Research Projects -
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セシウムがインフルエンザウイルス・RSウイルス感染に及ぼす影響
Grant number：20K08180 2020.04 - 2023.03
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)
山下 信子, 小川 寛人, 八代 将登, 難波 ひかる
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
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Investigation of host factors for severe influenza virus infection
Grant number：26461585 2014.04 - 2017.03
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)
Yamashita Nobuko
Grant amount：\4940000 （ Direct expense: \3800000 、 Indirect expense：\1140000 ）
Results: (i) Increased expression of IL-11, Fas L, Angiotensin II receptor genes was observed in vascular endothelial cells, when influenza virus infected the respiratory epithelium (in vitro）. It was thought to be an influenza virus specific response. (ii) VNN1 was newly identified as a gene involved in influenza virus replication in the respiratory epithelium. VNN1 is an enzyme that hydrolyzes pantetheine, and it was found that cysteamine which is a degradation product of pantetheine, has a virus replication inhibiting action.
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ウイルスプロモーターからの外来遺伝子発現に対する糖の促進効果に関する研究
Grant number：20656138 2008 - 2009
日本学術振興会 科学研究費助成事業 挑戦的萌芽研究 挑戦的萌芽研究
荒尾 雄二郎, 難波 ひかる, 木村 美幸
Grant amount：\3400000 （ Direct expense: \3400000 ）
1 外来遺伝子発現に対する糖鎖の促進効果
プルランを代表例として、CV-1細胞でのCMVプロモーター制御EGFP遺伝子の一過性発現に対する糖鎖の効果を調べると、濃度依存的に遺伝子発現を増加させ、12%で最大の効果を発揮した。プルラン以外の糖鎖である酵母マンナン、デキストラン、フルクトオリゴ糖、コンドロイチン硫酸でも有意な遺伝子発現増大効果が観察された。これらの知見は、本研究の応用性を広げるとともに、生物現象との共通性を示唆する点で重要である。
2 ヘキソーストランスポーター(GLUT)が糖センサーであるか否かの検討
GLUTを介して取込まれる蛍光グルコース類似体(2NBDG)を各種の糖の存在下でCV-1細胞に2時間取込ませ、2NBDG取込み量と各糖の外来遺伝子発現促進効果を比較したところ、糖濃度が100、500mMいずれの場合でも両者は相関しなかった。従って、GLUTは糖の遺伝子発現促進効果のセンサーではないと推定された。
3 阻害剤等を用いた糖の促進機構の解析
促進効果の高いラクチュロースと各種阻害剤を(1)の評価系で同時に作用させ、阻害剤の影響を検討した。ラクチュロースによる遺伝子発現増大は、G1阻害剤とG1/S阻害剤で低下し、G2/M阻害剤で相加的に増大した。従って、細胞周期、並びにそれ以外の因子の関与が推測され、その機構を解明する上で重要な手掛かりとなる。
4 糖添加後早期における細胞内遺伝子発現状態の解析
糖刺激早期(糖添加1時間後)におけるmRNAをマイクロアレイ法で解析した。その結果、RNAのスプライシング、結合、加工、代謝、並びにmRNAの代謝が活性化され、クロマチン、蛋白質-DNA複合体、及びヌクレオソームの集合状態が低下していると示唆された。添加後1時間以内にDNA構造体の乖離と転写の活性化が誘導されるというこの示唆は、その機構解明に大きな意義をもつ。 -
Imunoresponse against Human herpesvirus-6 and Human herpesvirus-7
Grant number：14570264 2002 - 2003
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)
YOSHIDA Mariko, INAMBA Hikaru, YAMADA Masao
Grant amount：\3500000 （ Direct expense: \3500000 ）
Plasmacytoid dendritic cell (DC) precursors in human blood are now recognized as identical to natural alpha interferon (IFN-α)-producing cells (IPCs) and are thought to play an important role in antiviral immunity. Therefore, We tried to investigate the susceptibility as well as the cellular responses of DCs to two variants of human herpesvirus 6 (HHV-6), namely A and B, and human herpesvirus 7 (HHV-7). DCs are isolated from cord blood mononuclear ells (CBMCs) by magnetic-bead separation. Although HHV-6A, HHV-6B and HHV-7 are Closely related in DNA sequence, each has distinctive genomic, antigenic, and biological properties. First, DCs are infected with these viruses at various multiplicity of infection and examined by Facs for defection of surface antigen, by ELISA for production of cytokines, and by the transmission electron microscopy (TEM) for replication of the viruses. Based on the observation of TEM, all of these viruses infect DCs and produce progeny viruses. The reticular inclusion bodies with a skein-like appearance were commonly observed, which are the particular structure in the nuclei of infected cells with these viruses. In the expression of surface antigens, CD80, CD83, CD86, and HLA-DR, which are marker antigen for maturation of DCs, are determined on infected cells with HHV-6 more intensive than on HHV-7 infected cells. Both of HHV-6A and HHV-6B infections trigger DCs to produce vast amounts of IFN-α and induces DCs to differentiate into mature DCs. In contrast, DCs infected with HHV-7 do not produce IFN-α neither differentiate into mature DCs. Second, naive CD4^+ T cells obtained from CBMCs are co-cultured with virus-infected DCs and measured the CD4^+ T cells-induced cytokines. HHV-6B infected DCs stimulate naive CD4^+ T cells to produce IFN-γ and interleukin-10 (IL-10). HHV-6A or HHV-7 infected DCs stimulate naive CD4^+_T cells to produce IL-4, IL-5, IL-10, and IFN-γ. These findings suggest that producing large amount or IFN-α from infected DCs dose not contribute to a critical link between innate and adaptive immunity. Viral specific proteins may be an important role on the differentiation of DCs and on the following events to dictate T cell mediated immunity.
