bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum immunoglobulin M antibody to hepatitis D as a surrogate marker of hepatitis D in interferon-treated patients and in patients who underwent liver transplantation
Authors: Borghesio Elisabetta , Rosina Floriano , Smedile Antonina , Lagget Marco , Niro Maria Grazia , Marinucci Giovanni , Rizzetto Mario
Published: July 2004

The kinetics of the immunoglobulin (Ig) M type antibody to the hepatitis D virus (IgM anti-HD) were investigated in hepatitis B surface antigen (HBsAg) carriers with chronic hepatitis D treated with interferon (IFN) and in patients with terminal hepatitis delta virus (HDV) cirrhosis who underwent liver transplantation. The IgM antibody disappeared in each of 8 patients who responded to IFN therapy with the persistent normalization of aminotransferases and with the clearance of serum HBsAg and HDV-RNA. The IgM reactivity did not decline in the 45 treated patients who did not respond to the cytokine or who experienced a relapse after responding while on therapy. The antibody rapidly disappeared from serum post-transplantation in each of 10 examined patients with HDV who underwent transplantation. In 5 patients who underwent transplantation and who became reinfected with HDV, the antibody remained undetectable during the early reinfection phase, as marked by HDV replication and by the absence of liver damage; however, it rapidly raised to pre-transplantation levels with the recurrence of hepatitis D (HD) in the liver graft. Monomeric 7S IgM anti-HD predominated over pentameric 19S antibody in each of the two patients examined for IgM anti-HD molecular species. The IgM antibody to HDV raises in response to HDV-induced damage and represents a valid surrogate marker of liver damage which is immunopathologically related to HDV infection. Besides providing diagnostic information, it provides the best predictor of impending resolution of chronic HDV disease, whether spontaneous or IFN-induced.
Quantitation of hepatitis G and C viruses in the liver: evidence that hepatitis G virus is not hepatotropic
Authors: Pessoa Mario G. , Terrault Norah A. , Detmer Jill , Kolberg Janice , Collins Mark , Hassoba Howayda M. , Wright Teresa L.
Published: July 2004

Hepatitis G virus (HGV) is prevalent in patients with chronic liver disease and has been previously detected in liver specimens. However, it is unknown whether the virus is replicating in the liver or is simply a contaminant from serum. We sought to determine whether HGV was hepatotropic and to determine whether coinfection with HGV and hepatitis C virus (HCV) influenced the level of either virus. Virus was quantitated using branched DNA (bDNA) assay for both HGV and HCV in the liver explants and pre-transplant serum samples from 30 transplant recipients: Group I, HGV/HCV coinfection (n = 10); group II, HCV infection alone, (n = 8); group III, HGV alone (n = 12). In patients with coinfection HCV (RNA) titers in liver were consistently higher than those for HGV RNA (median 1.13 × 108 and 360,000 Eq/g respectively, P < .01). The ratio of liver/serum viral RNA was significantly higher for HCV than for HGV (median 129 and 0.3 respectively, P < .01). Levels of HCV RNA were similar in patients with HCV infection alone versus those with HGV/HCV coinfection (median; liver = 1.15 × 107 vs. 1.13 × 108 Eq/g, serum = 500,000 vs. 200,000 Eq/mL) and levels of HGV RNA in liver and serum were similar in patients with HGV infection alone compared to those with HGV/HCV coinfection (median; liver = 1.2 × 106 vs. 4.0 × 105 Eq/g, serum = 4.5 × 106 vs. 2.6 × 106 Eq/mL). Levels of either virus appeared unaffected by the presence of an additional virus. The high ratio of HCV RNA levels in liver compared to serum is consistent with its known hepatotropism, but this pattern was not observed for HGV. The median liver/serum ratio of HGV RNA was less than unity, a finding consistent with serum contamination of liver tissue. Thus we conclude that the liver is not the main site of HGV replication.
Safety and immunogenicity of hepatitis A vaccine in patients with chronic liver disease
Authors: Keeffe Emmet B. , Iwarson Sten , McMahon Brian J. , Lindsay Karen L. , Koff Raymond S. , Manns Michael , Baumgarten Renate , Wiese Manfred , Fourneau Marc , Safary Assad , Clemens Ralf , Krause David S.
Published: July 2004

Acute hepatitis A superimposed on chronic liver disease (CLD) has been associated with severe or fulminant hepatitis. An open, multicenter study was performed to compare the safety and immunogenicity of an inactivated hepatitis A vaccine in patients with CLD with that in healthy subjects. A secondary objective was to compare the safety of the hepatitis A vaccine with that of a commercial hepatitis B vaccine in subjects with chronic hepatitis C. A total of 475 subjects over the age of 18 years were enrolled into 1 of 5 groups according to history, serological data, and previous diagnosis. Patients in groups 1 (healthy adults), 2 (chronic hepatitis B), 3 (chronic hepatitis C), and 5 (other CLD not caused by viral hepatitis) were vaccinated with two doses of inactivated hepatitis A vaccine, 6 months apart. Patients in group 4 (chronic hepatitis C) received 3 doses of a recombinant hepatitis B vaccine, according to a 0-, 1-, and 6-month schedule. Local injection-site symptoms were the most common reactions reported following vaccination in all groups (35.5% of all doses), with the hepatitis B vaccine eliciting fewer injection-site symptoms than the hepatitis A vaccine (19.8% compared with 37.5%). Although a higher percentage of healthy subjects (93%) seroconverted after a single dose of the hepatitis A vaccine than did subjects with chronic hepatitis C (73.7%) or CLD of nonviral etiologies (83.1%), more than 94% of all vaccinees were seropositive for anti-HAV after the complete vaccination course. At each time point, a lower geometric mean concentration of anti-HAV was observed for each group of CLD patients compared with the healthy control subjects. In conclusion, hepatitis A vaccine was well tolerated and induced a satisfactory immune response in patients with chronic hepatitis B, chronic hepatitis C, and miscellaneous CLD.
Anatomy of the human biliary system studied by quantitative computer-aided three-dimensional imaging techniques
Authors: Ludwig Jurgen , Ritman Erik L. , LaRusso Nicholas F. , Sheedy Patrick F. , Zumpe Günter
Published: July 2004

