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KANAI Kyosuke

    Department of Medicine　Research associate
Last Updated :2024/04/25

Researcher Information

URL

Research funding number

  • 20596621

ORCID ID

J-Global ID

Research Interests

  • Cytokine, Chemokine   Murine γ-herpesvirus 68   Human herpesvirus   Epstein-Barr virus   Virus   

Research Areas

  • Life sciences / Virology

Academic & Professional Experience

  • 2023/10 - Today  Tottori UniversityDivision of Virology, Department of Infection and immunity, Faculty of MedicineAssociate professor

Association Memberships

  • 日本免疫学会   日本ウイルス学会   

Published Papers

  • Kyosuke Kanai; Seiji Kageyama; Osamu Yoshie
    Journal of immunology (Baltimore, Md. : 1950) 2023/09 
    Extrahepatic viral infections are often accompanied by acute hepatitis, as evidenced by elevated serum liver enzymes and intrasinusoidal infiltration of CD8+ T cells, without direct infection of the liver. An example is infectious mononucleosis caused by primary infection with EBV. Previously, we demonstrated that airway infection of mice with murine γ-herpesvirus 68 (MHV68), a murine model of EBV, caused liver inflammation with elevated serum liver enzymes and intrahepatic infiltration of IFN-γ-producing CD8+ T cells and NK cells. Mechanistically, the expression of the CXCR3-ligand chemokines, which are commonly induced by IFN-γ and attract IFN-γ-producing Th1-type cells via CXCR3, was upregulated in the liver. Importantly, the liver inflammation was suppressed by oral neomycin, an intestine-impermeable aminoglycoside, suggesting an involvement of some products from the intestinal microbiota. In this study, we showed that the liver inflammation and the expression of the CXCR3-ligand chemokines in the liver were effectively ameliorated by i.p. administration of anti-TLR4 mAb or C34, a TLR4 blocker, as well as in TLR4-deficient mice. Conversely, intrarectal inoculation of Escherichia coli as an extraintestinal source of LPS aggravated liver inflammation in MHV68-infected mice with increased expression of the CXCR3-ligand chemokines in the liver. In contrast, the lung inflammation in MHV68-infected mice was not affected by oral neomycin, i.p. administration of C34, or TLR4 deficiency. Collectively, the LPS-TLR4 pathway plays a pivotal role in the liver inflammation of MHV68-infected mice at least in part by upregulating the CXCR3-ligand chemokines in the liver.
  • Alfredo A Hinay Jr; Kyosuke Kanai; Akeno Tsuneki-Tokunaga; Mizuki Komatsu; Elizabeth O Telan; Seiji Kageyama
    International journal of molecular sciences 23 (23) 2022/12 
    It has been considered that reduced susceptibility to antiretroviral drugs is influenced by drug adherence, drug tolerance and drug-resistance-related mutations in the HIV genome. In the present study, we assessed the intrinsic high viral growth capability as a potential viral factor that may influence their susceptibility to antiretroviral drugs using an in vitro model. Phytohemagglutinin-activated peripheral blood mononuclear cells (1.5 × 106 cells) were infected with HIV isolates (106 copies/mL). The culture was carried out at different concentrations (0.001-20 μM) of 13 synthetic antiretroviral compounds (six nucleoside/nucleotide reverse transcriptase inhibitors, one non-nucleoside reverse transcriptase inhibitor, four integrase inhibitors, and two protease inhibitors), and HIV production was assessed using HIV-RNA copies in culture. The 90% inhibitory concentration (IC90) and pharmacokinetics of an antiretroviral agent were used as parameters to determine the reduced antiretroviral drug susceptibility of HIV isolates with high growth capability to synthetic antiretroviral compounds. The high growth capability of HIV isolates without any known drug resistance-related mutation affected their susceptibility to tenofovir (IC90 = 2.05 ± 0.40 μM), lamivudine (IC90 = 6.83 ± 3.96 μM), emtricitabine (IC90 = 0.68 ± 0.37 μM), and efavirenz (IC90 = 3.65 ± 0.77 μM). These antiretroviral drugs showed IC90 values close to or above the maximum plasma concentration against HIV isolates with high growth capability without any known drug resistance-related mutation. Our results may contribute to the development of effective strategies to tailor and individualize antiretroviral therapy in patients harboring HIV isolates with high growth capability.
  • Alfredo A Hinay Jr; Sosuke Kakee; Seiji Kageyama; Akeno Tsuneki-Tokunaga; Waldy Y Perdana; Yui Akena; Shota Nishiyama; Kyosuke Kanai
    Vaccines 10 (9) 2022/09 
    In a previous study, we described the diverse growth capabilities of circulating seasonal influenza A viruses (IAVs) with low to high viral copy numbers in vitro. In this study, we analyzed the cause of differences in growth capability by evaluating pro-inflammatory cytokines (TNF-α, IL-6, IFN-β) and antiviral interferon-stimulated genes (ISG-15, IFIM1, and TRIM22). A549 cells (3.0 × 105 cells) were inoculated with circulating seasonal IAV strains and incubated for 6 and 24 h. In cells inoculated for 6 h, IAV production was assessed using IAV-RNA copies in the culture supernatant and cell pellets to evaluate gene expression. At 24 h post-infection, cells were collected for IFN-β and ISG-15 protein expression. A549 cells inoculated with seasonal IAV strains with a high growth capability expressed lower levels of IFN-β and ISGs than strains with low growth capabilities. Moreover, suppression of the JAK/STAT pathway enhanced the viral copies of seasonal IAV strains with a low growth capability. Our results suggest that the expression of ISG-15, IFIM1, and TRIM22 in seasonal IAV-inoculated A549 cells could influence the regulation of viral replication, indicating the existence of strains with high and low growth capability. Our results may contribute to the development of new and effective therapeutic strategies to reduce the risk of severe influenza infections.
  • Seiji Kageyama; Akeno Tsuneki-Tokunaga; Kyosuke Kanai
    Archives of virology Research Square Platform LLC 167 (1) 195 - 199 2021/08 
    Abstract Close observation of the local transmission of influenza A(H1N1) viruses enabled an estimation of the transmission period of the virus without a mutation. Of 4,448 isolates from 11 consecutive years, 237 isolates could be categorized into 57 strain groups with identical hemagglutinin genes. Transmission of these 57 strains was chased for the maximum duration of an epidemic season. In addition, 35 identical strains were recognized at the study site and other countries within 147 days. Consequently, it can be postulated that once an influenza virus enters a temperate country, the strain rarely mutates until the end of the season.
  • Akeno Tsuneki-Tokunaga; Kyosuke Kanai; Asao Itagaki; Hideaki Tsuchie; Takayoshi Okada; Masaaki Kasagi; Kiyoshi Tanaka; Miho Aoki; Alfredo Jr A Hinay; Seiji Kageyama
    Archives of virology 166 (4) 1193 - 1196 2021/04 
    The correlation of viral growth capability (n = 156) with the viral load in nasopharyngeal swabs (n = 76) was assessed. Epidemic influenza A/H1N1, A/H3N2, and B viruses showed a wide range of growth capability (104-1011 copies/mL) in Madin-Darby canine kidney cells. The growth was correlated with the nasopharyngeal viral load (r = 0.53). Six selected strains showed growth-dependent cell death (r = 0.96) in a growth kinetics assay. Epidemic influenza viruses exhibit a wide range of growth capability. Growth capability should be considered one of the key factors in disease prognosis.
  • Seiji Kageyama; Alfredo Amolong Hinay; Elizabeth Freda Omengan Telan; Genesis May Jopson Samonte; Prisca Susan Agustin Leano; Akeno Tsuneki-Tokunaga; Kyosuke Kanai
    Journal of the International Association of Providers of AIDS Care (JIAPAC) SAGE Publications 18 232595821985657 - 232595821985657 2325-9582 2019/01 
    Although drug-resistant HIV variants are considered to be less fit than drug-susceptible viruses, replication competence of these variants harbored by patients has not yet been elucidated in detail. We herein assessed the replication competence of strains obtained from individuals receiving antiretroviral therapy. Among 11 306 participants in a drug resistance surveillance in the Philippines, 2629 plasma samples were obtained from individuals after a 12-month treatment with zidovudine (ZDV)/lamivudine (3TC)/nevirapine (NVP). The replication competence of HIV isolates was then assessed by reinoculation into seronegative peripheral blood mononuclear cells in the absence of drugs in vitro. The drug resistance rate was estimated to be 9.2%. Drug-resistant strains were still a minority of closely related strains in a phylogenetic cluster. Among the available 295 samples, 37 HIV strains were successfully isolated. Progeny viruses were produced at a wide range (5.1 × 106 to 3.4 × 109 copies/mL) in primary culture of peripheral blood mononuclear cells. The viral yields were higher than the corresponding plasma viral load (1300 to 3.4 × 106 copies/mL) but correlated with those ( r = 0.4). These results suggest that strains with higher intrinsic replication competence are one of the primary targets of newly selected drugs at the increasing phase of the plasma viral load during antiretroviral therapy.
  • Kyosuke Kanai; Ah-Mee Park; Akiko Watanabe; Tomohiro Arikawa; Teruhito Yasui; Hiroki Yoshida; Ikuo Tsunoda; Osamu Yoshie
    Journal of Immunology American Association of Immunologists 200 (8) 2703 - 2713 1550-6606 2018/04 [Refereed]
     
