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Biomedical subjects

J Imanishi

Publications and source records attributed to J Imanishi.

At least 19 recordsLinked to original sources
PubMed1999

Biphasic translocation of a 70 kDa heat shock protein in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) reportedly induces the expression of a 70 kDa heat shock protein (hsp70) with no known function in the virus replication cycle. We report here a remarkably specific translocation pattern of hsp70 during HCMV infection of human diploid fibroblasts. Immunofluorescent observation and Western blotting of subcellular fractions revealed nuclear localization of hsp70 early in infection and predominantly cytoplasmic localization of hsp70 late in infection. Treatment of HCMV-infected cells with cycloheximide followed by treatment with actinomycin D allowed virus immediate-early gene expression but inhibited hsp70 nuclear localization. Phosphonoacetic acid and tunicamycin, both of which reportedly inhibit HCMV DNA replication, did not inhibit HCMV-induced nuclear localization of hsp70 but inhibited hsp70 translocation from the nucleus to the cytoplasm. These results indicate a correlation between HCMV multiplication and hsp70 localization, suggesting that hsp70 may play a role in HCMV multiplication.

Biological Transport↗
PubMed1999

Role of gamma interferon in Helicobacter pylori-induced gastric inflammatory responses in a mouse model.

The immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contribution of gamma interferon (IFN-gamma) to the immune responses, especially to the induction of gastric inflammation and to protection from H. pylori infection, was investigated with IFN-gamma gene knockout (IFN-gamma-/-) mice. We first examined the colonizing abilities of eight H. pylori strains with a short-term infection test in order to select H. pylori strains which could colonize the mouse stomach. Only three strains (ATCC 43504, CPY2052, and HPK127) colonized C57BL/6 wild-type mice, although all of the strains except for ATCC 51110 could colonize IFN-gamma-/- mice. The number of H. pylori organisms colonizing the stomach in wild-type mice was lower than that in IFN-gamma-/- mice. Oral immunization with the CPY2052 sonicate and cholera toxin protected against infection with strain CPY2052 in both types of mouse. These findings suggested that IFN-gamma may play a protective role in H. pylori infection, although the degree of its protective ability was estimated to be low. In contrast, in a long-term infection test done to examine the contribution of IFN-gamma to gastric inflammation, CPY2052-infected wild-type mice developed a severe infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium 15 months after infection, whereas CPY2052-infected IFN-gamma-/- mice showed no inflammatory symptoms. This result clearly demonstrated that IFN-gamma plays an important role in the induction of gastric inflammation caused by H. pylori infection.

Adjuvants, Immunologic↗
PubMed1998

Gene transfer in vitro and in vivo with Epstein-Barr virus-based episomal vector results in markedly high transient expression in rodent cells.

The EBV-based plasmid vectors, which carry oriP and EBNA1 gene from EBV genome, can be retained in the nucleus and replicate in human cells. Rodent cells are not permissive for the EBV plasmids, in terms of the plasmid replication. However, the EBV vector facilitates not only the long term maintenance of the plasmid but also high level gene expression at a transient phase after transfection. It has not been elucidated if rodent cells show this high level transient expression. We demonstrate that rodent cells transfected with an EBV vector expressed a marker gene more intensively than those with a conventional plasmid vector did. The high marker gene expression was also seen in rat myocardium injected in vivo with the EBV plasmid. The present data indicate that the EBNA1-oriP system functions in "non-permissive" rodent cells at transient phase, and may require different cellular factor(s) for the transient expression and plasmid replication.

Animals↗
PubMed1998

Successful transfer of ADA gene in vitro into human peripheral blood CD34+ cells by transfecting EBV-based episomal vectors.

We report a novel non-viral system for transfecting human immature hematopoietic cells in vitro. Epstein-Barr virus (EBV)-based episomal vectors carrying human adenosine deaminase (ADA) gene cDNA were transfected by electroporation into human peripheral blood (PB) CD34+ cells. The transgene-specific mRNA were detected from 37 to 100% of CFU-c (colony forming unit in culture) colonies derived from the transfected cells. A two-fold increase in enzyme activity was also found. These results indicate the successful transfer and expression of genes in human immature hematopoietic cells using the EBV-based episomal vector system.

Adenosine Deaminase↗
PubMed1998

Fas antigen expression and apoptosis of lymphocytes in macaques infected with simian immunodeficiency virus strain mac.