The branching geometry of the normal, cholangiographically identifiable human biliary tree was studied with an innovative computer-aided three-dimensional (3D) imaging technique. In addition, a serially sectioned conventional paraffin block from a normal donor liver was used to create and quantitatively study a microscopic 3D image. Finally, a geometric model was developed to estimate the enlargement of biliary surfaces imparted by microvilli. The images created by these techniques could be viewed in stationary modes or rotating around any preselected axis. Approximately 7 (±3) intrahepatic duct orders were cholangiographically identified. Computerized measurements of the images from three normal livers suggested that the mean total volume of duct orders 1 to 7 shown in the cholangiograms was 16.6 cm3. The volume of the entire macroscopic duct system was estimated to be between 14 and 24 cm3 (mean, 20.4 cm3), with an internal surface of 336 to 575 cm2 (mean, 398 cm2). A geometric model based on electron micrographs suggested that this surface is magnified approximately 5.5-fold by the presence of microvilli. Volume and surface area (SA) measurements of all ducts in the same orders increased nearly exponentially from the first toward the seventh branching order (i.e., from the hilus toward the periphery of the liver), and probably beyond. The microscopic computerized reconstruction of a septal bile duct with its tributaries also allowed volume measurements; the imaged duct system represented 2.7% of the portal tract volume. The data presented herein may help to better evaluate branching patterns of the biliary tree and, eventually, the quantitative aspects of site-restricted cholangiocyte function and their role in the development of biliary diseases.
Independent and combined action of hepatitis C virus infection and alcohol consumption on the risk of symptomatic liver cirrhosis
Authors: Corrao Giovanni , Aricò Sarino
Published: July 2004

Although alcohol intake and hepatitis C virus (HCV) infection are the major determinants of liver cirrhosis (LC) in Western countries, the joint effect of these two factors on LC risk has not yet been adequately studied. We used data from two hospital-based case-control studies performed in Italy. Cases were 285 cirrhotic patients admitted for the first time to district hospitals for liver decompensation. Controls were 417 patients admitted during the same period, and in the same hospitals as the cases, for acute diseases unrelated to alcohol. Alcohol consumption was expressed as lifetime daily alcohol intake (LDAI). Serum HCV antibodies (anti-HCV) were detected using a second-generation test and recombinant immunoblotting assay. We found a dose-effect relationship between LDAI and the risk of LC in both anti-HCV-negative and -positive subjects. Considering the extreme LDAI categories (LDAI = 0 g, lifetime teetotalers, and LDAI = 175 g), the LC odds ratios increased from 1.0 (reference category) to 15.0 (95% CI, 7.1-31.7) and from 9.2 (95% CI, 2.0-43.2) to 147.2 (95% CI, 42.1-514.3) in anti-HCV-negative and -positive patients respectively. The interaction between LDAI and HCV showed an additive structure for LDAI <50 g/day and a multiplicative structure for consumption >125 g/day. Alcohol intake and HCV infection are independent risk factors for symptomatic liver cirrhosis, each being sufficient to induce the disease. In subjects with high alcohol intake, the coexistence of HCV infection multiplies the alcohol-associated risk of cirrhosis. In subjects with low alcohol intake, other factors could be involved.
Hemodynamic effects of the somatostatin analog lanreotide in humans: Placebo-controlled, cross-over dose-ranging echo-doppler study
Authors: Mottet Christian , Sieber Cornel C. , Nauer André , Drewe Jürgen , Fried Ronald , Larsen Finn , Beglinger Christoph
Published: July 2004

Because of their vasoactive effects, somatostatin and its analogs are increasingly used in the management of complications of chronic liver diseases such as variceal bleeding. Postprandial hyperemia augments splanchnic blood flow, subsequently increasing portal pressure. The aim of this study was to explore effects of the somatostatin analog, lanreotide, on food-stimulated hemodynamic parameters in healthy human subjects. A dose-response curve was constructed in eight healthy male subjects in a placebo-controlled cross-over study. On 4 different days, either 0 (placebo), 50, 100, or 200 μg/h of lanreotide was infused intravenously in random order, starting at 45 minutes for 7 hours. On each day, a liquid test meal (Ensure plus, 1.5 kcal/mL) was perfused intraduodenally at a rate of 3 mL/min over 7 hours after a 45-minute basal period. Diastolic arterial pressure (dBP), heart rate (HR), superior mesenteric arterial (SMA) average flow velocity (SMA-V), SMA pulsatility index (SMA-PI), portal venous volume flow (PV-F), and renal artery (RA) resistance index (RA-RI) were measured on regular intervals (flows using Echo-Doppler technology). Lanreotide at all doses abolished food-stimulated splanchnic hyperemia both in the SMA and PV over 7 hours. The fall in dBP and increase in HR after food perfusion were blunted under lanreotide. Food as well as lanreotide did not modify RA-RI. In summary: 1) lanreotide inhibits food-induced splanchnic hyperemia in normal subjects; 2) in parallel, systemic hemodynamic alterations to food stimulation are abolished with lanreotide; and 3) renal vascular resistance is unchanged. Because of its persistent splanchnic vasoconstrictive effect, lanreotide should be tested in patients with portal hypertension.
Defining the outcome of immunosuppression withdrawal after liver transplantation
Authors: Devlin John , Doherty Derek , Thomson Linda , Wong Terence , Donaldson Peter , Portmann Bernard , Williams Roger
Published: July 2004

