Serum albumin is present at higher levels in alcoholic liver cirrhosis as compared to HCV-related cirrhosis.

Residual hepatic functional reserve in cirrhotic patients is generally evaluated by a multivariate scoring system (Child-Pugh classification), which includes serum albumin levels as a variable. However, several patients show discrepancies between serum albumin levels and the progression of liver fibrosis, especially those with alcoholic cirrhosis. To assess whether hepatic capacity of protein synthesis varies with the etiology of cirrhosis, serum albumin and cholinesterase levels, and prothrombin time were compared between alcoholic cirrhosis and hepatitis C virus (HCV)-related cirrhosis. To minimize the influence of malnutrition and extrahepatic platelet destruction, patients with hepatocellular carcinoma, uncontrolled diabetes, appetite loss and/or splenal longitudinal size >15 cm were excluded. The patients with compensated liver cirrhosis were divided into three groups as follows: alcohol(+)/HCV(+) (alcohol + HCV group; n=31), alcohol(-)/HCV(+) (HCV group; n=31) and alcohol(+)/HCV(-) (alcohol group; n=27). These groups were adjusted with respect to age, gender, body mass index and platelet count. Serum albumin levels in the alcohol group were significantly higher than those in the HCV group, with a difference of approximately 0.5 g/dl in every class of platelet count. The correlation of the alcohol + HCV group was intermediate between the alcohol and HCV groups. On the other hand, the correlations between serum cholinesterase levels and platelet counts were similar among the three groups. The prothrombin time was also comparable among the groups. Accordingly, serum albumin levels were higher in patients with alcoholic cirrhosis and alcohol consumption should be carefully considered when evaluating hepatic functional reserve.

Fertile females with nonalcoholic fatty liver disease (NAFLD) have higher levels of ALT than postmenopausal females: implications for the influence of fertility on NAFLD.

BACKGROUND/AIMS: Insulin resistance recently has been reported to play a major role in nonalcoholic fatty liver disease (NAFLD). We evaluated the influence of fertility on fatty liver injury in fertile and postmenopausal women with insulin resistance. METHODOLOGY: We investigated 152 patients with noninsulin-dependent diabetes mellitus without insulin treatment; 46 males, 52 fertile women and 54 postmenopausal women. All had liver damage and/or steatosis recognized by ultrasonography. We measured the fasting serum levels of C-peptide and insulin, as markers of insulin resistance, and the serum levels of ALT. The severity of liver steatosis was judged by ultrasonography. RESULTS: Fertile females had significantly higher levels of ALT and demonstrated a more significant correlation between serum levels of ALT and C-peptide or insulin than did the postmenopausal females or males. Fertile females with moderate to severe steatosis had significantly higher levels of ALT than those with mild or no steatosis, although such a significant difference was not found in postmenopausal females or males. CONCLUSIONS: We demonstrate that fertility is an important factor in fatty liver damage of NAFLD with insulin resistance, suggesting that estrogen may exacerbate nonalcoholic steatohepatitis.

Epigallocatechin-3-gallate, a polyphenol component of green tea, suppresses both collagen production and collagenase activity in hepatic stellate cells.

Catechins such as epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), and epigallocatechin (EGC) are polyphenol components of green tea. EGCG is the major component and has been reported to possess a wide range of biological properties including anti-fibrogenic activity. In hepatic fibrosis, activated hepatic stellate cells (HSCs) play a central role. In this study, we investigated the effect of catechins, including EGCG, on collagen production and collagenase activity in rat primary HSCs and activated human HSC-derived TWNT-4 cells. EGCG (50 microM) suppressed type I collagen production in rat HSCs more than ECG (50 microM) did; however, EGC (50 microM) did not show suppressive effects. EGCG also inhibited both collagen production and collagenase activity (active matrix metalloproteinase-1 [MMP-1]) in a dose-dependent manner, but did not affect the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production in TWNT-4 cells. Real-time PCR unexpectedly revealed that EGCG enhanced the transcription of type I collagen and TIMP-1, but did not affect the transcription of alpha-smooth muscle actin (alpha-SMA), and reduced the transcription MMP-1 in TWNT-4 cells. These findings demonstrated that EGCG inhibited collagen production regardless of enhanced collagen transcription and suppressed collagenase activity, and suggested that EGCG might have therapeutic potential for liver fibrosis.

