Biomedical subjects

Shigeru Morikawa

Publications and source records attributed to Shigeru Morikawa.

68 records · Page 4Linked to original sources

PubMed2002

Genetic diversity of the M RNA segment among Crimean-Congo hemorrhagic fever virus isolates in China.

The complete nucleotide sequences of the medium (M) segment of seven Chinese isolates of Crimean-Congo hemorrhagic fever (CCHF) virus were determined. The M-segment RNA of CCHF virus comprises 5356-5377 nucleotides depending on the isolate and encodes a protein comprising 1689-1697 amino acids in a viral complementary sense. Phylogenetic analysis of the M segment showed that the Chinese CCHF virus isolates were clustered into three groups, one of which was more closely related to a Nigerian isolate. Pairwise comparison of a precursor protein showed that amino-terminal regions comprising 250 amino acids were extraordinary heterogeneous, with a 22.4% identity in amino acids being observed between the most distantly related isolates. Since all the viruses were isolated from 1966 to 1988 within a restricted area in the Xinjiang Autonomous Region in western China, the results indicate that a multisource virus population is endemic in this region.

Animals↗

PubMed2002

Chimeric recombinant hepatitis E virus-like particles as an oral vaccine vehicle presenting foreign epitopes.

Many viral and bacterial pathogens establish infections through mucosal surfaces in their initial stage. However, only a few nonreplicating molecules successfully induce strong mucosal immune reaction without the addition of adjuvants by oral administration. To overcome this difficulty, we investigated whether hepatitis E virus-like particles (HEV-VLPs) could be utilized as a carrier molecule for foreign antigenic epitopes and to stimulate mucosal immunity without the need for adjuvants. To accomplish this goal, we incorporated a B cell epitope tag, consisting of 11 amino acids at the C-terminal of HEV-VLP. The chimeric VLP showed morphology similar to that of the mature HEV virion and VLP. The inserted epitope was reactive with a specific monoclonal antibody in the VLP form, suggesting that it was exposed on the surface of the VLP. After oral administration without adjuvant, this chimeric HEV induced significant levels of specific IgG and IgA to both the inserted epitope and HEV-VLP in intestinal secretions. These humoral immune responses were observed as early as 2 weeks after the first immunization. These results suggest the potential of HEV-VLP as a mucosal vaccine carrier vehicle for the presentation of antigenic epitopes through oral administration.

Administration, Oral↗

PubMed2002

Human T-cell leukemia virus type I Tax transactivates the matrix metalloproteinase-9 gene: potential role in mediating adult T-cell leukemia invasiveness.

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and of tropical spastic paraparesis/HTLV-I-associated myelopathy. Infiltration of various tissues by circulating leukemic cells is a characteristic of ATL. Matrix metalloproteinases (MMPs), which mediate the degradation of the basement membrane and extracellular matrix, play an important role in metastasis and tumor cell dissemination. The aim of this study was to explore whether expression of MMP-2 and MMP-9 was deregulated by HTLV-I infection. The data showed that HTLV-I-infected T-cell lines expressed high levels of MMP-9 compared with uninfected T-cell lines. In contrast, the levels of the related MMP-2 were not significantly altered by HTLV-I infection. In addition, the elevated expression of MMP-9 in HTLV-I-infected cells was attributable to the action of the viral transactivator protein Tax. The results show that Tax can activate the MMP-9 promoter and induce MMP-9 expression in T cells, indicating that the constitutive expression of MMP-9 in virus-infected cell lines is at least in part mediated by Tax. Activation of the MMP-9 promoter by Tax occurs mainly through the action of NF-kappaB and SP-1. The biologic significance of these observations was validated by the following 2 findings: MMP-9 expression was increased in primary ATL cells, and plasma MMP-9 levels were elevated in ATL patients. In addition, plasma levels of MMP-9 correlated with organ involvement in ATL patients. Together these data suggest that overexpression of MMP-9 in HTLV-I- infected cells may be in part responsible for the invasiveness of ATL cells.

Enzyme Induction↗

PubMed2002

Bone marrow transplantation in a child with Wiskott-Aldrich syndrome latently infected with acyclovir-resistant (ACV(r)) herpes simplex virus type 1: emergence of foscarnet-resistant virus originating from the ACV(r) virus.