Successful immunosuppression withdrawal should benefit the natural history of organ transplantation patients. To identify the clinical hazards of removing drug treatment and possible characteristics that predict a favorable outcome in long-term liver recipients, immunosuppression was withdrawn completely and the clinicopathological outcome documented in 18 liver recipients. Indication for transplantation, HLA matching, early rejection history, and presence of microchimerism were examined as predictors of outcome. Chimerism was determined by polymerase chain reaction-based examination for donor-specific HLA-DRB1 alleles and Y-gene-specific nucleotide sequences. At 3 years, 5 patients (28%) remained completely off immunosuppression; 12 patients (67%) experienced histological graft changes: acute rejection in 4, portal tract inflammation/hepatitis in 7, and necrosis in 1. Hepatitis B or C viral infections did not account for the nonrejection patterns. Unmasking of systemic disorders occurred. Chimerism, demonstrated in 7 patients (39%), with skin the optimal tissue, was not associated with tolerance. Parameters associated with successful drug withdrawal were transplantation for non-immune-mediated liver disorders, fewer donor-recipient HLA A, B, and DR mismatches, and a low incidence of early rejection. Immunosuppression withdrawal is a feasible option in a proportion of selected liver recipients, but identification of tolerant patients remains imprecise.
Occupancy of dipeptidyl peptidase IV activates an associated tyrosine kinase and triggers an apoptotic signal in human hepatocarcinoma cells
Authors: Gaetaniello Lucia , Fiore Michele , de Filippo Sergio , Pozzi Nicola , Tamasi Sonia , Pignata Claudio
Published: July 2004

Dipeptidyl peptidase IV (CD26/DPP-IV) is an ectoenzyme expressed on different cell types. Signaling properties and functional consequences of the CD26 triggering have been elucidated mostly on T cells, where the molecule delivers a costimulatory signal that potentiates T-cell activation through the T-cell receptor. We conducted studies in the human hepatocarcinoma-derived PLC/PRF/5 cell line to examine the signal transduction through CD26 and its functional properties in the absence of other T-cell-specific membrane molecules. Engagement of CD26 in PLC/PRF/5 cells through a specific antibody induces tyrosine phosphorylation of several proteins with maximal intensity 15 minutes after the stimulation. This effect was under the negative regulatory control of CD45 tyrosine phosphatase, in that the addition of orthovanadate clearly enhanced the phosphorylation events. Using in vitro kinase assays with CD26 immunoprecipitates, we observed that a protein or proteins with kinase activity are coprecipitated with the CD26 molecule. In addition, unlike Jurkat T cells, in which CD26 expression exerts a protective effect against apoptosis, in PLC/PRF/5 cells CD26 occupancy delivers a potent apoptotic signal. This effect was also observed in HepG2 cells, thus indicating that it represents a more general phenomenon occurring in different liver neoplastic cell lines.
New member of aldose reductase family proteins overexpressed in human hepatocellular carcinoma
Authors: Scuric Zorica , Stain Steven C. , Anderson French W. , Hwang Jung-Joo
Published: July 2004

The multistep process of liver carcinogenesis involves various genetic and phenotypic alterations. To identify genes whose expression is increased during hepatocarcinogenesis, differential-display polymerase chain reaction (DD-PCR) was used to examine differences in the mRNA composition of hepatocellular carcinoma (HCC) versus normal liver (nontumor) tissues. This approach identified 67 cDNAs that were preferentially expressed in HCC tissue. When these cDNAs were analyzed by reverse-Northern analysis, five were reproducibly expressed at high levels in HCC. Interestingly, Northern blot analysis revealed that one of the genes showed significantly increased mRNA levels in all five tested tumor samples, while its mRNA level in the nontumor samples was minimal. BLAST analysis revealed that this gene has high sequence identity with the genes from aldo-keto reductase family of proteins including the mouse fibroblast growth factor-induced gene (FR-1) (80% identity), mouse vas deferens protein (MVDP) (76%), and human aldose reductase (AR) (62%). Expression of this novel AR-related protein in all five tested HCCs suggests that this protein may play an important role in liver carcinogenesis.
Activation of mitogen-activated protein kinases/extracellular signal-regulated kinases in human hepatocellular carcinoma
Authors: Ito Yoshiki , Sasaki Yutaka , Horimoto Masayoshi , Wada Shigeo , Tanaka Yoshio , Kasahara Akinori , Ueki Takahiro , Hirano Tadamichi , Yamamoto Hidenao , Fujimoto Jiro , Okamoto Eizo , Hayashi Norio , Hori Masatsugu
Published: July 2004

