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Naoya Sakamoto

Publications and source records attributed to Naoya Sakamoto.

At least 19 recordsLinked to original sources
PubMed2007

[Case of severe ulcerative colitis successfully treated with intravenous cyclosporine without high dose corticosteroid].

A 44-year-old women developed marked myopathy one year earlier, when she was treated with intravenous prednisolone for acute severe exacerbation of ulcerative colitis. When she was admitted to our hospital for another severe exacerbation, intravenous cyclosporine A was administered as monotherapy because she could not tolerate corticosteroid. The treatment was successful and she obtained complete remission. Cyclosporine A monotherapy is considered to be a valuable alternative to proctocolectomy for severe ulcerative colitis patients who cannot tolerate corticosteroid.

Adult↗
PubMed2006

Effect of fluid shear stress on migration of vascular smooth muscle cells in cocultured model.

Migration of smooth muscle cells (SMCs) in hyperplasia is thought to have a correlation with blood flow conditions. In this study, the effect of shear stress applied to endothelial cells (ECs) on SMC migration was examined using a newly designed EC-SMC coculture model (CM), in which bovine SMCs and ECs were separated by a collagen layer and a membrane filter. After exposing the CM to shear stresses of 0.5, 1.0, or 1.5 Pa for 48 h, the number of SMCs migrating into the collagen layer was counted. Under static conditions, the migration of SMCs in the CM increased compared with SMCs cultured alone. Shear stress of 1.5 Pa significantly suppressed the SMC migration (p < 0.05) compared with the static CM. Media conditioned with the CM exposed to shear stress of 1.0 Pa (p < 0.05) and 1.5 Pa (p < 0.005) exhibited reduction in activated matrix metalloproteinase-2 (MMP-2) compared with the static CM, as analyzed by zymography. Addition of an inhibitor of nitric oxide (NO) synthase, N (omega)-nitro-L: -arginine methyl ester, to the media inhibited the effect of 1.5 Pa shear stress on SMC migration but MMP-2 activity was unaffected. These results suggest that physiological shear stress has protective roles in atherosclerogenesis.

Animals↗
PubMed2006

Negative regulation of intracellular hepatitis C virus replication by interferon regulatory factor 3.

BACKGROUND: Interferon regulatory factor (IRF)-3 plays an important role in initiating cellular interferon-stimulated gene-mediated antiviral responses. In the present study, we evaluated the effects of IRF-3 expression and activation on intracellular hepatitis C virus (HCV) replication using an HCV replicon system. METHODS: An HCV replicon was constructed that expressed a neomycin-selectable chimeric firefly luciferase reporter protein. A small interfering (si) RNA oligonucleotide directed against IRF-3 mRNA was designed and synthesized. A eukaryote expression plasmid vector was constructed that expressed IRF-3 mRNA under control of the cytomegalovirus early promoter/enhancer. To evaluate transcriptional activity of the interferon-stimulated genes, a reporter vector was used that expressed firefly luciferase under control of the interferon-stimulated response element (ISRE). RESULTS: The baseline expression of IRF-3 did not significantly differ between cells with and without expression of the replicon. Transfection of an IRF-3 expression plasmid into the cells raised the ISRE-luciferase activities. The increase of ISRE activity was significantly more potent in the replicon-expressing cells than in cells without replicon expression. Concomitantly, the overexpression of IRF-3 suppressed HCV replication levels. In contrast, siRNA knockdown of IRF-3 suppressed ISRE activity by 38% +/- 2%. Interestingly, the suppression of IRF-3 resulted in a significant increase of HCV replication, by up to twofold, depending on the IRF-3 suppression levels. CONCLUSIONS: IRF-3 negatively regulated intracellular HCV replication, and was partially activated in cells that expressed the HCV replicon. Thus, IRF-3 is a key molecule controlling HCV replication through modulation of host interferon gene responses.

Blotting, Western↗
PubMed2006

The wrapping method using biodegradable felt strips has a preventive effect on the thinning of the aortic wall: experimental study in the canine aorta.

