24), lung cancer (A549), and head and neck cancer (H891). In each cell line, we examined MK mRNA expression by TaqMan real-time quantitative polymerase chain reaction, MK promoter activity, after plasmid transfection, using a luciferase assay, and the transduction efficiency by co-transfection with the cytomegalovirus-beta-gal plasmid. In these cells, we assessed the cell type-specific replication of Ad-MK-Ela virus by measuring the Ela DNA copy number by real-time polymerase chain reaction and the cell growth inhibition due to this virus using the Alamar blue assay. In animal studies, nude mice were subcutaneously inoculated with KK47 cells and later intratumorally injected with phosphate-buffered saline or Ad5-CMV-LacZ or Ad-MK-Ela. RESULTS The MK mRNA expression level and MK promoter-driven luciferase activity were relatively greater and markedly increased, respectively, in the 5637, A549, and KK47 cells than in the T24 and H891 cells. After Ad-MK-EIa infection, the Ela DNA copy number increased more significantly in the KK47, 5637, and A549 cells than in the T24 and H891 cells. At a multiplicity of infection of 0.01, Ad-MK-Ela significantly inhibited KK47 and 5637 cell growth. In vivo, Ad-MK-Ela injection markedly inhibited KK47 tumor growth. CONCLUSIONS We have demonstrated the antitumor effect of Ad-MK-Ela in a human bladder cancer model overexpressing MK mRNA.
ELSEVIER SCIENCE INC, Nov. 2007, UROLOGY, 70(5) (5), 1009 - 1013, English
[Refereed]
Scientific journal

Carrier cell-mediated delivery of a replication-competent adenovirus for cancer gene therapy
Katsuyuki Hamada, Junzo Desaki, Kou Nakagawa, Ting Zhang, Toshiro Shirakawa, Akinobu Gotoh, Masatoshi Tagawa
Although replication-competent viruses have been developed to treat cancers, their cytotoxic effects are insufficient, as infection is inhibited by the generation of neutralizing antibodies. To address this limitation, we developed a carrier cell system to deliver a replication-competent adenovirus. Carrier cells infected with replication-competent adenovirus were incubated with target cancer cells in a high titer of anti-adenovirus antibody. Carrier cells were injected into syngeneic subcutaneous tumors after immunization with adenovirus. Carrier cell-derived cell fragments containing viral particles were engulfed by proliferative target cancer cells. This engulfment-mediated transfer of adenovirus was not inhibited by the anti-adenovirus antibody and enabled repetitive infection. After the induction of anti-adenoviral cytotoxic T-lymphocyte (CTL) responses by immunization with adenovirus, administration of carrier cells infected with a replication-competent adenovirus induced complete tumor regression. Adenovirus-GM-CSF augmented the anti-tumor effect of carrier cells by increasing anti-adenoviral and anti-tumoral CTL responses and decreased the number of injections of carrier cells required to induce complete tumor regression. This novel carrier cell mediated viral transfection system might prove useful in a variety of cancer therapies.
NATURE PUBLISHING GROUP, Jun. 2007, MOLECULAR THERAPY, 15(6) (6), 1121 - 1128, English
[Refereed]
Scientific journal

Induction of immunological antitumor effects by adenovirus-mediated gene transfer of B7-1 in a murine squamous cell carcinoma cell line
Yasuhiko Hoshitani, Haruhiko Ishida, Naoki Otsuki, Toshiro Shirakawa, Akinobu Gotoh, Ken-ichi Nibu
Objective: To evaluate the antitumor immune effects of B7-1 gene expression mediated by adenoviral vectors against squamous cell carcinoma. Transfection of the costimulatory molecule B7-1 gene into certain murine tumors increases antitumor immunity and suppresses tumor growth. Design: In vitro and in vivo study. Interventions: A murine squamous cell carcinoma cell line, KLN205, was infected with adenoviral vectors carrying either B7-1 (AdB7) or LacZ (AdCL) genes. Infected cells were injected subcutaneously into the flanks of DBA/2 mice. Main Outcome Measures: The growth of tumors infected with adenviral vectors was measured. Results: AdB7-infected cells grew significantly slower than AdCL-infected cells in vivo, while there was no significant difference in the growth rates between the 2 groups in vitro. Moreover, significant growth suppression of rechallenged noninfected parental cells was observed in the mice immunized with AdB7-infected cells but not in those immunized with AdCL-infected cells. Conclusion: These results suggest that the B7-1 gene has therapeutic potential for immunotherapy against head and neck squamous cell carcinoma.
AMER MEDICAL ASSOC, Mar. 2007, ARCHIVES OF OTOLARYNGOLOGY-HEAD & NECK SURGERY, 133(3) (3), 270 - 275, English
[Refereed]
Scientific journal

OP-318 ヒト膀胱癌細胞における選択的 Cox-2阻害剤、Etodolac のE-cadherin 発現増強および抗腫瘍効果の検討(第95回日本泌尿器科学会総会)
白川利朗, 岡本明子, 重村克巳, 寺尾秀治, 後藤章暢, 川端眞人, 藤澤正人
一般社団法人日本泌尿器科学会, 2007, 日本泌尿器科学会雑誌, 98(2) (2), 370 - 370, Japanese

PP-329 マウス前立腺癌モデルに対するPETを用いたAd-OC-TK/VAL遺伝子治療効果および遺伝子導入効率の評価に関する有用性の検討(第95回日本泌尿器科学会総会)
寺尾秀治, 白川利朗, 久保秀司, 田沢周作, 富永英之, 千田道雄, 武中篤, 藤澤正人, 後藤章暢
一般社団法人日本泌尿器科学会, 2007, 日本泌尿器科学会雑誌, 98(2) (2), 479 - 479, Japanese

PP-302 選択的 cyclooxygenase-II (COX-2) 阻害剤 etodolac のヒト前立腺がん細胞株における抗腫瘍効果の検討(第95回日本泌尿器科学会総会)
重村克巳, 白川利朗, 和田義孝, 後藤章暢, 藤澤正人
一般社団法人日本泌尿器科学会, 2007, 日本泌尿器科学会雑誌, 98(2) (2), 472 - 472, Japanese

Adenovirus-mediated transfer of human papillomavirus 16 E6/E7 antisense RNA and induction of apoptosis in cervical cancer
Katsuyuki Hamada, Toshiro Shirakawa, Akinobu Gotoh, Jack A. Roth, Michele Follen
Objective. In most cervical cancers, human papillornaviruses (HPVs) are identified. The E6 and E7 genes of HPVs encode proteins, that interfere with the function of the tumor suppressor proteins p53 and Rb. We are exploring the potential use of antisense HPV RNA transcripts for gene therapy for HPV-positive cervical cancers. Methods. Via a recombinant adenoviral vector, Ad5CMV-HPV 16 AS, we introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into human cervical cancer SiHa cells harboring HPV 16. We then analyzed the effects of expression of these genes on cell and tumor growth. Results. HPV 16 E6/E7 antisense RNA was detected for 14 days in Ad5CMV-HPV 16 AS-infected cells. After infection, E6 and E7 protein expression was suppressed, and p53 and Rb protein expression increased. The Ad5CMV-HPV 16 AS-infected cells underwent apoptosis in vitro and in vivo. Cell growth and tumorigenicity were greatly suppressed. Ad5CMV-HPV 16 AS treatment significantly reduced the volumes of established subcutaneous tumors. Conclusion. Transfection of cervical cancer cells with HPV 16 E61E7 antisense RNA in a form such as Ad5CMV-HPV 16 AS might be a potentially useful approach to the therapy of HPV 16-positive cervical cancer. Published by Elsevier Inc.
ACADEMIC PRESS INC ELSEVIER SCIENCE, Dec. 2006, GYNECOLOGIC ONCOLOGY, 103(3) (3), 820 - 830, English
[Refereed]
Scientific journal

Sccmec typing and detection of visa-related genes in methicillin-resistant staphylococcus aureus clinical strains from Kobe University Hospital, Japan
Tetsuo Takata, Toshiro Shirakawa, Jun Ito, Asako Okamoto, Muh Nasrum Massi, Shohiro Kinoshita, Mochammad Hatta, Masato Kawabata
A total of 50 clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) were collected from Kobe University Hospital in 2003. Molecular typing of SCCmec was performed by multiplex polymerase chain reaction (PCR) and the presence of six genes (vraR, vraG, vraA, vraF, fruA, and fruB) associated with vancomycin (VCM) resistance was examined by simple PCR analysis. Out of 50 MRSA strains isolated 47 strains contained Type II SCCmec and the remaining contained Type IV SCCmec. Thirty seven strains contained pUB110 plasmid. VraA was present in 69% of the strains, vraF in 10%, vraG in 53%, and vraR in 16%. Noteworthy, strains without pUB110 contained vraR in relatively higher frequency (31%) compared with strains with pUB110 (11%).
Nov. 2006, Southeast Asian Journal of Tropical Medicine and Public Health, 37(6) (6), 1149 - 1155, English
[Refereed]
Scientific journal

Genetically engineered Bifidobacterium animalis expressing the Salmonella flagellin gene for the mucosat immunization in a mouse model
Tetsuo Takata, Toshiro Shirakawa, Yoshiko Kawasaki, Shohiro Kinoshita, Akinobu Gotoh, Yasunobu Kano, Masato Kawabata
Background A critical component of the host defense against enteric infections is the immunological response of the mucosal membrane, a major starting point of infectious disease, such as typhoid fever. The mucosal immune system consists of an integrated network of lymphoid tissues, mucous membrane-associated cells, and effector molecules. In the present study, we developed a recombinant Bifidobacterium animalis (B. animalis) genetically modified with the Salmonella flagellin gene for mucosal immunization as an oral typhoid vaccine. Methods We constructed an oral vaccine against Salmonella typhimurium, consisting of recombinant B. animalis containing the flagellin gene of Salmonella. The recombinant B. animalis was administered orally to mice every other day for 6 weeks. Anti-flagellin antibodies in the serum and stools were measured by enzyme-linked immunosorbent assay (ELISA). Results We detected significantly higher levels of flagellin-specific IgA in the serum and stools of the mice treated with the recombinant B. animalis containing the flagellin gene than was seen in those treated with parental B. animalis. Conclusions Our findings suggest that an oral vaccination using recombinant B. animalis genetically modified with the flagellin gene of Salmonella may be effective against Salmonella infections. Copyright (c) 2006 John Wiley & Sons, Ltd.
JOHN WILEY & SONS LTD, Nov. 2006, JOURNAL OF GENE MEDICINE, 8(11) (11), 1341 - 1346, English
[Refereed]
Scientific journal

内分泌療法抵抗性前立腺癌に対するAd-OC-TK plus Valacyclovir遺伝子治療臨床研究におけるquality of lifeの評価
寺尾秀治, 白川利朗, 日向信行, 和田義孝, 宮田賢宏, 久保秀司, 田中一志, 武中篤, 荒川創一, 原勲, 藤澤正人, 後藤章暢
(一社)西日本泌尿器科学会, Oct. 2006, 西日本泌尿器科, 68(増刊) (増刊), 159 - 159, Japanese

Enumeration of bacterial cell numbers and detection of significant bacteriuria by use of a new flow cytometry-based device
Hiroshi Okada, Toshiro Shirakawa, Akinobu Gotoh, Yutaka Kamiyama, Satoru Muto, Hisamitsu Ide, Yukio Hamaguchi, Shigeo Horie
A new, automated How cytometry-based urine bacterium analyzer (UBA) was developed. We assessed the UBA for linearity of measurement, reproducibility of results, carryover rate, and correlation of measured results with those determined by urine culture. We also evaluated its ability to screen urine samples for significant bacteriuria. The UBA showed excellent linearity and a minor carryover rate. Results from the UBA were highly reproducible, and in between-run precision assays, the coefficients of variation for the UBA results were smaller than those for the urine culture results. Two hundred seventy-three urine specimens from patients attending the outpatient clinics of two university-based hospitals were examined. The results for the UBA were compared with those for urine culture. The UBA detected significant bacteriuria with a sensitivity of 96.6%, a specificity of 79.9%, a positive predictive value of 57.0%, a negative predictive value of 98.8%, a false-positive rate of 15.8%, a false-negative rate of 0.7%, and an accuracy of 83.5%. These results were comparable to or better than those obtained with previously reported screening procedures. The UBA can perform accurate enumeration of bacterial cells automatically in 90 seconds and can be used for the screening of significant bacteriuria.
AMER SOC MICROBIOLOGY, Oct. 2006, JOURNAL OF CLINICAL MICROBIOLOGY, 44(10) (10), 3596 - 3599, English
[Refereed]
Scientific journal

Study of AZFc partial deletion gr/gr in fertile and infertile Japanese males
Claudia Marcia Benedetto de Carvalho, Luciana Werneck Zuccherato, Masato Fujisawa, Toshiro Shirakawa, Andrea Kely Campos Ribeiro-dos-Santos, Sidney E. B. Santos, Sergio Danilo Junho Pena, Fabricio Rodrigues Santos
A recurrent partial azoospermia factor C (AZFc) deletion, called gr/gr, has been reported to be a male infertility risk factor. A specific type of Y chromosome observed in approximately 30% of Japanese males (haplogroup D derived at YAP+) is believed to have a fixed gr/gr deletion. A recent study claimed that spermatogenic failure is more likely in males with D Y chromosomes, because of the gr/gr deletion, the presence of which is not well characterized among D haplogroup chromosomes. We therefore decided to perform a systematic study of the frequency of the gr/gr deletion in the Japanese. We studied fertile and infertile males to investigate the possibility of different gr/gr frequencies. The deletions were detected by use of single tagged-sequences (STSs) and the D haplogroup sub-lineages typing were done by use of the biallelic markers M174, M64, M116.1, 12f2.2, M15, M151, and M125. Analysis of gr/gr deleted Y chromosomes showed that all are classified as haplogroup D2, suggesting a lineage association. The subtype D2b1 was most frequent among the Japanese, in control and infertile samples. The haplogroups D2b2, D*, and D1 were not found in any population group. Remarkably, we observed no statistical difference between haplogroup D sub-lineages of the infertile and control groups, although the statistical power of this study is low. This study suggests lack of significant evidence of increased infertility risk in haplogroup D Japanese males. We were also able to establish the ancestral chromosome that suffered a gr/gr deletion, and propose a new Y chromosome phylogeny for haplogroup D and its derivatives. In summary, we were able to define the frequency of gr/gr deletion in Japanese males and show that the gr/gr deletion was probably present in the ancestral Y chromosome that entered Japan at least 12,000 years ago.
SPRINGER TOKYO, Sep. 2006, JOURNAL OF HUMAN GENETICS, 51(9) (9), 794 - 799, English
[Refereed]
Scientific journal

Progress report on phase I/II clinical trial of Ad-OC-TK plus VAL therapy for metastatic or locally recurrent prostate cancer: Initial experience at Kobe University
Nobuyuki Hinata, Toshiro Shirakawa, Shuji Terao, Kazumasa Goda, Kazushi Tanaka, Yuji Yamada, Isao Hara, Sadao Kamidono, Masato Fujisawa, Akinobu Gotoh
There is no effective therapy for hormone-refractory prostate cancer and a novel therapeutic modality, such as a gene therapy, should be actively pursued. Previously, Gardner and Chung conducted a phase I clinical trial of Ad-OC-TK (recombinant adenoviral vector containing osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) for the treatment of hormone-refractory prostate cancer at the University of Virginia. We report on our ongoing phase I/II clinical trial of Ad-OC-TK plus VAL for the treatment of advanced prostate cancer at the Kobe University Hospital, Japan.
WILEY, Jun. 2006, INTERNATIONAL JOURNAL OF UROLOGY, 13(6) (6), 834 - 837, English
[Refereed]
Scientific journal

Effects of nafamostat mesilate on ADP-induced platelet aggregation and disaggregation in hemodialysis patients
M Miyata, T Shirakawa, B Acharya, S Terao, A Gotoh
Nafamostat mesilate (NM), a synthetic protease inhibitor, is the most commonly used anticoagulant in the setting of extracorporeal circulation (ECQ in patients with bleeding tendency. It inhibits both platelet aggregation and activation of coagulation factors. Although it has been reported that NM disaggregates aggregated platelets, little is known about such an effect in the setting of hemodialysis therapy (HD). We examined the effects of NM on adenosine 5'-diphosphate (ADP)-induced platelet aggregation and disaggregation using platelet-rich plasma obtained from 6 HD patients. The platelet aggregation was stimulated by 3 mu M ADP and change of aggregation was monitored by an aggregometer. NM adjusted to the final concentrations of 0.1 (1.9 x 10(-7)), 1.0 (1.9 x 10(-6)), 10, (1.9 x 10(-5)), and 100 (1.9 x 10(-4)) mu g/ml (M) or veronal-buffered saline (VBS) as control was added before or after to the stimulation of ADP. NM not only inhibited platelet aggregation, but also disaggregated already aggregated platelets at concentrations of 1.0 mu g/mln or higher. Moreover, NM almost completely disaggregated at 100 mu g/ml. This NM concentration of 1.0 mu g/ml was lower than the therapeutic concentration in ECC of HD (i.e., 10(-5)M). Both inhibitory and disaggregatory effects of NM expressed a dose-related dependency. Our results suggest that NM can exert both aggregation inhibitory and disaggregatory effects on platelets of HD patients within the therapeutic concentration.
LIPPINCOTT WILLIAMS & WILKINS, May 2006, ASAIO JOURNAL, 52(3) (3), 272 - 275, English
[Refereed]
Scientific journal

Decreased susceptibility to fluoroquinolones and gyrA gene mutation in the Salmonella enterica serovar Typhi and Paratyphi A isolated in Katmandu, Nepal, in 2003
T Shirakawa, B Acharya, S Kinoshita, S Kumagai, A Gotoh, M Kawabata
Typhoid fever is the most common clinical diagnosis among febrile patients presenting to hospital in Katmandu. Salmonella enterica serovar Typhi (S. enterica serovar Typhi) and Salmonella enterica serovar Paratyphi A (S. enterica serovar Paratyphi A) with decreased susceptibility to fluoroquinolones and resistance to nalidixic acid are common in recent years. In the present study, we examined the in vitro susceptibility to fluoroquinolones and the presence of gyrA gene mutations in 30 clinical strains of S. Typhi and 39 of S. Paratyphi A, all of which were isolated in Katmandu, Nepal, in 2003. In those strains, we found that 73.3% and 94.9% of S. Typhi and S. Paratyphi A strains contained gyrA gene mutation, and showed the resistance to a quinolone, nalidixic acid, and decreased susceptibility to fluoroquinolones, ciprofloxacin, and levofloxacin. Although fluoroquinolones may still be useful as antibiotics for the treatment of typhoid fever, clinicians should be aware of the possibility of treatment failures of infections with S. Typhi and S. Paratyphi A strains with decreased susceptibility to fluoroquinolones. (c) 2006 Elsevier Inc. All rights reserved.
ELSEVIER SCIENCE INC, Apr. 2006, DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 54(4) (4), 299 - 303, English
[Refereed]
Scientific journal

視床下部下垂体精巣軸におけるNeuropeptide YおよびPancreatic polypeptideの役割
合田上政, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 土橋正樹, 大岡均至, OKADA,Hiroshi, 上野尚彦, INUI,Akio, FUJISAWA, Masato
一般社団法人日本泌尿器科学会, Mar. 2006, 日本泌尿器科学会雑誌, 97巻, 2号, pp.378-378(2) (2), 378 - 378, Japanese
International conference proceedings

α1受容体遮断薬による前立腺肥大症の薬物療法についての排尿症状(I-PSS)を中心とした長期薬効の検討
SHIRAKAWA, Toshiro, 寺尾秀治, 大場健司, YAMADA,Yuji, 日向信之, WADA,Yoshitaka, 合田上政, OKADA,Hiroshi, GOTOH,Akinobu, FUJISAWA, Masato
(一社)日本泌尿器科学会, Mar. 2006, 日本泌尿器科学会雑誌, 97巻, 2号, pp.506-506(2) (2), 506 - 506, Japanese
International conference proceedings

ヒト膀胱癌細胞に対するミドカインプロモーターを組み込んだ増殖制限型アデノウイルスベクター(AD-MK-E1a)の有用性の検討
寺尾秀治, SHIRAKAWA, Toshiro, 合田上政, 濱田雄行, 田川雅敏, GOTOH,Akinobu, FUJISAWA, Masato
一般社団法人日本泌尿器科学会, Mar. 2006, 日本泌尿器科学会雑誌, 97巻, 2号, pp.513-513(2) (2), 513 - 513, Japanese
International conference proceedings

Rapid detection of the fluoroquinolone resistance-associated ParC mutation in Neisseria gonorrhoeae using TaqMan probes
K Shigemura, T Shirakawa, K Tanaka, S Arakawa, A Gotoh, M Fujisawa
Backgrounds: In fluoroquinolone-resistant Neisseria gonorrhoeae, the amino acid mutations in the fluoroquinolone-resistant determining region (QRDR) of the parC gene are an important factor. The aim of the present study was to develop a rapid detection method of a serine 88 to proline substitution in parC which we previously showed as having significantly higher fluoroquinolone minimal inhibitory concentrations (MIC) using the TaqMan discrimination system. Methods: We extracted DNA from 90 urine or urethral swab samples obtained from male patients with urethritis caused by N. gonorrhoeae. After DNA extraction, they were subjected to real-time polymerase chain reaction (PCR) using a TaqMan discrimination system and compared with the results of conventional DNA sequencing. Results: Of the 90 samples, the TaqMan technique result showed 13 samples that were classified as having a serine 88 to proline mutation in parC, and 77 samples that did not have a serine 88 to proline mutation in parC. The classifications of all samples completely corresponded to those determined by conventional DNA sequencing. We also found that N. gonorrhoeae with a serine 88 to proline mutation in parC have a significantly higher MIC to ciprofloxacin than that without a serine 88 to proline mutant in parC. Conclusions: The present genotyping method of real-time PCR using a TaqMan discrimination system could be applied to the rapid detection of a serine 88 to proline amino acid mutation in parC of N. gonorrhoeae. This point mutation is significant for the determination of fluoruquinolone resistance. This rapid detection system may lead to the prevention of use of noneffective antimicrobial agents and a decrease of resistant strains.
BLACKWELL PUBLISHING, Mar. 2006, INTERNATIONAL JOURNAL OF UROLOGY, 13(3) (3), 277 - 281, English
[Refereed]
Scientific journal

Mitochondrial DNA determines androgen dependence in prostate cancer cell lines
M Higuchi, T Kudo, S Suzuki, TT Evans, R Sasaki, Y Wada, T Shirakawa, Sawyer, JR, A Gotoh
Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is very common in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstrated that the androgen-independent cell line C4-2, established by inoculation of the androgen-dependent LNCaP cell line into castrated mice, has a greatly reduced amount of normal mitochondrial DNA and an accumulation of large-deletion DNA. Strikingly, the depletion of mitochondrial DNA from androgen-dependent LNCaP resulted in a loss of androgen dependence. Reconstitution of normal mitochondrial DNA to the mitochondrial DNA-depleted clone restored androgen dependence. These results indicate that mitochondrial DNA determines androgen dependence of prostate cancer cell lines. Further, mitochondrial DNA-deficient cells formed tumors in castrated athymic mice, whereas LNCaP did not. The accumulation of large deletion and depletion of mitochondrial DNA may thus play a role in the development of androgen independence, leading to progression of prostate cancers.
NATURE PUBLISHING GROUP, Mar. 2006, ONCOGENE, 25(10) (10), 1437 - 1445, English
Scientific journal

Antitumor effects of etodolac, a selective cyclooxygenase-II inhibitor, against human prostate cancer cell lines in vitro and in vivo
K Shigemura, T Shirakawa, Y Wada, S Kamidono, M Fujisawa, A Gotoh
Objectives. To investigate the effects of the selective cyclooxygenase-2 (COX-2) inhibitor etodolac on prostate cancer cell lines in vitro and in vivo and on E-cadherin expression in prostate cancer cells. Methods. We evaluated the cytotoxicity of etodolac on the three prostate cancer cell lines LNCaP, C4-2, and PC-3. We also performed quantitative real-time polymerase chain reaction to measure the mRNA expression of COX-2, Bcl-2, and E-cadherin in these cell lines after etodolac treatment. In addition, we investigated the in vivo antitumor effects of etodolac on a human prostate cancer xenograft model. Results. Etodolac exhibited significant antitumor effect in vivo and in vitro. The cytotoxicity of etodolac in LNCaP and C4-2 was markedly increased at a dose of 1000 nM in a time-dependent and dose-dependent manner. In the in vivo tumor growth study, the etodolac-treated mice exhibited more significant cytotoxicity than the phosphate-buffered saline-treated mice. Expression of E-cadherin after etodolac treatment tended to increase and that of Bcl-2 to decrease, but the expression of COX-2 had no definite tendency. Conclusions. The COX-2 inhibitor etodolac exhibited an antitumor effect on prostate cancer cell lines in vitro and in vivo, and it might be useful for the treatment of hormone-resistant prostate cancer.
ELSEVIER SCIENCE INC, Dec. 2005, UROLOGY, 66(6) (6), 1239 - 1244, English
[Refereed]
Scientific journal

腎臓癌におけるVEGF発現量と遺伝子型
田中久登, OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 山森元博, 瀬戸亮太, 中村任, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
Nov. 2005, 臨床薬理, 36巻, Suppl., pp.S268-S268, Japanese
International conference proceedings

前立腺肥大症に対するα1受容体遮断薬長期投与症例における排尿症状動態に関する検討
SHIRAKAWA, Toshiro, 寺尾秀治, 日向信之, 合田上政, OOBA, Takeshi, WADA,Yoshitaka, YAMADA,Yuji, GOTOH,Akinobu, OKADA,Hiroshi, FUJISAWA, Masato
(一社)日本排尿機能学会, Oct. 2005, 日本排尿機能学会誌, 16巻, 1号, pp.92-92(1) (1), 92 - 92, Japanese
International conference proceedings

視床下部下垂体精巣軸におけるNeuropeptide YおよびPancreatic polypeptideの役割に関する検討
合田上政, 寺尾秀治, 山口耕平, 土橋正樹, OOBA, Takeshi, KAMIDONO,Sadao, FUJISAWA, Masato, GOTOH,Akinobu, SHIRAKAWA, Toshiro, OKADA,Hiroshi
Oct. 2005, 日本不妊学会雑誌, 50巻, 4号, pp.321-321, Japanese
International conference proceedings

EGFR mRNA is upregulated, but somatic mutations of the gene are hardly found in renal cell carcinoma in Japanese patients
T Sakaeda, N Okamura, A Gotoh, T Shirakawa, S Terao, M Morioka, K Tokui, H Tanaka, T Nakamura, M Yagi, Y Nishimura, M Yokoyama, K Okumura
Purpose. Heterozygous somatic mutations of epidermal growth factor receptor (EGFR) in exons 18, 19, and 21 were recently reported to be associated with response to gefitinib in patients having nonsmall cell lung cancer. Such mutations are more frequently found among Japanese than Europeans. In this work, the frequency of mutations was investigated in renal cell carcinoma (RCC) samples obtained from Japanese subjects to examine the potential of gefitinib as a therapeutic agent for RCC. Methods. Nineteen patients with RCC, who gave written informed consent, were enrolled in this study. mRNA expression levels of EGFR were measured in RCC and its adjacent noncancerous renal tissue via the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Somatic mutations were determined using genomic DNA extracted from RCC by direct sequencing method. Results. mRNA expression was confirmed to be about 19 times higher in RCC than in adjacent noncancerous renal tissues, but no such mutations were detected in both. Conclusion. Results from this study do not support the validity of further clinical trials on gefitinib for RCC with genotyping even in Japanese patients, although EGFR plays a key role in tumor progression.
SPRINGER/PLENUM PUBLISHERS, Oct. 2005, PHARMACEUTICAL RESEARCH, 22(10) (10), 1757 - 1761, English
[Refereed]
Scientific journal

視床下部下垂体精巣軸におけるNeuropeptide YおよびPancreatic polypeptideの役割
合田上政, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 山口耕平, 土橋正樹, OOBA, Takeshi, OKADA,Hiroshi, 上野尚彦, INUI,Akio, KAMIDONO,Sadao, FUJISAWA, Masato
Sep. 2005, 日本内分泌学会雑誌, 81巻, 2号, pp.493-493, Japanese
International conference proceedings

ヒト膀胱癌細胞株に対するフラロデンドリマーを用いた光線力学療法の可能性
合田上政, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 高口豊, FUJISAWA, Masato
Sep. 2005, 日本癌治療学会誌, 40巻, 2号, pp.627-627, Japanese
International conference proceedings

ヒト膀胱癌細胞株に対するフラロデンドリマーを用いた光線力学療法の可能性
合田上政, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 高口豊, FUJISAWA, Masato
Sep. 2005, 日本癌学会64回総会記事, pp.393-393, Japanese
International conference proceedings

ヒト膀胱癌細胞に対するミドカインプロモーターを組み込んだ増殖制限型アデノウイルスベクター(AdMKE1a)の有用性の検討
寺尾秀治, SHIRAKAWA, Toshiro, 合田上政, FUJISAWA, Masato, GOTOH,Akinobu
Sep. 2005, 日本癌学会64回総会記事, pp.506-506, Japanese
International conference proceedings

バングラデシュ,ランガマティ県における熱帯熱マラリアのクロロキンプリマキン治療の有効性
松本安代, SHIRAKAWA, Toshiro, 高田哲男, KAWABATA, Masato
(一社)日本感染症学会, Sep. 2005, 感染症学雑誌, 79巻, 9号, pp.717-718(9) (9), 717 - 718, Japanese
International conference proceedings

Real-time PCR法を用いた腸チフスの血中細菌定量法の開発
SHIRAKAWA, Toshiro, 松本安代, 高田哲男, ARAKAWA, Souichi, 木下承晧, KUMAGAI, Syunichi, GOTOH,Akinobu, KAWABATA, Masato
(一社)日本感染症学会, Sep. 2005, 感染症学雑誌, 79巻, 9号, pp.794-794(9) (9), 794 - 794, Japanese
International conference proceedings

2003年臨床分離MRSAにおけるバンコマイシン耐性関連遺伝子保有状況
高田哲男, SHIRAKAWA, Toshiro, 松本安代, 木下承晧, KUMAGAI, Syunichi, GOTOH,Akinobu, KAWABATA, Masato
(一社)日本感染症学会, Aug. 2005, 感染症学雑誌, 79巻, 8号, pp.581-582(8) (8), 581 - 582, Japanese
International conference proceedings

Recombinant interleukin-2 enhanced the antitumor effect of ADV/RSV-HSV-tk/ACV therapy in a murine bladder cancer model
S Terao, T Shirakawa, K Goda, S Kamidono, M Fujisawa, A Gotoh
Background: Previous studies demonstrated the antitumor effects of IL-2 and ADV/RSV-HSV-tk in bladder tumor models. In our study, we employed the intramuscular injection of recombinant IL-2 combined with ADV/RSV-HSV-tk gene therapy in the MBT-2 murine bladder tumor model. Materials and Methods: In the in vitro study, after adenoviral gene transduction efficiency had been assessed, the cytotoxicity of ADV/RSV-HSV-tk/ACV was examined. In the in vivo study, ADV/RSV-HSV-tk was injected into MBT-2 subcutaneous tumors, ACV was injected intraperitoneally daily for 13 days and recombinant IL-2 was injected intramuscularly daily for 10 days. Results: The X-gal staining of MBT-2 cells infected with 125 multiplicity of injection (MOI) indicated > 20% adenoviral gene transduction efficiency. The cell growth of MBT-2 infected with 125 MOI was significantly inhibited by 40 mu M of ACV. In the in vivo study, the combination therapy significantly inhibited tumor growth in the MBT-2 tumor model. Conclusion: The systemic administration Of recombinant IL-2 in combination with HSV-tk gene therapy exhibited an enhanced antitumor effect.
INT INST ANTICANCER RESEARCH, Jul. 2005, ANTICANCER RESEARCH, 25(4) (4), 2757 - 2760, English
Scientific journal

A replication-selective adenoviral vector for head and neck cancers
H Tanaka, T Shirakawa, ZJ Zhang, K Hamada, A Gotoh, K Nibu
Objective: To test the oncolytic activity of cyclooxygenase 2 (COX-2) promoter-based conditional replication-selective adenovirus vector for squamous cell carcinoma cells of the head and neck. Design: In vitro study. Subjects: None. Interventions: A conditional replication-selective adenovirus vector in which the expression of E1a, required for viral replication, is controlled by the COX-2 promoter, Ad-COX2-E1a, was generated. Its oncolytic activity according to the levels of COX-2 and of Coxsackie and adenovirus receptor expression was tested in a series of human head and neck squamous cell carcinoma cell lines. Results: The respective COX-2 messenger RNA expression ratios of KB, H891, T891, T892, and L871 were 1.5, 60.0, 1.0, 14.6, and 1.3. The corresponding Coxsackie and adenovirus receptor messenger RNA expression ratios were 1, 1, 5, 3, and 1. In vitro assays showed significant growth suppression of cancer cell lines with strong expressions of COX-2. Conclusion: This study demonstrated the possibility of oncolytic therapy using the COX-2 promoter-based conditional replication-selective adenovirus for head and neck squamous cell carcinoma expressing COX-2.
AMER MEDICAL ASSOC, Jul. 2005, ARCHIVES OF OTOLARYNGOLOGY-HEAD & NECK SURGERY, 131(7) (7), 630 - 634, English
[Refereed]
Scientific journal

ラット副腎皮質細胞のMicroencapsulationによるホルモン補充の可能性
合田上政, 寺尾秀治, 山口耕平, 近藤有, 土橋正樹, KAMIDONO,Sadao, FUJISAWA, Masato, SHIRAKAWA, Toshiro, GOTOH,Akinobu, OKADA,Hiroshi
Jun. 2005, 泌尿器科紀要, 51巻, 6号, pp.429-429, Japanese
International conference proceedings

Quantitative detection of Salmonella enterica serovar Typhi from blood of suspected typhoid fever patients by real-time PCR
MN Massi, T Shirakawa, A Gotoh, A Bishnu, M Hatta, M Kawabata
We quantified the gene copies from Salmonella enterica serovar Typhi (S. Typhi) in the blood of patients suspected of having typhoid fever by using TaqMan-based real-time PCR (TaqMan assay) to target the S. Typhi flagellin gene in genomic DNAs isolated from blood samples. Of 55 blood samples taken from suspected typhoid fever patients, eight blood samples with a positive blood culture had S. Typhi loads ranging from 1.01 x 10(3) to 4.35 x 10(4) copies/ml blood, and from 47 blood samples with negative blood culture, there were 40 (85.1%) TaqMan assay-positive samples with loads ranging from 3.9 to 9.9 x 10(2) copies/ml blood. In the present study, the TaqMan assay detected more than 10(3) copies/ml blood of S. Typhi in all of the blood culture-positive samples, whereas less than 10(3) copies/ml blood of S. Typhi were detected in the blood culture-negative samples. Our findings suggest that a TaqMan assay may be useful for assessing S. Typhi loads, especially in cases of suspected typhoid fever with negative results from the standard blood culture test. (c) 2005 Elsevier GmbH. All rights reserved.
URBAN & FISCHER VERLAG, Jun. 2005, INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, 295(2) (2), 117 - 120, English
Scientific journal

前立腺癌骨転移巣に対する遺伝子治療臨床研究神戸大学医学部附属病院での実施例の報告
SHIRAKAWA, Toshiro, GOTOH,Akinobu, 寺尾秀治, 日向信之, HARA, Isao, KAMIDONO,Sadao
医学図書出版(株), May 2005, 泌尿器外科, 18巻, 臨増, pp.498-498(臨増) (臨増), 498 - 498, Japanese
International conference proceedings

Granulocyte-colony stimulating factor producing urothelial carcinoma of renal pelvis
S Terao, Y Yamada, T Shirakawa, Hara, I, N Kanomata, S Kamidono
We report a case of granulocyte-colony stimulating factor (G-CSF) producing urothelial carcinoma of the renal pelvis in a 39-year old man. The patient was admitted to Kobe University Hospital, Kobe, Japan, complaining of macrohematuria and a 6-month history of left abdominal swelling. Abdominal computed tomography showed a large mass in the left kidney and para-aortic lymph node enlargement. A remarkable degree of leukocytosis was detected without any acute infectious disease. Enzyme immunoassay of the serum demonstrated a remarkable high concentration of G-CSF. The patient underwent left nephroureterectomy and para-aortic lymphadenectomy. Histochemical examination revealed urothelial carcinoma. Immunohistochemical staining with an anti-G-CSF antibody demonstrated G-CSF secreting cells. The patient died 8 weeks after the surgical operation. To our knowledge, this is the second case of G-CSF producing urothelial carcinoma of renal pelvis reported in the English literature.
BLACKWELL PUBLISHING ASIA, May 2005, INTERNATIONAL JOURNAL OF UROLOGY, 12(5) (5), 500 - 502, English
[Refereed]
Scientific journal