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) is a key molecule in intracellular signal transducing pathways that transport extracellular stimuli from cell surface to nuclei. MAPK/ERK has been revealed to be involved in the physiological proliferation of mammalian cells and also to potentiate them to transform. However, its role in the outgrowth of human hepatocellular carcinoma (HCC) has yet to be clarified. Therefore, in this study, we investigated the activation of MAPK/ERK and its associated gene expression in HCC. MAPK/ERK was activated in 15 of 26 cases of HCC we examined (58%), and its activity level was significantly higher in HCC than in the adjacent non-cancerous lesions. Besides, MAPK/ERK activation in HCC was positively correlated with protein expression of transcription factor c-Fos. Furthermore, in 25 of 26 cases of HCC which genomic DNA was available, 22 cases without genomic DNA amplification exhibited positive correlation, not only between protein expression of c-Fos and cyclin D1, but also between MAPK/ERK activation and cyclin D1 expression. Concerning the relationship between MAPK/ERK activation and the clinicohistopathological features of HCC, the tumor (HCC) versus non-tumor (non-cancerous counterpart) ratio (T/N) of MAPK/ERK activity was positively correlated with tumor size, but neither with the stage of HCC nor the degree of differentiation of HCC. In conclusion, these findings suggest that MAPK/ERK activation in human HCC may play an important role in multistep hepatocarcinogenesis, especially in the progression of HCC; at least in part, through cyclin D1 up-regulation primarily induced by MAPK/ERK via c-Fos.
Inhibition by dexamethasone of transforming growth factor β1,-induced apoptosis in rat hepatoma cells: A possible association with Bcl-xL induction
Authors: Yamamoto Masahiro , Fukuda Kazunori , Miura Naoko , Suzuki Rie , Kido Toshitaka , Komatsu Yasuhiro
Published: July 2004

The authors previously reported that transforming growth factor β1 (TGF-β1) induces apoptosis in McA-RH7777 (7777) and McA-RH8994 (8994) rat hepatoma cell lines. Although these cell lines exhibit different responses to glucocorticoid treatment in various cellular functions and gene expression, dexamethasone (DEX) inhibited spontaneous and TGF-β1-induced apoptosis in both. Analysis of analogous hormones in TGF-β1-induced apoptosis in 8994 cells suggested the inhibitory effect to be glucocorticoid-specific. By cell-cycle analysis and DNA fragmentation assay using sodium butyrate, a G1-arrest-inducing reagent, regulation of apoptosis by TGF-β1 and DEX was shown independent of the cell cycle. For elucidation of the mechanisms of anti-apoptotic action of DEX, the effects of various chemical probes on this apoptosis model were examined, and various reagents known to exhibit anti-apoptotic activity in other experimental systems were found to be ineffective. The effect of TGF-β1 and DEX on cellular amounts of several apoptosis-related proteins, members of the Bcl-2 family, Bcl-2, Bcl-xL, Bcl-xS, Bad, and Bax was also examined. DEX drastically increased Bcl-xL in both cell lines irrespective of the presence of TGF-β1. Bcl-2 and Bcl-xS proteins were not detected, and Bax and Bad content did not change by treatment with TGF-β1 or DEX. Progesterone (Prog), a partial antagonist for glucocorticoid receptor, inhibited the effects of DEX on apoptosis and Bcl-xL expression in 8994 cells. Thus, Bcl-xL induction by DEX would appear closely associated with its inhibitory effect on spontaneous and TGF-β1-induced apoptosis in the hepatoma cell lines.
The mouse equivalent of the human p53ser249 mutation p53ser246 enhances aflatoxin hepatocarcinogenesis in hepatitis B surface antigen transgenic and p53 heterozygous null mice
Authors: Ghebranious Nader , Sell Stewart
Published: July 2004

The relative contribution to development of hepatocellular carcinoma of the mouse equivalent to the human p53ser249 mutation, found in human hepatocellular carcinoma associated with aflatoxin (AFB1) exposure, is compared with other major risk factors in a transgenic mouse model. Transgenic p53ser246 mice, expressing the mutant protein gene under the control of a truncated albumin promoter, were bred to mice lacking p53 (p53−/−) and to transgenic mice expressing hepatitis B surface antigen (HBsAg). AFB1 hepatocarcinogenesis was then determined in offspring with single or multiple risk factors by determination of the numbers of high-grade hepatic tumors at 13 months of age. In AFB1-treated male mice, expression of the p53ser246 mutation increases the incidence of high-grade tumors from 0% to 14% in HBsAg-negative, p53+/+ (wild-type homozygous) control mice; from 14% to 71% in HBsAg-negative, p53+/− (wild-type heterozygous) mice; and from 62% to 100% in HBsAg-positive, p53+/+ mice. Thus, whereas HBsAg expression and AFB1 together are strongly cocarcinogenic, the presence of the p53ser246 mutant not only significantly enhances this cocarcinogenic effect, it also increases tumorigenesis in AFB1-treated p53 heterozygous and homozygous mice not expressing HBsAg. The possibility that the p53ser246 mutant protein may act as a promoting agent for AFB1 hepatocarcinogenesis is discussed.
Variant liver estrogen receptor transcripts already occur at an early stage of chronic liver disease
Authors: Villa Erica , Dugani Aisha , Moles Anna , Camellini Lorenzo , Grottola Antonella , Buttafoco Paola , Merighi Annalisa , Ferretti Ilva , Esposito Patrizia , Miglioli Lucia , Bagni Alberto , Troisi Roberto , De Hemptinne Bernard , Praet Marlene , Callea Francesco , Manenti Federico
Published: July 2004

