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Kazuya Takizawa,
Tatsuo Nakashima,
Takuo Mizukami,
Madoka Kuramitsu,
Daiji Endoh,
Shigeto Kawauchi,
Kohsuke Sasaki,
Haruka Momose,
Yoshiharu Kiba,
Tetsuya Mizutani,
Rika A Furuta,
Kazunari Yamaguchi,
Isao Hamaguchi
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ABSTRACT: BACKGROUND: The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level. STUDY DESIGN AND METHODS: We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes. RESULTS: We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses. CONCLUSION: We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.
Transfusion 04/2013; · 3.22 Impact Factor
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Madoka Kuramitsu,
Aiko Sato-Otsubo,
Tomohiro Morio,
Masatoshi Takagi,
Tsutomu Toki,
Kiminori Terui,
RuNan Wang,
Hitoshi Kanno,
Shouichi Ohga,
Akira Ohara, [......],
Nobuo Mizue,
Michio Ozeki,
Atsuko Masumi,
Haruka Momose, Kazuya Takizawa,
Takuo Mizukami,
Kazunari Yamaguchi,
Seishi Ogawa,
Etsuro Ito,
Isao Hamaguchi
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ABSTRACT: Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.
Blood 03/2012; 119(10):2376-84. · 9.90 Impact Factor
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Atsuko Masumi,
Masahiko Ito,
Keiko Mochida,
Isao Hamaguchi,
Takuo Mizukami,
Haruka Momose,
Madoka Kuramitsu,
Momoka Tsuruhara, Kazuya Takizawa,
Atsushi Kato,
Kazunari Yamaguchi
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ABSTRACT: Chronic hepatitis C patients carry the risk of developing into B-cell non-Hodgkin's lymphoma (B-NHL). To clarify the mechanisms underlying this association, we first investigated the molecular markers of B cells from hepatitis C virus (HCV)-infected patients. CD19-positive cells were isolated as B cells from the peripheral blood mononuclear cells of patients infected with the hepatitis C virus and IFN-related gene expression was analyzed. We found that RIG-I and IRF-2 expression were up-regulated in CD19-positive cells from the infected patients. In vitro luciferase reporter analysis using human cell lines indicated that IRF-2 activates the human RIG-I promoter. IRF-2 expression levels were enhanced by HCV cDNA transfection in Huh7 cells. In addition, we observed much less induction in the interferon stimulated gene 15 (ISG15) after Sendai virus (SenV) stimulation of CD19-positive cells from infected patients versus healthy controls, thereby suggesting an impairment of RIG-I downstream signaling in HCV-infected patients. Hence, we found that the failure of the anti-viral response with enhanced IRF-2 oncogenic protein expression in blood B cells from HCV-infected patients. Our results provide important information to better understand the role of IRFs in the cause of HCV chronic infection.
Biochemical and Biophysical Research Communications 01/2010; 391(4):1623-8. · 2.48 Impact Factor
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Haruka Momose,
Jun-ichi Imai,
Isao Hamaguchi,
Mika Kawamura,
Takuo Mizukami,
Seishiro Naito,
Atsuko Masumi,
Jun-ichi Maeyama, Kazuya Takizawa,
Madoka Kuramitsu,
Nobuo Nomura,
Shinya Watanabe,
Kazunari Yamaguchi
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ABSTRACT: Transcriptomics is an objective index that reflects the overall condition of cells or tissues, and transcriptome technology, such as DNA microarray analysis, is now being introduced for the quality control of medical products. In this study, we applied DNA microarray analysis to evaluate the character of Japanese encephalitis (JE) vaccines. When administered into rat peritoneum, Vero cell-derived and mouse brain-derived JE vaccines induced similar gene expression patterns in liver and brain. Body weights and blood biochemical findings were also similar after administration of the two vaccines. Our results suggest that the two JE vaccines are likely to have equivalent characteristics with regard to reactivity in rats.
Japanese journal of infectious diseases. 01/2010; 63(1):25-30.
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ABSTRACT: Megakaryopoiesis is associated with inflammatory reactions. To investigate the role of interferon regulatory factors (IRFs) in inflammation-associated megakaryopoiesis, mouse bone marrow hematopoietic stem cells (HSCs) were analyzed. IFN-gamma treatment induced IRF-2 expression as well as the expression of CD41 and IRF-1 in HSCs. An in vitro clonogenic assay showed that IRF-2- but not IRF-1-overexpressing cells increased the number of megakaryocytic colonies. IRF-2 transfection up-regulated CD41 promoter activity in hematopoietic cell lines. The number of CD41-positive bone marrow cells increased in mice injected with IRF-2-expressing bone marrow cells. These findings suggest that IRF-2 plays an important role in megakaryopoiesis in inflammatory states.
