Tsuyoshi Yoshimoto

Nutritional investigation of non-obese patients with non-alcoholic fatty liver disease: the significance of dietary cholesterol.

Objective. The onset and progression of non-alcoholic fatty liver disease (NAFLD) seem to be affected by nutritive intake; however, detailed examinations have not been performed in non-obese NAFLD patients. The purpose of this study was to identify potential nutritive factors that affect NAFLD and its related nutritional problems. Material and methods. We investigated the distribution of abdominal fat, dietary intake, and biochemical data in patients with NAFLD and compared non-obese with obese patients. Results. There was no significant difference in the percentage of patients with diabetes or dyslipidemia between the obese and non-obese groups. Waist circumference, total abdominal fat levels, and subcutaneous fat levels were significantly higher in the obese group, while visceral fat levels were not significantly different between the two groups. Immunoreactive insulin (IRI) and homeostasis model assessment-insulin resistance (HOMA-IR) were significantly lower in the non-obese group, suggesting that the non-obese patients were not overtly insulin resistant. Although serum adiponectin and TNF-α levels were similar in both groups, leptin levels were significantly higher in the obese group. Total energy and carbohydrate intake tended to be higher in the obese group. A characteristic feature was that dietary cholesterol intake was significantly higher, while the intake of polyunsaturated fatty acids (PUFAs) was significantly lower in the non-obese group. Conclusions. In non-obese NAFLD patients: 1) although visceral fat was increased, insulin resistance and/or dysregulated secretion of adipocytokines was not necessarily shown; 2) intakes of total energy and carbohydrates were not excessive, although dietary cholesterol was superabundant and dietary PUFAs were significantly lower compared with those in obese patients; and 3) characteristic fat intake may be associated with the formation of NAFLD.

Expression profile of lipid metabolism‐associated genes in hepatitis C virus‐infected human liver

Aim:  Recent studies have shown that lipid metabolic pathways are required for the entry, replication and secretion of hepatitis C virus (HCV). Although little is known about the life cycle of HCV in humans, the activation of cholesterol and fatty acid biosynthesis may be critical for HCV proliferation.

Validity of FibroScan values for predicting hepatic fibrosis stage in patients with chronic HCV infection

OBJECTIVE:  The aim of this study was to validate the FibroScan system compared with liver histology and serum markers for the diagnosis of hepatic fibrosis. We also tried to determine the cut‐off levels and assess the feasibility of using FibroScan values to predict the fibrosis stage.

Expression profiles of genes associated with viral entry in HCV‐infected human liver

Recent studies have demonstrated that several cellular factors are involved in entry of hepatitis C virus (HCV) into host cells. Detailed gene expression profiles of these factors in HCV‐infected livers have not been reported for humans. Transcriptional levels of LDL receptor (LDLR), CD81, scavenger receptor class B type I (SR‐BI), claudin‐1, and occludin genes in liver samples from patients with chronic hepatitis C were investigated. Serum levels of LDL‐cholesterol (LDL‐C) and HCV core antigen were also evaluated, and expression of claudin‐1 and occludin were immunohistochemically analyzed. Compared with normal liver, transcription of LDLR and claudin‐1 genes was significantly suppressed (P < 0.0001) and occludin transcription was significantly up‐regulated in HCV‐infected livers (P < 0.0001). Significant positive correlations were found for LDLR versus occludin, LDLR versus claudin‐1, occludin versus claudin‐1, and CD81 versus SR‐BI in HCV‐infected (P = 0.0012, P < 0.0001, P = 0.0004, and P < 0.0001, respectively) and normal livers (P < 0.0001, P = 0.0051, P < 0.0001, and P < 0.0001, respectively). Positive correlation was observed between serum levels of HCV core antigen and LDL‐C (P = 0.0147), with their levels negatively correlated to LDLR (P = 0.0270 and P = 0.0021, respectively). Immunohistochemically, hepatocellular expression of claudin‐1 and occludin was increased in HCV‐infected livers. Different levels of expression were demonstrated at the mRNA and protein levels for occludin and claudin‐1 in HCV‐infected and normal livers. Correlation of elements associated with viral entry was comparable in HCV‐infected and normal livers. J. Med. Virol. 83:921–927, 2011.

Therapeutic effect of bezafibrate against biliary damage: a study of phospholipid secretion via the PPARalpha-MDR3 pathway.

