bluuebunny/crossref_metadata_2025 huggingface dataset:
Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/gastrin receptors via d -myo-inositol 1,4,5-triphosphate-, Ca2+-, and protein kinase C α-dependent mechanisms
Authors: Caligiuri Alessandra , LeSage Gene , Baiocchi Leonardo , Kanno Noriatsu , Chowdury Usha , Phinizy Jo Lynne , Glaser Shannon , Alpini Gianfranco , Benedetti Antonio , Marucci Luca , Alvaro Domenico , Papa Emanuela
Published: March 2004

We studied the role of gastrin in regulating cholangiocyte proliferation induced by bile duct ligation (BDL). In purified cholangiocytes, we evaluated (1) for the presence of cholecystokinin-B (CCK-B)/gastrin receptors, (2) the effect of gastrin on d -myo-Inositol 1,4,5-triphosphate (IP3) levels, and (3) the effect of gastrin on DNA synthesis and adenosine 3′, 5′-monophosphate (cAMP) levels in the absence or presence of CCK-A (L-364,718) and CCK-B/gastrin (L-365,260) receptor inhibitors, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetxymethyl ester) (BAPTA/AM; an intracellular Ca2+ chelator), and 2 protein kinase C (PKC) inhibitors, 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporin. To evaluate if gastrin effects on cholangiocyte proliferation are mediated by the isoform PKCα, we evaluated (1) for the presence of PKCα in cholangiocytes and (2) the effect of gastrin on the PKCα protein expression in a triton-soluble (containing cytoplasm + membrane) and a triton-insoluble (containing cytoskeleton) fraction. To evaluate the effects of gastrin in vivo, immediately following BDL, gastrin or bovine serum albumin (BSA) was infused by minipumps for 7 days to rats and we measured cholangiocyte growth and cAMP levels. We found CCK-B/gastrin receptors on cholangiocytes. Gastrin increased IP3 levels. Gastrin inhibited DNA synthesis and cAMP synthesis in cholangiocytes. Gastrin effects on cholangiocyte functions were blocked by L-365,260, BAPTA/AM, H7, and staurosporin but not by L-364,718. Gastrin induced translocation of PKCα from cholangiocyte cytoskeleton to membrane. In vivo, gastrin decreased cholangiocyte growth and cAMP synthesis compared with controls. We concluded that gastrin inhibits cholangiocyte growth in BDL rats by interacting with CCK-B/gastrin receptors through a signal transduction pathway involving IP3, Ca2+, and PKCα.
Hepatitis C-related cirrhosis: A predictor of diabetes after liver transplantation
Authors: Bigam Dave L. , Pennington Jason J. , Carpentier Andre , Wanless Ian R. , Hemming Alan W. , Croxford Ruth , Greig Paul D. , Lilly Leslie B. , Heathcote Jenny E. , Levy Gary A. , Cattral Mark S.
Published: March 2004

Hepatitis C virus (HCV) infection has recently been suggested to be a risk factor for the development of diabetes mellitus. The aim of our study was to investigate whether the prevalence of diabetes is increased among liver transplant recipients infected with HCV. We compared the prevalence of diabetes among 278 liver transplant recipients whose original cause of liver failure was HCV infection (110 patients), hepatitis B virus infection (HBV; 53 patients), and cholestatic liver disease (CLD; 115 patients). The pretransplantation prevalence of diabetes was higher in the HCV group (29%) compared with the HBV (6%) and CLD (4%) groups (P < .001). The prevalence of diabetes remained higher in the HCV group 1 year after transplantation: 37%, 10%, and 5% in the HCV, HBV, and CLD groups, respectively (P < .001). The cumulative steroid dose during the first year of transplantation was significantly lower in the HCV group compared with the CLD group. Multivariate analysis revealed that HCV-related liver failure (P = .002), pretransplantation diabetes (P < .0001), and male sex (P = .019) were independent predictors of the presence of diabetes 1 year after transplantation. The high prevalence of diabetes persisted in the HCV group, with 41% diabetic at 5 years. The majority of patients with diabetes mellitus (89%) required insulin therapy after transplantation. Patient and graft survival rates were similar among patients with and without diabetes. In conclusion, our study shows that there is a high prevalence of diabetes among liver transplant recipients infected with HCV both before and after transplantation.
A rho-specific exchange factor ect2 is induced from S to M phases in regenerating mouse liver
Authors: Sakata Hiromi , Rubin Jeffrey S. , Taylor William G. , Miki Toru
Published: March 2004

The ect2 oncogene was originally identified as a transforming complementary DNA (cDNA) from mouse epithelial cells in an expression cloning approach and encodes a product related to Rho-specific exchange factors and yeast cell cycle regulators. To explore the potential role of ect2 in the cell cycle, we examined the expression of the ect2 proto-oncogene in a liver regeneration model in mice after partial (two thirds) hepatectomy. We found that the expression of the ect2 transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases. The timing of ect2 expression matched that of proliferating cell nuclear antigen (PCNA) and partially overlapped cell division cycle 2 (Cdc2) expression. In situ hybridization analysis showed that ect2 was expressed at a high level in cells undergoing mitosis in regenerating liver. Moreover, expression of a dominant negative or an oncogenic mutant of ect2 in cultured mouse hepatocytes resulted in a large increase in the number of binucleated cells. These findings showed that Ect2 is expressed in a cell cycle-dependent manner during liver regeneration, and suggest that it has an important role in the regulation of cytokinesis.
High-lose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential
Authors: Wang Lu , Tang Zhao-You , Qin Lun-Xiu , Wu Xiao-Feng , Sun Hui-Chuan , Xue Qiong , Ye Sheng-Long
Published: March 2004