Variant estrogen receptors may be found in hepatocellular carcinoma and may influence its natural history. Because it is not known whether their occurrence is an early or a late event during the course of chronic liver disease or whether they cluster in some subgroups of patients, we investigated a series of patients in different stages of chronic liver disease. One hundred eleven consecutive patients were studied for variant estrogen receptor transcripts by reverse-transcription polymerase chain reaction of RNA extracted from liver biopsy specimens. In chronic active hepatitis, variant estrogen receptor transcripts were coexpressed with wild-type significantly more often in men than in women (P = .029) and in hepatitis B surface antigen (HBsAg)-positive subjects than in subjects positive for antibody to hepatitis C virus (P = .0006). In hepatocellular carcinoma, again in men (P = .004) and in HBsAg-positive patients (P = .0015), the variant estrogen receptor transcript was overexpressed or remained the only one expressed. Patients with liver cell dysplasia presented with the same estrogen receptor pattern than patients with hepatocellular carcinoma. This further reinforces the significance of liver cell dysplasia as a preneoplastic condition. The significantly higher occurrence of variant estrogen receptor in men (especially in HBsAg-positive men) already at an early stage of disease, like chronic active hepatitis, suggests that the alteration of estrogen receptors, favoring uncontrolled proliferation and development of hyperplasia, might constitute a prominent mechanism facilitating neoplastic transformation especially in men.
Analysis of CDKN2A, CDKN2B, CDKN2C, and cyclin Ds gene status in hepatoblastoma
Authors: Iolascon Achille , Giordani Lucia , Moretti Arcangela , Basso Giuseppe , Borriello Adriana , Della Ragione Fulvio
Published: July 2004

The status and the expression of cyclin-dependent kinase inhibitor A (CDKN2A) family genes, named CDKN2A, CDKN2B, and CDKN2C and of cyclin Ds (D1, D2, and D3) genes were investigated in 14 cases of human hepatoblastomas. These genes were selected because: 1) CDKN2A and CDKN2B are very frequently inactivated in human cancers; 2) cyclin Ds are overexpressed in several tumors and 3) CDKN2A is posttranscriptionally silenced in hepatocellular carcinomas. Structural analysis of the CDKN2A, CDKN2B, and CDKN2C genes in hepatoblastoma cases showed the absence of deletions and/or point mutations. Moreover, a detailed investigation of loss of heterozygosity at 9p21 and 1p32 (the chromosomal regions where CDKN2A genes are located) rules out the possible loss of one allele. Messenger RNA (mRNA) analysis showed that CDKN2C is expressed in all hepatoblastoma samples studied, while both CDKN2A and CDKN2B genes are not transcribed in the cancer specimens as well as in the matched normal liver tissues. Interestingly, an alternative mRNA expressed by the CDKN2A gene (β-transcript) is detectable in 100% of the samples investigated. The analysis of cyclin D genes expression revealed that cyclin D1 is highly transcribed in normal hepatic tissue while cyclin D2 or D3 genes were extensively expressed in the matched transformed samples. Investigation at protein level confirmed the data obtained on RNA analysis. Indeed, p16INK4A and p15INK4B (products of expression of CDKN2A and CDKN2B respectively) were not observable while p18INK4C (which is codified by CDKN2C) was clearly detectable in the samples analyzed. Moreover, a noticeable decrease of cyclin D1 content and increase of cyclin D3 level were observable in tumor tissues versus normal counterparts. Our findings demonstrated the following: 1) CDKN2A, CDKN2B, and CDKN2C genes are structurally unmodified in human hepatoblastoma, and 2) CDKN2A (α-transcript) and CDKN2B are transcriptionally silenced in normal liver whereas CDKN2A (β-transcript) and CDKN2C were clearly expressed. Finally, a clear shift in cyclin D type expression was observable during malignant transformation. These results show that CDKN2A gene family alterations are not involved in hepatoblastoma development, whereas changes in cyclin D types might play a role in this type of tumor. Furthermore, a highly regulated expression of CDKN2A seems to occur in normal hepatic tissue.
Cytomegalovirus infection is associated with increased inflammation and severe bile duct damage in rat liver allografts
Authors: Martelius Timi , Krogerus Leena , Höckerstedt Krister , Bruggeman Cathrien , Lautenschlager Irmeli
Published: July 2004

It has been suggested that cytomegalovirus (CMV) infection is involved in allograft rejection. In liver transplantation, it has been suggested that CMV is associated with the development of vanishing bile duct syndrome (VBDS), and persistent CMV has been found in liver grafts that develop chronic rejection. In this experimental study, the effect of rat CMV (RCMV) infection on intragraft changes was investigated in a rat model of acute liver allograft rejection. Liver transplantations were performed in a rat strain combination of PVG (RT1c) → BN (RT1n). No immunosuppression was given. One group of animals was infected with RCMV Maastricht Strain (105 plaque-forming units, intraperitoneally), and another group was left uninfected. The grafts were examined histologically after the rats were killed on postoperative days 7 through 9 at the early phase and days 20 through 30 at the late phase of rejection. Immunohistochemical studies were performed to demonstrate the immunological activation markers major histocompatibility complex class II and interleukin 2 receptors, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and their ligands. RCMV infection was demonstrated from the grafts by culture and direct antigen detection. In liver allografts undergoing acute rejection, CMV significantly increased portal inflammation and caused more severe bile duct damage than in the uninfected grafts. CMV was also linked to the induction of VCAM-1 in the endothelial cells. The ongoing infection was found to vary over time in the different structures of the liver grafts, including the vascular endothelium and bile ducts. Our results support an association between CMV infection and the immunological mechanisms of rejection, as well as the role of CMV in the development of bile duct damage in liver allografts.
(Latent) transforming growth factor β in liver parenchymal cells, its injury-dependent release, and paracrine effects on rat hepatic stellate cells
Authors: Roth Sylke , Michel Kristin , Gressner Axel M.
Published: July 2004

