A murine model of NKT cell-mediated liver injury induced by alpha-galactosylceramide/d-galactosamine.

Natural killer-T (NKT) cells are rich in the liver. However, their involvement in liver injury is not fully understood. We developed here a new murine model of NKT-cell-activation-associated liver injury, and investigated a role of tumor necrosis factor alpha (TNF-alpha) and Fas in pathogenesis. We injected intraperitoneally alpha-galactosylceramide (alpha-GalCer), an NKT-cell stimulant, into D-galactosamine (GalN)-sensitized mice. Survival rate, pathological changes of the liver, and plasma concentrations of cytokines were studied. Alpha-GalCer/GalN administration gave a lethal effect within 7 h, making pathological changes such as massive parenchymal hemorrhage, hepatocyte apoptosis, sinusoidal endothelial cell injury, and close apposition of lymphocytes to apoptotic hepatocytes. Anti-NK1.1 mAb-pretreated mice and Valpha14NKT knock out (KO) mice did not develop liver injury. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were elevated at 4 h in the plasma. These cytokines were produced by hepatic lymphocytes as demonstrated by in vitro stimulation with alpha-GalCer. The lethal effect was suppressed in TNF-alpha KO mice, TNF receptor-1 KO mice, and lpr/lpr (Fas deficient) mice, whereas it was not in IFN-gamma KO mice. These results indicate that the present liver injury is characterized by parenchymal hemorrhage and hepatocyte apoptosis, and mediated by TNF-alpha secretion and direct cytotoxicity of alpha-GalCer-activated NKT cells.

Clinicopathological significance of oxidative cellular damage in non-alcoholic fatty liver diseases.

Oxidative stress may play an important role in the progression of simple steatosis to non-alcoholic steatohepatitis (NASH). Oxidative stress is generated through multiple sources, including oxidation of free fatty acids, cytochrome P4502E1, iron overload, and necro-inflammatory cytokines including tumor necrosis factor-alpha. Oxidative stress may trigger damage to cellular membranes and nuclear DNA, which results in lipid peroxidation and oxidative DNA damage, respectively. Here, we present our data on intrahepatic localization and clinico-pathological significance of oxidative stress-induced cellular damage in the patients with non-alcoholic fatty liver diseases (NAFLD). Our subjects were 23 patients with non-alcoholic simple fatty liver, 17 with NASH, and 7 with normal liver (control). Hepatic expression of 4-hydroxy-2'-nonenal (HNE) and 8-hydroxydeoxyguanosine (8-OHdG), as reliable markers of lipid peroxidation and oxidative DNA damage, was in situ detected by using commercially available monoclonal antibodies. While no HNE adducts were observed in control livers, they were frequently detected in NAFLD. In NASH, the localization of the HNE adducts was in the cytoplasm of sinusoidal cells and hepatocytes with a predominance in zone 3. The grade of necro-inflammation as well as the stage of fibrosis significantly correlated with the HNE labeling index. With respect to 8-OHdG, although no 8-OHdG expression was observed in normal liver and only a few in non-alcoholic simple fatty liver, 11 of 17 patients with NASH (65%) exhibited nuclear expression of 8-OHdG in hepatocytes and sinusoidal cells in areas of active inflammation. The 8-OHdG index significantly correlated with the grade of necro-inflammation, but not with the stage of fibrosis. Our observations suggest that oxidative cellular damage occurs frequently in livers with NAFLD and may be associated with some clinico-pathological features of NAFLD including liver fibrosis and possibly, hepatocarcinogenesis.

Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.

Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries.

Expression of progenitor cell markers in livers with fulminant massive necrosis.

Studies of stem cells in various organs have greatly progressed, and progenitor cells have been confirmed even in liver by recognition of cytokeratin 14 (CK14), c-kit, flt-3, and CD34. We, therefore, immunohistochemically examined the expression of these progenitor cell markers in patients with confluent or massive necrosis, in addition to CK19, albumin, vimentin, and Ki-67. Our subjects were six survivors and 14 deceased patients. Expression of CK14 and c-kit was found in a small number of cells lining biliary ductule-like structures, and that of flt-3 was found in many lining cells in two deceased patients with multi-lobular necrosis. CK14 positive cells were positive for c-kit, flt-3, and CK19 in semi-serial sections, but were negative for albumin, Ki-67, and CD34. In conclusion, expression of CK14 and c-kit was found in a small number of cells lining biliary ductule-like structures, and that of flt-3 was found in many cells lining biliary ductule-like structures. CK14-positive cells were positive for c-kit, but negative for CD34. Since c-kit is a hematopoietic marker, our study suggests that CK14- and c-kit-positive cells may be derived from bone marrow in liver with fulminant hepatitis.

Fatty change in human hepatocellular carcinoma cell lines derived from patients with antibodies to hepatitis C virus.

