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Biomedical subjects

Masahiko Kaito

Publications and source records attributed to Masahiko Kaito.

At least 19 recordsLinked to original sources
PubMed2006

Expression of large tenascin-C splice variants by hepatic stellate cells/myofibroblasts in chronic hepatitis C.

BACKGROUND/AIMS: Earlier studies have suggested involvement of tenascin-C (TN-C) in liver fibrosis. Here, we examined expression of TN-C variants and types of alternatively spliced fibronectin-type III (FNIII) repeats in chronic hepatitis. METHODS: Using three monoclonal antibodies against TN-C variants, immunohistochemical staining was performed and the correlation with histological parameters of chronic hepatitis C was examined. The cellular source was also determined and variant expression and their types were tested using isolated rat hepatic stellate cells (HSCs), liver myofibroblasts, and/or LI90 cells. RESULTS: Large variants were not expressed in normal liver, but were up-regulated in chronic hepatitis, especially at sites of interface hepatitis and confluent necrosis, showing stronger correlations between staining intensity and these than with other parameters or fibrosis. TN-C deposition was closely correlated with increase in the number of alpha-smooth muscle actin-positive cells, i.e. activated HSCs/myofibroblasts, and in situ hybridization showed TN-C mRNA signals in the cells. Activated HSCs and myofibroblasts in culture highly expressed large variants of TN-C. In LI90 cells, sequencing of large variants revealed that the FNIII repeats D and A1/A4, followed by B, were preferentially included. CONCLUSIONS: TN-C and its variants are produced by HSCs/myofibroblasts, suggesting important roles in liver fibrogenesis.

Actins↗
PubMed2006

Hepatic oxidative DNA damage correlates with iron overload in chronic hepatitis C patients.

Hepatic oxidative stress occurs in chronic hepatitis C (CH-C), but little is known about its producing mechanisms and precise role in the pathogenesis of the disease. To determine the relevance of hepatic oxidatively generated DNA damage in CH-C, 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts were quantified in liver biopsy specimens by immunohistochemical staining, and its relationship with clinical, biochemical, and histological parameters, and treatment response was assessed in 40 CH-C patients. Hepatic 8-OHdG counts were significantly correlated with serum transaminase levels (r=0.560, p=0.0005) and histological grading activity (p=0.0013). Remarkably, 8-OHdG levels were also significantly related to body and hepatic iron storage markers (vs serum ferritin, r=0.565, p=0.0004; vs hepatic total iron score, r=0.403, p=0.0119; vs hepatic hepcidin messenger RNA, r=0.516, p=0.0013). Baseline hepatic oxidative stress was more prominent in nonsustained virological responder (non-SVR) than in SVR to interferon (IFN)/ribavirin treatment (50.8 vs 32.7 cells/10(5) microm2, p=0.0086). After phlebotomy, hepatic 8-OHdG levels were significantly reduced from 53.4 to 21.1 cells/10(5) microm2 (p=0.0125) with concomitant reductions of serum transaminase and iron-related markers in CH-C patients. In conclusion, this study showed that hepatic oxidatively generated DNA damage frequently occurs and is strongly associated with increased iron deposition and hepatic inflammation in CH-C patients, suggesting that iron overload is an important mediator of hepatic oxidative stress and disease progression in chronic HCV infection.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

Lactoferrin inhibits lipid peroxidation in patients with chronic hepatitis C.

Lactoferrin is a milk protein with inhibitory effect on lipid peroxidation induced by oxidative stress. Oxidative stress plays an important role in the pathogenesis of chronic hepatitis C (CHC). The aim of this study was to evaluate the effect of bovine lactoferrin (bLF) monotherapy on lipid peroxidation, hepatic inflammation and iron metabolism in patients with CHC. Ninety Japanese patients with CHC were randomly assigned to two groups: bLF group (n=47) treated with bLF at a dose of 3.6g/day and a control group (n=43) that remained untreated. Plasma 8-isoprostane levels and clinical laboratory data including iron metabolism parameters were measured. Plasma 8-isoprostatne level was significantly decreased from 8.6+/-3.7 to 6.9+/-2.1pg/ml in the bLF group (P<0.05). Plasma levels of 8-isoprostane did not significantly change in the control group. The decline in plasma 8-isoprostane levels was positively correlated with improvement in the level of ALT in the bLF group. No significant change in serum HCV RNA levels or iron metabolism markers was found after bLF treatment. Therapy with bLF was associated with improvement in lipid peroxidation and ALT levels in CHC. Administration of bLF is a promising therapeutic approach for suppressing oxidative stress in non-responders to antiviral therapy.