Cultured parenchymal liver cells (PC) were recently recognized to contain (latent) transforming growth factor β (TGF-β) while the expression of TGF-β mRNA remains controversial. This study was designed to analyze PC in different microenvironments (liver in situ, highly purified, isolated, and cultured PC) regarding the qualitative and quantitative content of mature and latent TGF-β protein (immunostainings, enzyme-linked immunosorbent assay [ELISA], and enzyme-labeled fluorescence [ELF] technique). The results were compared with its gene expression (reverse-transcription polymerase chain reaction [RT-PCR]). In all microenvironments, PC contained latent TGF-β, which was partially activated after cell isolation and culture. The amount of total TGF-β (mature plus latent) of latency-associated peptide (LAP) and of latent TGF-β binding protein (LTBP) were shown to decrease during culture. In contrast, TGF-β2 and TGF-β3 mRNA and LTBP-1 and -3 mRNA expression were first detectable after culture. Permeabilization of cell membranes in whole liver and of isolated PC with streptolysin O or carbon tetrachloride, respectively, released TGF-β, a part of which was integrated in the large latent complex as estimated by analytical gel filtration chromatography. The TGF-β released by damaged PC induces paracrine effects on hepatic stellate cell cultures. It stimulates hyaluronan synthesis and antagonizes the effect of mitogenic factor(s) of PC on [3H]thymidine incorporation. The results strongly suggest that the main part of hepatocellular TGF-β is not generated by de novo synthesis but from uptake into the liver in vivo. The immunodetection of preexisting mature TGF-β after isolation of the cells is probably caused by intracellular activation of latent TGF-β The injury-dependent discharge of TGF-β from PC might be an important mechanism for initiation and perpetuation of various forms of chronic human liver diseases.
Hypothyroidism minimizes liver damage and improves survival in rats with thioacetamide induced fulminant hepatic failure
Authors: Bruck Rafael , Oren Ran , Shirin Haim , Aeed Hussein , Papa Moshe , Matas Zipora , Zaidel Liliana , Avni Yona , Halpern Zamir
Published: July 2004

Recent data from animal studies suggest that induced hypothyroidism prevents the hyperdynamic circulation in portal vein ligated rats, liver cirrhosis in rats chronically treated with thioacetamide (TAA), and immune-mediated acute liver injury induced in mice by concanavalin A. Therefore, the aim of this present study is to determine whether hypothyroidism would likewise prevent fulminant hepatic failure (FHF) in rats. FHF was induced by 3 consecutive ip injections of TAA (400 mg/kg) at 24-hour intervals. Hypothyroidism was induced in rats by either methimazole (MMI) or propylthiouracil (PTU) and surgical thyroidectomy and was confirmed by elevated serum thyroid stimulating hormone levels. Serum levels of liver enzymes, blood ammonia, and prothrombin time were significantly lower in all 3 groups of hypothyroid rats. The stage of hepatic encephalopathy (HE) and the survival rates were significantly improved in the hypothyroid rats (P < .01); the histologic examination of their livers showed less necrosis and inflammation (P < .01). In the hypothyroid rats, the serum levels of malondialdehyde 48 hours after thioacetamide (TAA) administration were lower than in control rats (P < .01). Exogenous supplementation of hypothyroid rats with l -thyroxine started 48 hours before TAA administration abrogated the protective effects of hypothyroidism. The serum levels of tumor necrosis factor alfa (TNF-α), interleukin (IL) 2 and IL-6 after 24 hours were slightly lower in the hypothyroid rats, but the administration of soluble receptor of TNF (10-1,000 μg/rat) did not prevent the induction of fulminant liver failure by TAA. Oxygen extraction, studied in isolated perfused liver preparation, was significantly lower in livers of hypothyroid rats (P < .01). These results suggest that induced hypothyroidism decreases the development of liver injury in a rat model of FHF. The mechanism may involve diminished oxidative cell injury caused by decreased oxygen utilization and hypometabolism associated with hypothyroidism.
Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats
Authors: Petersen Bryon E. , Zajac Valerie F. , Michalopoulos George K.
Published: July 2004

Administration of 2-acetylaminofluorene (2-AAF) given before partial hepatectomy (PHx) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Our objective in this study was to examine the oval cell response and associated α-fetoprotein (AFP) gene expression by combining 2-AAF with selective damage of centrilobular regions (carbon tetrachloride [CCl4 ]) or periportal regions (allyl alcohol [AA]). Centrilobular damage results in a more enhanced oval cell response and AFP gene expression than periportal damage. Conversely, more intense proliferation of intraportal bile duct epithelia was seen with 2-AAF/AA than with 2-AAF/CCl4. The oval cell response and AFP gene expression was ranked as 2-AAF/CCl4 ≥ 2-AAF/PHx > 2-AAF/AA. AFP mRNA expression was also examined in an acute AA and CCl4 injury. We found very little AFP gene expression compared with the 2-AAF/hepatic injury models. To see a true oval cell response, the hepatocytes must be inhibited from proliferating. In addition, the results presented with the 2-AA/AA model suggest that the periportal matrix may be as important as the cells that populate the area.
Mitochondrial dysfunction and cytoskeletal disruption during chemical hypoxia to cultured rat hepatic sinusoidal endothelial cells: The pH paradox and cytoprotection by glucose, acidotic pH, and glycine
Authors: Nishimura Yoshiya , Romer Lewis H. , Lemasters John J.
Published: July 2004