    IL-27 is an immunoregulatory cytokine consisting of p28 and EBI3. Its receptor also has two subunits, WSX1 and gp130. Although IL-27 promotes Th1 differentiation in naive T cells, it also induces IL-10 expression in effector Th1 cells to curtail excessive immune responses. By using p28-deficient mice and WSX1-deficient mice (collectively called IL-27–deficient mice), we examined the role of IL-27 in primary infection by murine g-herpesvirus 68 (MHV68), a murine model of EBV. Upon airway infection with MHV68, IL-27–deficient mice had more aggravated lung inflammation than wild-type mice, although MHV68 infection per se was better controlled in IL-27–deficient mice. Although epithelial cells and alveolar macrophages were primarily infected by MHV68, interstitial macrophages and dendritic cells were the major producers of IL-27. The lung inflammation of IL-27–deficient mice was characterized by more IFN-g–producing CD8+ T cells and fewer IL-10–producing CD8+ T cells than that of wild-type mice. An infectious mononucleosis–like disease was also aggravated in IL-27–deficient mice, with prominent splenomegaly and severe hepatitis. Infiltration of IFN-g–producing effector cells and upregulation of the CXCR3 ligand chemokines CXCL9, CXCL10, and CXCL11 were noted in the liver of MHV68-infected mice. Oral neomycin effectively ameliorated hepatitis, with decreased production of these chemokines in the liver, suggesting that the intestinal microbiota plays a role in liver inflammation through upregulation of these chemokines. Collectively, IL-27 is essential for the generation of IL-10–producing effector cells in primary infection by MHV68. Our findings may also provide new insight into the mechanism of hepatitis associated with infectious mononucleosis.
  • Kyosuke Kanai; Ah-Mee Park; Hiroki Yoshida; Ikuo Tsunoda; Osamu Yoshie
    JOURNAL OF IMMUNOLOGY AMER ASSOC IMMUNOLOGISTS 198 (1) 119 - 127 0022-1767 2017/01 [Refereed]
     
