bluuebunny/crossref_metadata_2025 huggingface dataset:
Racial differences in responses to therapy with interferon in chronic hepatitis C
Authors: Reddy Rajender K. , Hoofnagle Jay H. , Tong Myron J. , Lee William M. , Pockros Paul , Heathcote Jenny E. , Albert Donald , Joh Tenshang
Published: July 2004

The likelihood of a sustained response to a course of interferon in patients with chronic hepatitis C correlates with several clinical and viral factors, including age, viral genotype and initial levels of hepatitis C virus (HCV) RNA in serum. The role of race and ethnicity has not been assessed. We evaluated the association of race with response to interferon in a large randomized, controlled trial using either consensus interferon (9 μg) or interferon alfa-2b (3 million units) given three times weekly for 24 weeks. African-American patients participating in the study were similar to white patients in mean age (43 vs. 42 years) and baseline levels of HCV RNA (3.6 vs. 3.0 million copies/mL) but had lower rates of cirrhosis (5% vs. 12%) and more frequently had viral genotype 1 (88% vs. 66%:P= .004). Most strikingly, the rates of end-of-treatment and sustained virological responses were lower among the 40 African-American patients (5% and 2%) than among the 380 white patients (33% and 12%) (P= .04 and .07). Rates of response among Hispanic and Asian-American patients were not statistically different than non-Hispanic white patients. Median viral levels decreased by week 24 of therapy by 2.5 logs in white patients (from 3.0 to 0.012 million copies/mL) but by only 0.5 logs among African- American patients (from 3.6 to 1.8 million copies/mL). Thus, there are marked racial differences in virological responses to interferon in hepatitis C that must be considered in assessing trials of interferon therapy and in counseling patients regarding treatment. The differences in response rates are as yet unexplained.
Redox manipulation using the thiol-oxidizing agent diethyl maleate prevents hepatocellular necrosis and apoptosis in a rodent endotoxemia model
Authors: Jones Julia J. , Fan Jie , Nathens Avery B. , Kapus Andras , Shekhman Mark , Marshall John C. , Parodo Jean , Rotstein Ori D.
Published: July 2004

Manipulation of the intracellular redox state has been shown to alter cell activation pathways with resultant changes in cellular function. Previous studies have suggested that thiol oxidation, using the glutathione-depleting agent diethyl maleate (DEM), was able to inhibit endothelial cell activation. We hypothesized that this agent might exert beneficial effects following endotoxemia in the rat, a model in which transendothelial migration of neutrophils is central to the development of hepatocellular injury. Sprague-Dawley rats treated intraperitoneally with lipopolysaccharide (LPS) (200 μg/kg) plus d -galactosamine (GalN) (600 mg/kg) developed hepatocellular necrosis, as evidenced by liver enzyme release and morphological changes. Pretreatment with DEM abrogated this injury in a dose-dependent fashion. Histology revealed reduced neutrophil accumulation in both the parenchyma and sinusoids, consistent with reduced neutrophil sequestration and transendothelial migration. This effect appeared to be related to the ability of DEM to prevent LPS-induced up-regulation of both vascular cell adhesion molecule-1 (VCAM-1) mRNA and intercellular adhesion molecule-1 (ICAM-1) mRNA in the liver, as well as reducing tumor necrosis factor (TNF) mRNA expression. In addition, DEM prevented hepatocyte apoptosis following LPS treatment. The effect was reproduced when TNF was used as an inflammatory stimulus, suggesting a direct protective effect on the hepatocyte. Taken together, these studies show that redox manipulation through thiol oxidation may represent a novel approach to preventing liver necrosis and apoptosis in inflammatory conditions.
Structure and expression of Tg737, a putative tumor suppressor gene, in human hepatocellular carcinomas
Authors: Bonura Celestino , Paterlini-Brechot Patrizia , Brechot Christian
Published: July 2004

Deletions of the Tg737 gene, whose product is involved in liver oval cell proliferation, differentiation, and ploidy control, have been recently shown in chemically induced rat liver tumors and in a limited series of patients with hepatocellular carcinoma (HCC). Thus, Tg737 has been proposed as a candidate new liver-specific tumor suppressor gene. To investigate this important issue, we analyzed the structure and expression pattern of the Tg737 gene in a group of 23 tumorous and adjacent nontumorous liver tissues, by combining polymerase chain reaction (PCR) and Southern and Northern blot-based analyses. We failed to identify deletions or gross alterations of the Tg737 gene by both PCR and Southern blot analyses. Northern blots showed comparable accumulation of normal Tg737 transcripts in both tumorous and nontumorous tissues. Collectively, therefore, our results do not support the hypothesis of frequent Tg737 genetic alterations in human HCC.
Current practice patterns of primary care physicians in the management of patients with hepatitis C
Authors: Shehab Thomas M. , Sonnad Seema S. , Jeffries Mark , Gunaratnum Naresh , Lok Anna S.
Published: July 2004