High glucose stimulates hepatic stellate cells to proliferate and to produce collagen through free radical production and activation of mitogen-activated protein kinase.

BACKGROUND: Nonalcoholic steatohepatitis is a clinicopathologic condition that may progress to liver fibrosis. Hyperglycemia is supposed to be one of the factors inducing hepatic fibrogenesis, but the mechanism has not been fully clarified. Oxidative stress is increasingly found in patients with diabetes/hyperglycemia in which conditions reactive oxygen species (ROS) are produced. METHODS: We performed experiments using hepatic stellate cells (HSCs) in culture in order to confirm the effect of high glucose concentrations on cell proliferation, type I collagen production, ROS production and activation of mitogen-activated protein (MAP) kinase pathway. RESULTS: High glucose stimulated cell growth of HSCs and up-regulated the levels of activated/phosphorylated extracellular signal-regulated kinase 1/2 and free radical production in HSCs. The MAP kinase phosphorylation and cell proliferation were suppressed by diphenylene iodonium chloride, an NADPH oxidase inhibitor, and by calphostin C, a protein kinase C (PKC)-specific inhibitor. Increased type I collagen mRNA and protein levels were also observed in HSCs at high glucose concentrations. CONCLUSIONS: Our findings indicate that high glucose concentrations may stimulate ROS production through PKC-dependent activation of NADPH oxidase, and induce MAP kinase phosphorylation subsequent to proliferation and type I collagen production by HSCs.

Beraprost sodium, a prostacyclin (PGI) analogue, ameliorates concanavalin A-induced liver injury in mice.

BACKGROUND/AIMS: Prostacyclin (PGI(2)) is a potent mediator in the inflammatory and coagulation processes. The aim of this study was to test whether beraprost sodium, a PGI(2) analogue, could prevent experimental hepatic injury induced by concanavalin A (Con A), which is a model of fulminant hepatic failure. METHODS: Beraprost (100 microg/kg) was administered intraperitoneally simultaneously with Con A (40 mg/kg) in C57B6J mice. Blood circulation in the liver was determined by laser-Doppler flowmetry. Plasma levels of alanine aminotransferase (ALT), tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6 were determined. Levels of TNF-alpha and IFN-gamma in culture supernatant of splenocytes were also determined. RESULTS: Beraprost administration reduced the incidence of death following hepatic failure (76.5% vs. 29.4%, P<0.05). Plasma levels of ALT were significantly lower in the beraprost-treated group than in the control group, and in the former, there was concomitant suppression of the histological features of injury. Beraprost significantly increased hepatic blood flow volume in Con A-treated mice. Plasma levels of TNF-alpha and IFN-gamma were significantly reduced at 6 and 12 h after Con A injection, respectively, but the levels of IL-6 were increased at 6 h. In vitro, beraprost also suppressed Con A-induced TNF-alpha production in splenocytes, while it stimulated IFN-gamma production. CONCLUSION: These findings imply that beraprost suppresses Con A-induced liver injury. These data also suggest that beraprost, which is clinically effective in treating pulmonary hypertension, may have therapeutic potential for preventing hepatic injury.

[A case of idiopathic portal hypertension complicated with sepsis caused by Aeromonas hydrophila].

A 53 year old man with idiopathic portal hypertension (IPH) was admitted because of high fever and diarrhea. Nineteen years before admission, he had received splenic hilar renal shunt operation with proximal flush ligation of splenic vein due to gastric varices. Three months before admission, he had been admitted to our hospital for evaluation of liver dysfunction. Liver biopsy examination had revealed peri-portal fibrosis consistent with IPH. Aeromonas hydrophila was isolated from blood. Although he was treated with antibiotics plus dopamine, glucose-insulin therapy, and mechanical ventilation, he had severe septic shock, and died 29 days after admission. We have to take notice of A. hydrophila infection in cases of portosystemic shunt because they fall in severe septic shock.