    EBI3 functions as the subunit of immune-regulatory cytokines, such as IL-27 and IL-35, by pairing with p28 and p35, respectively. We treated wild-type and EBI3-deficient mice with intratracheal administration of LPS and obtained bronchoalveolar lavage fluid (BALF) 24 h later. Although neutrophils were the predominant cells in BALF from both groups of mice, eosinophils were highly enriched and there was increased production of eosinophil-attracting chemokines CCL11 and CCL24 in BALF of EBI3-deficient mice. The bronchial epithelial cells and alveolar macrophages were the major producers of CCL11 and CCL24. Because no such increases in eosinophils were seen in BALF of p28/IL-27-deficient mice or WSX-1/IL-27Ra subunit-deficient mice upon intratracheal stimulation with LPS, we considered that the lack of IL-35 was responsible for the enhanced airway eosinophilia in EBI3-deficient mice. In vitro, IL-35 potently suppressed production of CCL11 and CCL24 by human lung epithelial cell lines treated with TNF-alpha and IL-1 beta. IL-35 also suppressed phosphorylation of STAT1 and STAT3 and induced suppressor of cytokine signaling 3. In vivo, rIL-35 dramatically reduced LPS-induced airway eosinophilia in EBI3-deficient mice, with concomitant reduction of CCL11 and CCL24, whereas neutralization of IL-35 significantly increased airway eosinophils in LPS-treated wild-type mice. Collectively, our results suggest that IL-35 negatively regulates airway eosinophilia, at least in part by reducing the production of CCL11 and CCL24.
  • Ah-Mee Park; Kyosuke Kanai; Tatsuki Itoh; Takao Sato; Tatsuya Tsukui; Yutaka Inagaki; Moises Selman; Kouji Matsushima; Osamu Yoshie
    PloS one 11 (2) e0148998  2016 [Refereed]
     
    Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases.
  • Keiko Nagata; Yuji Nakayama; Katsumi Higaki; Marika Ochi; Kyosuke Kanai; Michiko Matsushita; Satoshi Kuwamoto; Masako Kato; Ichiro Murakami; Takeshi Iwasaki; Eiji Nanba; Hiroshi Kimura; Kazuhiko Hayashi
    AUTOIMMUNITY TAYLOR & FRANCIS LTD 48 (5) 328 - 335 0891-6934 2015/08 [Refereed]
     
    Epstein-Barr virus (EBV) is a ubiquitous virus that infects most adults latently. It persists in B lymphocytes and reactivates occasionally. Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). We have reported that Graves' disease patients and healthy controls have EBV-infected lymphocytes that have TRAbs on their surface (TRAb(+)EBV(+) cells) in peripheral blood mononuclear cells (PBMCs). EBV reactivation is known to be associated with plasma cell differentiation and antibody production of B cells. In this study, we investigated whether TRAb(+) EBV(+) cells really produce TRAbs or not when persistent EBV is reactivated. We cultured PBMCs from 12 Graves' disease patients and 12 healthy controls for several days with cyclosporine A to expand the EBV-infected cell population, and then compared TRAb levels between EBV reactivation by 33 degrees C culture and EBV nonreactivation by 37 degrees C culture of PBMCs. Flow cytometry confirmed that all samples at day 0 (reactivation starting point) contained TRAb(+) EBV(+) cells. During 33 degrees C culture, EBV-reactivated cells with EBV-gp350/220 expression increased from about 1 to 4%. We quantified TRAb levels in culture fluids by radio-receptor assay, and detected an increased concentration for at least one sampling point at 33 degrees C (from days 0 to 12) for all patients and healthy controls. TRAb levels were significantly higher in supernatants of 33 degrees C culture than of 37 degrees C culture, and also significantly higher in supernatants from patients than those from controls. This study revealed TRAb production from TRAb(+) EBV(+) cells in response to reactivation induction of persistent EBV in different efficiencies between patients and controls.
  • Keiko Nagata; Katsumi Higaki; Yuji Nakayama; Hiromi Miyauchi; Yui Kiritani; Kyosuke Kanai; Michiko Matsushita; Takeshi Iwasaki; Hirotsugu Sugihara; Satoshi Kuwamoto; Masako Kato; Ichiro Murakami; Eiji Nanba; Hiroshi Kimura; Kazuhiko Hayashi
    AUTOIMMUNITY INFORMA HEALTHCARE 47 (3) 193 - 200 0891-6934 2014/05 [Refereed]
     
    Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein-Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves' disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves' disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves' disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves' disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves' disease.
  • Hitoshi Sano; Keiko Nagata; Kaoru Kato; Kyousuke Kanai; Kiyoshige Yamamoto; Keisuke Okuno; Satoshi Kuwamoto; Hiromi Higaki-Mori; Hirotsugu Sugihara; Masako Kato; Ichiro Murakami; Susumu Kanzaki; Kazuhiko Hayashi
    INTERVIROLOGY KARGER 56 (2) 114 - 121 0300-5526 2013 [Refereed]
     
