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Background Progranulin (GP88) is an 88-kDa glycoprotein growth factor with important biological effects in tumorigenesis and tumour survival. We investigated the usefulness of measuring serum GP88 concentrations as a predictive biomarker for hepatocellular carcinoma in patients with viral hepatitis C after treatment with direct-acting antiviral agents. Methods We measured the serum GP88 concentrations by using a sandwich enzyme-linked immunoassay from 67 healthy control subjects and 29 patients (20 patients who did not develop hepatocellular carcinoma and 9 patients who developed hepatocellular carcinoma after treatment) with viral hepatitis C after treatment with asunaprevir and daclatasvir. Results The serum GP88 concentrations of patients with chronic hepatitis C prior to antiviral treatment were significantly higher than those of healthy control subjects. After antiviral treatment, the serum GP88 concentrations of patients who eventually developed hepatocellular carcinoma were significantly higher than those who did not develop hepatocellular carcinoma. The changes in the serum GP88 concentrations before and after treatment in patients who developed hepatocellular carcinoma were significantly lower than those in patients who did not develop hepatocellular carcinoma. The cumulative incidence of hepatocellular carcinoma was significantly higher in either patients with high serum GP88 concentrations after treatment or those with small changes of serum GP88 concentrations pre- and post-treatment. Conclusions Sustained high concentrations of serum GP88 in patients treated with direct-acting antiviral agents are correlated with the risk of developing hepatocellular carcinoma.

We recently experienced a case of intestinal myiasis caused by maggots of fly, Sarcophagidae. A 61-year-old Japanese man presented in August 2015 with a 2-day his-tory of diarrhea and white maggots in his stool. Two days before his presentation, he took Chinese noodles and lettuce. At his presentation, we observed a number of discharged insect bodies in his stool. The insect bodies were identified as the larvae of the 3rd instar flesh fly Sarcophagidae based on macroscopic and microscopic examinations. We finally diagnosed as intestinal myiasis due to the fact that the patient had the insect bodies mixed in his diet, lettuce. A few days after the treatment with antibiotics, his symptoms disappeared. Accurate diagnosis of intestinal myiasis in developed countries is necessary to avoid ineffective treatment.

AIM This study aimed to evaluate the usefulness of early vascular phase images produced by contrast-enhanced ultrasonography (CE-US) with Sonazoid for the diagnosis of hypovascular hepatocellular carcinoma (HCC). METHODS Four hundred and seventeen patients with 674 hepatic nodules were evaluated using CE-US with Sonazoid between January 2007 and March 2010. Retrospective analysis was conducted on 49 histologically confirmed nodules showing hypovascularity relative to the surrounding liver tissue in the early vascular phase and no enhancement defect in the Kupffer phase of CE-US with Sonazoid. These nodules were classified according to early vascular phase image enhancement patterns as types I (largest avascular; avascular as a whole), II (second avascular; partially avascular), III (smallest avascular; not avascular, but faintly hypovascular relative to the surrounding liver) and IV (hypovascular as a whole with vessel-like structures passing inside nodules). RESULTS Among the 49 nodules, types I, II, III and IV were identified in 19 (38.8%), nine (18.4%), 15 (30.6%) and six (12.2%) cases, respectively. The proportion of tumorous nodules (well-differentiated HCC and high-grade dysplastic nodules) significantly decreased with a reduction of the avascular area (68.4% in the type I, 55.6% in the type II and 33.3% in the type III nodules; P

Despite widening interest in the possible association between infection/ inflammation and cancer development, knowledge of this issue in relation to oral cancer remains inadequate. This study aimed to determine the susceptibility of Apc-mutant Kyoto Apc Delta (KAD) rats, which are vulnerable to developing inflammation-associated colorectal carcinogenesis, to 4-nitroquinoline 1-oxide (4-NQO)-induced tongue carcinogenesis in order to clarify the role of inflammation in oral cancer. KAD (20 males and 22 females) and F344/NS1c (22 males and 23 females) rats received drinking water with or without 4-NQO (20 ppm) for eight weeks. Histopathological and immunohistochemical analyses of the tongue were performed at week 20. Additionally, the mRNA expression of inflammatory cytokines in the tongue mucosa was determined at week 8. Tongue squamous cell carcinoma (SCC) developed in the KAD and F344/NS1c rats that received 4-NQO. Regardless of gender, the incidence and multiplicity of tongue SCC were greater in the KAD rats than in the F344/NS1c rats. In addition, the multiplicity of tongue SCC in the female KAD rats was significantly greater than that observed in the male KAD (p &lt, 0.01) and female F344/NS1c rats (p &lt, 0.05). The levels of inflammation and the mRNA expression of inflammatory cytokines in the tongue in the 4-NQO-treated female KAD rats were the highest among the rats given 4-NQO. These results show that KAD rats, particularly females, are susceptible to 4-NQO-induced tongue carcinogenesis, suggesting the utility of models employing KAD rats for investigating the pathobiology of oral (tongue) carcinogenesis associated with inflammation.

Aim Accurate assessment of the coagulated area is imperative to achieve an excellent outcome from percutaneous radiofrequency ablation (PRFA) for the treatment of hepatocellular carcinoma (HCC). We evaluated the efficacy of contrast-enhanced ultrasonography (CEUS) with the contrast-enhancing agent Sonazoid for precisely assessing the therapeutic effect of PRFA for HCC. Methods We enrolled 87 consecutive patients with solitary naive HCC of less than 3 cm in diameter. PRFA treatment was performed with a 17-G cool-tip needle, and CEUS was performed to assess the ablative margin 3 h after the procedure, when the coagulated tumor outline was easiest to discern. The treatment was repeated until an ablative margin greater than 5 mm was confirmed. After CEUS assessment of the therapeutic response, the patients were followed to investigate local tumor recurrence. Results In 78 patients (89.7%), the outline of the coagulated tumors could be recognized by ultrasonography, and CEUS assessment of the ablative margin was successful. The remaining nine patients were assessed by computed tomography. The 5-year cumulative survival rate after the assessment of the treatment response with CEUS was 58.4%, and the 4-year cumulative total recurrence rate was 72.3%. The 5-year cumulative local tumor recurrence rate was very low (2.3%). Conclusion The assessment with CEUS at 3 h after the PRFA procedure was successful in the majority of the patients, and it yielded a very low rate of local recurrence.