BACKGROUND/AIMS: Evidence suggesting a relationship between fatty change in normal or malignant hepatocytes and hepatitis C virus has gradually accumulated, but less is known about the relationship between cell proliferation and fatty change in human hepatocellular carcinoma. METHODOLOGY: We studied the latter issue in two human hepatocellular carcinoma cell lines (OCUH-16 and Nuk-1) derived from hepatitis C virus-associated tumors. We examined the relationship between degree of fatty change assessed by oil-red-O staining and electron microscopy, actively proliferating cells counted using a monoclonal antibody to MIB-1 protein, and apoptotic cells counted using DNA nick-end labeling in the above two hepatocellular carcinoma cell lines with time lapse. RESULTS: On day 1 in culture, fatty change was present randomly in cytoplasm of some Nuk-1 cells, but was not found in OCUH-16 cells. Over time, fat droplets were found more frequently in large hepatocellular carcinoma cells in both Nuk-1 and OCUH-16 lines. Most of these cells were located in the periphery of hepatocellular carcinoma cell nests or islands as opposed to the small hepatocellular carcinoma cells located in the centers of nests in both lines. According to MIB-1 staining, these small cells proliferate more actively than the large, peripherally located hepatocellular carcinoma cells. Only a few apoptotic hepatocellular carcinoma cells were detected during culture. CONCLUSIONS: Fatty change in large hepatocellular carcinoma cells seems to be associated with less proliferative activity than was seen in small hepatocellular carcinoma cells without fatty change, located in more centrally cell nests, in these hepatocellular carcinoma cell lines.

Immunohistochemical detection of Fas and apoptosis in type-1 autoimmune hepatitis.

BACKGROUND/AIMS: Although Fas expression has been reported in liver with chronic viral hepatitis and primary biliary cirrhosis, little is known about Fas expression and apoptosis in type-1 autoimmune hepatitis. The aim of this study was to investigate whether the expression of Fas and apoptosis are found in liver with autoimmune hepatitis. The relationship between Fas expression and clinicopathological findings including the occurrence of apoptosis was also investigated. METHODOLOGY: Fas expression and apoptosis were immunohistochemically examined in liver tissues from 20 patients with autoimmune hepatitis and five control subjects using specific antibodies against Fas and single-stranded DNA. The grade of expression of Fas and apoptosis was evaluated and compared with histological findings and the results of liver function tests in each patient. RESULTS: Fas expression in hepatocytes was detected in all patients with autoimmune hepatitis, while Fas expression was not detected in control livers. The Fas-positive hepatocytes were particularly abundant in those areas facing piecemeal and confluent necrosis. In 30% of autoimmune hepatitis cases, bile-duct cells were faintly stained for Fas. A few hepatocytes positive for single-stranded DNA were found in the areas facing piecemeal necrosis and confluent necrosis. In 95% cases, many bile-duct cells were positive for single-stranded DNA. No relationship between the expression of Fas and single-stranded DNA was found in hepatocytes or bile-duct cells. However, the degree of Fas expression in hepatocytes significantly correlated with serum transaminase concentrations and was increased in parallel with the grade of activity but not with the stage of fibrosis. CONCLUSIONS: We have demonstrated that Fas expression is detected in hepatocytes of the liver with autoimmune hepatitis and that the level of Fas expression reflects the severity of inflammation in autoimmune hepatitis.

A new chemotherapeutic regimen for advanced unresectable hepatocellular carcinoma.

BACKGROUND/AIMS: The aim of this study was to evaluate the efficacy and toxicity of a novel chemotherapeutic regimen for advanced unresectable hepatocellular carcinoma. METHODOLOGY: Seventeen patients with unresectable hepatocellular carcinoma were treated by arterial infusion once a week of low-dose cisplatin (12 mg/m2) and doxorubicin (6 mg/m2) via a subcutaneously implanted injection port and by daily oral administration of 300 mg/day of UFT comprising 5-fluorouracil prodrug tegafur (FT) and uracil (U) at a ratio of 1:4. RESULTS: The median number of chemotherapy courses was 13 (range, 5-33). All patients were evaluated for response, toxicity, and survival. As assessed by conventional imaging criteria, there were 3 (17.6%) complete responses with disappearance of the primary tumor, tumor thrombosis of the portal vein and metastatic para-aortic lymph node swelling. In addition, there were 4 (23.5%) partial responses. Among 11 patients who had initially high alpha-fetoprotein levels (> 200 ng/mL), 5 (45%) had a > 50% drop after therapy. The overall tumor response rate (complete response + partial response) was 41% and the median survival was 7.1 (range; 4.2-25.1) months. As for toxicity, there was 1 treatment-related death due to septicemia caused by catheter-related infection. Myelosuppression and renal toxicity was relatively mild and transient. CONCLUSIONS: These results suggest that our low-dose chemotherapeutic regimen may be useful for the treatment of advanced unresectable hepatocellular carcinoma without worsening the quality of life of the patient.

Life-threatening severe immune thrombocytopenia during alpha-interferon therapy for chronic hepatitis C.