    Objectives: To clarify characteristics on rabbit in vivo infection with type 2 EBV nuclear antigen (EBNA-2)-deleted Epstein-Barr virus (P3HR-1-EBV) and compare infectious efficacy of P3HR-1-EBV with previously reported prototype type 1 EBV from B95-8. Methods: Twelve Japanese White rabbits were inoculated with P3HR-1-EBV via intranasal or intravenous routes and autopsied on day 70-84. Results: In only 2 of 12 P3HR-1-EBV-inoculated rabbits, EBV-DNA was detected in peripheral blood mononuclear cells (PBMCs). BamHI M rightward reading frame (BMRF)-1, EBNA-1 and BamHI Z leftward reading frame (BZLF)-1-mRNA were intermittently expressed in PBMCs. In 1 infected rabbit with continuous detection of EBV-DNA in PBMCs, many EBER-1-positive lymphocytes were observed in germinal centers and/or marginal zones in some follicles of the appendix, and for the first time a lymphocyte with EBER-1 expression infiltrating in the squamous cell layer of the tonsils was found. The other rabbit with a transient detection of EBV-DNA in PBMCs had no EBER-1-positive lymphocytes in the tissues examined. Few EBER-1-positive lymphocytes were detected in some rabbits without detection of EBV-DNA in PBMCs. Conclusions: P3HR-1-EBV showed less efficient infection in rabbits than EBV from the B95-8 cell line. However, a P3HR-1-EBV-inoculated animal model is meaningful because this is the first study of EBNA-2 function on in vivo EBV infection and it demonstrated the in vivo infectivity with lytic-type infection by EBNA-2-deleted EBV. Copyright (c) 2013 S. Karger AG, Basel
  • Kaoru Kato; Hitoshi Sano; Keiko Nagata; Hirotsugu Sugihara; Kyosuke Kanai; Satoshi Kuwamoto; Masako Kato; Ichiro Murakami; Kazuhiko Hayashi
    Journal of Vaccines and Vaccination 3 (6) 2157-7560 2012/10 [Refereed]
     
    Epstein-Barr virus (EBV) is a ubiquitous herpes virus that usually infects humans asymptomatically, occasionally inducing various EBV-associated diseases, including infectious mononucleosis (IM), chronic active EBV infection, and different types of malignant tumors. A history of IM is associated with a 3-fold increased risk for subsequent EBV-related Hodgkin lymphoma. In immunocompromised individuals or transplant patients, EBV presents a high risk for morbidity and mortality, although prophylactic vaccination against EBV might contribute to reduce this risk. In this study, we used a rabbit EBV infection model to determine whether vaccination with synthesized peptides based on gp350/220 sequences could effectively prevent EBV infection or decrease the rate or degree of EBV infection. Six rabbits vaccinated with EBV gp350-peptides and four controls were challenged with a minimum dose of EBV infection EBV-DNAs or EBV-RNAs were detected in 5/6 and 4/4 rabbits, respectively. This suggested that a gp350-peptide vaccine could not prevent primary EBV infections in rabbits and indicated the presence of EBV infection routes or mechanisms in rabbits other than the gp350-CD21 interaction observed in EBV infection of human B-cells. However, this vaccine probably has the efficacy to control the viral loads in inoculated rabbits, because 5 out of 6 vaccinated rabbits showed no detectable EBV-DNA in their blood and either no or only few EBER-1-positive lymphocytes in the lymphoid tissues. This vaccine was immunogenic however, developing other improved vaccines or adjuvants will be necessary to reduce EBV-related diseases. © 2012 Kato K, et al.
  • Kyosuke Kanai; Souichi Yamada; Yumiko Yamamoto; Yoshiko Fukui; Ichiro Kurane; Naoki Inoue
    JOURNAL OF GENERAL VIROLOGY SOC GENERAL MICROBIOLOGY 92 (Pt 5) 1005 - 1020 0022-1317 2011/05 [Refereed]
     
    Congenital infection by human cytomegalovirus (HCMV) is a major cause of birth defects and developmental abnormalities. Since guinea pig cytomegalovirus (GPCMV) crosses the placenta and causes infection in utero, GPCMV models are useful for studies of the mechanisms of transplacental transmission. During our characterization of a genomic locus required for GPCMV dissemination in animals, we found that the nucleotide sequence in and around the nearby immediate early genes in our lineage of GPCMV strain 22122 [designated GPCMV (ATCC-P5)] showed clear differences from that reported previously for the same strain [designated GPCMV (UMN)] passaged extensively in vitro. Since in vitro passaging of HCMV is known to result in genetic alterations, especially in the UL128-UL131A locus, and loss of growth ability in particular cell types, in this study we determined the complete genome sequence of GPCMV (ATCC-P5), which grows efficiently in animals. A total of 359 differences were identified between the genome. sequences of GPCMV (UMN) and GPCMV (ATCC-P5), and these resulted in structural differences in 29 protein-encoding regions. In addition, some genes predicted from our analysis but not from GPCMV (UMN) are well conserved among cytomegaloviruses. An additional 18 passages of GPCMV (ATCC-P5) in vitro generated no further marked alterations in these genes or in the locus corresponding to the HCMV UL128-UL131A. Our analyses indicate that the published sequence of GPCMV (UMN) contains a substantial number of sequencing errors and, possibly, some mutations resulting from a long history of passaging in vitro. Our re-evaluation of the genetic content of GPCMV will provide a solid foundation for future studies.
  • Satoshi Kuwamoto; Hiromi Higaki; Kyosuke Kanai; Takeshi Iwasaki; Hitoshi Sano; Keiko Nagata; Kaoru Kato; Masako Kato; Ichiro Murakami; Yasushi Horie; Osamu Yamamoto; Kazuhiko Hayashi
    HUMAN PATHOLOGY W B SAUNDERS CO-ELSEVIER INC 42 (5) 632 - 640 0046-8177 2011/05 [Refereed]
     