Fasudil hydrochloride hydrate, a Rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells.

BACKGROUND/AIMS: The Rho-ROCK signaling pathways play an important role in the activation of hepatic stellate cells (HSCs). We investigated the effects of fasudil hydrochloride hydrate (fasudil), a Rho-kinase (ROCK) inhibitor, on cell growth, collagen production, and collagenase activity in HSCs. METHODS: Rat HSCs and human HSC-derived TWNT-4 cells were cultured for studies on stress fiber formation and alpha-smooth muscle actin (alpha-SMA) expression. Proliferation was measured by BrdU incorporation, and apoptosis by TUNEL assay. The phosphorylation states of the MAP kinases (MAPKs), extra cellular signal -regulated kinase 1/2 (ERK1/2), c-jun kinase (JNK), and p38 were evaluated by western blot analysis. Type I collagen, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production and gene expression were evaluated by ELISA and real-time PCR, respectively. Collagenase activity (active MMP-1) was also evaluated. RESULTS: Fasudil (100 microM) inhibited cell spreading, the formation of stress fibers, and expression of alpha-SMA with concomitant suppression of cell growth, although it did not induce apoptosis. Fasudil inhibited phosphorylation of ERK1/2, JNK, and p38. Treatment with fasudil suppressed the production and transcription of collagen and TIMP, stimulated the production and transcription of MMP-1, and enhanced collagenase activity. CONCLUSION: These findings demonstrated that fasudil not only suppresses proliferation and collagen production but also increases collagenase activity.

Epigallocatechin-3-gallate, a green-tea polyphenol, suppresses Rho signaling in TWNT-4 human hepatic stellate cells.

Epigallocatechin-3-gallate (EGCG), a major constituent of the polyphenoids in green tea, has been reported to possess a wide range of biologic activities, including antifibrogenesis. Activated hepatic stellate cells (HSCs) are central to hepatic fibrosis, and Rho (a small GTPase)-signaling pathways have been implicated in the activation and proliferation of HSCs. In this study, we investigated the effect of EGCG on Rho-signaling pathways in activated human HSC-derived TWNT-4 cells. EGCG inhibited stress-fiber formation, an indicator of Rho activation, and changed the distribution of alpha-smooth-muscle actin. These inhibitory effects of EGCG were restored by overexpression of constitutively active Rho. A pull-down assay revealed that activated Rho (GTP-bound state) was strongly inhibited by ECGC and accompanied by suppressed phosphorylation of focal adhesion kinase, which is a regulator of Rho-signaling pathways. 5-Bromo-2'-deoxy-uridine incorporation demonstrated that ECGC (100 micromol/L suppressed cell growth by 80%, and terminal deoxynucleotidyl transferase viotin-deoxyruidine triphosphate nick end-labeling revealed that EGCG (100 micromol/L) caused apoptosis in half of the total cells. EGCG also strongly inhibited lysophoaphatidic acid (an activator of Rho) and induced phosphorylation of mitogen-activated protein kinases (Erk1/2, c-jun kinase, and p38). These findings demonstrate that EGCG regulates the structure and growth of HSCs by way of Rho-signaling pathways and suggest that EGCG has therapeutic potential in the setting of liver fibrosis.

Lemierre's syndrome: Porphyromonas asaccharolytica as a putative pathogen.