OBJECTIVE Bezafibrate (BF) has been used to treat biliary damage, particularly in patients with primary biliary cirrhosis (PBC), and its clinical efficacy has been demonstrated. The mechanism of action is thought to involve activation of the PPARalpha-MDR3-phospholipid (PL) secretion pathway. We tried to confirm this hypothesis in patients with hepatobiliary disease. METHODS The levels of serum gamma-glutamyl transpeptidase and alkaline phosphatase, and those of bile components were examined before and after BF administration in patients with obstructive jaundice undergoing percutaneous transhepatic biliary drainage (PTBD). Hepatic expression of PPARalpha and MDR3 was quantified by real-time PCR in patients with PBC or non-alcoholic fatty liver disease (NAFLD). RESULTS In patients with obstructive jaundice, BF decreased the serum levels of biliary enzymes and increased the bile concentration of PL. In patients with PBC or NAFLD, the expression levels of MDR3 were already up-regulated before starting the BF treatment. Although BF treatment did not further up-regulate MDR3 expression in NAFLD patients, PPARalpha expression was significantly increased. CONCLUSIONS BF enhanced the secretion of PL into bile in cholestatic patients undergoing PTBD. However, in patients with PBC or NAFLD, diseases that represent cholesterol overload, MDR3 was already expressed at a high level to compensate for bile acids overproduction, and its expression was hardly affected by BF. In patients with chronic liver diseases such as PBC, BF may induce clinical effects via mechanisms independent of PL secretion.

Add‐on therapy of pitavastatin and eicosapentaenoic acid improves outcome of peginterferon plus ribavirin treatment for chronic hepatitis C

Despite the use of pegylated‐interferon (peg‐IFN) plus ribavirin combination therapy, many patients infected with hepatitis C virus (HCV)‐1b remain HCV‐positive. To determine whether addition of pitavastatin and eicosapentaenoic acid (EPA) is beneficial, the “add‐on” therapy option (add‐on group) was compared retrospectively with unmodified peg‐IFN/ribavirin therapy (standard group). Association of host‐ or virus‐related factors with sustained virological response was assessed. In HCV replicon cells, the effects of pitavastatin and/or EPA on HCV replication and expression of innate‐immunity‐ and lipid‐metabolism‐associated genes were investigated. In patients infected with HCV‐1b, sustained virological response rates were significantly higher in the add‐on than standard group. In both groups, sustained virological response rates were significantly higher in patients with genotype TT of IL‐28B (rs8099917) than in those with non‐TT genotype. Among the patients with non‐TT genotype, sustained virological response rates were markedly higher in the add‐on than standard group. By multivariate analysis, genome variation of IL28B but not add‐on therapy remained as a predictive factor of sustained virological response. In replicon cells, pitavastatin and EPA suppressed HCV replication. Activation of innate immunity was obvious in pitavastatin‐treated cells and EPA suppressed the expression of sterol regulatory element binding protein‐1c and low‐density lipoprotein receptor. Addition of pitavastatin and EPA to peg‐IFN/ribavirin treatment improved sustained virological response in patients infected with HCV‐1b. Genotype variation of IL‐28B is a strong predictive factor in add‐on therapy. J. Med. Virol. 85:250–260, 2013.

The state of cholesterol metabolism in the liver of patients with primary biliary cirrhosis: the role of MDR3 expression.

AimBecause dyslipidemia, such as hypercholesterolemia, is a characteristic of primary biliary cirrhosis (PBC), hepatic lipid metabolism may be disturbed in PBC patients. We examined the expression of lipid metabolism-associated genes in PBC liver.MethodsAll of the patients examined were in stage I or II PBC and without medication. RNA was isolated from liver specimens by needle biopsies of PBC patients and controls. The expression levels of various genes were measured by real-time RT-PCR. Multidrug resistance 3 (MDR3) expression was examined immunohistochemically. Statistical correlations between the gene expression levels and indices of blood testing were calculated.ResultsThe expression levels of sterol regulatory element-binding protein (SREBP) 2 and LDL receptor were significantly lower, and those of apolipoprotein B, microsomal triglyceride transfer protein, ATP-binding cassette G5, and liver X receptor α (LXRα) were significantly higher in the PBC liver than in the normal control liver. The expression levels of bile acid synthesis- and excretion-associated genes did not change, and those of farnesoid X receptor, peroxisome proliferator-activated receptor α, and SREBP-1c were similar between the PBC and normal liver. MDR3 gene expression levels in the PBC liver were more than 4-fold higher than those in the control liver. Immunohistochemically, strong canalicular staining for MDR3 was observed in the PBC liver. LXRα expression was positively correlated with MDR3 levels. Serum levels of γ-glutamyl transpeptidase (GGT) and IgM were negatively correlated with MDR3 levels.ConclusionsHepatocellular cholesterol metabolism was at least partially disturbed, even in the early stage of PBC. The most characteristic finding was a distinct elevation of MDR3 expression, and the MDR3 levels were negatively correlated with GGT and IgM levels.