    Recently, it has been shown that approximately 80% of Merkel cell carcinomas harbor a novel polyomavirus named Merkel cell polyomavirus, thought to be a carcinogenic agent. However, it is not fully elucidated whether Merkel cell carcinomas differ with regard to the presence or absence of Merkel cell polyomavirus. To address this, we investigated morphologic differences between Merkel cell polyomavirus positive and negative Merkel cell carcinomas by morphometry. Using polymerase chain reaction and real-time quantitative polymerase chain reaction, Merkel cell polyomavirus was detected in 20 (77%) of 26 Merkel cell carcinoma cases, including 4 Merkel cell carcinomas combined with squamous cell carcinomas. Interestingly, Merkel cell polyomavirus was detected only in ordinary (pure) Merkel cell carcinomas; none of the 4 combined Merkel cell carcinomas + squamous cell carcinomas was positive for Merkel cell polyomavirus (P = .001). Morphometric analyses revealed that Merkel cell polyomavirus negative Merkel cell carcinomas had more irregular nuclei (P < .001) and more abundant cytoplasm (P = .001) than Merkel cell polyomavirus positive Merkel cell carcinomas, which had uniform round nuclei and scant cytoplasm. Reliability of the morphometry was confirmed using intraobserver and interobserver reliability tests. These results demonstrated statistically significant differences in tumor cell morphology between Merkel cell polyomavirus positive and negative Merkel cell carcinomas and reconfirmed the absence of Merkel cell polyomavirus in combined tumors. Furthermore, the results strongly suggest fundamental biological differences between Merkel cell polyomavirus positive and negative Merkel cell carcinomas, supporting that Merkel cell polyomavirus plays an important role in the pathogenesis of Merkel cell polyomavirus positive Merkel cell carcinoma. (C) 2011 Elsevier Inc. All rights reserved.
  • Keiko Nagata; Shuji Fukata; Kyosuke Kanai; Yukio Satoh; Takaya Segawa; Satoshi Kuwamoto; Hirotsugu Sugihara; Masako Kato; Ichiro Murakami; Kazuhiko Hayashi; Takeshi Sairenji
    VIRAL IMMUNOLOGY MARY ANN LIEBERT, INC 24 (2) 143 - 149 0882-8245 2011/04 [Refereed]
     
    In Graves' disease, the IgG class autoantibody against thyrotropin receptor (TRAb) is produced excessively and induces hyperthyroidism. Epstein-Barr virus (EBV) is one of the human herpesviruses that persists for life, mainly in B lymphocytes, and is occasionally reactivated. Therefore, EBV may affect the antibody production of B lymphocytes that would normally produce TRAb. The purpose of the present study was to evaluate the association of EBV reactivation with the etiology of Graves' disease. Serum levels of EBV antibodies and IgE were determined by ELISA. TRAb levels were determined by radioreceptor assay. We performed in-situ hybridization (ISH) of EBV-encoded small RNA (EBER)1 on the thyroid tissue of one of our patients. In Graves' disease patients with TRAb levels >= 10%, EA antibody levels, which indicate EBV reactivation, were moderately but significantly correlated with the levels of TRAb, and weakly but significantly correlated with IgE. EBER1-ISH revealed that one of our patients had EBV-infected lymphocytes infiltrating the thyroid gland. EBV reactivation may stimulate antibody-producing B lymphocytes predisposed to make TRAb, and this may contribute to or exacerbate the disease.
  • Kyosuke Kanai; Kaoru Kato; Hitoshi Sano; Keiko Nagata; Keisuke Okuno; Satoshi Kuwamoto; Hiromi Higaki; Hirotsugu Sugihara; Masako Kato; Ichiro Murakami; Kazuhiko Hayashi
    INTERVIROLOGY KARGER 54 (1) 17 - 24 0300-5526 2011 [Refereed]
     
    Most humans become lifelong carriers of Epstein-Barr virus (EBV) by adulthood. Primary EBV infection in adolescents causes infectious mononucleosis. EBV infection is associated with various diseases, neoplasms and hematological disorders. Recently, we reported that EBV can infect rabbits by intravenous, intranasal and/or peroral inoculation, which caused primary EBV infection in rabbits with heterogeneous host reactions. Some rabbits showed chronic and lifelong EBV infection with hemophagocytosis. In this study, to reveal detailed mechanisms in rabbit EBV infection, an in vitro investigation was performed. We elucidated that: (1) EBV can infect rabbit peripheral blood mononuclear cells and splenic lymphocytes in vitro, because EBV gene expressions were confirmed. (2) It is highly likely that the B cell is the main target cell of rabbit EBV infection and is immortalized similar to humans. (3) CD8+ T cells increased in the rabbit in vivo model after EBV inoculation, whereas an increase of B cells occurred after their transient decrease. These data suggest that EBV-infected B cells were proliferated, while CD8+ T cells increased to recognize and kill them. This system may explain the paths of rabbit EBV infection and host reaction, simulating human EBV infection. In vitro studies will be helpful to reveal the pathogenesis of rabbit EBV infection and EBV-associated diseases. Copyright (c) 2010 S. Karger AG, Basel
  • Kanai K; Yamada S; Inoue N
    Uirusu 60 (2) 197 - 207 0042-6857 2010/12
  • Kyosuke Kanai; Kazuaki Takashima; Keisuke Okuno; Kaoru Kato; Hitoshi Sano; Satoshi Kuwamoto; Hiromi Higaki; Keiko Nagata; Hirotsugu Sugihara; Masako Kato; Ichiro Murakami; Kazuhiko Hayashi
    VIRUS RESEARCH ELSEVIER SCIENCE BV 153 (1) 172 - 178 0168-1702 2010/10 [Refereed]
     