Approximately 4 million Americans are infected with the hepatitis C virus (HCV). Most patients with hepatitis C have no symptoms until cirrhosis is established. Thus, initial diagnosis and management of hepatitis C rely on primary care physicians identifying and screening high-risk individuals. We administered a survey to 1,233 primary care physicians in a health maintenance organization (HMO) in April 1997 to assess their knowledge of the risk factors for HCV infection and approach to the management of 2 hypothetical HCV antibody-positive patients, 1 with elevated and the other with normal alanine transaminase (ALT). Four hundred four (33%) physicians returned the survey. Ninety percent of respondents correctly identified the risk factors for HCV infection, but 20% still considered blood transfusion in 1994 as a significant risk factor for HCV infection. Sixty-two percent of respondents would refer HCV antibody-positive patients with abnormal transaminase levels, but 33% would follow these patients themselves, even though none of the respondents had treated any hepatitis C patient on their own. Forty-three percent of respondents overestimated, while 29% did not know the efficacy of interferon treatment. Sixty-five percent of respondents would retest patients for HCV antibody, regardless of risk factors and transaminase levels. We found that most primary care physicians correctly identified the significant risk factors for HCV infection and appropriately managed the 2 hypothetical patients, but there was considerable confusion about the use of HCV tests and the effectiveness of treatment. Educational programs for primary care physicians are needed to implement hepatitis C screening and to initiate further evaluation and management of those who test positive.
Consistent infiltration of thymus-derived T cells into the parenchymal space of the liver in normal mice
Authors: Yamamoto Satoshi , Sato Yoshinobu , Shimizu Takao , Halder Ramesh C. , Oya Hiroshi , Bannai Makoto , Suzuki Kenji , Ishikawa Hiromichi , Hatakeyama Katsuyoshi , Abo Toru
Published: July 2004

We previously reported that extrathymic T cells (intermediate T-cell receptor cells [TCRint cells]) are in situ generated in the parenchymal space of the liver in mice. They subsequently migrate to the sinusoidal lumen. In this study, we characterized how such extrathymic T cells, natural killer (NK) cells, and thymus-derived T cells (high T-cell receptor cells [TCRhigh cells]) localized in the parenchymal space or the sinusoidal lumen of mice. To this end, liver irrigation with physiological saline from the portal vein was performed and the distribution of lymphocyte subsets was compared between the liver (i.e., lymphocytes in the parenchymal space) and the irrigation solution (i.e., lymphocytes in the sinusoidal lumen). Extrathymic T cells and NK cells were found to be abundant in both the liver and sinusoidal lumen. As expected, thymus-derived T cells were abundant in the sinusoidal lumen. However, a significant proportion of thymus-derived T cells were always present in the parenchymal space, even after intensive irrigation with or without collagenase. These results suggest that thymus-derived T cells may consistently infiltrate the parenchymal space from the sinusoidal lumen in normal mice. This possibility was confirmed by (1) the injection of B6 splenic cells (TCRhigh cells) or the thymus graft into B6-nu/nu mice (presence of only TCRint cells) and by (2) using parabiotic mice of B6.Ly5.1 and B6.Ly5.2 strains (sharing circulation) in conjunction with immunofluorescence tests and immunohistochemical staining. In other words, inverted routes of migration and homing between extrathymic T cells and thymus-derived T cells exist in the liver.
Bile acid structure and selective modulation of murine hepatic cytochrome P450-linked enzymes
Authors: Paolini Moreno , Pozzetti Laura , Piazza Francesco , Cantelli-Forti Giorgio , Roda Aldo
Published: July 2004

We examined the effects of the administration of different bile acids onin vivohepatic murine cytochrome P450 (CYP) content, nicotinamide adenine dinucleotide phosphate (NADPH)-CYP-reductase, and individual mixed-function oxidases (MFOs). Neither CYP level nor reductase were appreciably affected by single intraperitoneal administration of taurodeoxycholic acid (TDCA) (12.2 or 24.4 mg × kg−1bw). MFO to various isoenzymes were slightly reduced 24 hours after treatment. Taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) both induced CYP, reductase, and MFOs. CYP3A1/2-linked activity (i.e., testosterone 6β-hydroxylase, andN-demethylation of aminopyrine) in a dose-dependent fashion was enhanced (˜2-3-fold). CYP2E1- (hydroxylation ofp-nitrophenol), CYP1A2-(O-demethylation of methoxyresorufin), CYP2A1/2- and CYP2B1/2-(6α-hydroxylase), and CYP2B9- (16α-hydroxylase) dependent MFOs, as well as 7α-, 16β-, 2α-, and 2β-hydroxylations, were all significantly induced by THDCA. Apart from alkoxyresorufin metabolism and a modest CYP2E1 increase, TUDCA behaved like THDCA. A generalized induction was also recorded after ursodeoxycholic acid (UDCA) administration. THDCA and TDCA did not show substantial differences in theN-demethylation of aminopyrine when different species (rat vs. mouse) and administration route (intraperitoneal vs. intravenous) were compared. Results on the most affected isoenzymes, CYP3A1/2 (THDCA, TUDCA, and UDCA) and CYP2E1 (UDCA), were sustained by means of Western immunoblotting. CYP3A induction was paralleled by a corresponding increase in mRNA. These data could partially explain the therapeutic mechanism of UDCA, TUDCA, and THDCA in chronic cholestatic liver disease. CYP3A induction, which is linked to P-glycoprotein (Pgp) family overexpression, may enhance hepatic metabolism, transport, and excretion of toxic endogenous lipophilic bile acids.
Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat
Authors: Nanji Amin A. , Jokelainen Kalle , Rahemtulla Amir , Miao Lili , Fogt Franz , Matsumoto Hiroshi , Tahan Steven R. , Su Grace L.
Published: July 2004

