Biomedical subjects

Isao Sakaida

Publications and source records attributed to Isao Sakaida.

54 records · Page 3Linked to original sources

PubMed2003

Leptin receptor-deficient Zucker (fa/fa) rat retards the development of pig serum-induced liver fibrosis with Kupffer cell dysfunction.

The aim of this study was to investigate the role of leptin in the development of liver fibrosis with Kupffer cell function using leptin receptor deficient rats. Male Zucker (fa/fa) and control (fa/-) rats received pig serum for 8 weeks. Animals were sacrificed to estimate the degree of liver fibrosis and stellate cell activation with the expression of alpha smooth muscle actin (alphaSMA). Microarray analysis was performed. Isolated Kuppfer cells of Zucker and control rats were treated with LPS. LPS uptake and TNF-alpha production were examined. Stellate cells were also isolated from Zucker and control rats. The expression of procollagen type I mRNAs was examined. Control rats developed liver fibrosis 8 weeks after injection of pig serum and showed an increased liver hydroxyproline content of 348 +/- 34 microg/g (n = 10) compared with Zucker rats (225 +/- 13, n = 10, P < 0.01). The procollagen type I mRNA level and alphaSMA expression of Zucker rats were also significantly reduced. Microarray analysis indicated significantly reduced expression of TNF-alpha, LPS-binding protein, urokinase-type plasminogen activator (uPA), IGF, IGF-binding protein (IGFBP)-3,5, and increased expression of apolipoprotein IV. Isolated Kupffer cells of Zucker rats showed significantly reduced LPS uptake as well as TNF-alpha production compared with control rats. However, no significant change was observed in procollagen type I mRNA levels of isolated stellate cells after 4 days of culture on plastic dishes. These results suggest that leptin receptor deficiency retards the development of liver fibrosis due to the dysfunction of Kuppfer cells.

Actins↗

PubMed2003

L-cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation.

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.

Animals↗

PubMed2003

Gadolinium chloride reverses dimethylnitrosamine (DMN)-induced rat liver fibrosis with increased matrix metalloproteinases (MMPs) of Kupffer cells.

The aim of this study was to investigate whether matrix metalloproteinases (MMP-13, 9) of Kupffer cells induced by gadolinium chloride (GdCl(3)) treatment can reverse dimethylnitrosamine (DMN)-induced liver fibrosis (in vivo) and the effect of GdCl(3) on MAP kinase activity (in vitro). Male Wistar rats 6 weeks of age received DMN (10 mg/kg) three successive days a week for 4 weeks. Then two groups of rats (n = 6 each) were treated twice weekly with either GdCl(3) (7 mg/kg) or saline solution intravenously for the next 4 weeks. Animals were sacrificed to estimate the degree of liver fibrosis. Isolated Kuppfer cells were treated with GdCl(3) and the expressions of MMPs, MAP kinase activity (ERK, SAPK/JNK, P38) as well as apoptosis were also examined. Rats that received DMN for 4 weeks followed by GdCl(3) injection for 4 weeks showed an reduced liver hydroxyproline content compared to rats treated with DEN followed by saline (277 +/- 22 VS 348 +/- 34 microg/g, n = 6 each, P < 0.01). There were significantly increased MMP-13 mRNA levels in GdCl(3)-treated rats. However, no significant change was observed in procollagen type I mRNA levels. Isolated Kuppfer cells treated with GdCl(3) showed increased MAP kinase activity, especially P38 pathway as well as MMP-13, 9 mRNA and type I collagen-degrading activity leading to apoptosis. SB203580, inhibitor of P38 pathway diminished these activation and prevented apoptosis. These results suggest that Kuppfer cells can reverse liver fibrosis via the expression of MMPs mainly through P38 pathway.

Animals↗

PubMed2003

Proteomic profiling of heat shock protein 70 family members as biomarkers for hepatitis C virus-related hepatocellular carcinoma.

To identify proteins linked to the pathogenesis of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV), we profiled protein expression levels in samples of HCC. To identify essential proteins, ten samples of HCV-related HCC were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These experiments revealed increased levels of nine proteins in cancerous tissues compared to levels in corresponding noncancerous liver tissues. We focused on four members of the heat shock protein 70 family: 78 kDa glucose-regulated protein (GRP78), heat shock cognate 71 kDa protein (HSC70), 75 kDa glucose-regulated protein (GRP75), and heat shock 70 kDa protein 1 (HSP70.1). These results were confirmed by immunoblot analysis. In an additional 11 samples, the same expression patterns of these four proteins were observed. In total, 21 samples showed statistically significant up-regulation of GRP78, GRP75 and HSP70.1 in cancerous tissues. HSC70 showed a tendency toward overexpression. There has been no report describing overexpression of these four proteins simultaneously in HBV-related HCC as well as nonviral HCC. Our results suggest that these four proteins play important roles in the pathogenesis of HCV-related HCC and could be molecular targets for diagnosis and treatment of this disease.