Aim Protein–energy malnutrition is frequently observed in patients with liver cirrhosis (LC). Non-protein respiratory quotient (npRQ) measured by indirect calorimetry is a good marker to estimate energy malnutrition, and predicts the prognosis of patients with LC. However, measurement of npRQ is limited because of the high cost of indirect calorimetry. Our aim was to find out an alternative marker to npRQ that can be used in the routine clinical setting. Methods One hundred and fifty-six patients with LC were enrolled in this study. Indirect calorimetry and blood examinations were conducted after overnight fasting, and anthropometry was performed by an expert dietician. The correlation between npRQ and other parameters were calculated by simple and multiple regression analysis. Receiver–operator curve (ROC) analysis was used to identify the cut-off value that would best predict the threshold npRQ of 0.85. Results Plasma levels of free fatty acid (FFA) was significantly correlated with npRQ value by simple (r = −0.39, P

AIM Combination chemoprevention is a promising strategy to improve the prognosis of hepatocellular carcinoma (HCC). A malfunction of retinoid X receptor-α (RXR-α) due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and acyclic retinoid (ACR) can prevent HCC development by inhibiting RXR-α phosphorylation. The present study examined the possible combined effects of ACR plus branched-chain amino acids (BCAA), which can also prevent the development of HCC in obese patients with liver cirrhosis, in human HCC xenografts in nude mice. METHODS This study examined the effects of the combination of ACR plus BCAA on the growth of Huh7 human HCC xenografts in nude mice. The effects of the combination on the phosphorylation of RXR-α, extracellular signal-regulated kinase (ERK), Akt and insulin-like growth factor-1 receptor (IGF-1R) proteins, and on the expression levels of retinoic acid receptor-β (RAR-β) and p21(CIP1) mRNA, were also examined by western blot and real-time reverse transcription polymerase chain reaction analyses, respectively. RESULTS The combined treatment with ACR plus BCAA significantly inhibited the growth of Huh7 xenografts. The combination of these agents caused a marked inhibition of the phosphorylation of RXR-α, ERK, Akt and IGF-1R proteins in the xenografts. In addition, the expression levels of RAR-β and p21(CIP1) mRNA significantly increased by these agents. CONCLUSION The combination of ACR and BCAA restores the function of RXR-α by inhibiting its phosphorylation and increasing the level of RAR-β, a heterodimeric partner for RXR-α, and thus suppresses the growth of HCC xenografts. Therefore, this combination might be an effective regimen for the treatment and, probably, chemoprevention of HCC.

Aim: In Japan, the indication for liver transplantation in patients with acute liver failure (ALF) is currently determined according to the guideline published in 1996. However, its predictive accuracy has fallen in recent patients. Thus, we attempted to establish a new guideline. Methods: The subjects were 1096 ALF patients enrolled in a nationwide survey. All patients showed a prothrombin time

The diagnostic criteria of fulminant hepatitis in Japan are different from those of acute liver failure in Europe and the United States, both in regard to the histological features in the liver and the cutoff values of the prothrombin time. Thus, the Intractable Hepato-Biliary Disease Study Group established novel diagnostic criteria for “acute liver failure” in Japan based on the demographic and clinical features of the patients. Patients showing prothrombin time values of 40% or less of the standardized values or international normalized ratios of 1.5 or more caused by severe liver damage within 8 weeks of onset of the symptoms are diagnosed as having “acute liver failure”, where the liver function prior to the current onset of liver damage is estimated to be normal. Acute liver failure is classified into “acute liver failure without hepatic coma” and “acute liver failure with hepatic coma,” depending on the severity of the hepatic encephalopathy; the latter is further classified into two types, the “acute type” and the “subacute type”, in which grade II or more severe hepatic coma develops within 10 days and between 11 and 56 days, respectively, after the onset of disease symptoms. Patients without histological findings of hepatitis, such as those with liver damage caused by drug toxicity, circulatory disturbance or metabolic disease, are also included in the disease entity of “acute liver failure”, while acute-on-chronic liver injuries, such as liver injury caused by alcohol, are excluded. A nationwide survey of “acute liver failure” in Japan based on the novel criteria is proposed.

Obesity and related adipocytokine disbalance increase the risk of hepatocellular carcinoma. To determine the impact of increased levels of leptin, an obesity-related adipocytokine, on the recurrence of hepatocellular carcinoma, we conducted a prospective case-series analysis. Eighty-five consecutive primary hepatocellular carcinoma patients at our hospital from January 2006 to December 2008 were analyzed. Serum leptin level significantly correlated with Body Mass Index, total body fat, and the amount of subcutaneous fat. They included 33 with stage I/II, who underwent curative treatment. The factors contributing to recurrence of hepatocellular carcinoma, including leptin, were subjected to univariate and multivariate analyses using the Cox proportional hazards model. Body Mass Index (p = 0.0062), total body fat (p = 0.0404), albumin (p = 0.0210), α-fetoprotein (p = 0.0365), and leptin (p = 0.0003) were significantly associated with the recurrence of hepatocellular carcinoma in univariate analysis. Multivariate analysis suggested that leptin (hazard ratio 1.25, 95% CI 1.07–1.49, p = 0.0035) was a sole independent predictor. Kaplan-Meier analysis showed that recurrence-free survival was lower in patients with greater serum leptin concentrations (>5 ng/mL, p = 0.0221). These results suggest that the serum leptin level is a useful biomarker for predicting the early recurrence of hepatocellular carcinoma.

The Intractable Liver Diseases Study Group of Japan, supported by the Ministry of Health, Labor and Welfare, established novel diagnostic criteria for "acute liver failure" in 2011. In these criteria, patients without histological findings of hepatitis are included in the disease entity of "acute liver failure", as in Europe and the USA. In this report, classification criteria for the etiologies of "acute liver failure" in Japan are proposed.