Inflammatory stimuli and lipid peroxidation activate nuclear factor kappa B (NF-κB) and upregulate proinflammatory cytokines and chemokines. The present study evaluated the relationship between pathological liver injury, endotoxemia, lipid peroxidation, and NF-κB activation and imbalance between pro- and anti-inflammatory cytokines. Rats (5 per group) were fed ethanol and a diet containing saturated fat, palm oil, corn oil, or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in control rats. Pathological analysis was performed and measurements of endotoxin were taken, lipid peroxidation, NF-κB, and messenger RNA (mRNA) levels of proinflammatory cytokines (tumor necrosis factor-α [TNFα], interleukin-1 β [IL-1β], interferon-γ, [IFN-γ], and IL-12), C-C chemokines (regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein [MCP]-1, macrophage inflammatory protein [MIP]-1α), C-X-C chemokines (cytokine induced neutrophil chemoattractant (CINC), MIP-2, IP-10, and epithelial neutrophil activating protein [ENA]-78), and anti-inflammatory cytokines (IL-10, IL-4, and IL-13). Activation of NF-κB and increased expression of proinflammatory cytokines C-C and C-X-C chemokines was seen in the rats exhibiting necroinflammatory injury (fish oil-ethanol [FE] and corn oil-ethanol[CE]). These groups also had the highest levels of endotoxin and lipid peroxidation. Levels of IL-10 and IL-4 mRNA were lower in the group exhibiting inflammatory liver injury. Thus, activation of NF-κB occurs in the presence of proinflammatory stimuli and results in increased expression of proinflammatory cytokines and chemokines. The Kupffer cell is probably the major cell type showing activation of NF-κB although the contribution of endothelial cells and hepatocytes cannot be excluded. Downregulation of anti-inflammatory cytokines may additionally exacerbate liver injury.
Activation of rat hepatic stellate cells leads to loss of glutathion S -transferases and their enzymatic activity against products of oxidative stress
Authors: Whalen Richard , Rockey Don C. , Friedman Scott L. , Boyer Thomas D.
Published: July 2004

Oxidative stress, mediated partly by lipid peroxidation products, may lead to increased collagen synthesis by hepatic stellate cells (HSC). Stellate cells are protected from oxidative stress by enzymes of detoxication such as the glutathioneS-transferases (GSTs), which form glutathione conjugates with lipid peroxidation products (e.g., 4-hydroxy-2-nonenal [HNE]). To better understand the role of GSTs in stellate cell biology, we examined the expression and enzymatic activity of GSTs in normal and activated (both culture- and in vivo-activated) stellate cells. Normal stellate cells contained numerous isoforms of GST including those that detoxify HNE. High levels of enzymatic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) and HNE were present in normal stellate cells and were similar to levels present in whole liver. Following activation by growth in culture, the expression of several GSTs (rGSTA1/A2, A3, and M1) was lost. Also, enzymatic activities toward CDNB and HNE fell ˜90%. However, expression of rGSTP1 was maintained. A similar loss of rGSTA1/A2, A3, and M1 with persistent expression of rGSTP1 was present after activation in vivo. Furthermore, we identified 2 subpopulations of activated stellate cells with different GST phenotypes from injured livers. In summary, activated stellate cells lose most forms of GST and associated enzymatic activities that are present in normal stellate cells. The findings raise the possibility that activated stellate cells have less ability to detoxify lipid peroxidation products and may be susceptible to oxidative stress. Additionally, we propose that the phenotypic change in GSTs is a sensitive marker of stellate cell activation.
Mutations in nonstructural protein 5A gene and response to interferon in hepatitis C virus genotype 2 infection
Authors: Murakami Takeshi , Enomoto Nobuyuki , Kurosaki Masayuki , Izumi Namiki , Marumo Fumiaki , Sato Chifumi
Published: July 2004

An association has been reported between mutations in the amino acid residues 2209-2248 of the nonstructural protein 5A (NS5A) gene (interferon-sensitivity determining region [ISDR]) and interferon efficacy in hepatitis C virus (HCV)-1b infection. This relationship was analyzed in chronic HCV-2 infection. Forty patients with HCV-2a and 35 with HCV-2b were treated with interferon alfa for 6 months with a total dose of 468 to 860 million units. Pretreatment NS5A sequences were determined by direct sequencing. A higher complete and sustained response rate was observed in HCV-2a than in HCV-2b (70% vs. 34%; P = .003). Serum HCV-RNA levels were lower in complete responders than nonresponders in HCV-2a (P= .049) and HCV-2b (P= .02). The number of amino acid mutations was greater in complete responders than nonresponders in NS5A2193-2228 (the region corresponding to the ISDR of HCV-1b) alone (P= .049), or NS5A2163-2228 consisting of NS5A2193-2228 plus its upstream region (P= .02) in HCV-2a, but not in HCV-2b. A significant inverse correlation was observed between serum HCV-RNA levels and the number of amino acid mutations in NS5A2193-2228 (P= .003) or NS5A2163-2228 (P= .005) in HCV-2a. With multivariate analysis, the number of substitutions in NS5A was an independent predictor for complete response in HCV-2a (odds ratio: 6.4;P = .03). Interferon efficacy is associated with amino acid variations in the NS5A protein in HCV-2a infection.
Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by In Vivo liver injury in the rat
Authors: Marra Fabio , Arrighi Maria Cristina , Fazi Marilena , Caligiuri Alessandra , Pinzani Massimo , Romanelli Roberto G. , Efsen Eva , Laffi Giacomo , Gentilini Paolo
Published: July 2004