To investigate the role of apoptosis in the pathogenesis of HIV infection we used macaques infected with simian immunodeficiency virus (SIV) as a primate model and examined the characteristics of the apoptosis of lymphocytes in SIV mac-infected macaques. In vitro apoptosis was more strongly induced in peripheral blood mononuclear cells (PBMC) from SIV mac239-infected macaques than those from uninfected controls. We found that the frequency of Fas antigen-positive cells was higher in PBMC from SIV mac-infected macaques than from uninfected controls, and in vitro apoptosis of PBMC was suppressed by an inhibitor of the interleukin-1 beta converting enzyme (ICE) family proteases. In biopsied lymph nodes, the number of apoptotic nuclei in T cell-dependent areas was higher in SIV mac-infected macaques than in uninfected controls. A higher number of apoptotic nuclei in lymph nodes of SIV mac-infected macaques was observed in the stage of persistent general lymphadenopathy than in those with AIDS-related complex, while there was no significant difference in the extent of apoptosis of cultured PBMC among the SIV mac-infected macaques. These results suggest that in vitro apoptosis is mediated by the Fas/Fas ligand and ICE system and that apoptosis in lymph nodes may be more closely related to the stage of SIV mac infection than is that of cultured PBMC.

Animals↗
PubMed1998

Relationship of vacA genotypes of Helicobacter pylori to cagA status, cytotoxin production, and clinical outcome.

BACKGROUND: Mosaicism in vacA alleles with three distinct families of vacA signal sequences (s1a, s1b and s2) and two distinct families of middle region alleles (m1 and m2) has been reported. It was suggested that the vacA s1a genotype was closely associated with duodenal ulcer disease and with high cytotoxin production. The aim of this study was to evaluate the role of vacA genotyping with respect to gastric inflammation and injury, cytotoxin activity, and clinical presentation. METHODS: H. pylori from patients with gastritis, peptic ulcer disease, or gastric cancer were characterized by vacA typing by polymerase chain reaction (PCR) and DNA sequencing. In vitro cytotoxin activity was assessed by vacuolation assay using Vero cells as well as with Hela cells. RESULTS: Four hundred ninety-one strains were tested. vacA genotype s1a/m1 was present in more than 95% of strains independent of presentation with gastritis, peptic ulcer, or gastric cancer. No vacA genotype was associated with high average cytotoxin activity. The s2/m2 isolates had low or absent cytotoxin activity. All cagA negative strains (n = 18) were s1a strains and both s2/m2 strains were cagA positive. One strain that was a recombinant of m1 and m2 strains was identified and had low cytotoxin activity. The nucleotide and amino acid sequences between original m1 strains and Japanese m1 strains (new m1 strains) were about 85% and 81%, respectively. Strains with the new m1 genotype had nucleotide and amino acid sequences similarity of more than 96%. There was no difference in cytotoxin activity between strains with the Western type m1 and the new type m1 genotype. CONCLUSION: In this as in other reported studies ( approximately 1500 patients overall) vacA genotype was strongly but not exclusively associated with the presence of cagA. Overall, the studies did not support a role for vacA genotyping in relation to cytotoxin activity, virulence, histologic finding, or risk of a particular H. pylori disease. vacA genotype s1 is likely to be a surrogate marker for the presence of the cag pathogenicity island.

Adult↗
PubMed1998

Induced expression and localization to nuclear-inclusion bodies of hsp70 in varicella-zoster virus-infected human diploid fibroblasts.

The expression and subcellular localization of cellular heat-shock protein hsp70 were examined in varicella-zoster virus (VZV)-infected human diploid fibroblasts. Infection with VZV elevated the steady-state levels of hsp70 mRNA by 24 hr post-infection (hpi). Western blotting analysis revealed an increase in accumulation of hsp70 from 24 hpi. Subcellular localization of the hsp70 in VZV-infected cells was examined by indirect immunofluorescence. In most VZV-infected cells, hsp70 was localized to inclusion bodies induced in the cell nucleus by infection with VZV. In some cells, however, the remaining parts of the cell nucleus and the cytoplasm were also stained with anti-hsp70 antibody. These results indicate that infection with VZV induces the expression of hsp70 and its localization to VZV-specific inclusion bodies, which suggests the involvement of hsp70 in molecular events within inclusion bodies.