Postoperative recurrence of human hepatocellular carcinoma (HCC) is the major issue that must be addressed to further improve prognosis. This study was undertaken to investigate the effects of interferon-alfa-1b (IFN-α-1b) on recurrent tumor and metastasis after curative resection in nude mice bearing an HCC xenograft with high metastatic potential. Tumor tissues from LCI-D20, a metastatic model of HCC in nude mice, were orthotopically implanted in 105 nude mice. Eleven days later, 64 mice underwent curative resection of liver tumors. IFN-α at different doses was administered subcutaneously to mice with or without resection. In mice without resection, when comparison was made among control, IFN 7.5 × 106 U/kg/day, 1.5 × 107 U/kg/day for treated groups, and 3 × 107 U/kg/day; tumor volume was 8,475 mm3 ± 2,636 mm3, 7,963 mm3 ± 3,214 mm3, 769 mm3 ± 287 mm3, and 13 mm3 ± 9 mm3; incidence of lung metastasis being 100%, 80%, 40%, and 0%; life span was 45 ± 4 days, 53 ± 8 days, 81 ± 6 days, and 105 ± 24 days, respectively. In mice with curative resection, when comparison was made among control, IFN 5 × 105 U/kg/day, 1 × 106 U/kg/day, 4 × 106 U/kg/day, 7.5 × 106 U/kg/day, 1.5 × 107 U/kg/day, and 3 × 107 U/kg/day for treated groups; incidence of recurrent tumor was 100%, 100%, 87.5%, 100%, 87.5%, 62.5%, and 12.5%; lung metastasis being 100%, 75%, 87.5%, 50%, 62.5%, 0%, and 0%, respectively. IFN-α inhibited neovascularization induced by LCI-D20 tumor specimens implanted into the micropocket of nude mice corneas. In conclusion, high-dose and long-term therapy with IFN-α dose-dependently inhibits tumor growth and recurrence after resection of HCC. The effect of IFN-α may be attributed to antiangiogenesis in this experiment. These results provide potential clinical implication, particularly for the prevention of recurrence after curative resection of HCC.
Hepatic cytochrome P450 down-regulation during aseptic inflammation in the mouse is interleukin 6 dependent
Authors: Siewert Elmar , Bort Roque , Kluge Reinhart , Heinrich Peter C. , Castell José , Jover Ramiro
Published: March 2004

Expression of cytochromes P450 (CYP) is markedly reduced during inflammatory processes. In vitro studies with hepatocytes have shown that cytokines generated during these processes down-regulate CYP. However, it is not clear to what extent each individual cytokine contributes to the overall reduced expression of the various CYP isoenzymes in vivo. Interleukin 6 (IL-6), a major player during inflammatory processes, is recognized as the most important cytokine modulating the hepatic expression of acute-phase protein (APP) genes. For this reason, we selected the IL-6−/− mouse as a model to investigate the role of IL-6 in the down-regulation of hepatic CYP during experimental inflammation. Our results show that the reduction in messenger RNA (mRNA) levels of CYP1A2, CYP2A5, and CYP3A11 during turpentine-induced inflammation was abrogated in IL-6-deficient mice, confirming that IL-6 is an indispensable player for the down-regulation of hepatic CYP during aseptic inflammation. Moreover, the different CYP isoenzymes showed a variable grade of dependence on IL-6, CYP2A5 being the most sensitive one. In the case of CYP2E1, differences between IL-6−/− and wild-type mice were no longer maintained after 24 hours, suggesting a delayed, rather than abrogated, CYP down-regulation in the absence of IL-6. As opposed to that, hepatic CYP repression took place in IL-6-deficient mice during lipopolysaccharide (LPS)-mediated inflammation. This contrasting behavior observed for CYP is surprisingly similar to the one seen for extracellular (serum amyloid A, β-fibrinogen) and intracellular (metallothionein-1) APPs and points to the fact that, in the model of bacterial inflammation (LPS), the effects of IL-6 on CYP down-regulation are likely to be substituted by other cytokines or mediators.
Growth control of human biliary epithelial cells by interleukin 6, hepatocyte growth factor, transforming growth factor β1, and activin a: Comparison of a cholangiocarcinoma cell line with primary cultures of non-neoplastic biliary epithelial cells
Authors: Yokomuro Shigeki , Tsuji Hirokazu , Lunz John G. , Sakamoto Toshiki , Ezure Tsukasa , Murase Noriko , Demetris Anthony J.
Published: March 2004