Summary Background The benefits of prolonging peginterferon and ribavirin after 48 weeks of treatment to maximize sustained virological responses (SVR) in hepatitis C virus (HCV) genotype 1-infected patients remain to be understood. Aim To investigate whether extended treatment longer than 72 weeks may be superior to 72-week treatment. Methods A total of 120 treatment-naive or retreated patients with HCV genotype 1 were treated with peginterferon-alpha-2b (1.5 μg/kg/week) plus weight-based ribavirin. We had 34 late responders, in whom HCV RNA first became undetectable at week 12–48, and randomized them into three groups receiving standard-dose peginterferon-alpha-2b plus low-dose ribavirin (200 mg/day) for extended 24 weeks (group A), receiving low-dose peginterferon-alpha-2b (0.75 μg/kg/week) plus low-dose ribavirin for extended 48 weeks (group B) or no extended treatment (group C), and evaluated the outcome according to their virological response. Results Multivariate analysis showed that the treatment for 96 weeks was identified as a significant, independent factor associated with SVR in HCV genotype 1-infected late responders in comparison with group A [odds ratio (OR), 10.002; P = 0.080] and group C (OR, 17.748; P = 0.025). Conclusion Extending the treatment duration from 48 weeks to 96 weeks improves SVR rates in genotype 1-infected patients with late virological response to peginterferon-alpha-2b and ribavirin.

AIM: To evaluate the correlation between hepatitis C virus (HCV) specific cytotoxic T lymphocytes (CTLs) and viral clearance in antiviral treated patients, we examined the number and function of HCV epitope-specific CTLs and the viral load in 12 HLA-A2-positive patients with chronic hepatitis C, after undergoing interferon therapy. METHODS: Peripheral blood mononuclear cells (PBMC) were analyzed on days 0, 3, 7, 14 and 28 of undergoing antiviral therapies. To investigate the quantity of the antigen specific CTLs, CD8-positive T cells were isolated using microbeads and were stained for HLA-A*0201 tetramers. To investigate the function of CTLs, PBMC were stimulated with the same synthetic epitope peptides and analyzed to determine their interferon (IFN)-gamma expression. RESULTS: In seven patients, HCV-RNA became undetectable 4 weeks after antiviral therapies (EVR), but five patients were non-responders (NR). In peptide NS3 1406 on day 3 and day 7 of therapy and in NS3 1073 on day 3 of therapy, the level of IFN-gamma expression on CD8+ T cells was significantly higher in the EVR group than in the NR group. In other peptides, the number of and cytokine production from the CTLs in the EVR group were also higher than in the NR group, but not significantly. CONCLUSION: After antiviral therapy, analysis of the number and function of antigen-specific CTLs in the early phase was thus found to be useful for predicting viral clearance in chronic hepatitis C patients.

This study aimed to evaluate the usefulness of early vascular phase images produced by contrast-enhanced ultrasonography (CE-US) with Sonazoid for the diagnosis of hypovascular hepatocellular carcinoma (HCC).Four hundred and seventeen patients with 674 hepatic nodules were evaluated using CE-US with Sonazoid between January 2007 and March 2010. Retrospective analysis was conducted on 49 histologically confirmed nodules showing hypovascularity relative to the surrounding liver tissue in the early vascular phase and no enhancement defect in the Kupffer phase of CE-US with Sonazoid. These nodules were classified according to early vascular phase image enhancement patterns as types I (largest avascular; avascular as a whole), II (second avascular; partially avascular), III (smallest avascular; not avascular, but faintly hypovascular relative to the surrounding liver) and IV (hypovascular as a whole with vessel-like structures passing inside nodules).Among the 49 nodules, types I, II, III and IV were identified in 19 (38.8%), nine (18.4%), 15 (30.6%) and six (12.2%) cases, respectively. The proportion of tumorous nodules (well-differentiated HCC and high-grade dysplastic nodules) significantly decreased with a reduction of the avascular area (68.4% in the type I, 55.6% in the type II and 33.3% in the type III nodules; P 0.05). All nodules demonstrating the type IV enhancement pattern were non-tumorous.Based on the size of avascular area in the early vascular phase of CE-US with Sonazoid, we can predict the malignant potential of the nodules. CE-US with Sonazoid is very useful for evaluation of hypovascular hepatic nodules.

Cell adhesion to the extracellular matrix (ECM) plays vital roles in both morphogenesis and regulation of gene expression in cells of adult organisms. How intracellular, cytoskeletal, and signaling factors connect and communicate with the ECM is a fundamental question. Using a cDNA microarray analysis, we identified phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) phosphatase mRNA as being up-regulated in hepatocytes cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, which led to the finding that the PI(4,5)P2 levels of hepatocytes decreased on EHS gel. These changes in hepatocytes on EHS gel were accompanied by promotion of actin depolymerization and differentiated phenotypes of the hepatocytes. Treatment with PI(4,5)P2 or a phospholipase C inhibitor, U73122, resulted in decreased mRNA expressions of albumin and hepatocyte nuclear factor 4 (HNF-4) in hepatocytes. In contrast, actin-disrupting agent gelsolin increased mRNA expressions of albumin and HNF-4. In conclusion, organization of the actin cytoskeleton via PI(4,5)P2 is involved in the regulation of hepatocyte differentiation by the ECM.

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 α-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 100 kIU/ml, at day 7 above 50 kIU/ml, and at day 14 above 10 kIU/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.

Hepatocyte nuclear factor 4alpha (HNF-4alpha) is a transcription factor that controls embryonal liver development and that maintains and regulates gene expression in adult liver cells. We have previously demonstrated that transient overexpression of HNF-4alpha up-regulates a number of liver-specific genes in hepatoma cell lines. In this study, we extend these studies by assessing the functional role of HNF-4alpha in regulating cellular viability and liver-specific functions of primary rat hepatocytes. In cells transfected with an adenovirus vector carrying rat HNF-4alpha cDNA, induction and maintenance of liver-specific genes and functions were observed over a long-term culture, which might be associated with the prevention of a rapid loss of the mitochondrial membrane potential. In addition, we demonstrated that transthyretin mRNA was up-regulated by HNF-4alpha in primary hepatocytes, but not in hepatoma cells. These results indicate that HNF-4alpha plays a role in the maintenance of morphologically and biochemically functional hepatocytes and that the difference in expression of liver-specific genes induced by HNF-4alpha may depend on a differentiation state of cells.