Journal Article↗
PubMed2006

Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens.

Various genotypes of the hepatitis B virus (HBV) induce liver disease of distinct severity, but the underlying virological differences are not well defined. Huh7 cells were transfected with plasmids carrying 1.24-fold the HBV genome of different genotypes/subgenotypes (2 strains each for Aa/A1, Ae/A2, Ba/B2 and D; 3 each for Bj/B1 and C). HBV DNA levels in cell lysates, determined by Southern hybridization, were the highest for C followed by Bj/Ba and D/Ae (P < .01), and the lowest for Aa (P < .01), whereas in culture media, they were the highest for Bj, distantly followed by Ba/C/D and further by Ae/Aa (P < .01). The intracellular expression of core protein was more than 3-fold lower for Ae/Aa than the others. Hepatitis B e antigen (HBeAg) was excreted in a trend similar to that of HBV DNA with smaller differences. Secretion of hepatitis B surface antigen (HBsAg) was most abundant for Ae followed by Aa, Ba, Bj/C and remotely by D, which was consistent with mRNA levels. Cellular stress determined by the reporter assay for Grp78 promoter was higher for C and Ba than the other genotypes/subgenotypes (P < .01). Severe combined immunodeficiency mice transgenic for urokinase-type plasminogen activator (uPA/SCID), with the liver replaced for human hepatocytes, were inoculated with virions passed in mouse and recovered from culture supernatants. HBV DNA levels in their sera were higher for C than Ae by 2 logs during 4-7 weeks after inoculation. In conclusion, virological differences among HBV genotypes were demonstrated both in vitro and in vivo. These differences may influence HBV infections with distinct genotypes in clinical and epidemiological settings.

Adolescent↗
PubMed2006

Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy.

We performed indirect immunogold electron microscopy (EM) for immunological identification and characterization of hepatitis C virus (HCV). To clarify the morphology of HCV, an indirect immunogold EM of two plasma samples from patients with high HCV RNA titers was carried out using antibodies specific for the putative HCV envelope protein (E) 1. Spherical virus particles 55-65 nm in diameter with delicate spike projections were detected in the 1.14-1.16 g/ml fractions after sucrose density gradient centrifugation. Polyclonal and monoclonal antibodies to the putative HCV E1 specifically recognized these particles. In addition, immunogold EM of the samples was also performed to uncover the morphology of HCV core particles. Spherical particles 33-40 nm in diameter (average, 37 nm) were detected in the 1.22- to 1.25-g/ml fractions by conventional EM after sucrose density gradient centrifugation. Immunogold EM using rabbit polyclonal antibody (RR8) specific for the putative HCV core protein and colloidal gold-labeled goat antirabbit IgG showed binding of the gold particles with RR8. Some of the HCV core particles showed icosahedric morphology. Optical rotation technique showed that the HCV core particles exhibit sixfold symmetry and that the length of the regular hexagon side is approximately 20 nm, suggesting that they have an icosahedric structure. Further, the detection limit of the indirect immunogold EM was evaluated in 11 plasma samples from chronic hepatitis B patients with different degrees of hepatitis B virus (HBV) DNA titers using antihepatitis B surface antigen antibody. The study showed that the detection limit of virus using this method is 10(7) virions/ml.

DNA, Viral↗
PubMed2006

The ultrastructural morphology of native hepatitis B virus.