Upon liver injury, hepatic stellate cells (HSC) show increased proliferation, motility, and extracellular matrix (ECM) production. The extracellular signal-regulated kinases (ERK) control different functions in a cell-specific manner. In this study, we evaluated the role of ERK activation in cultured HSC stimulated with platelet-derived growth factor (PDGF) and after induction of liver injuryin vivo. HSC were isolated from normal human liver tissue, cultured on plastic, and used in their myofibroblast-like phenotype. Inin vivoexperiments, HSC were isolated from normal rats or at different time points after a single intragastric administration of CCl4. Nontoxic concentrations of PD98059, a specific inhibitor of ERK activation, reduced PDGF-induced activation of ERK in a dose-dependent fashion. Suppression of ERK activation was associated with complete inhibition of HSC proliferation and with a 57% reduction in chemotaxis. In the presence of the ERK inhibitor, binding of the AP-1 complex and of STAT1 to the related regulatory elements was inhibited. The inhibition of the DNA binding activity of STAT1 was mediated by a reduction in PDGF-induced tyrosine phosphorylation. Expression ofc-fosin response to PDGF was also reduced, but not suppressed, by treatment with PD98059. In HSC isolated from CCl4-treated rats, ERK activity increased as early as 6 hours following liver damage, and declined thereafter. The results of this study indicate that ERK activation regulates proliferation and chemotaxis of HSC, and modulates nuclear signaling. Acute liver damagein vivoleads to activation of ERK in HSC.
Hepatic iron and nonalcoholic fatty liver disease
Authors: Younossi Zobair M. , Gramlich Terry , Bacon Bruce R. , Matteoni Christi A. , Boparai Navdeep , O'Neill Rosemary , McCullough Arthur J.
Published: July 2004

Increased iron is suspected to enhance hepatic injury associated with nonalcoholic fatty liver disease (NAFL). We evaluated the impact of iron accumulation on the outcome of NAFL. Patients with NAFL were identified from our database. Twenty-two clinicodemographic and 19 pathological features were available for each patient. Histological staining (Perls' Prussian blue), hepatic iron concentration (HIC), and hepatic iron index (HII) were determined. Data on follow-up, mortality, and cause of death were analyzed. In 65 patients with available liver biopsy blocks, HIC and HII were 1,171 ± 717 μg/g dry weight and 0.43 ± 0.30 μmol/g/yr, respectively. Males had more iron accumulation (HIC: 1,514 ± 836 vs. 859 ± 389,P= .0001; and HII: 0.58 ± 0.35 vs. 0.29 ± 0.16,P=.0001). In type II diabetics, both HIC (977 ± 769 vs. 1,301 ± 659;P< .05) and HII (0.30 ± 0.23 vs. 0.52 ± 0.32;P< .05) were lower. Iron accumulation was not related to other variables analyzed. Increased iron was not seen in those with higher grades of fibrosis or other pathological features associated with the aggressive form of NAFL (hepatocyte necrosis, fibrosis, ballooning degeneration, and Mallory hyaline). Iron accumulation was not associated with increased overall mortality, liver-related mortality, or development of cirrhosis. In summary, in most patients with NAFL, significant iron accumulation is not seen. Additionally, in our series of patients with NAFL, iron is not associated with poor clinical or pathological outcomes.
Fetal microchimerism alone does not contribute to the induction of primary biliary cirrhosis
Authors: Tanaka Atsushi , Lindor Keith , Gish Robert , Batts Kenneth , Shiratori Yasushi , Omata Masao , Nelson Lee J. , Ansari Aftab , Coppel Ross , Newsome Margaret , Gershwin Eric M.
Published: July 2004

Microchimerism has been implicated in the etiology of autoimmune diseases. It has also been implicated in the induction/maintenance of fetal tolerance. We used polymerase chain reaction (PCR) analysis to determine whether microchimerism occurred in patients who subsequently developed primary biliary cirrhosis (PBC), and thus may be involved in its etiology. We performed PCR amplification of sequences unique to both the X and Y chromosomes from the livers of 37 women with PBC and 39 female controls using WAVE technology; a very sensitive technology based on an ion-pair reverse-phase high-performance liquid chromatography system. All patients were known to have had at least 1 son and it was confirmed that PBC was diagnosed after the birth of the son. Data were analyzed for both detection of the Y chromosome gene and the ratio of the yield of the Y chromosome PCR products to the X chromosome. The prevalence of Y chromosome detection in PBC was 26 of 37 (70%) compared with 28 of 39 (72%) in controls, and the ratio of Y chromosome to X chromosome was similar between the PBC and control groups, 0.402 ± 0.143 vs. 0.271 ± 0.055, respectively. Our results, using our more sensitive technology, showed that microchimerism is a very common event in human liver and supported the thesis that this may contribute to the induction/maintenance of fetal tolerance. However, although we cannot exclude the possibility that select fetal major histocompatibility complex (MHC) haplotypes might contribute to disease susceptibility, our data suggest that microchimerism by itself does not play a significant role in the development of PBC.
Is serum bilirubin concentration the only valid prognostic marker in primary biliary cirrhosis?
Authors: Krzeski Piotr , Zych Wlodzimierz , Kraszewska Ewa , Milewski Bohdan , Butruk Eugeniusz , Habior Andrzej
Published: July 2004