Rapid detection and differentiation of Gram-negative and Gram-positive pathogenic bacteria in urine using TaqMan probe
K Shigemura, T Shirakawa, H Okada, K Tanaka, S Kamidono, S Arakawa, A Gotoh
Urinary tract infection has been shown to be quite complicated and often difficult to diagnose and treat. For appropriate diagnosis, it is very important to find the correct Gram stain classification as soon as possible, especially in severe cases where there is a possibility of severe sepsis developing. In order to solve this problem, we developed a new method to detect a Gram stain of bacteria obtained from 1 ml of urine from urinary tract infection patients using a consensus real-time PCR protocol with a TaqMan probe that allows detection of spiked bacterial 16S DNA from urine. We extracted DNA of 55 urine samples obtained from patients with complicated urinary tract infection and at the same time performed urine culture testing. After DNA extraction, they were subjected to real-time PCR using a TaqMan discrimination system. Sixteen kinds of bacteria were cultured from the urine culture testing. Of these bacteria, eight were classified as Gram-positive bacteria and the other eight were classified as Gram-negative bacteria. Of the 55 samples, the TaqMan technique result showed 27 samples that were classified as Gram-negative bacteria; 11 samples that were Gram-positive, 10 that included both Gram-negative and -positive bacteria, and 7 that showed no amplification. The classifications of all samples corresponded exactly to those determined by urine culture testing. The present genotyping method of real-time PCR using a TaqMan discrimination system could be applied to the rapid detection of Gram-positive or -negative bacteria in urine of urinary tract infection patients. This assay can differentiate those species tested, but whether the presence of other (untested) bacteria could lead to misinterpretation is unknown. For further investigation, it is important to test other (untested) bacteria in the near future.
SPRINGER, Mar. 2005, CLINICAL AND EXPERIMENTAL MEDICINE, 4(4) (4), 196 - 201, English
[Refereed]
Scientific journal

腎癌における各種遺伝子のmRNA発現変動及び遺伝子型
OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 中村任, 盛岡正志, 徳井健次, 田中久登, YAGI, Mariko, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
Mar. 2005, 薬剤学, 65巻, Suppl., pp. 189-189, Japanese
International conference proceedings

マウス膀胱癌におけるADV/RSV-HSV-TK遺伝子治療とRecombinant IL-2免疫療法との併用療法
寺尾秀治, SHIRAKAWA, Toshiro, 合田上政, 日向信之, KAMIDONO,Sadao, GOTOH,Akinobu
(一社)日本泌尿器科学会, Mar. 2005, 日本泌尿器科学会雑誌, 96巻, 2号, pp. 284-284(2) (2), 284 - 284, Japanese
International conference proceedings

マウス造精機能障害モデルにおけるTRAIL発現抑制の効果
合田上政, FUJISAWA, Masato, SHIRAKAWA, Toshiro, 寺尾秀治, 山口耕平, 近藤有, 土橋正樹, OOBA, Takeshi, GOTOH,Akinobu, OKADA,Hiroshi, KAMIDONO,Sadao
日本生殖内分泌学会, Mar. 2005, 日本泌尿器科学会雑誌, 96巻, 2号, pp. 220-220, 37 - 42, Japanese
International conference proceedings

バングラデシュ,ランガマティ県における熱帯熱マラリアのクロロキンプリマキン治療の有効性
松本安代, SHIRAKAWA, Toshiro, 高田哲男, KAWABATA, Masato
(一社)日本感染症学会, Mar. 2005, 感染症学雑誌, 79巻, 臨増, pp. 214-214(臨増) (臨増), 214 - 214, Japanese
International conference proceedings

Real-time PCR法を用いた腸チフスの血中細菌定量法の開発
SHIRAKAWA, Toshiro, 松本安代, 高田哲男, ARAKAWA, Souichi, 木下承晧, KUMAGAI, Syunichi, GOTOH,Akinobu, KAWABATA, Masato
(一社)日本感染症学会, Mar. 2005, 感染症学雑誌, 79巻, 臨増, pp. 303-303(臨増) (臨増), 303 - 303, Japanese
International conference proceedings

2003年臨床分離MRSAにおけるバンコマイシン耐性関連遺伝子保有状況
高田哲男, SHIRAKAWA, Toshiro, 松本安代, 木下承晧, KUMAGAI, Syunichi, GOTOH,Akinobu, KAWABATA, Masato
(一社)日本感染症学会, Mar. 2005, 感染症学雑誌, 79巻, 臨増, pp. 141-141(臨増) (臨増), 141 - 141, Japanese
International conference proceedings

[Gene therapy].
Akinobu Gotoh, Toshiro Shirakawa, Yoshitaka Wada, Nobuyuki Hinata, Shuji Terao, Isao Hara, Soichi Arakawa, Sadao Kamidono, Hiroshi Okada, Atsushi Takenaka, Masato Fujisawa
We selected bone-metastatic prostate cancer as the target form of recurrent prostate cancer and developed a suicide-gene therapy based on an adenovirus vector with an organ-specific osteocalcin promoter. Related clinical studies have already been conducted in the United States at the University of Virginia, where results so far have established the safety of this therapy. In the present paper, in addition to presenting the results of these gene-therapy studies from the basic research to the clinical stage, we discuss the clinical studies begun by our group in August 2003. In the 21st century, therapeutic systems in use are undergoing major changes. Gene therapy is likely to become an important therapeutic option in recurrent prostate cancer. In terms of theory and technology however, this form of treatment is still at a very immature stage of development. We look forward to evolution in this field to provide an established treatment for recurrent prostate cancer and are committed to actively continuing with the development of gene therapy through translational research.
Feb. 2005, Hinyokika kiyo. Acta urologica Japonica, 51(2) (2), 75 - 9, Japanese, Domestic magazine
[Refereed]

ホルモン抵抗性前立腺癌転移巣に対する治療法について
GOTOH,Akinobu, SHIRAKAWA, Toshiro, WADA,Yoshitaka, 日向信之, 寺尾秀治, HARA, Isao, ARAKAWA, Souichi, KAMIDONO,Sadao, OKADA,Hiroshi, TAKENAKA, Atsushi, FUJISAWA, Masato
ホルモン抵抗性前立腺癌転移巣に対する治療法について報告した.著者等の企図する遺伝子治療は,1)HSV-TKタンパク質の全ての翻訳領域を含む遺伝子を用いた「自殺遺伝子治療」であり,TSV-TK遺伝子により最終的に三リン酸化された代謝産物は癌細胞のDNA合成を阻害し,DNA鎖の分裂およびアポトーシスを引き起こした.2)HSV-TK遺伝子の上流に臓器特異性のマウスOCプロモーターを組み込んだアデノウイルスベクター(ad-OC-TKベクター)を前立腺癌の局所再発巣へ直接,CT・超音波ガイド下に局所注入した.OCプロモーターを活性できない正常細胞ではHSV-TKタンパクは発現せず,ad-OC-TKベクターには感染しなかった.3)我が国において,2名の患者にad-OC-TKベクター投与を実施し,投与後1日目の血中と1週間後の組織中にウイルスDNAを検出し,組織中にのみHSV-TKmRNAが検出された.2例とも重篤な副作用は認めなかった
泌尿器科紀要刊行会, 2005, 泌尿器科紀要, 51巻, pp.75-79(2) (2), 75 - 79, Japanese
Scientific journal

Identification and sequencing of Salmonella Enterica serotype typhi isolates obtained from patients with perforation and non-perforation typhoid fever
Muhammad Nasrum Massi, Toshiro Shirakawa, Akinobu Gotoh, Mochammad Hatta, Masato Kawabata
We describe the characterization of Salmonella enterica serovar Typhi, isolated from the blood of patients with perforation and non-perforation typhoid fever, by a combination of conventional microbiological tests, 16S rRNA gene sequencing, and flagellin gene and CDP-tyvelose epimerase (rfbE) gene sequencing. The 16S rRNA gene sequencing showed that there were four base mutations from perforation samples and only three from non-perforation samples. These findings indicated that the isolates were a strain of Salmonella enterica. The flagellin gene sequences from the two groups were 100% identical to that of the H1-d flagellin gene of serovar Typhi. Sequences of the rfbE from both groups were also 100% identical.
Jan. 2005, Southeast Asian Journal of Tropical Medicine and Public Health, 36(1) (1), 118 - 122, English
Scientific journal

Measles encephalitis in early pregnancy and after delivery
Yasuyo Matsumoto, Ippei Morimoto, Takahiro Shibutani, Masaaki Mukubou, Toshiro Shirakawa, Akinobu Gotoh, Masato Kawabata
We present two cases of measles encephalitis, one in early pregnancy and one after delivery. In case 1, the patient became unconscious 6 days after the appearance of a rash and was treated with glycerol and immunoglobulin. In case 2, the patient became unconscious 6 days after the appearance of a rash and was also treated with glycerol and immunoglobulin. Both of them recovered without any neurological sequelae. Pregnancy is a risk factor for severe measles complications, and vaccination should be promoted much more in countries with poor measles control, such as Japan. © Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2005.
Springer Japan, 2005, Journal of Infection and Chemotherapy, 11(2) (2), 97 - 100, English
[Refereed]
Scientific journal

Presence of a mutation in ponA1 of Neisseria gonorrhoeae in numerous clinical samples resistant to various β-lactams and other, structurally unrelated, antimicrobials
Katsumi Shigemura, Toshiro Shirakawa, Nasrum Massi, Kazushi Tanaka, Soichi Arakawa, Akinobu Gotoh, Masato Fujisawa
The number of resistant strains in patients with Neisseria gonorrhoeae urethritis has been increasing, making effective treatment difficult. Chromosomally mediated penicillin-resistant N. gonorrhoeae arise through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. To understand the occurrence of penicillin resistance in patients with N. gonorrhoeae infection, we performed this study. In addition, we studied minimum inhibitory concentrations (MICs) of antimicrobials against N. gonorrhoeae strains. We measured the MICs of penicillin G, other β-lactams, and other kinds of antimicrobials against 53 clinical N. gonorrhoeae isolates from male patients with urethritis in Hyogo and Osaka, Japan. The ponA genes, encoding PBP 1 of these isolates, were sequenced. Of the 53 isolates tested, 41 strains showed some resistance to penicillin G. A mutation in the ponA (ponA1) gene was identified in 46 isolates. There was a tendency that ponA mutant (ponA1) in N. gonorrhoeae led to higher antimicrobial MICs of β-lactam antimicrobial agents (including penicillin) than those of non-ponA mutants. However, we found lower than expected MICs of penicillin and β-lactams even in ponA mutants. Therefore, we consider that detailed investigations for the further understanding of the effect of other genes, such as penC (which is reported to be related to ponA1 in achieving high-level penicillin resistance) should be our next step. © Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2005.
Springer Japan, 2005, Journal of Infection and Chemotherapy, 11(5) (5), 226 - 230, English
[Refereed]
Scientific journal

遺伝子治療が奏功した進行性前立腺癌の1例
SHIRAKAWA, Toshiro, GOTOH,Akinobu, 寺尾秀治, 日向信之, 重村克己, 合田上政, WADA,Yoshitaka, MURAMAKI,Mototsugu, TANAKA, Kazushi, YAMADA,Yuji, HARA, Isao, KAMIDONO,Sadao
泌尿器科紀要刊行会, Dec. 2004, 泌尿器科紀要, 50巻, 12号, pp. 911-911(12) (12), 911 - 911, Japanese
International conference proceedings

Rapid detection of gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae by denaturing high-performance liquid chromatography
K Shigemura, T Shirakawa, H Okada, K Tanaka, T Udaka, S Kamidono, S Arakawa, A Gotoh
The detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that is used to detect mutations in human DNA. This is the first report that this technique is used as a tool to detect mutations in genes encoding fluoroquinolone resistance in Neisseria gonorrhoeae. Eighty-one strains of N. gonorrhoeae were used in this study. Genomic DNA from each strain was subjected to PCR amplification of 225 bp in gyrA and 166 bp in parC spanning the fluoroquinolone-resistance determining regions (QRDRs). After we per-formed DNA sequencing of these amplicons and identification of mutations in the QRDRs, DHPLC was undertaken to investigate whether its results correlate the distinctive chromatogram with their DNA mutations pattern. The profilings detected by DHPLC completely corresponded to the results of the DNA sequencing in mutation patters in gyrA and parC genes. They resulted in the following amino acid substitutions: Ser-91Phe, Asp-95Gly, and Asp-95Asn in gyrA; and Gly-85Asp, Asp-86Asn, Ser-87Arg, and Ser-88Pro in parC, respectively. These mutations existed alone or as combinations. and we identified five mutations patterns in gyrA and six in parC including wild-type. These mutations and their patterns could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This novel detection system facilitates the detection of resistance.
ELSEVIER SCIENCE BV, Dec. 2004, JOURNAL OF MICROBIOLOGICAL METHODS, 59(3) (3), 415 - 421, English
[Refereed]
Scientific journal

Association of VEGF genotype with mRNA level in colorectal adenocarcinomas
M Yamamori, T Sakaeda, T Nakamura, N Okamura, T Tamura, N Aoyama, T Kamigaki, M Ohno, T Shirakawa, A Gotoh, Y Kuroda, M Matsuo, M Kasuga, K Okumura
The mRNA expression of vascular endothelial growth factor (VEGF) was evaluated in colorectal adenocarcinomas and adjacent noncancerous colorectal tissues in IS Japanese patients. The expression was confirmed to be up-regulated in the colorectal adenocarcinomas, when compared with the noncancerous tissues. Twelve genotypes of VEGF: six positions in the promoter region, two in the 5'UTR, and four in the 3'UTR, and their association with the expression of VEGF mRNA were evaluated. While G-1877A, T-1455C, G-1154A, C702T, and G1612A were not detected, C-2578A, T-1498C, G-1190A,C-634G, C-7T, C936T, and C1451T were found at allele frequencies of 4/36, 15/36, 15/36, 20/36, 8/36, 6/36, and 6/36, respectively, suggesting that C-2578A, G-1154A, and G1612A were associated with a decreased-risk for colorectal adenocarcinoma. T-1498C (G-1190A) and C-7T were found to be associated with higher levels of VEGF mRNA, and may be a risk factor for the development of liver metastasis and/or prognosis of colorectal adenocarcinoma. (C) 2004 Elsevier Inc. All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, Dec. 2004, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 325(1) (1), 144 - 150, English
[Refereed]
Scientific journal

Inhibition mechanism of Gosha-jinki-gan on the micturition reflex in rats
A Gotoh, K Goto, A Sengoku, T Shirakawa, Y Akao, M Fujisawa, H Okada, S Arakawa, S Kamidono
We investigated the actions of Gosha-jinki-gan, a traditional Japanese medicine containing processed Aconiti tubers, on urinary bladder function in anesthetized rats. In cystometrical investigations, Gosho-jinki-gan (1.0 g/kg, i.d.) increased bladder capacity as well as micturition threshold pressure. In addition, it decreased the frequency of distension-induced rhythmic bladder contractions. However, it did not influence the amplitude of bladder contractions induced by electrical stimulation of the pontine micturition center. The inhibitory effect of Gosha-jinki-gan on bladder motility was abolished by pretreatment with nor-binaltorphimine (10 mg/kg, s.c.), and was diminished by the concomitant use of anti-dynorphin A antiserum (10 mug, i.t.), yohimbine (10 mug, i.t.), or methysergide (20 mug, i.t.). Processed Aconiti tuber extract (27 mg/kg, i.d.) also suppressed bladder motility, and the effect was abolished by nor-binaltorphimine. These results suggest that Gosha-jinki-gan attenuates bladder sensation via the kappa-opioid receptor-stimulating action of processed Aconiti tuber. Gosha-jinki-gan may be a useful anti-pollakiuria agent that does not influence bladder contractility at micturition.
JAPANESE PHARMACOLOGICAL SOC, Oct. 2004, JOURNAL OF PHARMACOLOGICAL SCIENCES, 96(2) (2), 115 - 123, English
[Refereed]
Scientific journal

膀胱癌におけるAd-RSV-tk遺伝子治療とIL-2免疫療法との併用療法(Interleukin-2 enhanced the anti-tumor effect of adenoviral-mediated HSV-tk gene therapy in a murine bladder cancer model)(英語)
Shirakawa Toshiro, Hinata Nobuyuki
日本癌学会, Sep. 2004, 日本癌学会総会記事, 63回(63回) (63回), 518 - 518, Japanese
Research society

膀胱癌におけるAd-RSV-tk遺伝子治療とIL-2免疫療法との併用療法(Interleukin-2 enhanced the anti-tumor effect of adenoviral-mediated HSV-tk gene therapy in a murine bladder cancer model)(英語)
寺尾秀治, KAMIDONO,Sadao, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 日向信之, 合田上政
日本癌学会, Sep. 2004, Cancer Science, 95巻, Suppl., pp. 518-518, 518 - 518, Japanese
International conference proceedings

前立腺癌治療戦略ホルモン抵抗性前立腺癌転移巣に対する遺伝子治療臨床研究
GOTOH,Akinobu, SHIRAKAWA, Toshiro, HARA, Isao, KAMIDONO,Sadao
Sep. 2004, 日本癌治療学会誌, 39巻, 2号, pp. 315-315, Japanese
International conference proceedings

マウス造精機能障害モデルにおけるTRAIL発現抑制の効果
合田上政, FUJISAWA, Masato, SHIRAKAWA, Toshiro, 近藤有, 土橋正樹, GOTOH,Akinobu, OKADA,Hiroshi, KAMIDONO,Sadao
Sep. 2004, 日本内分泌学会雑誌, 80巻, 2号, pp. 454-454, Japanese
International conference proceedings

ヒト前立腺小細胞癌株(SO-MI)に対するp53遺伝子導入の効果
合田上政, SHIRAKAWA, Toshiro, GOTOH,Akinobu, OKADA,Hiroshi, HARA, Isao, KAMIDONO,Sadao, FUJISAWA, Masato
Sep. 2004, Cancer Science, 95巻, Suppl., pp. 515-515, Japanese
International conference proceedings

Adenoviral-mediated HGF expression inhibits germ cell apoptosis in rats with cryptorchidism
K Goda, M Fujisawa, T Shirakawa, M Dobashi, G Shiota, ZJ Zhang, A Gotoh, S Kamidono
Background Prior studies have shown that the hepatocyte growth factor (HGF), as known for its multiple biological effects, possibly regulates spermatogenesis or tubulogenesis in the testis. To clarify the effect of HGF on restoration of spermatogenesis, or testicular weight, we transferred the HGF gene into the testis of the rat experimental cryptorchid model. Methods Replication-deficient recombinant adenoviral vectors containing the CAG promoter driving rat HGF (pAxCAHGF) and LacZ (pAxCALacZ) were constructed. Sprague-Dawley rats surgically induced with unilateral cryptorchidism and subsequent orchidopexy were divided into three groups: control (PBS), pAxCALacZ and pAxCAHGF by intratesticular injection. At 2 and 4 weeks after subsequent orchidopexy, testes were removed and weighed. These specimens were analyzed histopathologically, and examined for cell apoptosis. HGF expression in these specimens associated with c-Met receptor-mediated signal molecules was examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot or immunohistochemical study. Results Adenovirus-mediated HGF gene transfer induced overexpression of HGF in some seminiferous epithelial cells and interstitial cells, increased the phosphorylation of ERK and Akt, and decreased numbers of apoptotic cells of germ cells. HGF transduction also significantly increased the numbers of germ cells and testicular weight by 4 weeks compared with the other control groups. Conclusions Adenoviral-mediated HGF gene transfer into the testis in the cryptorchidism rats inhibited germ cell apoptosis and restored spermatogenesis. Copyright (C) 2004 John Wiley Sons, Ltd.
JOHN WILEY & SONS LTD, Aug. 2004, JOURNAL OF GENE MEDICINE, 6(8) (8), 869 - 876, English
[Refereed]
Scientific journal

Serum testosterone levels in patients with nonmosaic Klinefelter syndrome after testicular sperm extraction for intracytoplasmic sperm injection
H Okada, T Shirakawa, T Ishikawa, K Goda, M Fujisawa, S Kamidono
We measured testosterone levels in 24 patients with nonmosaic Klinefelter syndrome before and at 6 and 12 months after conventional or microdissection testicular sperm extraction. Testosterone levels decreased after surgery by either technique, and they did not recover to baseline concentrations, even when using less invasive microdissection techniques.
ELSEVIER SCIENCE INC, Jul. 2004, FERTILITY AND STERILITY, 82(1) (1), 237 - 238, English
[Refereed]
Scientific journal

Antiproliferative activity of Rhinacanthus nasutus (L.) kurz extracts and the active moiety, rhinacanthin C
A Gotoh, T Sakaeda, T Kimura, T Shirakawa, Y Wada, A Wada, T Kimachi, Y Takemoto, A Iida, S Iwakawa, M Hirai, H Tomita, N Okamura, T Nakamura, K Okumura
Rhinacanthus nasutus (L.) KURZ (Acanthaceae) is a shrub widely distributed in South China and India. In this study, the antiproliferative activity of the ethanol extract of root and aqueous extract of leaves of R. nasutus, and the supposed active moiety rhinacanthin C was assessed in vitro using the human cervical carcinoma cell line HeLa, its MDR1-overexpressing subline Hvr100-6, human prostate carcinoma PC-3 cells and human bladder carcinoma T24 cells. Rhinacanthin C was chemically synthesized and its content in the R. nasutus extracts was determined by HPLC with a photodiode array detector. The antiproliferative activity of the R. nasutus extracts was also assessed in vivo using sarcoma 180-bearing mice. It was suggested that 1) the in vitro antiproliferative activity of rhinacanthin C was comparable with or slightly weaker than that of 5-FU, 2) rhinacanthin C showed antiproliferative activity for MDR1-overexpressing Hvr100-6 cells, similarly to parent HeLa cells, 3) the in vitro antiproliferative activity of the ethanol extract of root R. nasutus was due to rhinacanthin C, whereas that of the aqueous extract of leaves of R. nasutus was due to constituents other than rhinacanthin C, and 4) both of the R. nasutus extracts showed in vivo antiproliferative activity after oral administration once daily for 14 d.
PHARMACEUTICAL SOC JAPAN, Jul. 2004, BIOLOGICAL & PHARMACEUTICAL BULLETIN, 27(7) (7), 1070 - 1074, English
[Refereed]
Scientific journal

A Cox-2 promoter-based replication-selective adenoviral vector to target the Cox-2-expressing human bladder cancer cells
T Shirakawa, K Hamada, ZJ Zhang, H Okada, M Tagawa, S Kamidono, M Kawabata, A Gotoh
Purpose: Cyclooxygenase-2 (Cox-2), an enzyme that catalyzes the synthesis of prostaglandins, is overexpressed in a variety of premalignant and malignant conditions, including urinary bladder cancer. In the present study, we examined the feasibility of using Cox-2 promoter-based replication-selective adenovirus for targeting bladder cancer cells that express Cox-2 transcriptional activity. Experimental Design: A series of human cancer cell lines, including three bladder cancer cell lines (KK47, T24, and 5637), were evaluated for their Cox-2 and CAR (the Coxsackievirus and adenovirus receptor) mRNA expression levels by quantitative real-time PCR. AdE3-cox2-327, a replication-selective adenovirus in which the expression of E1a is controlled by the Cox-2 promoter, was generated, and its tissue-specific activity was tested in vitro and in vivo. Results: Three bladder cancer cell lines express higher levels of Cox-2 mRNA than does the human prostate cancer cell line PC3, the primary cultured human benign prostatic fibroblast, PF cells, and the human colon cancer cell line Colo320. Relatively higher expression of CAR mRNA was detected in the KK47, 5637, respectively, and Colo320 than in the T24, PC-3, and PF cells. In vitro assays revealed significant growth suppression of both Cox-2- and CAR-expressing bladder cancer cells KK47 and 5637 in comparison with the other cells that lack Cox-2 expression and/or CAR expression. Conclusions: The present study demonstrated both specificity and efficacy of AdE3-cox2-327, a selectively replicated adenovirus, toward the Cox-2-expressing bladder cancer cells in vitro and in vivo. We also found that CAR expression in the target cancer cells is an important factor for the efficacy of selectively replicated adenovirus-based gene therapy.
AMER ASSOC CANCER RESEARCH, Jul. 2004, CLINICAL CANCER RESEARCH, 10(13) (13), 4342 - 4348, English
[Refereed]
Scientific journal

薬剤耐性淋菌性尿道炎の分子生物学的検討およびDHPLCを用いた迅速遺伝子診断法の開発
重村克巳, OKADA,Hiroshi, TANAKA, Kazushi, ARAKAWA, Souichi, KAMIDONO,Sadao, SHIRAKAWA, Toshiro, GOTOH,Akinobu, 木下承皓
May 2004, 泌尿器科紀要, 50巻, 5号, pp. 381-381, Japanese
International conference proceedings

nafamostat mesilateの血小板凝集解離作用
Shirakawa Toshiro, Hinata Nobuyuki
(一社)日本透析医学会, May 2004, 日本透析医学会雑誌, 37(Suppl.1) (Suppl.1), 856 - 856, Japanese
Research society

Long-term outcome of postoperative interferon-alpha adjuvant therapy for non-metastatic renal cell carcinoma
A Gotoh, T Shirakawa, N Hinata, Y Wada, Hara, I, M Fujisawa, G Kawabata, H Okada, S Akakawa, S Kamidono
Aim: To investigate the long-term efficacy of postoperative interferon-alpha (IFN-alpha) adjuvant therapy in preventing recurrence in non-metastatic renal cell carcinoma treated with radical nephrectomy and to identify related prognostic markers. Methods: Long-term follow-up was conducted to study rates of survival and non-recurrence in 88 subjects following radical nephrectomy for non-metastatic disease. Results: The overall survival rate was 90% at 5 years and 88% at 10, with corresponding non-recurrence rates of 81% and 74%. Survival rates reviewed by preadministration pT stage showed a falling tendency from T1 through to T3 in line with pathological progression; when cases at stage pT1b or below were compared with those at stage pT2 or above, the latter showed a tendency to lower survival rates (P = 0.0966, Breslow-Gehan-Wilcoxon). Similarly, non-recurrence rates tended to fall in line with pathological progression, with a significant difference found in the comparison of cases at stage pT1b or below with those at stage pT2 or above (P = 0.0265, log-rank, Mantel-Cox). Duration of IFN-alpha administration showed a tendency to positive correlation with long-term survival (P = 0.3765, Breslow-Gehan-Wilcoxon). Non-recurrence rate was not found to differ according to duration of administration. Comparison of groups with normal and abnormal preadministration inummosuppressive acidic protein values showed that the normal group tended to have higher rates of survival and non-recurrence (P = 0.3371, Breslow-Gehan-Wilcoxon). Conclusions: Immunosuppressive acidic protein values appear to be a useful predictive marker for recurrence. A randomized trial, examining long-term outcome according to tumor stage and variables such as duration of administration, dose, administration time, and dosing schedule is required.
BLACKWELL PUBLISHING ASIA, May 2004, INTERNATIONAL JOURNAL OF UROLOGY, 11(5) (5), 257 - 263, English
[Refereed]
Scientific journal

泌尿器科における漢方医学漢方薬の基礎研究と臨床応用頻尿に対する牛車腎気丸の効果
GOTOH,Akinobu, SHIRAKAWA, Toshiro, 日向信之, WADA,Yoshitaka, KAMIDONO,Sadao
牛車腎気丸の作用機序に関する基礎的研究および,臨床応用として前立腺肥大症における頻尿の改善効果を検討した.消化管投与による牛車腎気丸の効果を独自に検討し,抗コリン作用ではなく,機序の一つとして下行性抑制系の活性化により律動的膀胱収縮頻度を抑制し,さらにκオピオイド受容体を介して作用することなどを明らかにした.頻尿を主訴とする前立腺肥大症に対する牛車腎気丸の治療効果を検討し,頻尿を主訴とする前立腺肥大症患者に使いやすい治療法と考えられた
(株)医学書院, Apr. 2004, 臨床泌尿器科, 58巻, 5号, pp. 301-306(5) (5), 301 - 306, Japanese
[Refereed]
Scientific journal

Susceptibilities of Neisseria gonorrhoeae to fluoroquinolones and other antimicrobial agents in Hyogo and Osaka, Japan
K Shigemura, H Okada, T Shirakawa, K Tanaka, S Arakawa, S Kinoshita, A Gotoh, S Kamidono
Objectives: Decreasing susceptibility of Neisseria gonorrhoeae to fluoroquinolones has been reported in several countries. Knowledge of local N gonorrhoeae susceptibilities to various antimicrobials is important for establishing a rational treatment strategy in each region. Methods: Isolates of N gonorrhoeae from male urethritis patients attending four urological clinics in Hyogo and Osaka prefectures in Japan were collected during 2002. The MICs for nine antimicrobials: penicillin G, tetracycline, cefixime, ceftriaxone, levofloxacin, gatifloxacin, ciprofloxacin, moxifloxacin, and spectinomycin were determined for each isolate. All isolates were also tested for beta lactamase producing profiles. Results: Among the 87 isolates obtained, only one isolate was revealed to produce beta lactamase. MIC90 values for ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin were over 8 mug/ml, over 8 mug/ml, 4 mug/ml, and 2 mug/ml, respectively. The proportion of isolates resistant to fluoroquinolones was over 60% (ciprofloxacin, 70.1%; levofloxacin, 65.5%; gatifloxacin, 70.1%). Chromosomally mediated penicillin and tetracycline resistance was identified in 12.6% and 33.3% of the isolates. MIC90 values for cefixime and ceftriaxone and were 0.5 mug/ml and 0.0063 mug/ml. All isolates were sensitive to ceftriaxone and 90.8% of them were sensitive to cefixime. MIC90 for spectinomycin was 32 mug/ml and all isolates were sensitive to it. Fluoroquinolone resistance correlated significantly with MICs for penicillin G but not tetracycline. Conclusion: Ceftriaxone and spectinomycin demonstrated lower MICs and so are recommended for N gonorrhoeae. Susceptibilities of N gonorrhoeae should be monitored periodically by region.
B M J PUBLISHING GROUP, Apr. 2004, SEXUALLY TRANSMITTED INFECTIONS, 80(2) (2), 105 - 107, English
[Refereed]
Scientific journal

Mutations in the gyrA and parC genes and in vitro activities of fluoroquinolones in 91 clinical isolates of Neisseria gonorrhoeae in Japan
K Shigemura, T Shirakawa, H Okada, N Hinata, B Acharya, S Kinoshita, T Kofuku, M Kawabata, S Kamidono, S Arakawa, A Gotoh
Background and Objectives: Fluoroquinolone resistance in Neisseria gonorrhoeae has been associated with alternations in the quinolone-resistance determining regions in the gyrA and parC genes. Goal: The goal of this study was to investigate the correlation between fluoroquinolone minimum inhibitory concentrations (MICs) and mutations in the gyrA and parC genes of 91 N. gonorrhoeae clinical isolates from Japan. Study Design: The MICs of fluoroquinolones ciprofloxacin, levofloxacin, and gatifloxacin for 91 clinical isolates from male gonococcal urethritis in Hyogo or Osaka, Japan, were measured, and the gyrA and parC genes of these isolates were sequenced. Results: Among 91 isolates tested, over 70% isolates were resistant to ciprofloxacin. We found that 4 mutations (Ser-91-Phe, Ser-91-Ile, Asp-95-Gly in gyrA, and Ser-88-Pro in parC) had significant correlation to MICs of fluoroquinolone (ciprofloxacin, levofloxacin, and gatifloxacin). Conclusion: Some mutations in QRDR had a significant relationship to the fluoroquinolone resistance of 91 N. gonorrhoeae clinical isolates from Japan.
LIPPINCOTT WILLIAMS & WILKINS, Mar. 2004, SEXUALLY TRANSMITTED DISEASES, 31(3) (3), 180 - 184, English
[Refereed]
Scientific journal

Development of human chorionic gonadotropin subunit-beta promoter-based toxic gene therapy for testicular cancer
T Shirakawa, A Gotoh, ZJ Zhang, CH Kao, LWK Chung, TA Gardner
Objectives. To develop a new toxic gene therapy using the tissue-specific human chorionic gonadotropin-beta (hCG-beta) promoter for testicular cancer. Although most patients presenting with disseminated testicular tumor are cured through the use of chemotherapy with or without surgery, those patients with relapse after initial therapy present a difficult clinical problem. The serum tumor marker hCG-beta is frequently elevated in patients with testicular cancer, and the pretreatment and post-treatment levels of serum hCG-beta are highly predictive of treatment outcome. Methods. Human testicular embryonal carcinoma cell line, NEC 8, a human prostate cancer cell line, PC-3, and a human bladder cancer cell line, WH, were used in this study. A transient expression experiment was used to analyze the activity of a 729-bp hCG-beta promoter in all three cell lines. A recombinant adenovirus carrying thymidine kinase (Ad-hCG-beta-TK) under control of the hCG-beta promoter was generated. The tissue-specific activity of Ad-hCG-beta-TK was tested in vitro and in vivo. Results. The hCG-beta promoter had significantly greater activity in the hCG-beta-producing cell line (NEC 8) than in the non-hCG-beta-producing cell lines (PC-3 and WH). In vitro, Ad-hCG-beta-TK with acyclovir significantly inhibited NEC 8 growth but not PC-3 or WH cell growth. In vivo, Ad-hCG-beta-TK with acyclovir significantly inhibited NEC 8 subcutaneous tumor growth in nude mice. Conclusions. In this study, we explored the possibility of developing a new therapeutic agent to target and induce the killing of testicular germ cell tumor selectively by using tissue-specific hCG-beta promoters. UROLOGY 63: 613-618, 2004. (C) 2004 Elsevier Inc.
ELSEVIER SCIENCE INC, Mar. 2004, UROLOGY, 63(3) (3), 613 - 618, English
[Refereed]
Scientific journal

Chemosensitivity assessed by collagen gel droplet embedded culture drug sensitivity test, and MDR1, MRP1, and MRP2 mRNA expression in human colorectal adenocarcinomas
T Nakahara, T Sakaeda, T Nakamura, T Tamura, C Nishioka, N Aoyama, N Okamura, T Shirakawa, A Gotoh, T Kamigaki, M Ohno, Y Kuroda, M Matsuo, M Kasuga, K Okumura
Purpose. To evaluate chemosensitivity and its correlation with expression levels of the multidrug resistant transporter (MDR1) and the multidrug resistance-associated proteins 1 and 2 (MRP1, MRP2) mRNA in human colorectal adenocarcinomas. Methods. Colorectal adenocarcinomas were obtained as surgical samples from 25 patients. The chemosensitivity of 12 anticancer drugs was assessed by the collagen gel droplet embedded culture drug sensitivity test (CD-DST). The expression levels of MDR1, MRP1, and MRP2 mRNA in colorectal adenocarcinomas were also evaluated by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results. The chemosensitivity was successfully evaluated for 16 of 25 patients, and the anticancer drugs were effective against the samples showing a relatively high growth rate. Gemcitabine hydrochloride was found to be more promising than those often prescribed for the treatment of colorectal adenocarcinoma. There was no correlation of the mRNA expression levels of MDR1 and MRP1 with the chemosensitivity of any anticancer drugs tested, but mitomycin C was found to be more effective for the colorectal adenocarcinoma with relatively high expression of MRP2 mRNA.
KLUWER ACADEMIC/PLENUM PUBL, Mar. 2004, PHARMACEUTICAL RESEARCH, 21(3) (3), 406 - 412, English
[Refereed]
Scientific journal

外傷性膀胱・直腸破裂術後,回腸Blind loop部に小腸膀胱瘻を生じた1例
熊野晶文, SHIRAKAWA, Toshiro, TANAKA, Kazushi, HARA, Isao, KAWABATA,Gaku, OKADA,Hiroshi, KAMIDONO,Sadao, 生田繁, FUJISAWA, Masato
Feb. 2004, 泌尿器科紀要, 50巻, 2号, pp. 140-140, Japanese
International conference proceedings

PP4-143 ラットSertoli細胞へのErythropoietin遺伝子導入とその生理学的意義(一般演題(ポスター))
合田上政, 藤澤正人, 白川利朗, 土橋正樹, 近藤有, 岡田弘, 守殿貞夫
一般社団法人日本泌尿器科学会, 2004, 日本泌尿器科学会雑誌, 95(2) (2), 566 - 566, Japanese

