Liver injury induced by lipopolysaccharide is mediated by TNFR-1 but not by TNFR-2 or Fas in mice.

Endotoxemia causes liver injury in which tumor necrosis factor (TNF)-alpha plays a significant role by inducing hepatic apoptosis. We here examined if such apoptosis is strictly dependent on TNF-alpha and which type of TNF receptor (TNFR) is involved, employing TNFR-1- and -2-knockout mice. Lipopolysaccharide (LPS) dose-dependently induced liver injury in both wild-type (WT) and TNFR-2-knockout mice as indicated by plasma ALT activities, whereas the injury was absent in TNFR-1-knockout mice. Similarly, apoptotic hepatocyte death was observed in WT and TNFR-2-knockout mice after LPS-injection, but not in TNFR-1-knockout mice. Plasma levels of TNF-alpha, interleukin (IL)-6, IL-10 and interferon-gamma as well as hepatic TNF-alpha levels increased equally in mice with either genotype after LPS-injection. LPS also enhanced equally the mRNA expression of Fas but not Fas ligand irrespective of either genotype, as measured by RNase protection assay. These findings suggest that apoptotic liver injury induced by LPS depends on TNF-alpha signaling through TNFR-1 but not via TNFR-2 or Fas-Fas ligand pathway.

Prospective study on early virologic response to treatment with interferon alpha-2b plus ribavirin in patients with chronic hepatitis C genotype 1b.

To evaluate the predictive value of early virologic response (EVR) of achieving a sustained virologic response (SVR), an open, prospective trial including 42 patients with chronic hepatitis C genotype 1b was performed with directly observed 24-week treatment with interferon alpha-2b plus ribavirin. We assessed the predictive values of EVR at days 3, 7, 14, and weeks 4, 8, and 12 of the SVR. The SVR in an intention-to-treat analysis was 19.0%. Patients who reached SVR presented a significantly faster reduction in plasma viral load. Stepwise multiple logistic regression analysis of the factors (gender, age, IFN dosage, ribavirin dosage, HCV RNA, ISDR, and loss of HCV RNA at week 4) revealed that loss of HCV RNA at week 4 was the only independent variable of treatment outcome (P=0.0039). A viral load at treatment day 3 above 100kIU/ml, at day 7 above 50kIU/ml, and at day 14 above 10kIU/ml was 100% predictive for virologic non-response in all except 1 patient. The cutoff levels for HCV RNA at days 3 and 14 of treatment were associated with an algorithm of the failure to detect HCV RNA after 12 weeks of treatment. In conclusions, a very early virologic response assessment could be useful for prediction of later outcome of combination therapy in chronic hepatitis C genotype 1b.

Prognostic value of energy metabolism in patients with viral liver cirrhosis.

The effect of energy malnutrition on survival in patients with non-alcoholic viral liver cirrhosis has not been well defined. We characterized energy metabolism at study entrance and prospectively analyzed its effect on subsequent survival in cirrhotics. One hundred nine consecutive patients with viral liver cirrhosis and 22 healthy control subjects participated in the study. By indirect calorimetry after overnight bedrest and fasting, resting energy expenditure (REE) was measured and non-protein respiratory quotient (npRQ) was calculated. Survival of cirrhotics were followed for up to 8 y. Survival rate was estimated with the Kaplan-Meier method. REE at entrance was significantly higher than the predicted basal metabolic rate (BMR) in cirrhotics (P < 0.001). NpRQ was significantly lower in cirrhotics than in controls (P < 0.001). Survival rate was significantly lower in patients with low npRQ ( < 0.85) than in patients with scores above 0.85 (P < 0.01) and was significantly higher in normal metabolic patients (0.9 < REE/BMR < 1.1) than in hypometabolic (REE/BMR < 0.9) or hypermetabolic (1.1 < REE/BMR) patients (P < 0.05). The proportional hazards model showed that npRQ (relative risk = 0.0003, 95% confidence interval = 0.0000-0.0970), REE/BMR (0.0199, 0.0007-0.5652), prothrombin time, and ammonia were independent significant factors determining survival. Thus evaluation of energy metabolism can be used to predict survival in patients with viral liver cirrhosis.

Genotyping of hepatitis C virus by melting curve analysis with SYBR Green I.