Biomarkers, Tumor↗

PubMed2003

Herbal medicine Inchin-ko-to (TJ-135) prevents liver fibrosis and enzyme-altered lesions in rat liver cirrhosis induced by a choline-deficient L-amino acid-defined diet.

BACKGROUND/AIMS: The herbal medicine Inchin-ko-to (TJ-135), extract power from three herbs, has recently been reported possessing anti-apoptotic activity. The aim of this study was to investigate whether TJ-135 has any influence on the development of preneoplastic lesions as well as liver fibrosis. METHODS: The effects of the TJ-135 were examined using the choline-deficient L-amino acid-defined diet-induced liver fibrosis model. In addition, the effect of TJ-135 on mitogen-activated protein (MAP) kinase, type III procollagen mRNA expression and the medium N-terminal procollagen III propeptide (PIIINP) concentration in a hepatic stellate cell line (LI90) were examined. RESULTS: TJ-135 prevented fibrosis in a dose-dependent manner up to 1.5% (w/w). TJ-135 also reduced the expression of type III procollagen mRNA in the liver, as well as the number of activated stellate cells. Furthermore, TJ-135 reduced the area of preneoplastic lesions in the liver. With LI90 cells, TJ-135 reduced MAP kinase (ERK and JNK but not P38) activities resulting in reduced type III procollagen mRNA and PIIINP concentrations in the medium in a dose-dependent manner. CONCLUSIONS: These results indicate that although TJ-135 has anti-apoptotic activity, TJ-135 does not increase preneoplastic lesions but significantly reduces liver fibrosis through the inhibition of stellate cell activation without a reduction of hepatocyte cell death.

Amino Acids↗

PubMed2003

Effect of a late evening snack on the blood glucose level and energy metabolism in patients with liver cirrhosis.

BACKGROUND/AIM: Patients with liver cirrhosis suffer from energy malnutrition. Late evening snacks (LESs) have been recently reported to be effective for this. However, it is known that a significant proportion of patients with liver cirrhosis have glucose intolerance as a complication. For this reason, the influence of LES on the blood glucose level should be examined. SUBJECTS/METHOD: We administered an oral supplement with branched-chain amino acids (Aminoleban EN) to patients with liver cirrhosis at 10 P.M. to investigate the changes of the blood glucose level and energy metabolism with an indirect calorimeter. Ten patients (average age, 70; Child A/B/C, 5/4/1) participated in this study. The administration period was 7 days. Blood glucose levels were examined before and after breakfast, lunch, supper and at 10 P.M. RESULTS: (1) The fat oxidation rate was significantly decreased and the carbohydrate oxidation rate significantly increased. As a result, RQ was significantly improved. (2) With many cases, an increase of glucose level after meals seemed to reduce with LES administration for 1 week. (3) BTR was significantly improved. CONCLUSIONS: LES could improve energy malnutrition, correct amino acid imbalance, and ultimately may improve glucose intolerance in patients with liver cirrhosis.

Journal Article↗

PubMed2003

Arterial infusion chemotherapy using cisplatin, 5-fluorouracil, and isovorin for patients with advanced hepatocellular carcinoma, pilot study: Is a high dose of the biochemical modulator effective?

The authors report the efficacy of arterial infusion chemotherapy using cisplatin (CDDP), 5-fluorouracil (5-FU), and leucovorin (LV) for patients with advanced hepatocellular carcinoma (HCC). In this study, we evaluated the efficacy of our regimen with high-dose LV, using isovorin (IV) (high dose group), comparing the previous regimen (low-dose LV; low dose group). This is a retrospective, historical, and non-controlled trial. In the high dose group (n=15), one course of chemotherapy consisted of the daily administration of CDDP (10 mg/1 h, for 5 days) and IV (12.5 mg/10 min, for 5 days) followed by 5-FU (250 mg/5 h, for 5 days). In the low dose group (n=9), changing to the administration of LV (12 mg/day), the same regimen was employed. In principle, we did this 20 times. In the high dose group, complete response (CR) was found in two patients, and partial response (PR) in six patients. Thus, the response rate was 53%. In the low dose group, CR was found in two patients, and PR in three patients. Thus, the response rate was 56%. There were no significant differences in the response rate (P=0.71), the survival rate (P=0.29) and the toxicity between the two groups. We considered the recommended dose of LV to be 12 mg/day in our regimen, although this is a preliminary study.