Cell Line↗
PubMed1998

Hydrostatic pressure induces expression of interleukin 6 and tumour necrosis factor alpha mRNAs in a chondrocyte-like cell line.

OBJECTIVES: To clarify the effect of pressure on the expressions of proteoglycan core protein and metabolism related cytokines in a chondrocyte-like cell line, HCS-2/8. METHODS: HCS-2/8 cells were exposed to 1, 5, 10, or 50 MPa of hydrostatic pressure (HP) for two hours, and mRNA expressions of interleukin 6 (IL6) and tumour necrosis factor alpha (TNFalpha) were examined by using reverse transcription-polymerase chain reaction (RT-PCR) method with specific primer sets; and mRNA of proteoglycan core protein, stromelysin, and tissue inhibitor of metalloproteinase 1 (TIMP1) were measured with northern blotting. RESULTS: HP exposure caused temporal morphological changes of the cells, but did not affect cellular viability, IL6 and TNFalpha mRNA expressions were not observed in the control cells under the atmospheric pressure, whereas in the cells treated with HP, pressure dependent enhancement of IL6 mRNA expression was observed between 30 minutes and four hours after HP release. TNFalpha mRNA expression also increased 30 minutes after the exposure to 50 MPa of HP and disappeared four hours later. Proteoglycan core protein mRNA levels increased between 30 minutes and four hours after the exposure to 1 or 5 MPa of HP, whereas the levels decreased after 10 or 50 MPa of HP. Stromelysin and TIMP1 mRNA signals did not respond to HP. CONCLUSION: HP at excessively high levels induced IL6 and TNFalpha expression and reduced the expression of proteoglycan core protein, while physiological levels of HP increased the expression of proteoglycan core protein. These findings are important when considering the pathology of osteoarthritis.

Aggrecans↗
PubMed1998

Chemokines in the gastric mucosa in Helicobacter pylori infection.

BACKGROUND: Although chemokines have been suggested to play an important role in Helicobacter pylori associated gastritis, few studies have investigated the role of chemokines other than interleukin 8 (IL-8) in gastric mucosa. AIMS: To investigate the expression and production patterns of various chemokines using gastric biopsy specimens. METHODS: In 192 patients, expression patterns of C-X-C chemokines (IL-8 and growth regulated alpha (GRO alpha)) and C-C chemokines (regulated on activation, normal T cell expressed and presumably secreted (RANTES), monocyte chemotactic and activating factor (MCAF), macrophage inflammatory protein 1 alpha (MIP-1 alpha), and MIP-1 beta) were examined using reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). cagA gene was identified using PCR. RESULTS: H pylori infection was associated with increased rates of expression of mRNA for IL-8, GRO alpha, RANTES, and MIP-1 alpha and with increased levels of mucosal IL-8 and GRO alpha. IL-8 and GRO alpha levels correlated with the density of H pylori in both the antrum and corpus. The levels of these chemokines correlated with cellular infiltration in the antrum but not the corpus. cagA gene positive H pylori infection was associated with increased rates of expression of mRNA for IL-8 and GRO alpha and with increased levels of these chemokines. CONCLUSION: H pylori infection is associated with increased expression rates and production of C-X-C chemokines (IL-8 and GRO alpha), but not with increased production of C-C chemokines. Although H pylori infection is associated with increased C-X-C chemokines in the antrum and corpus, there is a difference in the inflammatory response between these two areas of the stomach.

Adult↗
PubMed1998

Effects of PDGF on the migration of rabbit corneal fibroblasts and epithelial cells.

PURPOSE: We investigated the effects of human platelet-derived growth factor (PDGF) isomers on the migration of rabbit corneal fibroblasts and epithelial cells in the presence or absence of fibronectin (FN). METHODS: A modified Boyden's chamber method was used to detect cell migration. Cells placed in the inner chamber were incubated with PDGF in the outer chamber at 37 degrees C at 24 h. In addition, epithelial cells were incubated in the presence or absence of FN. The migrated cells were stained and the cell number counted under a microscope. Checkerboard analysis was used to distinguish chemotaxis from chemokinesis. RESULTS: Natural PDGF and PDGF-BB enhanced the migration of corneal fibroblasts, whereas PDGF-AA did not. As for corneal epithelial cells, all the isomers of PDGF enhanced the migration, but only in the presence of FN, as in the absence of FN or at a low concentration gradient of FN, there was no enhanced cell migration. A checkerboard assay demonstrated that PDGF-BB had a chemotactic effect on the migration of corneal fibroblasts and epithelial cells, whereas PDGF-AA had a chemotactic effect on only corneal epithelial cells. CONCLUSION: These results suggest that PDGF is involved in corneal wound healing by stimulating the migration of corneal epithelial cells in the presence of FN and fibroblasts.