A case of Lemierre's syndrome is reported in which metastatic abscesses resulted from septic thrombophlebitis of the internal jugular vein secondary to bacterial pharyngitis. A 32-year-old male suffering from a painful left-sided neck mass, sore throat, and fever was admitted to our hospital. Computed tomography revealed thrombosis of the left internal jugular vein, septic pulmonary emboli, and a liver abscess. Blood culture showed Porphyromonas asaccharolytica. Although empyema occurred transiently during the treatment, the patient recovered following prolonged antimicrobial therapy. Although Fusobacterium species are a well-known cause of Lemierre's syndrome, cases in whom Porphyromonas species was isolated have scarcely been reported. Moreover, case reports from Japan have been few.

A multi-step, incremental expansion method for radio frequency ablation: optimization of the procedure to prevent increases in intra-tumor pressure and to reduce the ablation time.

BACKGROUND/AIMS: Radio frequency ablation (RFA) has been accepted clinically as a useful local treatment for hepatocellular carcinoma (HCC). However, intra-hepatic recurrence after RFA has been reported. We initially hypothesized that recurrence was attributable to increases in intra-tumor pressure during RFA, and we subsequently measured the pressure and optimized the procedure. METHODS: A block of pig liver sealed in a rigid plastic case was used as a model of an HCC tumor with a capsule. We compared the pressure between a single-step full expansion of the needle (single-step method) and incremental, stepwise expansion (multi-step method), and evaluated the effect of varying the electrical power. Finally, we performed a preliminary comparison of the ablation times for these methods in HCC cases. RESULTS: The multi-step method resulted in a significantly lower pressure and shorter total ablation time than the single-step method. Furthermore, incremental expansion in 10 steps resulted in a lower pressure and shorter ablation time than four steps. Seventy W-ablation resulted in a lower pressure and shorter time than 30- or 50 W-ablation. In HCC cases, the multiple-step method had a significantly shorter ablation time than the single-step method. CONCLUSION: The multi-step method can be recommended to reduce the ablation time, and suppress the increase in pressure.

High relative fat-free mass is important for maintaining serum albumin levels in patients with compensated liver cirrhosis.

AIM: In patients with liver cirrhosis, hypoalbuminemia causes edema and ascites, and a reduction in the quality of life. Since musculature is catabolized to supply amino acids for albumin synthesis in malnutritional cirrhotic patients, muscular volume is hypothesized to play an important role in albumin production. Therefore, we investigated the correlation between serum albumin levels and the fat-free mass (FFM) in cirrhotic patients. METHODS: Fifty-seven patients (26 males and 31 females) with compensated liver cirrhosis were evaluated. Patients with edema or ascites were excluded from the study. Healthy volunteers (n = 104; 48 males and 56 females) were also evaluated as controls. FFM was measured using 5-500 kHz multifrequency bioelectric impedance analysis. To minimize the difference in FFM distribution between males and females, we introduced a new marker, relative FFM (rFFM), which represents the ratio of FFM in a patient relative to that in a volunteer of the same height. Following FFM measurement, the serum albumin levels of patients were assayed monthly. RESULTS: In patients with active cirrhosis (alanine aminotransaminase (ALT) > 50 U/L), both albumin (the difference between maximum and minimum levels) and the standard deviation of albumin levels (SD-albumin) during the observation period showed a significant correlation with rFFM. Multiple linear regression analysis using variables such as rFFM, platelet number, and serum cholesterol levels, choline esterase, albumin, bilirubin, and ALT revealed that rFFM and ALT were significant and independent factors that influenced albumin or SD-albumin in cirrhotic patients. CONCLUSION: Our results indicate that cirrhotic patients with high rFFM showed less of a decrease in albumin levels, and that the muscle volume is one of the most important factors for maintaining serum albumins level in active cirrhosis. Exercise and protein-rich nutrition at the early stage of liver cirrhosis may be advisable for maintaining or increasing muscular volume.

Clinical significance of serum levels of vascular endothelial growth factor and its receptor in biliary disease and carcinoma.