Journal Article↗

PubMed2003

An in vivo model for monitoring trans-differentiation of bone marrow cells into functional hepatocytes.

The plasticity of bone marrow cells (BMCs) remains controversial. The present study found that persistent injury induces efficient trans-differentiation of BMCs into functional hepatocytes. Mice with liver cirrhosis induced by carbon tetrachloride were injected with 1 x 10(5) non-treated green fluorescent protein (GFP)-positive BMCs via the tail vein. In these mice, transplanted GFP-positive BMCs efficiently migrated into the peri-portal area of liver lobules after one day, repopulating 25% of the recipient liver by 4 weeks. In contrast, no GFP-positive BMCs were detected following transplantation into control mice with undamaged livers. BMCs trans-differentiated into functional mature hepatocytes via immature hepatoblasts. Serum albumin levels were significantly elevated to compensate for chronic liver failure in BMC transplantation. These results reveal that recipient conditions and microenvironments represent key factors for successful cell therapy using BMCs.

Albumins↗

PubMed2002

A new cell therapy using bone marrow cells to repair damaged liver.

Hepatic stem cells can transdifferentiate into hepatocytes, bile duct cells, pancreatic cells, or intestinal cells. Hepatic stem cells had been thought to be located in the canal of Hering in the liver, but recent work has demonstrated the existence of hepatic stem cells in bone marrow as well. Cell therapy using autologous bone marrow cells has few ethical problems and many applications for treating severe liver disease. Further in vitro and in vivo analysis is crucial to develop the therapy for clinical use.

Animals↗

PubMed2002

Increased expression of tumor necrosis factor-alpha messenger RNA in the intestinal mucosa of inflammatory bowel disease, particularly in patients with disease in the inactive phase.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha), may be involved in the pathogenesis of inflammatory bowel diseases (IBDs). The aim of this study was to evaluate the effect of TNF-alpha on the inflammatory activity of IBD. METHODS: TNF-alpha mRNA expression in intestinal mucosal biopsy specimens from IBD patients [ulcerative colitis (UC), n = 54; and Crohn's disease (CD), n = 11] was analyzed using a competitive polymerase chain reaction. The degree of macrophage infiltration was analyzed by immunohistochemistry, using an antihuman CD68 antibody. RESULTS: TNF-alpha mRNA expression was increased in UC patients, corresponding to the inflammatory activity. However, in CD, TNF-alpha mRNA expression was not correlated with the endoscopic findings. CONCLUSIONS: We clarified that TNF-alpha mRNA expression was responsible for the inflammatory activity in UC. However, TNF-alpha mRNA expression was not correlated with the mucosal injury in CD.

Adolescent↗

PubMed2002

Effects of secretin on TCDCA- or TDCA-induced cholestatic liver injury in the rat.

Secretin, a gastrointestinal hormone, is known to act on bile duct epithelial cells and has been thought to have no effects on the bile acid transport in the liver. However, secretin was proved recently to stimulate biliary secretion of taurocholic acid (TCA) and elevate the maximum hepatic transport capacity of TCA. In this study, to evaluate the effect of secretin on the biliary secretion of taurochenodeoxycholic acid (TCDCA) or taurodeoxycholic acid (TDCA), which are known as cytotoxic bile acids, changes in bile flow, biliary excretions of bile acids and serum levels of TCDCA or TDCA were studied in a TCDCA- or TDCA-induced cholestatic rat model with and without secretin administration. Secretin prevented the decrease in bile flow and enhanced biliary excretions of bile acids and bicarbonate, but serum levels of TCDCA or TDCA at the end of the study showed no significant changes in the secretin group as compared with controls. Serum levels of alanine and asparate aminotransferases were highly elevated in all rats given TCDCA or TDCA. These data indicate secretin is a possible treatment for patients with prolonged intrahepatic cholestasis.

Journal Article↗

PubMed2002

Prolyl 4-hydroxylase inhibitor is more effective for the inhibition of proliferation than for inhibition of collagen synthesis of rat hepatic stellate cells.

The aim of this study was to investigate whether a prolyl 4-hydroxylase inhibitor (HOE 077) prevents the proliferation and collagen synthesis of rat hepatic stellate cells (HSCs). Rat HSCs were isolated and cultured with 100, 500, 1000 or 2000 &mgr;g/ml of HOE 077 with or without hepatocytes. After 4 day culture, the cell cycle of HSCs was examined by flow cytometry along with messenger RNA expression of procollagen type I. After 4 days of culture, HSCs had DNA synthesis and a high concentration (2000 &mgr;g/ml) of HOE 077 significantly reduced this DNA synthesis. However, HOE 077 incubated with hepatocytes could significantly reduce mitosis at a lower concentration of HOE077 (500 &mgr;g/ml) without significant reduction of alpha(2)(I) procollagen mRNA expression. These results indicate that the prolyl 4-hydroxylase inhibitor is more effective for the inhibition of proliferation than for inhibition of collagen synthesis of HSCs.