A human leukocyte antigen (HLA)-matched unrelated bone marrow transplantation (BMT) was performed in a 13-year-old patient with the congenital immunodeficiency syndrome, Wiskott-Aldrich syndrome. The patient had a history of acyclovir (ACV)-resistant (ACV(r)) herpes simplex virus type 1 (HSV-1) infections prior to BMT. After BMT, the skin lesions caused by HSV-1 relapsed on the face and genito-anal areas. Ganciclovir (GCV) therapy was initiated, but the mucocutaneous lesions worsened. An HSV-1 isolate recovered from the lesions during this episode was resistant to both ACV and GCV. The ACV(r) isolate was confirmed to have the same mutation in the viral thymidine kinase (TK) gene as that of the previously isolated ACV(r) isolates from the patient. After treatment switch to foscarnet (PFA), there was a satisfactory remission but not a complete recovery. Although the mucocutaneous lesions improved, a PFA-resistant (PFA(r)) HSV-1 was isolated 1 month after the start of PFA therapy. The PFA(r) HSV-1 isolate coded for the same mutation in the viral TK gene as the ACV(r) HSV-1 isolates. Furthermore, the PFA(r) isolate also expressed a mutated viral DNA polymerase (DNA pol) with an amino acid (Gly) substitution for Val at position 715. This is the first report on the clinical course of a BMT-associated ACV(r) HSV-1 infection that subsequently developed resistance to foscarnet as well.

Acyclovir↗

PubMed2002

Importance of C-terminus of herpes simplex virus type 1 thymidine kinase for maintaining thymidine kinase and acyclovir-phosphorylation activities.

We previously isolated an acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1), strain TAR, from a child with Wiskott-Aldrich syndrome. An acyclovir-sensitive HSV-1, strain TAS, had been isolated from the same patient before the isolation of HSV-1 TAR. The TK protein of ACV-sensitive HSV-1 TAS was composed of 376 amino acids, while that of HSV-1 TAR was composed of 407 amino acids with altered amino acid residue between positions 355-407. The elongation of TK was caused by a single nucleotide deletion of cytosine from a homopolymer stretch of 4 cytosine residues between positions 1061-1064. There was no viral TK activity in HSV-1 TAR-infected Vero cells, indicating the importance of the C-terminal portion of TK protein from positions 355-376. Recombinant TK polypeptides with amino acid deletions at the C-terminus were prepared, and TK and ACV-phosphorylation activities were examined. Deletion of 5 and 6 amino acids from the C-terminus of the TK polypeptide of HSV-1 TAS resulted in a reduction of TK activity by approximately 75% and 100%, respectively. These mutant TK polypeptides did not phosphorylate ACV. These results indicate that amino acid residues from positions 371-376 in the C-terminal portion of HSV-1 TK protein are essential for keeping TK and ACV-phosphorylation activities.

Acyclovir↗

PubMed2002

Genotypic and phenotypic characterization of the thymidine kinase of ACV-resistant HSV-1 derived from an acyclovir-sensitive herpes simplex virus type 1 strain.

Twenty-four strains of acyclovir (ACV)-resistant (ACV(r)) herpes simplex virus type 1 (HSV-1) were generated from the HSV-1 TAS strain by exposure to ACV, and the genotype and phenotype of the thymidine kinase (TK) from these mutants were analyzed. The TK polypeptide of the ACV(r) HSV-1 strains was examined by Western blot using an anti-HSV-1 TK rabbit serum. The sensitivity of each strain to ACV, foscarnet and cidofovir (CDV) was also determined. A single guanine (G) insertion or a single cytosine (C) deletion was detected in 12 of the 24 ACV(r) strains at the G or C homopolymer stretches within the TK gene. Genotypic analysis predicted that two thirds of the ACV(r) HSV-1 strains expressed truncated TK polypeptides, while one third expressed viral TK polypeptide with a single amino acid substitution at various sites. Western blot abnormalities in the viral TK polypeptides were identified in 21 ACV(r) strains. There was an inverse correlation between the susceptibility of the HSV-1 mutant strains to ACV and that to CDV. Nucleotide sequencing of the TK gene and Western blot analysis of the viral TK polypeptides are considered to be one of the methods for predicting virus sensitivity to ACV and CDV.