Aims/background Tumor necrosis factor-alpha (TNF-alpha) is known as a proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes. TNF-alpha also influences liver repair following hepatotoxic damage, and regeneration following partial hepatectomy (PH). The aim of this study was to assess the mechanism by which TNF-alpha influences liver cell apoptosis and regeneration following PH in TNF-alpha-deficient (TNF-alpha(-/-)) mice. Methods PH was performed in wild mice and TNF-alpha(-/-) mice. Results In both groups, serum alanine aminotransferase and serum total bilirubin levels comparably peaked at 6 and 48 h after PH, respectively. No differences were observed in hepatocyte proliferation, as determined by mitotic and the proliferating cell nuclear antigen labeling indices, between TNF-alpha(+/+) and TNF-alpha(-/-) mice. Few terminal deoxynucleotidyl transferase nick end-labeling-positive hepatocytes were seen in either type of mice. Nuclear factor-kappa B DNA binding activity in the remaining liver of TNF-alpha(-/-) mice after PH was similar to that of control mice. Ribonuclease protection assay showed that transforming growth factor beta1 mRNA was up-regulated comparably in the livers of the two groups, and that other cytokines were hardly seen in either. Interleukin-6/ signal transducer and activator of transcription-3-dependent pathway was not affected in TNF-alpha(-/-) mice. Conclusions These findings suggest that TNF-alpha has little influence on liver regeneration and liver cell apoptosis after PH in mice.

Leflunomide is a novel immunosuppressive and anti-inflammatory agent for the treatment of autoimmune disease. The aim of this study was to investigate whether leflunomide protects from liver injury induced by concanavalin A (Con A), a T-cell-dependent model of liver damage. BALB/c mice were injected with 25 mg/kg Con A in the presence or absence of 30 mg/kg leflunomide. Liver injury was assessed biochemically and histologically. Levels of circulating cytokines and expressions of cytokine messenger RNA (mRNA) in the liver and the spleen were determined. Treatment with leflunomide markedly reduced serum transaminase activities and the numbers of dead liver cells. Leflunomide significantly inhibited increases in plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 2 concentrations, and also reduced TNF-alpha mRNA expression in the liver after administration of Con A. These findings were supported by the results in which leflunomide administration decreased the number of T lymphocytes infiltrating the liver as well as inhibiting their production of TNF-alpha. Activation of nuclear factor kappaB (NF-kappaB), which regulates TNF-alpha production, was inhibited in the liver of mice treated with leflunomide, resulting in a reduction of TNF-alpha production from lymphocytes infiltrating the liver. In conclusion, leflunomide is capable of regulating T-cell-mediated liver injury in vivo and that this event may depend on the decrease of TNF-alpha production in the liver through inhibition of NF-kappaB activation caused by leflunomide.

Background/aims Tumor necrosis factor (TNF)-alpha itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF-alpha/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)-kappaB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF-alpha. Here, we focused on the roles of NF-kappaB and phosphatidylinositol 3-kinase (PI3K)-regulated serine/threonine kinase Akt. Methods TNF-alpha was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IkappaBalpha (Ad5IkappaB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl-2 family members and caspase activity were examined. Results In the mice livers, treatment with Ad5IkappaB or the wortmannin suppressed the activation of NF-kappaB or Akt, respectively. Suppression of either NF-kappaB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF-alpha administration. However, in mice treated with both Ad5IkappaB and wortmannin, TNF-alpha administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IkappaB, wortmannin, and TNF-alpha markedly increased the activation of caspase-3 and -9, and activated caspase-8 to a lesser degree, suggesting that TNF-alpha-induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF-kappaB and PI3K/Akt pathways had no effect on expression of Bcl-2 families. Conclusion The inducible activation of NF-kappaB and constitutive activation of Akt regulate hepatocyte survival against TNF-alpha, which occurs independent of Bcl-2 families.

Hepatocyte nuclear factor 4alpha (HNF-4alpha), a liver-specific transcription factor, plays a significant role in many liver-specific functions, including lipid, glucose, drug, and ammonia metabolism, and also in embryonal liver development. However, its functions and regulation are not yet clearly understood. In this study, we constructed an adenovirus vector carrying rat HNF-4alpha cDNA and transfected the adenovirus to human hepatoma cells, HuH-7, to enforce expression of the exogenous HNF-4alpha gene. We analyzed HNF-4alpha-induced genes using cDNA microarray technology, which included over 9000 genes. As a result, 62 genes showed a greater than 2.0-fold change in expression level after the viral transfection. Fifty-six genes were consistently induced by HNF-4alpha overexpression, and six genes were repressed. To assess HNF-4alpha function, we attempted to classify the genes, which had been classified by their encoding protein functions in a previous report. We could classify 45 genes. The rest of the HNF-4alpha-sensitive genes were unclassified (4 genes) or not identified (13 genes). Among the classified genes, almost half of the induced genes (26 of 40) were related to metabolism genes and particularly to lipid metabolism-related genes. This cDNA microarray analysis showed that HNF-4alpha is one of the central liver metabolism regulators.

Accurate assessment of the coagulated area is imperative to achieve an excellent outcome from percutaneous radiofrequency ablation (PRFA) for the treatment of hepatocellular carcinoma (HCC). We evaluated the efficacy of contrast-enhanced ultrasonography (CEUS) with the contrast-enhancing agent Sonazoid for precisely assessing the therapeutic effect of PRFA for HCC.We enrolled 87 consecutive patients with solitary naïve HCC of less than 3 cm in diameter. PRFA treatment was performed with a 17-G cool-tip needle, and CEUS was performed to assess the ablative margin 3 h after the procedure, when the coagulated tumor outline was easiest to discern. The treatment was repeated until an ablative margin greater than 5 mm was confirmed. After CEUS assessment of the therapeutic response, the patients were followed to investigate local tumor recurrence.In 78 patients (89.7%), the outline of the coagulated tumors could be recognized by ultrasonography, and CEUS assessment of the ablative margin was successful. The remaining nine patients were assessed by computed tomography. The 5-year cumulative survival rate after the assessment of the treatment response with CEUS was 58.4%, and the 4-year cumulative total recurrence rate was 72.3%. The 5-year cumulative local tumor recurrence rate was very low (2.3%).The assessment with CEUS at 3 h after the PRFA procedure was successful in the majority of the patients, and it yielded a very low rate of local recurrence.