BACKGROUND: Recent studies have focused on whether different hepatitis C virus (HCV) genotypes are associated with different profiles of pathogenicity, infectivity, and response to antiviral therapy. We needed to develop a convenient screening test for HCV genotypes 1 and 2. METHOD: We tested 55 patients with known chronic HCV infection. Viral RNA was extracted from serum samples using an automatic viral RNA purification system, and HCV genotypes were determined by reverse transcriptase-polymerase chain reaction using LightCycler melting curve analysis with SYBR Green I. RESULTS: HCV RNA was detected in all samples and each genotype was determined. The mean (standard deviation) melting temperatures for subtypes 1b (n = 32), 2a (n = 15) and 2b (n = 8) were 93.14 degrees C (0.51 degrees C), 91.08 degrees C (0.49 degrees C) and 91.77 degrees C (0.27 degrees C), respectively. Genotypes 1 and 2 were differentiated within 3 h by this method. CONCLUSIONS: Our melting curve analysis is a rapid and convenient screening test for differential identification of HCV genotypes 1 and 2.

Increased serum soluble Fas in patients with acute liver failure due to paracetamol overdose.

BACKGROUND/AIMS: Experimental studies have suggested that apoptosis via the Fas/Fas Ligand signaling system may play an important role in the development of acute liver failure. The aim of the study was to investigate the soluble form of Fas in patients with acute liver failure. METHODOLOGY: Serum levels of sFas (soluble Fas) were measured by ELISA in 24 patients with acute liver failure and 10 normal control subjects. Serum levels of tumor necrosis factor-alpha and interferon-gamma were also determined by ELISA. RESULTS: Serum sFas was significantly increased in patients with acute liver failure (median, 26.8 U/mL; range, 6.9-52.7 U/mL) compared to the normal controls (median, 8.6 U/mL; range, 6.5-12.0 U/mL, P < 0.0001). Levels were significantly greater in patients with acute liver failure due to paracetamol overdose (median, 28.7 U/mL; range, 12.8-52.7 U/mL, n = 17) than those due to non-A to E hepatitis (median, 12.5 U/mL; range, 6.9-46.0 U/mL, n = 7, P < 0.01). There was no relationship of sFas to eventual outcome in the patients. A significant correlation was observed between serum sFas levels and aspartate aminotransferase (r = 0.613, P < 0.01). CONCLUSIONS: The increased concentration of sFas in serum of patients with acute liver failure may reflect activation of Fas-mediated apoptosis in the liver and this together with increased tumor necrosis factor-alpha may be an important factor in liver cell loss.

Fas-mediated apoptosis in acute alcoholic hepatitis.

BACKGROUND/AIMS: The Fas-mediated apoptosis pathway has been implicated in liver diseases. The aim of the study was to investigate the role of this system in alcoholic hepatitis. METHODOLOGY: The expression of Fas, Fas ligand, and intracellular signaling molecules for apoptosis were determined by immunoblot analysis in fresh frozen liver samples from 19 patients with alcoholic liver disease. RESULTS: Fas and Fas ligand expression was significantly increased in the liver of patients with alcoholic hepatitis (n = 11) as compared with alcoholic liver disease patients without hepatitis (n = 8). Similarly, there were significant differences in the expression of FADD, ICE, and CPP32 in the liver between the two groups. There were significant positive correlations between the Fas ligand and the FADD, ICE, or CPP32 levels in the liver. The expression of Fas, Fas ligand, FADD, ICE, or CPP32 correlated with serum markers of hepatic injury. Plasma soluble Fas levels in patients with alcoholic hepatitis (median: 15.1 U/mL, range: 9.7-19.2 U/mL) were significantly higher than in normal controls (n = 9) (median: 2.8 U/mL, range: 1.9-3.7 U/mL; p < 0.001). There was a significant positive correlation between plasma soluble Fas levels and the hepatic expression of FADD in these patients. CONCLUSIONS: These results indicate that Fas-mediated apoptosis may play an important role in alcoholic hepatitis.

Effects of plasma from patients with fulminant hepatic failure on function of primary rat hepatocytes in three-dimensional culture.