Acyclovir↗

PubMed2002

Characterization of granulocyte colony-stimulating factor receptor expressed on human lymphocytes.

We have studied the characterization of granulocyte colony-stimulating factor receptor (G-CSFR) in human lymphocytes. About one-third to one-quarter of the B lymphocytes from peripheral B-cell sources displayed G-CSF binding on the two-colour immunofluorescence study. The rate of G-CSFR-expressing (G-CSFR+) B cells was higher in bone marrow and cord blood than in peripheral blood, spleen and tonsil. G-CSFR expression was greater in the surface immunoglobulin D (IgD)-positive (sIgD+) B-cell population, but scarce in the sIgD- B-cell population. In tonsil, G-CSFR+ B cells were present among the cells with naive B and germinal-centre B phenotypes, but those with memory B phenotype were rarely found on triple-colour immunofluorescence analysis. Mitogen-activated, but not resting, T lymphocytes also showed G-CSF binding. Several continuous T- and B-cell lines expressed functional G-CSFR, because the addition of G-CSF enhanced the proliferative response of these cell lines. A sequence analysis of G-CSFR mRNA isoforms obtained from the T and B cells revealed that G-CSFR was derived from class I and class IV mRNA. Our results indicated that G-CSFR was constitutively expressed on the B-cell surface and was inducible in T cells.

B-Lymphocytes↗

PubMed2002

MD41, a novel T helper 0 clone, mediates mast-cell dependent delayed-type hypersensitivity in mice.

In a previous study on mouse, we have shown that delayed-type hypersensitivity (DTH) could be classified into two types according to MC requirement. The first type of DTH could be elicited by sensitization with methylated human serum albumin (MHSA) in complete Freund's adjuvant (CFA) in both wild type and mast-cell deficient (W/Wv) mice. The second type could be elicited by MHSA in incomplete Freund's adjuvant (IFA) sensitization in wild type but not W/Wv mice. While the former was related to classic tuberculin (tbc)-type DTH, the latter appeared to be a novel mast-cell dependent DTH (MD-DTH). In order to investigate the mechanism of MD-DTH, in this study, we generated an effector T-cell clone (MD41) from lymph node cells of MHSA in IFA-sensitized mice and analysed its pattern of cytokine production. Our results from cytokine assays show that following antigen stimulation, MD41 cells produce significant amounts of the T helper 1 (Th1) cytokine interferon-gamma (IFN-gamma) as well as the Th2 cytokines interleukin (IL)-4 and IL-10. In addition, double staining for IL-4 and IFN-gamma revealed that MD41 cells produce both Th1- and Th2-type cytokines simultaneously, which suggest that MD41 represents a Th0 clone rather than a mixture of Th1 and Th2 clones. Adoptive transfer of MD41 cells into wild-type mice resulted in the development of DTH skin reactions similar to those produced by active sensitization, with very similar histological findings. However, DTH skin reactions could not be induced in W/Wv mice unless first reconstituted with normal bone marrow MC (BM-MC). Therefore, our study suggests that in conjunction with tissue MC, MD41, a less-polarized MD-DTH-derived Th0 clone, is capable of developing murine DTH to the same extent as strongly polarized Th1 cells and mediates MD-DTH rather than tbc-type DTH.

Adoptive Transfer↗

PubMed2002

Development of an immunofluorescence method for the detection of antibodies to Ebola virus subtype Reston by the use of recombinant nucleoprotein-expressing HeLa cells.

An indirect immunofluorescent assay (IFA) to detect Ebola virus subtype Reston (EBO-R) antibodies was developed by the use of a HeLa cell line stably expressing EBO-R nucleoprotein (NP). This IFA has a high specificity for the detection of EBO-R IgG antibodies in both hyperimmune rabbit sera and monkey sera collected during an EBO-R outbreak in the Philippines in 1996. Furthermore, this IFA showed a higher sensitivity for the detection of EBO-R antibodies than did the IFA using HeLa cells expressing the NP of Ebola virus subtype Zaire. These results suggest that this new IFA is useful for seroepidemiological studies of EBO-R infection among monkeys.

Animals↗

PubMed2002

Immunofluorescence technique using HeLa cells expressing recombinant nucleoprotein for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus.

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.

Animals↗

PubMed2002

Recombinant nucleoprotein-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus.