Animals↗
PubMed1998

Adenovirus mediated gene delivery to the joints of guinea pigs.

OBJECTIVE: To clarify in vivo applicability of adenovirus mediated gene delivery to examine a gene therapy for human joint diseases. METHODS: We directly injected vectors harbouring beta-galactosidase gene and transforming growth factor (TGF)-beta1 gene into the joints of Hartley guinea pigs. Expressions of delivered LacZ were examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside staining and reverse transcription-polymerase chain reaction. The levels of TGF-beta1 that were delivered to the joint and then transferred to the joint fluid were assessed by ELISA. RESULTS: LacZ expression was observed in almost all synovial tissue samples and in chondrocytes on the surface of degenerated cartilage. In the other organs, expression of delivered genes was not observed. For 2 weeks following gene delivery TGF-beta1 levels in joint fluid were significantly higher than the levels in the controls for 2 weeks. CONCLUSION: Direct gene delivery into the joint cavity is feasible with the in vivo gene delivery method using adenovirus vector and would be clinically applicable.

Adenoviridae↗
PubMed1998

[Role of cytokines in the pathogenesis of gastrointestinal diseases associated with Helicobacter pylori infection].

It is well known that Helicobacter pylori can cause gastritis, gastroduodenal ulcers and malignant diseases. The infiltration of polymorphonuclear leukocytes is recognized in the lesions of these diseases, and the infiltration disappears by antibiotic therapy. However, it is not yet clarified how Helicobacter pylori induces the formation of lesions including leukocyte infiltration. Recently, we have confirmed that several kinds of cytokines are expressed in the gastric biopsy specimens of gastroduodenal diseases. Especially, it is conjectured that chemokines such as interleukin-8 (IL-8) which are expressed in the specimens, induce leukocyte infiltration, gastric mucosal inflammation and gastroduodenal ulcers. It is possible that Helicobacter pylori CagA gene is closely related with IL-8 expression because this cytokine is more strongly expressed in the specimens from the patients infected with CagA-positive Helicobacter than those with CagA-negative one.

Anti-Bacterial Agents↗
PubMed1997

Use of EBV-based Vector/HVJ-liposome complex vector for targeted gene therapy of EBV-associated neoplasms.

Targeted suicide gene therapy for Epstein-Barr virus (EBV)-associated neoplasms was attempted by using EBV-based plasmid vectors coupled with hemagglutinating virus of Japan (HVJ)-liposome in vitro. Expression of EBV nuclear antigen (EBNA)1 is a common feature of the neoplasms associated with EBV. When various leukemic cell lines were transduced with a vector carrying a marker gene and EBV replication origin of plasmid (oriP), the marker gene product was exclusively detected in cells expressing EBNA1. Transduction of herpes simplex virus (HSV)-1 thymidine kinase (Tk) gene resulted in a marked reduction in viable cell number by ganciclovir (GCV) specifically in EBNA1 positive cells. The results demonstrate that this virus-free system may be applicable to gene therapy of EBV-associated neoplasms.

Antiviral Agents↗
PubMed1997

Efficient gene transduction by Epstein-Barr-virus-based vectors coupled with cationic liposome and HVJ-liposome.

We show here a novel non-viral strategy to transduce human cells by using an EBV-based vector system. The EBV-based vectors, the plasmid vectors carrying EBV oriP (origin for plasmid replication) and EBNA (EBV nuclear antigen) 1 gene from EBV genome, were combined with 2 gene delivery systems, i.e., cationic liposome and HVJ-liposome. By both methods, EBV-based vectors could be more efficiently transfected into HeLa cells than non-EBV, conventional plasmid vectors. When human primary fibroblasts were transfected, EBV-based vectors coupled with cationic liposome but HVJ-liposome resulted in successful gene transduction, while human bone marrow cells were transduced with both HVJ-liposome- and cationic liposome-EBV vectors. These results suggest the potential applications of the EBV-based vector system for gene therapy.