From the many prognostic models for primary biliary cirrhosis (PBC) patients based on Cox's regression analysis, the Mayo model has gained the most popularity and was successfully validated in some centers. The aim of our study was to validate the Mayo survival model for Polish PBC patients and, in case of its inapplicability, to select prognostic variables and to create time-fixed and time-dependent survival models for the patients. We used database information on patients from 6 medical centers in Poland, fulfilling clinical, serological, and/or pathological criteria of PBC. The Mayo model was validated using data from 116 PBC patients. The time-fixed and time-dependent models were created using data on clinical and biochemical variables used in the Mayo model from 162 and 208 patients, respectively. The Mayo model validation was performed graphically and by one-sample log-rank tests after dividing the study sample into 3 groups of high, medium, and low risk. The survival analysis was performed using Cox's proportional hazards regression method on clinical and biochemical variables used in the Mayo model. Treatment with ursodeoxycholic acid (UDCA) was included in the time-dependent analysis. Validation showed that the Mayo model overestimated death risk in Polish PBC patients. Of the variables used in the Mayo model, serum bilirubin concentration appeared to be the only variable of prognostic importance. The analysis shows that serum bilirubin concentration holds most of the prognostic information for our PBC patients irrespective of prior treatment with UDCA.
Cell motility mediated by rho and rho-associated protein kinase plays a critical role in intrahepatic metastasis of human hepatocellular carcinoma
Authors: Genda Takuya , Sakamoto Michiie , Ichida Takafumi , Asakura Hitoshi , Kojiro Masamichi , Narumiya Shuh , Hirohashi Setsuo
Published: July 2004

Human hepatocellular carcinoma (HCC) can invade the portal vein and metastasizes to other parts of the liver even at a relatively early stage of the disease, with less tumor spread occurring outside the liver. This intrahepatic metastasis is the main cause of liver failure and death in HCC patients. To analyze the mechanisms of intrahepatic metastasis we have constructed metastatic models using orthotopic implantation of human HCC cell lines. Five HCC cell lines formed liver tumors after injection into the livers of SCID mice, and of those 5 cell lines, Li7 and KYN-2 cells also resulted in vascular tumor thrombi and intrahepatic metastasis. These 2 cell lines had markedly higher cell motilities than the other 3 cell lines in vitro. Their motilities appeared to be Rho-mediated; serum and lysophosphatidic acid (LPA) evoked actin reorganization and motility of Li7 cells, and C3 exoenzyme exposure reduced the motility of both serum-stimulated Li7 cells and KYN-2 cells. Dominant negative and active forms of p160 Rho-associated coiled-coil forming protein kinase (p160ROCK), one of the downstream effectors of Rho, were separately and stably introduced into Li7 cells. Dominant active p160ROCK transfectants showed increased motility that was independent of serum and LPA, and dominant negative p160ROCK transfectants showed reduced motility under stimulation. Furthermore, implantation of dominant negative p160ROCK transfectants resulted in a reduced metastatic rate in vivo compared with the parent cells or a control transfectant. These findings indicate that cell motility mediated by the Rho/p160ROCK signaling pathway plays a critical role in intrahepatic metastasis of human HCC.
Expression of inositol 1,4,5-trisphosphate receptor isoforms in rat cirrhosis
Authors: Dufour Jean-François , Lüthi Michael , Forestier Marc , Magnino Fabrice
Published: July 2004

Ca2+ signals mediate the hepatic effects of numerous hormones and growth factors. Hepatic Ca2+ signals are elicited by the inositol trisphosphate receptor, an intracellular Ca2+ channel. Three isoforms of this receptor have been identified; they are expressed and regulated differently. We investigated the effect of liver fibrosis and cirrhosis on the hepatic expression of the inositol trisphosphate receptor isoforms. Two different rat models were used: bile duct ligation (fibrosis) and chronic exposure to CCl4/phenobarbital (cirrhosis). Messenger RNA levels were determined by ribonuclease protection assay (RPA), competitive polymerase chain reaction (PCR) followed by Southern blotting, and real-time quantitative PCR. Protein expression was assessed by Western blotting; tissue distribution was assessed by immunohistology. In control animals, isoform 2 was the predominant isoform, isoform 1 represented less than one third, and isoform 3 less than 1%. After bile duct ligation, expression of types 1 and 3 increased 1.9- and 5.7-fold, and expression of type 2 decreased 2.5-fold at the protein level. After exposure to CCl4/phenobarbital, expression of types 1, 2, and 3 were 2.4-, 0.9-, and 4.2-fold their expression in control animals. Type 2 was localized to the apical domain of hepatocytes, consistent with a role for Ca2+ signals in canalicular function. Type 3 was detectable in intrahepatic bile duct epithelial cells and not in hepatocytes, suggesting that Ca2+ signals may be regulated differently in these cells. Signaling through inositol trisphosphate receptor participates in the pathogenesis of cirrhosis, because this process affects the expression of its isoforms.
Loss of wild-type carrier-mediated L-carnitine transport activity in hepatocytes of juvenile visceral steatosis mice
Authors: Yokogawa Koichi , Yonekawa Masayuki , Tamai Ikumi , Ohashi Rikiya , Tatsumi Yasuaki , Higashi Yasuhiko , Nomura Masaaki , Hashimoto Noriyoshi , Nikaido Hiroko , Hayakawa Jun-ichiro , Nezu Jun-ichi , Oku Asuka , Shimane Miyuki , Miyamoto Ken-ichi , Tsuji Akira
Published: July 2004