Effects of Gosha-Jinki-Gan on the pollakisuria
Gotoh A, Shirakawa T, Hinata N, Wada Y, Kamidono S
2004, Japanese Journal of Clinical Urology, 58(5) (5), 301 - 306
[Refereed]

Quantitative detection of Escherichia coli from urine of patients with bacteriuria by real-time PCR.
Nobuyuki Hinata, Toshiro Shirakawa, Hiroshi Okada, Katsumi Shigemura, Sadao Kamidono, Akinobu Gotoh
INTRODUCTION: Compared with the classical urine culture method, PCR is more rapid, and can detect smaller numbers of bacteria, however it is inferior for quantification. Because of the lack of quantification in routine PCR, the meaning of a positive PCR test result has not been validated for all infections. We report on the development of a novel quantitative detection system for the urinary tract infection (UTI) Escherichia coli using real-time PCR. PATIENTS: We enrolled 200 patients with suspected bacteriuria. METHODS: The gene encoding the universal stress protein (uspA) was found to be highly specific for E. coli. We quantified the copy numbers of E. coli in the urine of patients with UTI by using a real-time PCR assay (the TaqMan system) targeting uspA genes in genomic DNAs isolated from urine samples (n=200). To evaluate the feasibility of this method, the results were compared with those of a standard urine culture. RESULTS: The incidence of positive urine cultures was 75% (150 of 200), and various doses of E. coli were detected in 84 of 150 specimens. The real-time PCR method also detected 84 cases of urinary infections of E. coli in the same specimens. Furthermore, the result of the quantification of E. coli using real-time PCR strongly correlated (r2=0.925) with the result of urine culture. CONCLUSION: Our results suggest that using quantitative-PCR means a faster and simpler diagnosis of E. coli urinary infection can be made compared with the traditional urine culture method.
2004, Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology, 8(3) (3), 179 - 84, English, International magazine
[Refereed]
Scientific journal

Quantitative analysis of the levels of expression of muscarinic receptor subtype RNA in the detrusor muscle of patients with overactive bladder.
Nobuyuki Hinata, Toshiro Shirakawa, Hiroshi Okada, Bishnu Achaya, Sadao Kamidono, Akinobu Gotoh
INTRODUCTION: Antimuscarinic drugs have frequently been used for the treatment for patients with an overactive bladder (OAB) and there have been many studies on the distribution of muscarinic receptor subtypes in the bladder. However, the distribution of muscarinic receptor subtypes in OAB patients has not been well investigated. In this study we investigated the distribution of muscarinic receptor subtypes with mRNA and protein expressions in patients with and without OAB, and investigated both the dome and trigone area. METHODS: Samples of bladder smooth muscle were obtained from 10 individuals, five patients with OAB and a non-OAB group consisting of five patients who received radical cystectomy. RESULTS: The M2 receptor was predominant, but there was no significant difference in the level of M2 expression between the groups in the dome area. M5 expression in the dome area was significantly higher in the OAB group than in the non-OAB group. In the trigone area, the level of M2 mRNA expression was the highest in the non-OAB group, and was significantly lower in the OAB group. The levels of M1 and M5 mRNA expression were also observed in samples obtained from the trigone area. CONCLUSION: The multiformity of the muscarinic receptor subtypes in human bladder smooth muscle was confirmed, and our results suggest that the efficacy of a given pharmacologic therapy differs from patient to patient.
2004, Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology, 8(1) (1), 17 - 22, English, International magazine
[Refereed]
Scientific journal

Messenger RNA levels and enzyme activities of 5 alpha-reductase types 1 and 2 in human benign prostatic hyperplasia (BPH) tissue
T Shirakawa, H Okada, B Acharya, ZJ Zhang, N Hinata, Y Wada, T Uji, S Kamidono, A Gotoh
BACKGROUND. Benign prostatic hyperplasia (BPH) development requires testicular androgens and aging. The principle prostatic androgen is dihydrotestosterone (DHT). Testosterone is converted to DHT by the enzyme 5 alpha-reductase. Two distinct 5 alpha-reductase enzymes, types 1 and 2, have been identified. While some studies have suggested that type 2 isoenzyme predominates in the prostate, studies on the prostatic localization of the two isoenzymes are controversial. The purpose of this study was to determine the quantitative expressions of 5 alpha-reductase types 1 and 2 in BPH tissues. METHODS. We examined the localizations of types 1 and 2 isoenzymes in BPH tissues using immunohistochemical staining and a real-time quantitative RT-PCR assay using the TaqMan system. We measured the enzyme activities of types 1 and 2 at pH values of 7.5 and 5.0, respectively. RESULTS. Our immunohistochemical study showed that type 1 isoenzyme was expressed predominantly in epithelial cells, whereas type 2 isoenzyme was expressed in both stromal and epithelial cells. The real-time RT-PCR assay demonstrated that the copy numbers of type 1 isoenzyme mRNA were significantly higher than those of type 2 isoenzyme mRNA. There were significant associations between enzyme activity at pH 7.5 and type 1 isoenzyme mRNA expression, and between the activity at pH 5.0 and type 2 mRNA expressions. CONCLUSIONS. We demonstrated that 5 alpha-reductase type 1 had a specific enzyme activity in the prostate, which supports the hypothesis that the type I isoenzyme may play a significant role in maintaining prostate enlargement along with the type 2 isoenzyme. (C) 2004 Wiley-Liss, Inc.
WILEY-LISS, Jan. 2004, PROSTATE, 58(1) (1), 33 - 40, English
[Refereed]
Scientific journal

Radiation induces p53-dependent cell apoptosis in bladder cancer cells with wild-type-p53 but not in p53-mutated bladder cancer cells
N Hinata, T Shirakawa, ZJ Zhang, A Matsumoto, M Fujisawa, H Okada, S Kamidono, A Gotoh
Purpose. It has been reported in several studies that the absence in cancer cells of the p53 tumor suppressor gene, mutations of which are frequently found in bladder cancer, increases their resistance to ionizing radiation. Other studies, however, suggest that mutations of the p53 gene could increase the radiosensitivity of cancer cells, although the evidence is still inconclusive. In the present study, we investigated the relationship between p53 status and radiation response in five different bladder cancer cell lines. Materials and Methods. Five different human bladder cancer cell lines (KK47: with wt-p53, RT4: with wt-p53, T24: with mutated p53, 5637: with mutated p53, UM-UC-3: with mutated p53) were used in the study. Cells were irradiated with 0, 2, 4, 6 or 8 Gy, then trypsinized and re-plated for clonogenic survival assay, quantitative RT-PCR assay, flow-cytometry analysis and TUNEL assay. Results. The clonogenic assay demonstrated that KK47 and RT4 had significantly higher radiosensitivity than other cell lines. Quantitative RT-PCR analysis showed that radiation induced increased expression of p53, Bax, and p21 mRNA in KK47 and RT4. After irradiation, G1 cell-cycle arrest was observed in KK47 and RT4 under flow cytometry analysis, while T24, 5637, and UM-UC-3 showed an increase in the proportion of G2 cells. Increased cell apoptosis was also observed under TUNEL assay in KK47 and RT4, but not in other cell lines. Conclusions: It was demonstrated that ionizing radiation induces p53-dependent cell apoptosis in bladder cancer cells with wt-p53 but not in those with mutated p53.
SPRINGER-VERLAG, Dec. 2003, UROLOGICAL RESEARCH, 31(6) (6), 387 - 396, English
[Refereed]
Scientific journal

前立腺癌骨転移巣におけるオステオカルシン発現の意義
Shirakawa Toshiro, Hinata Nobuyuki
泌尿器科紀要刊行会, Nov. 2003, 泌尿器科紀要, 49(11号) (11号), 696 - 696, Japanese
Research society

Combination with CD/5-FC gene therapy enhances killing of human bladder-cancer cells by radiation
ZJ Zhang, T Shirakawa, N Hinata, A Matsumoto, M Fujisawa, H Okada, S Kamidono, M Matsuo, A Gotoh
Background Resistance to radiation and chemotherapy is a significant obstacle to the treatment of advanced bladder cancer. Gene therapy combined with radiation represents a new approach to cancer treatment. In the present study, we investigated whether adenovirally directed, cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy could induce cell toxicity and radiosensitization through the intracellular production of 5-fluorouracil (5-FU) in bladder-cancer cells. Methods Three human bladder-cancer cell lines, KK47 (wild-type p53+), T24 (p53 mutated) and 5637 (p53 mutated), were investigated. A recombinant adenovirus vector containing the CD gene (Ad-RSV-CD) was used. Cells were infected with Ad-RSV-CD and treated with 5-FC. Forty-eight hours after infection, the cells were irradiated and cytotoxicity assays performed to determine the extent of increase in in vitro cytotoxicity. A KK47 subcutaneous tumor-xenografts model was used in an animal study to examine the tumor growth inhibitory effect of this combination therapy. Ad-RSV-CD was directly injected into the tumor and daily 5-FC was intraperitoneally injected. Forty-eight hours after injection of Ad-RSV-CD, the tumor was irradiated. The tumor volume was measured every day. Results in all three cell lines, the combination treatment enhanced the cell killing of human bladder-cancer cells in vitro. It also enhanced the tumor-growth inhibition in the KK47 tumor model. Conclusions In the present study, we demonstrated that CD/5-FC gene therapy combined with radiation therapy enhances cell killing of human bladder-cancer cells in in vitro and in vivo animal models. Copyright (C) 2003 John Wiley Sons, Ltd.
JOHN WILEY & SONS LTD, Oct. 2003, JOURNAL OF GENE MEDICINE, 5(10) (10), 860 - 867, English
[Refereed]
Scientific journal

前立腺癌細胞内の亜鉛分布状態の変化ホルモンと亜鉛の関係
SHIRAKAWA, Toshiro, GOTOH,Akinobu, 北村ゆり, SUGIMURA, Kazuro, 川上拓男, 井手亜里
Sep. 2003, Biomedical Research on Trace Elements, 14巻, 3号, pp. 215-218, Japanese
[Refereed]
Scientific journal

前立腺癌骨転移巣に対する遺伝子治療臨床研究の現況
Shirakawa Toshiro, Hinata Nobuyuki, Hara Isao
(一社)日本癌治療学会, Sep. 2003, 日本癌治療学会誌, 38(2号) (2号), 239 - 239, Japanese
Research society

ホルモン抵抗性前立腺癌に対するEstramustine,COX-2阻害剤併用療法の治療効果の検討
Shirakawa Toshiro, Hinata Nobuyuki
(一社)日本癌治療学会, Sep. 2003, 日本癌治療学会誌, 38(2号) (2号), 659 - 659, Japanese
Research society

irritative symptomを主症状とするBPH患者に対する塩酸プロピベリンの効果
Hinata Nobuyuki, Shirakawa Toshiro
(一社)日本排尿機能学会, Sep. 2003, 日本排尿機能学会誌, 14(1) (1), 86 - 86, Japanese
Research society

Investigations on Distribution and Quantity of Zinc in Prostate Cancer Using Synchrotron Radiation Microbeams
Takuo Kawakami, Ari Ide-Ektessabi, Kazurou Sugimura, Yuri Kitamura, Akinobu Gotoh, Toshiro Shirakawa
Synchrotron radiation (SR) micro beams were used to investigate ultra-Trace elements in a single cell. X-ray fluorescence (XRF) spectroscopy using SR was applied to determine the distribution and density of the ultra-Trace elements in incubated prostate cancer cells. Metallic elements such as zinc, iron and copper play an important role in the metabolism and regulatory dynamics of cells. In this study, we focused on zinc because the prostate tissues contain the highest levels of zinc compared to any other tissues in the body and the level of zinc is a factor that influences the progress of malignancy. We measured two types of cells (LNCaP and C4-2) that were incubated in the normal culture medium, zinc-contained one or testosterone-contained one. The distribution and density of zinc, calcium, copper and iron have close relations to the existence of zinc and androgen in the culture medium.
American Institute of Physics Inc., Aug. 2003, AIP Conference Proceedings, 680, 526 - 529, English
[Refereed]
International conference proceedings

The growth inhibitory effect of p21 adenovirus on androgen-dependent and -independent human prostate cancer cells
A Gotoh, T Shirakawa, Y Wada, M Fujisawa, H Okada, S Kamidono, K Hamada
To assess the potential of p21 as a gene therapy treatment for prostate cancer, by introducing p21 into both androgen-dependent (AD) and -independent (AI) human prostate cancer cell lines via a recombinant adenoviral vector, Ad5CMV-p21, carrying human p21 cDNA. The LNCaP, DU145 and PC-3 human prostate cancer cell lines were cultured and infected with Ad5CMV-p21. Cell growth, cell-cycle progression and tumorigenicity were then assessed by thymidine incorporation into cellular DNA, and cell number, flow cytometry, and tumour growth after inoculating the cells into nude mice. Growth was inhibited in Ad5CMV-p21 viral-infected AD and AI prostate cancer cells. The effects were dose-dependent, regardless of the androgen status of the cell lines. Flow cytometric analysis showed that Ad5CMV-p21 arrested cell-cycle progression at G1/S with no appreciable effect on the levels of apoptotic cells. The tumorigenicity of cancer cells infected with Ad5CMV-p21 was greatly reduced in athymic mice. These results suggest that Ad5CMV-p21 may be a new therapeutic agent for human prostate cancer gene therapy.
BLACKWELL PUBLISHING LTD, Aug. 2003, BJU INTERNATIONAL, 92(3) (3), 314 - 318, English
[Refereed]
Scientific journal

Establishment of a prostatic small-cell carcinoma cell line (SO-MI)
H Okada, T Shirakawa, H Miyake, A Gotoh, M Fujisawa, S Arakawa, S Kamidono
BACKGROUND. Prostatic small-cell carcinoma is an extremely rare, highly aggressive disease. We established a cell line from this tumor. MATERIALS AND METHODS. Tumor tissue obtained from a 24-year-old Japanese man was used to establish the cell line. Cultured cells and tumors transplanted into nude mice were characterized by histologic, immunohistologic, immunocytologic, and molecular biologic methods. RESULTS. An immortal culture cell line (SO-MI) was successfully established. SO-MI cells adhered weakly to plastic surfaces in vitro, showing a 52- to 72-hr doubling time. SO-MI cells were heterotopically and orthotopically transplantable in nude mice. The cells were immunoreactive for NSE, chromogranin A, and NCAM, but not for ACTH, calcitonin, serotonin, gastrin, insulin, glucagons, LCA, EMA, PAP, PSA, androgen receptor, and p53. SO-MI cells secreted NSE in vitro and in vivo. SO-MI cells at passage 30 contained 50-59 chromosomes with a modal number of 55. PCR suggested that the p53 gene was deleted in SO-MI cells. RT-PCR detected no mRNA encoding androgen receptor in these cells. CONCLUSIONS. SC-MI cells retain the neuroendocrine nature of the original tumor, and should be useful in studying possible etiologies and new treatments. (C) 2003 Wiley-Liss, Inc.
WILEY-LISS, Aug. 2003, PROSTATE, 56(3) (3), 231 - 238, English
[Refereed]
Scientific journal

Blockade of TGF-beta action ameliorates renal dysfunction and histologic progression in anti-GBM nephritis
AH Zhou, H Ueno, M Shimomura, R Tanaka, T Shirakawa, H Nakamura, M Matsuo, K Iijima
Background. We tested whether the entire soluble extracellular domain of the human transforming growth factor-P (TGF-beta) type II receptor, fused to the Fc portion of human immunoglobulin G (IgG1) (Tbeta-ExR) and expressed in skeletal muscles by adenovirus-mediated gene transfer (AdTbeta-ExR), can ameliorate renal dysfunction and histologic progression in a rat experimental anti-glomerular basement membrane (GBM) nephritis. Methods. Anti-GBM nephritis was induced in Wistar Kyoto rats by an intravenous injection of anti-rat glomerular basement membrane (GBM) sera. At day 1 (24 hours after induction), AdTbeta-ExR (1 X 10(9) pfu/mL) was injected into the femoral muscle in the treatment group, and an adenovirus vector-expressing bacterial P-galactosidase (AdLacZ) was injected into the control group. Then, clinical and histologic changes were examined for 3 weeks after the induction of anti-GBM nephritis. Results. Tbeta-ExR was detected in the serum at day 7, but the serum concentration of Tbeta-ExR had decreased below the detectable level by day 14. Although blood pressure and the degree of proteinuria were similar in both groups, the deterioration of renal function was significantly blunted in the treatment group. Crescent formation and interstitial fibrosis were also ameliorated in the treatment group. These histologic improvements were accompanied by the decreased interstitial infiltration of macrophages and the decreased a-smooth muscle actin (alpha-SMA)-positive cells in the glomeruli and the interstitium. Conclusion. This study demonstrated for the first time that the blockade of TGF-beta action by AdTbeta-ExR in the early stage of anti-GBM nephritis ameliorates the clinical and histologic progression. In addition, this study shed light on the development of a specific gene therapy for human crescentic glomerulonephritis.
BLACKWELL PUBLISHING INC, Jul. 2003, KIDNEY INTERNATIONAL, 64(1) (1), 92 - 101, English
[Refereed]
Scientific journal

尿中分離菌に対するフローサイトメトリーを用いた迅速薬剤感受性試験について
Shigemura Katsumi, Tanaka Kazushi, Hinata Nobuyuki, Shirakawa Toshiro, Arakawa Soichi
(一社)日本感染症学会, May 2003, 感染症学雑誌, 77(5号) (5号), 363 - 364, Japanese
Research society

尿中分離菌に対するフローサイトメトリーを用いた迅速薬剤感受性試験について
Shigemura Katsumi, Tanaka Kazushi, Hinata Nobuyuki, Shirakawa Toshiro, Arakawa Soichi
尿路感染症研究会, Apr. 2003, 尿路感染症研究会記録集, (11号) (11号), 18 - 18, Japanese
Research society

Complete resection of synovial sarcoma of prostatic fascia
SHIRAKAWA TOSHIRO, FUJISAWA MASATO, Gotoh Akinobu
Mar. 2003, Urology, Vol. 61, No. 3, pp. 644-644, English
[Refereed]
Scientific journal

定量的PCR法を用いた細菌尿中の大腸菌の定量
Hinata Nobuyuki, Shirakawa Toshiro, Shigemura Katsumi
(一社)日本泌尿器科学会, Feb. 2003, 日本泌尿器科学会雑誌, 94(2号) (2号), 384 - 384, Japanese
Research society

Gene expression profiles of ABC transporters and cytochrome P450 3A in Caco-2 and human colorectal cancer cell lines
T Nakamura, T Sakaeda, N Ohmoto, Y Moriya, C Komoto, T Shirakawa, A Gotoh, M Matsuo, K Okumura
Purpose. The mRNA levels of MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), cytochrome P450 3A (CYP3A) and villin in human colorectal cell lines (HCT-15, LoVo, DLD-1, HCT-116 and SW620) were quantitatively compared with those in Caco-2 cells. Methods. The mRNA levels were determined by real time quantitative polymerase chain reaction and expressed as the relative concentrations of MDR1 mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Results. MDR1 mRNA was expressed in HCT-15 LoVo and DLD-1 cells at similar or lower level to Caco-2. The expression of MRP1 mRNA in the cell lines tested was comparable with Caco-2. MRP2 mRNA was detected only in HCT-116 and SW620 at significantly lower level than Caco-2. CYP3A mRNA was detected in HCT-15, LoVo, DLD-1 and SW620 at similar level to Caco-2. Conclusions. HCT-15 LoVo and DLD-1 cells express proteins important for regulating the intestinal absorption of drugs, i.e., MDR1, MRP1 and CYP3A, whereas HCT-116 and SW620 cells were not acceptable for evaluation of absorption properties of drug candidates.
KLUWER ACADEMIC/PLENUM PUBL, Feb. 2003, PHARMACEUTICAL RESEARCH, 20(2) (2), 324 - 327, English
[Refereed]
Scientific journal

Lack of association between Y chromosome haplogroups and male infertility in Japanese men
Cláudia M.B. Carvalho, Masato Fujisawa, Toshiro Shirakawa, Akinobu Gotoh, Sadao Kamidono, Tatiana Freitas Paulo, Sidney E.B. Santos, Juliane Rocha, Sérgio D.J. Pena, Fabrício R. Santos
The Y chromosome carries several genes involved in spermatogenesis, which are distributed in three regions in the euchromatic part of the long arm, called AZFa (azoospermia factor a), AZFb, and AZFc. Microdeletions in these regions have been seen in 10-15% of sterile males with azoospermia or severe oligozoospermia. The relatively high de novo occurrence of these microdeletion events might be due to particular chromosome arrangements associated with certain Y chromosome haplogroups. To test whether there is any association between Y chromosome types and male infertility, we studied a sample of 84 Japanese oligozoospermic or azoospermic males. The patients were analyzed for the presence of Yq microdeletions and also typed with a battery of unique event polymorphisms (UEPs) to define their Y haplogroups. Six of the infertile patients presented likely pathological microdeletions detectable with the sequence tagged sites (STS) markers used. There was no significant association between Y chromosome haplogroups and the microdeletions. We also compared the Y haplogroup frequencies in our subset sample of 51 idiopathic azoospermia patients with 57 fertile control Japanese males, and did not observe any significant differences. Contrary to previous reports, our data suggest that Y microdeletions and other molecular events causally associated with male infertility in Japan occur independently of the Y chromosome background. © 2002 Wiley-Liss, Inc.
Jan. 2003, American Journal of Medical Genetics, 116(2) (2), 152 - 158, English
[Refereed]
Scientific journal

ラット造精機能障害モデルにおけるHepatocyte Growth Factor遺伝子導入の効果
合田上政, 藤澤正人, 土橋正樹, 山崎隆文, 張竹君, 白川利朗, 後藤章暢, 岡田弘, 荒川創一, 守殿貞夫
一般社団法人日本泌尿器科学会, 2003, 日本泌尿器科学会雑誌, 94(2) (2), 173 - 173, Japanese

Lack of association between Y chromosome haplogroups and male infertility in Japanese men
CMB Carvalho, M Fujisawa, T Shirakawa, A Gotoh, S Kamidono, TF Paulo, SEB Santos, J Rocha, SDJ Pena, FR Santos
The Y chromosome carries several genes involved in spermatogenesis, which are distributed in three regions in the euchromatic part of the long arm, called AZFa (azoospermia factor a), AZFb, and AZFc. Microdeletions in these regions have been seen in 10-15% of sterile males with azoospermia or severe oligozoospermia. The relatively high de novo occurrence of these microdeletion events might be due to particular chromosome arrangements associated with certain Y chromosome haplogroups. To test whether there is any association between Y chromosome types and male infertility, we studied a sample of 84 Japanese oligozoospermic or azoospermic males. The patients were analyzed for the presence of Yq microdeletions and also typed with a battery of unique event polymorphisms (UEPs) to define their Y haplogroups. Six of the infertile patients presented likely pathological microdeletions detectable with the sequence tagged sites (STS) markers used. There was no significant association between Y chromosome haplogroups and the microdeletions. We also compared the Y haplogroup frequencies in our subset sample of 51 idiopathic azoospermia patients with 57 fertile control Japanese males, and did not observe any significant differences. Contrary to previous reports, our data suggest that Y microdeletions and other molecular events causally associated with male infertility in Japan occur independently of the Y chromosome background. (C) 2002 Wiley-Liss, Inc.
WILEY-LISS, Jan. 2003, AMERICAN JOURNAL OF MEDICAL GENETICS PART A, 116A(2) (2), 152 - 158, English
[Refereed]
Scientific journal

Effects of mefloquine usage on genetic polymorphism of Plasmodium falciparum in Thai-Myanmarese border
Matsuo Toshiaki, SHIRAKAWA, Toshiro, Singhasivanon Pratap, Looareesuwan Sornchai, KAWABATA, Masato
We studied the polymorphism of msp-1, which encodes a major surface protein onthe merozoite, isolated from blood samples from western Thailand in 1999. Our studyarea was a low-transmission area for malaria, where mefloquine has been used as anantimalarial drug since 1994. Forty-nine patients were confirmed to have contractedfalciparum malaria twice within 24 weeks. The number of detected haplotypes in 49patients was 89 at the first diagnosis and 68 at the second diagnosis. The mean numberof haplotypes per patient significantly decreased from 1.82 to 1.39 but the frequencydistributions of msp-1 haplotypes did not change significantly with the use ofmefloquine. Our study strongly suggests that the antigenic diversity of Plasmodium falciparum is retained during mefloquine therapy in low-transmission areas.
Kobe University, 2003, The Kobe Journal Of Medical Sciences, Vol. 49, No. 5-6, pp. 143-151(5) (5), 143 - 151, English
[Refereed]
Scientific journal
Link to Kobe Univ. Repository (Kernel)

Rapid diagnosis of typhoid fever by PCR assay using one pair of primers from flagellin gene of Salmonella typhi
Muhammad Nasrum Massi, Toshiro Shirakawa, Akinobu Gotoh, Acharya Bishnu, Mochammad Hatta, Masato Kawabata
Using the polymerase chain reaction (PCR) assay, we developed a rapid diagnosis method for Salmonella typhi infection in blood specimens from patients with typhoid fever. Primers were designed from the flagellin gene sequence, which would give an amplification product of 367 base pairs. In this study, the specificity of the assay, with no amplification, was seen for the other Salmonella strains with the flagellin gene, and not for non-Salmonella bacteria. For the sensitivity test, the protocol described allowed the detection of two to three copies of the Salmonella typhi genome, as determined by serial dilution of genomic DNA from Salmonella typhi. With the PCR technique, genomic DNA of Salmonella typhi was detected in 46 of 73 blood samples collected from patients with clinically suspected typhoid fever who had fever within 3 days of admission to the General Hospital, Makassar, South Sulawesi, Indonesia, and who had had no prior antibiotic treatment. The PCR results (63% positive cases) were compared with those of blood culture (13.7% positive cases) and the Widal test (35.6% positive cases), using the same samples from each of the 73 patients admitted to the General Hospital in Makassar. The time taken for PCR analysis of each sample was less than 12 h, compared with 3 to 5 days for blood or clot culture. The PCR with one pair of primers can be used as a novel, rapid diagnotic method for typhoid fever, particularly when results of standard culture assays are negative.
Springer Japan, 2003, Journal of Infection and Chemotherapy, 9(3) (3), 233 - 237, English
[Refereed]
Scientific journal

Inhibition of urinary bladder motility by a spinal action of U-50488H in rats
A Gotoh, K Goto, A Sengoku, T Shirakawa, Y Akao, M Fujisawa, H Okada, S Arakawa, H Sasaki, S Kamidono
We examined the effect of a kappa agonist, U-50488H, upon the bladder motility of anaesthetized rats. The frequency of distension-induced rhythmic bladder contractions was reduced by the intravenous (10 mg kg(-1)) or intrathecal (10-100 mug) administration of U-50488H. The effect of intravenous U-50488H was inhibited by pre-treatment with nor-binaltorphimine (10 mg kg(-1), s.c.). The inhibition of bladder contractions by intrathecal U-50488H (30 mug) was eliminated with the concomitant use of nor-binaltorphimine (10 mg kg(-1), s.c.), and diminished by reserpine (4 mg kg(-1), i.p.), yohimbine (10 mug, i.t.) or methysergicle (20 mug, i.t.). The amplitude of bladder contractions induced by an electrical stimulation of the pontine micturition centre was not inhibited by intrathecal U-50488H (30 and 100 mug). These results suggested that a kappa agonist could inhibit micturition reflex as well as other opioids, and at least part of the inhibition was due to the diminishment of bladder sensation based on the activation of the descending monoaminergic systems through the spinal kappa-opioid receptors.
ROYAL PHARMACEUTICAL SOC GREAT BRITAIN, Dec. 2002, JOURNAL OF PHARMACY AND PHARMACOLOGY, 54(12) (12), 1645 - 1650, English
[Refereed]
Scientific journal

[Gene therapy for prostate cancer].
Akinobu Gotoh, Toshiro Shirakawa, Yoshitaka Wada, Nobuyuki Hinata, Shigeji Matsubara, Isao Hara, Masato Fujisawa, Hiroshi Okada, Soichi Arakawa, Sadao Kamidono
The substantial advances made in recent years in the molecular biology of malignant urological tumors and the associated progressive analysis of these conditions at a molecular level have spurred research aimed at gene-based treatment. In the field of prostate cancer, while there have been many ground-breaking studies particularly in the United States, none has yet led to a revolutionary treatment for recurrent prostate cancer. Gene-based treatment is being applied seriously in clinical settings, especially in the United States, but so far without significant effect. Many researchers worldwide are devoting energy to the development of effective vectors. By adjusting the promoter, which has the function of directing the vector, we have developed organ-specific vectors for the treatment of prostate cancer. In the present study, which targeted prostate cancer with bone metastasis, we developed a suicide-gene therapy using an adenovirus vector with an organ-specific osteocalcin promoter. Clinical trials of this vector have already been conducted at the University of Virginia in the United States and have so far confirmed the safety of the therapy. In the present paper we present the results of this gene-therapy research from the basic to the clinical phase alongside an outline of related research at our institution. Gene therapy for cancer is now being targeted not only against the primary tumor but systemic cancers including distant metastases; systemic administration of adenovirus vectors with organ-specific promoters may become one of the most promising systemic anti-tumor therapies of the next-generation.
Nov. 2002, Hinyokika kiyo. Acta urologica Japonica, 48(11) (11), 729 - 32, Japanese, Domestic magazine
[Refereed]

Effects of polymorphisms of MDR1, MRP1, and MRP2 genes on their mRNA expression levels in duodenal enterocytes of healthy Japanese subjects
Y Moriya, T Nakamura, M Horinouchi, T Sakaeda, T Tamura, N Aoyama, T Shirakawa, A Gotoh, S Fujimoto, M Matsuo, M Kasuga, K Okumura
In the present study, we examined whether polymorphisms in the ATP-binding cassette (ABC) transporter genes, MDR1, MRP1 and MRP2, were associated with their respective mRNA expression levels in duodenal enterocytes of 13 healthy Japanese volunteers. MDR1 genotypes of T-129C, G2677(A,T) and C3435T, MRP1 genotypes of G128C, C218T, G2168A and G3173A, and MRP2 genotypes of C-24T, G1249A, C2302T, C2366T and G4348A were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or direct sequencing. Mutations T-129C, G2677(A,T) and C3435T of MDR1 gene were found at allele frequencies of 2/26, 16/26 and 12/26, respectively. Mutations G2168A of the MRP1 gene and C-24T of the MRP2 gene were also found at allele frequencies of 1/26 and 6/26, respectively, whereas other mutations were not detected in MRP1 and MRP2 genes. The relative concentrations (mean+/-S.E.) of MDR1 mPNA to villin mRNA were 0.38+/-0.15, 0.56+/-0.14 and 1.13+/-0.42 in the subjects with C/C-3435, C/T-3435 and T/T-3435, respectively, which supported the lower serum concentrations of digoxin after single oral administration in the subjects with the mutant T-allele at position 3435. Genetic collaboration between positions 3435 and 2677 was suggested, and those in G/G(2677), G/(A,T)(2677) and T/(A,T)(2677) were 0.16+/-0.05, 1.10+/-0.40, and 0.63+/-0.16, respectively (p = 0.107). However, there was no remarkable effect of the G2168A of the MRP1 gene or of C-24T of the MRP2 gene on the relative MRP1 or MRP2 mRNA concentrations, respectively.
PHARMACEUTICAL SOC JAPAN, Oct. 2002, BIOLOGICAL & PHARMACEUTICAL BULLETIN, 25(10) (10), 1356 - 1359, English
[Refereed]
Scientific journal

MDR1 up-regulated by apoptotic stimuli suppresses apoptotic signaling
T Sakaeda, T Nakamura, M Hirai, T Kimura, A Wada, T Yagami, H Kobayashi, S Nagata, N Okamura, T Yoshikawa, T Shirakawa, A Gotoh, M Matsuo, K Okumura
Purpose. Recently, MDR1 (P-glycoprotein) and related transporters have been suggested to play a fundamental role in regulating apoptosis, but little information is available concerning the role of MDR1. Here, the effect of apoptotic stimuli on the MDR1 mRNA and apoptotic signaling was examined in MDR1-overexpressing cells. Methods. The expression levels of mRNA for MDR1, MRP1, MRP2, p53, p21, Bax, and Bcl-2 were measured by real time quantitative polymerase chain reaction in HeLa and its MDR1-overexpressing sublines. The effects of apoptotic stimuli by cisplatin (CDDP) on their levels were also assessed as well as on caspase 3, 8, and 9 activities. Results. MDR1 was rapidly upregulated when the cells were exposed to apoptotic stimuli by CDDP. The increase in Bax mRNA to Bcl-2 mRNA ratio after treatment with CDDP was suppressed in MDR1-overexpressing cells. The increases in caspase 3 and 9 activities after treatment with CDDP were suppressed in MDR1-overexpression cells. Conclusion. MDR1 is upregulated by apoptotic stimuli suppressed apoptotic signaling presumably via the mitochondrial pathway.
KLUWER ACADEMIC/PLENUM PUBL, Sep. 2002, PHARMACEUTICAL RESEARCH, 19(9) (9), 1323 - 1329, English
[Refereed]
Scientific journal

Flavonoids differentially regulate IFN gamma-induced ICAM-1 expression in human keratinocytes: molecular mechanisms of action
T Bito, S Roy, CK Sen, T Shirakawa, A Gotoh, M Ueda, M Ichihashi, L Packer
The effect of plant flavonoids on intercellular adhesion molecule-1 (ICAM-1) expression in human keratinocyte was investigated. ICAM-1 is known to mediate skin inflammation. Among the flavonoids tested, taxifolin was the most potent in inhibiting interferon gamma (IFNgamma)-induced ICAM-1 protein as well as mRNA expression in human keratinocytes. Much smaller dosages of taxifolin were required in primary keratinocytes compared to HaCaT (immortalized cell) to achieve similar levels of inhibition in the inducible ICAM-1 expression. Regulation of inducible ICAM-1 expression by taxifolin was at transcriptional level by inhibiting the activation of signa transducers and activators of transcription (STAT)1 and protein tyrosine phosphorylation of Janus kinase (JAK)1 suggesting that the JAK-STAT pathway may be the molecular site of action of taxifolin. Finally, taxifolin pre-treatment also potently inhibited IFNgamma-induced ICAM-1 expression in a reconstructed human skin equivalent suggesting therapeutic potential of taxifolin in skin pathological conditions related to increased cell adhesion and inflammation. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
ELSEVIER SCIENCE BV, Jun. 2002, FEBS LETTERS, 520(1-3) (1-3), 145 - 152, English
[Refereed]
Scientific journal

Estrogen levels influence beta-3-adrenoceptor-mediated relaxation of the female rat detrusor muscle
S Matsubara, H Okada, T Shirakawa, A Gotoh, T Kuno, S Kamidono
Objectives. To observe the expression of beta-3-adrenoceptor in the detrusor muscle in female rats and investigate the relaxant effect of beta-adrenoceptor agonists on detrusor muscle in ovariectomized rats with or without estrogen replacement therapy. Methods. We first performed reverse transcriptase-polymerase chain reaction to demonstrate mRNA encoding the beta-3-adrenoceptor in the detrusor muscle from female rats. We then performed pharmacologic and physiologic studies to determine the effect of estrogen replacement therapy on the beta-adrenoceptor-mediated relaxation of the detrusor muscle of the ovariectomized rats. Results. Beta-3-adrenoceptor was expressed in the detrusor muscle in female rats with or without ovariectomy. A nonselective beta-adrenoceptor agonist relaxed precontracted detrusor muscle irrespective of ovariectomy or estrogen replacement in a dose-dependent manner; and a selective beta-3-adrenoceptor agonist relaxed the detrusor muscle more in ovariectomized rats than in ovariectomized rats with estrogen replacement or in control rats. Conclusions. Selective beta-3-adrenoceptor agonists relaxed the detrusor muscle of female rats with low estrogen levels. This result may give a clue to the treatment of frequent urination or incontinence in postmenopausal women who are not receiving hormonal replacement therapy.
ELSEVIER SCIENCE INC, Apr. 2002, UROLOGY, 59(4) (4), 621 - 625, English
[Refereed]
Scientific journal

