bluuebunny/crossref_metadata_2025 huggingface dataset:
The monoethylglycinexylidide test does not correctly evaluate lidocaine metabolism after ischemic liver injury in the rat
Authors: Leclercq I , Saliez A , Wallemacq P E , Horsmans Y , Lambotte L
Published: July 2004

Although the monoethylglycinexylidide (MEGX) test defined as a single determination of MEGX plasma concentration after lidocaine injection has been proposed as a liver function test, some discrepancies appeared in assessing the quality of liver donor for transplantation as well as the severity of liver disease. The present study used a severe ischemia- reperfusion liver injury (IRI) in rat to evaluate the various factors able to influence the level of MEGX. The metabolism of lidocaine was studied on microsomes isolated from intact rats and from rats submitted to this liver injury. A significant reduction of the various pathways transforming lidocaine but also MEGX was demonstrated. Lidocaine inhibited the MEGX transformation both in intact and injured liver microsomes. In vivo, plasma MEGX concentrations, determined by high-performance liquid chromatography (HPLC), were lower in IRI than in controls up to 80 minutes after lidocaine injection but not later. By contrast, using the usual commercial fluorescence polarization immunoassay (FPIA), MEGX concentrations were paradoxically higher in IRI than in controls. Moreover, MEGX values obtained using FPIA were threefold higher in controls and ninefold higher in IRI than with HPLC. It was shown that these differences were related to the detection by FPIA of free and mainly of conjugated hydroxy-MEGX that accumulated in plasma from rats submitted to an IRI. These data emphasize the complexity of factors influencing the appearance and disappearance of MEGX because of delayed MEGX formation with liver injury but also to inhibition of its further metabolization. The choice of the sampling time for MEGX determination is critical and has to be optimized in every type of liver injury. Moreover, a specific technique, such as HPLC, will avoid cross-reactivity with other metabolites, which may be particularly abundant when the biliary excretion is impaired.
Effect of a xanthine analog on human hepatocellular carcinoma cells (Alexander) in culture and in xenografts in SCID mice
Authors: Marucci L , Varticovski L , Arias I M
Published: July 2004

Hepatocellular carcinoma (HCC) frequently overexpresses the MDR1 gene and is resistant to drugs transported by the multidrug-resistance efflux pump. A xanthine analog, 1-(11-dodecylamino-10-hydroxyundecyl)- 3,7-dimethylxanthine (CT-2584,CTI), is cytotoxic to many tumors in culture and was four times more effective than verapamil in inhibiting Rhodamine 123 secretion in MDR1-overexpressing Chinese hamster ovary cells. However, studies using PRF/PLC/5 (Alexander) cells revealed that CT-2584 is cytotoxic by another mechanism not involving inhibition of MDR1 function. Alexander cells have integrated the hepatitis B surface antigen (HBsAg) gene and quantitatively secrete HBsAg. The parent cell line, Alex 0, has low MDR1 expression and is drug-sensitive, whereas a derived line, Alex 0.5, is drug-resistant and overexpresses MDR1 100 times. Both cell lines were similarly killed within 24 or 48 hours by CT-2584. Freshly isolated rat and human hepatocytes were considerably more resistant to killing by CT-2584. In vivo, CT-2584 significantly reduced tumor growth in SCID mice bearing Alex 0 or 0.5 xenografts as determined by serial measurements of HBsAg. Hepatic parenchyma was normal, whereas apoptosis and cellular loss were observed in xenografts. The xenograft model is useful for testing pharmacological therapy of HCC.
Central thyrotropin-releasing hormone stimulates hepatic DNA synthesis in rats
Authors: Yoneda M , Tamori K , Sato Y , Yokohama S , Nakamura K , Kono T , Makino I
Published: July 2004