We investigated mechanisms underlying death of cultured rat liver sinusoidal endothelial cells exposed to chemical hypoxia with KCN (2.5 mmol/L) to simulate the adenosine triphosphate (ATP) depletion and reductive stress of anoxia. During chemical hypoxia, acidotic pH prevented cell death. Glucose (0.3-10 mmol/L) also prevented cell killing. Cytoprotection by glucose but not acidosis was associated with prevention of ATP depletion. After 4 hours of chemical hypoxia at pH 6.2 (simulated ischemia), rapid cell death occurred when pH was restored to pH 7.4 with or without washout of KCN (simulated reperfusion). This pH-dependent reperfusion injury (pH paradox) was prevented after KCN washout at pH 6.2. Glycine (0.3-3 mmol/L) also prevented the pH paradox, but glucose did not. The initial protection by acidotic pH and glycine during simulated reperfusion was lost when pH was later restored to 7.4 or glycine was subsequently removed. Mitochondria depolarized during chemical hypoxia. After washout of cyanide, mitochondrial membrane potential (△Ψ) did not recover in cells that subsequently lost viability. Conversely, those cells that repolarized after cyanide washout did not subsequently lose viability. The actin cytoskeleton and focal adhesions became severely disrupted during chemical hypoxia at both pH 6.2 and 7.4 and did not recover after cyanide washout under any condition. Glucose during chemical hypoxia prevented cytoskeletal disruption. In conclusion, endothelial cell damage during simulated ischemia/reperfusion involves mitochondrial dysfunction, ATP depletion, and ATP-dependent cytoskeletal disruption. Glycine and acidotic pH prevented cell killing after reperfusion but did not reverse mitochondrial injury or the profound disruption to the cytoskeleton.
Use and outcome of liver transplantation in acetaminophen-induced acute liver failure
Authors: Bernal William , Wendon Julia , Rela Mohamed , Heaton Nigel , Williams Roger
Published: July 2004

Once defined clinical criteria are fulfilled in acetaminophen-induced hepatotoxicity, prognosis without orthotopic liver transplantation (OLT) may be very poor. In the present study, we examined the application and outcome of OLT in 548 patients admitted to a single center between 1990 and 1996. Four hundred twenty-four (77%) of the patients studied did not fulfill transplantation criteria, and 396 of these (93%) survived. The majority of the 28 nonsurvivors (7%) in this group fulfilled two of three combined criteria, and the finding of a high APACHE III score could be used as an indicator for the need for OLT. Of the 56 patients (45%) not listed, in only a small proportion was this caused by psychiatric reasons, and in the majority, it was a consequence of the rapid development of multiple organ failure and cerebral edema. This also applied to 24 (35%) of the 68 listed patients in whom the rapidity of clinical deterioration, reflected in increasing APACHE III scores, was such that even with the prompt availability of donor organs, OLT was not possible. In the final event, only 44 (35%) of those who fulfilled criteria underwent OLT, of whom 33 (75%) survived to leave the hospital. Survival was greatest in those receiving unreduced grafts, and markers of early graft function differed significantly between survivors and nonsurvivors. Liver transplantation is an effective treatment in a relatively small number of patients with acetaminophen-induced hepatotoxicity, and for a substantial proportion, transplantation was never an option because of the rapidity of clinical deterioration. APACHE III scoring may be of value in decision making and in better defining patients in clinical trials.
Contribution of the murine mdr1a P-glycoprotein to hepatobiliary and intestinal elimination of cationic drugs as measured in mice with an mdr1a gene disruption
Authors: Smit Johan W. , Schinkel Alfred H. , Müller Michael , Weert Betty , Meijer Dirk K. F.
Published: July 2004

In the mouse, both the mdr1a and the mdr1b gene encode drug-transporting P-glycoproteins. The mdr1a P-glycoprotein is expressed in epithelial cells of, among others, the liver and the intestine. Furthermore, the mdr1b gene product is found in the liver but is not detectable in the intestine. To establish the potential involvement of P-glycoprotein in the elimination of cationic amphiphilic drugs from the body, we investigated biliary, intestinal, and urinary excretion in mice with a homozygous disruption of the mdr1a gene (mdr1a(−/−) mice). These mice are fully viable under laboratory conditions and have normal bile flow. Cumulative biliary excretion (expressed as percent of the intravenously administered dose excreted over a 1-hour period) of several cationic compounds was decreased as follows in mdr1a(−/−) mice compared with the wild-type animals: tri-n-butylmethylammonium (TBuMA), 0.7% versus 2.1%; azidoprocainamide methoiodide (APM), 3.8% versus 7.6%; and vecuronium, 22.7% versus 41.3% The luminal secretion of both TBuMA and APM in the small intestine was profoundly decreased, respectively 4.6-fold (1.8% vs. 8.2% in the wild-type) and 7.9-fold (1.6% vs. 10.3% in the wild-type) in mdr1a(−/−) mice. Thus mdr1a P-glycoprotein contributes substantially to the removal of a wide variety of cationic agents from the body through intestinal and hepatobiliary secretion, but it evidently acts in concert with other transport system(s). These processes probably provide a protective mechanism limiting the overall rate of absorption as well as the bioavailability of potentially toxic organic amines.
p21Ras downstream effectors are increased in activity or expression in mouse liver tumors but do not differ between Ras -mutated and Ras -wild-type lesions
Authors: Kalkuhl Arno , Troppmair Jacob , Buchmann Albrecht , Stinchcombe Stefan , Buenemann Christoph Lars , Rapp Ulf R. , Kaestner Klaus , Schwarz Michael
Published: July 2004