AIM: To investigate the clinical significance of serum vascular endothelial growth factor (VEGF) and soluble VEGF receptor-1 (VEGFR1/Flt-1) (sVEGFR1) levels in biliary diseases. METHODS: We analyzed the serum levels of these proteins in patients with acute cholangitis (group 1), biliary malignancies (group 2), and primary biliary cirrhosis or primary sclerosing cholangitis (group 3), and in healthy donors (group 4). The influence of inflammation was also analyzed. Serum VEGF levels were expressed as VEGF per platelet (VEGF/PLT, pg/10(6)) in order to exclude the influence of platelet counts. RESULTS: sVEGFR1 levels were significantly higher in groups 1 and 2 than in the control group, but did not correlate with inflammatory markers. VEGF/PLT levels were generally higher in patients with active inflammation than in those with carcinoma. C-reactive protein strongly correlated with the levels of serum VEGF independently of platelet and leukocyte counts, even in cancer patients. In cancer patients, VEGF/PLT and sVEGFR1 levels might be indicators for evaluating the effect of medical treatment or the disease progression. CONCLUSION: Serum VEGF and VEGFR1 might be useful markers for gauging the clinical effect of various treatments on patients.

Effect of IL-4 and IL-13 on collagen production in cultured LI90 human hepatic stellate cells.

BACKGROUND: Recently, it has been reported that interleukin 4 (IL-4) and 13 (IL-13) directly activate fibroblasts and promote fibrosis. In the process of hepatic fibrosis, the effects of these cytokines on hepatic stellate cells (HSCs) are not well known. METHODS: We evaluated the effects of IL-4 and IL-13 on the collagen production and the proliferation of LI90, a hepatic stellate cell line. We also examined whether interferon (IFN) interferes with the expression of collagen, since IFN has been reported to clinically suppress hepatic fibrosis. RESULTS: The receptor complex for IL-4 and IL-13 was IL-4Ralpha/IL-13Ralpha1 on LI90 cells, and the phosphorylation of Stat6 was induced by IL-4 and IL-13. The treatment of LI90 cells with IL-4 or IL-13 increased the production of collagen I protein levels by nearly three times in comparison with untreated cells. Collagen mRNA levels were increased roughly 10-fold by IL-4 and 100-fold by IL-13. Interestingly, BrdU incorporation in LI90 cells was decreased by IL-4 or IL-13 treatment. Furthermore, induction of collagen I production by these cytokines was blocked by IFNalpha or IFNbeta treatment, although neither treatment alone suppressed collagen production. CONCLUSIONS: Our data suggested that IL-4 and IL-13 directly affected HSCs by increasing collagen production and suppressing cell proliferation. The anti-fibrogenetic effect of IFN may be due in part to the blockade of IL-4 and IL-13 stimulation of HSCs.

Adipose differentiation related protein induces lipid accumulation and lipid droplet formation in hepatic stellate cells.

The function of adipose differentiation-related protein (ADRP) is known to be the uptake of long-chain fatty acids and formation of lipid droplets in lipid-accumulating cells. We hypothesized that ADRP might stimulate activated hepatic stellate cells (HSCs) to accumulate lipids, resulting in their transition to the quiescent state. In this study, cultured HSCs in fifth passages isolated from rat were infected by adenovirus vector expressing ADRP (Ad.GFP-ADRP), and morphologic and functional changes were evaluated in comparison with control HSCs infected by recombinant adenovirus-expressing beta-galactosidase (Ad.LacZ). In Ad.GFP-ADRP-infected cells only, many tiny lipid droplets were apparent in the cytoplasm, while the outline of the cells was not changed. The ADRP was detected around the lipid droplets. In HSCs with intracellular actin filaments, the staining pattern of the filaments before and after infection with Ad.GFP-ADRP or Ad.LacZ did not differ. The cell proliferation rate was not influenced by infection with Ad.LacZ or Ad.GFP-ADRP. Type I collagen secretion from cells overexpressing ADRP was not significantly different from that of Ad.LacZ-infected cells. In our in vitro study, ADRP overexpression induced the formation of cytoplasmic lipid droplets in activated HSCs but could not convert other characteristics of the activated form into those of the quiescent form.