Effect of the mutation (C3435T) at exon 26 of the MDR1 gene on expression level of MDR1 messenger ribonucleic acid in duodenal enterocytes of healthy Japanese subjects
T Nakamura, T Sakaeda, M Horinouchi, T Tamura, N Aoyama, T Shirakawa, M Matsuo, M Kasuga, K Okumura
The effect of the C3435T mutation at exon 26 of the MDR1 gene on the expression levels of MDR1 messenger ribonucleic acid (mRNA) was evaluated by means of real-time polymerase chain reaction in 51 biopsy specimens of duodenum obtained from 13 healthy Japanese subjects. The mRNA levels of MDR1 were 0.38 +/- 0.15, 0.56 +/- 0.14, and 1.13 +/- 0.42 (mean value +/- SE) in the subjects with the homozygote of wild-type allele (C/C), compound heterozygote with mutant T allele (C/T), and the homozygote of the mutant allele (T/T), respectively, reasonably explaining the lower digoxin serum concentration after administration of a single oral dose to subjects harboring a mutant T allele. Good correlation (r =.797; P <.01) was observed between the mRNA concentrations of MDR1 and CYP3A4 in the individual biopsy specimens. This finding suggested a lower plasma concentration of the substrates for CYP3A4 in subjects harboring the C3435T mutation of the MDR1 gene.
MOSBY, INC, Apr. 2002, CLINICAL PHARMACOLOGY & THERAPEUTICS, 71(4) (4), 297 - 303, English
[Refereed]
Scientific journal

Gene therapy
Gotoh, A., Shirakawa, T., Wada, Y., Hinata, N., Matsubara, S., Hara, I., Fujisawa, M., Okada, H., Arakawa, S., Kamidono, S.
2002, Acta Urologica Japonica, 48(11) (11), 729 - 732
[Refereed]
Scientific journal

尿路生殖器へ浸潤をきたした骨盤内他臓器疾患に対する泌尿器科術式について
藤澤正人, 白川利朗, 田中一志, 後藤章暢, 原勲, 川端岳, 岡田弘, 荒川創一, 八尾昭久, 松本修, 原口貴裕, 山中望, 守殿貞夫
一般社団法人日本泌尿器科学会, 2002, 日本泌尿器科学会雑誌, 93(2) (2), 245 - 245, Japanese

Real-time quantitative polymerase chain reaction for MDR1, MRP1, MRP2, and CYP3A-mRNA levels in Caco-2 cell lines, human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas
T Nakamura, T Sakaeda, N Ohmoto, T Tamura, N Aoyama, T Shirakawa, T Kamigaki, T Nakamura, KI Kim, Kim, SR, Y Kuroda, M Matsuo, M Kasuga, K Okumura
The expression levels of mRNAs for MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), and cytochrome P450 3A (CYP3A) in Caco-2 cells were quantitatively compared with those in human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas. Caco-2 cells (passages 36-38) were kindly supplied by several laboratories in Japan. Human duodenal enterocytes were obtained from five healthy male volunteers. Normal colorectal tissues and colorectal adenocarcinomas were simultaneously obtained from seven patients with primary colorectal adenocarcinoma. MDR1, MRP1, MRP2, and CYP3A mRNA levels were determined by real-time quantitative polymerase chain reactions (PCR). Relative concentrations of mRNAs for target proteins (MDR1, MRP1, MRP2, and CYP3A) and glyceraldehyde-3-phosphate dehydrogenase in Caco-2 cells were 1.00 +/- 0.15, 1.02 +/- 0.06, 0.94 +/- 0.10, and 0.68 +/- 0.60, respectively, and those in human enterocytes were about 12-, 3-, 7-, and 8000-fold higher than in the Caco-2 cells, respectively. In contrast, MDR1, MRP1, and CYP3A mRNA levels in Caco-2 cells were comparable to those in normal colorectal tissue and colorectal adenocarcinoma.
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, Jan. 2002, DRUG METABOLISM AND DISPOSITION, 30(1) (1), 4 - 6, English
[Refereed]
Scientific journal

Additional gene therapy with Ad5CMV-p53 enhanced the efficacy of radiotherapy in human prostate cancer cells
R Sasaki, T Shirakawa, ZJ Zhang, A Tamekane, A Matsumoto, K Sugimura, M Matsuo, S Kamidono, A Gotoh
Purpose: The aim of this study was. to investigate the efficacy of combination therapy of ionizing radiation (IR) and adenoviral p53 gene therapy and to evaluate its molecular mechanisms. Methods and Materials: Two human prostate cancer cell lines, DU145 and PC-3 cells, containing different types of p53 gene mutations, were investigated. The recombinant adenovirus vector containing the wild-type p53 gene (Ad5CMV-p53) was used for this study. Cells were irradiated (in 0, 2, 4, and 6 Gy, 300 cGy/min) and after 12 h of irradiation, the cells were infected with various doses of Ad5CMV-p53 (0-40 multiplicity of infection [MOI]). Cytotoxicity was determined by clonogenic assay. The molecular mechanisms were evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), apoptotic cell detection, and cell cycle analysis. Results: The cell growth inhibition in DU145 (p53-mutated) cells by IR was strongly enhanced by additional Ad5CMV-p53 infection in a viral dose-dependent manner. In DU145 cells, IR alone induced minimal p53 mRNA expression. However, IR combined with Ad5CMV-p53 infection stimulated significant increase in p53 mRNA expression supplemented with Bax and p21 mRNA expressions. In PC-3 (p53-null), IR induced Bax and p21 mRNA expression, while the combination effects were observed in p53, Bax, and p21 mRNA expression. Apoptotic cell deaths were rarely observed after IR alone (DU145: 3%, PC-3: 5%). However, after combination therapy, the proportion of apoptotic cells greatly increased (sevenfold in DU145 cells, and twice in PC-3 cells). G1 cell cycle arrest was observed after Ad5CMV-p53 infection and the combination in both cell lines. Conclusion: In this study, we demonstrated that the combination of IR and Ad5CMV-p53 gene therapy resulted in remarkable synergistic effects in human prostate cancer cells. This combination therapy could be one of the optimal treatment strategies for radioresistant prostate cancer. (C) 2001 Elsevier Science Inc.
ELSEVIER SCIENCE INC, Dec. 2001, INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS, 51(5) (5), 1336 - 1345, English
[Refereed]
Scientific journal

[Prospects for molecular research in urological oncology: gene therapy].
A Gotoh, T Shirakawa, Y Wada, N Hinata, I Hara, M Fujisawa, H Okada, S Arakawa, S Kamidono
The recent great advances in genetic engineering are now making possible the identification and isolation of the trigger genes of many hereditary illnesses, and the clarification of the relevant molecular mechanisms. The idea that if the genetic abnormalities responsible for illness could be established at a DNA level, treatment at the genetic level repairing damaged genes or supplying absent ones would also be possible was the incentive for the recent boom in gene therapy. Clinical research into gene therapy began in 1990 and currently over 3,000 patient cases are being studied. Some 70% of these are cancer patients. This is not simply because such patients are relatively numerous, but is also a sign of the wish, held earnestly by many researchers and clinicians as well as cancer patients and their families, to at last overcome this intractable disease. Gene therapy, so far conducted mainly in the United States, has hitherto not lived up to initial expectations in its concrete results. The reason for this results mainly in technical factors, such as the rate of success in implanting genes into target cells, the rate of successful expression of the implanted genes, and the successful achievement of specific expression at the target site. Gene therapy in the form of clinical research into renal cancer and lung cancer is now under way in Japan. It is too early at this stage to evaluate this work, but the present paper takes this opportunity to give an outline of gene therapy, and to examine its current state, future prospects and problem areas with particular reference to cancer.
Nov. 2001, Hinyokika kiyo. Acta urologica Japonica, 47(11) (11), 829 - 32, Japanese, Domestic magazine
[Refereed]
Scientific journal

Drug-resistant human bladder-cancer cells are more sensitive to adenovirus-mediated wild-type p53 gene therapy compared to drug-sensitive cells
T Shirakawa, R Sasaki, TA Gardner, CH Kao, ZJ Zhang, K Sugimura, M Matsuo, S Kamidono, A Gotoh
We investigated the therapeutic potential and molecular mechanism of adenovirus-mediated wt p53 gene therapy for drug-resistant human bladder cancers. KK47, a human bladder-cancer cell line, along with the drug-resistant sublines KK47/DDP10, KK47/DDP20 (cisplatin-resistant) and KK47/ADM (doxorubicin-resistant) were used for the experiments. All 4 KK47 cell lines had genetically normal p53 genes. Using an in vitro cytotoxicity assay, the drug-resistant cell lines were more sensitive to Ad-CMV-p53 cell killing than the KK47 parental cell line. Ad-CMV-p53 induced higher levels of p53 protein and mRNA in the drug-resistant cell lines than in the parental cell line and, consequently, higher levels of p21 and Box mRNA, which resulted in higher percentages of G(1) cell-cycle arrest and apoptosis. The higher efficiencies of adenoviral gene transfer in the drug-resistant cell lines were confirmed by X-gal staining after infection with Ad-CMV-beta -gal. In conclusion, adenovirus-mediated wt p53 gene therapy was more effective in the drug-resistant bladder-cancer cell lines than in the drug-sensitive bladder-cancer cell line. (C) 2001 Wiley-Liss, Inc.
WILEY-LISS, Oct. 2001, INTERNATIONAL JOURNAL OF CANCER, 94(2) (2), 282 - 289, English
[Refereed]
Scientific journal

Y-chromosome microdeletion and phenotype in cytogenetically normal men with idiopathic azoospermia
M Fujisawa, T Shirakawa, M Kanzaki, H Okada, S Arakawa, S Kamidono
Objective: To determine the prevalence of microdeletions of the long ann of chromosome Y within the AZFa, AZFb, and AZFc subregions in patients with idiopathic azoospermia, and then correlate the microdeletions with clinical phenotypes to determine the most important subregion for screening. Design: Controlled clinical study. Setting: Male infertility clinic, Kobe University Hospital. Patient(s): Among 89 consecutive azoospermic patients, those whose infertility was related to known hereditary, endocrine, or obstructive causes or a cytogenetic abnormality were excluded; 54 remaining patients were studied using a polymerase chain reaction (PCR). Of these patients, 33 had Sertoli cell only syndrome, 10 had maturation arrest, and 11 had hypospermatogenesis. Intevention(s): Blood and semen samples and testicular biopsies were obtained from all of the participants. Main Outcome Measure(s): We performed semen analysis, polymerase chain amplification of 28 DNA loci on the Iona arm of the Y chromosome involving the DAZ (deleted in azoospermia), and measured the plasma FSH, LH, testosterone, prolactin, and estradiol levels. Result(s): Microdeletions were detected in 14 of the 54 patients (nine with Sertoli cell only, three with maturation arrest, and two with hypospermatogenesis). Most microdeletions involved AZFb or AZFc. Patients with hypospermatogenesis or maturation arrest showed deletion only in AZFc. The DAZ gene was deleted in four patients with Sertoli cell only and one patient with maturation arrest. The RBM gene was deleted in two patients with Sertoli cell only who had particularly large deletions, but in no patients with arrest or hypospermatogenesis. Conclusion(s): Cytogenetically azoospermic patients should be examined for microdeletions before undertaking assisted reproduction. AZFc may be the most important subregion to screen. In addition, intact AZFa and AZFb subregions may be important for the presence of germ cells. (Fertil Steril(R) 2001;76:491-5. (C) 2001 by American Society for Reproductive Medicine.).
ELSEVIER SCIENCE INC, Sep. 2001, FERTILITY AND STERILITY, 76(3) (3), 491 - 495, English
[Refereed]
Scientific journal

Adenovirus-mediated p53 gene transfer to rat testis impairs spermatogenesis
M Fujisawa, T Shirakawa, H Fujioka, A Gotoh, H Okada, S Arakawa, S Kamidono
The tumor suppressor protein p53 participates in normal cell differentiation as well as induction of programmed cell death. The authors investigated the effect of p53 overexpression on spermatogenesis by transferring p53 gene into the rat testes. Replication-deficient recombinant adenovirus vectors were constructed to include cytomegalovirus (CMV) promoter driving wild-type p53 (Ad-CMV-p53) or beta -galactosidase (Ad-CMV-beta -gal). Virus was delivered to cells of the tubules by slow retrograde injection through the rete testis. At 0, 4. 7, and 14 days, testes were removed, weighed, and analyzed histopathologically, including immunohistochemistry for p53, Bcl-2. Bar. and interleukin-1 beta converting enzyme (ICE). Testicular weight was decreased in Ad-CMV-p53 group at 14 days after injection. while no change occurred in phosphate-buffered saline-injected controls or Ad-CMV-beta -gal-infected testes. Beyond 4 days, cell degradation in tubules interfered with immunohistochemical observation in the Ad-CMV-p53 group. At 4 days, p53 was expressed mostly in spermatocytes. Bar showed greater expression in the p53 group than in the control or Ad-CMV-beta -gal group. ICE, expressed mostly in spermatids. was more abundant in the p33 group than in controls. Overall, p53 overexpression in the testis impaired spermatogenesis.
HEMISPHERE PUBL CORP, May 2001, ARCHIVES OF ANDROLOGY, 46(3) (3), 223 - 231, English
[Refereed]
Scientific journal

Breast cancer working group
R. Sasaki, H. Nishimura, T. Soejima, Y. Ejima, E. Yoden, T. Shirakawa, A. Gotoh, Y. Ota, A. Matsumoto, K. Sugimura
2001, International Journal of Radiation Oncology Biology Physics, 51, 56 - 57, English
[Refereed]
Scientific journal

Adenovirus-mediated p53 gene transfer to rat testis impairs spermatogenesis
M. Fujisawa, T. Shirakawa, H. Fujioka, A. Gotoh, H. Okada, S. Arakawa, S. Kamidono
The tumor suppressor protein p53 participates in normal cell differentiation as well as induction of programmed cell death. The authors investigated the effect of p53 overexpression on spermatogenesis by transferring p53 gene into the rat testes. Replication-deficient recombinant adenovirus vectors were constructed to include cytomegalovirus (CMV) promoter driving wild-type p53 (Ad-CMV-p53) or β-galactosidasc (Ad-CMV-β-gal). Virus was delivered to cells of the tubules by slow retrograde injection through the rete testis. At 0, 4, 7, and 14 days, testes were removed, weighed, and analyzed histopathologically, including immunohistochemistry for p53, Bcl-2, Bax, and interleukin-1b converting enzyme (ICE). Testicular weight was decreased in Ad-CMV-p53 group at 14 days after injection, while no change occurred in phosphate-buffered saline-injected controls or Ad-CMV-β-gal-infected testes. Beyond 4 days, cell degradation in tubules interfered with immunohistochemical observation in the Ad-CMV-p53 group. At 4 days, p53 was expressed mostly in spermatocytes. Bax showed greater expression in the p53 group than in the control or Ad-CMV-β-gal group. ICE, expressed mostly in spermatids, was more abundant in the p53 group than in controls. Overall, p53 overexpression in the testis impaired spermatogenesis. © 2001 Informa UK Ltd All rights reserved.
2001, Systems Biology in Reproductive Medicine, 46(3) (3), 223 - 231, English
[Refereed]
Scientific journal

Gene therapy for bladder cancer using adenoviral vector
Y. Wada, A. Gotoh, T. Shirakawa, K. Hamada, S. Kamidono
Background and Purpose: Bladder cancer is common. Current treatment for patients with superficial bladder cancer involves transurethral resection followed by adjuvant bacillus Calmette-Guérin (BCG) administration. Adjuvant BCG has been reported to be effective in 38% to 68% of patients however, more than 30% of patients do not respond. Because p53 mutations are common among superficial bladder cancers, we tested the feasibility of using p53 as a gene therapy agent for targeting superficial tumors, which are easily accessible using an intravesical approach. Materials and Methods: Wild-type p53 was transduced into various human and murine bladder cancer cell lines (HTB9, KU-1, and MBT-2) using a recombinant adenoviral vector (Ad5CMV-p53) in vitro. Also, subcutaneous tumors were established and then treated with intratumoral injection of Ad5CMV-p53 or control viruses. Results: In vitro assays revealed significant growth suppression of target cells by Ad5CMV-p53 in comparison with those receiving the control Ad5-CMV-PA vector or untreated control cells. In vivo studies using subcutaneous bladder tumor models established in syngeneic mice demonstrated that the rate of tumor growth and volume was reduced to a greater extent by 14 days of intratumoral injection of Ad5CMV-p53 rather than Ad5CMV-PA. Furthermore, the survival of host animals bearing tumors that were infected with Ad5CMV-p53 was significantly longer than that of the control group treated with Ad5CMV-PA (P < 0.01). Conclusion: Our data suggest that Ad5CMV-p53 is effective in suppressing bladder cancer growth and improving host survival.
Mary Ann Liebert Inc., 2001, Molecular Urology, 5(2) (2), 47 - 52, English
[Refereed]
Scientific journal

Gene therapy for prostate cancer; Development of tissue specific promoter-based gene therapy
A Gotoh, T Shirakawa, Y Wada, SC Ko, CH Kao, LWK Chung, S Kamidono
We have defined the androgen responsiveness and status of prostate specific antigen (PSA) secretion of prostate cancer cells as well as non-prostatic cells transduced with PSA promoter reporter constructs. Androgen-independent (Al) human prostate cancer remains a lethal phenotype for which there is no effective therapy. AI prostate cancer produces and secretes large amounts of PSA at both primary and metastatic sites. The aim of this investigation is to explore the use of the PSA promoter as a mean of prostate cell specific expression of a toxic gene thymidine kinase (TK) to an AI PSA-producing human prostate cancer cell line. An adenovirus vector carrying human herpes simplex thymidine kinase (TK) gene under control of the PSA promoter (Ad-PSA-TK) was generated to target PSA-producing Al prostate cancer cells. Upon the administration of acyclovir, Ad-PSA-TK efficiently killed the AI PSA-producing human prostate cancer cells. This paper discusses the importance of tissue specific promoter system,for using gene therapy of prostate cancer, and summarizes recent gene therapy strategies developed to target this process. This strategy can potentially be used in combination with current therapeutic modalities to achieve more effective tumor cell-kill with much reduced toxicity.
WORLD SCIENTIFIC PUBL CO PTE LTD, 2001, FRONTIERS IN HUMAN GENETICS: DISEASES AND TECHNOLOGIES, 363 - 376, English
[Refereed]
International conference proceedings

p53 Adenoviral vector (Ad-CMV-p53) induced prostatic growth inhibition of primary cultures of human prostate and an experimental rat model
T Shirakawa, A Gotoh, TA Gardner, CH Kao, ZJ Zhang, S Matsubara, Y Wada, N Hinata, M Fujisawa, K Hanioka, M Matsuo, S Kamidono
Background Benign prostatic hyperplasia (BPH) is the most common proliferative disease affecting men. Numerous minimally invasive technologies are being developed or are currently in use to obviate the need for transurethral surgery. The goal of the present study was to develop a novel molecular based approach for the treatment of BPH using recombinant p53 adenoviral vector. The over-expression of wt-p53 can cause cell apoptosis or cell growth arrest, thus preventing the uncontrolled cell proliferation underlying BPH pathophysiology. Methods Ad-CMV-p53, a replication-deficient recombinant adenovirus containing cytomegalovirus promoter driving p53 gene, was used. Human prostate stromal CPS) cells were evaluated for apoptosis (TUNEL assay), mRNA levels of key cell cycle regulators influencing apoptosis (p-53, Bax and Bcl-2) using quantitative RT-PCR and cytotoxicity after Ad-CMV-p53. Ad-CMV-p53 was unilaterally injected into rat ventral prostates and growth inhibition was measured by prostate weight 3 weeks after injection. Results In vitro exposure to Ad-CMV-p53 significantly inhibited the proliferation of PS cells, induced mRNA over-expression of both wt-p53 and Bax, and increased the proportion of apoptotic cells. A 30% decrease in average prostate weight was demonstrated in rodents after Ad-CMV-p53 injection. Conclusions The results suggest that further investigation of molecular gene therapy with recombinant wt-p53 adenovirus for the treatment of BPH is warranted. Copyright (C) 2000 John Wiley & Sons, Ltd.
JOHN WILEY & SONS LTD, Nov. 2000, JOURNAL OF GENE MEDICINE, 2(6) (6), 426 - 432, English
[Refereed]
Scientific journal

Tissue-specific promoters in gene therapy for the treatment of prostate cancer
T. Shirakawa, A. Gotoh, Y. Wada, S. Kamidono, S. C. Ko, C. Kao, T. A. Gardner, L. W.K. Chung
Delivery of therapeutic toxic genes to and their expression in tumor cells through the use of tissue-specific promoters could decrease their toxic effect on neighboring normal cells when virus-mediated gene delivery results in their infection. We have demonstrated the utility of two prostate cancer- specific promoters, long PSA and osteocalcin, for tissue-specific toxic gene therapy for prostate cancer. The two promoters were highly active in both androgen-dependent and androgen-independent prostate cancer cells. We also introduce the Phase I trial of osteocalcin promoter-based toxic gene therapy for bone metastases of prostate cancer, which is in progress at the University of Virginia.
Mary Ann Liebert Inc., 2000, Molecular Urology, 4(2) (2), 73 - 82, English
[Refereed]
International conference proceedings

Cytotoxicity of adenoviral-mediated cytosine deaminase plus 5-fluorocytosine gene therapy is superior to thymidine kinase plus acyclovir in a human renal cell carcinoma model
T Shirakawa, TA Gardner, SC Ko, N Bander, S Woo, A Gotoh, S Kamidono, LWK Chung, C Kao
Purpose: An estimated 11,600 Americans will die of renal cell carcinoma in 1998. The lack of effective chemotherapy or radiotherapy requires the investigation of novel treatment modalities. We compared two forms of toxic gene therapy, cytosine deaminase (CD) plus 5-fluorocytosine (5-FC) and thymidine kinase (TK) plus acyclovir (ACV), in pre-clinical models of human renal cell carcinoma. Materials and Methods: Replication-deficient recombinant adenoviral vectors containing the Rous sarcoma virus promoter driving CD (Ad-RSV-CD) or TK (Ad-RSV-TK) gene expression were constructed and tested for in vitro cell-killing assays at various viral multiplicity of infection (MOI) and in vivo for growth inhibition of a human renal cell carcinoma, SK-RC-29 models. Subcutaneous tumors of SK-RC-29 were examined by electron microscopy for presence of intercellular gap junctions. Levels of expression of the gap junctional associated connexin 43 protein in SK-RC-29, 31, 38, 42, 52 human RCC cell lines were examined by western immunoblotting. Results: In vitro cell-killing assay comparing Ad-RSV-CD/5F-C and Ad-RSV-TK/ACV at a wide range of MOI (2.5 to 20) revealed superior cell-kill by Ad-RSV-CD/5-FC over Ad-RSV-TK/ACV. Consistent with these results, we observed that Ad-RSV-CD/5-FC but not Ad-RSV-TKIACV demonstrated a significant in vivo tumor growth inhibition. These results are corroborated by the lack of gap junctions in SK-RC-29 subcutaneous tumors by the electron microscopy and the absence of connexin-43 in all five human RCC cell lines by western immunoblotting. Conclusion: We have demonstrated in this study that Ad-RSV-CD/5-FC is superior to Ad-RSV-TK/ACV for the treatment of human RCC in cell culture and animal models. The results are supported by the lack of gap junctional communication between RCC cells assessed by connexin-43 expression.
LIPPINCOTT WILLIAMS & WILKINS, Sep. 1999, JOURNAL OF UROLOGY, 162(3) (3), 949 - 954, English
[Refereed]
Scientific journal

In vivo suppression of osteosarcoma pulmonary metastasis with intravenous osteocalcin promoter-based toxic gene therapy
T Shirakawa, SC Ko, TA Gardner, J Cheon, T Miyamoto, A Gotoh, LWK Chung, CH Kao
Pulmonary metastases are the main cause of death of patients with several types of cancer, including osteosarcoma, renal cell carcinoma, malignant melanoma, and breast cancer. Previously, we demonstrated that intralesional injection of the recombinant adenovirus (Ad) vector containing the herpes simplex virus thymidine kinase (TK) gene driven by an osteocalcin (OC) promoter (Ad-OC-TK) effectively suppressed the growth of osteosarcoma cells in vitro and tumors in vivo in a tumor-specific manner when supplemented with the prodrug acyclovir (ACV). In this communication, we studied the potential efficacy of the treatment of osteosarcoma pulmonary metastases with a systemic delivery route of Ad-OC-TK supplemented with ACV. We established osteosarcoma lung metastases in nude mice by the intravenous injection of rat osteosarcoma cells, ROS 17/2.8. These cells colonized and formed tumor nodules within 1 week in the lungs of nude mice. Whereas systemic delivery of a recombinant Ad vector containing the Escherichia coli beta-galactosidase (beta-gal) gene driven by a Rous sarcoma virus universal promoter (Ad-RSV-beta-gal) resulted in the nonspecific expression of beta-gal activity in the lung parenchyma, Ad-OC-beta-gal administration resulted in specific beta-gal expression in tumor cells deposited in the lung. When nude mice bearing ROS 17/2.8 lung tumors were treated with systemic Ad-OC-TK through tail vein administration, subsequent intraperitoneal ACV treatment significantly decreased the number of tumor nodules (P < .0001) and the net lung wet weight (P = .0005) while significantly increasing (.005 < P < .01) the survival of animals, when compared with untreated and Ad-OC-TK- or ACV-treated control groups. These results suggest that Ad-OC-TK/ACV may be used as a systemic therapy for the treatment of osteosarcoma lung metastasis.
APPLETON & LANGE, Sep. 1998, CANCER GENE THERAPY, 5(5) (5), 274 - 280, English
[Refereed]
Scientific journal

Development of prostate-specific antigen promoter-based gene therapy for androgen-independent human prostate cancer
A Gotoh, SC Ko, T Shirakawa, J Cheon, CH Kao, T Miyamoto, TA Gardner, LJ Ho, CBJ Cleutjens, J Trapman, FL Graham, LWK Chung
Purpose: The goal of this study is to develop a tissue-specific toxic gene therapy utilizing the prostate specific antigen (PSA) promoter for both androgen-dependent (AD) and androgen-independent (AI) PSA-secreting prostate cancer cells. Ideally this gene therapy would be effective without the necessity of exposing the target cells to circulating androgens. Materials and Methods: An AI subline of LNCaP, an AD PSA-secreting human prostate cancer cell line, C4-2, was used in this study. Castrated mice bearing C4-2 tumors secrete PSA. A transient expression experiment was used to analyze the activity of two PSA promoters, a 5837 bp long PSA promoter and a 642 bp short PSA promoter, in C4-2 cells. A recombinant adenovirus (Ad-PSA-TK) carrying thymidine kinase under control of the long PSA promoter was generated. The tissue-specific activity of Ad-PSA-TK was tested in vitro and in vivo. Results: The long PSA promoter had superior activity over short PSA promoter, and higher activity in C4-2 cells than in LNCaP cells. High activity of Ad-PSA-TK was observed in C4-2 cells in an androgen deprived condition. In vitro, Ad-PSA-TK was further demonstrated to induce marked C4-2 cell-kill by acyclovir in medium containing 5% FBS. No cell-kill was observed in control WH cells (a human bladder cancer cell line). In vivo, Ad-PSA-P-TK with acyclovir significantly inhibited subcutaneous C4-2 tumor growth and PSA production in castrated animals. Conclusion: The 5837 bp long PSA promoter was active in the androgen free environment and could be used to target both androgen-dependent and independent PSA-producing prostate cancer cells in vitro, and prostate tumors in castrated hosts.
LIPPINCOTT WILLIAMS & WILKINS, Jul. 1998, JOURNAL OF UROLOGY, 160(1) (1), 220 - 229, English
[Refereed]
Scientific journal

Osteocalcin Promoter を用いたAdenovirus Vector (Ad-OC-TK) による前立腺癌に対する遺伝子治療 : 第86回日本泌尿器科学会総会
白川利朗, 後藤章暢, Chung Leland W.K., 守殿貞夫
一般社団法人日本泌尿器科学会, 1998, 日本泌尿器科学会雑誌, 89(2) (2), 303 - 303, Japanese

Assessment of proliferating cell nuclear antigen expression and prognosis in patients with renal cell carcinoma with pulmonary metastases
Akinobu Gotoh, Keisuke Hanioka, Toshiro Shirakawa, Yoshitaka Wada, Kazuo Gohji, Hiroshi Okada, Soichi Arakawa, Sadao Kamidono
Background: The presence of proliferating cell nuclear antigen (PCNA) has been suggested as a more important prognostic marker than either grade or mitotic index in the prognosis of patients with renal cell carcinoma. We assessed the immunoreactivity of PCNA in primary lesions and pulmonary metastases from patients with renal cell carcinoma and correlated the results with various histopathologic features and prognostic factors. Methods: We studied the relationship between PCNA expression and clinical prognostic factors from resected primary lesions and pulmonary metastases from 10 patients and primary lesions from 32 patients with renal cell carcinoma without metastases. The cells were immunohistochemically stained with PCNA monoclonal antibody (PC-10) and 1000 nuclei were counted. The results were expressed as a ratio of stained to total cells (PCNA labeling index, LI%). Results: The PCNA LI of pulmonary metastatic nuclei was significantly higher than the PCNA LI of renal lesions either from patients with (P < 0.05) or without (P < 0.01) metastases. Also, the mean PCNA LI of the pulmonary lesions in patients dying within 3 years of diagnosis was higher than the mean PCNA LI of patients surviving greater than 3 years. Conclusion: Our findings suggest that the PCNA LI, which was determined by immunohistochemical analysis, is an important marker reflecting the biologic behavior of renal cell carcinomas. The degree of PCNA expression in this study was of prognostic significance.
Blackwell Publishing, 1998, International Journal of Urology, 5(3) (3), 214 - 218, English
[Refereed]
Scientific journal

Chemogene therapy: Osteocalcin promoter-based suicide gene therapy in combination with methotrexate in a murine osteosarcoma model
J Cheon, SC Ko, TA Gardner, T Shirakawa, A Gotoh, C Kao, LWK Chung
We previously reported that the recombinant adenovirus (Ad) vector containing the thymidine kinase (TK) gene driven by the osteocalcin (OC) promoter (Ad-OC-TK), when delivered concurrently with acyclovir (ACV), is highly selective in blocking the growth of osteosarcoma in experimental models (Cancer Res. 1996;56:4614-4619). To investigate the possible additive effects of the combined treatment of gene therapy and conventional chemotherapy (chemogene therapy), we compared the effect of low dose (IC10) methotrexate (MTX) and OC promoter-based toxic gene therapy with either of these single modalities alone. We choose low dose MTX with the intent of determining whether chemosensitization of the osteosarcoma may be possible in combination with gene therapy with an overall reduced toxicity profile and enhanced therapeutic efficacy when compared to a single agent alone. In vitro, the combined treatments of MTX (3 ng/mL) and Ad-OC-TK (20 multiplicity of infection (MOI)/target cell) plus ACV (10 mg/mL) had an additive therapeutic effect over that of either MTX (P < 0.05) or Ad-OC-TK plus ACV treatment alone (P < 0.05). In vivo, nude mice with subcutaneous tumors of either human osteosarcoma (MG-63) or rat osteosarcoma (ROS) received three intratumoral injections of Ad-OC-TK (5 X 10(8) PFU) plus daily intraperitoneal ACV (40 mg/kg body weight) for 2 weeks in combination with five weekly bolus intraperitoneal MTX (87.5 mg/kg). Osteosarcoma tumor growth was inhibited more efficiently than by either Ad-OC-TK plus ACV (P < 0.05) or MTX treatment (P < 0.005) alone. At day 45 in the ROS group, 100% of the animals survived when treated with chemogene therapy, whereas 80% survived with gene therapy and no animals survived in the MTX-treated or untreated controls. In summary, we developed a novel therapeutic strategy for the treatment of osteosarcoma employing both chemotherapy and gene therapy. Chemogene therapy could potentially achieve better antitumor effects with reduced toxicity than the conventional chemotherapy or gene therapy protocols alone.
APPLETON & LANGE, Nov. 1997, CANCER GENE THERAPY, 4(6) (6), 359 - 365, English
[Refereed]
Scientific journal

Y chromosome (Yq11) microdeletions in idiopathic azoospermia
Toshiro Shirakawa, Masato Fujisawa, Masanori Kanzaki, Hiroshi Okada, Soichi Arakawa, Sadao Kamidono
Background: Cytogenetic anomalies and molecular deletions of the Y chromosome in idiopathically sterile men suggest that genetic factor(s) controlling spermatogenesis are located in the distal portion of Yq11. We studied Y chromosome microdeletions in the Yq11.23 region in idiopathic azoospermia. Methods: We studied 25 azoospermic male patients with a cytogenetically normal 46XY karyotype 13 exhibited Sertoli-cell-only syndrome and 12 exhibited maturation arrest. Microdeletions in the Yq11 region were examined using the PCR technique with 4 pairs of primers from DNA loci in Yq11.23. Results: Microdeletions in Yq11.23 were detected in 4 of the 25 azoospermic men. The most common deletion was of the Y6HP52pr sequence, which was detected in 3 of 13 men with Sertoli-cell-only syndrome but in only 1 of 12 with maturation arrest. Conclusion: Detection of microdeletions within the Yq11 sequence is an important clue to the genetic factor(s) underlying azoospermia.
Blackwell Publishing, 1997, International Journal of Urology, 4(2) (2), 198 - 201, English
[Refereed]
Scientific journal

Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models
SC Ko, J Cheon, CH Kao, A Gotoh, T Shirakawa, RA Sikes, G Karsenty, LWK Chung
Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed in high levels by osteoblasts. To determine whether the OC promoter mediates cell-specific gene expression in cells of osteoblast lineage, we constructed a recombinant adenovirus, Ad-OC-TK, which contains the OC promoter that drives the expression of herpes simplex virus thymidine kinase (TK). We tested the expression of TK by this virus in osteoblast cell lines as well as in non-osteoblastic cell lines by assessing the enzyme activity of TK in vitro. Whereas the OC promoter failed to drive the expression of the TK gene in several non osteoblastic cell lines such as WH, a human bladder transitional carcinoma, and NIH 3T3, an embryonic mouse fibroblast cell line, the OC promoter mediated high levels of expression in osteoblast cell lines including murine ROS and human MG-63 cells. The addition of acyclovir (ACV), a pro-drug for the inhibition of cell proliferation, resulted in the induction of osteoblast-specific cell death in vitro. Intratumoral injection of Ad-OC-TK into murine ROS osteosarcoma abolished tumor growth in a host treated with subsequent i.p. ACV injection in vivo. The Ad-OC-TK virus plus ACV treatment appears to be highly selective in blocking the growth of both murine and human osteosarcoma cell lines in vitro and murine osteosarcoma in vivo.
AMER ASSOC CANCER RESEARCH, Oct. 1996, CANCER RESEARCH, 56(20) (20), 4614 - 4619, English
[Refereed]
Scientific journal

Hyperprolactinaemia among infertile patients and its effect on sperm functions
H Okada, T Iwamoto, H Fujioka, T Shirakawa, N Tatsumi, M Kanzaki, K Minayoshi, K Ohya, M Fujisawa, S Arakawa, S Kamidono, J Ishigami
The effects of hyperprolactinaemia on sperm function were investigated in 264 men with oligozoo-, asthenozoo-, or teratozoospermia and who were attending a male infertility clinic. None of the patients exhibited galactorrhea or complained of impotence. There was no correlation between abnormal values in spermiogram and hyperprolactinaemia. After multiple measurements of serum prolactin concentration, 15 cases (5.7%) were diagnosed as hyperprolactinaemic (greater than or equal to 10 ng ml(-1)). Six of these patients were taking cimetidine and six were taking anti-anxiety drugs. Serum prolactin returned to the normal level after discontinuation of these drugs; thus these 12 cases were considered as drug-induced hyperprolactinaemia. The other three patients were diagnosed as having pituitary microadenomas and received bromocriptine treatment; the serum prolactin levels normalized within 1 month. No changes in sperm concentration, motility or morphology were found after normalization of serum prolactin levels. Sperm fertilizing ability was monitored by the hamster test for 10 months in the three patients with pituitary microadenoma, and no improvement was observed. Results suggest that hyperprolactinaemia, which does not cause symptoms, has little effect on the impairment of sperm functions. Measurement of serum prolactin in infertile men could be justified, however, for early detection of pituitary adenomas.
BLACKWELL WISSENSCHAFTS-VERLAG GMBH, Jul. 1996, ANDROLOGIA, 28(4) (4), 197 - 202, English
[Refereed]
Scientific journal

不妊患者にみられたImmotile spermatozoaに関する臨床的検討
岡田弘, 藤岡一, 龍見昇, 白川利朗, 神崎正徳, 岩本孝弘, 藤澤正人, 荒川創一, 守殿貞夫
一般社団法人日本泌尿器科学会, 1996, 日本泌尿器科学会雑誌, 87(2) (2), 208 - 208, Japanese

特発性男性不妊のY染色体遺伝子異常について
白川利朗, 藤澤正人, 藤岡一, 龍見昇, 神崎正徳, 岩本孝弘, 岡田弘, 荒川創一, 守殿貞夫
一般社団法人日本泌尿器科学会, 1996, 日本泌尿器科学会雑誌, 87(2) (2), 479 - 479, Japanese