Journal Article↗

PubMed2002

Antioxidant, N-acetyl-L-cysteine inhibits the expression of the collagen alpha2 (I) promoter in the activated human hepatic stellate cell line in the absence as well as the presence of transforming growth factor-beta.

Although recent studies suggest the inhibitory property of N-acetyl-L-cysteine (NAC) on activation of hepatic stellate cell (HSC), the effects on once-activated HSCs are not well clarified. We investigated the influences of NAC on human-derived once-activated HSC line, LI90 with a focus on the collagen alpha2 (I) (COL1A2) promoter expression. Plasmid containing whole length of COL1A2 promoter linked to firefly luciferase gene and its various 5'-deletions were transiently transfected to LI90. The luciferase activity was determined with or without 10 mM of NAC in the absence or presence of 1 ng/ml of transforming growth factor (TGF)-beta. The effects of NAC on generation of intracellular reactive oxygen species (ROS) in LI90 were also analyzed. As a result, NAC significantly (P<0.05) suppressed the COL1A2 promoter expression in the absence or presence of TGF-beta. The expression was much more inhibited when used the deletion containing only AP-1/NF-kappaB binding sites than that including only three SP-1 binding sites. The ROS production was also comparably inhibited by NAC in both condition. These results indicated NAC suppressed, through its anti-oxidative action, the COL1A2 promoter expression in once-activated HSCs in the absence or presence of TGF-beta at least partly by affecting the signal transduction cascade encompassing AP-1/NF-kappaB activation.

Journal Article↗

PubMed2002

Th1 down-regulation at the single-lymphocyte level in HCV-related liver cirrhosis and the effect of TGF-beta on Th1 response: possible implications for the development of hepatoma.

Patients with hepatitis C virus (HCV) related-liver cirrhosis (LC) often develop hepatoma. The type 1 helper T cell (Th1) response presents an antitumor effect. We evaluated the Th1 response in patients with HCV-related LC at the single-cell level and examined the influence of transforming growth factor (TGF)-beta, an immunosuppressive cytokine, on the Th1 response. We determined the ratios of Th1-type cytokine (IFN-gamma, IL-2)-producing cells to CD3-positive cells in 14 patients (LC group) and in 16 normal controls using flow cytometry and measured serum TGF-beta(1) and TGF-beta(2) levels by ELISA. We then incubated, peripheral blood mononuclear cells from seven healthy volunteers with recombinant TGF-beta(1) or TGF-beta(2) for 48 h, and determined the ratio of IFN-gamma producing cells to CD3-positive cells. The IFN-gamma ratio was significantly lower in the LC group (29.7+/-0.3 vs. 44.2+/-15.0%, P<0.01). The serum TGF-beta(2) level was significantly increased in the LC group (601+/-232 vs. 329+/-118 pg/ml, P<0.001). TGF-beta(2) significantly suppressed IFN-gamma production at the single-cell level (10.0+/-4.3 vs. 7.3+/-2.0%, P<0.05). These findings indicated that the enhanced down-regulation of Th1 by TGF-beta(2) in patients with HCV-related LC might be effective against hepatoma.

Journal Article↗

PubMed2002

Effects on liver functions of gastric variceal therapy by endoscopic ligation using a detachable snare and sclerotherapy with O-ring ligation.

Although various effects of therapy for gastric varices, a complication of portal hypertension, on portal hemodynamics have been suggested, the effects of the therapy on liver functions have not yet been clarified. The aim of this study was to examine the effects of endoscopic gastric variceal therapy on liver functions in patients with hepatic cirrhosis. The subjects were 18 hepatic cirrhosis patients with the complication of gastric varices, who could be monitored for a long term (at least 1-year after therapy). They consisted of patients receiving prophylactic treatments after being confirmed to have a risk of rupture of gastric varices (16 prophylactic cases) and those with a history of bleeding and receiving palliative treatments to prevent rebleeding (two palliative cases). To evaluate liver functions, blood tests before the therapy, just after the therapy and at 1, 3, 6 and 12 months after the therapy were retrospectively examined and the levels of albumin, total bilirubin (T.Bil), ALT, choline esterase (ChE), platelets and the Child-Pugh classification were assessed. The albumin levels tended to increase after the therapy, and the level at 6 months after the therapy was significantly higher than the pre-treatment value (P=0.0311). At 1 year after the therapy, the level was higher than the pre-treatment value, but there was no significant difference. The ChE level tended to decrease just after the therapy, but it tended to be increased at 3 and 6 months after the therapy. Although significant improvement was not seen in T.Bil, ALT, platelet levels or the Child-Pugh classification after the therapy, none of these worsened. Endoscopic gastric variceal ligation therapy significantly improved liver functions, or at least albumin synthesis.