Arg-Gly-Asp (RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity.

Liver cirrhosis is caused by a relative imbalance between synthesis and degradation of collagens. Arg-Gly-Asp (RGD) peptide is a major adhesive domain of several extracellular matrix (ECM) components, such as that involved in the binding of fibronectin to the alpha5beta1 integrin receptor. We previously reported that RGD peptide increased the expression of matrix metalloproteinase in hepatic stellate cells (HSCs) which play a major role in hepatic fibrosis. We evaluated whether RGD-peptides inhibit the progression of liver fibrosis in an animal model of carbon tetrachloride-induced hepatotoxicity. RGD peptide (GRGDS) (1 mg/kg body weight) was injected intraperitoneally (i.p.) 3 times a week for one month. The group treated with control peptide (GRGES) showed pathologically typical hepatic fibrosis, while the RGD-treated group showed minimal fibrotic changes. The liver contents of collagen and hydroxyproline in the RGD-treated group was significantly lower than that of the control group. Collagenase activity measured in liver homogenates was significantly higher in the treated group than in the control group. In an in vitro study using TWNT-4 cells derived from human HSCs, RGD peptide (100 mug/ml) reduced the expression of type I collagen and tissue inhibitor of matrix metalloproteinase-1, and increased that of matrix metalloproteinase-1. These results indicated that RGD peptides inhibited liver fibrosis associated with both decreased collagen production and increased collagenase acitivity, and suggested that RGD peptide might be useful for the therapy of hepatic fibrosis.

[Case of NASH exacerbated after testectomy due to seminoma].

A 41-year-old Japanese male was admitted to our hospital because of the increased levels of serum AST, ALT, and gamma-GTP on December 18, 2002. He was diagnosed with right testis seminoma in 1994 and had received right high orchiectomy and radiation therapy. At that time, liver dysfunction was not pointed out. As weight was increased in 2001, liver dysfunction was pointed out. He was diagnosed as left testis seminoma in June, 2002, left high orchiectomy and chemotherapy was performed. Abdominal ultrasonography showed the moderate fatty liver, and hepatic histopathology revealed a typical and remarkable steatohepatitis with lymphocyte infiltration. He had no life history of alcohol-consumption, and he was diagnosed as non-alcoholic steatohepatitis (NASH). He was treated with a low-calorie diet, which showed favorable effects on his serum levels of AST, ALT, gamma-GTP, and LDH. This case suggests that the altered sex hormone balance might exacerbate NASH because liver dysfunction was particularly worsened after bilateral high orchiectomy.

Serum levels of soluble molecules associated with evasion of immune surveillance: a study in biliary disease.

BACKGROUND: Biliary carcinoma cells produce the transmembrane proteins, Fas, FasL, and RCAS1. It has been demonstrated that the Fas/FasL and RCAS1 systems induce apoptosis of activated immune cells and that the soluble isoforms of these proteins (sFas, sFasL, and sRCAS1) also exhibit this function. METHODS: We measured serum levels of these soluble-types in patients with biliary disease by ELISA and investigated their clinical significance. RESULTS: In some cases of cholangitis and autoimmune biliary disease, serum sFasL values were over 0.1 ng/ml but the protein was undetectable in any patients with biliary carcinoma. sFas levels were significantly higher in the autoimmune disease (mean, 6.83 ng/ml) and cancer (mean, 6.42 ng/ml) groups than in the cholangitis group (mean, 4.23 ng/ml) and normal controls (mean, 2.93 ng/ml). However, the sFas values in malignancy did not correlate with the progression of clinical stage. The percentage positive for serum sRCAS1 was 9.7% in benign disease but was 63.4% in cancer. CONCLUSIONS: Our data suggest that serum sFasL in biliary disease may be derived predominantly from activated immune cells and not from cancer cells and that autoimmune biliary disease may be mediated by the Fas/FasL apoptotic system. sRCAS1 is highly tumor-specific and may be of value in the diagnosis of malignancy.