Cell lines (2.2.15 cells) capable of supporting the replication of hepatitis B virus (HBV) DNA and intact viral particles have been established by HBV DNA transfection into HepG2 cells. The purpose of this study was to determine the ultrastructural morphology of native HBV particles without purification in the culture supernatants and in sera from patients. Electron microscopy (EM) and immunogold EM of the samples were carried out using polyclonal and monoclonal anti-hepatitis B surface antigen antibodies. HBV particles in the purified samples from the culture supernatants by density-gradient centrifugation were examined to compare the morphology with that of unpurified samples. EM and immunogold EM studies demonstrated the presence of Dane particles (41.8 nm in diameter), cobra-shaped (head diameter, 42.4 nm), and horn-shaped (head diameter, 43.5 nm) particles in the culture supernatants and in the sera from two patients. The tail of the cobra-like particles had a diameter of 21.0 nm and a length of 214 nm. The hornlike particles had a long branch 20.1 nm in diameter with a length of 189 nm, and a short branch 21.4 nm in diameter with a length of 112 nm. The ratio of Dane particles and cobra- and horn-shaped particles in the supernatants was 5 : 4 : 1. After ultracentrifugation, the cobra- and horn-shaped particles completely disappeared; there were only Dane particles together with spheres of 22 nm and filaments. In conclusion, this study showed for the first time that the native replicative form of HBV is cobra- and horn-shaped.

Cell Line↗
PubMed2006

Decreased protein C activation in patients with fulminant hepatic failure.

OBJECTIVE: Abnormalities of the blood coagulation system have an influence on outcome in patients with fulminant hepatic failure (FHF). The protein C (PC) pathway is one of the main modulators of the blood coagulation system. The role of the PC pathway in FHF is not clear. In the present study, we evaluated endothelial cell injury and the grade of activated protein C (APC) generation in FHF patients. MATERIAL AND METHODS: The effect of APC on the expression of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1 from LI90 stellate cells was also evaluated. This study comprised 5 patients with FHF, 6 with acute hepatitis (AH), 12 with chronic hepatitis (CH) and 20 healthy subjects. RESULTS: The plasma concentrations of thrombin-antithrombin complex and thrombomodulin were significantly increased in FHF patients compared with those in AH patients and healthy subjects. The circulating levels of activated protein C-protein C inhibitor (APC-PCI) complex and the APC-PCI/PC ratio were significantly decreased in patients with FHF compared to healthy controls. APC significantly inhibited in vitro the expression of TNFalpha and MCP-1 from LI90 stellate cells. CONCLUSIONS: This study demonstrated enhanced endothelial cell injury in association with decreased PC activation and hypercoagulability in FHF.

Antithrombin III↗
PubMed2006

siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations.

RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.

Base Sequence↗
PubMed2006

Role of transferrin receptor 2 in hepatic accumulation of iron in patients with chronic hepatitis C.

BACKGROUND AND AIM: Iron deposition in the liver is a common finding in patients with chronic hepatitis C (CH-C). The mechanism of this hepatic accumulation of iron is not completely understood. This study assessed if the protein expression of transferrin receptor 2 (TfR2) is upregulated in the liver of patients with CH-C and if TfR2 protein mediates iron accumulation during hepatitis C virus (HCV) infection. METHOD: Liver specimens from patients with CH-C that underwent interferon (IFN) therapy (n=23) and from patients with CH-B (n=18) were evaluated. Hepatic expression of TfR2 protein was analyzed by immunohistochemistry. Total hepatic iron score (THIS) was evaluated by Prussian blue staining. RESULTS: TfR2 protein was expressed in the cell membrane and cytosol of hepatocytes. Cytosol TfR2 protein was found to co-localize with Tf. THIS (P=0.0198) and hepatic TfR2 (P=0.0047) expression were significantly higher in CH-C than in CH-B. The change in THIS values (rho=0.580, P=0.0079) and the grade of histological activity (rho=0.444, P=0.0373) were significantly correlated with changes in TfR2 expression after IFN therapy. CONCLUSIONS: The protein expression of TfR2 is significantly associated with iron deposition in the liver in patients with CH-C. HCV infection may affect the hepatic expression of TfR2, leading to iron accumulation in the liver.

Adult↗
PubMed2006

Increased lipid peroxidation in patients with non-alcoholic fatty liver disease and chronic hepatitis C as measured by the plasma level of 8-isoprostane.