Sertoli cellにおけるNeuropeptide Yの発言とその制御
神崎正徳, 藤澤正人, 白川利朗, 龍見昇, 源吉顕治, 岩本孝弘, 岡田弘, 荒川創一, 守殿貞夫
一般社団法人日本泌尿器科学会, 1995, 日本泌尿器科学会雑誌, 86(3) (3), 557 - 557, Japanese

Sertoli cellにおけるInterleukin 6 receptor(IL-6-R)mRNAの発現とその制御
奥田喜啓, 藤澤正人, 白川利朗, 神崎正徳, 源吉顕治, 岩本孝弘, 岡田弘, 荒川創一, 守殿貞夫
一般社団法人日本泌尿器科学会, 1995, 日本泌尿器科学会雑誌, 86(3) (3), 763 - 763, Japanese

乏精子症患者におけるLH-RH testの意義について
藤澤正人, 白川利朗, 龍見昇, 神崎正徳, 岩本孝弘, 源吉顕治, 岡田弘, 荒川創一, 守殿貞夫
一般社団法人日本泌尿器科学会, 1995, 日本泌尿器科学会雑誌, 86(3) (3), 760 - 760, Japanese

Image cytometryによるヒト精巣DNAの定量的解析
岩本孝弘, 藤澤正人, 白川利朗, 神崎正徳, 源吉顕治, 岡田弘, 荒川創一, 守殿貞夫, 日中宏和, 松本修
一般社団法人日本泌尿器科学会, 1995, 日本泌尿器科学会雑誌, 86(3) (3), 761 - 761, Japanese

MISC

インドネシアにおけるヒト及び環境由来のESBL産生Escherichia coliの分布調査
坂本夏那, 大澤佳代, 重村克巳, 重村克巳, 北川孝一, 木下承晧, 亀岡正典, 楠木まり, 楠木まり, 宮良高維, 藤沢正人, 白川利朗, 白川利朗
2021, 日本化学療法学会雑誌, 69(Supplement-A) (Supplement-A)

Preclinical Study of Oral Cancer Vaccine Using Recombinant Bifidobacterium Expressing WT1 Protein in Murine Bladder Cancer Model and Non-Human Primate
Koichi Kitagawa, Mako Kato, Shota Komai, Ryota Sako, Hazuki Doi, Yoshiko Hashii, Takane Katayama, Toshiro Shirakawa
CELL PRESS, Apr. 2020, MOLECULAR THERAPY, 28(4) (4), 35 - 36, English
Summary international conference

Genetic diagnosis of urinary tract infection
Toshiro Shirakawa, Masato Fujisawa
Lead, Feb. 2020, 泌尿器外科, 33(2) (2), 117 - 120, Japanese
[Invited]
Introduction scientific journal

The anti-tumor effect of oral cancer vaccine using Bifidobacterium as a platform for displaying Wilms' tumor 1 protein.
Hikaru Minagawa, Yoshiko Hashii, Natsuki Nakagawa, Hiroko Nakajima, Yoshihiro Oka, Takane Katayama, Toshiro Shirakawa, Keiichi Ozono
AMER SOC CLINICAL ONCOLOGY, Mar. 2019, JOURNAL OF CLINICAL ONCOLOGY, 37(8) (8), English
Summary international conference

Cancer immunotherapy combining oral vaccination of recombinant Bifidobacterium longum displaying human Wilms' tumor 1 protein and anti-PD-1 checkpoint blockade for solid tumors in mice experimental model
Koichi Kitagawa, Maho Tatsumi, Mako Kato, Shota Komai, Yoshiko Hashii, Takane Katayama, Toshiro Shirakawa
AMER ASSOC CANCER RESEARCH, Feb. 2019, CANCER IMMUNOLOGY RESEARCH, 7(2) (2), English
Summary international conference

Combination of Oral WT1 Cancer Vaccine and Anti-PD-1 Antibody Induced the Synergistic Anti-Tumor Effect in Mouse Prostate Cancer Model
Koichi Kitagawa, Reina Gonoi, Hiroki Saito, Maho Tatsumi, Yoshiko Hashii, Takane Katayama, Toshiro Shirakawa
CELL PRESS, May 2017, MOLECULAR THERAPY, 25(5) (5), 156 - 156, English
Summary international conference

INTERNATIONAL COMPARISON OF CAUSATIVE BACTERIA AND ANTIMICROBIAL SUSCEPTIBILITIES OF URINARY TRACT INFECTIONS BETWEEN DEVELOPED AND DEVELOPING COUNTRIES
Katsumi Shigemura, Koichi Kitagawa, Kuntaman Kuntaman, Toshiro Shirakawa, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2017, JOURNAL OF UROLOGY, 197(4) (4), E295 - E295, English
Summary international conference

Development of Novel Cancer Therapy Combination of Ad-SOCS Gene Therapy and LAK Cell Immunotherapy for the Treatment of Prostate Cancer
Hiroki Saito, Koichi Kitagawa, Tetsuji Naka, Satoshi Serada, Toshiro Shirakawa
NATURE PUBLISHING GROUP, May 2016, MOLECULAR THERAPY, 24, S49 - S49, English
Summary international conference

Development of the Novel Oral Tumor Vaccine Using Bifidobacterium longum Displaying Wilms' Tumor 1 Protein
Koichi Kitagawa, Tsugumi Oda, Ayame Araki, Reina Gonoi, Hiroki Saito, Yoshiko Hashii, Takane Katayama, Keiichi Ozono, Toshiro Shirakawa
NATURE PUBLISHING GROUP, May 2016, MOLECULAR THERAPY, 24, S157 - S157, English
Summary international conference

MOLECULAR CHARACTERISTICS OF EXTENDED-SPECTRUM ?-LACTAMASE-PRODUCING ESCHERICHIA COLI ISOLATED URINARY TRACT INFECTION IN A UNIVERSITY TEACHING HOSPITAL
Katsumi Shigemura, Kayo Osawa, Kazushi Tanaka, Yuzo Nakano, Toshiro Shirakawa, Soichi Arakawa, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2016, JOURNAL OF UROLOGY, 195(4) (4), E274 - E274, English
Summary international conference

The Combination of p53 Dendritic Cell Vaccine and Ad-p53 Gene Therapy Against p53 Overexpressing and p53 Deleted Tumor Cell Lines
Hiroki Saito, Koichi Kitagawa, Risa Yamasaki, Nami Katai, Naoya Morishita, Masato Kawabata, Dody Bautista, Dante Dator, Toshiro Shirakawa
NATURE PUBLISHING GROUP, May 2015, MOLECULAR THERAPY, 23, S167 - S167, English
Summary international conference

Development of Combination Therapy of Bifidobacterium-Based Oral Vaccine Displaying HCV-NS3 with Interferon-alpha
Koichi Kitagawa, Hiroki Ishikawa, Tsugumi Oda, Hiroki Saito, Naoya Morishita, Kouske Shimamoto, Norihisa Nishida, Yoichi Matsuura, Hak Hotta, Toshiro Shirakawa
NATURE PUBLISHING GROUP, May 2015, MOLECULAR THERAPY, 23, S259 - S259, English
Summary international conference

神戸大学インドネシア拠点のあゆみと実績
内海孝子, 清水一史, 小瀧将裕, 亀岡正典, 白川利朗, HOTTA HAK, 林祥剛
Apr. 2015, 最新医学, 74(4) (4), 745 - 743, Japanese
[Refereed][Invited]
Others

Indonesia-Kobe University Collaborative Research Center for Emerging and Reemerging Infectious Diseases (CRC-ERID) J-GRID (Japan initiative for Global Research Network on Infectious Diseases)
Toshiro Shirakawa, Kazufumi Shimizu, Takako Utsumi, Masanori Kameoka, Hak Hotta, Yoshitake Hayashi
The Center for Infectious Diseases (CID), Kobe University Graduate School of Medicine, has led an Asia-related medical research program for over 50 years. The Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) established the Indonesia-Kobe University Collaborative Research Center for Emerging and Reemerging Infectious Diseases (CRC-FRID), which is staffed by Japanese researchers from the CID, Kobe University Graduate School of Medicine, and Indonesian researchers from the Institute of Tropical Disease (ITD) of Airlangga University, Surabaya, Indonesia. There they focus on five disease types – influenza, infectious hepatitis, dengue fever, HIV/AIDS, and infectious diarrheal diseases – in collaborative research. This paper summarizes research results for these 5 diseases as published in previous papers.
Fuji Technology Press, 01 Oct. 2014, Journal of Disaster Research, 9(5) (5), 828 - 835, English
Book review

META-ANALYSIS OF EFFICACY AND SAFETY OF AD-P53 and E1B 55KD REGION DELETED ADENOVIRUS GENE THERAPIES
Yasuhiro Endo, Toshiro Shirakawa
WILEY-BLACKWELL, Jul. 2014, JOURNAL OF GENE MEDICINE, 16(7-8) (7-8), 275 - 276, English
Summary international conference

E10A, AN ADENOVIRUS CARRYING ENDOSTATIN GENE, DRAMATICALLY INCREASED THE TUMOR DRUG CONCENTRATION OF METRONOMIC CHEMOTHERAPY WITH LOW DOSE CISPLATIN IN A XENOGRAFT MOUSE MODEL FOR HEAD AND NECK SQUAMOUS CELL CARCINOMA
Zainal Adhim, Xubin Lin, Wenlin Huang, Tsutomu Nakamura, Hiroyuki Yasui, Naoki Otsuki, Ken-ichi Nibu, Masato Fujisawa, Toshiro Shirakawa
WILEY-BLACKWELL, Jul. 2014, JOURNAL OF GENE MEDICINE, 16(7-8) (7-8), 238 - 239, English
Summary international conference

REGULATORY PATHWAY FOR THE DEVELOPMENT OF ADVANCED THERAPY
Toshiro Shirakawa, Yasuhiro Endo
WILEY-BLACKWELL, Jul. 2014, JOURNAL OF GENE MEDICINE, 16(7-8) (7-8), 269 - 270, English
Summary international conference

SIGNIFICANT RECEPTORS OR BIOMARKERS FOR THE SYMPTOMS OF OVERACTIVE BLADDER
Fukashi Yamamichi, Katsumi Shigemura, Toshiro Shirakawa, Hosny Behnsawy, Masuo Yamashita, Hideaki Miyake, Kazushi Tanaka, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2014, JOURNAL OF UROLOGY, 191(4) (4), E50 - E50, English
Summary international conference

【C型肝炎治療update】基礎研究の進歩ビフィズス菌を利用したC型肝炎経口ワクチンの開発(解説/特集)
Shirakawa Toshiro
Feb. 2014, 日本臨床, 73(2) (2), 239 - 242, Japanese
Introduction scientific journal

SIGNIFICANT BIOMARKER FOR LOWER URINARY TRACT SYMPTOMS OR PAIN FROM CHRONIC PROSTATITIS
Fukashi Yamamichi, Minori Matsumoto, Katsumi Shigemura, Toshiro Shirakawa, Hideaki Miyake, Soichi Arakawa, Kazushi Tanaka, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2013, JOURNAL OF UROLOGY, 189(4) (4), E480 - E481, English
Summary international conference

DOES MUTATION IN GYRA OR PARC OR EFFLUX PUMP EXPRESSION PLAY THE MAIN ROLE IN FLUOROQUINOLONE-RESISTANT ESCHERICHIA COLI URINARY TRACT INFECTIONS?
Katsumi Shigemura, Kazushi Tanaka, Toshiro Shirakawa, Soichi Arakawa, Hideaki Miyake, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2013, JOURNAL OF UROLOGY, 189(4) (4), E472 - E472, English
Summary international conference

MUTATIONS IN THE GYRA AND PARC GENES AND IN VITRO ACTIVITIES OF FLUOROQUINOLONES IN CLINICAL ISOLATES OF PSEUDOMONAS AERUGINOSA DERIVED FROM URINARY TRACT INFECTIONS AND THEIR RAPID DETECTION BY DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
Minori Matsumoto, Katsumi Shigemura, Toshiro Shirakawa, Hideaki Miyake, Soichi Arakawa, Kazushi Tanaka, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2013, JOURNAL OF UROLOGY, 189(4) (4), E472 - E472, English
Summary international conference

Post-operative infection and prophylactic antibiotic administration after radical cystectomy with orthotopic neobladder urinary diversion
Katsumi Shigemura, Kazushi Tanaka, Minori Matsumoto, Yuzo Nakano, Toshiro Shirakawa, Masahiro Miyata, Masuo Yamashita, Soichi Arakawa, Masato Fujisawa
The purpose of this study was to investigate the association between prophylactic antibiotic administration (PAA) and post-operative infection in radical cystectomy with orthotopic neobladder urinary diversion carried out for patients with bladder cancer. Fifty-seven consecutive cases were analyzed retrospectively. Post-operative infections were categorized as urinary tract, wound, and remote infections. We used the antibiotics tazobactam/piperacillin (TAZ/PIPC), sulbactam/ampicillin (SBT/ABPC), flomoxef (FMOX), cefazolin (CEZ), cefotiam (CTM), and cefmetazole (CMZ). Twenty-five (43.9%) patients had post-operative infections. Five of these (8.77%) patients had wound infections, 22 (38.6%) patients had urinary tract infections, and 2 (3.51%) had remote infections. Our statistical analysis demonstrated that the patients with TAZ/PIPC used for PAA (5/18: 27.8%) had a significantly lower post-operative infection rate than patients with other antibiotics (24/39: 61.5%) (p = 0.0442). In addition, the patients with a shorter-duration PAA (within 72 h after the operation (48-72 h)) had a significantly lower rate of post-operative infections (12/33: 36.4%) than those with longer-duration PAA (longer than 72-96 h after the operation) (16/24: 66.7%) (p = 0.0239). Taken together, these results suggest that TAZ/PIPC with shorter PAA duration (within 72 h) might lead to a lower rate of post-operative infections. In conclusion, our data showed that PAA with TAZ/PIPC with a shorter duration PAA (within 72 h) might be recommended for radical cystectomy with orthotopic neobladder reconstruction. A prospective study based on our data is desirable to establish or revise guidelines for prophylactic medication for preventing post-operative infection after radical cystectomy with orthotopic neobladder urinary diversion.
SPRINGER JAPAN KK, Aug. 2012, JOURNAL OF INFECTION AND CHEMOTHERAPY, 18(4) (4), 479 - 484, English
[Refereed]
Introduction scientific journal

Sonic hedgehog signaling and Androgens are linked in tumor-stromal interaction through Epithelial-Mesenchymal transition (EMT) in prostate cancer progression
Katsumi Shigemura, Fatma Meligy, Hosny Behnsawy, Fukashi Yamamichi, Wen-Chin Haung, Xiangyan Li, Leland Chung, Kunito Yamanaka, Keisuke Hanioka, Masuo Yamashita, Toshiro Shirakawa, Kazushi Tanaka, Soichi Arakawa, Masato Fujisawa
AMER ASSOC CANCER RESEARCH, Apr. 2012, CANCER RESEARCH, 72, English
Summary international conference

SONIC HEDGEHOG SIGNALING AND ANDROGENS ARE LINKED IN TUMOR-STROMAL INTERACTION THROUGH EPITHELIAL-MESENCHYMAL TRANSITION (EMT) IN PROSTATE CANCER PROGRESSION
Meligy Fatma, Katsumi Shigemura, Hosny Behnsawy, Fukashi Yamamichi, Wen-chin Haung, Xiangyan Li, Leland Chung, Masuo Yamashita, Masato Fujisawa, Masato Kawabata, Toshiro Shirakawa
ELSEVIER SCIENCE INC, Apr. 2012, JOURNAL OF UROLOGY, 187(4) (4), E395 - E395, English
Summary international conference

POSSIBLE ROLE OF SONIC HEDGEHOG SIGNALING AND THE LINK WITH EPITHELIAL-MESENCHYMAL TRANSITION (EMT) IN RENAL CANCER PROGRESSION
Fatma Meligy, Katsumi Shigemura, Hosny Behnsawy, Fukashi Yamamichi, Masuo Yamashita, Wen-chin Haung, Li Xiangyan, Leland Chung, Masato Fujisawa, Masato Kawabata, Toshiro Shirakawa
ELSEVIER SCIENCE INC, Apr. 2012, JOURNAL OF UROLOGY, 187(4) (4), E60 - E61, English
Summary international conference

THE MECHANISMS AND RISK FACTORS FOR FLUOROQUINOLONE-RESISTANCE IN ENTEROCOCCUS FAECALIS STRAINS CLINICALLY ISOLATED FROM URINARY TRACT INFECTION PATIENTS
Tomihiko Yasufuku, Katsumi Shigemura, Minori Matsumoto, Yuzo Nakano, Toshiro Shirakawa, Kazushi Yanaka, Masato Kawabata, Soichi Arakawa, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2012, JOURNAL OF UROLOGY, 187(4) (4), E372 - E372, English
Summary international conference

Rapid detection of the gyrA and parC mutations in fluoroquinolone-resistant pseudomonas aeruginosa strain by denaturing high-performance liquid chromatography in UTI patients in Japan
M. Matsumoto, K. Shigemura, T. Shirakawa, T. Yasufuku, Y. Nakano, K. Tanaka, S. Kinoshita, M. Kawabata, S. Arakawa, M. Fujisawa
ELSEVIER SCIENCE BV, Feb. 2012, EUROPEAN UROLOGY SUPPLEMENTS, 11(1) (1), E295 - E295, English
Summary international conference

Gene therapy for the treatment of prostate cancer using osteocalcin promoter
後藤章暢, 寺尾秀治, 白川利朗
日本臨床社, Jun. 2011, Japanese journal of clinical medicine, 69, 550 - 553, Japanese

エンドスタチン遺伝子導入による腫瘍内プラチナ濃度の増大
中村任, Adhim Zainal, Lin Xubin, Huang Wenlin, 安井裕之, 大月直樹, 丹生健一, 藤澤正人, 白川利朗
(公社)日本薬剤学会, May 2011, 日本薬剤学会年会講演要旨集, 26年会, 229 - 229, Japanese

前立腺癌患者末梢血中Circulating tumor cells(CTCs)の検出法の開発
山道深, 森下真一, 白川利朗, 藤澤正人, 松岡孝幸, 川端眞人
泌尿器科紀要刊行会, May 2011, 泌尿器科紀要, 57(5) (5), 275 - 275, Japanese

CORRELATION OF OVEREXPRESSION OF EFFLUX PUMP GENES WITH ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI STRAINS CLINICALLY ISOLATED FROM URINARY TRACT INFECTION PATIENTS
Tomihiko Yasufuku, Katsumi Shigemura, Toshiro Shirakawa, Minori Matsumoto, Yuzo Nakano, Kazushi Tanaka, Soichi Arakawa, Shouhiro Kinoshita, Masato Kawabata, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2011, JOURNAL OF UROLOGY, 185(4) (4), E544 - E545, English
Summary international conference

INTRAVENOUS INJECTION OF THE CARRIER-CELL BASED ONCOLYTIC ADENOVIRUS SUPPRESSES THE GROWTH OF MULTIPLE LUNG TUMORS IN MOUSE SQUAMOUS CELL CARCINOMA MODEL
Toshiro Shirakawa, Katsuyuki Hamada, Shunichi Kitajima, Chihomi Mitsuoka, Naya Saito, Tadayuki Matsuoka, Zainal Adhim, Shuji Terao, Akinobu Gotoh, Ken-ichi Nibu, Masato Fujisawa, Kyung-Mi Lee
JOHN WILEY & SONS LTD, Dec. 2010, JOURNAL OF GENE MEDICINE, 12(12) (12), 1034 - 1035, English
Summary international conference

RISK FACTORS FOR URINARY TRACT INFECTION WITH FLUOROQUINOLONE-RESISTANT ESCHERICHIA COLI STRAINS
Tomihiko Yasufuku, Katsumi Shigemura, Toshiro Shirakawa, Minori Matsumoto, Yuzo Nakano, Kazushi Tanaka, Soichi Arakawa, Shouhiro Kinoshita, Masato Kawabata, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2010, JOURNAL OF UROLOGY, 183(4) (4), E236 - E236, English
Summary international conference

ヒト膀胱癌細胞におけるCox‐2阻害剤,3剤の抗腫瘍効果の比較検討
白川利朗, アドヒムザイナル, 松岡孝幸, 重村克己, 丹生健一, 川端眞人, 藤澤正人
(一社)日本泌尿器科学会, Feb. 2010, 日本泌尿器科学会雑誌, 101(2) (2), 250 - 250, Japanese

Intravenous Injection of Allogeneic Tumor Cell Vaccine Carrying Oncolytic Adenovirus (AdE3-IAI.3B) Suppresses the Growth of Multiple Lung Tumors in Mouse Squamous Cell Carcinoma Model
Toshiro Shirakawa, Katsuyuki Hamada, Shunichi Kitajima, Chihomi Mitsuoka, Naoya Morishita, Norie Yamaoka, Aya Saito, Tadayuki Matsuoka, Zainal Adhim, Shuji Terao, Akinobu Gotoh, Ken-ichi Nibu, Masato Fujisawa, Kyung-Mi Lee, Wenlin Huang
NATURE PUBLISHING GROUP, May 2009, MOLECULAR THERAPY, 17, S159 - S159, English
Summary international conference

Pharmacological and biosafety test of cancer gene therapy by oncolytic adenovirus-infected carrier cell
Katsuyuki Hamada, Ting Zhang, Toshiro Shirakawa, Wenlin Huang
AMER ASSOC CANCER RESEARCH, May 2009, CANCER RESEARCH, 69, English
Summary international conference

EMERGENCE OF FLUOROQUINOLONE-RESISTANT E. COLT STRAIN AND THEIR RELATED MUTATIONS OF THE GYRA AND PARC GENES IN UTI PATIENTS IN JAPAN
Tomihiko Yasufuku, Katsumi Shigemura, Toshiro Shirakawa, Yuzo Nakano, Kazushi Tanaka, Soichi Arakawa, Shouhiro Kinoshita, Masato Kawabata, Masato Fujisawa
ELSEVIER SCIENCE INC, Apr. 2009, JOURNAL OF UROLOGY, 181(4) (4), 140 - 140, English
Summary international conference

CLINICAL TRIAL DESIGN FOR ADENOVIRAL GENE THERAPY PRODUCTS
Toshiro Shirakawa
Two adenoviral gene therapy products have now been approved for clinical use in China, and elsewhere at least four products are in phase III clinical trials. The adenovirus vector is the most commonly used vector for clinical gene therapy due to its high rate of gene transfer in vivo. However, the clinical efficacy of this vector has yet to be proven, despite over 300 clinical trials that have shown it to be well-tolerated and efficient in gene transfer. Recent studies in China have reported improved efficacy of an adenoviral gene therapy product in combination with radiotherapy and/or chemotherapy for the treatment of several types of cancer. There are now at least three phase III clinical trials for adenoviral cancer gene therapy that are ongoing in the U.S., and two of these call for combination therapy with either chemotherapy or radiation. In addition, in the European Union, one adenoviral gene therapy product for malignant glioma in combination with surgery showed a therapeutic benefit in the preliminary results of a phase III trial. This product, when applied as just one component of a multimodality approach, along with surgery, radiation and/or chemotherapy, holds some promise for proving the clinical efficacy of adenovirus vectors.
PROUS SCIENCE, SA, Apr. 2009, DRUG NEWS & PERSPECTIVES, 22(3) (3), 140 - 145, English
[Refereed]
Introduction scientific journal

【がん化学療法個別化の現状と展望】泌尿器科腫瘍の遺伝子診断と個別化治療を目指した分子標的療法
Shirakawa Toshiro
医薬ジャ-ナル社, Dec. 2008, 医薬ジャーナル, 44巻, 12号, pp. 83-86(12) (12), 67 - 71, Japanese
Introduction scientific journal

腎臓がん患者におけるsorcinの発現低下(Suppression of sorcin mRNA in patients with renal cell carcinoma)
川上恵, 中村任, 岡村昇, 寺尾秀治, 後藤章暢, 白川利朗, 藤澤正人, 山森元博, 栄田敏之
日本癌学会, Sep. 2008, 日本癌学会総会記事, 67回, 390 - 391, English

The current status of adenovirus-based cancer gene therapy
Toshiro Shirakawa
Adenoviruses are the most commonly used gene-delivery vectors due to the efficiency of their in vivo gene transfer. Since 1993, about 300 protocols using an adenoviral vector have been performed, although they have yet to be proven effective in clinical trials. The adenovirus-based vector has been continuously improved by modification of the adenoviral genome and capsid, and novel adenovirus-delivery systems, such as the carrier-cell delivery system, have been recently proposed. Adenovirus-based cancer gene therapy is fast becoming one component of a multi-modality treatment approach to advanced cancer, along with surgery, radiotherapy, and chemotherapy.
KOREAN SOC MOLECULAR & CELLULAR BIOLOGY, Jun. 2008, MOLECULES AND CELLS, 25(4) (4), 462 - 466, English
[Refereed]
Book review

Therapeutic efficacy of midkine promoter-based conditionally replicative adenovirus vector for targetting the midkine-expressing human bladder cancer cells
Shuji Terao, Toshiro Shirakawa, Kazushi Tanaka, Atsushi Takenaka, Sadao Kamidono, Masato Fujisawa, Akinobu Gotoh
ELSEVIER SCIENCE INC, Apr. 2008, JOURNAL OF UROLOGY, 179(4) (4), 374 - 374, English
Summary international conference

腎癌におけるSorcin発現の検討とVEGF発現に及ぼす影響
寺尾秀治, 岡村昇, 白川利朗, 中村任, 栄田敏之, 奥村勝彦, 武中篤, 藤澤正人, 後藤章暢
(一社)日本泌尿器科学会, Feb. 2008, 日本泌尿器科学会雑誌, 99(2) (2), 485 - 485, Japanese

Gene therapy in prostate cancer: past, present and future
Toshiro Shirakawa, Masato Fujisawa, Akinobu Gotoh
The absence of effective therapies for hormone refractory prostate cancer establishes the need to develop novel therapeutic modality, such as a gene therapy, that can be applied either separately or in conjunction with current treatment modalities for the treatment of advanced prostate cancer. About 80 protocols for prostate cancer gene therapy have been practiced since 1994. The gene therapy modality is ideal for the treatment of prostate cancer. The disease progress can be precisely monitored by serum-PSA level, the local access is easy by ultra-sound guidance, and prostate as an accessory organ is highly immunogenic. Consequently, the number of prostate cancer gene therapy trials is increasing now. In this paper, we review the previous clinical trials of prostate cancer gene therapy in the chronologic order, and predict the future prospects.
FRONTIERS IN BIOSCIENCE INC, Jan. 2008, FRONTIERS IN BIOSCIENCE-LANDMARK, 13, 2115 - 2119, English
[Refereed]
Introduction scientific journal

Human gene therapy for prostate cancer: present and future
後藤章暢, 寺尾秀治, 白川利朗
日本臨床社, Dec. 2007, Japanese journal of clinical medicine, 65, 517 - 521, Japanese

Ad-OC-TK gene therapy for the treatment of prostate cancer
白川利朗, 後藤章暢, 藤澤正人
日本臨床社, Dec. 2007, Japanese journal of clinical medicine, 65, 528 - 532, Japanese

ホルモン不応性再燃前立腺癌の治療骨転移を有するホルモン不応性前立腺癌に対する遺伝子治療臨床研究の長期成績
白川利朗, 後藤章暢, 寺尾秀治, 日向信之, 田中一志, 武中篤, 原勲, 守殿貞夫, 藤澤正人
(一社)日本癌治療学会, Sep. 2007, 日本癌治療学会誌, 42(2) (2), 337 - 337, Japanese

腎癌におけるSorcinおよびVEGFの発現に関する検討(Effect of sorcin on VEGF expression in renal cell carcinoma)
寺尾秀治, 岡村昇, 白川利朗, 中村任, 栄田敏之, 武中篤, 藤澤正人, 後藤章暢
日本癌学会, Aug. 2007, 日本癌学会総会記事, 66回, 182 - 182, English

新しい抗癌剤の臨床研究ホルモン療法抵抗性の転移性前立腺癌の患者に対するAd-OC-TK遺伝子療法の臨床試験成績(Clinical Studies of New Anticancer Drugs Results of clinical study of Ad-OC-TK gene therapy for the patients with hormone refractory metastatic prostate cancer)
白川利朗, 後藤章暢, 寺尾秀治, 日向信之, 田中一志, 武中篤, 原勲, 守殿貞夫, 藤澤正人
日本癌学会, Aug. 2007, 日本癌学会総会記事, 66回, 71 - 71, English

プロテオーム解析による腎臓癌に対する新規診断マーカー・創薬ターゲットの探索
松本隼, 岡村昇, 増田太郎, 金子直樹, 渡辺真, 後藤章暢, 白川利朗, 寺尾秀治, 瀬戸亮太, 中村任, 矢上達郎, 藤澤正人, 栄田敏之, 西村紀, 奥村勝彦
(公社)日本薬学会, Mar. 2007, 日本薬学会年会要旨集, 127年会(3) (3), 232 - 232, Japanese

Improvement of quality of life of patients with metastatic or local recurrent prostate cancer after phase I/II clinical trial of Ad-OC-TK plus valacyclovir
Shuji Terao, Toshiro Shirakawa, Masahiro Miyata, Shuji Kubo, Masato Fujisawa, Akinobu Gotoh
JOHN WILEY & SONS LTD, Dec. 2006, JOURNAL OF GENE MEDICINE, 8(12) (12), 1470 - 1470, English
Summary international conference

腎臓癌のタンパク発現プロファイル解析
瀬戸亮太, 岡村昇, 増田太郎, 西村紀, 後藤章暢, 白川利朗, 寺尾秀治, 中村任, 田中久登, 栄田敏之, 奥村勝彦
(一社)日本TDM学会, Jul. 2006, TDM研究, 23(3) (3), s138 - s138, Japanese

Genetically Engineered Bifidobacterium animalis Expressing Salmonella flagellin Gene for the Mucosal Immunization in Mouse Model
Toshiro Shirakawa, Tetsuo Takata, Akinobu Gotoh, Yasunobu Kano, Masato Kawabata
NATURE PUBLISHING GROUP, May 2006, MOLECULAR THERAPY, 13, S424 - S424, English
Summary international conference

Phase I/II clinical trial of gene therapy for hormone refractory metastatic prostate cancer
A Gotoh, S Terao, T Shirakawa
JOHN WILEY & SONS LTD, Mar. 2006, JOURNAL OF GENE MEDICINE, 8(3) (3), 371 - 372, English
Summary international conference

Therapeutic efficacy of midkine promoter-based replication-selective adenovirus vector to target the midkine-expressing human bladder cancer cells
S Terao, T Shirakawa, K Goda, M Fujisawa, A Gotoh
JOHN WILEY & SONS LTD, Mar. 2006, JOURNAL OF GENE MEDICINE, 8(3) (3), 389 - 389, English
Summary international conference

【再燃前立腺癌に対する治療戦略】遺伝子治療の現状と展望
SHIRAKAWA, Toshiro, GOTOH,Akinobu
Feb. 2006, Urology View, 4巻, 1号, pp.71-75, Japanese
Introduction scientific journal

内分泌療法抵抗性前立腺癌に対するAd-OC-TK plus Valacyclovir遺伝子治療臨床研究におけるquality of lifeの評価
寺尾秀治, 白川利朗, 日向信行, 和田義孝, 宮田賢宏, 久保秀司, 田中一志, 武中篤, 荒川創一, 原勲, 藤澤正人, 後藤章暢
2006, 西日本泌尿器科, 68

連載講座、なくならない感染症⑫、クラミジア
SHIRAKAWA, Toshiro
福田商店広告部, Dec. 2005, Circles, 7巻, 3, pp.21-23(3) (3), 21 - 23, Japanese
Introduction scientific journal

P-492 腎臓癌におけるEGFR発現量およびゲフィチニブ感受性関連体細胞変異(3.医薬品適正使用5,医療薬学の未来へ翔(はばた)く-薬剤師の薬剤業務・教育・研究への能動的関わり-)
大松秀明, 岡村昇, 後藤章暢, 白川利朗, 寺尾秀治, 中村任, 徳井健次, 田中久登, 八木麻理子, 大石美惠, 西口工司, 栄田敏之, 奥村勝彦
日本医療薬学会, 01 Sep. 2005, 日本医療薬学会年会講演要旨集, 15, 351 - 351, Japanese

マウス造精機能障害モデルにおけるTRAIL発現抑制の効果
合田上政, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 土橋正樹, OKADA,Hiroshi, KAMIDONO,Sadao, FUJISAWA, Masato
日本生殖内分泌学会, Sep. 2005, 日本生殖内分泌学会雑誌, 10巻, pp.37-42, 37 - 42, Japanese
Introduction scientific journal

NAFAMOSTAT MESILATE NOT ONLY INHIBITS PLATELET AGGREGATIONS BUT ALSO DISAGGREGATES ALREADY AGGREGATED PLATELETS
Masahiro Miyata, Toshiro Shirakawa, Bishnu Acharya, Kengou Nagamitsu, Masateru Horimoto, Akinobu Gotoh
WILEY-BLACKWELL, Jun. 2005, NEPHROLOGY, 10, A181 - A181, English
Summary international conference

COX-2臓器特異性プロモーターを組み込んだ増殖型アデノウイルスベクターによる頭頸部扁平上皮癌に対する治療法の検討
田中博紀, NIBU, Kenichi, ZhangZhujun, SHIRAKAWA, Toshiro, 後藤彰暢
May 2005, 日本耳鼻咽喉科学会会報, 108巻, 5増刊, pp.595-595, Japanese
Introduction scientific journal

Overexpression of erythropoietin increases intratesticular testosterone levels in rats with unilateral cryptorchidism
K Goda, M Dobashi, T Shirakawa, H Maruyama, S Terao, Y Kondo, H Okada, A Gotoh, M Fujisawa
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2005, JOURNAL OF UROLOGY, 173(4) (4), 410 - 410, English
Summary international conference

Establishment of a human prostate small cell cancer cell line and p53 adenoviral gene therapy
K Goda, T Shirakawa, A Gotoh, S Terao, M Dobashi, H Okada, Hara, I, S Kamidono, M Fujisawa
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2005, JOURNAL OF UROLOGY, 173(4) (4), 122 - 122, English
Summary international conference

Microencapsulation of adrenocortical cells for corticosteroid supplementation in rat bilateral adrenalectomized model
K Goda, T Shirakawa, M Dobashi, S Terao, H Okada, A Gotoh, S Kamidono, M Fujisawa
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2005, JOURNAL OF UROLOGY, 173(4) (4), 142 - 142, English
Summary international conference

Interleukin-2 enhanced the anti-tumor effect of adenoviral-mediated HSV-tk gene therapy in murine bladder cancer model.
S Terao, T Shirakawa, K Goda, S Kamidono, A Gotoh
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2005, JOURNAL OF UROLOGY, 173(4) (4), 214 - 214, English
Summary international conference

Polymorphism of the interleukin-4, interleukin-13, and signal transducer and activator of transcription 6 genes in Indonesian children with minimal change nephrotic syndrome
Bishnu Acharya, Toshiro Shirakawa, Ardanykusuma Pungky, Parlin Damanik, Muh. Nasrum Massi, Masahiro Miyata, Masafumi Matsuo, Akinobu Gotoh
Background/Aims: Minimal change nephrotic syndrome (MCNS) in children is frequently associated with allergy and immunoglobulin E (IgE) production. T-helper sub-type 2 cytokines, such as interleukin (IL)-4and IL-13, have been implicated in the regulation of IgE production. We investigated the associations of gene polymorphisms of IL-4, IL-13, and signal transducer and activator 6 (STAT6) in Indonesian children with MCNS (n = 84) and controls with neither allergic nor renal disease (n = 61). Methods: Polymerase chain reaction-restriction fragment length polymorphism was used to determine the IL-4 promoter gene polymorphism (-590C/T) and IL-13 gene polymorphism (4257G/A), and direct sequencing was used for the STAT6 3S untranslated region (2964G/A) polymorphism. Results: There was a significant difference between the MCNS group and the controls in the genotypic distribution of IL-4 and IL-13 gene polymorphism. In the case of the IL-4 promoter gene, the frequency of the CC homozygote was significantly lower in the MCNS group than in the controls, while, in the case of IL-13, the frequency of the GG homozygote was significantly lower in the MCNS group. However, there was no difference between the MCNS group and the controls in the STAT6 gene polymorphism. Conclusion: The genetic variations in the IL-4 and IL-13 genes may be associated with predisposition to MCNS. Copyright © 2005 S. Karger AG.
2005, American Journal of Nephrology, 25(1) (1), 30 - 35, English
[Refereed]
Introduction scientific journal