A well characterized human cholangiocarcinoma (CC) cell line, SG231, was compared with primary cultures of normal human biliary epithelial cells (BECs) for alterations in interleukin 6 (IL-6) and hepatocyte growth factor (HGF)-mediated stimulation and transforming growth factor β1 (TGF-β1) and activin A-mediated inhibition of growth. Results were compared with immunolabeling of the original tumor and after injection of SG231 into the liver of BALB/cByJ-scid mice. In vitro, both BECs and CCs expressed met, gp80, and gp130 messenger RNA (mRNA) and protein, but the levels of expression were higher in the CCs than in the BECs. In both the CCs and BECs, exogenous HGF or IL-6 induced phosphorylation of met or gp130, respectively, and a concentration-dependent increase in DNA synthesis. However, the CCs but not BECs, continued to grow in basal serum-free medium (SFM) and spontaneously produced both IL-6 and HGF under these conditions, which resulted in auto-phosphorylation of gp130 and met, respectively; and neutralizing anti-HGF or anti-IL-6 alone inhibited CC growth, indicative of autocrine growth control circuits. Conversely, activin A inhibits the growth of both BECs and CCs, but does not significantly increase apoptosis. Activin-A-induced growth inhibition of both CCs and BECs can be reversed by 100 ng/mL exogenous IL-6, but not by 10 to 100 ng/mL HGF. TGF-β1 inhibited the growth of BECs but had no mitoinhibitory or proapoptotic effects on CCs. Immunolabeling of the original tumor and after inoculation into scid mice showed positive staining for met, gp130, gp80, and IL-6. This study contributes to a further understanding of BEC growth control and derangements that can occur during cholangiocarcinogenesis.
Short- and long-term outcome of severe alcohol-induced hepatitis treated with steroids or enteral nutrition: A multicenter randomized trial
Authors: Cabré Eduard , Rodríguez-Iglesias Pilar , Caballería Joan , Quer Joan C. , Sánchez-Lombraña José L. , Parés Albert , Papo Michel , Planas Ramon , Gassull Miquel A.
Published: March 2004

Steroids are recommended in severe alcohol-induced hepatitis, but some data suggest that artificial nutrition could also be effective. We conducted a randomized trial comparing the short- and long-term effects of total enteral nutrition or steroids in these patients. A total of 71 patients (80% cirrhotic) were randomized to receive 40 mg/d prednisolone (n = 36) or enteral tube feeding (2,000 kcal/d) for 28 days (n = 35), and were followed for 1 year or until death. Side effects of treatment occurred in 5 patients on steroids and 10 on enteral nutrition (not significant). Eight enterally fed patients were prematurely withdrawn from the trial. Mortality during treatment was similar in both groups (9 of 36 vs. 11 of 35, intention-to-treat) but occurred earlier with enteral feeding (median 7 vs. 23 days; P = .025). Mortality during follow-up was higher with steroids (10 of 27 vs. 2 of 24 intention-to-treat; P = .04). Seven steroid patients died within the first 1.5 months of follow-up. In contrast to total enteral nutrition (TEN), infections accounted for 9 of 10 follow-up deaths in the steroid group. In conclusion, enteral feeding does not seem to be worse than steroids in the short-term treatment of severe alcohol-induced hepatitis, although death occurs earlier with enteral nutrition. However, steroid therapy is associated with a higher mortality rate in the immediate weeks after treatment, mainly because of infections. A possible synergistic effect of both treatments should be investigated.
Hepatocellular carcinoma occurring in nonfibrotic liver: Epidemiologic and histopathologic analysis of 80 French cases
Authors: Bralet Marie-Pierre , Régimbeau Jean-Marc , Pineau Pascal , Dubois Sylvie , Loas Gwenolé , Degos Françoise , Valla Dominique , Belghiti Jacques , Degott Claude , Terris Benoît
Published: March 2004

Hepatocellular carcinoma (HCC) occurring in nonfibrotic liver represents a rare, ill-defined subgroup of HCC without cirrhosis in which mechanisms of hepatocarcinogenesis remain unclear. The aim of our study was to assess epidemiological factors and detailed histopathologic changes in the nontumoral liver of patients developing such tumors. Of 330 HCCs resected in our institution between 1985 and 1998, we retrospectively analyzed 80 cases (53 men, 27 women; mean age, 51 ± 16 years) in which the nontumoral liver showed no (n = 28) or minimal (n = 52) portal fibrosis without any septal fibrosis. In the group with no portal fibrosis there was no male predominance, and patients were significantly younger (44 ± 19 years vs. 54 ± 14 years) than those with minimal portal fibrosis. Sixty-seven tumors were typical HCCs, 8 were of fibrolamellar type, and 5 were hepatocholangiocarcinomas. Mean tumor size was 10 ± 5 cm. Risk factors for HCC development were found in 30 patients: hepatitis B (n = 17) or C (n = 2) virus infections, alcohol consumption (n = 11), and hemochromatosis (n = 1). In the nontumoral liver, periportal and lobular necrosis, mild portal inflammation, steatosis, and iron overload were present in 15%, 57%, 52%, and 54% of cases, respectively. Liver cell changes were noted in 6%. This study emphasizes the need for strict criteria to classify HCC without cirrhosis. HCC in nonfibrotic liver is a distinct subgroup in which nontumoral liver shows nonspecific minimal changes without regeneration or premalignant lesion. Etiologic factors are often unidentified, although presence of HBV infection in 21% suggests a direct oncogenic role of this virus.
Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats
Authors: Nakamura Toru , Sakata Ryuichiro , Ueno Takato , Sata Michio , Ueno Hikaru
Published: March 2004