The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni(2+) column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previously determined to be antibody positive or negative by immunofluorescence tests on CCHFV-infected Vero E6 cells. We found very good correlation between the two tests: 87% for the positive sera (13 of 15) and 99% for the negative sera (107 of 108). These results indicate that the new IgG ELISA using His-CCHFV rNP has high sensitivity and specificity for detecting CCHFV antibodies. The CCHF patients' sera with high titers reacted only with the NP fragment containing amino acid residues between 201 and 306 in Western blotting. It is known that amino acid homologies are high in this region among various isolates. Thus, it is expected that this ELISA can detect antibodies not only for Chinese strains of CCHFV but also for other strains circulating in the world. These results suggest that the IgG ELISA system developed with the recombinant CCHFV NP is a valuable tool for diagnosis and epidemiological investigations of CCHFV infections.

Antibodies, Viral↗

PubMed2002

Histopathology of natural Ebola virus subtype Reston infection in cynomolgus macaques during the Philippine outbreak in 1996.

We investigated the livers, spleens, kidneys and lungs collected from 24 cynomolgus macaques (Macaca fascicularis) naturally infected with Ebola virus subtype Reston (EBO-R) during the Philippine outbreak in 1996, in order to reveal the histopathologic findings. These macaques showed necrotic hepatocytes with inclusions, slight to massive fibrin deposition in splenic cords, depletion of lymphoid cells in the white pulp of the spleen, and fibrin thrombi in some organs. Immunohistochemical analysis using anti-leukocyte antigen L1 antibody revealed an increase in blood-derived macrophages/monocytes in the livers, kidneys and lungs of EBO-R infected macaques. EBO-R NP antigens were detected in the macrophages/monocytes, endothelial cells and fibroblasts in the liver, spleen, kidney and lung. These results indicate that EBO-R infection is characterized by systemic coagulopathy and an increase in blood-derived macrophages/monocytes in accordance with the EBO-R propagation in macrophages/monocytes.

Animals↗

PubMed2002

The effect of statins on mRNA levels of genes related to inflammation, coagulation, and vascular constriction in HUVEC. Human umbilical vein endothelial cells.

Large-scale clinical trials have demonstrated significant reductions in cardiovascular events following statin therapy. The observed benefit of statin therapy, however, may be greater in these trials than is to be expected from lowering lipid levels alone. In order to clarify the mechanism by which statins prevent cardiovascular events in vascular wall cells, we investigated the changes in gene expression profiles after incubation with atorvastatin or pitavastatin in cultured human umbilical vein endothelial cells using DNA microarrays. Statins affected the expression levels of genes involved in inflammation, coagulation, and vascular constriction. The mRNA levels for interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) decreased after statin treatment. Statins reduced mRNA levels of plasminogen activator inhibitor-1 (PAI-1) and increased the mRNA levels of thrombomodulin. Statins reduced the mRNA levels of endothelin-1 and increased the mRNA levels of nitric oxide synthase-3 (eNOS). These results show that, statins are clinically effective because of their ability to change the gene expression profile of endothelial cells thereby preventing vascular events.

Atorvastatin↗

PubMedDate not supplied

An autopsy case of Pena-Shokeir syndrome: severe retardation of skeletal muscle development compared with neuronal abnormalities.

An infant with multiple joint ankyloses, facial anomalies, and pulmonary hypoplasia, features similar to the phenotype of Pena-Shokeir syndrome, was examined at autopsy. Histological examination of the skeletal muscles revealed many small muscle fibers in a mixed, not group, distribution, although the structure of them was normally arranged. Histochemical assessment of adenosine triphosphatase (ATPase) activity of the iliopsoas muscle demonstrated the failure of the differentiation into type I fibers and the retardation of the skeletal muscle. At the same time, severe pulmonary hypoplasia, which was the likely cause for the retardation of the respiratory system, was found. In contrast to these numerous pathologic changes in the skeletal muscles, no significant abnormalities were observed in the central nervous system except for a somewhat immature external appearance; however, an examination of the spinal cord could not be carried out. Overall, this pattern of pathology suggests the possibility that developmental disorders of the mesenchyme are the primary contributors to the pathogenesis of Pena-Shokeir syndrome, while the immaturity of the central nervous system is involved to a lesser degree.

Abnormalities, Multiple↗