Phase I/II clinical trial of Ad-OC-TK plus VAL for the patients with metastatic or local recurrent prostate cancer: Initial experience in Japan
T Shirakawa, N Hinata, S Terao, K Shigemura, N Taniguch, K Fukushima, K Sugimoto, K Sugimura, M Matsuo, S Maeda, S Kamidono, CH Kao, TA Gardner, LWK Chung, A Gotoh
ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2004, MOLECULAR THERAPY, 9, S228 - S229, English
Summary international conference

Overexpression of erythropoietin in sertoli cells enhances testosterone production in leydig cells through activation of JAK2/STAT5 but not map kinases
K Goda, M Fujisawa, T Shirakawa, M Dobashi, Y Kondo, A Gotoh, H Okada, S Kamidono
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2004, JOURNAL OF UROLOGY, 171(4) (4), 362 - 362, English
Summary international conference

Propiverine hydrochloride relieves irritative symptoms of benign prostatic hyperplasia
H Okada, T Shirakawa, S Mato, T Iizumi, N Hinata, A Gotoh, S Kamidono, S Horie
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2004, JOURNAL OF UROLOGY, 171(4) (4), 357 - 358, English
Summary international conference

Age is the limiting factor for successful sperm retrieval in nonmosaic Klinefelter syndrome
H Okada, T Shirakawa, T Ishikawa, K Goda, Y Kondo, Y Yamamoto, N Sofikitis, M Fujisawa, Miyagawa, I, S Kamidono, S Horie
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2004, JOURNAL OF UROLOGY, 171(4) (4), 511 - 511, English
Summary international conference

Rapid detection of GYRA and PARC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae urethritis by denaturing high-performance liquid chromatography (DHPLC)
K Shigemura, T Shirakawa, H Okada, K Tanaka, S Arakawa, S Kamidono, A Gotoh
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2004, JOURNAL OF UROLOGY, 171(4) (4), 31 - 31, English
Summary international conference

【泌尿器科ガイドラインの背景と現況】学会とガイドライン
KAMIDONO,Sadao, SHIRAKAWA, Toshiro, GOTOH,Akinobu, HARA, Isao
Nov. 2003, Urology View, 1巻, 6号, pp. 45-49, Japanese
Introduction scientific journal

GBS distribution of Kobe city
TAKATA Tetsuo, SHIRAKAWA Toshiro, KIMURA Hiroyuki, KINOSHITA Shohiro, KAWABATA Masato
30 Sep. 2003, 地理情報システム学会講演論文集 = Papers and proceedings of the Geographic Information Systems Association, 12, 63 - 66, Japanese

A conditional replication-competent adenoviral vector, Ad-COX2-E1a, to target the COX-2 expressing human bladder cancer cells
ZJ Zhang, T Shirakawa, K Hamada, S Kamidono, M Matsuo, A Gotoh
ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2003, MOLECULAR THERAPY, 7(5) (5), S356 - S357, English
Summary international conference

A new rapid bacteral drug susceptibility testing method in the urinary tract infection using flow-cytometry
H Okada, K Shigemura, K Tanaka, N Hinata, N Hinata, T Shirakawa, A Gotoh, Y Hamaguchi, S Arakawa, S Kamidono
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2003, JOURNAL OF UROLOGY, 169(4) (4), 7 - 7, English
Summary international conference

Quantitative detection of Escherichia coli from urine samples of urinary tract infection(UTI) patients by real-time PCR method
N Hinata, T Shirakawa, A Gotoh, H Okada, S Kamidono
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2003, JOURNAL OF UROLOGY, 169(4) (4), 10 - 10, English
Summary international conference

Messnger RNA levels and enzyme activities of 5 alpha-reductase type 1 and 2 in the human benign prostatic hyperplasia (BPH) tissue
T Shirakawa, H Okada, A Gotoh, N Hinata, Y Wada, S Kamidono, T Uji, A Yamamoto
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2003, JOURNAL OF UROLOGY, 169(4) (4), 284 - 284, English
Summary international conference

In vivo gene transfer of hepatocyte growth factor accelerates restoration of spermatogenesis in rat cryptorchid model
K Goda, M Fujisawa, M Dobashi, T Yamazaki, T Shirakawa, ZJ Zhang, A Gotoh, H Okada, S Arakawa, S Kamidono, G Shiota
LIPPINCOTT WILLIAMS & WILKINS, Apr. 2003, JOURNAL OF UROLOGY, 169(4) (4), 411 - 411, English
Summary international conference

前立腺癌に対するカテキン及びアントシアニンの抗腫瘍効果の比較検討
後藤章暢, 和田義孝, 松本浩彦, 白川利朗, 日向信之, 荒川創一, 守殿貞夫, 藤澤正人
日本腎泌尿器疾患予防医学研究会, Mar. 2003, 日本腎泌尿器疾患予防医学研究会誌, 11(1) (1), 90 - 91, Japanese

前立腺肥大症組織における5-Alpha-Reductaseのサブタイプ、Type-1およびType-2の発現と酵素活性に関する検討
白川利朗, 岡田弘, 後藤章暢, 日向信之, 和田義孝, 守殿貞夫, 宇治達哉, 山本明良
社団法人日本泌尿器科学会, 15 Feb. 2003, 日本泌尿器科學會雜誌, 94(2) (2), 133 - 133, Japanese

前立腺癌に対する遺伝子治療臨床研究--臓器特異性プロモーターを用いて (第1土曜特集遺伝子治療--現状とその近未来) -- (現在審査中の遺伝子臨床研究)
後藤章暢, 白川利朗
医歯薬出版, 02 Nov. 2002, 医学のあゆみ, 203(5) (5), 344 - 348, Japanese

定量的PCR法を用いた細菌尿中の大腸菌の定量
日向信之, 白川利朗, 和田義孝, 守殿貞夫, 後藤章暢
西日本泌尿器科学会, Nov. 2002, 西日本泌尿器科, 64(増刊) (増刊), 174 - 174, Japanese

難治性前立腺癌に対する治療の現状と問題点遺伝子治療
後藤章暢, 白川利朗, 和田義孝, 日向信之, 松原重治, 原勲, 藤澤正人, 岡田弘, 荒川創一, 守殿貞夫
泌尿器科紀要刊行会, Nov. 2002, 泌尿器科紀要, 48(11) (11), 729 - 732, Japanese

MDR1遺伝子多型に依存したジゴキシン体内動態
堀之内正則, 中村任, 栄田敏之, 阪井俊介, 森田圭紀, 田村孝雄, 青山伸郎, 白川利朗, 松尾雅文, 春日雅人
(一社)日本臨床薬理学会, Mar. 2002, 臨床薬理, 33(2) (2), 255S - 256S, Japanese

ヒト十二指腸上皮におけるCYP3A4ならびにMDR1遺伝子発現量に対するMDR1遺伝子型の影響
守屋友加, 中村任, 栄田敏之, 堀之内正則, 田村孝雄, 青山伸郎, 白川利朗, 松尾雅文, 藤本貞毅, 春日雅人
(一社)日本臨床薬理学会, Mar. 2002, 臨床薬理, 33(2) (2), 377S - 378S, Japanese

CD/5-FC遺伝子治療による,膀胱癌の放射線感受性増強効果についての基礎的検討
白川利朗, 後藤章暢, 張竹君, 日向信之, 和田義孝, 原勲, 藤澤正人, 川端岳, 岡田弘, 荒川創一
社団法人日本泌尿器科学会, 20 Feb. 2002, 日本泌尿器科学会雑誌, 93(2) (2), 168 - 168, Japanese

膀胱癌細胞株におけるp53変異と放射線感受性に関する検討
日向信之, 白川利朗, 後藤章暢, 原勲, 藤澤正人, 川端岳, 岡田弘, 荒川創一, 守殿貞夫
社団法人日本泌尿器科学会, 20 Feb. 2002, 日本泌尿器科学会雑誌, 93(2) (2), 321 - 321, Japanese

泌尿器科腫瘍学における分子研究の展望遺伝子治療
後藤章暢, 白川利朗, 和田義孝, 日向信之, 原勲, 藤澤正人, 岡田弘, 荒川創一, 守殿貞夫
泌尿器科紀要刊行会, Nov. 2001, 泌尿器科紀要, 47(11) (11), 829 - 832, Japanese

自殺遺伝子を用いた癌遺伝子治療における臓器特異性プロモーターの有用性についての基礎研究
後藤章暢, 和田義孝, 白川利朗, 日向信之, 藤澤正人, 岡田弘, 守殿貞夫
日本癌学会, Sep. 2001, 日本癌学会総会記事, 60回, 619 - 619, Japanese

膀胱癌に対するCD/5-FC遺伝子治療及び放射線療法の併用療法の有用性についての基礎的検討
白川利朗, 後藤章暢, 日向信之, 藤澤正人, 岡田弘, 守殿貞夫
日本癌学会, Sep. 2001, 日本癌学会総会記事, 60回, 618 - 618, Japanese

ヒト大腸がん及び大腸ポリープにおける薬物輸送担保のリアルタイムPCR解析
角本幹夫, 中村任, 大本暢子, 栄田敏之, 森田圭紀, 田村孝雄, 青山伸郎, 春日雅人, 白川利朗, 松尾雅文
(公社)日本薬学会, Mar. 2001, 日本薬学会年会要旨集, 121年会(3) (3), 28 - 28, Japanese

Caco-2細胞及びヒト十二指腸上皮細胞における薬物輸送担体・代謝酵素発現量のリアルタイムPCR解析
大本暢子, 角本幹夫, 中村任, 栄田敏之, 森田圭紀, 田村孝雄, 青山伸郎, 白川利朗, 松尾雅文, 春日雅人
(公社)日本薬学会, Mar. 2001, 日本薬学会年会要旨集, 121年会(3) (3), 113 - 113, Japanese

前立腺癌細胞に対する放射線・アデノウイルスp53遺伝子併用療法の検討
佐々木良平, 白川利朗, 後藤章暢, 松尾雅文, 杉村和朗
(公社)日本医学放射線学会, Feb. 2001, 日本医学放射線学会雑誌, 61(2) (2), S201 - S201, Japanese

カテキン及びアントシアニンの前立腺癌に対する抗腫瘍効果の比較検討
松本浩彦, 後藤章暢, 白川利朗, 和田義孝, 日向信之, 藤澤正人, 荒川創一, 守殿貞夫
日本癌学会, Sep. 2000, 日本癌学会総会記事, 59回, 607 - 607, Japanese

【がん遺伝子治療の進展と現状での問題点】前立腺癌に対する新しい遺伝子治療法の確立
和田義孝, 後藤章暢, 白川利朗, 日向信之, 岡田弘, 荒川創一, 守殿貞夫, Kao Chinghai, Gardner Thomas A, Chung Leland
(一社)日本癌治療学会, Sep. 2000, 日本癌治療学会誌, 35(2) (2), 249 - 249, Japanese

癌遺伝子治療におけるアデノウイルスベクターの毒性比較
和田義孝, 後藤章暢, 白川利朗, 日向信之, 原勲, 藤澤正人, 岡田弘, 荒川創一, 守殿貞夫, Gardner Thomas
日本癌学会, Sep. 2000, 日本癌学会総会記事, 59回, 404 - 405, Japanese

膀胱癌に対する放射線療法及びp53遺伝子治療による併用療法の検討
日向信之, 白川利朗, 佐々木良平, 後藤章暢, 和田義孝, 藤澤正人, 荒川創一, 松尾雅文, 守殿貞夫
日本癌学会, Sep. 2000, 日本癌学会総会記事, 59回, 63 - 64, Japanese

抗癌剤抵抗性ヒト膀胱腫瘍に対するp53アデノウイルスベクターを用いた遺伝子治療の検討
白川利朗, 後藤章暢, 佐々木良平, 日向信之, 和田義孝, 藤澤正人, 岡田弘, 荒川創一, 松尾雅文, 守殿貞夫
日本癌学会, Sep. 2000, 日本癌学会総会記事, 59回, 407 - 407, Japanese

前立腺肥大症に対する遺伝子治療の検討
日向信之, 白川利朗, 後藤章暢, 松原重治, 和田義孝, 藤澤正人, 荒川創一, 守殿貞夫
泌尿器科紀要刊行会, Sep. 2000, 泌尿器科紀要, 46(9) (9), 681 - 681, Japanese

抗癌剤抵抗性ヒト膀胱腫瘍に対するp53アデノウイルスベクターを用いた遺伝子治療の検討
白川利朗, 後藤章暢, 佐々木良平, 日向信之, 和田義孝, 藤澤正人, 岡田弘, 荒川創一, 内藤誠二, 松尾雅文
社団法人日本泌尿器科学会, 20 Mar. 2000, 日本泌尿器科学会雑誌, 91(3) (3), 250 - 250, Japanese

前立腺癌骨転移モデルを想定した癌遺伝子治療
和田義孝, 後藤章暢, 白川利朗, 日向信之, Chung Leland, Gardner Thomas, 守殿貞夫
社団法人日本泌尿器科学会, 20 Mar. 2000, 日本泌尿器科学会雑誌, 91(3) (3), 354 - 354, Japanese

ぼうこう癌に対する放射線療法およびp53遺伝子治療による併用療法の検討
日向信之, 白川利朗, 佐々木良平, 後藤章暢, 和田義孝, 藤沢正人, 荒川創一, 松尾雅文, 守殿貞夫
2000, Japanese Journal of Cancer Research, 91(Supplement (Sept)) (Supplement (Sept))

カテキンおよびアントシアニンの前立腺癌に対する抗腫よう効果の比較検討
松本浩彦, 後藤章暢, 白川利朗, 和田義孝, 日向信之, 藤沢正人, 荒川創一, 守殿貞夫
2000, Japanese Journal of Cancer Research, 91(Supplement (Sept)) (Supplement (Sept))

癌遺伝子治療における,アデノウイルスベクターの毒性比較
和田義孝, 後藤章暢, 白川利朗, 日向信之, 原勲, 守殿貞夫, GARDNER T, KAO C, CHUNG L
2000, Japanese Journal of Cancer Research, 91(Supplement (Sept)) (Supplement (Sept))

抗癌剤抵抗性ヒトぼうこう腫ように対するp53アデノウイルスベクターを用いた遺伝子治療の検討
白川利朗, 後藤章暢, 佐々木良平, 日向信之, 和田義孝, 藤沢正人, 岡田弘, 荒川創一, 守殿貞夫
2000, Japanese Journal of Cancer Research, 91(Supplement (Sept)) (Supplement (Sept))

Ablative gene therapy treatment of human renal cell carcinoma: Cytosine deaminase plus 5-fluorocytosine has superior bystander effect over thymidine kinase plus acyclovir.
T Shirakawa, TA Gardner, C Kao, SC Ko, T Miyamoto, LWK Chung, NH Bander, S Woo, A Gotoh, S Kamidono
LIPPINCOTT WILLIAMS & WILKINS, May 1998, JOURNAL OF UROLOGY, 159(5) (5), 147 - 147, English
Summary international conference

A PATHOLOGICAL STUDY OF PRIMARY AND METASTATIC LESIONS IN CASES OF RENAL CELL CARCINOMA
GOTOH AKINOBU, GOHJI KAZUO, SHIRAKAWA TOSHIRO, WADA YOSHITAKA, MIZUNO YOSHIHITO, OKADA HIROSHI, ARAKAWA SOICHI, KAMIDONO SADAO, HANIOKA KEISUKE
20 Dec. 1997, 西日本泌尿器科, 59(12) (12), 881 - 884, Japanese

FUNDAMENTAL AND CLINICAL STUDIES ON THE EFFECT OF COMBINATION THERAPY WITH IMIPENEM/CILASTATIN AND VANCOMYCIN IN PATIENTS WITH MRSA INFECTIONS IN THE UROLOGICAL FIELD
IMAI TOSHIO, TANAKA KAZUSHI, CHOKYU HIROFUMI, MIYAZAKI SHIGENORI, MATSUI TAKASHI, ARAKAWA SOICHI, KAMIDONO SADAO, SAITO HIROSHI, HARADA KENJI, TACHIBANA YUJI, KATAOKA NOBUO, TAKENAKA ATSUSHI, KOBAYASHI MAKI, OMAE HIROSHI, SHINOZAKI MASAFUMI, MIZUNO YOSHIHITO, TATSUMI NOBORU, OGAWA TAKAYOSHI, YAMANAKA KUNITO, KONDO KANEYASU, NAKAGAWA HIROSHI, SHIRAKAWA TOSHIRO, GOTO KIYOHIKO
20 Mar. 1997, 西日本泌尿器科, 59(3) (3), 192 - 198, Japanese

Boari変法による尿管再建術 : 一般演題 : 第43回中部総会
川端岳, 下垣博義, 山中望, 近藤兼安, 中川泰始, 白川利朗, 岡泰彦, 岡本恭行, 斎藤宗吾
社団法人日本泌尿器科学会, 20 Jun. 1994, 日本泌尿器科學會雜誌, 85(6) (6), 1039 - 1039, Japanese

Books And Other Publications

Development of Cell Surface Engineering
Toshiro Shirakawa, Koichi Kitagawa
Contributor, 第2章医療応用２ビフィズス菌を用いた新規経口ワクチンの開発, 株式会社CMC出版, Apr. 2020

先端治療技術の実用化と開発戦略
SHIRAKAWA TOSHIRO, KITAGAWA KOICHI
Others, (株)技術情報協会, Apr. 2017, Japanese
Scholarly book

第7章経口ワクチン
SHIRAKAWA TOSHIRO
Others, 株式会社情報機構, Oct. 2013, Japanese
Scholarly book

Cancer Gene Therapy / 14.Current therapeutic strategies in gene therapy for prostate cancer
Shirakawa Toshiro
Joint work, Research Signpost, Jan. 2010, English
Scholarly book

Welcome to ゲノムワールド / 5-3 遺伝子治療 5-4 再生医療
Shirakawa Toshiro
Joint work, 廣川書店, Oct. 2009, Japanese
Textbook

Gene Therapy 2007 / Gene Therapy in prostate cancer: past, present and future
SHIRAKAWA TOSHIRO, FUJISAWA MASATO
Joint work, 21st Century's Center of Excellence Program of Japanese Ministry of Education and Science, Jan. 2007, English
Scholarly book

Lectures, oral presentations, etc.

WT1 oral cancer vaccine combined with anti-PD-1 antibody completely suppressed tumor growth in a murine bladder cancer model
SHIRAKAWA TOSHIRO, KITAGAWA KOICHI, 辰巳真帆, 門脇雅英, 片山高嶺, 橋井佳子, FUJISAWA MASATO
CIMT 2019 Annual Meeting, May 2019, English, ドイツ, International conference
Oral presentation

Cross-resistance and the mechanisms of cephalosporine-resistant urinary tract infection (UTI)-causative bacteria isolated in Indonesia
katsumi Shigemura, Koichi Kitagawa, Toshiro Shirakawa, Masato Fujisawa
34th Annual EAU Congress, Mar. 2019, English, Barcelona, International conference
Poster presentation

Comparison of molecular characteristics of carbapenem-resistant urinary tract infection- causing pathogens between Japan, Taiwan and Indonesia
katsumi Shigemura, Kento Nishimoto, Kayo Osawa, Kuntaman K, Sung S, Chen K, Kitagawa K, Huang T, Toshiro Shirakawa, Masato Fujisawa
34th Annual EAU Congress, Mar. 2019, English, Barcelona, International conference
Poster presentation

尿路感染症におけるカルバペネム耐性腸内細菌科細菌の分子生物学的検討ならびに迅速診断法の確立
SHIGEMURA KATSUMI, OSAWA KAYO, 江夏徳寿, 北川孝一, SHIRAKAWA TOSHIRO, NAKANO YUZO, FUJISAWA MASATO
第28回泌尿器科分子・細胞研究会, Feb. 2019, Japanese, 下関, Domestic conference
Poster presentation

日本（神戸）とインドネシア（Yogyakarta）における尿路感染症の国際間比較
KITAGAWA KOICHI, SHIGEMURA KATSUMI, 西本健人, 山田尚輝, Prahara Yuri, Andy Zulfiqqar, OSAWA KAYO, 宇田篤史, SHIRAKAWA TOSHIRO, ARAKAWA SOICHI, MIYARA TAKAYUKI, FUJISAWA MASATO
第34回日本環境感染学会総会・学術集会, Feb. 2019, Japanese, 神戸, Domestic conference
Poster presentation

改変型ヒトWilms’ tumor 1タンパク表層発現ビフィズス菌を用いた経口癌ワクチンの各種固形癌に対する抗腫瘍免疫誘導効果に関する検討
辰巳真帆, KITAGAWA KOICHI, 加藤真子, 駒井翔太, 橋井佳子, 片山高嶺, SHIRAKAWA TOSHIRO
第22回日本ワクチン学会学術集会, Dec. 2018, Japanese, 神戸, Domestic conference
Oral presentation

遺伝子組換えビフィズス菌を用いた次世代型経口結核ワクチンの開発
古田拓也, 門脇雅英, KITAGAWA KOICHI, 土井教生, 片山高嶺, SHIRAKAWA TOSHIRO
第22回日本ワクチン学会学術集会, Dec. 2018, Japanese, 神戸, Domestic conference
Oral presentation

Cancer immunotherapy combining oral vaccination of recombinant Bifidobacterium longum displaying human Wilms’ tumor 1 protein and anti-PD-1 checkpoint blockade for solid tumors in mice experimental model.
KITAGAWA KOICHI, 辰巳真帆, 加藤真子, 駒井翔太, 橋井佳子, 片山高嶺, SHIRAKAWA TOSHIRO
CRI-CIMT-EATI-AACR Fourth International Cancer Immunotherapy Conference, Sep. 2018, English, New York, USA, International conference
Poster presentation

インドネシアの尿路感染症患者より分離されたカルバペネマーゼ産生グラム陰性桿菌の分子疫学的調査
OSAWA KAYO, SHIGEMURA KATSUMI, KITAGAWA KOICHI, FUJISAWA MASATO, SHIRAKAWA TOSHIRO
第13回日本臨床検査学教育学会学術大会, Aug. 2018, Japanese, 札幌, Domestic conference
Oral presentation

インドネシアで分離されたCTX-M-15型ESBL産生Escherichia coliにおける染色体性およびプラスミド性についての分子疫学調査
OSAWA KAYO, SHIGEMURA KATSUMI, FUJISAWA MASATO, SHIRAKAWA TOSHIRO
第13回日本臨床検査学教育学会学術大会, Aug. 2018, Japanese, 札幌, Domestic conference
Oral presentation

Overexpression of SOCS3 mediated by adenovirus vector in prostate cancer cells increased the sensitivity to lymphokine-activated killer cells in vitro and in vivo.
KITAGAWA KOICHI, 米田智美, 石河求己, 門脇雅英, OTSUKI NAOKI, NIBU KENICHI, FUJISAWA MASATO, 世良田聡, 仲哲治, SHIRAKAWA TOSHIRO
第24回日本遺伝子細胞治療学会, Jul. 2018, Japanese, 東京, Domestic conference
Oral presentation

Analysis of antimicrobial resistance mechanism in successive infections of Pseudomonas aeruginosa
Noriko Nakanishi, Ryohei Nomoto, Kanako Sato, Chihiro Koike, Mari Kusuki, Tatsuya Nakamura, Shigemura Katsumi, Shirakawa Toshiro, Tokimatsu Issei, Osawa Kayo
第91回日本細菌学会総会, Mar. 2018, Japanese, 日本細菌学会, 福岡, Domestic conference
Poster presentation

Anti-tumor effect against prostate cancer induced by oral cancer vaccine using recombinant Bifidobacterium displaying Wilms' tumor 1 protein in mice
五ノ井玲菜, 北川孝一, 古田拓也, 辰巳真帆, 斉藤大樹, 橋井佳子, 片山高嶺, Shirakawa Toshiro
第40回日本分子生物学会年会, Dec. 2017, Japanese, The Molecular Biology Society of Japan, 神戸, Domestic conference
Poster presentation

Anti-tumor effect and suppression of immune escapeby adenovirus carrying SOCS3 gene in prostate cancer
米田智美, 斉藤大樹, 福井悠夏, 北川孝一, 世良田聡, 仲哲治, Shirakawa Toshiro
第40回日本分子生物学会年会, Dec. 2017, Japanese, The Molecular Biology Society of Japan, 神戸, Domestic conference
Poster presentation

Enhanced anti-tumor effect by combining oral cancer vaccine using Bifidobacterium displaying WT1 protein with anti-PD-1 antibody therapy in mouse prostate cancer model.
KITAGAWA KOICHI, 斉藤大樹, 五ノ井玲菜, 辰巳真帆, 古田拓也, 橋井佳子, 片山高嶺, SHIRAKAWA TOSHIRO
Third CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference 2017, Sep. 2017, English, Mainz, Germany, International conference
Poster presentation

Enhanced anti-tumor effect by combining oral cancer vaccine using Bifidobacterium displaying WT1 protein with anti-PD-1 antibody therapy in mouse prostate cancer model.
北川孝一, 斉藤大樹, 五ノ井玲菜, 辰巳真帆, 古田拓也, 橋井佳子, 片山高嶺, Shirakawa Toshiro
Third CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference, Sep. 2017, English, Association for Cancer Immunotherapy, Mainz, ドイツ, International conference
Poster presentation

Development of novel oral cancer vaccine using Bifidobacterium
Shirakawa Toshiro
Hwasun Adenoviral Cancer Therapeutics Symposium, Sep. 2017, English, Chonnam National University, 光州, 韓国, International conference
[Invited]
Nominated symposium

兵庫県内で分離されたPseudomonas aeruginosaの薬剤耐性機構の解析
佐藤加奈子, Osawa Kayo, Shigemura Katsumi, 中村竜也, Fujisawa Masato, Tokimatsu Issei, Shirakawa Toshiro
第12回日本臨床検査学教育学会学術大会, Aug. 2017, Japanese, 日本臨床検査学教育学会, 埼玉, Domestic conference
Oral presentation

Clostridium difficileの薬剤感受性と遺伝子解析
衣川真矢, Osawa Kayo, Shigemura Katsumi, 中村竜也, Tokimatsu Issei, Fujisawa Masato, Shirakawa Toshiro
第12回日本臨床検査学教育学会学術大会, Aug. 2017, Japanese, 日本臨床検査学教育学会, 埼玉, Domestic conference
Oral presentation

腸管免疫を利用した新規経口がんワクチンと免疫チェックポイント阻害剤の併用療法の開発
Shirakawa Toshiro, 北川孝一, Fujisawa Masato
Development of combinational cancer immunotherapy comprising WT1 oral cancer vaccine and immune checkpoint inhibitor, Jul. 2017, English, Japan Society of Gene and Cell Therapy, 岡山, Domestic conference
Public symposium

Wilms’ tumor 1タンパク発現ビフィズス菌を用いた経口癌ワクチンの抗腫瘍免疫誘導効果に関する検討
北川孝一, 辰巳真帆, 五ノ井玲菜, 斉藤大樹, 橋井佳子, 片山高嶺, Shirakawa Toshiro
第21回腸内細菌学会, Jun. 2017, Japanese, The Japan Bifidus Foundation, 神戸, Domestic conference
Poster presentation

The level of urinary titin of DMD patients is >100-times higher than that of healthy control
Hiroyuki Awano, Matsumoto M, Nagai M, Shirakawa Toshiro, Takasaki T, Maruyama N, Nabeshima Y, Matsuo M, Iijima Kazumoto
第59回日本小児神経学会学術集会, Jun. 2017, Japanese, 日本小児神経学会, 大阪, Domestic conference
Oral presentation

Urinaty titin reveals persistent proteolysis in Duchenn Muscular Dystrophy
Hiroyuki Awano, Matsumoto M, Nagai M, Shirakawa Toshiro, Maruyama K, Iijima Kazumoto, Nabeshima Y, Matsuo M
22nd International Congress of the World Muscle society,, May 2017, English, The World Muscle Society, Saint-Malo, France, International conference
Poster presentation

Urinary exceation of 8-OHdG, a biomarker of oxdative DNA damage, increases with age in DMD patients
Matsumoto M, Hiroyuki Awano, Nagai Takayuki, Shirakawa Toshiro, Iijima Kazumoto, Matsuo M
22nd International Congress of the World Muscle society,, May 2017, English, The World Muscle Society, Saint-Malo, France, International conference
Poster presentation

International comparison of causative bacteria and antimicrobial susceptibilities of UTIs between developed and developing countries
SHIGEMURA KATSUMI, KITAGAWA KOICHI, K Kuntaman, NAKANO YUZO, SHIRAKAWA TOSHIRO, TOKIMATSU ISSEI, FUJISAWA MASATO
American Urological Association (AUA) 2017 Annual Meeting 112th, May 2017, English, Boston, USA, International conference
Poster presentation

International comparison of causative bacteria and antimicrobial susceptibilities of UTIs between developed and developing countries
Shigemura Katsumi, Koichi Kitagawa, Kuntman Kuntaman, Nakano Yuzo, Shirakawa Toshiro, Tokimatsu Issei, Fujisawa Masato
The112nd American Urological Association Annual Meeting, May 2017, English, American Urological Association, ボストン, アメリカ, International conference
Poster presentation

Combination of Oral WT1 Cancer Vaccine and Anti-PD-1 Antibody Induced the Synergistic Anti-tumor Effect in Mouse Prostate Cancer Model
北川孝一, 五ノ井玲菜, 辰巳真帆, 斉藤大樹, 橋井佳子, 片山高嶺, Shirakawa Toshiro
American Society of Gene & Cell Therapy 20th Annual Meeting, May 2017, English, American Society of Gene & Cell Therapy, Washington DC, USA, International conference
Poster presentation

尿路感染症における原因菌と薬剤感受性の国際比較
Shigemura Katsumi, Osawa Kayo, Nakano Yuzo, 小瀧将広, L. Alimsardjono, D. Rahadjo, U. Hadi, F. Setiawan, E.B.Wasito, Ni Made Merta Niasih, K. Kuntaman, M.Rusli, Tokimatsu Issei, 荒川創一, Shirakawa Toshiro, Fujisawa Masato
第64回日本化学療法学会西日本支部総会第86回日本感染症学会西日本地方会学術集会, Nov. 2016, Japanese, 日本化学療法学会, 沖縄, Domestic conference
Oral presentation

Development of the novel oral cancer vaccine using Bifidobacterium longum displaying Wilms’ tumor 1 protein
KITAGAWA KOICHI, 小田麗未, 荒木綾芽, 五ノ井玲菜, 斉藤大樹, 橋井佳子, 片山高嶺, SHIRAKAWA TOSHIRO
第22回日本遺伝子細胞治療学会学術集会, Jul. 2016, Japanese, 東京, Domestic conference
Oral presentation

Molecular characteristics of extended-spectrum ?-lactamase-producing Escherichia coli isolated urinary tract infection in a university teaching hospital
Shigemura Katsumi, Osawa Kayo, Kazushi Tanaka, Nakano Yuzo, Shirakawa Toshiro, Soichi Arakawa, Fujisawa Masato
American Urological Association 2016 Annual Meeting L74:AA74, May 2016, English, American Urological Association, San Diego, USA, International conference
Poster presentation

Development of the novel oral tumor vaccine using Bifidobacterium longum displaying Wilms’ tumor 1 protein.
KITAGAWA KOICHI, 小田麗未, 荒木綾芽, 五ノ井玲菜, 斉藤大樹, 橋井佳子, 片山高嶺, SHIRAKAWA TOSHIRO
American Society of Gene & Cell Therapy 19th Annual Meeting, May 2016, English, Washington DC, USA, International conference
Poster presentation

Azithromycinに対する Neisseria gonorrhoeae薬剤感受性の最近7年間の変遷について
Osawa Kayo, Shigemura Katsumi, 吉田弘之, Shirakawa Toshiro, Fujisawa Masato, Arakawa Soichi
第28回日本性感染症学会学術大会, Dec. 2015, Japanese, 日本性感染症学会, 東京, Domestic conference
Oral presentation

Development of Combination Therapy of Bifidobacterium-based Oral Vaccine Displaying HCV-NS3 with Interferon-α
KITAGAWA KOICHI, 石川博規, 小田麗未, 斉藤大樹, 森下直矢, 島本康介, 西田典永, 松浦洋一, HOTTA HAK, SHIRAKAWA TOSHIRO
American Society of Gene & Cell Therapy 18th Annual Meeting, May 2015, English, New Orleans, Louisiana, USA, International conference
Poster presentation

兵庫県における淋菌の薬剤感受性の最近13年間の変遷についての検討
OSAWA KAYO, SHIRAKAWA TOSHIRO
第89回日本感染症学会学術講演会, Apr. 2015, Japanese, 京都, Domestic conference
Poster presentation

経尿道的前立腺レーザー核出術(HoLEP)における術後尿失禁に関連する因子についてのurodynamic studyを用いた検討
Shigemura Katsumi, Tanaka Kazushi, 桃園宏之, 長富俊孝, 江夏徳寿, Muramaki Mototsugu, Shirakawa Toshiro, Miyake Hideaki, Fujisawa Masato
第103回日本泌尿器科学会総会, Apr. 2015, Japanese, 日本泌尿器科学会, 金沢, Domestic conference
Poster presentation

ビフィズス菌を応用した新規経口ワクチン･プラットフォームの開発
Shirakawa Toshiro
第88回日本細菌学会総会, Mar. 2015, Japanese, 日本細菌学会, 岐阜, Domestic conference
[Invited]
Invited oral presentation

兵庫県下におけるアジスロマイシン耐性淋菌の分子遺伝学的解析
三浦真希子, Shigemura Katsumi, Osawa Kayo, 吉田弘之, 藤原美樹, 澤村暢, 奈須聖子, Arakawa Soichi, Fujisawa Masato, Shirakawa Toshiro
第28回日本性感染症学会学術大会, Dec. 2014, Japanese, 日本性感染症学会, 神戸, Domestic conference
Oral presentation

兵庫県におけるアジスロマイシン(AZM)耐性淋菌の分子遺伝学的解析
OSAWA KAYO, SHIRAKAWA TOSHIRO
第27回日本性感染症学会学術大会, Dec. 2014, Japanese, Domestic conference
Oral presentation

セフェム系薬剤感受性低下の遺伝解析
Osawa Kayo, Shigemura Katsumi, 額田雪絵, 吉田弘之, 藤原美樹, 奈須聖子, Shirakawa Toshiro, Fujisawa Masato, Arakawa Soichi
第27回日本性感染症学会学術大会, Dec. 2014, Japanese, 日本性感染症学会, 神戸, Domestic conference
Oral presentation

セフェム系薬剤感受性低下Neisseria gonorrhoeaeの遺伝解析
OSAWA KAYO, SHIRAKAWA TOSHIRO
第27回日本性感染症学会学術大会, Dec. 2014, Japanese, Domestic conference
Oral presentation

Prenatal diagnosis of holoprosencephaly with proboscis and synophthalmia caused by monosomy 18p
Yamasaki Y, Shirakawa Toshiro, Miyahara Yoshiya, Morita Hiroki, Yamada Hideto
20th World Congress on Controversies in Obstetrics, Gynecology & Infertility (COGI), Nov. 2014, English, Ho Chi Minh, Vietnam, International conference
Poster presentation

Expression of epithelial-mesenchymal transition-related factors in adherent placenta
Shirakawa Toshiro, Miyahara Yoshiya, Yamasaki Y, Morita Hiroki, Yamada Hideto
20th World Congress on Controversies in Obstetrics, Gynecology & Infertility (COGI), Nov. 2014, English, Ho Chi Minh, Vietnam, International conference
Poster presentation

Diagnosis and post-operative management of the giant ovarian tumors
Miyahara Yoshiya, Yamasaki Y, Shirakawa Toshiro, Morita Hiroki, Yamada Hideto
20th World Congress on Controversies in Obstetrics, Gynecology & Infertility (COGI), Nov. 2014, English, Ho Chi Minh, Vietnam, International conference
Poster presentation

前立腺肥大症患者の下部尿路症状（LUTS）に対するシロドシンおよびナフトピジルの臨床効果に関する比較検討
Shirakawa Toshiro, Shigemura Katsumi, 原口貴裕, 江夏徳寿, 森下真一, 源吉顕治, 宮崎治郎, Miyake Hideaki, Tanaka Kazushi, Fujisawa Masato
第21回日本排尿機能学会総会, Sep. 2014, Japanese, 日本排尿機能学会, 岡山, Domestic conference
Poster presentation

Development of the novel cancer therapeutic strategy incorporating the gene therapy and immune cellular therapy
Shirakawa Toshiro
CNUH International Symposium for Urogenital Translational Research, Sep. 2014, English, Chonnam National University Hospital, Gwangju, Korea, International conference
[Invited]
Invited oral presentation