We investigated whether anti-transforming growth factor β (TGF-β) molecular intervention can halt the progression of liver fibrosis in rats. To block TGF-β action in a specific manner, we prepared an adenovirus expressing a truncated type II TGF-β receptor (AdTβ-TR), which specifically inhibits TGF-β signaling as a dominant-negative receptor. We also used an adenovirus expressing bacterial β-galactosidase (AdLacZ) as a control adenovirus. Rats were treated with dimethylnitrosamine (DMN) for 3 weeks; then, AdTβ-TR, AdLacZ, or saline was intravenously applied once, followed by an additional 3-week DMN treatment. The ratio between the truncated receptor and the wild-type receptor at the mRNA level was 15 at 1 week and 10 at 3 weeks after gene transfer. Immunohistostaining analysis showed that the truncated receptor was expressed mainly in septal cells including hepatic stellate cells. Liver fibrosis, as assessed by histology, hydroxyproline content, and the serum level of hyaluronic acid, progressed during the additional 3-week DMN treatment. However, in rats infected with AdTβ-TR, the fibrosis remained at the level seen in rats given DMN for only 3 weeks. All AdTβ-TR-treated rats remained alive, whereas DMN-treated rats infused with either AdLacZ or saline died of liver dysfunction. In the livers of AdTβ-TR-treated rats, electron microscopy showed: 1) less accumulation of extracellular matrix proteins in the Disse’s spaces; 2) regenerated hepatocytes; and 3) fat droplet-rich “quiescent” hepatic stellate cells. Our results demonstrate that TGF-β plays a critical role in the progression of liver fibrosis, and suggest that anti-TGF-β intervention should be therapeutic in already-established fibrotic livers, not only by suppressing fibrosis, but by facilitating hepatocyte regeneration.
Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes
Authors: Larkin Janet M. , Woo Bonnie , Balan Vijayan , Marks David L. , Oswald Barbara J. , LaRusso Nicholas F. , McNiven Mark A.
Published: March 2004

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of ˜25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.
Expression of cystic fibrosis transmembrane conductance regulator in liver tissue from patients with cystic fibrosis
Authors: Kinnman Nils , Lindblad Anders , Housset Chantal , Buentke Eva , Scheynius Annika , Strandvik Birgitta , Hultcrantz Rolf
Published: March 2004

The authors examined the expression of cystic fibrosis transmembrane conductance regulator (CFTR) and its relationship to histopathological changes in cystic fibrosis (CF) liver tissue. Immunohistochemistry was used to examine expression of CFTR, intercellular adhesion molecule-1 (ICAM-1) and liver cell-type markers in liver cryosections in 11 patients with CF-associated liver disease, and non-CF controls with (n = 17) and without (n = 3) liver disease. In CF patients prominent inflammatory infiltrates were not found, yet hepatic stellate cells were identified within fibrotic areas around bile ducts. Proliferating bile ducts displayed ICAM-1 immunoreactivity in 3 cases, but bile ducts were otherwise negative. In 2 patients homozygous for R764X and for 1112delT no CFTR immunoreactivity was detected. Bile-duct epithelial cells in patients carrying the ΔF508 mutation displayed aberrant cytoplasmic immunolocalization of CFTR, as determined with confocal laser scanning microscopy, in contrast to the distinct CFTR expression at the luminal surface seen in controls. No clear relationship between CFTR expression and fibrosis or inflammation was evidenced in CF patients. In conclusion, these findings are consistent with an impairment of ΔF508 CFTR processing in intrahepatic biliary epithelium. ICAM-1 expression on bile-duct epithelial cells and inflammatory infiltrates were rare findings in CF liver tissue, indicating that immunological mechanisms are unlikely to be involved in initiation of CF-associated liver disease.
Evaluation of a new enzyme immunoassay for hepatitis C virus (HCV) core antigen with clinical sensitivity approximating that of genomic amplification of HCV RNA
Authors: Tanaka Eiji , Ohue Chiharu , Aoyagi Katsumi , Yamaguchi Kenjiro , Yagi Shintaro , Kiyosawa Kendo , Alter Harvey J.
Published: March 2004

The aim of this study was to analyze the clinical performance of a new enzyme immunoassay (EIA) for hepatitis C virus (HCV) core antigen in comparison with the reverse transcription polymerase chain reaction (RT-PCR). A total of 310 patients with acute or chronic hepatitis C, and 132 HCV-negative controls were studied. Chemiluminescence EIA with monoclonal anti-HCV core antigen was used, and qualitative and quantitative commercial RT-PCRs and an in-house nested RT-PCR were performed. Compared with nested RT-PCR, the core antigen assay showed 97% sensitivity and 100% specificity in 75 patients with chronic hepatitis C and 132 controls. HCV core antigen was positive in 16 (94%) of 17 patients with acute hepatitis C at initial consultation. In 3 persons prospectively followed, core antigen was detected in the first available (1-3 weeks) post-transfusion sample. In 167 anti-HCV-positive individuals, 129 (77%) were viremic; core antigen was detected in 126 (98%) compared with 129 (100%) for nested RT-PCR and 121 (94%) for the commercial RT-PCR. In 48 patients with chronic hepatitis C treated with interferon alfa, the concentration of core antigen before treatment was significantly (P < .002) lower in patients with sustained response than in nonresponders. All responders had a sustained loss of core antigen, whereas all nonresponders remained core antigen positive. The concentrations of HCV core antigen and HCV RNA correlated significantly (n = 48, r = .627, P < .001). In conclusion, the HCV core antigen assay is useful for the diagnosis of acute and chronic hepatitis C, and for predicting and monitoring the effect of interferon alfa treatment.
Superparamagnetic iron oxide-enhanced magnetic resonance images of hepatocellular carcinoma: Correlation with histological grading
Authors: Imai Yasuharu , Murakami Takamichi , Yoshida Shigeyuki , Nishikawa Masahiro , Ohsawa Masahiko , Tokunaga Koh , Murata Masaru , Shibata Kunitaka , Zushi Shinichiro , Kurokawa Masanori , Yonezawa Takeshi , Kawata Sumio , Takamura Manabu , Nagano Hiroaki , Sakon Masato , Monden Morito , Wakasa Kenichi , Nakamura Hironobu
Published: March 2004