多施設症例を用い, retrospective に多変量解析にて劇症肝炎の肝移植適応基準を作成し, 新たな集計症例に適用して prospective に検討した. その結果, 肝性脳症発現時に, 1) 年齢が45歳以上, 2) 初発症状から脳症発現までの日数が11日以上, 3) PTが10%未満, 4) 総ビリルビン濃度が18.0mg/dl以上, 5) 直接/総ビリルビン比が0.67以下の5項目中2項目以上満たす場合は予後不良と予測して肝移植の登録を行い可能なら移植を行う. 移植が施行できない場合は5日後の経過すなわち, 1) 脳症がI度以内に覚醒あるいは昏睡度でII度以上の改善, 2) PTが50%以上に改善の2項目を満たす場合は生存と再予測し, 1項目以下の場合は予後不良と再予測し肝移植の登録を継続する「劇症肝炎における肝移植適応のガイドライン」を提案した. Prospective study の正診率は0.82であり, このガイドラインは劇症肝炎に対する肝移植の適応を判断する際に有用と考えられる.

The Intractable Liver Diseases Study Group of Japan, supported by the Ministry of Health, Labor and Welfare, established novel diagnostic criteria for "acute liver failure" in 2011. In these criteria, patients without histological findings of hepatitis are included in the disease entity of "acute liver failure", as in Europe and the USA. In this report, classification criteria for the etiologies of "acute liver failure" in Japan are proposed.

Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) initiates apoptosis and simultaneously activates the transcription factor, nuclear factor-kappaB (NF-kappaB), which suppresses apoptosis by an unknown mechanism. Pretreatment with TNF-alpha or interleukin-1beta (IL-1beta), which activated NF-kappaB in the liver, dramatically prevented TNF-alpha-induced liver-cell apoptosis in D-galactosamine (GalN)-sensitized mice, but not anti-Fas antibody-induced hepatotoxicity. This protective effect of TNF-alpha continued for 5 hours after TNF-alpha administration, a time course similar to that found in NF-kappaB activation after TNF-alpha administration. In mice treated with adenoviruses expressing a mutant form of IkappaB, the antiapoptotic effect of TNF-alpha was inhibited in part. Prior TNF-alpha administration was not found to block the activation of caspase-8, although caspase-3 was inhibited in mice treated with TNF-alpha plus GalN/TNF-alpha compared with mice treated with GalN/TNF-alpha. These results indicate that TNFR and Fas independently regulate murine apoptotic liver failure, and that a rapid defense mechanism induced by the activation of NF-kappaB blocks death-signaling at the initiation stage of hepatic apoptosis mediated by TNFR, probably downstream of caspase-8, but not by Fas.

Background/Aims: The extracellular matrix plays an essential role in the regulation of cell proliferation in different cell types. However, the regulation of cell cycle control in hepatocytes in response to growth factors and extracellular matrix signals is not well understood. The aims of this study were to investigate the expression of key cell cycle control elements, including cyclins, A and D1, and cyclin-dependent kinase inhibitors, p21 and p27, in rat hepatocytes in primary culture on dried collagen or Engelbreth-Holm-Swarm in the presence of epidermal growth factor. Methods: Hepatocytes prepared from Wistar rats were cultured on various extracellular matrix in Williams medium E in the presence or absence of 20 ng/ml epidermal growth factor. DNA synthesis was measured by [ 3 H]thymidine uptake and mRNA expression of cell cycle-related genes was determined by reverse transcription polymerase chain reaction. Results: Cyclins D1 and A mRNA levels were high at the G1/S boundary in epidermal growth factor-stimulated hepatocytes cultured on dried collagen. In contrast to spread cells, hepatocytes cultured on an Engelbreth-Holm-Swarm gel that were prevented from spreading failed to progress through the G1 phase and enter the S phase. This shape-dependent blockage of cell cycle progression correlated with the up-regulation of the cell cycle inhibitors p21 and p27. Conclusions: Changes in hepatocyte-extracellular matrix interactions may control hepatocyte growth within the local microenvironment by modulating cell shape and regulating cyclins and the cyclin-dependent kinase inhibitors p21 and p27.

A 61-year-old female was admitted to our hospital with severe jaundice and anemia. She was diagnosed with severe acute hepatitis secondary to autoimmune hepatitis (AIH) on the basis of positive anti-nuclear antibody titers, high serum IgG levels, and liver biopsy. Autoimmune hemolytic anemia (AIHA) was diagnosed because of the presence of reticulocytosis, decreased haptoglobin, positive direct Coombs test, and erythroid hyperplasia in the bone marrow. Although AIH occurs in association with various immunological disorders, an association with AIHA is rarely reported. We report a rare case of severe AIH associated with AIHA.

Protein-energy malnutrition is frequently observed in patients with liver cirrhosis (LC). Non-protein respiratory quotient (npRQ) measured by indirect calorimetry is a good marker to estimate energy malnutrition, and predicts the prognosis of patients with LC. However, measurement of npRQ is limited because of the high cost of indirect calorimetry. Our aim was to find out an alternative marker to npRQ that can be used in the routine clinical setting.One hundred and fifty-six patients with LC were enrolled in this study. Indirect calorimetry and blood examinations were conducted after overnight fasting, and anthropometry was performed by an expert dietician. The correlation between npRQ and other parameters were calculated by simple and multiple regression analysis. Receiver-operator curve (ROC) analysis was used to identify the cut-off value that would best predict the threshold npRQ of 0.85.Plasma levels of free fatty acid (FFA) was significantly correlated with npRQ value by simple (r = -0.39, P 0.0001) and multiple regression analysis (t = -2.96, P = 0.0052). Free fatty acid rose in parallel with the increasing disease severity as defined by Child-Pugh classification (P 0.05). FFA was also correlated with increasing oxidation rate of fat (r = 0.38, P 0.0001) and decreasing oxidation rate of carbohydrate (r = -0.39, P 0.0001). The cut-off value of FFA to predict npRQ = 0.85 was 660 μEq/L by ROC analysis.FFA is a useful alternative marker to represent npRQ in patients with LC.