Juvenile visceral steatosis (JVS) mice, which show systemic l-carnitine deficiency, may be an animal model of Reye's syndrome because of its phenotype of fat deposition and mitochondrial abnormalities in the liver. In this study, we compared the characteristics of the L-carnitine transport in isolated hepatocytes from wild-type and JVS mice. The uptake of l-carnitine by wild-type hepatocytes was saturable and the Eadie-Hofstee plot showed 2 distinct components. The apparent Michaelis constant (K
m) and the maximum transport rate (V
max) were 4.6 μmol/L and 59.5 pmol/15 min/106 cells, respectively, for the high-affinity component, and 404 μmol/L and 713 pmol/15 min/106 cells, respectively, for the low-affinity component. The high-affinity l-carnitine uptake occurred via an active carrier-mediated transport mechanism, which is characterized by Na+-, energy-, and pH-dependency. On the other hand, the high-affinity uptake was absent in JVS hepatocytes, and the values of K
m and V
max for the low-affinity uptake were 475 μmol/L and 557 pmol/15 min/106 cells, respectively. The hepatic carnitine transport properties in wild-type hepatocytes were similar to those of high-affinity mouse Octn2-transfected HEK293 cells. This study suggests that Octn2-type carnitine transporter is dysfunctional in hepatocytes of JVS mice.
Expression of functional CD40 in human hepatocellular carcinoma
Authors: Sugimoto Kazushi , Shiraki Katsuya , Ito Takeshi , Fujikawa Katsuhiko , Takase Koujiro , Tameda Yukihiko , Moriyama Masami , Nakano Takeshi
Published: July 2004

CD40, a member of the tumor necrosis factor receptor (TNFR) family, plays a crucial role in the survival, proliferation, and differentiation in B cells. However, the expression of CD40 other than in B cells has not been well studied. Therefore, we investigated the expression and function of CD40 in hepatocellular carcinomas (HCCs). Expression of CD40 mRNA in 6 established HCC cell lines was analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and CD40 expression on cell surface was examined by flow cytometrical analysis. We also examined the expression of CD40 in human HCC tissues (45 cases) and nontumor liver tissues (30 cases) by immunohistochemistry. To examine the function of CD40 in HCC cells, we investigated the effect of CD40 signaling on anti-Fas antibody and TNF-α-induced apoptosis in HepG2 cells. In addition, intracellular levels of cysteine protease P32 (CPP32) protein in HepG2 cells were also determined by Western blotting. We have shown that 6 HCC cell lines constitutively expressed CD40 mRNA and membrane-bound CD40 antigen, which was slightly up-regulated by interferon gamma (IFN-γ). In addition, 60% of human HCC tissues demonstrated positive staining for CD40, whereas nontumor tissues showed little detectable staining. In HepG2 cells, CD40 stimulation does not affect cell viability, but significantly inhibited Fas and TNFR-mediated apoptosis in a dose-dependent manner by blocking the activation of CPP32. From these results, we conclude that CD40 expression in HCCs plays an important role in tumor biology, especially the resistance against Fas and TNFR-mediated apoptosis.
Expression of connective tissue growth factor in experimental rat and human liver fibrosis
Authors: Paradis Valerie , Dargere Delphine , Vidaud Michel , de Gouville Anne-Charlotte , Huet Stephane , Martinez Vincent , Gauthier Jean-Michel , Ba Nathalie , Sobesky Rodolphe , Ratziu Vlad , Bedossa Pierre
Published: July 2004

Connective tissue growth factor (CTGF) stimulates in vitro fibroblast proliferation and extracellular matrix synthesis. The aim of this study was to assess the role of CTGF in liver fibrogenesis. CTGF expression was investigated both at the protein and mRNA level in biopsies of chronic liver diseases, in experimental models of liver fibrosis, and in hepatic stellate cells in culture. CTGF immunostaining was observed in most human liver biopsies with significant fibrosis. An increase of CTGF immunostaining was associated with a higher score of fibrosis both in the group of chronic hepatitis C (χ2 = 9.3;P< .01) and in the non-hepatitis C group (χ2 = 7.2;P< .02). In situ hybridization showed CTGF mRNA expression in spindle cells in both the fibrous septa and sinusoidal lining. In experimental models of liver fibrosis, CTGF accumulated in parallel with the development of septal fibrosis and cirrhosis. Quantification of CTGF mRNA by a real-time reverse-transcription polymerase chain reaction (RT-PCR) assay showed a significant increase of CTGF mRNA in both CCl4-induced and bile duct-ligated rat models of liver fibrosis. Expression of CTGF protein and mRNA was definitively assigned to hepatic stellate cells, because CTGF was detected by Western blot both in lysate and supernatant of a hepatic stellate cell line derived from rats. These cells also displayed CTGF protein and mRNA as shown by immunohistochemistry and in situ hybridization. In conclusion, this study shows that CTGF is strongly expressed during liver fibrogenesis, and hepatic stellate cells seem to be the major cellular sources of CTGF in the liver.
Primary disease recurrence after liver transplantation for alveolar echinococcosis: Long-term evaluation in 15 patients
Authors: Bresson-Hadni Solange , Koch Stéphane , Beurton Isabelle , Vuitton Dominique-Angèle , Bartholomot Brigitte , Hrusovsky Stephan , Heyd Bruno , Lenys Danièle , Minello Anne , Becker Marie C. , Vanlemmens Claire , Mantion Georges-André , Miguet Jean-Philippe
Published: July 2004