OBJECTIVES: Wrapping methods have been widely used to reinforce the anastomotic site in vascular surgery; however, postoperative changes in the aortic wall wrapped by nonbiodegradable felt have not been well characterized. The purposes of this investigation are to elucidate the sequelae of wrapping with nonbiodegradable felt on the aortic wall and to modify those changes by using biodegradable felt with or without basic fibroblast growth factor (bFGF). METHODS: The descending thoracic aortas of 15 beagles were wrapped with three different materials: nonbiodegradable polytetrafluoroethylene (PTFE) felt, biodegradable polyglycol acid (PGA) material, and PGA with 100 microg bFGF (n = 5 in each group). The descending thoracic aorta was resected after 3 months. The thickness of the aortic wall, vessel density in the media and the adventitia, and the wall strength were assessed. Untreated native aortic wall served as a normal control. RESULTS: The thickness of the media of the PTFE group was lower than that of the PGA + bFGF group (66% +/- 5% vs 85% +/- 6% of control, P < .05). The adventitia-media ratio in the PTFE group decreased compared with controls (59.1% of normal, P < 0.05), whereas those in the PGA and PGA + bFGF groups increased (172.1% and 189.6% of normal, respectively, P < .01). The collagen-smooth muscle ratio in the media was higher in the PTFE group than in the controls (0.14 +/- 0.02 vs 0.07 +/- 0.01, P < .01). The number of vessels in the adventitia was higher in the PGA + bFGF group than those in PTFE or PGA groups (29.6 +/- 2.5/mm2 vs 6.4 +/- 0.8/mm2, 19.0 +/- 1.1/mm2, P < .01). The PGA + bFGF group demonstrated larger failure force than the PTFE group (4.0 +/- 0.3 kgf vs 1.6 +/- 0.3 kgf, P < .01). The failure stress in the PGA and PGA + bFGF groups was larger than that in PTFE group (PTFE:PGA + bFGF = 5.3 +/- 0.9 x10(2) kPa:11.7 +/- 1.7 x 10(2) kPa, P < .01; PTFE:PGA = 5.3 +/- 0.9 x 10(2) kPa:11.2 +/- 1.2 x 10(2) kPa, P < .05). CONCLUSION: The aortic wall wrapped with nonbiodegradable PTFE felt showed a reduced thickness and diminished vessels in the adventitia. Biodegradable felt (PGA), with or without bFGF, modified these histologic changes. The vessel-rich thickened adventitia, after wrapping by PGA with bFGF, was associated with increased aortic wall strength. CLINICAL RELEVANCE: This investigation was conducted in an attempt to elucidate mechanisms underlying the occurrence of late postoperative false aneurysm after aortic surgery. We hypothesized that sustaining compression of the aorta by the felt strip may cause structural derangement and local ischemia on the aortic wall. We used a simple wrapping of the aorta with a felt strip rather than a felt strip at anastomotic sites to simplify the experimental model and to exclude confounding factors brought by technical inconsistency on the surgical anastomosis. We further attempted to find a clue for preventing adverse effects of wrapping with a conventional felt strip. Practically, we pursued a possible application of a biodegradable felt strip to aortic wrapping in our experimental model before we proceed in a clinical application of the new material.

Aneurysm, False↗
PubMed2006

Adhesive force of human hepatoma HepG2 cells to endothelial cells and expression of E-selectin.

Expression of adhesion molecules may play an important role in the interaction of tumor cells with vascular endothelial cells during tumor invasion and metastasis. In this study, the adhesive force of human hepatoma HepG2 cells to human umbilical vein endothelial cells (HUVECs) was investigated using a micropipette aspiration technique. Expression of an adhesion molecule, E-selectin, was also observed by immunofluorescence microscopy. In particular, the adhesive force after stimulation of HUVECs with recombinant human interleukin-1beta (rhIL-1beta) was examined. The results demonstrated that the adhesive force of HepG2 cells to stimulated HUVECs is significantly higher than that of unstimulated control cells, and that immunofluorescence of E-selectin in stimulated HUVECs showed a higher fluorescent intensity compared to control cells. Moreover, addition of monoclonal anti-human E-selectin decreased the adhesive force of HepG2 cells to stimulated HUVECs by 50%. These results suggest that endothelial E-selectin may be a main mediator of carcinoma metastasis of malignant tumor through blood circulation, possibly increasing the adhesive force of human hepatoma HepG2 cells to HUVECs in the early stage of metastases.

Antibodies, Monoclonal↗
PubMed2006

[A case of sacroiliitis complicated by ulcerative colitis that was successfully treated with steroid].

A 23-year-old woman was admitted in November, 2002, complaining pain of the left side and buttock. She had ulcerative colitis when she was 16 and received medical treatment. Based on physical examination and findings of magnetic resonance imaging and bone scintigrapy, as sacroiliitis complicated by ulcerative colitis. was diagnosed Reports on sacroiliitis and ankylosing spondylitis complicated by inflammatory bowel diseases (IBD) are relatively rare in Japan, whereas they are common complications of IBD in Western countries. The efficacy of steroids on pain relief of sacroiliitis and ankylosing spondylitis is unclear.

Adult↗
PubMed2006

[A case report: Severe bone marrow suppression caused by 6-mercaptopurin in Crohn's disease patient].