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of chronic liver diseases. The plasma level of 8-isoprostane, a product of lipid peroxidation, is a marker of oxidative stress in vivo. The aim of the present study was to clarify whether the degree of lipid peroxidation, as measured by the plasma level of 8-isoprostane, influences the progression of chronic liver diseases and hepatocarcinogenesis. METHODS: Plasma 8-isoprostane levels were investigated in 14 patients with non-alcoholic fatty liver disease (NAFLD), 75 with chronic hepatitis C (CH-C), 14 with cured CH-C, 14 with HCV-positive hepatocellular carcinoma (HCC-C) and 38 healthy volunteers. 8-Isoprostane was measured by enzyme immunoassay after affinity column purification. RESULTS: Plasma 8-isoprostane was significantly elevated in NAFLD (11.9 [3.8-56.8] pg/mL), CH-C (10.1 [4.2-134.5] pg/mL) as compared to controls (6.3 [3.6-11.1] pg/mL). Plasma 8-isoprostane values were positively correlated with body mass index in NAFLD (P < 0.05) and with total cholesterol in cured CH-C (P < 0.01). 8-Isoprostane levels were not significantly related to sex, age, biochemical data or iron metabolism markers in all liver diseases. In addition, after the administration of peg-interferon, the values of 8-isoprostane improved in almost all patients, reaching values of healthy subjects. CONCLUSIONS: 8-Isoprostane values are elevated in patients with NAFLD and CH-C as compared to healthy controls. Oxidative stress caused by increased lipid peroxidation is involved in the pathogenesis of NAFLD and CH-C.

Adolescent↗
PubMed2006

Circulating level of large splice variants of tenascin-C is a marker of piecemeal necrosis activity in patients with chronic hepatitis C.

BACKGROUND: It has been reported that histological activity index and piecemeal necrosis are good factors to evaluate the prognosis in patients with chronic hepatitis C (CHC). Thus, there is a need for simple and noninvasive means to assess disease activity and piecemeal necrosis in patients with CHC. In this study, we measured the serum concentrations of large splice variants of tenascin-C (cTN-C) in patients with CHC, and examined their correlation with the degree of inflammatory activity and fibrosis as evaluated in liver biopsy specimens. METHODS: The serum levels of cTN-C in 150 patients with CHC and 50 healthy volunteers were measured by enzyme-linked immunosorbent. The histology of liver biopsy specimens was also evaluated following the Desmet's grading/staging system and the Ishak's classification. Liver specimens obtained by biopsy were also immunohistochemically evaluated with anti-human tenascin-C antibodies. RESULTS: Serum cTN-C concentrations were significantly higher in CHC patients than in healthy volunteers (P < 0.0001). The levels of cTN-C showed no significant difference among the fibrosis stages as assessed by the Desmet's grading/staging system and Ishak's classification scores. However, the concentration of cTN-C was significantly correlated with the grade of activity. According to the Ishak's classification, the concentration of cTN-C was increased in proportion to the degree of piecemeal necrosis. Specific immunostaining of cTN-C was observed in limited areas of piecemeal necrosis but not in fibrotic areas. CONCLUSION: The measurement of serum levels of cTN-C is a useful marker of the activity of CHC, in particular of the degree of piecemeal necrosis.

Adult↗
PubMed2006

Morphological identification of hepatitis C virus E1 and E2 envelope glycoproteins on the virion surface using immunogold electron microscopy.

It is known that hepatitis C virus (HCV) particles are spherical, 55-65 nm particles with fine surface projections of about 6 nm in length and with a 30-35 nm inner core. We have reported that free HCV particles labeled with gold particles specific to the HCV E1 glycoprotein are located in 1.14-1.16 g/ml fractions from plasma samples with high HCV RNA titers after sucrose density gradient centrifugation. However, the morphology of the HCV E2 glycoprotein on the virion has not yet been elucidated. To visualize HCV E2 localization on the virion, we used the same plasma samples where HCV particles were clearly shown. An indirect immunogold electron microscopic study was carried out using monoclonal and polyclonal anti-HCV E2 antibodies. HCV-like particles specifically reacted with the anti-HCV E2 antibodies. Moreover, to evaluate the localization of the HCV E1 and E2 glycoproteins on the virion surface, an immunogold electron microscopic study using double labeling with anti-HCV E1 antibodies and anti-HCV E2 antibodies was also performed. These particles also specifically reacted with both anti-E1 and E2 antibodies. This is the first report showing the presence of both HCV E1 and E2 glycoproteins on HCV virion surface in human plasma samples.

Hepacivirus↗