Combination chemoprevention is a promising strategy to improve the prognosis of hepatocellular carcinoma (HCC). A malfunction of retinoid X receptor-α (RXR-α) due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and acyclic retinoid (ACR) can prevent HCC development by inhibiting RXR-α phosphorylation. The present study examined the possible combined effects of ACR plus branched-chain amino acids (BCAA), which can also prevent the development of HCC in obese patients with liver cirrhosis, in human HCC xenografts in nude mice. This study examined the effects of the combination of ACR plus BCAA on the growth of Huh7 human HCC xenografts in nude mice. The effects of the combination on the phosphorylation of RXR-α, extracellular signal-regulated kinase (ERK), Akt and insulin-like growth factor-1 receptor (IGF-1R) proteins, and on the expression levels of retinoic acid receptor-β (RAR-β) and p21(CIP1) mRNA, were also examined by western blot and real-time reverse transcription polymerase chain reaction analyses, respectively. The combined treatment with ACR plus BCAA significantly inhibited the growth of Huh7 xenografts. The combination of these agents caused a marked inhibition of the phosphorylation of RXR-α, ERK, Akt and IGF-1R proteins in the xenografts. In addition, the expression levels of RAR-β and p21(CIP1) mRNA significantly increased by these agents. The combination of ACR and BCAA restores the function of RXR-α by inhibiting its phosphorylation and increasing the level of RAR-β, a heterodimeric partner for RXR-α, and thus suppresses the growth of HCC xenografts. Therefore, this combination might be an effective regimen for the treatment and, probably, chemoprevention of HCC.

Oxidative stress plays an important role in liver carcinogenesis. To determine the impact of oxidative stress on the recurrence of stage I/II hepatocellular carcinoma (HCC) after curative treatment, we conducted a prospective case series analysis.This study included 45 consecutive patients with stage I/II HCC, who underwent curative treatment by surgical resection or radiofrequency ablation at Gifu Municipal Hospital from 2006 to 2007. In these 45 cases, recurrence-free survival was estimated using the Kaplan-Meier method. The factors contributing to HCC recurrence, including the serum levels of derivatives of reactive oxygen metabolites (d-ROM) as an index of oxidative stress, were subjected to univariate and multivariate analyses using the Cox proportional hazards model.The serum levels of d-ROM (P = 0.0231), α-fetoprotein (AFP, P = 0.0274), and fasting plasma glucose (P = 0.0400) were significantly associated with HCC recurrence in the univariate analysis. Multivariate analysis showed that the serum levels of d-ROM (hazard ratio [HR] 1.0038, 95 % confidence interval [CI] 1.0002-1.0071, P = 0.0392) and AFP (HR 1.0002, 95 % CI 1.0000-1.0003, P = 0.0316) were independent predictors of HCC recurrence. Kaplan-Meier analysis showed that recurrence-free survival was low in patients with high serum d-ROM (≥570 Carr U, P = 0.0036) and serum AFP (≥40 ng/dL, P = 0.0185) levels.The serum levels of d-ROM and AFP can be used for screening patients with a high risk for HCC recurrence. Patients who show increased levels of these factors require careful surveillance.

To summarize the annual nationwide survey on fulminant hepatitis (FH) and late-onset hepatic failure (LOHF) between 2004 and 2009 in Japan.The annual survey was performed in a two-step questionnaire process to detail the clinical profile and prognosis of patients in special hospitals.Four hundred and sixty (n = 227 acute type; n = 233 subacute type) patients had FH and 28 patients had LOHF. The mean age of patients with FH and LOHF were 51.1 ± 17.0 and 58.0 ± 14.4 years, respectively. The causes of FH were hepatitis A virus in 3.0%, hepatitis B virus (HBV) in 40.2%, other viruses in 2.0%, autoimmune hepatitis in 8.3%, drug allergy-induced in 14.6% and indeterminate etiology in 29.6% of patients. HBV reactivation due to immunosuppressive therapy was observed in 6.8% of FH patients. The short-term survival rates of patients without liver transplantation (LT) were 48.7% and 24.2% for the acute and subacute type, respectively, and 13.0% for LOHF. The prognosis was poor in patients with HBV reactivation. The implementation rate for LT in FH patients was equivalent to that in the previous survey. The short-term survival rates of total patients, including LT patients, were 54.2% and 40.8% for the acute and subacute type, respectively, and 28.6% for LOHF.The demographic features and etiology of FH patients has gradually changed. HBV reactivation due to immunosuppressive therapy is problematic. Despite advances in therapeutic approaches, the prognosis of patients without LT has not improved.

To describe a case of autosomal-dominant (AD)-chronic mucocutaneous candidiasis (CMC) with a signal transducer and activator of transcription (STAT) 1 gene mutation, and some of the important complications of this disease such as chronic hepatitis. We present a 23-year-old woman with CMC, chronic active hepatitis, and hypothyroidism. Her father also had CMC. We performed several immunological analyses of blood and liver samples, and searched for gene mutations for CMC in the patient and her father. We identified the heterozygous substitution c.821 G > A (p.Arg274Gln) in the STAT1 gene of both the patient and her father. The level of β-glucan induced interferon (IFN)-γ in her blood cells was significantly low. Immunoblot analysis detected serum anti-interleukin (IL)-17 F autoantibody. She was found to have increased (low-titer) antibodies related to her hypothyroidism and hepatitis. Her serum IL-18 levels fluctuated with her AST and ALT levels. Liver biopsy revealed CD68-positive cell infiltration and IL-18 expression in the sinusoidal regions. These results suggest that the chronic active hepatitis in this patient may be exacerbated by the excessive IL-18 accumulation caused by recurrent mucocutaneous fungal infection, and decreased IFN-γ production. AD-CMC is known to be caused by a gain-of-function mutation of the STAT1 gene. Chronic active hepatitis is a rare complication of AD-CMC, with currently unknown pathogenesis. It seems that the clinical phenotype in this patient is modified by autoimmune mechanisms and cytokine dysregulation. AD-CMC can be complicated by various immune disorders including autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.

