Wilson disease protein ATP7B is localized in the late endosomes in a polarized human hepatocyte cell line.

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. The gene responsible for Wilson disease has been cloned, however, the precise localization of this gene product ATP7B, a copper-transporting ATPase, is still controversial. We examined the localization of ATP7B by expressing a chimeric protein, ATP7B-tagged with green fluorescent protein (GFP) (GFP-ATP7B), in HEK293, Hep3B and a highly polarized human hepatocyte line (OUMS29). Intracellular organelles were visualized by immunofluorescence microscopy. The effects of bathocuproine disulfonate, a copper chelator, and copper sulfate were examined. GFP-ATP7B colocalized with a late endosome marker, but not with endoplasmic reticulum, Golgi, or lysosome markers in a copper-depleting condition. Treatment with copper sulfate did not affect the localization of ATP7B. ATP7B is localized in the late endosomes in both copper-depleting and copper-loaded conditions. ATP7B seems to translocate copper from the cytosol into the late endosomes, and copper may be excreted to bile via lysosomes. We believe that the disturbed incorporation of copper into the late endosomes caused by mutated ATP7B is the main defect in Wilson disease.

Expression of human glucocorticoid receptor in lymphocytes of patients with autoimmune hepatitis.

Background and aim: Alternative splicing of human glucocorticoid receptor (hGR) premessenger RNA (mRNA) generates two highly homogenous isoforms, termed hGRalpha and hGRbeta. hGRalpha is a ligand-activated transcription factor, which, in the hormone-bound state, modulates the expression of glucocorticoid-responsive genes by binding to specific glucocorticoid response element DNA sequences. In contrast, hGRbeta may be an endogenous inhibitor of glucocorticoid action and transcriptionally inactive. hGRbeta protein has been known to correlate with the development of glucocorticoid resistance. Glucocorticoids can effectively relieve autoimmune hepatitis (AIH), but some patients with this disease are refractory even when glucocorticoids are administered. The aim of this study was to determine the incidence of hGRbeta mRNA in patients with AIH by reverse transcription polymerase chain reaction (RT-PCR), and to compare the clinical characteristics of hGRbeta-positive and -negative patients with AIH. Materials and methods: RNA was obtained from peripheral blood mononuclear cells (PBMCs) of 62 patients, consisting of 26 with AIH, 10 with primary biliary cirrhosis (PBC), seven with chronic viral hepatitis (CVH), 10 with ulcerative colitis (UC), six with pemphigus, and three with systemic lupus erythematosus (SLE), and 10 healthy volunteers. The total RNA obtained was reverse transcribed, the resulting complementary DNA amplified using specific primers for hGRalpha and hGRbeta. Results: The hGRalpha mRNA was detected in RNA from PBMCs of all patients and healthy volunteers. The hGRbeta mRNA was detected in 15 (57.6%) patients with AIH. This incidence was significantly higher than that for patients with PBC (0%) or CVH (28.6%) or for healthy volunteers (20.0%) ( [Formula: see text] ). Of the hGRbeta-positive and -negative groups, serum ALT and total bilirubin (TB) levels were significantly higher in the positive group ( [Formula: see text] ). The total dose of glucocorticoid was higher in the positive group, but the difference was not statistically significant. However, the average monthly dose was significantly higher in the positive group ( [Formula: see text] ). The rate of relapse of AIH was significantly higher in the positive group (60.0%) than in the negative (10.0%) ( [Formula: see text] ). The rate of usage of immunosuppressive drugs was higher in the positive group (33.3%) than in the negative (18.2%), but the difference was not statistically significant. Conclusions: These data show that hGRbeta expression in PBMCs of patients with AIH assessed by RT-PCR is closely associated with resistance to glucocorticoids which affects the outcome of therapy with this drug.

A case of merged idiopathic portal hypertension in course of mixed connective tissue disease.

We report a case of idiopathic portal hypertension (IPH) complicated with mixed connective tissue disease (MCTD). The patient, a 41-year-old woman, was admitted to another hospital because of tarry stools in March 2000. Emergency endoscopy revealed bleeding from the esophageal varices, and she was referred to our hospital. Twelve years before, she had been diagnosed as having MCTD, because she exhibited Raynaud's phenomenon, pleuritis, pericarditis, and had high titers of anti nuclear antibody (ANA) and anti-U1 ribonucleoprotein (RNP). Laboratory examinations, imaging examinations, and liver biopsy indicated that the esophageal varices were caused by IPH. The association of IPH and MCTD is very rare; to the best of our knowledge, only five cases of MCTD, associated with pulmonary hypertension (PH), have been reported.