Development of Combination Therapy of Bifidobacterium Displaying HCV-NS3 Oral Vaccine with Interferon-Alpha
Koichi Kitagawa, Chika Omoto, Hiroki Ishikawa, Tsugumi Oda, Naoya Morishita, Hotta Hak, Shirakawa Toshiro
第20回日本遺伝子治療学会, Aug. 2014, Japanese, 日本遺伝子治療学会, 東京, Domestic conference
Oral presentation

COMBINATION THERAPY OF BIFIDOBACTERIUM DISPLAYING HCV-NS3 ORAL VACCINE WITH INTERFERON-ALPHA IN MICE
KITAGAWA KOICHI, 大本知佳, 石川博規, 小田麗未, 森下直矢, HOTTA HAK, SHIRAKAWA TOSHIRO
第20回日本遺伝子治療学会学術集会, Aug. 2014, Japanese, Domestic conference
Oral presentation

兵庫県下で分離されたメタロβラクタマーゼ産生腸内細菌の解析
OSAWA KAYO, SHIRAKAWA TOSHIRO
第88回日本感染症学会学術講演会, Jun. 2014, Japanese, 福岡, Domestic conference
Poster presentation

当院で分離されたESBLｓ産生Escherichia coli の遺伝子解析
OSAWA KAYO, SHIRAKAWA TOSHIRO
第88回日本感染症学会学術講演会, Jun. 2014, Japanese, 福岡, Domestic conference
Poster presentation

SIGNIFICANT RECEPTORS OR BIOMARKERS FOR THE SYMPTOMS OF OVERACTIVE BLADDER
Yamamichi F, Shigemura Katsumi, Shirakawa Toshiro, Behnsawy H, Yamashita M, Miyake Hideaki, Tanaka Kiwamu, Fujisawa Masato
109thAUA(American Urological Association)annual meeting, May 2014, English, American Urological Association, オーランド, アメリカ, International conference
Poster presentation

前立腺癌増悪において上皮ならびに間質でのSonic hedgehogとandrogenシグナル伝達が上皮間葉移行を作動させる
山道深, Shigemura Katsumi, Hosny M. Behnsawy, Fatma Y. Meligy, Wen-chin Huang, Xiangyan Li, Lel, W.K.Chung, 川端眞人, 後藤章暢, Miyake Hideaki, Tanaka Kazushi, Shirakawa Toshiro, Fujisawa Masato
第102回日本泌尿器科学会総会, Apr. 2014, Japanese, 日本泌尿器科学会, 神戸, Domestic conference
Poster presentation

Does mutation in gyrA or parC or efflux pump expression play the main role in fluoroquinolone-resistant Escherichia coli causing urinary tract infections?
Shigemura Katsumi, Tanaka Kiwamu, Shirakawa Toshiro, Arakawa Soichi, Miyake Hideaki, Fujisawa Masato
29thEAU(European Association of Urology) Annual Congress, Apr. 2014, English, European Association of Urology, ストックホルム, スウェーデン, International conference
Poster presentation

Expression of epithelial-mesenchymal transition-related factors in placenta accreta
Shirakawa Toshiro, Mihayara Y, Niiya Kiyoshi, Morita Hiroki, Ebina Yasuhiko, Yamada Hideto
19th World Congress on Controversies in Obstetrics, Gynecology & Infertility (COGI), Feb. 2014, English, Macau, China, International conference
Poster presentation

Bacterial identification using the ssrA gene encoding tmRNA
OSAWA KAYO, SHIRAKAWA TOSHIRO
Asian-African Research Forum on Emerginｇ and Reemerging Infections 2014, Jan. 2014, English, 仙台, International conference
Poster presentation

Bacterial identification using the ssrA gene encoding tmRNA
Ayaka Kayo, Osawa Kayo, Shigemura Katsumi, Hiroyuki Yoshida, Miki Fujiwara, Yoshio Iijima, Fujisawa Masato, Dadik Raharjo, Subijanto Marto Sudarmo, Kawabata Masato, Shirakawa Toshiro
Asian-African Research Forum on Emerginｇ and Reemerging Infections 2014, Jan. 2014, English, 仙台, International conference
Public symposium

Active surveillance of children's acute diarrheal disease using a novel molecular diagnostic multiplexing system in Surabaya, Indonesia
OSAWA KAYO, SHIRAKAWA TOSHIRO
Asian-African Research Forum on Emerginｇ and Reemerging Infections 2014, Jan. 2014, English, 仙台, International conference
Poster presentation

Current status and perspective of personalized cancer therapy incorporating the gene therapy and immune-cellular therapy
SHIRAKAWA TOSHIRO
第17回国際個別化医療学会, Nov. 2013, English, Domestic conference
[Invited]
Invited oral presentation

Current status and perspective of personalized cancer therapy incorporating the gene therapy and immune-cellular therapy
Shirakawa Toshiro
The 17th International Congress of Personalized Medicine, Nov. 2013, Japanese, International Society of Personalized Medicine, 神戸, Domestic conference
[Invited]
Invited oral presentation

The p53-specific CTL induced by Ad-p53 infected dendritic cells showed the high cytotoxicity in p53-overexpressed and Ad-53 infected tumor cell lines
斉藤大樹, 山崎里沙, 森下直矢, Dody Bautista, Dante Dator, Kawabata Masato, Shigemura Katsumi, Fujisawa Masato, Shirakawa Toshiro
The 17th International Congress of Personalized Medicine, Nov. 2013, Japanese, International Society of Personalized Medicine, 神戸, Domestic conference
Poster presentation

Synergistic antitumor effect of the combination of adenoviral gene therapy and immunotherapy for head and neck cancer
安藤聡志, 斉藤大樹, 森下直矢, Kawabata Masato, Ohtsuki Naoki, Nibu Ken-ichi, Fujisawa Masato, Shirakawa Toshiro
The 17th International Congress of Personalized Medicine, Nov. 2013, Japanese, International Society of Personalized Medicine, 神戸, Domestic conference
Poster presentation

BPH/LUTSに対するデュタステリドの有用性に関する検討
Haraguchi Takahiro, Miyake Hideaki, 江夏徳寿, Shirakawa Toshiro, Tanaka Kazushi, Fujisawa Masato
第20回日本排尿機能学会, Sep. 2013, Japanese, 日本排尿機能学会, 静岡, Domestic conference
Poster presentation

The p53-specific CTL induced by Ad-p53 infected dendritic cells showed the high cytotoxicity in p53-overexpressED and Ad-p53 infected tumor cell lines.
斉藤大樹, 山崎里沙, 森下直矢, Kawabata Masato, Dody Bautista, Dante Dator, Shirakawa Toshiro
19th Annual Meeting of Japan Society of Gene Therapy, Jul. 2013, Japanese, Japan Society of Gene Therapy, 岡山, Domestic conference
Oral presentation

SYNERGISTIC ANITITUMOR EFFECT OF THE COMBINATIION OF ADENOVIRAL GENE THERAPY AND IMMUNOTHERAPY FOR HEAD AND NECK CANCER
安藤聡志, 斉藤大樹, 森下直矢, Kawabata Masato, Ohtsuki Naoki, Nibu Ken-ichi, Fujisawa Masato, Shirakawa Toshiro
19th Annual Meeting of Japan Society of Gene Therapy, Jul. 2013, Japanese, Japan Society of Gene Therapy, 岡山, Domestic conference
Poster presentation

ORAL ADMINISTRATION OF GENETICALLY MODIFIED BIFIDOBACTERIUM DISPLAYING HCV-NS3 MULTI-EPITOPE FUSION PROTEIN CAN INDUCE THE HCV-NS3 SPECIFIC SYSTEMIC IMMUNE RESPONSE IN MICE
大本知佳, 北川孝一, 竹井咲希, 森下直矢, 片山高嶺, Hotta Hak, Kawabata Masato, Shirakawa Toshiro
19th Annual Meeting of Japan Society of Gene Therapy, Jul. 2013, Japanese, Japan Society of Gene Therapy, 岡山, Domestic conference
Oral presentation

DEVELOPMENT OF THE ORAL UNIVERSAL TYPE A INFLUENZA VACCINE USING BIFIDOBACTERIUM DISPLAYING INFLUENZA-M2e PROTEIN
尾崎鈴佳, 北川孝一, 代崎千尋, 森下直矢, 片山高嶺, Kawabata Masato, Shirakawa Toshiro
19th Annual Meeting of Japan Society of Gene Therapy, Jul. 2013, Japanese, Japan Society of Gene Therapy, 岡山, Domestic conference
Oral presentation

尿路感染症患者より分離したPseudomonas aeruginosa 薬剤耐性株におけるefflux pump遺伝子の発現についての検討
OSAWA KAYO, SHIRAKAWA TOSHIRO
第87回日本感染症学会学術講演会, Jun. 2013, Japanese, 横浜, Domestic conference
Poster presentation

尿路感染症患者より分離したPseudomonas aeruginosa 薬剤耐性株におけるefflux pump遺伝子の発現についての検討
加藤綾香, Osawa Kayo, Shigemura Katsumi, Tanaka Kazushi, Arakawa Soichi, Fujisawa Masato, Shirakawa Toshiro
第87回日本感染症学会学術講演会, Jun. 2013, Japanese, 日本感染症学会, 横浜, Domestic conference
Poster presentation

Candida urinary tract isolation and Candida species susceptibilities to anti-fungus medication in Kobe University Hospital
OSAWA KAYO, SHIRAKAWA TOSHIRO
28th International Congress of Chemotherapy and Infection, Jun. 2013, English, 横浜, International conference
Poster presentation

Candida urinary tract isolation and Candida species susceptibilities to anti-fungus medication in Kobe University Hospital
Shigemura Katsumi, Osawa Kayo, Tanaka Kazushi, Hiroyuki Yoshida, Shirakawa Toshiro, Fujisawa Masato, Arakawa Soichi
28th International Congress of Chemotherapy and Infection, Jun. 2013, English, 日本感染症学会他, 横浜, International conference
Poster presentation

Does mutation in gyrA or parC or efflux pump expression play the main role in fluoroquinolone-resistant Escherichia coli causing urinary tract infections?
Shigemura Katsumi, Tanaka Kiwamu, Shirakawa Toshiro, Arakawa Soichi, Miyake Hideaki, Fujisawa Masato
108thAUA(American Urological Association)annual meeting, May 2013, English, AUA(American Urological Association)annual meeting, サンディエゴ, アメリカ, International conference
Poster presentation

Development of the Oral Universal Type A Influenza Vaccine Using Bifidobacterium Displaying Influenza-M2e Protein
尾崎鈴佳, 森下直矢, 北川孝一, 代崎千尋, 斉藤大樹, Shigemura Katsumi, Fujisawa Masato, Kawabata Masato, Shirakawa Toshiro
American Society of Gene & Cell Therapy 16th Annual Meeting, May 2013, English, American Society of Gene & Cell Therapy, Salt Lake City, America, International conference
Poster presentation

Combination of CD3+CD56+ (NKT) Cells Immunotherapy and Adenovirus-p53 Therapy for Head and Neck Squamous Cell Carcinoma
斉藤大樹, 森下直矢, Kyung-Mi Lee, Dante Dator, Kawabata Masato, Fujisawa Masato, Shirakawa Toshiro
American Society of Gene & Cell Therapy 16th Annual Meeting, May 2013, English, American Society of Gene & Cell Therapy, Salt Lake City, America, International conference
Poster presentation

Sonic hedgehog signaling and Androgens are linked in tumor-stromal interaction through Epithelial-Mesenchymal transition (EMT) in prostate cancer progression
Shigemura Katsumi, Shirakawa Toshiro, Yamamichi F, Matsumoto M, Behnsawy HM, Meligy FY, Miyake Hideaki, Tanaka Kiwamu, Fujisawa Masato
28thEAU(European Association of Urology), Mar. 2013, English, EAU(European Association of Urology), ミラノ, イタリア, International conference
Poster presentation

Frequency of Diarrheagenic Escherichia coli among Children in Surabaya, Indoneshia
OSAWA KAYO, SHIRAKAWA TOSHIRO
Asian-African Research Forum on Emerginf and Reemerging Infections, Jan. 2013, English, International conference
Oral presentation

Development of a Multiplex PCR for the Rapid Identification of Salmonella Serotypes.
OSAWA KAYO, SHIRAKAWA TOSHIRO
Asian-African Research Forum on Emerginf and Reemerging Infections, Jan. 2013, English, International conference
Poster presentation

tmRNAマーカーを用いたPCRによる細菌同定法の検討
OSAWA KAYO, SHIRAKAWA TOSHIRO
第65回日本細菌学会関西支部総会, Nov. 2012, Japanese, Domestic conference
Oral presentation

Multiplex PCRによるSalmonella 血清型の迅速スクリーニング法の開発
OSAWA KAYO, SHIRAKAWA TOSHIRO
第65回日本細菌学会関西支部総会, Nov. 2012, Japanese, Domestic conference
Oral presentation

阪神地区における91 淋菌臨床株のgyrA, parC のアミノ酸変異とキノロン系
Shigemura Katsumi, Tanaka Kazushi, Shirakawa Toshiro, Miyake Hideaki, Arakawa Soichi, Fujisawa Masato
第41回薬剤耐性菌研究会, Oct. 2012, Japanese, 薬剤耐性菌研究会, 岐阜県, Domestic conference
Poster presentation

Sonic hedgehog signaling and Androgens are linked in tumor-stromal interaction through Epithelial-Mesenchymal transition (EMT) in prostate cancer progression
Shigemura Katsumi, Fatma Y. Meligy, Hosny M. Behnsawy, Fukashi Yamamichi, Wen-Chin Haung, Xiangyan Li, Kunito Yamanaka, Keisuke Hanioka, Miyake Hideaki, Kawabata Masato, Shirakawa Toshiro, Fujisawa Masato
107th AUA(American Urological Association)annual meeting, May 2012, English, AUA(American Urological Association)annual meeting, アトランタ, International conference
Poster presentation

Significant biomarker for lower urinary tract symptoms in chronic prostatitis
Shigemura Katsumi, Matsumoto M, Shirakawa Toshiro, Yamamichi F, Arakawa Soichi, Tanaka Kiwamu, Miyake Hideaki, Fujisawa Masato
107th AUA(American Urological Association)annual meeting, May 2012, English, AUA(American Urological Association)annual meeting, アトランタ, International conference
Poster presentation

Possible role of sonic hedgehog signaling and the link with Epithelial-Mesenchymal transition (EMT) in renal cancer progression
Shigemura Katsumi, Fatma Y. Meligy, Hosny M. Behnsawy, Fukashi Yamamichi, Wen-Chin Haung, Xiangyan Li, Lela, d Chung, Masuo Yamashita, Kawabata Masato, Shirakawa Toshiro, Fujisawa Masato
107th AUA(American Urological Association)annual meeting, May 2012, English, AUA(American Urological Association)annual meeting, アトランタ, International conference
Poster presentation

Mechanisms of and risk factors for fluoroquinolone resistance in clinical Enterococcus faecalis isolates from patients with urinary tract infections.
Shigemura Katsumi, Yasufuku T, Shirakawa Toshiro, Matsumoto M, Nakano Y, Tanaka Kiwamu, Arakawa Soichi, Kawabata Masato, Fujisawa Masato
107th AUA(American Urological Association)annual meeting, May 2012, English, AUA(American Urological Association)annual meeting, アトランタ, International conference
Poster presentation

Antimicrobial resistance in Salmonella strains clinically isolated in Hyogo, Japan (2009-2011)
大澤佳代, SHIRAKAWA TOSHIRO
第86回日本感染症学会総会, Apr. 2012, Japanese, 長崎, Domestic conference
Poster presentation

Antimicrobial resistance in Salmonella strains clinically isolated in Hyogo, Japan (2009-2011)
大澤佳代, SHIRAKAWA TOSHIRO
Asian-African Research Forum n Emerging and Reemerging, Jan. 2012, English, 神戸, International conference
Poster presentation

Characterization of wzz genes in Escherichia coli O157
大澤佳代, SHIRAKAWA TOSHIRO
International Union of Microbiological Societies 2011 Congress, Sep. 2011, English, Sapporo, International conference
Poster presentation

尿路感染症由来緑膿菌臨床株における高速液体クロマトグラフィー法によるフルオロキノロン系抗菌薬耐性の迅速診断法確立への検討
松本穣, Shigemura Katsumi, Shirakawa Toshiro, 安福富彦, 中野雄造, Tanaka Kazushi, 木下承皓, Kawabata Masato, Arakawa Soichi, Fujisawa Masato
第99回日本泌尿器科学会総会, Apr. 2011, Japanese, 日本泌尿器科学会総会, 名古屋, Domestic conference
Poster presentation

尿路感染症由来緑膿菌株における高速液体クロマトグラフィー法によるフルオロキノロン系抗菌薬耐性の迅速診断法に関する検討
松本穣, Shigemura Katsumi, Shirakawa Toshiro, 安福富彦, 中野雄造, Tanaka Kazushi, 木下承晧, Kawabata Masato, Arakawa Soichi, Fujisawa Masato
第85回日本感染症学会総会・学術講演会, Apr. 2011, Japanese, 日本感染症学会総会・学術講演会, 東京, Domestic conference
Poster presentation

帳球菌の尿路感染症臨床株におけるgyrA,parC遺伝子変異とフルオロキノロン系抗菌薬耐性の相関についての検討
安福富彦, Shigemura Katsumi, Shirakawa Toshiro, 松本穣, 中野雄造, Tanaka Kazushi, 木下承皓, Kawabata Masato, Arakawa Soichi, Fujisawa Masato
第99回日本泌尿器科学会総会, Apr. 2011, Japanese, 日本泌尿器科学会総会, 名古屋, Domestic conference
Oral presentation

泌尿器科領域悪性腫瘍に対する抗癌化学療法中に生じた発熱性好中球減少症（ＦＮ)の検討
安福富彦, Shigemura Katsumi, 松本穣, 中野雄造, Shirakawa Toshiro, Tanaka Kazushi, Arakawa Soichi, Fujisawa Masato
第58回日本化学療法学会西日本支部総会, Nov. 2010, Japanese, 日本化学療法学会総会, 大分, Domestic conference
Oral presentation

尿路感染症由来大腸菌の臨床株における薬剤排出ポンプ遺伝子の発現と各種抗菌薬耐性との関連の解析
Shigemura Katsumi, 安福富彦, Shirakawa Toshiro, 木下承晧, 松本穣, 中野雄造, Tanaka Kazushi, Kawabata Masato, Arakawa Soichi, Fujisawa Masato
第62回日本泌尿器科学会西日本総会, Nov. 2010, Japanese, 日本泌尿器科学会, 鹿児島, Domestic conference
Oral presentation

前立腺癌患者末梢血中circulating tumor cells(CTCs)の検出法の開発
山道深, 松岡孝幸, Kawabata Masato, 森下真一, Shirakawa Toshiro, Fujisawa Masato
第60回日本泌尿器科学会中部総会, Nov. 2010, Japanese, 日本泌尿器科学会, 名古屋, Domestic conference
Oral presentation

尿路感染症由来緑膿菌臨床株におけるgyrA,parC遺伝子変異とキノロン系抗菌薬耐性についての検討
松本穣, Shigemura Katsumi, Shirakawa Toshiro, 安福富彦, 中野雄造, Tanaka Kazushi, 武中篤, Arakawa Soichi, 木下承皓, Kawabata Masato, Fujisawa Masato
第19回日本腎泌尿器疾患予防医学研究会, Jul. 2010, Japanese, 日本腎泌尿器疾患医学研究会, 千葉, Domestic conference
Oral presentation

尿路感染症由来緑膿菌臨床株におけるgyrA、parC遺伝子変異とキノロン系抗菌薬耐性の関連についての検討
松本穣, Shigemura Katsumi, Shirakawa Toshiro, 安福富彦, 中野雄造, Tanaka Kazushi, 武中篤, Arakawa Soichi, 木下承皓, Kawabata Masato, Fujisawa Masato
第58回日本化学療法学会総会, Jun. 2010, Japanese, 日本化学療法学会総会, 長崎, Domestic conference
Oral presentation

尿路感染症由来緑膿菌臨床株におけるgyrA、parC遺伝子変異とキノロン系抗菌薬耐性の関連についての検討
松本穣, Shigemura Katsumi, Shirakawa Toshiro, 安福富彦, 中野雄造, Tanaka Kazushi, 武中篤, Arakawa Soichi, 木下承晧, Kawabata Masato, Fujisawa Masato
第98回日本泌尿器科学会総会, Apr. 2010, Japanese, 日本泌尿器科学会, 盛岡, Domestic conference
Oral presentation

尿路感染症由来大腸菌の臨床株における薬剤排出ポンプ遺伝子の発現と各種抗菌薬耐性との関連の解析
安福富彦, Shigemura Katsumi, Shirakawa Toshiro, 中野雄造, Tanaka Kazushi, 武中篤, Arakawa Soichi, 木下承晧, Kawabata Masato, Fujisawa Masato
第98回日本泌尿器科学会総会, Apr. 2010, Japanese, 日本泌尿器科学会, 盛岡, Domestic conference
Poster presentation

「薬剤耐性（1）」尿路感染症由来大腸菌の臨床株における薬剤排出ポンプ遺伝子の発現と各種抗菌薬耐性との関連の解析
安福富彦, Shigemura Katsumi, Shirakawa Toshiro, 中野雄造, Tanaka Kazushi, 武中篤, Arakawa Soichi, 木下承皓, Kawabata Masato, Fujisawa Masato
第84回日本感染症学会総会・学術講演会, Apr. 2010, Japanese, 日本感染症学会, 京都, Domestic conference
Others

ヒト膀胱癌細胞におけるCox-2阻害剤、3剤の攻腫瘍効果の比較検討
Shirakawa Toshiro, ザイナルアドヒム, 松岡孝幸, Shigemura Katsumi, Nibu Ken-ichi, Kawabata Masato, Fujisawa Masato
第98回日本泌尿器科学会総会, Apr. 2010, Japanese, 日本泌尿器科学会, 盛岡, Domestic conference
Oral presentation

尿路感染症起因大腸菌の臨床株におけるｇｙｒA、parC遺伝子変異とフルオロキノン系抗菌薬耐性の相関についての検討
安福富彦, Shigemura Katsumi, Shirakawa Toshiro, 中野雄造, Tanaka Kazushi, 武中篤, Arakawa Soichi, 木下承晧, 原田益善, Kawabata Masato, Fujisawa Masato
第52回日本感染症学会中日本地方会学術集会, Nov. 2009, Japanese, 日本感染症学会, 名古屋, Domestic conference
Oral presentation

A case of intrauterine fetal death assosiated with multiple anomalry
Shirakawa Toshiro, Makihara Natsuko, Amano Mariko, Morimoto Noriyuki, Morizane Mayumi, Yamasaki Mineo, Yamada Hideto, Kitazawa Riko
第121回近畿産婦人科学会学術集会, Nov. 2009, Japanese, 近畿産婦人科学会, 神戸, Domestic conference
Oral presentation

Risk factors and the mechanisms for fluoroquinolone resistance in the 156 clinically isolated Escherrichia colistrains of urinary tract infections
Tomihiko Yasufuku, Shigemura Katsumi, Shirakawa Toshiro, Yuzo Nakano, Tanaka Kazushi, Arakawa Soichi, Shouhiro Kinoshita, Kawabata Masato, Fujisawa Masato
第25回腎移植・血管外科研究会, Nov. 2009, English, 尿路感染症研究会, 東京, Domestic conference
Others

EMERGENCE OF FLUOROQUINOLONE-RESISTANT E. COLI STRAIN AND THEIR RELATED MUTATIONS OF THE GYRA AND PARC GENES IN UTI PATIENTS IN JAPAN
Tomihiko Yasufuku, Shigemura Katsumi, Shirakawa Toshiro, Yuzo Nakano, Tanaka Kazushi, Arakawa Soichi, Shouhiro Kinoshita, Kawabata Masato, Fujisawa Masato
UTI研究会, Nov. 2009, English, UTI研究会, 東京, International conference
Oral presentation

大腸菌の臨床株におけるgyrA, parC遺伝子変異とフルオロキノロン系抗菌薬耐性の相関についての検討
安福富彦, Shigemura Katsumi, Shirakawa Toshiro, 中野雄造, Tanaka Kazushi, 武中篤, Arakawa Soichi, 木下承皓, Kawabata Masato, Fujisawa Masato
第97回日本泌尿器科学会総会, Apr. 2009, Japanese, 日本泌尿器科学会, 岡山, Domestic conference
Oral presentation

前立腺肥大症患者におけるシロドシンおよびナフトピジルの臨床効果に関する比較検討
Shirakawa Toshiro, Haraguchi Takahiro, 松本穣, 竹田雅, 森下真一, 山道深, 源吉顕治, Harada Kenichi, Tanaka Kazushi, 武中篤, Fujisawa Masato
第97回日本泌尿器科学会総会, Apr. 2009, Japanese, 日本泌尿器科学会, 岡山, Domestic conference
Oral presentation

EMERGENCE OF FLUOROQUINOLONE-RESISTANT E. COLI STRAIN AND THEIR RELATED MUTATIONS OF THE GYRA AND PARC GENES IN UTI PATIENTS IN JAPAN
Tomihiko Yasufuku, Shigemura Katsumi, Shirakawa Toshiro, Yuzo Nakano, Tanaka Kazushi, Arakawa Soichi, Shouhiro Kinoshita, Kawabata Masato, Fujisawa Masato
第104回ＡＵＡ（Amerigcan Urological Association）, Apr. 2009, English, 米国泌尿器科学会, シカゴ, アメリカ, International conference
Poster presentation

腎臓がん患者におけるsorcinの発現低下
Nakamura Tsutomu, Okamura Noboru, Gotoh Akinobu, Shirakawa Toshiro, Fujisawa Masato, Sakaeda Toshiyuki
第67回日本癌学会, Oct. 2008, Japanese, 日本癌学会, 名古屋, Domestic conference
Oral presentation

ヒト膀胱癌細胞に対するミドカインプロモーターを組み込んだ増殖制限型アデノウイルスベクター（ADMKE1a）の有用性の検討
Shirakawa Toshiro, Nomi Masashi, Tanaka Kazushi, Takenaka Atsushi, Fujisawa Masato, Gotoh Akinobu
第46回日本癌治療学会, Oct. 2008, Japanese, 日本癌治療学会, 名古屋, Domestic conference
Oral presentation

Suppression of sorcin mRNA in patients with renal cell carcinoma
川上恵, Nakamura Tsutomu, Okamura Noboru, 寺尾秀治, Gotoh Akinobu, Shirakawa Toshiro, Fujisawa Masato, 山森元博, Sakaeda Toshiyuki
第67回日本癌学会学術総会, Oct. 2008, Japanese, 日本癌学会, 名古屋, Domestic conference
Poster presentation

Current status of gene therapy for prostate cancer
Shirakawa Toshiro
9th International Congress on Cell Biology, Oct. 2008, English, Korean Society for Molecular and Cellular Biology, Seoul, Korea, International conference
[Invited]
Invited oral presentation

Pre-clinical studies of GMJ2.1: carrier cell-based adenoviral oncolytic virotherapy for head and neck aquamous cell carcinoma
Shirakawa Toshiro
2008 International Society for Cell and Gene Therapy of Cancer, China Conference, Sep. 2008, English, International Society for Cell and Gene Therapy of Cancer, Shijiazhuang, China, International conference
Oral presentation

Therapeutic efficacy of midkine promoter-based conditionally replicative adenovirus vector for targetting the midkine-expressing human bladder cancer cells
Shirakawa Toshiro, Tanaka Keiko, Takenaka Atsushi, Kamidono Sadao, Fujisawa Masato, Gotoh Akinobu
第103回ＡＵＡ（American Urological Association）ａｎｎｕａｌｍｅｅｔｉｎｇ, May 2008, English, 米国泌尿器科学会, オーランド, アメリカ, International conference
Poster presentation

腎癌におけるSorcin発現の検討とVEGF発現に及ぼす影響
Okamura Noboru, Shirakawa Toshiro, Nakamura Tsutomu, Sakaeda Toshiyuki, Okumura Katsuhiko, Takenaka Atsushi, Fujisawa Masato, Gotoh Akinobu
第96回日本泌尿器科学会, Apr. 2008, Japanese, 日本泌尿器科学会, 横浜, Domestic conference
Poster presentation

感染症分野における分子疫学および分子診断の進歩について
Shirakawa Toshiro
第30回日本臨床検査専門医会総会, Nov. 2007, Japanese, 日本臨床検査専門医会, 大阪, Domestic conference
Invited oral presentation

Effect of sorcin on VEGF expression in renal cell carcinoma
Okamura Noboru, Shirakawa Toshiro, Nakamura Tsutomu, Sakaeda Toshiyuki, Takenaka Atsushi, Fujisawa Masato, Gotoh Akinobu
第66回日本癌学会総会, Oct. 2007, Japanese, 日本癌学会, 横浜, Domestic conference
Oral presentation

Clinical Studies of New Anticancer Drugs Results of clinical study of Ad-OC-TK gene therapy for the patients with hormone refractory metastatic prostate cancer
Shirakawa Toshiro, Gotoh Akinobu, Tanaka Kazushi, Takenaka Atsushi, Hara Isao
第66回日本癌学会総会, Oct. 2007, Japanese, 日本癌学会, 横浜, Domestic conference
Oral presentation

骨転移を有するホルモン不応性前立腺癌に対する遺伝子治療臨床研究の長期成績
Shirakawa Toshiro, Gotoh Akinobu, Tanaka Kazushi, Takenaka Atsushi, Hara Isao, Kamidono Sadao, Fujisawa Masato
第45回日本癌治療学会, Oct. 2007, Japanese, 日本癌治療学会, 京都, Domestic conference
Public symposium

ホルモン不応性再燃前立腺癌の治療骨転移を有するホルモン不応性前立腺癌に対する遺伝子治療臨床研究の長期成績
Shirakawa Toshiro, Gotoh Akinobu, Tanaka Kazushi, Takenaka Atsushi, Hara Isao, Kamidono Sadao, Fujisawa Masato
第45回日本癌治療学会総会, Oct. 2007, Japanese, 日本癌治療学会, 京都, Domestic conference
Oral presentation

The impact and perspective on chronic kidney disease in Vietnam
Ito Jun, Kawabata Masato, Shirakawa Toshiro
第22回日本国際保健医療学会総会, Oct. 2007, Japanese, 日本国際保健医療学会, 大阪, Domestic conference
Oral presentation

Long-term outcome of phase I/II clinical trial of Ad-OC-TK/VAL gene therapy for hormone-refractory metastatic prostate cancer
Shirakawa Toshiro, Gotoh Akinobu, Tanaka Kazushi, Takenaka Atsushi, Hara Isao, Kamidono Sadao, Fujisawa Masato
第66回日本ガン学会学術総会, Oct. 2007, English, 日本癌学会, 横浜, Domestic conference
[Invited]
Invited oral presentation

ホルミウムレーザー前立腺核出術(HoLEP)22例の臨床的検討
Ooba Takeshi, Soga Hideo, Shirakawa Toshiro, Fujisawa Masato
第28回日本レーザー医学会, Sep. 2007, Japanese, 日本レーザー医学会, 北海道, Domestic conference
Oral presentation

Evaluation of protein expression in human renal cell carcinoma by proteome analysis using the NBS method
Nakamura Tsutomu, Okamura Noboru, Gotoh Akinobu, Shirakawa Toshiro, Fujisawa Masato, Sakaeda Toshiyuki, Okumura Katsuhiko
日本薬物動態学会ビジョン・シンポジウム, Jul. 2007, English, 日本薬物動態学会, 東京, Domestic conference
Poster presentation

Results of a phase I/II study of Ad-OC-TK/VAL gene therapy for the patients with metastatic or local recurrent prostate cancer
Shirakawa Toshiro, Gotoh Akinobu, Tanaka Kazushi, Takenaka Atsushi, Hara Isao, Kamidono Sadao, Fujisawa Masato
第13回日本遺伝子治療学会, Jun. 2007, Japanese, 日本遺伝子治療学会, 名古屋, Domestic conference
Public symposium

Long-term results of a phase I/II study of Ad-OC-TK/VAL gene therapy for the patients with metastatic or local recurrent prostate cancer
Shirakawa Toshiro, Gotoh Akinobu, Tanaka Kazushi, Takenaka Atsushi, Hara Isao, Kamidono Sadao, Fujisawa Masato
第10回米国遺伝子治療学会, Jun. 2007, English, 米国遺伝子治療学会, シアトル, アメリカ, International conference
Oral presentation

The impact and perspective on chronic kidney disease in Vietnam
Ito Jun, Fujisawa Masato, Kawabata Masato, Shirakawa Toshiro
第50回日本腎臓学会総会（第１回アジアフォーラムCKDイニシアチブ）, May 2007, English, 日本腎臓学会、アジア太平洋腎臓学会、国際腎臓学会, 浜松, International conference
Oral presentation

選択的cyclooxygenase-II(COX-2)阻害剤etodolacのヒト前立腺がん細胞株における抗腫瘍効果の検討
Shigemura Katsumi, Shirakawa Toshiro, Gotoh Akinobu, Fujisawa Masato
第95回日本泌尿器科学会総会, Apr. 2007, Japanese, 日本泌尿器科学会総会, 神戸, Domestic conference
Oral presentation

マウス前立腺癌モデルに対するPETを用いたAd-OC-TK/VAL遺伝子治療効果および遺伝子導入効率の評価に関する有用性の検討
Shirakawa Toshiro, Takenaka Atsushi, Fujisawa Masato, Gotoh Akinobu
第95回日本泌尿器科学会総会, Apr. 2007, Japanese, 日本泌尿器科学会総会, 神戸, Domestic conference
Oral presentation

ヒト膀胱癌細胞における選択的Cox-2阻害剤、EtodolacのE-cadherin発現増強および抗腫瘍効果の検討
Shirakawa Toshiro, Shigemura Katsumi, Gotoh Akinobu, Kawabata Masato, Fujisawa Masato
第95回日本泌尿器科学会総会, Apr. 2007, Japanese, 日本泌尿器科学会, 神戸, Domestic conference
Oral presentation

内分泌療法抵抗性前立腺癌に対するAd-OC-TK plus Valacyclovir遺伝子治療臨床研究におけるquality of lifeの評価
SHIRAKAWA TOSHIRO, TANAKA KAZUSHI, TAKENAKA ATSUSHI, ARAKAWA SOICHI, Hara Isao, FUJISAWA MASATO
第58回日本泌尿器科学会西日本大会, Nov. 2006, Japanese, 日本泌尿器科学会, 長崎, Domestic conference
Oral presentation

Phase I/II clinical trial of Ad-OC-TK plus VAL for the patients with metastatic or local recurrent prostate cancer: Kobe University Experience
SHIRAKAWA TOSHIRO
2006 International Society for Cell and Gene Therapy of Cancer, Japan Conference, Oct. 2006, English, International Society for Cell and Gene Therapy of Cancer, 千葉, International conference
Oral presentation

Ad-OC-TK/VAL遺伝子治療臨床試験における肝機能障害に対する検討
SHIRAKAWA TOSHIRO, TAKENAKA ATSUSHI, Hara Isao, ARAKAWA SOICHI, FUJISAWA MASATO
第４４回日本癌治療学会総会, Oct. 2006, Japanese, 日本癌治療学会, 東京, Domestic conference
Oral presentation

【18F】FDGを用いたPETによるAd-OC-TK/VAL遺伝子治療評価方法の有用性の検討
SHIRAKAWA TOSHIRO, FUJISAWA MASATO
第６５回日本癌学会学術総会, Sep. 2006, Japanese, 日本癌学会, 横浜, Domestic conference
Oral presentation

Improvement of Quality of life of patients with metastatic or local recurrent prostate cancer after Phase Ⅰ/Ⅱ clinical trial of Ad-OC-TK plus Valacyclovir.
SHIRAKAWA TOSHIRO, FUJISAWA MASATO
第１２回日本遺伝子治療学会, Aug. 2006, English, 日本遺伝子治療学会, 東京, Domestic conference
Oral presentation

ホルミウムレーザー前立腺核手術(HoLEP)２２例の臨床的検討
Ooba Takeshi, SOGA HIDEO, SHIRAKAWA TOSHIRO, FUJISAWA MASATO
日本レーザー医学会関西地方会, Jul. 2006, Japanese, 日本レーザー医学会, 神戸, Domestic conference
Oral presentation

Genetically engineered Bifidobacterium animalis expressing the Salmonella flagellin gene for the mucosal immunization in a mouse model
SHIRAKAWA TOSHIRO, KAWABATA MASATO
American Society of Gene Therapy 9th annual meeting, May 2006, English, 米国遺伝子治療学会, バルチモア, International conference
Oral presentation