    Most humans become lifelong carriers of Epstein-Barr virus (EBV) by adulthood. Primary EBV infection in adolescents causes in one to two-third of cases infectious mononucleosis. EBV infection is associated with various diseases, neoplasms and hematological disorders. Recently we reported that EBV can infect rabbits frequently by intravenous, intranasal or/and peroral inoculation, which caused primary EBV infection in rabbits with heterogeneous host reactions. Here we presented follow up data that of six primary EBV-infected rabbits out of seven inoculated intravenously with EBV, two out of six EBV-infected rabbits showed lifetime EBV infection. (1) EBV-DNA were detected in blood through life. (2) High antibody titers against EA-D were maintained over 1000 days. (3) A focal mass lesion was transiently observed by ultrasonography in the spleen of one rabbit. (4) Two lifelong EBV-detected rabbits died on day 1522 or 1400, and autopsy revealed proliferation of lymphocytes expressing EBER1 or LMP1 accompanied with mild hemophagocytosis in the spleen or lymph nodes. We hypothesized some EBV-infected rabbits could not eliminate EBV for life and showed somewhat similar features to persistent EBV infection, mild CAEBV and/or mild sublethal hemophagocytosis. These lifelong EBV-infected rabbits might be a new useful animal model for studying lifelong persistent EBV infection, taking place in almost all adults. (C) 2010 Elsevier B.V. All rights reserved.
  • Keisuke Okuno; Kazuaki Takashima; Kyosuke Kanai; Makoto Ohashi; Ryosuke Hyuga; Hirotsugu Sugihara; Satoshi Kuwamoto; Masako Kato; Hitoshi Sano; Takeshi Sairenji; Susumu Kanzaki; Kazuhiko Hayashi
    JOURNAL OF MEDICAL VIROLOGY WILEY-LISS 82 (6) 977 - 986 0146-6615 2010/06 [Refereed]
     
    Epstein Barr virus (EBV) is spread universally in humans, and it causes infectious mononucleosis and sometimes induces serious EBV-associated disease. The detailed mechanism of primary infection in humans has remained unclear, because it is difficult to examine the dynamics of EBV in vivo. In this study, a natural EBV-infection rabbit model by intranasal or peroral inoculation is described. Ten male rabbits were examined for EBV-DNA or mRNA expression and anti-EBV antibodies in blood. Four of 10 rabbits showed the evidence of EBV infection; detection of EBV-DNA or EBV-related genes mRNA in peripheral blood mononuclear cells, increased EBV antibodies in the plasma, and the presence of lymphocytes expressing EBER1 and EBV-related gene proteins in the lymphoid tissues of a rabbit. Three of four infected rabbits were detected transiently EBV-DNA and/or mRNA of EBV-related genes such as EBNA1, EBNA2, BZLF1, and EA in blood, while in one of four, EBV-DNA and/or mRNA were detected for more than 200 days after viral inoculation The level of EA-IgG increased and its level was maintained in all infected rabbits, whereas those of VCA-IgM and VCA-IgG increased transiently, and EBNA-IgG was not elevated. Pathological examination of a rabbit infected transiently revealed some scattered lymphocytes expressing EBER1, LMP1, and EBNA2 in the spleen and lymph nodes. EA expression was also observed in the spleen. These findings suggest that EBV can infect the rabbit by the intranasal or peroral route, and that this rabbit model is useful for examining the pathophysiology of natural primary EBV infection in humans. J. Med. Virol. 82:977-986, 2010. (C) 2010 Wiley-Liss. Inc
  • Okuno K; Horie Y; Kanai K; Kato M; Kuwamoto S; Okazaki T; Hayashi K
    Journal of clinical and experimental hematopathology : JCEH 49 (1) 45 - 51 1346-4280 2009/05 [Refereed]
     
    Post-transplant lymphoproliferative disorder (PTLD) is one of the most important complications of solid organ transplantation or hematopoietic stem cell transplantation. Most PTLDs are associated with Epstein-Barr virus (EBV) infection. Although post-transplant Hodgkin lymphoma (HL) is included in PTLD, there have been no studies in the literature on adult cases of post-transplant HL after cord blood stem cell transplantation (CBSCT). This is due to the fact that EBV infection of cord blood cells usually does not occur, and EBV-infected lymphocytes of the recipient should be eradicated by preconditioning therapy. We report a 26-year-old woman case of post-transplant HL, which occurred after CBSCT for relapsed acute lymphoblastic leukemia. Three years and eight months after CBSCT, the enlarged cervical lymph node was histologically diagnosed as EBV associated post-transplant HL, which showed immunophenotypes of classical HL and latency type II EBV infection. She underwent chemotherapy, and has survived 4 years and 6 months after CBSCT. Differential diagnosis of post-transplant HL with good prognosis and HL-like PTLD with aggressive behavior is important, and immunohistochemical methods were useful and essential for it. The source of EBV associated HL in this case will be discussed.
  • Asako Kawaguchi; Kyosuke Kanai; Yukio Satoh; Chizu Touge; Keiko Nagata; Takeshi Sairenji; Yoshitsugu Inoue
    VIRUS GENES SPRINGER 38 (2) 215 - 223 0920-8569 2009/04 [Refereed]
     