FEBS letters 10/2009; 583(21):3493-500. · 3.54 Impact Factor
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Jumpei Yamazaki,
Takuo Mizukami, Kazuya Takizawa,
Madoka Kuramitsu,
Haruka Momose,
Atsuko Masumi,
Yasushi Ami,
Hideki Hasegawa,
William W Hall,
Hajime Tsujimoto,
Isao Hamaguchi,
Kazunari Yamaguchi
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ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is a malignant lymphoproliferative disorder caused by HTLV-I infection. In ATL, chemotherapeutic responses are generally poor, which has suggested the existence of chemotherapy-resistant cancer stem cells (CSCs). To identify CSC candidates in ATL, we have focused on a Tax transgenic mouse (Tax-Tg) model, which reproduces ATL-like disease both in Tax-Tg animals and also after transfer of Tax-Tg splenic lymphomatous cells (SLCs) to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Using a limiting dilution transplantation, it was estimated that one CSC existed per 10(4) SLCs (0.01%). In agreement with this, we have successfully identified candidate CSCs in a side population (0.06%), which overlapped with a minor population of CD38(-)/CD71(-)/CD117(+) cells (0.03%). Whereas lymphoma did not develop after transplantation of 10(2) SLCs, 10(2) CSCs could consistently regenerate the original lymphoma. In addition, lymphoma and CSCs could also be demonstrated in the bone marrow and CD117(+) CSCs were observed in both osteoblastic and vascular niches. In the CSCs, Tax, Notch1, and Bmi1 expression was down-regulated, suggesting that the CSCs were derived from Pro-T cells or early hematopoietic progenitor cells. Taken together, our data demonstrate that CSCs certainly exist and have the potential to regenerate lymphoma in our mouse model.
Blood 08/2009; 114(13):2709-20. · 9.90 Impact Factor
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Isao Hamaguchi,
Jun-ichi Imai,
Haruka Momose,
Mika Kawamura,
Takuo Mizukami,
Seishiro Naito,
Jun-ichi Maeyama,
Atsuko Masumi,
Madoka Kuramitsu, Kazuya Takizawa,
Hiroshi Kato,
Tetsuya Mizutani,
Yoshinobu Horiuchi,
Nobuo Nomura,
Shinya Watanabe,
Kazunari Yamaguchi
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ABSTRACT: Although vaccines are routinely used to prevent infectious diseases, little is known about the comprehensive influences caused by vaccines. In this study, we showed, using comprehensive gene expression analysis, that pertussis vaccine affected many genes in multiple organs of vaccine-treated animals. In particular, lung was revealed to be the most suitable target to evaluate pertussis vaccine toxicity. The 13 genes identified from the analysis of vaccine-treated lung at day 1 showed a clear dendrogram corresponding to pertussis vaccine toxicity. Furthermore, quantitative analysis of these genes revealed a positive correlation between their respective expression levels and the degree of toxic effects observed in samples that had been treated with various doses of reference pertussis vaccines. The quantification of this 13 gene-set is an indicator of the vaccine toxicity-related reaction.
Vaccine 08/2008; 26(36):4686-96. · 3.77 Impact Factor
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Takuo Mizukami,
Jun-ichi Imai,
Isao Hamaguchi,
Mika Kawamura,
Haruka Momose,
Seishiro Naito,
Jun-ichi Maeyama,
Atsuko Masumi,
Madoka Kuramitsu, Kazuya Takizawa,
Nobuo Nomura,
Shinya Watanabe,
Kazunari Yamaguchi
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ABSTRACT: We propose that DNA microarray analysis can be used in the quality control of pandemic and endemic influenza vaccine. Based on the expression profiles of 76 genes in the rat lung one day after inoculation of influenza vaccine, we can distinguish whole-virion influenza vaccine (PDv: pandemic influenza vaccine and WPv: whole virion-particle vaccine) and sub-virion vaccine (HA vaccine) from saline. Among these 76 genes, we found genes up-regulated by influenza infection, as well as genes involved in the immune response, and interferon. Hierarchical clustering of each influenza vaccine by the expression profiles of these 76 genes matched data from current quality control tests in Japan, such as the abnormal toxicity test (ATT) and the leukopenic toxicity test (LTT). Thus, it can be concluded that DNA microarray technology is an informative, rapid and highly sensitive method with which to evaluate the quality of influenza vaccines. Using DNA microarray system, consistent with the results of the ATT and LTT, it was clarified that there was no difference in vaccine quality between PDv and WPv.
Vaccine 05/2008; 26(18):2270-83. · 3.77 Impact Factor
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Takuo Mizukami,
Madoka Kuramitsu, Kazuya Takizawa,
Haruka Momose,
Atsuko Masumi,
Seishiro Naito,
Atsushi Iwama,
Takehiko Ogawa,
Toshiaki Noce,
Isao Hamaguchi,
Kazunari Yamaguchi
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ABSTRACT: Germ-line stem cells (GSCs) constitute a stem cell population with remarkable stability and proliferative potential in vitro and are a useful model for studying the mechanism of self-renewal and "stemness" function of committed tissue stem cells. To identify GSC-specific genes, we performed subtractive hybridization using cDNA from GSCs, testis, and embryonic stem (ES) cells, and successfully identified 11 genes highly expressed in GSCs. Histological analysis confirmed expression of Cry alpha b, Mcpt8, Cxcl5, Fth1, Ctla2 alpha, and Spp1 in undifferentiated spermatogonia on the basement membrane area of the seminiferous epithelium of the testis, where the GSC niche is thought to be located. Among GSC-specific genes, quantitative PCR analysis showed seven genes-Fth1, Cry alpha b, Spp1, Bcap31, Arhgap1, Ctla2 alpha, and Serpina3g-to be common transcripts highly expressed in hematopoietic stem cells (HSCs). Histological analysis confirmed that Ctla2 alpha-, Serpina3g-, and Spp1-expressing cells were observed in the trabecular bone region of the bone marrow, where the HSC niche is located. Furthermore, histological analysis revealed that only Spp1 was expressed in the hair follicle bulge in the area of the hair follicle stem cell niche. Thus, identifying stemness genes by comparative analysis to GSCs is a powerful tool with which to explore the fundamental commonalities of HSCs and other stem cell types.