Journal Article↗

PubMed2002

Ubiquitin is a possible new predictive marker for the recurrence of human hepatocellular carcinoma.

BACKGROUND/AIM: Ubiquitin (Ub)-dependent degradation of regulatory proteins controls many cellular processes such as cell cycle progression, morphogenesis and signal transduction. In this study, we evaluated the meaning of ubiquitination in chronic liver diseases, especially human hepatocellular carcinoma, with regard to recurrence. METHODS: A total 74 of liver tissues (8 of chronic hepatitis [CH], 9 of liver cirrhosis [LC], 7 of dysplastic nodule low grade (DSL), or dysplastic nodule high grade (DSH)and 50 of hepatocellular carcinoma [HCC]) were analyzed for ubiquitination by immunohistochemistry. Cell proliferation was also analyzed using Ki-67 staining. As a comparative marker for progression of HCC, PIVKA-II (protein induced by vitamin K absence-II) was employed to examine the recurrence rate of HCC. RESULTS: Ubiquitin (Ub) was positive in nuclei and cytoplasm of HCC in immunohistochemistry. The labeling index (L.I.) of ubiquitination was significantly higher with HCC than with other chronic liver diseases and tended to correlate with the lack of poorly-differentiated of HCC. The L.I. of Ki-67 staining was also correlated (P < 0.0001) with that of ubiquitination. The hepatocellular carcinoma (HCC) samples from potentially curatively operated patients having a ubiquitination L.I. of more than 20% suffered significantly higher recurrence of HCC than did patients with an L.I. of less than 20%. On the other hand, PIVA-II did not show such a difference. CONCLUSION: Ubiquitin (Ub) may reflect the growth activity of neoplasms and will be a possible new predictive marker for the recurrence of human hepatocellular carcinoma after potentially curative operation.

Biomarkers↗

PubMed2002

Current strategies for chemoprevention of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) develops frequently in the liver of patients with chronic hepatitis and cirrhosis caused by persistent infection with hepatitis B virus or hepatitis C virus in Asia. Several studies including ours have revealed that the risk of HCC increases in parallel with the progression of hepatic fibrosis associated with chronic hepatitis and liver cirrhosis. It may be delineated reasonably, therefore, that suppression of fibrosis by chemical (or biological) agents would be able to decrease the risk of HCC. We have demonstrated that antifibrotic agents, such as HOE 077, TJ-9 and interferon, can reduce the risk of HCC. Accordingly, antifibrotic agents are promising candidates for chemoprevention of HCC. The results of our study are discussed within the scope of the current state of the art as regards chemopreventing HCC.

Animals↗

PubMedDate not supplied

Elevated levels of circulating cell-free DNA in the blood of patients with hepatitis C virus-associated hepatocellular carcinoma.

BACKGROUND: Circulating cell-free DNA is present in increased amounts in the blood of patients with one of several forms of cancer. MATERIALS AND METHODS: A real-time PCR assay with glutathione S-transferase pi (GSTP1) gene was used to measure cell-free DNA levels in the sera of 52 patients with hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV), which included 30 HCV carriers without known HCC and 16 HCV-negative non-cancer patients (controls). RESULTS: Cell-free DNA levels were significantly higher in the sera from HCC patients than in the sera from HCV carriers or the control subjects. Cell-free DNA levels were associated with the degree of tumor differentiation and size but not patient age, gender, TNM stage or levels of alpha-fetoprotein (AFP) or protein induced by vitamin K absence (PIVKA-II). The cell-free DNA assay had a sensitivity of 69.2% and a specificity of 93.3% in discriminating HCC and HCV carriers at the optimal cut-off value of 73.0 ng/ml, with an area of 0.90 (95% CI, 0.83-0.96) under the receiver operating characteristic curve. The discriminative power of cell-free DNA was superior to that of AFP or PIVKA-II. CONCLUSION: Our results showed that levels of circulating cell-free DNA are significantly increased in sera of patients with HCV-associated HCC, suggesting that circulating cell-free DNA may be a good biomarker specific for HCV-associated HCC.

Aged↗