    To study variations of Epstein-Barr virus (EBV), we analyzed the gp350/220 gene for several cell lines and Japanese wild isolates using direct sequencing. The N-terminal region was highly conserved in all EBVs except for Jijoye/P3HR-1 and a few isolates. The variation of the region coincided with EBV types A and B (also referred to as types 1 and 2) and were, respectively, designated as the types a and b. The type A/a was detected in most Japanese cell lines and wild isolates, and was classified as China1 type with latent membrane protein (LMP) 1 gene. The type B/b was detected in only a few wild isolates with the Med and China2 types. The C-terminus had more diversity than the N-terminus and lacked the divergence between types A/a and B/b. The phylogenetic analyses of the gp350/220 and LMP1 genes may suggest a mode of EBV evolution into types A/a and B/b and then to LMP1 subtypes.
  • 長島賢典; 高島一昭; 安藤健介; 奥野啓介; 金井亨輔; 西連寺剛; 林一彦
    米子医学雑誌 59 (2) 53 - 61 0044-0558 2008/03 [Refereed]
  • 長田佳子; 大橋誠; 金井亨輔; 長嶋賢典; 西連寺剛
    日本内分泌学会雑誌 83 (4) 1077 - 1077 0029-0661 2008/03
  • Kyosuke Kanai; Yukio Satoh; Hiroyuki Yamanaka; Asako Kawaguchi; Kazutaka Horie; Kenji Sugata; Yoshiko Hoshikawa; Tetsutaro Sata; Takeshi Sairenji
    VIRUS GENES SPRINGER 35 (3) 563 - 569 0920-8569 2007/12 [Refereed]
     
    A gene of the Epstein-Barr virus (EBV), BamHI-C fragment rightward reading frame I (BCRF1), codes viral interleukin-10 (vIL-10), which is a close homolog to human IL-10. EBV strain variations are known at EBV latent membrane protein 1 (LMP1), and the distinct forms of LMP1 have been identified. In order to further elucidate the variations of EBV strains, the BCRF1 (vlL-10) gene was analyzed using PCR-direct sequencing in African Burkitt's lymphoma (BL) cell lines Raji, P3HR-1, EB1 and Daudi, Japanese BL cell line Akata, lymphoblastoid cell line OB and 22 wild EBV isolates from eight gastric carcinoma tissues and 14 throat washes. We found only five variations of the vIL-10 gene in them with one silent mutation and three non-silent mutations. Raji had no mutation to the prototype gene of B95-8. EB1 and P3HR-1 had non-silent mutations in the sequences leading to the arginine/serine and threonine/proline interchanges at residues 4 and 166, respectively. The silent mutation was detected at valine 102 in Daudi and also in the Japanese cell lines Akata, OB and 20 (90.9%) of the wild EBV isolates. The type of variations in the vIL-10 gene had a common relationship with those in the LMP1 gene. All of the variants of valine 102 had Chinal-type LMP1 sequences except for Daudi with Med-type LMP1 and other minorities with B95-8 type LMP1. The conservativeness of vIL-10 with a few variations suggests the indispensability of the vIL-10 gene in EBV and that the variations of the vIL-10 gene may depend upon the geographical prevalence of the EBV strains. This is the first report regarding the variations of the vIL-10 gene in cell lines and other wild isolates.
  • Kyosuke Kanai; Yukio Satoh; Yuriko Saiki; Haruo Ohtani; Takeshi Sairenji
    VIRUS GENES SPRINGER 34 (1) 55 - 61 0920-8569 2007/01 [Refereed]
     
    Sequence variations in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene have been described in many EBV-isolates. To characterize the genomic relationship between Japanese EBV and the EBV isolates of other countries, we analyzed the LMP1 nucleotide sequences in EBV positive cell lines and clinical specimens, including five African Burkitt's lymphoma (BL) cell lines, a Japanese BL cell line, a B-lymphoblastoid cell line, a nasopharyngeal carcinoma hybrid cell line, six gastric carcinoma tissues, two peripheral blood mononuclear cells, and a B95-8 cell line, which contained the prototype EBV genome. We determined the C-terminal nucleotide sequences of LMP1 by PCR-direct sequencing analysis and characterized the sequence variation of Japanese isolates, made a phylogenetic tree from the sequence patterns of LMP1 by a neighbor-joining method. The results indicate that the Japanese EBV isolates are greatly different from the African BL isolates but are closely related to the China 1, which is a strain of Chinese EBV isolates.
  • Yun Wang; Kyosuke Kanai; Yukio Satoh; Bing Luo; Takeshi Sairenji
    INTERVIROLOGY KARGER 50 (3) 229 - 236 0300-5526 2007 [Refereed]
     
    Objectives: To elucidate variations of latent membrane protein 1 (LMP1) in Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) and explore the LMP1 variations of neighboring countries, China and Japan. Methods: In 12 and 8 EBVaGCs from eastern China and Japan, respectively, the C-termini of LMP1 were analyzed using PCR and sequencing. The sequences were compared with previously published strains and were characterized on a phylogenetic tree. The difference between Chinese and Japanese isolates was characterized. Results: Ten of 12 Chinese GC isolates (83.3%) and all of the 8 (100%) Japanese GC isolates belonged to the China 1 strain. Also, B95-8 type isolates were found in 2 of 12 Chinese GC. In the 18 China 1 type isolates, additional mutations outside the signature sequence changes were found. All Japanese isolates (100%) had two or more additional mutations, whereas only 5 of 10 (50%) Chinese isolates had two or more additional mutations. The difference was statistically significant (p = 0.0359). Conclusions: China 1 is the dominant strain in GC from eastern China and Japan. The similarity to that of nasopharyngeal carcinoma (NPC) from China supports the view that China 1 strain represents a geographic-associated polymorphism rather than an NPC-associated polymorphism. Japanese isolates show more mutations than Chinese isolates, suggesting a geographic difference between Chinese and Japanese isolates in GC.