Stem Cells and Development 03/2008; 17(1):67-80. · 4.46 Impact Factor
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ABSTRACT: The gene encoding ribosomal protein S19 (RPS19) is one of the responsible genes for Diamond-Blackfan anaemia (DBA), a congenital erythroblastopenia. Although haplo-insufficiency of RPS19 has been suggested to be the onset mechanism underlying the pathogenesis of DBA, the sequential mechanism has not been elucidated. In order to analyse the consequences of the missense mutation of RPS19 specific for DBA patients, we made mutated RPS19 expression vectors. Twelve C-terminally Flag-tagged missense mutants were exogenously expressed from retroviral vectors and analysed by Western blot analysis and flow cytometry. When these 12 mutants were expressed in the erythro-leukaemic cell lines K562 and human bone marrow CD34(+) cells, almost all of the mutant proteins (except for G120R) were unstable, and the levels of mutated RPS19 protein were significantly low. To address the effect of deficient RPS19 expression on cell proliferation, RPS19 was downregulated by siRNA. Repressive expression of RPS19 in human CD34(+) cells produced an elevated number of cells at G0 and induced erythroid progenitor-specific defects in BM cells. These results suggest that abnormal ribosomal biogenesis causes inadequate cell cycle arrest in haematopoietic progenitors, and that, subsequently, erythroid progenitors are specifically hampered. These in vitro phenotypes of genetically manipulated CD34(+) cells mimic DBA pathogenesis.
British Journal of Haematology 03/2008; 140(3):348-59. · 4.94 Impact Factor
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Isao Hamaguchi,
Jun-ichi Imai,
Haruka Momose,
Mika Kawamura,
Takuo Mizukami,
Hiroshi Kato,
Seishiro Naito,
Jun-ichi Maeyama,
Atsuko Masumi,
Madoka Kuramitsu, Kazuya Takizawa,
Masayo Mochizuki,
Masaki Ochiai,
Akihiko Yamamoto,
Yoshinobu Horiuchi,
Nobuo Nomura,
Shinya Watanabe,
Kazunari Yamaguchi
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ABSTRACT: Conventional animal tests such as leukocytosis promoting tests have been used for decades to evaluate toxicity of pertussis vaccine. Here, we examined gene expression in relation to the vaccine toxicity using a DNA microarray. Comparison of conventional animal test data with the DNA microarray-based gene expression data revealed a gene expression pattern highly correlated with leukocytosis in animals. Of 10,490 rat genes analyzed, two genes, alpha1-acid-glycoprotein (Agp) and hemopexin (Hpx), were found up-regulated by the toxin administration in a dose-dependent manner (assayed by a quantitative PCR based on the microarray). Variation of the gene expression was very small amongst the test animals, and the results were highly reproducible. These findings suggest that gene expression analysis of vaccine-treated animals can be used as an accurate and simple method of pertussis vaccine safety assessment.
Vaccine 05/2007; 25(17):3355-64. · 3.77 Impact Factor
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Takuo Mizukami,
Atsuko Masumi,
Haruka Momose,
Madoka Kuramitsu, Kazuya Takizawa,
Seishiro Naito,
Jun-ichi Maeyama,
Keiko Furuhata,
Momoka Tsuruhara,
Isao Hamaguchi,
Kazunari Yamaguchi
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ABSTRACT: Vaccines differ from other pharmaceutical products. The quality and safety of batches are regulated to high standards by national regulatory authorities. Various quality control and safety tests have been developed, including the abnormal toxicity test (ATT), which is described in the World Health Organization (WHO) guidelines and in each country's pharmacopoeia. However, the criteria for abnormal results are not well defined in these guidelines. In addition, the animal grade to be used in ATT, classified on the basis of microbial colonization, was not designated in either guideline.In this study, we report a new and improved method of performing ATT, including statistical, histopathological analysis and hematological findings. It is based on the observation that there are body weight changes characteristic to each vaccine, and such standardized changes can be used as references for evaluating test vaccines. In addition, histopathological data are useful for determining vaccine quality and safety. Combined with histopathological examination, the improved ATT will be of great use for evaluating the consistency, quality and safety of different batches of vaccine. The results of these analyses were similar using either ‘clean’ or specific pathogen-free guinea pigs.
Biologicals.