A case of hepatic inflammatory pseudotumor with primary biliary cirrhosis.

Inflammatory pseudotumor (IPT) of the liver is an unusual non-neoplastic benign lesion. A 75-year-old man was hospitalized for esophageal varices and gastric cancer. Three years before admission, he had been diagnosed as having primary biliary cirrhosis (PBC) without Sjögren's syndrome. Computed tomography (CT) and magnetic resonance imaging (MRI) scans showed multiple masses (S3, S5, S6) less than 2 cm in diameter in the liver. Since these masses were difficult to distinguish from hepatocellular carcinoma, or metastatic liver carcinoma, one of the masses (S5) was removed during an operation for gastric cancer. Histological examination demonstrated marked infiltration of plasma cells and some histiocytes, findings consistent with the histological features of IPT. The coexistence of hepatic IPT and PBC in this case may have been an accidental event. However, the immunological and environmental factors associated with PBC are thought to be involved in the development of IPT; in addition, cholangitis associated with PBC could have contributed to the development of IPT.

Autoimmune hepatitis accompanied by systemic lupus erythematosus.

We report a series of five patients with autoimmune hepatitis (AIH) accompanied by systemic lupus erythematosus (SLE) (AIH-SLE overlap). Serologic tests showed that all patients were positive for antinuclear antibody and double-stranded DNA antibody. Histological examination of the liver showed that three of the patients had chronic hepatitis with severe activity. One of the other two had acute and severe hepatitis with submassive necrosis in both portal and lobular areas. The last patient already had liver cirrhosis. All patients had a mild form of SLE and showed a rapid response to corticosteroid. There was no serious involvement of organs other than the liver in any of the patients, and the prognoses were comparatively good in all patients.

Autoimmune hepatitis overlapping with primary sclerosing cholangitis.

A case of autoimmune hepatitis (AIH) complicated by primary sclerosing cholangitis (PSC) in a 36-year-old woman is reported. AIH overlapping with PSC has been rarely reported, and to the best of our knowledge, there have been no reports in Japan. Based on the criteria for diagnosis of AIH and on typical endoscopic retrograde cholangiograph (ERC) findings, the patient was diagnosed as having AIH overlapping with PSC. Her transaminase levels normalized within 1 month after administration of prednisone, azathioprine and ursodeoxycholic acid, but her cholestatic enzyme level remained elevated. The effect of treatment on the cholestatic features is thought to be an important factor for predicting the prognosis of AIH overlapping with PSC.

Adhesion molecules and CXC chemokines in endotoxin-induced liver injury.

Interactions between leukocytes and sinusoidal endothelial cells are known to be involved in the pathogenesis of acute liver injury. Various adhesion molecules and chemokines play key roles in these cell-to-cell interactions, and the expression of these adhesion molecules and the production of chemokines are regulated by inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interferon-gamma (IFN-gamma). We have shown that the expression of intercellular adhesion molecule-1 (ICAM-1) on cultured rat sinusoidal endothelial cells stimulated with TNF-alpha increases in a dose-dependent manner. The number of neutrophils that adhered to sinusoidal endothelial cells pretreated with TNF-alpha also increased in a dose-dependent manner and significantly decreased upon incubation with an anti-ICAM-1 antibody. In endotoxin-induced rat liver injury, the number of neutrophils infiltrating the sinusoids increased after serum TNF-alpha, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) reached their peak levels. In addition, the level of ICAM-1 expression on sinusoidal endothelial cells greatly increased from 8 h after exposure to endotoxin, and these cells were adhered to neutrophils that expressed both LFA-1 and Mac-1. Moreover, lipo-prostaglandin E1 (PGE1) reduced the extent of liver injury, and also reduced the number of neutrophils that infiltrated the liver, was reduced the production of MIP-2 and CINC, but not that of TNF-alpha, in rats injected with endotoxin.

HLA-A2-restricted cytotoxic T lymphocyte activity during interferon beta therapy in patients with chronic hepatitis C.