Superparamagnetic iron oxide (SPIO)-enhanced magnetic resonance (MR) imaging has been used for the detection of hepatic tumors. However, little is known about this technique in relation to hepatocellular carcinoma (HCC). The aim of this study was to investigate whether SPIO-enhanced MR imaging can be useful in assessing histological grades of HCC. The authors studied histologically proven tumors including 31 HCCs and 6 dysplastic nodules. The ratio of the Kupffer-cell count in the tumorous tissue relative to that in the nontumorous tissue (Kupffer-cell-count ratio) decreased as HCCs became less well differentiated. The ratio of the intensity of the tumorous lesion to that of the nontumorous area on SPIO-enhanced MR images (SPIO intensity ratio) correlated inversely with Kupffer-cell-count ratio in HCCs and dysplastic nodules (r = −.826, P< .001) and increased as the degree of differentiation of HCCs decreased, indicating that the uptake of SPIO in HCCs decreased as the degree of differentiation of HCCs declined. All of the dysplastic nodules and some well-differentiated HCCs showed hypointense or isointense enhancement, relative to the surrounding liver parenchyma, indicating greater or similar uptake of SPIO in the tumor when compared with nontumorous areas. These results suggest that SPIO-enhanced MR imaging reflects Kupffer-cell numbers in HCCs and dysplastic nodules, and is useful for estimation of histological grading in HCCs, although uncertainties persist in differentiating dysplastic nodules from well-differentiated HCCs.
Concanavalin A simultaneously primes liver hematopoietic and epithelial progenitor cells for parallel expansion during liver regeneration after partial hepatectomy in mice
Authors: Sakamoto Toshiki , Ezure Tsukasa , Lunz John , Murase Noriko , Tsuji Hirokazu , Fung John J. , Demetris Anthony J.
Published: March 2004

Liver hematopoietic progenitor cells (LHPC) and liver epithelial progenitor cells (LEPC) share a remarkable number of growth and differentiation-controlling receptor-ligand signaling systems. These likely account for the ability of the liver to support hematopoiesis in fetal life, and possibly for suggestions that LHPC can differentiate into hepatocytes. In these experiments, the kinetics and magnitude of LHPC and LEPC activation and expansion were studied by using a concanavalin A (Con A) liver injury model followed by partial hepatectomy (PH). Studies were performed in interleukin 6-deficient (IL-6−/−) mice and wild-type (IL-6+/+) controls, which show equal susceptibility to Con A- induced injury, because IL-6/gp130 signaling has been implicated in both LHPC and LEPC expansion. Con A pretreatment primed LHPC and LEPC for a rapid and parallel expansion after PH in IL-6+/+ mice, which was significantly blunted and delayed in the IL-6−/− mice. Exogenous IL-6 given immediately before PH after Con A, augmented both LHPC and LEPC expansion in the IL-6−/− mice. Thus, the proinflammatory cytokine IL-6, commonly produced in liver injury and inflammatory disease, is an important growth factor involved in the expansion of LHPC and LEPC. This observation has implications for both hepatic carcinogenesis and transplantation.
Hepatitis C-induced hepatic allograft injury is associated with a pretransplantation elevated viral replication rate
Authors: Pelletier Shawn J. , Raymond Daniel P. , Crabtree Traves D. , Berg Carl L. , Iezzoni Julia C. , Hahn Young S. , Sawyer Robert G. , Pruett Timothy L.
Published: March 2004

Hepatitis C virus (HCV) allograft infection after liver transplantation follows a variable but accelerated course compared with the nontransplantation population. Predictors of outcome and mechanisms of reinfection remain elusive. The accelerated HCV-induced allograft injury associated with a 10- to 20-fold increase in serum viral quantity posttransplantation was hypothesized to be the result of elevated intrahepatic viral replication rates. Patients (N = 23) with HCV-induced end-stage liver disease who underwent liver transplantation between October 1995 and December 1998 were prospectively studied. HCV-induced allograft injury was defined by posttransplantation persistent biochemical hepatitis or allograft fibrosis not explained by other diagnoses. Liver biopsies (N = 92) were obtained by protocol and when clinically indicated. Negative-strand HCV RNA (putative intermediate for replication) was detected by a strand-specific reverse-transcription polymerase chain reaction (RT-PCR) assay and semiquantatively compared with constitutively expressed 18S rRNA. Recipients with increased pretransplantation replication were at increased risk for the development of posttransplantation biochemical hepatitis (P = .03), an increased rate of allograft fibrosis (P = .006), and increased mortality rate (40.0% vs. 0.0%; P = .02). There was no correlation with quantities of genomic HCV RNA in the serum with relative intrahepatic viral replication either before or after liver transplantation. The relative rate of HCV replication within the allograft was not elevated in the posttransplantation period compared with that seen within the explanted liver. Accelerated allograft injury caused by HCV may be predicted by viral replication rates within the explanted liver. The stable intrahepatic replication rate after transplantation suggests that elevated serum viral loads are the result of decreased viral clearance, possibly secondary to immunosuppressive therapy.
Regulation of multidrug resistance 2 P-glycoprotein expression by bile salts in rats and in primary cultures of rat hepatocytes
Authors: Gupta Seema , Stravitz Todd R. , Pandak William M. , Müller Michael , Vlahcevic Reno Z. , Hylemon Phillip B.
Published: March 2004