Mouse liver tumors frequently harbor activating ras gene mutations. Downstream effector molecules of p21Ras include Raf-1 kinase which mediates external signals via kinase signaling pathways to nuclear transcription factors including c-Fos and c-Jun. Mouse liver tumors with differing ras-mutational status were analyzed for alterations in Ras/Raf-1 signal transduction. Tumors were characterized with respect to the presence of base substitutions in the 3 known hot-spot positions at codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras. Ha-ras codon 61 or Ki-ras codon 13 mutations, but no N-ras mutations, were detected in 23 out of 33 tumors analyzed, while no ras-mutations were found in 10 of the tumors. There was no significant difference in the expression of p21Ras proteins between ras-mutated tumors and tumors without detectable ras mutations. To allow for determination of Raf-1 kinase activity in tumors, a sensitive and specific assay was developed for measurements with tissue homogenates. Raf-1 kinase activity was increased about four-fold in liver tumors as compared with normal liver tissue. No significant differences in kinase activity, however, were evident between ras-mutated and ras-wild-type tumors. The same was true with respect to the levels of c-fos and c-jun mRNAs. Moreover, there were no significant differences in cell division (5-bromo-2′-deoxyuridine-labeling indices) of hepatocytes from ras-mutated and ras-wild-type tumors. The similar degree of constitutive activation of the Ras/Raf-1 signaling pathway in liver tumors, with and without detectable ras mutations, suggests that other molecules within the signaling pathway may substitute for ras-mutations during oncogenic conversion of ras-wild-type hepatocytes.
Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?
Authors: Zegers Mirjam M. P. , Zaal Kristien J. M. , Hoekstra Dick
Published: July 2004

A photoreactive sphingolipid precursor was used to investigate the potential involvement of protein-lipid interactions that may convey specificity to sphingolipid transport in the human hepatoma cell line, HepG2. A 125I-labeled, photoreactive ceramide, 125I-N3-Cer, was incubated with the cells and became incorporated into two sphingolipid products. The major product was photoreactive sphingomyelin (125I-N3-SM) (25% of total radioactivity), while only minor amounts of photoreactive glucosylceramide (125I-N3-GlcCer) were formed (<2%). After photoactivation, a restricted number of proteins was labeled. Given the absolute amounts of the newly synthesized, photoreactive lipids and their precursor present in the cells, labeling of the proteins can be assumed to be derived from interaction with either ceramide (Cer) or sphingomyelin (SM), or both. To discriminate between these possibilities, photoactivation and protein analysis was performed in cells treated with D-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol (PDMP), an inhibitor of sphingolipid biosynthesis. In treated cells, the radioactive SM pool was reduced by ≈80% Concomitantly, labeling of a 60-kd protein, seen in control cells, decreased. Furthermore, the 60-kd protein is membrane-associated and insoluble in detergent at low temperature. Moreover, when cells containing photoreactive sphingolipids after a preincubation with the photoreactive Cer were photoactivated and subsequently incubated with fluorescent sphingolipid analogs, transport of the latter to the bile canalicular membrane, as observed in control cells, was inhibited. Taken together, the data suggest that distinct proteins, among them a 60-kd protein, may play a specific and functional role in sphingolipid transport to the bile canalicular membrane.
Expression of hepatitis B virus X protein in HBV-infected human livers and hepatocellular carcinomas
Authors: Su Qin , Schröder Claus H. , Hofmann Walter J. , Otto Gerd , Pichlmayr Rudolf , Bannasch Peter
Published: July 2004

Transactivation of cellular genes and functional inactivation of p53 by the hepatitis B virus (HBV) X gene-encoded protein (HBx) are proposed as alternative mechanisms for induction of hepatocellular carcinomas (HCCs) in chronic HBV infection. Using an immunohistochemical approach, we studied the expression of HBx in 39 explanted livers with HBV-associated disease. Because the data reported previously have been inconsistent, possibly due to the application of different antibodies, we compared results with 5 polyclonal and 6 monoclonal anti-HBx antibodies from five laboratories. Ten of the 11 antibodies reacted with recombinant HBx by Western blotting, but only 1 polyclonal and 2 monoclonal antibodies reacted specifically with HBx in tissue, and were thus suitable for immunohistochemistry. Three other polyclonal antibodies reacted with tissue components in addition to HBx. One polyclonal and 4 monoclonal antibodies did not recognize the HBx in the tissue. HBx was demonstrated in 16 of 30 (53.3%) cirrhotic livers and 10 of 18 (58.8%) HCCs by all specific antibodies. The expression of HBx, among three HBV antigens examined, was found to be preferentially maintained in HCC and the surrounding liver parenchyma, including focal or nodular preneoplastic lesions. However, the immunoreactivity was always limited to the cytoplasm of a small number of parenchymal and neoplastic cells. The role of X gene expression in HBV-associated human hepatocarcinogenesis remains to be established.