RCAS1-expressing macrophages in inflammatory liver diseases

To the editor: The FAS/Fas ligand (FasL) system is one of the main apoptosis-inducing systems and is involved deeply in pathologic changes in many diseases including cancers and autoimmune diseases. Recently, FasL expression in macrophages has been reported, and macrophage is considered to be an important component in liver diseases. In primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH), FasL-positive macrophages were identified and it was considered that the expression was involved in apoptosis of hepatocytes and bile duct cells (1, 2). RCAS1 is also considered to be a mediator for another apoptosis-inducing system. RCAS1 was first identified as a tumor-associated antigen in gynecological cancer (3); however, the antigen was expressed in various kinds of carcinoma cells including hepatocellular carcinoma and cholangiocarcinoma (4, 5). RCAS1 exists both in membrane-bound and soluble forms. The receptor for RCAS1 is expressed on the surface of lymphocytes, especially in their activated state, and RCAS1 binding to the receptor results in apoptosis of lymphocytes (6). Therefore, RCAS1 is thought to function in the evasion of immune surveillance and in promoting tumor progression. We have reported that RCAS1, in addition to cancer cells, can also be produced by target cells in autoimmune liver diseases (5, 7). In PBC, biliary epithelial cells are RCAS1-positive, especially in active inflammatory areas, and may defend themselves from immune attack by inducing apoptosis of lymphocytes. Here, we report on another new and noteworthy finding in inflammatory liver diseases; namely that infiltrating macrophages and/or Kupffer cells can also be positive for RCAS1. In addition, based on previous in vitro studies and the results of immunohistochemical staining in this study, we infer the functional difference between RCAS1 and FasL. We immunohistochemically assessed expression of RCAS1, FasL, and a specific marker for macrophages/Kupffer cells, LN-5 (8), in liver biopsy samples from patients with chronic viral hepatitis (n5 13, HBV/HCV5 3/10), acute or acute on chronic hepatitis (n5 12, HBV/druginduced/unknown etiology5 3/3/6), AIH (n5 8), graft vs. host disease (GVHD, n58), PBC (n514), and primary sclerosing cholangitis (PSC, n5 6). Informed consent was obtained from each patient prior to their inclusion in the study. In our samples of chronic viral hepatitis, Kupffer cells were reactive for LN-5 and, generally, LN-5 were positive with not very large number of infiltrated mononuclear cells in the portal/periportal areas regardless of serum alanine transaminase (ALT) levels in individuals. Serum ALT levels of these patients on the day of the biopsy were 27–365 IU/l (median 147 IU/l). Several FasL-positive cells were also seen mainly in the portal areas and many of them appeared to be positive for LN-5. On the other hand, we could not find RCAS1 positivity in mononuclear cells, hepatocytes, biliary epithelial cells, and other components. In case of acute or acute on chronic hepatitis, serum ALT levels of the patients on the day of the biopsy were 274–4660 IU/l (median 1131 IU/l). Generally, there were many LN-5-positive mononuclear cells in the hepatic lobules and portal areas. In the individual case, various percentages of LN-5-positive cells were also FasL-positive, and especially in the portal areas most of the infiltrated macrophages were FasL-positive. The positive cell number or percentage for LN-5 or FasL was not related to the serum ALT levels of the patients. By contrast, RCAS1-positive cells tended to be found in the patients with higher serum ALT levels (4647 IU/l), although the number was not many. RCAS1-positive cells were mainly shown at the areas of massive hepatocytic death, and most of the cells showed positivity for FasL and LN-5 (data not shown). Immunohistochemical staining for RCAS1, FasL, and LN-5 was tested on the tissue samples from patients with AIH (n5 8, ALT levels: 26–609 IU/l, median 88 IU/l), GVHD (n5 8, ALT levels: 48–998 IU/l, median 204.5 IU/l), Liver International 2006: 26: 385–387 r 2006 The Author Journal compilation r 2006 Blackwell Munksgaard

Comparison of tissue pressure and ablation time between the LeVeen and cool-tip needle methods

BackgroundRadio frequency ablation (RFA) has been accepted clinically as a useful local treatment for hepatocellular carcinoma (HCC). However, intrahepatic recurrence after RFA has been reported which might be attributable to increase in intra-tumor pressure during RFA. To reduce the pressure and ablation time, we developed a novel method of RFA, a multi-step method in which a LeVeen needle, an expansion-type electrode, is incrementally and stepwise expanded. We compared the maximal pressure during ablation and the total ablation time among the multi-step method, single-step method (a standard single-step full expansion with a LeVeen needle), and the method with a cool-tip electrode. Finally, we performed a preliminary comparison of the ablation times for these methods in HCC cases.ResultsA block of pig liver sealed in a rigid plastic case was used as a model of an HCC tumor with a capsule. The multi-step method with the LeVeen electrode resulted in the lowest pressure as compared with the single-step or cool-tip methods. There was no significant difference in the ablation time between the multi-step and cool-tip ablation methods, although the single-step methods had longer ablation times than the other ablation procedures. In HCC cases, the multi-step method had a significantly shorter ablation time than the single-step or cool-tip methods.ConclusionWe demonstrated that the multi-step method was useful to reduce the ablation time and to suppress the increase in pressure. The multi-step method using a LeVeen needle may be a clinically applicable procedure for RFA.

Decreased portal flow volume increases the area of necrosis caused by radio frequency ablation in pigs.

Background/aims: Although radio frequency ablation (RFA) has been widely accepted as an effective treatment for hepatocellular carcinoma (HCC), severe complications are not uncommon. Major complications seem to occur as a result of over‐ablation beyond the intended area. As most patients with HCC have underlying cirrhosis, we speculated that decreased portal flow might cause the necrosis associated with RFA. To confirm this hypothesis, we examined the area of necrosis resulting from RFA under varying conditions of portal flow in a porcine model.