Biliary phospholipid secretion is tightly coupled to the secretion of free cholesterol and bile salts. The secretion of phospholipids across the canalicular membrane of hepatocytes occurs via the multidrug resistance 2 (mdr2) P-glycoprotein (Pgp). The mechanism underlying the coupling of bile salt and phospholipid secretion has not been elucidated. The aims of this study were to determine the effects of bile acid structure on the expression of mdr2 in vitro and in vivo. Under optimal culture conditions, taurine-conjugated bile acids (50 μmol/L) increased mdr2 messenger RNA (mRNA) levels in the following order: taurocholate (TCA) (288 ± 36%, P < .005) = taurodeoxycholate (TDCA) (276 ± 36%, P < .025) > taurochenodeoxycholate (TCDCA) (216 ± 34%, P < .025) > tauroursodeoxycholate (TUDCA) (175 ± 28%, P < .05) of control levels. The increase in mdr2 mRNA levels by TCA was both time and concentration dependent. Cholate feeding to rats with intact enterohepatic circulation increased mdr2 transcriptional activity by 4-fold and protein mass by 1.9-fold. Chronic biliary diversion (CBD) decreased mdr2 mRNA levels to 66 ± 9% (P < .025) of sham-operated controls. Intraduodenal infusion of TCA for 48 hours in CBD rats caused a significant increase in mdr2 mRNA levels (224%) as compared with CBD controls. A diet high in cholesterol (4%) decreased mdr2 mRNA levels to 57% ± 2 (P < .001) of pair-fed controls. Squalestatin (1 μmol/L), an inhibitor of cholesterol biosynthesis, increased mdr2 mRNA levels by 8.8-fold (P < .005) in hepatocyte cultures after 24 hours. In conclusion, in the rat, bile acids up-regulated mdr2 transcriptional activity whereas cholesterol decreased mdr2 mRNA both in vitro and in vivo.
Liver-Derived Hepatitis C Virus (HCV)-Specific CD4+ T Cells Recognize Multiple HCV Epitopes and Produce Interferon Gamma
Authors: Schirren Carl Albrecht , Jung Maria-Christina , Gerlach Joern Tilman , Worzfeld Thomas , Baretton Gustavo , Mamin Maxim , Gruener Norbert Hubert , Houghton Michael , Pape Gerd Rudolf
Published: March 2004

Virus-specific CD4+ T-cell response at the site of inflammation is believed to play a decisive role for the course of viral disease. In hepatitis C virus (HCV) infection, the majority of studies focused on the peripheral blood T-cell response. In this study we analyzed intrahepatic virus-specific CD4+ T-cell response and compared this with that in the peripheral blood. Liver and blood-derived T-cell lines were studied in 36 patients (18 with chronic hepatitis C and 18 with HCV-associated cirrhosis). Virus-specific interferon gamma (IFN-γ) production at a single cell level to various HCV-proteins (core, nonstructural [NS] 3/4, NS5) were determined by enzyme-linked immunospot (ELIspot). Phenotyping was done by fluorescent-activated cell sorter analysis. In approximately half (16 of 36 [44%]) of intrahepatic T-cell lines a significant number of IFN-γ spots were observed, whereas this was the case in only 19% (7 of 36 T-cell lines) in the blood. In relative terms, core and nonstructural proteins were recognized with the same frequency in both compartments, but HCV-specificity was significantly more often detected in liver tissue compared with the blood. Hepatitis activity index, viral load, and alanine transaminase levels did not correlate with the detection of HCV-specific CD4+ T cells. All T-cell lines were dominated by CD4+ T cells. In conclusion, HCV-specific CD4+ T cells are multispecific, compartmentalize to the liver, and produce IFN-γ. We speculate that our data would support the concept of compartmentalization of specific T cells at the site of inflammation and that a low frequency of specific T cells is associated with failure to clear the virus and a chronic course of disease.
Assessment of Long-Term Outcomes of Community-Acquired Hepatitis C Infection in a Cohort With Sera Stored From 1971 to 1975
Authors: Rodger Alison J. , Roberts Stuart , Lanigan Anna , Bowden Scott , Brown Tracy , Crofts Nick
Published: March 2004