A 23-year-old man was admitted for treatment of acute exacerbation of ileitis and perianal abscess caused by Crohn's disease. After incision and drainage of the abscess, coupled with antibiotic therapy, 6-mercaptopurine (6-MP) was commenced. His white blood cell (WBC) count on day 12 after initiation of 6-MP was not decreased. However, on day 24 he was re-admitted because of severe myelosuppression (WBC: 300/microl), which was complicated by the recurrence of the perianal abscess. Myelosuppression was prolonged and required the administration of granulocyte colony stimulating factor (G-CSF). G-CSF was continued for 17 days to achieve recovery of his WBC count to a normal level.

Abscess↗
PubMed2005

Divergent activities of interferon-alpha subtypes against intracellular hepatitis C virus replication.

BACKGROUNDS: Interferon (IFN)-alpha is represented by several structurally related subtypes that show different antiviral and anti-tumor effects. Here, we analyzed differential effects of IFN-alpha subtypes on intracellular hepatitis C virus (HCV) replication using HCV subgenomic replicon system as a model. METHODS: Huh7 and HeLa cells supporting expression of HCV replicon were treated with various concentrations of five recombinant human IFN-alpha subtypes 1, 2, 5, 8, and 10, and with IFN-alpha con1. The effects of IFNs on various cell-signaling pathways were assayed by using ISRE-, GAS-, AP1-, NF-kappa B-, CRE-, and SRE-luciferase reporter plasmids. RESULTS: Each IFN-alpha subtype suppressed HCV replication in a dose-dependent manner. Among them, IFN-alpha8 was the most effective, while IFN-alpha1 was the least effective with 50% inhibitory concentrations of 0.123IU/ml versus 0.375IU/ml, respectively. These differential effects against HCV replication did not correlate with levels of the IFN-responsive ISRE or GAS reporter activities, nor they did activate the other reporters, AP1, NF-kappa B, CRE and SRE. CONCLUSION: There were divergent effects of IFN-alpha subtypes against HCV replication that may be through JAK-STAT-independent pathways. Exploring further mechanisms of action may elucidate IFN-mediated cellular antiviral mechanisms.

Journal Article↗
PubMed2005

Enhancement of mitochondrial gene expression in the liver of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is one of the most important autoimmune liver diseases but the etiology and pathogenesis remain unknown. In this study, we analyzed differential mRNA expression in the liver of a patient with PBC using suppression subtractive hybridization to identify overexpressed genes. Overexpression of mRNA transcripts from mitochondrial DNA was observed in the PBC liver, compared to normal liver. To explore the mechanism of increased mitochondrial transcription, we investigated the mRNA levels of nuclear DNA-encoded regulator molecules of mitochondrial gene expression in 60 liver biopsy samples from various diseases, including PBC, using competitive RT-PCR. Increased expression of mitochondrial transcriptional factor A (mtTFA) and mitochondrial nuclear respiratory factor 1 (NRF-1) mRNA was demonstrated in PBC liver compared to other liver diseases, while NRF-1 coactivator 1, PGC-1 was suppressed. Mitochondrial DNA-encoded mRNA molecules are overexpressed in the PBC liver, and this is associated with up-regulation of mitochondrial transcription factor mtTFA and its transactivator NRF-1. Further studies are needed to focus on the relevance of this perturbation of mitochondrial gene expression in the pathogenesis of PBC.

Journal Article↗
PubMed2005

Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins.

BACKGROUND & AIMS: Cyclosporin A specifically suppresses hepatitis C virus (HCV) replication in vitro at clinically achievable concentrations. In this study, we investigated the mechanisms of action of cyclosporin A against HCV replication. METHODS: The in vitro effects of cyclosporin A on HCV replication were analyzed using an HCV replicon system that expresses chimeric luciferase reporter protein. RESULTS: The significant effects of cyclosporin A on expression of an HCV replicon and the absence of such effects of FK506, which shares mechanisms of action with cyclosporin A, suggested the involvement of intracellular ligands of cyclosporin A, the cyclophilins. Transient and stable knockdown of the expression of cytoplasmic cyclophilins A, B, and C by short hairpin RNA-expressing vectors suppressed HCV replication significantly. A cyclosporin analogue, cyclosporin D, which lacks immunosuppressive activity but exhibits cyclophilin binding, induced a similar suppression of HCV replication. Furthermore, cyclosporin A treatment of Huh7 cells induced an unfolded protein response exemplified by expression of cellular BiP/GRP78. Treatment of cells with thapsigargin and mercaptoethanol, which induce the unfolded protein responses, suppressed HCV replication, suggesting that the cyclosporin-induced unfolded protein responses might contribute to the suppression of HCV protein processing and replication. CONCLUSIONS: The anti-HCV activity of cyclosporin A is mediated through a specific blockade of cyclophilins, and these molecules may constitute novel targets for anti-HCV therapeutics.