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Inhibitory Effects of Beta-herpesviruses on Hematopoiesis
Grant number：13670299 2001 - 2002
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)
YAMADA Masao, NAMBA Hikaru, ISOMURA Hiroki, YOSHIDA Mariko, ARAO Yujirou
Grant amount：\3600000 （ Direct expense: \3600000 ）
Human herpesvirus 6 (HHV-6), as well as human cytomegalovirus, is related to various severe complications after hematopoietic stem cell transplantation (SCT) under extensive use of immunosuppressive drugs. We monitored the activity of five herpesviruses including three beta-herpesviruses after allogeneic SCT, and showed that HHV-6 is associated with delayed engraftment of hematopoietic cells after SCT. In vitro models, however, have not proven the relationship between beta-herpesviruses and the clinical manifestations.
We established the in vitro systems to assess the effects of HHV-6 and related beta herpesviruses on hematopoietic colony formation. We comparatively examined HHV-6A, HHV6B, and human herpesvirus 7 (HHV-7). We showed that both type of HHV-6 suppresses all three lineages of hematopoietic colony formation of erythroid (BFU-E), granulocyte /macrophage (CFU-GM), and megakaryocyte (CFU-Meg) in vitro. On the other hand, HHV-7 did not have any suppressive effect on in vitro hematopoietic colony formation..
We focused on HHV-6B and further examined the interaction with human CD34+ cells, which are a major source of hematopoietic progenitor cells. CD34+ cells were immunomagnetically isolated from cord blood mononuclear cells using anti-CD34+ antibodies coated onto either Dynabeads or MACS beads. The CD34+ population selected with Dynabeads showed a broad range of fluorescence. The population selected with MACS beads showed a narrow range of fluorescence. After infection with HHV-6, Two transcripts of the immediate early genes were detected with both cell populations. HHV-6 suppressed colony formation of BFU-E, CFU-GM, and CFU-Meg. HHV-6 suppressed cell growth after 3 to 7 days culture in the presence of thrombopoietin (TPO). More differentiated CD34+ cells were more susceptible to the effects of HHV-6. These data indicate that the targets for hematopoietic suppression by HHV-6 are relatively differentiated cells that express a lower amount of the CD34 antigen (dim cells) among a heterogeneous cell population. -
Analysis of antigenic properties of human herpesvirus 7 and the host immune responses
Grant number：10670285 1998 - 1999
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)
YAMADA Masao, ISOMURA Hiroki, NAMBA Hikaru, YOSHIDA Mariko, OHUCHI Reiko
Grant amount：\2400000 （ Direct expense: \2400000 ）
To elucidate antigenic properties of human herpesviruses 6 and 7 (HHV-6 and -7), three monoclonal antibodies (Mabs) against HHV-6 and 13Mabs against HHV-7 were established and characterized by radio-immunoprecipitation. The 40-kDa phosphoprotein by recognized by a Mab (TK17) was the product of HHV-7 U27 gene. Among these Mabs, a Mab(IK3) which recognizes the 135-kDa late polypeptide of HHV-6 and several Mabs (TK3 and others) which recognize the 125-kDa late polypeptide of HHV-7 were selected to monitor virus growth by a dot blot antigen-detection method. Using the dot blot method, we established a neutralizing antibody assay for these viruses. The dot-blot neutralization assay is easy to perform, is highly reproducible and objective when compared with the conventional methods based on cytopathology or immunofluorescence for determining neutralization endpoints. Using this method, 55 sera from healthy adults were examined. In most individuals, neutralizing antibody titers against HHV-7 were much higher than those against HHV-6. Elevated neutralizing titers against HHV-7 might be attributed to continuous booster effects by persistent HHV-7 production in saliva. Furthermore, we monitored neutralizing antibody titers against these viruses in 15 children with documented history of exanthem subitum. Transferred antibody titers against these viruses from mothers were readily demonstrated by the neutralization test. Transferred antibody titers against HHV-7 were higher and remained longer after birth than those of HHV-6, and these findings were in accord with clinical observation that HHV-6 infection usually occurs earlier than HHV-7 infection. It is notable that no cross-reactive elevation of heterologous neutralizing antibody titer was observed.