The aim of this study was to examine the long-term effects of hepatitis C virus (HCV) infection on a cohort of patients admitted with acute viral hepatitis from 1971 through 1975. The availability of stored sera from this time enabled testing to identify those who were anti-HCV-positive on admission. Sixteen percent (n = 238) of the cohort tested anti-HCV-positive. The unexposed group was selected from those who were anti-HCV-negative. Systematic approaches were used to locate the cohort and health outcomes assessed by a study-specific questionnaire and clinical, serological, virological, and biochemical assessment. Complete follow-up was achieved on 98 anti-HCV-positive individuals and 201 negatives. Injecting drug use (IDU) was the presumed route of infection. At a mean of 25 years’ follow-up, 54% of the anti-HCV-positive group had evidence of chronic HCV infection (both anti-HCV- and HCV-RNA-positive); the remainder were HCV-RNA-negative. Sixty-nine percent of those chronically infected had elevated serum alanine transaminase (ALT) levels, but only 8% had progressed to overt cirrhosis, and no cases of hepatocellular carcinoma (HCC) were identified. In summary, anti-HCV-positive subjects were 8 times more likely to have died from suicide or drug overdose than from HCV-related disease. Anti-HCV-positive study subjects were at increased risk of liver-related pathology after 25 years’ follow-up, but few had progressed to overt cirrhotic liver disease. Excess mortality in this group was not the result of liver disease. This suggests that the natural history of community-acquired HCV may be more benign than previously thought.
Altered Gene Expression in the Liver of γ-Glutamyl Transpeptidase-Deficient Mice
Authors: Habib Geetha M. , Shi Zheng-zheng , Ou Ching-nan , Kala Geeta , Kala Subbarao V. , Lieberman Michael W.
Published: March 2004

We used mice deficient in γ-glutamyl transpeptidase (GGT) to analyze the effects of GGT deficiency and altered thiol levels on gene expression in liver. GGT-deficient mice have markedly reduced levels of glutathione (GSH), cysteine, methionine, and cysteinylglycine in liver. Steady-state RNA levels of the catalytic subunit of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in GSH synthesis, are elevated 4-fold in these mice, while those for glutathione synthetase (GSH syn) are elevated 2-fold. RNA levels of cystathionase (cystathionine γ-lyase), a key enzyme in the synthesis of cysteine from methionine, are elevated ˜3.5-fold. In contrast, levels of RNA coding for multidrug resistance protein 2 (MRP2), which transports GSH into bile, are half wild-type values. We found no change in RNA levels of enzymes related to oxidative injury (CuZn and Mn superoxide dismutases [SOD], catalase, and glutathione peroxidase). Similarly, RNA levels of glutathione reductase and ribonucleotide reductase were unchanged. Furthermore, in contrast to previous in vitro results, methyl methanesulfonate did not induce stress-activated signal transduction as measured by c-jun phosphorylation in livers of GGT-deficient mice, despite further depletion of GSH by buthionine sulfoximine. Our findings indicate that GGT deficiency itself and/or altered thiol levels regulate expression of genes involved in GSH metabolism, but have no effect on the expression of other antioxidant genes.
Epidermal Growth Factor Impairs the Cytochrome C/Caspase-3 Apoptotic Pathway Induced by Transforming Growth Factor β in Rat Fetal Hepatocytes Via a Phosphoinositide 3-Kinase-Dependent Pathway
Authors: Fabregat Isabel , Herrera Blanca , Fernández Margarita , Álvarez Alberto M. , Sánchez Aránzazu , Roncero César , Ventura Juan-José , Valverde Ángela M. , Benito Manuel
Published: March 2004

Transforming growth factor β (TGF-β)-mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-β-induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-β disrupts the mitochondrial transmembrane potential (ΔΨm) and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase-dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-β produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-xL, an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-xL levels even in the presence of TGF-β. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-β-induced bcl-xL down-regulation. We conclude that PI 3-kinase mediates the survival effect of EGF on TGF-β-induced death by acting upstream from the mitochondrial changes, i.e., preventing bcl-xL down-regulation, cytochrome c release, and activation of caspase-3.
Subclinical Seizure Activity and Prophylactic Phenytoin Infusion in Acute Liver Failure: A Controlled Clinical Trial
Authors: Ellis Antony J. , Wendon Julia A. , Williams Roger
Published: March 2004

Subclinical seizure activity in the patient with encephalopathy and on ventilation with acute liver failure (ALF) is a poorly recognized entity. Its importance lies in the likely exacerbation of cerebral hypoxia and the contribution of such seizure activity to the development of cerebral edema. The aim of the present study was to document the frequency of subclinical seizure activity in a cohort of patients with ALF by using a cerebral function and activity monitor that allows continuous recording of electroencephalogram activity at the bedside and to determine whether the prophylactic administration of the antiepileptic agent phenytoin would reduce its occurrence. Forty-two patients were enrolled in a controlled clinical trial: 20 patients were given phenytoin and 22 acted as controls. Subclinical seizure activity was recorded in 3 and 10 patients, respectively, of the treated and control groups. Pupillary abnormalities indicative of seizure activity and/or raised intracranial pressure (ICP) were also seen less frequently in the phenytoin-treated group compared with the controls (5 and 11 patients, respectively). Autopsy examinations available in 19 patients showed signs of cerebral edema in only 2 (22%) of the phenytoin-treated patients compared with 7 (70%) of the controls (P < .033). Based on these findings, we recommend that patients with ALF, on reaching the stage of grade III or IV encephalopathy, should be routinely monitored for subclinical seizure activity. In this study, prophylaxis with phenytoin reduced the frequency of such seizure activity and its effects, and proved to be safe with the regimen used.
Vaccination of Chimpanzees With Plasmid DNA Encoding the Hepatitis C Virus (HCV) Envelope E2 Protein Modified the Infection After Challenge With Homologous Monoclonal HCV
Authors: Forns Xavier , Payette Paul J. , Ma Xiaoying , Satterfield William , Eder Gerald , Mushahwar Isa K. , Govindarajan Sugantha , Davis Heather L. , Emerson Suzanne U. , Purcell Robert H. , Bukh Jens
Published: March 2004

Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. Development of vaccines to prevent HCV infection, or at least prevent progression to chronicity, is a major goal. In mice and rhesus macaques, a DNA vaccine encoding cell-surface HCV-envelope 2 (E2) glycoprotein stimulated stronger immune responses than a vaccine encoding intracellular E2. Therefore, we used DNA encoding surface-expressed E2 to immunize chimpanzees 2768 and 3001. Chimpanzee 3001 developed anti-E2 after the second immunization and antibodies to hypervariable region 1 (HVR1) after the third immunization. Although chimpanzee 2768 had only low levels of anti-E2 after the third immunization, an anamnestic response occurred after HCV challenge. CTL responses to E2 were not detected before challenge, but a strong response was detected after HCV challenge in chimpanzee 2768. An E2-specific CD4+ response was detected in chimpanzee 2768 before challenge and in both chimpanzees postchallenge. Three weeks after the last immunization, animals were challenged with 100 50% chimpanzee-infectious doses (CID50) of homologous monoclonal HCV. As a control, a naive chimpanzee was inoculated with 3 CID50 of the challenge virus. The vaccine did not generate sterilizing immunity because both vaccinated chimpanzees were infected. However, both vaccinated chimpanzees resolved the infection early whereas the control animal became chronically infected. Compared with the control animal, hepatitis appeared earlier in the course of the infection in both vaccinated chimpanzees. Therefore, DNA vaccine encoding cell surface-expressed E2 did not elicit sterilizing immunity in chimpanzees against challenge with a monoclonal homologous virus, but did appear to modify the infection and might have prevented progression to chronicity.
High-Throughput Quantitative Analysis of Hepatitis B Virus DNA in Serum Using the TaqMan Fluorogenic Detection System
Authors: Loeb Keith R. , Jerome Keith R. , Goddard James , Huang Meei-li , Cent Anne , Corey Lawrence
Published: March 2004

Reproducible quantitative assays to detect viral nucleic acids have proven useful in defining disease progression and following response to therapy in a wide variety of viral infections. We describe the development of a quantitative assay to detect hepatitis B virus (HBV) DNA using real-time fluorescent-probe polymerase chain reaction (PCR) (TaqMan). The assay is highly reproducible, highly automated, and much more sensitive than the currently used branched-chain DNA (bDNA) assay for HBV. The quantitative PCR assay accurately detected samples ranging from 10 to 109 copies of HBV DNA per milliliter. Of 157 serum samples submitted for HBV quantitation, 119 were positive by TaqMan PCR versus only 55 by bDNA (P < .001). All 55 bDNA-positives were positive by TaqMan. Of the 77 samples with detectable HBV-DNA titers below 3.75 × 105 copies by TaqMan, only 13 were detected by bDNA. We tested 119 patients negative for all HBV serologic markers, and all tested negative in the TaqMan assay. HBV DNA was detected by TaqMan in 164 of 195 (84%) of hepatitis B surface antigen (HBsAg)-positive samples. Among hepatitis B e antigen (HBeAg)-positive samples, median titers were 4.3 × 106 copies/mL versus 322 copies/mL in HBeAg-negative samples (P = .012). The TaqMan assay for HBV DNA is highly sensitive and reproducible and thus appears useful in accurately defining levels of viral replication among persons with HBV infection.
Furosemide–Induced Natriuresis As A Test to Identify Cirrhotic Patients With Refractory Ascites
Authors: Spahr Laurent , Villeneuve Jean-Pierre , Tran Hanh Khiem , Pomier-Layrargues Gilles
Published: March 2004

The diagnosis of refractory ascites in cirrhotic patients carries a poor prognosis and liver transplantation should always be considered in this situation. Identification of patients who will not respond to diuretic therapy usually requires several weeks of observation during which a trial of diuretics is instituted using stepwise increases in dosage in order to classify ascites as refractory. In the present study we evaluated the effect of a single dose of 80 mg intravenous furosemide on urinary sodium excretion over 8 hours in cirrhotic patients with ascites responsive to diuretic treatment (group 1; n = 14) and patients with refractory ascites (group 2; n = 15). The test was performed after 3 days without diuretics and patients were on a 80 mEq sodium/day diet. Refractory ascites was defined by the absence of response after 3 months of high doses of diuretics (spironolactone 200 mg/d + furosemide 80 mg/d + metolazone 2.5 mg/d) and the need for repeated paracentesis. The two groups had similar degrees of liver and renal dysfunction as assessed by the Pugh score and creatinine clearance. The effects of furosemide on 8–hour natriuresis was much higher in patients with responsive ascites as compared with patients with refractory ascites (125 ± 46 vs. 30 ± 16 mEq; mean ± SD; P < .0001). A natriuresis lower than 50 mEq/8 hours was observed in all group–2 patients as compared with none from group 1. The present study shows that patients with refractory ascites can be identified quickly and accurately by using this simple furosemide–induced natriuresis test, which could be very useful to select patients for liver transplantation.