In Japan, the indication for liver transplantation in patients with acute liver failure (ALF) is currently determined according to the guideline published in 1996. However, its predictive accuracy has fallen in recent patients. Thus, we attempted to establish a new guideline. The subjects were 1096 ALF patients enrolled in a nationwide survey. All patients showed a prothrombin time40% of the standardized value and grade II or more severe hepatic encephalopathy. A multiple logistic regression analysis and receiver operating characteristic analysis were performed in 698 patients seen between 1998 and 2003 to identify significant parameters determining the outcome of patients. The extracted parameters were graded as numerical scores. An established scoring system was validated in patients seen between 2004 and 2008. Six parameters were identified and graded as 0, 1 and/or 2; the interval between disease onset and development of hepatic encephalopathy, prothrombin time, serum total bilirubin concentration, the ratio of direct to total bilirubin concentration, peripheral platelet count and the presence of liver atrophy. When the prognosis of the patients with total score of 5 or more was judged as "death", the predictive accuracy was 0.80 with sensitivity, specificity, positive predictive value and negative predictive value greater than 0.70. The values were similarly high in patients for validation. Novel scoring system for predicting the outcome of ALF patients may be useful to determine the indication of liver transplantation, since the system showed high predictive accuracy even after validation.

The diagnostic criteria of fulminant hepatitis in Japan are different from those of acute liver failure in Europe and the United States, both in regard to the histological features in the liver and the cutoff values of the prothrombin time. Thus, the Intractable Hepato-Biliary Disease Study Group established novel diagnostic criteria for "acute liver failure" in Japan based on the demographic and clinical features of the patients. Patients showing prothrombin time values of 40% or less of the standardized values or international normalized ratios of 1.5 or more caused by severe liver damage within 8 weeks of onset of the symptoms are diagnosed as having "acute liver failure", where the liver function prior to the current onset of liver damage is estimated to be normal. Acute liver failure is classified into "acute liver failure without hepatic coma" and "acute liver failure with hepatic coma," depending on the severity of the hepatic encephalopathy; the latter is further classified into two types, the "acute type" and the "subacute type", in which grade II or more severe hepatic coma develops within 10 days and between 11 and 56 days, respectively, after the onset of disease symptoms. Patients without histological findings of hepatitis, such as those with liver damage caused by drug toxicity, circulatory disturbance or metabolic disease, are also included in the disease entity of "acute liver failure", while acute-on-chronic liver injuries, such as liver injury caused by alcohol, are excluded. A nationwide survey of "acute liver failure" in Japan based on the novel criteria is proposed.

Aim: Several studies have reported that insulin resistance raises the risk of primary hepatocellular carcinoma (HCC). We conducted a prospective, case series study to test the impact of insulin resistance on the recurrence after curative radiofrequency ablation (RFA) of stage I HCC in HCV-positive patients. Methods: From January 2006 to December 2007, 226 consecutive patients underwent treatment for primary HCC at our institutions, including 37 stage I cases. Among them, 33 were HCV-positive, and three, six and 24 received curative surgery, transarterial chemoembolization or RFA, respectively. In the 24 patients treated with RFA, recurrence-free survival was analyzed using the Kaplan–Meier method. The factors contributing to recurrence of HCC were subjected to univariate and multivariate analyses using the Cox proportional hazards model. Insulin resistance was estimated by the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). Results: Kaplan–Meier analysis showed that the recurrence-free survival was lower in patients with higher HOMA-IR (>2.3, P = 0.0252) or with lower serum albumin level (

Clinical impact of protein-energy malnutrition (PEM) on the outcome of liver cirrhosis is well documented. As a candidate interventional modality to improve PEM in cirrhosis, effects of branched-chain amino acid (BCAA) supplementation on event-free survival and quality of life (QOL) was first reported by Yoshida et al. in 1989. Although critical arguments still continue regarding the effects of BCAA, several randomized trials in the last 5 years have brought positive results, and seem to have settled the discussion in a favorable direction for the efficacy of BCAA in liver cirrhosis. Actually, The European Society for Clinical Nutrition and Metabolism (ESPEN) upgraded the recommendation of BCAA supplementation in decompensated liver cirrhosis in the latest revision of its guidelines in 2006, by referring to the literatures from Italy and Japan. Particularly in these two long-term randomized studies with 1-2 years-supplementation, event-free survival was estimated by employing composite endpoints such as aggravation of hepatic failure (ascites, peripheral edema, hepatic encephalopathy, and jaundice), rupture of esophageal or gastric varices, development of liver cancer, and death from any cause. Both trials agreed on the effect of BCAA to reduce the incidence of hepatic failure, thus contributing to the rise in the event-free survival. Quality of life is another essential marker of outcome survey. Marchesini, Muto, and Nakaya reported the improved QOL in cirrhotics with BCAA supplementation. In particular, quantitative analysis of QOL measured by Short Form 36 (SF-36) questionnaire demonstrated a significant recovery of general heath perception score in BCAA supplemented patients in a randomized trial. In this article, the long-term outcome of BCAA treatment in liver cirrhosis will be reviewed with its action mechanisms. In addition, the effects of BCAA treatment on the incidence of liver cancer in obese patients with type C liver cirrhosis, significance of obesity as a risk factor for type C liver cancer, and a possible role of Body Mass Index to estimate the histological grade of fat deposition in the liver will be briefly discussed.

Primary adenoid cystic carcinoma (ACC) of the peripheral lung is a rare entity. Here we report two cases of primary ACC. Case 1 is an 84-year-old male with a past-medical history of cecal cancer presented with a 10 mm left upper lung nodule. Case 2 is a 40-year-old female who presented with 30 mm right upper lobe. Intraoperative (Case 1) and pre-operative (Case 2) histopathologic and cytologic diagnoses were consistent with a primary peripheral lung ACC. An upper lobectomy±mediastinal lymph node dissection was performed and immunohistochemical staining with thyroid transcription factor (TTF)-1, c-KIT and MYB on the excision specimen confirmed our diagnosis.