Between 1986 and 1991, 21 patients received liver grafts in our center for incurable alveolar echinococcosis (AE). The aim of this study was to analyze the long-term results in 15 of these 21 patients who survived more than 1 year after undergoing a liver transplantation (LT). The follow-up, mainly aimed at the diagnosis of recurrence, consisted of repeated radiological and specific immunological investigations. The role of pre- and post-LT benzimidazole (BZM) therapy was also evaluated. Among the 15 patients, 8 patients had a palliative LT related to previously known pulmonary AE metastases and/or inextirpable abdominal parasitic foci. In the 7 remaining patients, LT was considered curative. In June 1998, the mean follow-up duration was 96 months (range: 28-138 months). Five late deaths occurred, 2 of them were directly related to residual AE. A reinfection of the graft was observed in 4 patients. Preoperative BZM therapy seemed useful in preventing or delaying the parasitic recurrence. Post-LT BZM was able to stabilize and even to reduce residual AE. The anti-Em2 enzyme-linked immunosorbent assay (ELISA), which is the standard test used in patient follow-up after partial liver resection for AE, did not appear useful in detecting recurrence here; however, an ELISA, using a crude heterologous antigen (Echinococcus granulosus) allowed early diagnosis of residual AE. In conclusion, primary disease recurrence is not rare after LT for AE. Immunosuppressive therapy may favor larval growth in extrahepatic sites; therefore, an extensive extrahepatic radiological check-up has to be performed before LT. BZM therapy seems to stabilize residual foci. Anti-Eg immunoglobulin G (IgG) follow-up is the most useful test for early diagnosis of parasite recurrence.
Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates
Authors: Sabile Abdelmajid , Perlemuter Gabriel , Bono Fulvia , Kohara Kyoko , Demaugre France , Kohara Michinori , Matsuura Yoshiharu , Miyamura Tatsuo , Bréchot Christian , Barba Giovanna
Published: July 2004

Several lines of evidence suggest that hepatitis C virus (HCV) core protein may modulate cellular transduction signals and alter lipid metabolism. We have investigated the binding of HCV core protein to cellular proteins by combining 2 yeast hybrid, confocal, and surface plasmon resonance assays. Our results show the direct binding of the viral protein to apolipoprotein AII (apoAII) and map the interaction domain to the C-terminal of HCV core protein. To investigate the biological relevance of the interaction between HCV core and lipid metabolism, we took advantage of the well-established increase in apoAII expression caused by fibrates in HepG2 cells. After fenofibric acid treatment, we show a parallel increase in apoAII and core protein secretion, this effect being abolished by brefeldin A. Our study identifies apoAII as one of the cellular targets for HCV core protein. We also show that the intervention of fenofibric acid in cellular lipid metabolism directly affects the expression pattern of HCV core protein.
Progelatinase A is produced and activated by rat hepatic stellate cells and promotes their proliferation
Authors: Benyon Christopher R. , Hovell Christopher J. , Da Gaça Marianna , Jones Emma H. , Iredale John P. , Arthur Michael J. P.
Published: July 2004

Activated hepatic stellate cells (HSCs) are a potential source of gelatinase A, which accumulates in fibrotic livers. Progelatinase A activation requires its binding to a complex of membrane-type matrix metalloproteinase (MT-MMP) and tissue inhibitor of metalloproteinases (TIMP)-2. These studies examine gelatinase A, MT1-MMP, and TIMP-2 synthesis by HSCs during activation in vitro and the potential role of gelatinase A in promoting HSC proliferation. Gelatinase A, MT1-MMP, and TIMP-2 messenger RNA (mRNA) were all upregulated in HSCs activated on plastic over 5 to 14 days. Gelatinase A expression was maximal at 7 days of culture, coinciding with the peak of HSC proliferation and the onset of procollagen I and α-smooth muscle actin (α-SMA) mRNA expression. Active forms of gelatinase A of 62 kd and 66 kd were secreted by activated HSCs and reached a maximum of 12.1% of total enzyme in 14-day culture supernatants. Treatment of HSCs with concanavalin A (con A) induced activation of MT1-MMP and enhanced secretion of activated gelatinase A, which reached a maximum of 44.4% of the total enzyme secreted into culture supernatants using 30 μg/mL con A. [14C]-gelatin degradation assays confirmed the presence of gelatinolytic activity in activated HSC supernatants, which reached a maximum level at 7 days of culture. Antisense oligonucleotide inhibition of endogenous progelatinase A production, or the MMP inhibitor 1,10-phenanthroline inhibited 3H-thymidine incorporation into HSC DNA by greater than 50%. We conclude that HSCs produce progelatinase A during activation in vitro and activate this enzyme coincident with MT1-MMP and TIMP-2 synthesis. Gelatinase A activity is required for maximal proliferation of HSCs in vitro suggesting this metalloproteinase is an autocrine proliferation factor for HSCs.
Chronic ethanol consumption causes alterations in the structural integrity of mitochondrial DNA in aged rats
Authors: Cahill Alan , Stabley Gabriel J. , Wang Xiaolan , Hoek Jan B.
Published: July 2004

Chronic ethanol consumption adversely affects the respiratory activity of rat liver mitochondria. It causes increased cellular production of oxygen radical species and selectively decreases mitochondrial glutathione (GSH) levels. Here we show, using Southern hybridization techniques on total rat genomic DNA, that long-term (11-13 months) ethanol feeding, using the Lieber-DeCarli diet, results in a 36% (P < .05; n = 4) decrease in hepatic mitochondrial DNA (mtDNA) levels when compared with paired controls. UV quantitation of mtDNA isolated from hepatic mitochondria showed that chronic ethanol intake (11-13 months) causes a 44% (P < .01; n = 6) decrease in the amount of mtDNA per milligram of mitochondrial protein. No significant decline in mtDNA levels was seen in ethanol-fed animals maintained on the diet for 1 to 5 months. Ethanol feeding caused a 42% (P < .01; n = 4) and a 132% (P < .05; n = 3) increase in 8-hydroxydeoxyguanosine (8-OHdG) formation in mtDNA in animals maintained on the diet for 3 to 6 months and 10 to 11 months, respectively. In addition, agarose gel electrophoresis revealed a 49% increase (P < .05; n = 3) in mtDNA single-strand breaks (SSB) in animals fed ethanol for more than 1 year. These findings suggest that chronic ethanol consumption causes enhanced oxidative damage to mtDNA in older animals along with increased strand breakage, and that this results in its selective removal/degradation by mtDNA repair enzymes.
Conserved hepatitis C virus sequences are highly immunogenic for CD4+ T cells: Implications for vaccine development
Authors: Lamonaca Vincenzo , Missale Gabriele , Urbani Simona , Pilli Massimo , Boni Carolina , Mori Cristina , Sette Alessandro , Massari Marco , Southwood Scott , Bertoni Roberto , Valli Antonietta , Fiaccadori Franco , Ferrari Carlo
Published: July 2004