視床下部下垂体精巣軸におけるNeuropeptide YおよびPancreatic polypeptideの役割
Gotoh Akinobu, SHIRAKAWA TOSHIRO, FUJISAWA MASATO
第94回日本泌尿器科学会, Apr. 2006, Japanese, 日本泌尿器科学会, 福岡, Domestic conference
Oral presentation

α１遮断薬による前立腺肥大症の薬物療法についての排尿症状（ＩＰＳＳ）を中心とした長期薬効の検討
SHIRAKAWA TOSHIRO
第９４回日本泌尿器科学会総会, Apr. 2006, Japanese, 日本泌尿器科学会, 福岡, Domestic conference
Poster presentation

α1受容体遮断薬による前立腺肥大症の薬物療法についての排尿症状(IPSS)を中心とした長期薬効の検討
SHIRAKAWA TOSHIRO, Ooba Takeshi, Yamada Yuji, FUJISAWA MASATO
第94回日本泌尿器科学会, Apr. 2006, Japanese, 日本泌尿器科学会, 福岡, Domestic conference
Oral presentation

ヒト膀胱癌細胞に対するミドカインプロモーターを組み込んだ増殖制限型アデノウイルスベクター(AD-MK-E1a)の有用性の検討
SHIRAKAWA TOSHIRO, Gotoh Akinobu, FUJISAWA MASATO
第94回日本泌尿器科学会, Apr. 2006, Japanese, 日本泌尿器科学会, 福岡, Domestic conference
Oral presentation

No association of VEGF genotype with mRNA expression in renal cell carcinoma
田中久登, OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 山森元博, 瀬戸亮太, 中村任, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
第26回日本臨床薬理学会年会, Dec. 2005, Japanese, 日本臨床薬理学会, 別府, Domestic conference
Poster presentation

腎臓癌におけるVEGF発現量と遺伝子型
田中久登, OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 山森元博, 瀬戸亮太, 中村任, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
第26回日本臨床薬理学会, Dec. 2005, Japanese, 日本臨床薬理学会, 別府, Domestic conference
Poster presentation

Correlation of expression levels of various genes with EGFR in renal cell carcinoma
徳井健次, OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 中村任, 田中久登, YAGI, Mariko, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
第55回日本薬学会近畿支部, Nov. 2005, Japanese, 日本薬学会, 西宮, Domestic conference
Oral presentation

Intra-muscular infjection of rb-IL-2 enhanced the antitumor effect of Ad-OC-TK plus ACV Suicide-gene therapy on murine prostate concer model
SHIRAKAWA, Toshiro, 張竹君, 寺尾秀治, WADA,Yoshitaka, FUJISAWA, Masato, GOTOH,Akinobu
第７回泌尿器遺伝子・細胞治療研究会, Nov. 2005, Japanese, 泌尿器遺伝子・細胞治療研究会, 岡山, Domestic conference
Oral presentation

前立腺肥大症に対するα１受容体遮断薬長期投与症例における排尿症状動態に関する検討
SHIRAKAWA, Toshiro, 寺尾秀治, 日向信之, 合田上政, OOBA, Takeshi, WADA,Yoshitaka, YAMADA,Yuji, GOTOH,Akinobu, OKADA,Hiroshi, FUJISAWA, Masato
第１２回日本排尿機能学会, Oct. 2005, Japanese, 日本排尿機能学会, 松本, Domestic conference
Oral presentation

腎臓癌におけるEGFR発現量と各種遺伝子の発現相関
徳井健次, OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 中村任, 田中久登, YAGI, Mariko, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
第55回日本薬学会近畿支部総会・大会, Oct. 2005, Japanese, 日本薬学会, 西宮, Domestic conference
Poster presentation

腎臓癌におけるEGFR発現量およびゲフィチニブ感受性関連体細胞変異
大松秀明, OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 中村任, 徳井健次, 田中久登, YAGI, Mariko, 大石美惠, 西口工司, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
第15回医療薬学会年会, Oct. 2005, Japanese, 日本医療薬学会, 岡山, Domestic conference
Poster presentation

Multi-Functional GMP Facility for Cell and Gene Therapy, in Kobe City, Japan
SHIRAKAWA, Toshiro
The Williamsburg BioProcessing Conference Asia-Pacific 2nd Annual Meeting, Sep. 2005, English, The Williamsburg BioProcessing, シンガポール, International conference
[Invited]
Invited oral presentation

Real-Time PCR法を用いた腸チフスの血中細菌定量法の開発
SHIRAKAWA, Toshiro, 松本安代, 高田哲男, GOTOH,Akinobu, KAWABATA, Masato, 木下承晧, KUMAGAI, Syunichi, ARAKAWA, Souichi
第７９回日本感染症学会, Apr. 2005, Japanese, 日本感染症学会, 名古屋, Domestic conference
Poster presentation

2003年臨床分離MRSAにおけるバンコマイシン耐性関連遺伝子保有状況
高田哲男, SHIRAKAWA, Toshiro, 松本安代, 木下承晧, KUMAGAI, Syunichi, GOTOH,Akinobu, KAWABATA, Masato
第７９回日本感染症学会, Apr. 2005, Japanese, 日本感染症学会, 名古屋, Domestic conference
Oral presentation

腎癌における各種遺伝子のmRNA発現変動及び遺伝子型
OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 中村任, 盛岡正志, 徳井健次, 田中久登, YAGI, Mariko, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
日本薬剤学会第20年会, Mar. 2005, Japanese, 日本薬剤学会, 東京, Domestic conference
Poster presentation

Change of mRNA levels of several genes in renal cell carcinoma
OKAMURA, Noboru, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 寺尾秀治, 中村任, 盛岡正志, 徳井健次, 田中久登, YAGI, Mariko, SAKAEDA, Toshiyuki, OKUMURA, Katsuhiko
日本薬剤学会第20回記念大会, Mar. 2005, Japanese, 日本薬剤学会, 東京, Domestic conference
Oral presentation

前立腺癌骨転移巣に対する遺伝子治療
SHIRAKAWA, Toshiro
第２１回神戸UGカンファレンス, 2005, Japanese, 神戸UGカンファレンス, 神戸, Domestic conference
[Invited]
Invited oral presentation

泌尿器科病棟における血液培養陽性例の検討
合田上政, FUJISAWA, Masato, SHIRAKAWA, Toshiro, 土橋正樹, 近藤有, OKADA,Hiroshi, KAMIDONO,Sadao
第54回日本泌尿器科学会中部総会, Nov. 2004, Japanese, 第54回日本泌尿器科学会中部総会, 泉佐野, Domestic conference
Oral presentation

前立腺癌骨転移巣に対する遺伝子治療臨床研究
SHIRAKAWA, Toshiro
第６９回日本泌尿器科学会東部総会, Sep. 2004, Japanese, 日本泌尿器科学会東部総会, 東京, Domestic conference
[Invited]
Invited oral presentation

間歇的アンドロゲン除去療法の休薬中に性交渉が可能となった前立腺癌の一例
GOTOH,Akinobu, SHIRAKAWA, Toshiro, HARA, Isao, KAMIDONO,Sadao
第15回日本性機能学会学術総会・西部総会, Sep. 2004, Japanese, 第15回日本性機能学会学術総会・西部総会, 徳島, Domestic conference
Oral presentation

原発巣の診断に苦慮した甲状腺癌陰嚢皮下転移の1例
合田上政, SHIRAKAWA, Toshiro, GOTOH,Akinobu, OKADA,Hiroshi, HARA, Isao, KAMIDONO,Sadao, FUJISAWA, Masato
第187回日本泌尿器科学会関西地方会, Jun. 2004, Japanese, 第187回日本泌尿器科学会関西地方会, 神戸, Domestic conference
Oral presentation

経直腸的前立腺針生検後の感染阻止化学療法に関する検討
寺尾秀治, KAMIDONO,Sadao, GOTOH,Akinobu, SHIRAKAWA, Toshiro, 日向信之, 合田上政
第52回日本化学療法学会総会, Jun. 2004, Japanese, 第52回日本化学療法学会総会, 沖縄, Domestic conference
Oral presentation

院内感染制御における泌尿器科医の役割
SHIRAKAWA, Toshiro, GOTOH,Akinobu, 寺尾秀治, 日向信之, 重村克巳, 合田上政, WADA,Yoshitaka, MURAMAKI,Mototsugu, TANAKA, Kazushi, YAMADA,Yuji, HARA, Isao, KAMIDONO,Sadao
第92回日本泌尿器科学会総会, Apr. 2004, Japanese, 第92回日本泌尿器科学会総会, 大阪, Domestic conference
Oral presentation

ヒト精巣組織におけるTRAILおよびDR4、DR5、DcR1、DcR2の発現に関する免疫組織学的検討
合田上政, FUJISAWA, Masato, SHIRAKAWA, Toshiro, 寺尾秀治, 山口耕平, 近藤有, 土橋正樹, OKADA,Hiroshi, GOTOH,Akinobu, KAMIDONO,Sadao
第92回日本泌尿器科学会総会, Apr. 2004, Japanese, 第92回日本泌尿器科学会総会, 大阪, Domestic conference
Oral presentation

前立腺癌の遺伝子治療
SHIRAKAWA, Toshiro
ＣＯＥシンポジウム千葉大学大学院「消化器扁平上皮癌に対する最先端多戦略治療拠点形成」Cancer Gene Therapy Symposium, Feb. 2004, Japanese, ＣＯＥシンポジウム, 千葉, Domestic conference
[Invited]
Invited oral presentation

Process development of gene therapies in Japan
SHIRAKAWA, Toshiro
The williamsburg BioProcessing Conference Asia-Pacific 1st annual meeting, Sydney, Australia, 2004, English, The williamsburg BioProcessing Conference Asia-Pacific 1st annual meeting, Sydney, Australia, シドニー, International conference
[Invited]
Invited oral presentation

薬剤耐性淋菌性尿道炎の分子生物学的検討およびDHPLCを用いた迅速遺伝子診断法の開発
重村克己, SHIRAKAWA, Toshiro, OKADA,Hiroshi, TANAKA, Kazushi, ARAKAWA, Souichi, 木下承酷, GOTOH,Akinobu, KAMIDONO,Sadao
・第５３回日本泌尿器科学会中部総会, Nov. 2003, Japanese, 日本泌尿器科学会中部総会, 金沢, Domestic conference
[Invited]
Invited oral presentation

放射線併用抗癌化学療法が有用であった女性尿道癌の2例
日向信之, SHIRAKAWA, Toshiro, WADA,Yoshitaka, 重村克巳, OKADA,Hiroshi, GOTOH,Akinobu, KAMIDONO,Sadao
第53回日本泌尿器科学会中部総会, Oct. 2003, Japanese, 第53回日本泌尿器科学会中部総会, 金沢, Domestic conference
Oral presentation

当科における最近の腹腔鏡下手術
重村克巳, SHIRAKAWA, Toshiro, OKADA,Hiroshi, TANAKA, Kazushi, ARAKAWA, Souichi, 木下承晧, GOTOH,Akinobu, KAMIDONO,Sadao
第53回日本泌尿器科学会中部総会, Oct. 2003, Japanese, 第53回日本泌尿器科学会中部総会, 金沢, Domestic conference
Oral presentation

前立腺癌骨転移巣に対する遺伝子治療臨床研究の現況
合田上政, FUJISAWA, Masato, 土橋正樹, 山崎隆文, 張竹君, SHIRAKAWA, Toshiro, GOTOH,Akinobu, OKADA,Hiroshi, ARAKAWA, Souichi, KAMIDONO,Sadao
第41回日本癌治療学会総会, Oct. 2003, Japanese, 日本癌治療学会, 札幌, Domestic conference
[Invited]
Invited oral presentation

精巣腫瘍晩期再発症例の臨床的検討
GOTOH,Akinobu, SHIRAKAWA, Toshiro, KAMIDONO,Sadao
第53回日本泌尿器科学会中部総会, Oct. 2003, Japanese, 第53回日本泌尿器科学会中部総会, 金沢, Domestic conference
Oral presentation

後腹膜鏡下右腎尿管全摘除術および腹腔鏡下膀胱全摘除術を併施した1症例
SHIRAKAWA, Toshiro, OKADA,Hiroshi, GOTOH,Akinobu, 日向信之, WADA,Yoshitaka, KAMIDONO,Sadao, 宇治達哉, 山本明良
第53回日本泌尿器科学会中部総会, Oct. 2003, Japanese, 第53回日本泌尿器科学会中部総会, 金沢, Domestic conference
Oral presentation

CD44v8-10分子遺伝子導入は膀胱癌細胞株の生物学的悪性度を亢進させる
重村克巳, OKADA,Hiroshi, TANAKA, Kazushi, ARAKAWA, Souichi, KAMIDONO,Sadao, SHIRAKAWA, Toshiro, GOTOH,Akinobu, 木下承晧
第62回日本癌学会学術総会, Sep. 2003, Japanese, 第62回日本癌学会学術総会, 名古屋, Domestic conference
Oral presentation

放射光による微量元素分析と生命科学・医学への応用前立腺癌細胞内の亜鉛分布状態の変化―ホルモンと亜鉛の関係
SHIRAKAWA, Toshiro, SUGIMURA, Kazuro, 北村ゆり, 敷根俊輔, 川上拓男, 井手亜里, GOTOH,Akinobu
第１４回日本微量元素学会, Jul. 2003, Japanese, 日本微量元素学会, 大阪, Domestic conference
[Invited]
Invited oral presentation

新しい迅速直接抗菌剤感受性測定装置の開発とそのUTIにおける有用性
OKADA,Hiroshi, 重村克巳, SHIRAKAWA, Toshiro, TANAKA, Kazushi, GOTOH,Akinobu, KAWABATA,Gaku, ARAKAWA, Souichi, 浜口行雄, KAMIDONO,Sadao
第51回日本化学療法学会総会, May 2003, Japanese, 第51回日本化学療法学会総会, 横浜, Domestic conference
Oral presentation

前立腺癌に対する腹腔鏡下前立腺全摘除術の治療成績とQOL
SHIRAKAWA, Toshiro, GOTOH,Akinobu, 日向信之, WADA,Yoshitaka, HARA, Isao, OKADA,Hiroshi, KAMIDONO,Sadao
第91回日本泌尿器科学会総会, Apr. 2003, Japanese, 第91回日本泌尿器科学会総会, 徳島, Domestic conference
Oral presentation

CD44R1分子遺伝子導入は膀胱癌細胞株の生物学的悪性度を亢進させる
熊野晶文, SHIRAKAWA, Toshiro, TANAKA, Kazushi, HARA, Isao, KAWABATA,Gaku, OKADA,Hiroshi, KAMIDONO,Sadao, 生田繁, FUJISAWA, Masato
第91回日本泌尿器科学会総会, Apr. 2003, Japanese, 第91回日本泌尿器科学会総会, 徳島, Domestic conference
Oral presentation

Affiliated Academic Society

アメリカ遺伝子治療学会

日本癌治療学会

日本癌学会

日本感染症学会

日本遺伝子治療学会

日本泌尿器科学会

Research Themes

腸管細胞の獲得免疫における動態評価
橋井佳子, 戸村道夫, 香山尚子, 白川利朗, 片山高嶺, 皆川光
日本学術振興会, 科学研究費助成事業基盤研究(C), 基盤研究(C), 地方独立行政法人大阪府立病院機構大阪国際がんセンター(研究所), 01 Apr. 2021 - 31 Mar. 2024

尿路感染症における網羅的薬剤耐性機構の研究
重村克巳, 大澤佳代, 宮良高維, 白川利朗
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 01 Apr. 2019 - 31 Mar. 2022

Development of a mucosal long peptide cancer vaccine using a membrane transport polymer as a carrier.
白川利朗, 佐久間信至
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 01 Apr. 2019 - 31 Mar. 2022

Development of a novel oral vaccine against COVID-19
Toshiro Shirakawa
AMED, ワクチン開発推進事業, 新型コロナウイルス感染症（COVID-19）に対するワクチン開発（アカデミア主導）, Kobe Univeristy, Oct. 2020 - Mar. 2022, Principal investigator

【AMED】腸管免疫を利用した新規経口がんワクチンの開発
白川利朗
国立研究開発法人日本医療研究開発機構, 橋渡し研究戦略的推進プログラム, Apr. 2019 - Mar. 2022, Principal investigator
Competitive research funding

Clarification of the mechanism of effectiveness of the oral cancer vaccine a platform expressing a cancer antigen
橋井佳子, 白川利朗, 片山高嶺
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Osaka University, 01 Apr. 2017 - 31 Mar. 2020

Midkine-Promoter based Conditionally Replicative Adenovirus expressing siRNA against EGFR for Head and Neck Cancer
Nibu Ken-ichi, SHIRAKAWA toshiro
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 01 Apr. 2016 - 31 Mar. 2019
Background: Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinomas (HNSCCs). Midkine expression is restricted in adult tissues, but is increased in several malignant tumors, including HNSCCs. Here we evaluated the antitumor effect of Midkine-promoter based conditionally replicative adenovirus expressing siRNA against EGFR for targeting HNSCCs expressing Midkine. Methods: A conditionally replicative adenovirus vector controlled by the Midkine promoter, Ad-MK-siEGFR, was generated by integrating gene-expressing siRNA against EGFR. Antitumor effect of Ad-MK-siEGFR was tested in vitro using established HNSCC cell line, T891 with strong Midkine expression. Results: Expression of EGFR in T891 infected with Ad-MKsiEGFR was significantly lower than that of T891 infected with control. Cytotoxicity assays showed significant growth suppression of Ad-MK-siEGFR in T891 cells.

Gene therapy for head and neck cancer by introducing suppressor of cytokine signaling (SOCS)
Otsuki Naoki, Shirakawa Toshiro
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 01 Apr. 2016 - 31 Mar. 2019
The JAK / STAT signaling pathway, which is one of the cytokine signaling pathways activated in head and neck cancer, is suppressed by suppressor of cytokine signaling (SOCS). By overexpression of SOCS3 mediated by adenovirus vector in a head and neck cancer cell line, it was shown that the growth of tumor cells is suppressed.

尿路感染症における多剤耐性菌の迅速診断・迅速疫学診断法の確立
重村克巳
学術研究助成基金助成金／基盤研究(C), Apr. 2016 - Mar. 2019
Competitive research funding

腸管免疫を利用した新規経口がんワクチンの開発
白川利朗
AMED, 橋渡し研究戦略的推進プログラム：シーズB, Apr. 2017 - Mar. 2018, Principal investigator
Competitive research funding

ステルス型カルバペネム耐性菌の分子疫学的解析及び効果的検出法の確立
大澤佳代
学術研究助成基金助成金／基盤研究(C), Apr. 2015 - Mar. 2018
Competitive research funding

Development of novel HCV treatment using oral vaccine
SHIRAKAWA TOSHIRO, KATAYAMA Takane
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2014 - Mar. 2017, Principal investigator
Currently, there are about 170 million HCV (hepatitis C virus) carriers worldwide. Although the treatment outcomes for chronic HCV infection have been improved with interferon and antiviral drugs, the curative rate is still not satisfactory. Recently, we have generated a novel oral HCV vaccine displaying HCV-NS3 protein on the cell surface of bifidobacteria and confirmed that it could induce the HCV-specific cellular immunity in a mouse experimental model. In this study, we have developed novel combinational oral vaccine therapy for HCV infection and confirmed its synergistic effect with interferon using a mouse syngeneic tumor model expressing NS3 protein.
Competitive research funding

【AMED】染色体性薬剤耐性遺伝子を保持する薬剤耐性菌の分子疫学的解析
白川利朗
国立研究開発法人日本医療研究開発機構, 医療分野国際科学技術共同研究開発推進事業戦略的国際共同研究プログラム（ベトナム・インドネシア）, 2017, Principal investigator
Competitive research funding

頭頸部癌に対するヒト乳頭腫ウイルス遺伝子を標的とした集学的治療の開発
大月直樹
学術研究助成基金助成金／基盤研究(C), Apr. 2013 - Mar. 2016
Competitive research funding

悪性胸膜中皮腫に対する新規治療法の開発及び実用化に関する研究
白川利朗
革新的がん医療実用化研究事業, 2016, Principal investigator
Competitive research funding

メタロβラクタマーゼ産生腸内細菌の分布調査及び早期診断法の検討
大澤佳代
科学研究費補助金／基盤研究(C), Apr. 2012 - Mar. 2015
Competitive research funding

Development of a novel oral therapeutic HCV vaccine using bifidobacterium displaying antigen
SHIRAKAWA Toshiro, HOTTA Haku, KATAYAMA Takane
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 2011 - 2013, Principal investigator
More than 170 million people worldwide are chronic HCV (Hepatitis C virus) carriers. A combination of pegylated interferon-alpha with ribavirin, the standard treatment for HCV infection, has been effective in fewer than 50 percent of patients infected with HCV genotype 1. A strong T cell response against the nonstructural protein 3 (NS3) is important for recovery from acute HCV infection, and an early multi-specific CD4 helper and CD8 cytotoxic T cell response is critical for HCV clearance. In the present study, we successfully constructed a genetically modified Bifidobacterium longum displaying recombinant HCV-NS3 peptides containing some CD4 and CD8 epitopes located in the HCV-NS3 region as an oral vaccine against chronic HCV infection. The oral administration of this vaccine could induce NS3-specific immune responses in mice through intestinal mucosal immunity.
Competitive research funding

Genetherapy for Head and Neck Cancer using carrier cell-based delivery of replication-selective adenoviral vector
NIBU Ken-ichi, OTSUKI Naoki, SHIRAKAWA Toshiro
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 2009 - 2011
While multimodal treatments have been performed for the treatment of head and neck cancers, the prognoses of the patients with advanced head and neck cancer are still poor. The estimated survival rates of those patients are around 30%. New strategies for the treatment for head and neck cancer are desirable. To address this issue, we developed the midkine promoter-driven replication-selective adenoviral vector containing siRNA for EGFR or VEGF. With this adenoviral vector, synergistic effect of oncolytic adenovirus and inhibition of oncogene via RNA interference is effect.

Novel oral vaccine using genetically engineering bifidobacterium displaying antigen on its cell-surface
SHIRAKAWA Toshiro, KATAYAMA Tkakane
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 2008 - 2010
Genetically modified probiotic bacteria show promise as an antigen delivery vehicle for mucosal immunization. We developed a novel vaccine platform utilizing Bifidobacterium as an antigen delivery vehicle for mucosal immunization. Genetically modified Bifidobacterium longum displaying Salmonella-flagellin on the cell surface was constructed for the oral typhoid vaccine. We confirmed the efficacy of this oral vaccine in a murine typhoid fever model.

癌細胞ワクチンによるオンコリティック・アデノウイルスの新規ドラッグデリバリー
白川利朗
2010, Principal investigator
Competitive research funding

ヒト乳頭腫ウイルス遺伝子を標的とした頭頸部癌に対する遺伝子治療
大月直樹
科学研究費補助金／基盤研究(C), 2008
Competitive research funding

Discovery of targets for treatment with renal cell carcinoma by gene and protein expression profile analysis
OKUMURA Katsuhiko, SAKAEDA Toshiyuki, GOTOH Akinobu, SHIRAKAWA Toshiro, OKAMURA Noboru, NAKAMURA Tsutomu
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 2004 - 2005
Renal cell carcinoma (RCC) is one of the most chemotherapy or radiotherapy-resistant malignant tumors, and therefore, the standard therapy is surgical treatment. Also, there is no efficient biomarker for diagnosis. To elucidate resistant mechanisms and discovery novel targets for treatment and biomarker for diagnosis, transcriptome and proteome analysis were carried out using RCC and adjacent normal tissues. Since lower expression of sorcin in RCC was found, we investigated its role in cancer proliferation by RNA interference. Up-regulation of vascular endothelial growth factor (VEGF), a potent angiogenesis factor contributing to cancer proliferation, was found by knock-down of sorcin using siRNA in renal cell carcinoma cells, Caki-1. This suggested that lower expression of sorcin caused up-regulation of VEGF, thereby lead cancer proliferation. To comprehensive analysis of protein expressions in RCC and adjacent normal tissues, samples were labeled with 2-nitrobeazenesulfonyl chloride with six ^<12>C or ^<13>C atoms. Then, they were applied MALDI-TOF/Ms and pair peak of 6 Da difference were analyzed and identified. Unknown proteins were identified in addition to known proteins, which have been reported to be up-regulated in RCC.

増殖型アデノウイルスベクターおよびキャリアー細胞を用いた膀胱癌に対する遺伝子治療
白川利朗
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), Kobe University, 2004 - 2005
膀胱壁筋層に浸潤する膀胱癌の約50%は、すでに癌の微小転移を有しており、1年以内に臨床上、明らかな転移巣を呈するといわれている。近年の医学の進歩、特に多剤併用抗癌化学療法の確立にも関わらず、それらの患者のほとんどは2年以内に死亡する。現在、遺伝子治療を代表とする分子標的療法が大きな注目を集めているが、膀胱癌を対象としたそれらの新しい治療法の開発も切望されるところである。従来の非増殖型ウイルスベクターについては、その遺伝子導入効率において限界が指摘されており、癌細胞においてのみ複製される腫瘍特異的な増殖型ウイルスベクターが盛んに研究されている。本研究で用いる増殖型ウイルスベクターはウイルスの複製に必要な遺伝子、E1aを腫瘍特異的プロモーターにより制御し、腫瘍内でのみ増殖可能としたアデノウイルスベクターである。 Cox-2はサイトカインや発癌プロモーターなどの刺激により、マクロファージ、血管内皮細胞、癌細胞などにおいて誘導され、炎症反応、血管新生、発癌などに関与すると言われ、膀胱癌や大腸癌など多くのヒト癌細胞での発現増強が確認されている。我々はCox-2プロモーターを組み込んだ増殖型アデノウイルスベクター、AdE3-cox-2-327を既に作製した。本ベクターはCox-2を強発現する膀胱癌などの癌細胞においてのみ増殖可能であり、癌細胞内で複製を繰り返すことにより細胞融解を引き起こし癌細胞を特異的に殺傷する。平成16年度、われわれはAdE3-cox-2-327の膀胱癌に対する抗癌作用をIn vitro, In vivo双方で確認した。また平成17年度、AdE2-cox-2-327をキャリアー細胞、A549細胞に感染させヒト膀胱癌細胞KK47を接種して作成したマウスの皮下腫瘍に直接、投与することにより抗腫瘍効果が高まることを確認した。

Clinical research of gene therapy using tissue-specific promoter for the treatment of metastatic prostate cancer.
KAMIDONO Sadao, GOTOH Akinobu, MAEDA Sakan, SUGIMURA Kazuro, MATSUO Masafumi, SHIRAKAWA Toshiro
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A), Grant-in-Aid for Scientific Research (A), Kobe University, 2002 - 2005
Background : The absence of effective therapies for hormone refractory prostate cancer establishes the need to develop novel therapeutic modality, such as a gene therapy, that can be applied either separately or in conjunction with current treatment modalities for the treatment of advanced prostate cancer. The phase I/II clinical trial of the Ad-OC-TK (recombinant adenoviral vector containing osteocalcin promoter driven herpes-simplex-virus thymidine kinase gene) plus VAL (Valacyclovior) for the treatment of hormone-refractory prostate cancer was performed at the Kobe University Hospital, Japan.. Method : To date, six patients with localized recurrence or bone metastasis of hormone refractory prostate cancer were enrolled. The doses of a Ad-OC-TK injected directly to localized recurrent tumor or bone metastatic lesion was 2.5 x 10^9 or 10^<10> plaque-forming unit (PFU) / Day 1 and Day 8. Patient was given one gram of VAL twice daily for 21 days. Results : The initial patients tolerated this therapy without serious adverse events. Despite intralesional injections, both the hsvTK and adenoviral genes were able to detect in peripheral blood samples. Biopsies of each lesion demonstrated hsvTK, CAR and OC proteins after treatment demonstrating transcriptional expression in these specimens and increased apoptosis post-treatment observed by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay. Initially, serum PSA levels declined in 4 out of 6 patients coincident with valacyclovir pro-drug activation of hsv-TK. The quantitative analysis of bone scintigraphy showed the decreased RI accumulation only in injected lesion. Conclusions : The Ad-OC-TK plus VAL therapy as a tissue-specific OC promoter-based toxic gene therapy has demonstrated the localized anti-tumor effect without any serious adverse events in the initial Japanese patients with hormone-refractory prostate cancer.

前立腺癌転移巣に対する臓器特異性プロモーターを用いた遺伝子治療臨床研究
守殿貞夫
科学研究費補助金／基盤研究(A), 2005
Competitive research funding

前立腺癌骨転移巣に対する遺伝子治療における、ＰＥＴを用いた評価法の確立
後藤章暢
科学研究費補助金／基盤研究(B), 2005
Competitive research funding

骨肉腫再発巣および骨転移に対する臓器特異性プロモーターを用いた遺伝子治療臨床研究
秋末敏宏
科学研究費補助金／基盤研究(B), 2005
Competitive research funding

遺伝子治療用アデノウイルスベクターの高生産バイオリアクタープロセスの開発
福田秀樹
科学研究費補助金／萌芽研究, 2005
Competitive research funding

遺伝子治療による、膀胱癌の放射線および抗癌剤の感受性増強効果の研究
白川利朗
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), Kobe University, 2002 - 2003
膀胱壁筋層に浸潤した膀胱癌に対する治療法の、現在における第一選択は膀胱全摘出術である。尿路変更術の進歩などにより、膀胱全摘出術を受ける患者さんの術後のQOL(Quality of Life)は著しく改善されたが、膀胱機能の温存やさらなるQOLの向上を目指して、現在、様々な膀胱温存療法が検討されている。アメリカにおいて行われた大規模な臨床試験では、著しい局所浸潤、または全身状態の不良などで手術不可能な症例、あるいは患者さん自身が膀胱を摘出することを拒否した症例に対して、経尿道的膀胱腫瘍切除術、放射線療法、および抗癌化学療法の三者併用による膀胱温存療法が施行され、病期によっては膀胱全摘出術と同等の治療成績が確認された。本研究では、これらの三者併用療法に加えて、p53遺伝子や自殺遺伝子の導入による遺伝子治療をさらに併用し、膀胱癌の放射線や抗癌剤に対する感受性を増強することによる、進行性膀胱癌に対する膀胱温存根治療法の実現の可能性を検討した。最初に種々のP53遺伝子の変異を持つ膀胱癌細胞と持たない膀胱癌細胞での放射線感受性を検討した。結果、正常のP53遺伝子を持つ膀胱癌細胞では、放射線照射によりP53依存性の細胞死を顕著に認めたが、P53遺伝子に変異を持つ膀胱癌細胞では認められず放射線感受性も比較的低かった。以上の結果よりP53遺伝子治療と放射線療法の併用療法の可能性が示唆された。次にアデノウイルスベクターを用いたCD/5-FC遺伝子治療と放射線療法の併用療法の膀胱癌に対する有用性をヒト膀胱癌細胞株、KK47細胞を用いて検討した。結果、本併用療法は細胞培養系、および動物実験双方において相乗的な抗癌作用を示した。本研究により膀胱癌に対する遺伝子治療と放射線療法の併用療法の可能性が示唆された。

Efficacy of Combination Therapy of Ionizing Radiation and Gene Therapy in Prostate Cancer in vitro
SOEJIMA Toshinori, SHIRAKAWA Toshiro, GOTOH Akinobu, SUGIMURA Kazuro
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University Graduate School of Medicine, 2000 - 2002
Several studies have demonstrated that the function of the p53 gene is one of the major determinants of intrinsic cellular sensitivity to the cytotoxic effects of ionizing radiation (IR). Moreover, a strategy to combine adenovirus-mediated wild-type p53 gene transfer with DNA-damaging treatments, such as IR and chemotherapeutic agents, has also been studied in glioblastoma, lung, colorectal, ovarian, and head-and-neck cancers, and additional cytotoxicity or tumor-suppressing activity has been consistently observed. In prostate cancer cells, the significant cytotoxicity has been previously reported with the induction of recombinant wild-type p53 adenovirus (Ad5CMV-p53). In this study, the interactions between IR and Ad5CMV-p53 gene therapy in two androgen-independent prostate cancer cell lines (DU145 and PC-3) were evaluated. We demonstrated that this combination therapy resulted in remarkable synergistic effects and also assessed its molecular mechanisms. The cell growth inhibition in DU145 (p53-mutated) cells by IR was strongly enhanced by additional Ad5CMV-p53 infection in a viral dose-dependent manner. In DU145 cells, IR alone induced minimal p53 mRNA expression. However, IR combined with Ad5CMV-p53 infection stimulated significant increase in p53 mRNA expression supplemented with Bax and p21 mRNA expressions. In PC-3 (p53-null), IR induced Bax and p21 mRNA expression, while the combination effects were observed in p53, Bax, and p21 mRNA expression. Apoptotic cell deaths were rarely observed after IR alone (DU145: 3%, PC-3: 5%). However, after combination therapy, the proportion of apoptotic cells greatly increased (sevenfold in DU145 cells, and twice in PC-3 cells). G1 cell cycle arrest was observed after Ad5CMV-p53 infection and the combination in both cell lines. In conclusion, Ad5CMV-p53 infection enhances the effect of radiation therapy by efficient induction of apoptosis and cell cycle arrest. This combination therapy could be one of the optimal treatment strategies for radioresistant prostate cancer.

Development of testicular tumor gene therapy using human testicular tumor cell-specific promoters
GOTOH Akinobu, KAMIDONO Sadao, SHIRAKAWA Toshiro
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University School of Medicine, 2000 - 2002
One of the most basic issues in gene therapy for cancer is how to express the relevant genes efficiency and specifically in cancer cells. In recent years, there has been an upsurge in basic research into gene therapy for cancer using organ-specific promoters specifically activated in cancer cells and in related clinical trials, with particular attention focused on therapy involving a combination of organ-specific promoters and suicide genes. Motivated by the finding that serum β-HCG (human chorionic gonadotropin) is elevated in over 70% of patients with advanced testicular tumor resistant to chemotherapy, the present study was designed to develop a gene therapy based on the application of a β-HCG promoter as an organ-specific promoter and to investigate its usefulness. It is intended to select a β-HCG promoter with DNA size of 729 base pairs, but we also cloned the DNA of β-HCG promoters of various other sizes, and measured the activity of each promoter in testicular tumor cells in order to determine the optimal size of a β-HCG promoter for gene therapy. It was found that the cloned β-HCG promoter DNA with 729 base pairs had the best specificity, and this was inserted into a plasmid vector integrated into a luciferase gene. This vector was introduced genetically into a variety of cells-choriocarcinoma cells (JAR), embryonal cancer cells (NEC8, NEC14), prostate cancer cells (PC-3, DU145), and bladder cancer cells (WH), and luciferase activity measured to confirm cell-specific activity in the β-HCG promoter. High levels of promoter activity were observed only in the β-HCG producing choriocarcinoma cells (JAR) and embryonal cancer cells (NEC8, NEC14). Based on these findings, we created an Ad-β-HCG -TK and undertook a similar experiment, in which specific anti-tumor activity was found only in the choriocarcinoma cells (JAR) and the embryonal cancer cells (NEC8, NEC14). No cell-damaging action was observed in normal tissue. Next, in order to explore the development of gene therapy using replication-competent adenovirus vectors, we created one integrating an E1A gene controlled by a ? -HCG promoter. At present, we are engaged in in vivo and in vitro therapy studies using this vector to investigate the optimal gene therapy for human testicular tumors, with the eventual aim of conducting a comparison with gene therapy using suicide genes.

Industrial Property Rights

免疫原性ポリペプチド表層発現ビフィズス菌（米国）
SHIRAKAWA TOSHIRO, 堀田博, 片山高嶺
14/768508, 18 Aug. 2015, 大学長, 9925259, 27 Mar. 2018
Patent right

免疫原性ポリペプチド表層発現ビフィズス菌
SHIRAKAWA TOSHIRO, HOTTA HAK, 片山高嶺
特願2015-501435, 14 Feb. 2014, 大学長, 特許6213969, 29 Sep. 2017
Patent right

腫瘍免疫誘導剤
SHIRAKAWA TOSHIRO, 斎藤彩, 松岡孝幸
特願2011-508225, 29 Mar. 2010, 大学長, 特許5582542, 25 Jul. 2014
Patent right

ビフィズス菌表層提示融合タンパク質発現遺伝子
SHIRAKAWA TOSHIRO, 山本さくら
特願2011-531985, 17 Sep. 2010, 大学長, 特許5561681, 20 Jun. 2014
Patent right

経口ワクチン
SHIRAKAWA TOSHIRO, KAWABATA MASATO, 高田哲男, 谷口道子, 岡本明子
特願2009-505269, 19 Mar. 2008, 大学長, 特許5187642, 01 Feb. 2013
Patent right