A congenital malformation of the bronchial tree, known as bronchial cyst, is mostly found in the mediastinum or lung. Intracardiac bronchogenic cysts are quite rare and only two cases of right ventricular bronchogenic cyst have so far been reported. We herein report a case in which a cyst developed in the right ventricle of a 65-year-old Japanese male who presented with aphasia due to cerebral infarction and pulmonary embolism. The cyst was successfully removed and the patient successfully recovered following surgery.

Tumor necrosis factor-alpha (TNF-alpha) is known as a proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes. TNF-alpha also influences liver repair following hepatotoxic damage, and regeneration following partial hepatectomy (PH). The aim of this study was to assess the mechanism by which TNF-alpha influences liver cell apoptosis and regeneration following PH in TNF-alpha-deficient (TNF-alpha(-/-)) mice.PH was performed in wild mice and TNF-alpha(-/-) mice.In both groups, serum alanine aminotransferase and serum total bilirubin levels comparably peaked at 6 and 48 h after PH, respectively. No differences were observed in hepatocyte proliferation, as determined by mitotic and the proliferating cell nuclear antigen labeling indices, between TNF-alpha(+/+) and TNF-alpha(-/-) mice. Few terminal deoxynucleotidyl transferase nick end-labeling-positive hepatocytes were seen in either type of mice. Nuclear factor-kappa B DNA binding activity in the remaining liver of TNF-alpha(-/-) mice after PH was similar to that of control mice. Ribonuclease protection assay showed that transforming growth factor beta1 mRNA was up-regulated comparably in the livers of the two groups, and that other cytokines were hardly seen in either. Interleukin-6/ signal transducer and activator of transcription-3-dependent pathway was not affected in TNF-alpha(-/-) mice.These findings suggest that TNF-alpha has little influence on liver regeneration and liver cell apoptosis after PH in mice.

Tumor necrosis factor (TNF)-alpha itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF-alpha/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)-kappaB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF-alpha. Here, we focused on the roles of NF-kappaB and phosphatidylinositol 3-kinase (PI3K)-regulated serine/threonine kinase Akt.TNF-alpha was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IkappaBalpha (Ad5IkappaB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl-2 family members and caspase activity were examined.In the mice livers, treatment with Ad5IkappaB or the wortmannin suppressed the activation of NF-kappaB or Akt, respectively. Suppression of either NF-kappaB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF-alpha administration. However, in mice treated with both Ad5IkappaB and wortmannin, TNF-alpha administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IkappaB, wortmannin, and TNF-alpha markedly increased the activation of caspase-3 and -9, and activated caspase-8 to a lesser degree, suggesting that TNF-alpha-induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF-kappaB and PI3K/Akt pathways had no effect on expression of Bcl-2 families.The inducible activation of NF-kappaB and constitutive activation of Akt regulate hepatocyte survival against TNF-alpha, which occurs independent of Bcl-2 families.

We encountered a case of left hepatic duct cancer that developed 7 years after surgical resection of early-stage adenocarcinoma of the gallbladder. A 65-year-old woman was hospitalized with high fever and general fatigue. She also had elevated serum levels of alkaline phosphatase, gamma-glutamyltranspeptidase, and carbohydrate antigen 19-9. Seven years earlier, she had undergone extended cholecystectomy and resection of the extrahepatic bile duct for early-stage mucinous adenocarcinoma of the gallbladder. Conventional examinations did not reveal any responsible lesions. Magnetic resonance (MR) cholangiography, however, showed a tumor obstructing the left hepatic duct, and dynamic MR images revealed multiple foci of bacterial abscess in the liver. Surgically resected tissue again revealed mucinous adenocarcinoma. The present case is rare in that metachronous mucinous adenocarcinoma of the biliary system occurred after a long interval. This case suggests the usefulness of MR imaging in the postsurgical monitoring of patients with gallbladder carcinoma.

A 55-year-old Japanese man presented with rapidly growing tumor in the left parotid region without any symptoms. Based on the fine-needle aspiration cytology report of parotid epithelial malignant tumor (suggestive of salivary duct carcinoma), the left parotidectomy was performed. Histopathology and immunohistochemistry examinations revealed features of salivary duct carcinoma. Although salivary duct carcinoma comprising a small proportion of salivary gland tumors and is known to be aggressive, he is free from recurrence and metastases 36 months after the surgery and radiation therapy.

Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.

Tumor necrosis factor-α (TNFα) and Fas ligand are death factors that bind to their receptors, two TNF receptors (TNFR), TNFR1 and TNFR2, and Fas, respectively, and induce apoptosis in a variety of cell types. In galactosamine (GalN)-sensitized mice, hepatocyte apoptosis and liver failure were observed after injection of TNFα. On the contrary, neither apoptotic cell nor liver injury was shown in mice treated with TNFα alone. Histological analyses revealed that administration of GalN/TNF-α caused massive hemorrhagic liver damage and fragmented nuclei in hepatocytes, which were similar to those observed after the treatment with anti-Fas antibody. In contrast to hepatotoxicity by TNFα, however, GalN hardly sensitized the liver to injury by anti-Fas antibody. Compared with the GalN/TNFα model, most of the changes occurred earlier in the anti-Fas model. The expression of TNFR1 mRNA in the liver was up-regulated and reached a peak within 2h after GalN administration. However, the change of TNFR2 mRNA in the liver was less than that of TNFR1 mRNA. GalN treatment failed to affect TNFα-induced nuclear factor-κB activation. These results indicate that unlike apoptosis through Fas, TNFR-mediated liver cell apoptosis requires sensitization of the parenchymal cells. In the sensitization of hepatocytes to apoptosis, up-regulation of TNFR1 on hepatocytes could play an important role.

Serum pro- and anti-inflammatory mediators in patients with acute liver diseases were assessed to clarify the clinical significance of these measurements in relation to disease severity. Concentrations of circulating tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, IL-12, and soluble TNF receptors (sTNFR) p55 and p75 were measured at admission in patients with fulminant hepatitis (FH; n=19), severe acute hepatitis (AHS, n=15), or acute hepatitis (AH, n=7). Serum concentrations of TNF-alpha, IL-10, and sTNFR-55 were significantly higher in patients with FH than in those with AHS (P