Base Sequence↗
PubMed2005

Mutations in the NS5B region of the hepatitis C virus genome correlate with clinical outcomes of interferon-alpha plus ribavirin combination therapy.

BACKGROUND AND AIM: Combination treatments of interferon-alpha (IFN) and ribavirin (RBV) are more effective than those of IFN alone in hepatitis C virus (HCV) infection. However, mechanisms of the action of the combination regimen are not well understood. To elucidate the viral genetic basis of IFN plus RBV combination therapy, genetic variabilities of HCV-1b were analyzed. METHODS: We performed pair-wise comparisons of full-length HCV genomic sequences in three patients' sera before and after initiation of IFN plus RBV treatment. Subsequently, we analyzed amino acid sequences of the NS5B region, which codes for the viral RNA-dependent RNA polymerase, and compared these with the outcomes of the therapy in 81 patients. RESULTS: Analysis of the entire HCV sequence in patients who received IFN plus RBV therapy did not show consistent amino acid changes between before and after the initiation of the therapy. NS5B sequence analyses revealed that mutations at positions 300-358 of NS5B, including polymerase motif B to E, occurred more frequently in a group of patients exhibiting a sustained viral response (SVR) or an end-of-treatment response (ETR) compared with a group of patients exhibiting a non-response (NR). Closer examination revealed that mutations at aa 309, 333, 338 and 355 of NS5B occurred significantly more frequently in the SVR plus ETR group than in the NR group (P = 0.0004). Multivariate analysis showed that the number of mutations at these four sites was an independent predictor of SVR plus ETR versus NR. CONCLUSIONS: Particular amino acid changes in the NS5B region of HCV may correlate with outcomes of IFN plus RBV combination therapy.

Antiviral Agents↗
PubMed2004

Down-regulation of p27Kip1 promotes cell proliferation of rat neonatal cardiomyocytes induced by nuclear expression of cyclin D1 and CDK4. Evidence for impaired Skp2-dependent degradation of p27 in terminal differentiation.

Mammalian cardiomyocytes lose their capacity to proliferate during terminal differentiation. We have previously reported that the expression of nuclear localization signal-tagged cyclin D1 (D1NLS) and its partner cyclin-dependent kinase 4 (CDK4) induces proliferation of rat neonatal cardiomyocytes. Here we show that the D1NLS/CDK4 cells, after their entry into the cell cycle, accumulated cyclin-dependent kinase inhibitor p27 in the nuclei and decreased the cyclin-dependent kinase 2 (CDK2) activity, leading to early cell cycle arrest. Biochemical analysis demonstrated that Skp2-dependent p27 ubiquitylation was remarkably suppressed in cardiomyocytes, whereas Skp2, a component of Skp1-Cullin-F-box protein ubiquitin ligase, was more actively ubiquitylated compared with proliferating rat fibroblasts. Specific degradation of p27 by co-expressing Skp2 or p27 small interfering RNA caused an increase of CDK2 activity and overrode the limited cell cycle. These data altogether indicate that the impaired Skp2-dependent p27 degradation is causally related to the loss of proliferation in cardiomyocytes. This provides a novel insight in understanding the molecular mechanism by which mammalian cardiomyocytes cease to proliferate during terminal differentiation.

Animals↗
PubMed2004

Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon- alpha.

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Amino Acid Sequence↗
PubMed2004

Specific inhibition of hepatitis C virus replication by cyclosporin A.

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.

Antiviral Agents↗
PubMed2004

Changes of HCV quasispecies during combination therapy with interferon and ribavirin.

Treatment of chronic hepatitis C virus (HCV) infection with interferon (IFN) and ribavirin improves the rate of eradication of HCV, but only about 13-14% of non-responders (NR) with HCV of genotype 1b previously treated with IFN achieve a sustained virological response (SVR). To determine whether HCV quasispecies diversity correlates with the outcome of therapy with IFN and ribavirin, we studied 13 patients undergoing combination therapy with IFN-alpha2b and ribavirin after failure of IFN monotherapy. HCV quasispecies diversity was assessed by cloning and sequencing before and during combination therapy. During therapy, quasispecies diversity diminished in NR patients, both in the hypervariable region (HVR) 1 of the envelope 2 (E2) domain and in the interferon sensitivity-determining region (ISDR) in the NS5A. Pre-treatment nucleotide quasispecies diversity was lower in SVR and end-of-therapy viral response (ETR) patients than in NR patients. Resistance to ribavirin was associated with high pre-treatment heterogeneity and the selection of quasispecies of the HCV genome. HVR quasispecies may be a predictor of efficacy of combination therapy with IFN and ribavirin.

Journal Article↗
PubMed2004

Regulation of hepatitis C virus replication by interferon regulatory factor 1.

Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses.

Base Sequence↗