BACKGROUND/AIM: As biotechnology continues to advance, a bioartificial liver is expected to be developed for the treatment of patients with fulminant hepatic failure (FHF) whose liver dysfunction is potentially reversible or for providing liver support as a bridge to liver transplantation. While monolayer-cultured hepatocytes rapidly lose their capacity to express many liver-specific functions over time when cultured, spherical-shaped hepatocytes in three-dimensional culture with the use of extracellular matrix components sustain long-term survival by maintaining differentiated hepatocyte functions. The aim of this study was to investigate whether sufficient functions of viable spherical-shaped hepatocytes could be maintained in plasma of patients with FHF in order to use these cells in an extracorporeal system. METHODS: Hepatocyte functions were evaluated under monolayer or three-dimensional culture in FHF plasma. RESULTS: Primary rat hepatocytes on poly-N-p-vinylbenzyl-D-lactonamide (PVLA) formed spheroids even in FHF plasma and maintained their spherical shapes in FHF plasma as long as in medium. Spherical-shaped hepatocytes on PVLA cultured in FHF plasma showed higher activity in albumin secretion, urea formation, and gluconeogenesis than those in normal human plasma or medium. As being cultured in medium, hepatocytes on PVLA cultured in plasma were also superior to cells on collagen in regard to albumin secretion, amino acid metabolism, and gluconeogenesis. CONCLUSIONS: These findings demonstrated that FHF plasma is not toxic to rat hepatocyte spheroids and that hepatocyte spheroids have potential use in the development of a bioartificial liver.

Hypoplastic myelodysplastic syndromes can be distinguished from acquired aplastic anaemia by bone marrow stem cell expression of the tumour necrosis factor receptor.

It is often difficult to distinguish hypoplastic myelodysplastic syndrome (h-MDS) from acquired aplastic anaemia (AA), because of the considerable clinical, cytological and histological similarities between these two disorders. The distinction between AA and h-MDS is important because there is a higher risk of progression to acute leukaemia in patients with h-MDS compared with AA. Recent studies suggest that tumour necrosis factor-alpha (TNF-alpha) plays an important role in the development of AA. In order to determine the potential importance of TNF-alpha in the differential diagnosis of hypoplastic bone marrow (BM) disorders, we examined whether analysis ofTNF-receptor expression could be used as a tool to differentiate AA from h-MDS. Flow cytometric analysis revealed that BM stem cells (CD34+) from AA patients have markedly greater TNF receptor (R) 1 and TNFR2 expression than those from patients with MDS and h-MDS. We suggest that the BM stem cells with a high expression of TNFR in patients with AA may be potently sensitive to TNF-alpha stimulation of differentiation. Thus, we propose that quantification of TNFR expression in BM stem cellsmay be a useful method to distinguish AA from h-MDS.

Lack of tumor necrosis factor alpha induces impaired proliferation of hepatitis B virus-specific cytotoxic T lymphocytes.

Recent studies have shown that tumor necrosis factor alpha (TNF-alpha) plays critical roles in not only viral clearance but also lymphoid tissue development and stem cell differentiation. In this study, we attempted to induce hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) by immunization of TNF-alpha knockout (TNF-alpha(-/-)) mice with HBsAg-encoding plasmid DNA. An immunization with the HBV plasmid failed to induce CTL responses in TNF-alpha(-/-) mice, although CTLs were readily induced in wild-type mice by the same protocol. Weak CTL responses were produced in TNF-alpha(-/-) mice after two sessions of immunization with the HBV plasmid; however, TNF-alpha was required to maintain the responses of these CTL lines to in vitro stimulation and, even then, the responses were lost after 3 weeks. Interestingly, a limiting dilution of a CTL line showed that HBV-specific CTL clones with high specific cytotoxicity were present in TNF-alpha(-/-) mice, but these clones again failed to proliferate for more than 3 weeks. Furthermore, since exogenously added TNF-alpha enhanced the proliferation of a TNF-alpha(-/-) clone but suppressed that of a TNF-alpha(+/+) clone in vitro, TNF-alpha also has a direct effect on the proliferation of CTLs. In conclusion, TNF-alpha is essential rather than important for the proliferation of HBV-specific CTLs both in vivo and in vitro and this effect is not only due to the activation of dendritic cells but is also induced by the direct effect on CTLs.