BACKGROUND AND AIMS: Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) may contribute to viral clearance and liver cell injury in patients with chronic hepatitis C. In the present study, we attempted to determine the serial HCV-specific CTL activity during interferon-beta (IFN-beta) therapy in patients with chronic hepatitis C and whether there is any relationship between the CTL response and clinical response to IFN-beta therapy. METHODS: Eight HLA-A2-positive patients with chronic hepatitis C were treated initially with 6 million U/ml of IFN-beta every day for 8 weeks and then 3 times weekly for the subsequent 16 weeks. Peripheral blood mononuclear cells (PBMC) were collected before the start, 4 weeks after the start, and after the end of IFN treatment and were stimulated with 2 peptides corresponding to core sequences, which were previously reported to have an HLA-A2 restricted-CTL epitopes. Cytolytic activity was determined by a standard 51Cr-release assay using allogenic HLA-matched EBV-transformed B lymphoblastoid cell lines (B-LCL). RESULTS: HCV-specific CTL responses were detected in 2 of the 8 patients before treatment with IFN-beta. One of 2 patients was not observed HCV-specific CTL responses after 4 weeks of IFN-beta treatment, however these two patients showed CTL responses at the end of IFN-beta treatment, and finally HCV-RNA was negative. In addition, HCV-specific CTL responses were observed in 4 patients after 4 weeks of IFN-beta treatment. Three of these 4 patients showed CTL responses only at 4 weeks after IFN-beta treatment. However, there were no differences between clinical parameters or between IFN efficacy in HCV specific CTL response-positive (n = 4) and -negative (n = 4) patients at 4 weeks after the start of IFN-beta treatment. CONCLUSIONS: These findings suggest that there are few relations between peripheral HCV-specific CTL response and clinical response to IFN therapy in patients with chronic hepatitis C, although IFN enhances the host immune response against HCV synergistically with antiviral activities.

Expression of uncoupling protein-2 in biliary epithelial cells in primary biliary cirrhosis.

BACKGROUND/AIMS: Uncoupling proteins are thought to protect cells from oxidative stresses. Because uncoupling protein-2 is expressed in liver and reactive oxygen species are involved in pathogenesis of various liver diseases, this protein may protect liver cells from disease-associated oxidative stress. However, uncoupling protein-2 expression in human liver has not been examined. METHODS: We investigated hepatic uncoupling protein-2 distribution in various liver diseases including primary biliary cirrhosis, autoimmune hepatitis, chronic viral hepatitis, and histologically normal liver by immunohistochemistry. RESULTS: Uncoupling protein-2 was expressed in some hepatocytes, however, the degree of hepatocytic uncoupling protein-2 expression did not differ significantly among liver diseases and normal liver. Uncoupling protein-2 was abundant in biliary epithelial cells in primary biliary cirrhosis but not in other liver specimens. Enhanced uncoupling protein-2 expression in biliary epithelial cells was specific for primary biliary cirrhosis and did not result simply from cholestasis. The percentage of uncoupling protein-2 positive bile ducts in primary biliary cirrhosis patients treated with ursodeoxycholic acid was significantly lower than in untreated patients. CONCLUSIONS: These results suggest that uncoupling protein-2 is involved in the pathogenesis of primary biliary cirrhosis.

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes.

Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazone-treated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (DeltaPsi(m)) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2',7'-dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electron-dense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the DeltaPsi(m) per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity.

Troglitazone induces p27Kip1-associated cell-cycle arrest through down-regulating Skp2 in human hepatoma cells.

Increasing evidence has confirmed that ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) exhibit antitumoral effects through inhibition of cell proliferation and induction of cell differentiation in several malignant neoplasms. Recently, we have documented the accumulation of a cyclin-dependent kinase inhibitor, p27(Kip1), as well as an unexpected accumulation in cyclin E in G1-arrested human hepatoma cells treated with the PPARgamma ligand troglitazone. Simultaneous accumulations in both p27(Kip1) and cyclin E are known to be characteristic phenotypes in cells derived from mice lacking Skp2, an F-box protein component of the SCF ubiquitin-ligase complex. Thus, the aim of the present study was to assess whether Skp2 might be involved in the down-regulation of p27(Kip1) in troglitazone-treated human hepatoma cells. A striking decrease in Skp2 expression and a reciprocal increase in p27(Kip1) expression were found in troglitazone-treated hepatoma cells but not in those cells treated with other PPARgamma ligands such as pioglitazone and ciglitazone. Quantitative real-time RT-PCR analysis showed that troglitazone down-regulated Skp2 at the mRNA levels. Consistently, ectopic overexpression in Skp2 brought resistance to troglitazone, resulting in a decreased population of arrested cells at the G1 phase compared with that in the mock-transfected cells. In surgically resected hepatocellular carcinoma (HCC) tissue, an increased expression in Skp2 was found in both the moderately differentiated HCCs and the poorly differentiated HCCs. In conclusion, troglitazone attenuated Skp2 expression, thereby promoting p27(Kip1) accumulation in human hepatoma cells. This therapeutic potential of the ligand may lead to new cell-cycle-based antitumor strategies for advanced HCCs.