Although mild thrombocytopenia is a common adverse effect of interferon therapy, severe life-threatening thrombocytopenia is extremely rare. Here, we report a case of chronic hepatitis C patient that developed severe thrombocytopenia during alpha-interferon therapy, possibly due to an autoimmune mechanism. A 24-year-old female presented chronic hepatitis C in May, 1998. Based on the clinicopathological findings including a liver biopsy, administration of alpha-interferon was begun. In the fourth week of therapy, she experienced mild dyspnea and general fatigue. Complete blood count demonstrated thrombocytopenia (48,000/microL). Despite the immediate withdrawal of interferon, her platelet count further decreased to 1,100/microL. Bone marrow aspirate and elevated platelet-associated IgG antibodies were suggestive of immune thrombocytopenia. She was treated with intravenous and oral administration of steroids. Her platelet count returned to normal level 5 days later. Response to steroid treatment was consistent with the diagnosis of alpha-interferon-induced immune thrombocytopenia in this patient.

A clinicopathological study of inflammatory pseudotumors of the liver with special reference to vessels.

BACKGROUND/AIMS: Inflammatory pseudotumor of the liver is rare, and patients with inflammatory pseudotumor frequently undergo unnecessary surgical resection as a result of misdiagnosis of malignancy. In this study, we therefore investigated inflammatory pseudotumor clinicopathologically to clarify its characteristics. METHODOLOGY: Twenty patients including 3 with inflammatory pseudotumor and 17 with various malignant liver tumors were studied. We further investigated tumor vessels by means of immunohistochemistry using monoclonal antibodies against CD34, factor VIII-related antigen and alpha-smooth muscle actin. RESULTS: Although serum levels of alkaline phosphatase were significantly higher in inflammatory pseudotumor patients than in other patients, the laboratory data alone could not precisely distinguish inflammatory pseudotumor from other hepatic tumors. On imaging studies such as ultrasonography and computed tomography, significant changes in tumor size, especially size reduction, during relatively short follow-up periods were often observed in inflammatory pseudotumor but not in other liver tumors. An enhancement of the peripheral regions of inflammatory pseudotumor was frequently observed in the early phase of contrast-medium dynamic computed tomography. This might be due to abnormal vessels located in the peripheral regions of inflammatory pseudotumor which might result from obliteration of some pre-existing vessels in portal tracts within inflammatory pseudotumor. Immunohistochemical analysis further revealed that abnormal vessels in the peripheral regions of inflammatory pseudotumor were positively stained with CD34, factor VIII-related antigen and alpha-smooth muscle actin as were tumor sinusoids within hepatocellular carcinoma and tumor capillaries in other malignant liver tumors. CONCLUSIONS: Although inflammatory pseudotumor seems to have some features in imaging studies, a biopsy is needed for a correct diagnosis of inflammatory pseudotumor.

Expression of Fas and Bcl-2 proteins and induction of apoptosis in human hepatocellular carcinoma cell lines.

Because the induction of apoptosis in cancer cells is very important in clinical management, it is useful to examine the association with the Fas-Fas ligand pathway and Bcl-2 protein family in apoptosis. We morphologically examined the expression of Fas and Bcl-2 proteins and induction of apoptosis by anti-Fas in four human hepatocellular carcinoma cell lines, PLC/PRF/5, Huh-6, and Huh-7, as well as OCUH-16, which was originally established in our university. Fas protein was expressed in 96% of OCUH-16 cells in cytoplasm, 24% of PLC/PRF/5 cells, 20% of Huh-6 cells, and no Huh-7 cells. Bcl-2 protein was expressed in 43%-72% of cells in cytoplasm and nuclei of the four lines examined. Administration of anti-Fas induced apoptosis in about 40% of OCUH-16 cells, but did not induce apoptosis in the other three cell lines. In conclusion, an original cell line, OCUH-16 cells, expressed Fas and Bcl-2 proteins and underwent apoptosis following treatment with anti-Fas, but the other three cell lines examined did not undergo apoptosis. OCUH-16 cells are thus very useful for the study of apoptosis and molecules related to apoptosis at the levels of cell-surface receptors and intracytoplasmic regulation of apoptosis.

In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases.

BACKGROUND/AIMS: Although oxidative stress is an important candidate in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), the localization and pathological significance of oxidative stress-induced cellular damage in NAFLD remains unclear. METHODS: Hepatic expression of 4-hydroxy-2'-nonenal (HNE) and 8-hydroxydeoxyguanosine (8-OHdG), as reliable markers of lipid peroxidation and oxidative DNA damage, respectively, was immunohistochemically investigated in NAFLD and the results were compared with histological findings. RESULTS: While no HNE adducts were observed in control livers, they were frequently detected in NAFLD. In NASH, the localization of the adducts was in the cytoplasm of sinusoidal cells and hepatocytes with a predominance in zone 3. The grade of necro-inflammation as well as the stage of fibrosis significantly correlated with the HNE index. Regarding 8-OHdG, although no 8-OHdG expression was observed in normal liver and only a few in fatty liver, 11 of 17 cases (64.7%) with NASH exhibited nuclear expression of 8-OHdG in hepatocytes and sinusoidal cells in areas of active inflammation. The 8-OHdG index significantly correlated with the grade of necro-inflammation. CONCLUSIONS: Oxidative cellular damage occurs frequently in livers with NAFLD and may be associated with some clinico-pathological features of NAFLD including liver fibrosis and possibly, hepatocarcinogenesis.