Bone Marrow Cells↗
PubMed1997

Extremely efficient gene transfection into lympho-hematopoietic cell lines by Epstein-Barr virus-based vectors.

We have estimated the efficiency of Epstein-Barr virus (EBV)-based vectors in transfecting genes into cell lines of lympho-hematopoietic lineages. The transfection efficiency was estimated both at transient and stable phases, in terms of expression of a marker gene and acquisition of drug resistance, respectively. Plasmid vectors carrying EBV oriP (replication origin of plasmid), EBNA (EBV nuclear antigen)-1 and as the marker genes, murine CD8 alpha cDNA and neoR (neomycin resistant) genes were transfected into various cell lines by electroporation. When cell lines constitutively expressing EBNA-1 were transduced, virtually all the cells expressed CD8 alpha on day 3 and acquired G418 resistance thereafter. In the case of K562 cells, which do not express EBNA-1, approximately 40% of cells expressed the marker gene product on day 3 posttransfection, and 30% of cells became stable transfectants. These data suggest a broader application of the EBV vector system in basic immunology and medicine.

Cell Line↗
PubMed1997

Hydrostatic pressure influences mRNA expression of transforming growth factor-beta 1 and heat shock protein 70 in chondrocyte-like cell line.

The present study investigated the influence of hydrostatic pressure on the expression of cytokines and heat shock protein 70 in a chondrocyte-like cell line. Chondrocyte-like cells (HCS-2/8) were exposed to hydrostatic pressure by a special pressure apparatus. Total RNA for cytokines (interleukin-1 beta, basic fibroblast growth factor, insulin-like growth factor-I, and transforming growth factor-beta 1) and for heat shock protein 70 was extracted and was analyzed by a polymerase chain reaction method and Northern blotting. An assay for incorporation of [35S]sulfate was performed to assess proteoglycan synthesis. The expression of transforming growth factor-beta 1 mRNA was enhanced after exposure to 5-MPa of hydrostatic pressure and was reduced after 50 MPa, whereas the expression of heat shock protein 70 was enhanced following exposure to 50 MPa of hydrostatic pressure. The incorporation of [35S]sulfate into the cultured cells increased following exposure to 1-5 MPa of hydrostatic pressure and decreased following 10-50 MPa of pressure. These results suggest that hydrostatic pressure at physiologic levels enhances the expression of transforming growth factor-beta 1 mRNA in addition to increasing proteoglycan synthesis in chondrocytes and that excessively high hydrostatic pressure reduces the expression of transforming growth factor-beta 1 mRNA and increases the expression of heat shock protein 70 mRNA while decreasing proteoglycan synthesis.

Cartilage, Articular↗
PubMed1997

Analysis of heat shock proteins and cytokines expressed during early stages of osteoarthritis in a mouse model.

OBJECTIVE: Osteoarthritis (OA) is a debilitating disease of the joints. The joints of affected individuals are characterized by a progressive degeneration of articular cartilage leading to inflammation and pain. The expression of heat shock proteins (HSPs) is a ubiquitous self-protective mechanism of all cells under stress, furthermore, the synovium of osteoarthritic individuals contains high levels of cytokines. This study seeks to establish the role of HSPs and cytokines in OA. METHODS: We have investigated the presence of HSPs and cytokines in articular cartilage during early stages of OA in a mouse that is known to develop spontaneous OA lesions (C57 black mouse). The articular cartilage from closely related mice (C57BL/6) was used as control. Messenger RNAs (mRNAs) for HSPs (HSP32, HSP47, HSP60, HSP70, HSP84 and HSP86) and cytokines [interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The mRNA levels of HSP47, HSP70, HSP86, IL-6, and IFN-gamma were up-regulated in the cartilage of C57 black mice, whereas, the level of expression of HSP32, HSP60, HSP84 and IL-1 beta remained unchanged. Furthermore, the expression of IL-1 beta, IL-6, TNF-alpha and IFN-gamma mRNA was associated with expression of HSP60, HSP47, HSP70 and HSP70/HSP86 mRNA, respectively. CONCLUSIONS: The findings in this study suggest that chondrocytes are conditioned under non-physiological stress during early stages of OA, In addition, among HSPs, HSP70 was associated with two different highly expressed cytokines in C57 black mice, indicating the possible role of HSP70 as a characteristic indicator of early stage of OA.

Animals↗