MISC

Research Grants & Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : KANAI Kyosuke
     
    EB virus causes infectious mononucleosis (IM) with hepatitis in primary infected adults, but the mechanism of pathogenesis is not well understood. We have previously shown that intestinal bacterial products may be associated with the hepatitis using MHV68-infected mice. In the present study, we evaluated the impact of lypopolysaccaride (LPS) and peptideglycan (PDG) as candidates of intestinal bacterial products. As the result, Neutralizing antibodies to TLR4, LPS receptor, suppressed hepatitis. However, neutralizing antibodies to TLR2, PDG receptor, did not inhibit. Furthermore, the TLR4 inhibitor C34 suppressed hepatitis. These results suggest that the LPS-TLR4 pathway may be associated with the hepatitis in IM.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2015/04 -2018/03 
    Author : Kageyama Seiji; Kanai Kyosuke; Tsuneki Akeno
     
    The drug resistance rate was estimated to be 9.2% among the 2,629 referred samples from patients receiving antiretroviral treatment in 2015-2016 in the Philippines. Thirty-seven HIV strains were isolated from 142 HIV-positive serum/plasma samples of patients harboring drug-resistant strains with detectable viral load after a 12-month treatment of drug-naive patients. The replication competence was then assessed by re-inoculation into seronegative peripheral blood mononuclear cells in vitro. The viral yield correlated with the plasma viral load. However, more progeny viruses were produced after the in vitro re-inoculation than the corresponding plasma viral load. Intrinsic replication competence could be assessed by the in vitro re-inoculation system, but not be estimated from in vivo plasma viral load. These results suggest that the replication competence of HIV become an important marker especially when a drug regimen should be altered.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : YOSHIE Osamu; NAKAYAMA Takashi; TSUNODA Ikuo
     
    CCL28 is expressed in the mucosal tissues and to attract IgA-antibody secreting cells (IgA-ASCs) via CCR10. CCL28 has an antimicrobial activity against microbes in vitro. However, in vivo evidence for the role of CCL28 in the mucosal immunity remains scanty. We generated CCL28-deficient mice and demonstrated that CCL28-deficient mice showed reduced numbers and altered distribution of IgA-ASCs in the colon. The IgA contents in the fecal extracts were low. The average amounts of IgA secreted by a single IgA-ASC isolated from the lamina propria of the colon was reduced. Furthermore, the 16S rRNA sequencing analysis of feces revealed an increase of the Class Bacilli. Consistent with the low IgA production and altered microbiota in the colon, CCL28-deficient mice had aggravated colitis upon treatment with dextran sulfate, which was ameliorated by oral antibiotics. Therefore, CCL28 has an role in the mucosal immunity of the colon as a chemoattractant with a direct antimicrobial activity.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2012/04 -2014/03 
    Author : KANAI Kyosuke
     
    Cell-mediated immunity (CMI) is thought to be crucial for protection against Varicella-Zoster virus (VZV) infection. We screened for major CMI-inducible VZV protein by Enzyme-Linked Immuno-Spot (ELISPOT) assay. Because already-known CMI antigens of other herpesviruses are localized in cell nuclei, nuclear distributed VZV proteins were examined as candidates for major CMI antigens. VZV protein(s) inducing CMI were searched among peripheral blood mononuclear cell specimens derived from healthy volunteers. As the result, ORF8, 61 and 66 induced CMI in more than 80% of specimens. About ORF61, epitope sequences were identified using oligo peptides. Our findings contribute to establish a new standardized CMI-quantification system.
  • 日本学術振興会：科学研究費助成事業 特別研究員奨励費
    Date (from‐to) : 2009 -2009 
    Author : 金井 亨輔
     
    EBウイルス関連疾患の病態・病因解明および治療法開発のため、EBウイルス感染モデル動物による実験系の構築を目的とした研究が、様々な動物種を用いてなされている。ウサギ(Japanese White種)は有望な動物種候補のひとつであり、EBウイルスが静脈注射により感染することがこれまでに知られている。EBウイルス感染ウサギは一過性の脾腫、末梢血中でのEBV-DNAの検出がなされ、これらの症状は次第に緩和する。本研究は、EBウイルス感染ウサギにおけるEBウイルスの感染状態のより詳細な解析を目的とした以下の検討を行った. EBウイルス感染ウサギの末梢血におけるEBV遺伝子発現の経時的解析を行ったところ、EBウイルス遺伝子の発現が長期間継続して確認された。また、死後の剖検で脾臓からウイルス遺伝子の発現が確認されただけでなく、血球貪食像や異型リンパ球が認められた。このことから、ウサギにおけるEBウイルス感染症状は一時的なものだけではなく、終生継続していることが示され、またEBウイルスが長期的な影響を宿主に及ぼしている可能性が示唆された。 試験管内における末梢血リンパ球、脾臓リンパ球のEBV感染の解析を行ったところ、EBウイルス遺伝子発現が確認され、試験管内でも感染が成立することが示された。また、これまで検討に用いられたB95-8株EBVとは異なるEBV株であるP3HR-1株EBVを用いてウサギへの感染の検討を行い、P3HR-1株EBVでもウサギへの感染が成立することが明らかにした。これらの事実は、ウサギを用いてのEBウイルスの病態・病因解析が、試験管内で行える可能性を示す。 本研究により得られた事実は、EBウイルス感染モデル動物としてのウサギの有用性が改めて支持した。今後、これらEBウイルス感染ウサギが、EBウイルス関連疾患の病態・病因の解析に役立つことが期待される。

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