The HLA class II-restricted T-cell response to hepatitis C virus (HCV) antigens is believed to influence the final outcome of hepatitis C, because it is vigorous in patients who recover from acute hepatitis C, but it is weak in those who develop a chronic infection. For this reason, exogenous stimulation of T-cell responses in chronic HCV infection may represent a strategy to cure patients with chronic hepatitis C by approximating the vigor of their T-cell reactivity to that of patients who succeed in recovering from hepatitis. It may also be a preventive approach to avoid spread of the virus by facilitating the development of a vigorous protective response at the very early stages of infection. T-cell-based vaccines composed of immunodominant, promiscuous, and conserved T-cell epitopes may represent a powerful tool to achieve optimal stimulation of the T-cell reactivity. To identify HLA class II-restricted T-cell epitopes useful for this purpose, 22 subjects with acute HCV infection were studied and followed for an average time of 29 months. Eight of them recovered from hepatitis, and 14 developed a chronic infection. Overlapping 20-mer peptides covering the entire core and NS4 antigens and a panel of peptides representing highly conserved regions of core, NS3, NS4, and NS5 were used. By direct peripheral blood T-cell stimulation and by fine-specificity analysis of HCV-specific T-cell lines and clones, highly immunogenic T-cell epitopes were identified within core, NS3, and NS4. All these epitopes are immunodominant and highly conserved among the known HCV isolates. Moreover, they are promiscuous, because they can be presented to T cells by different HLA class II molecules. Immunodominance, sequence conservation, and promiscuity make these epitopes ideal components of preventive or therapeutic T-cell-based vaccines against HCV.
The structural and functional polarity of the hepatic human/rat hybrid WIF-B is a stable and dominant trait
Authors: Bender Virginie , Bravo Pilar , Decaens Catherine , Cassio Doris
Published: July 2004

WIF-B cells were generated previously to obtain a good in vitro model expressing the structural and functional polarity of hepatocytes. Here we tested the stability and the strength of the WIF-B polarized phenotype. WIF-B cells stayed polarized and formed functional bile canaliculi even after 3 months in culture or after injection in nude mice and culture of the resulting tumors. WIF-B was subcloned and 10,000 colonies were examined; all (except for 3) were composed of bile canaliculi forming cells. Some subclones were characterized; the polarized ones presented the same properties and karyotype as the WIF-B cells; the 3 unpolarized subclones had a lower level of E-cadherin and different karyotypes. WIF-B cells were fused with their nonpolarized hepatic parental cells. The polarity state of the resulting FWIF hybrids was studied from day 11 to day 38 after fusion, by immunolocalization of hepatocyte domain-specific plasma membrane proteins. Most FWIF colonies (>80%) were composed of polarized cells. Soon after fusion these cells were exclusively polarized as simple epithelial cells. The percent of colonies containing cells expressing the typical hepatocyte polarity increased with time and reached 80% at day 38. This result confirms the two-step polarization process previously described for WIF-B. Chromosomally complete FWIF hybrids were examined several months after fusion. As shown by the study of bile acid transport and by confocal analysis of the localization of membrane domain markers, FWIF cells expressed a functional and fully polarized hepatic phenotype. In conclusion, polarity is a stable and dominant trait of WIF-B.
Differential regulation of extracellular matrix synthesis during liver regeneration after partial hepatectomy in rats
Authors: Rudolph Lenhard K. , Trautwein Christian , Kubicka Stefan , Rakemann Tim , Bahr Matthias J. , Sedlaczek Nick , Schuppan Detlef , Manns Michael P.
Published: July 2004

Little is known about the modulation of the extracellular matrix (ECM) during liver regeneration. We studied the temporospatial expression of procollagens and of matrix metalloproteinases (MMPs) and their physiological antagonists, the tissue inhibitors of metalloproteinases (TIMPs) after two-thirds partial hepatectomy (PH) by Northern blot analysis and in situ hybridization. The entry of hepatocytes into the S-phase at 24 hours after PH was accompanied by a peak (sixfold induction) of hepatic TIMP-1 RNA levels that steadily declined thereafter to reach normal levels 144 hours after PH. Moderate MMP-2 and TIMP-2 RNA levels remained constant up to 144 hours after PH, and MMP-1 and -13 RNA were always undetectable. In situhybridization showed a dramatic upregulation of TIMP-1 RNA transcripts in mesenchymal cells of portal, perisinusoidal and, to a lesser extent, pericentral areas. In contrast, scattered hepatocytes represented only a minor fraction (below 10%) of TIMP-1 RNA positive cells. When hepatocytes stopped DNA synthesis at 72 hours after PH, an upregulation of procollagen α1(I) and α2(III) transcripts was observed paralleled by threefold increased PIIINP levels in the sera. Our data reveal a tightly regulated program of de novo matrix synthesis after PH. Whereas interstitial procollagens appear to participate in the induction and maintenance of the quiescent hepatocyte phenotype, the early and localized expression of TIMP-1 indicates a role unrelated to its function as a general MMP-antagonist, e.g., as a growth promoting agent for hepatocytes.