Central neuropeptides play a role as physiological regulators in the autonomic nervous system. One of these neuropeptides, thyrotropin- releasing hormone (TRH), is distributed throughout the central nervous system (CNS) and acts as a neurotransmitter to regulate gastric functions through the vagus nerve. However, the autonomic nervous system is also involved in hepatic regeneration, but the effect of TRH is unknown. Therefore, the CNS's effect of TRH on hepatic DNA synthesis was studied in rats. Hepatic DNA synthesis was assessed by [Methyl-3H]thymidine incorporation 6, 12, 24, 48, and 72 hours after intracisternal injection of the TRH analog, RX 77368 (1, 5, 10, and 100 ng), and by 5-bromo-2′-deoxyuridine (BrdU) labeling of the liver section. Hepatic DNA synthesis was stimulated by intracisternal TRH analog (10 ng), with a peak response at 24 hours after peptide injection, and returned to baseline by 72 hours. This stimulatory effect by central TRH analog on hepatic DNA synthesis was dose-related, ranging from 1 ng to 10 ng (dpm/microg DNA at 24 hours [mean ± SE]: saline, 95 ± 6; 1 ng, 114 ± 14; 5 ng, 318 ± 57; 10 ng, 693 ± 78; 100 ng, 710 ± 135). Hepatocytes were randomly labeled by BrdU 24 hours after intracisternal TRH analog (10 ng). Intravenous TRH analog (10 ng) did not influence hepatic DNA synthesis. The stimulatory effect of TRH analog was blocked by hepatic branch vagotomy and atropine, but not by hepatic sympathectomy, 6-hydroxydopamine, insulin antibody, or hypophysectomy. These results indicate that TRH acts in the CNS to stimulate hepatic DNA synthesis through vagal and cholinergic mechanisms, and that TRH may be the chemical messenger involved in brain regulation of hepatic proliferation.
Quantitation of sinusoid-like vessels in hepatocellular carcinoma: Its clinical and prognostic significance
Authors: Tanigawa N , Lu C , Mitsui T , Miura S
Published: July 2004

Angiogenesis is crucial for tumor growth and metastasis. Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. However, the relationship between tumor vascularity and the outcome of patients with HCC has not been evaluated. To clarify whether tumor angiogenesis is related to the prognosis of patients, immunohistochemical staining, using anti-von Willebrand factor (vWF) and anti-CD34, was applied in resected specimens from 43 cases of HCC. In nonmalignant tissue, staining was confined to vessels in the portal tract and to a few periportal sinusoids with both of the endothelial markers applied. In tumor tissue, however, sinusoid-like vessels reacted intensively with anti-CD34 but not with anti-vWF. The intratumor microvessel density (MVD) highlighted by anti-CD34 was 297 ± 88 (per 0.74 mm2), which was significantly higher than that highlighted by anti-vWF (4 ± 7). When only the MVD highlighted by anti-CD34 was analyzed, tumor diameter larger than 2 cm, poor differentiation (Edmondson's II to IV), and portal invasion were significantly related to the subgroup with MVD > or = 290. Overall survival curves of patients with MVD < 290 were better, and these patients were more likely to remain tumor free. Cox hazards model revealed intratumor MVD and Edmondson's grade to be independent prognostic factors for the overall survival of patients. These results demonstrated for the first time that tumor angiogenesis assessed by anti-CD34 was correlated with the outcome of patients with HCC, suggesting a potential role for anti-CD34 in the diagnosis and treatment of HCC.
The application of image analysis and neural network technology to the study of large-cell liver-cell dysplasia and hepatocellular carcinoma
Authors: An C S , Petrovic L M , Reyter I , Tolmachoff T , Ferrell L D , Thung S N , Geller S A , Marchevsky A M
Published: July 2004

Liver cell dysplasia (LCD) is considered a preneoplastic lesion, whose characterization and differentiation from hepatocellular carcinoma (HCC) and from the reactive changes seen in cirrhosis has been controversial. We studied 12 cases of LCD (large cell type) with image analysis techniques (IA) and compared the findings with those of HCC (n = 40), and a spectrum of non-neoplastic hepatic lesions including normal liver and cirrhosis (n = 49). A minimum of 200 Feulgen-stained nuclei were measured from each lesion with the CAS 200 image analysis system. The data were collected with the aid of CellSheet software. Thirty-four variables were measured, including geometric, textural, and photometric nuclear features and DNA ploidy. The data were analyzed with multivariate statistics and a backpropagation neural network (NN). Stepwise statistical analysis selected 22 variables that were statistically significant in the three groups with P values <.05. Various NN architectures were developed using these variables. The best NN architecture included a sigmoidal transfer function, 14 input, 16 hidden, and 3 output neurons. It trained to completion after 8,887 runs using 90% of the lesions. This NN yielded a 100% cross-validation rate for unknown cases. These data support the concept of LCD (large cell type) as a lesion that can be objectively distinguished from HCC and non-neoplastic liver. Our study also demonstrates the potential usefulness of IA for the evaluation of difficult histopathological problems.
Paracrine interaction between hepatocytes and macrophages after extrathyroidal proteolysis of thyroglobulin
Authors: Brix K , Wirtz R , Herzog V
Published: July 2004

Thyroglobulin (Tg), the precursor of the thyroid hormones triiodothyronine (T3) and thyroxine (T4), is known to derive from thyroid epithelial cells. Part of Tg reaches the circulation as an intact molecule by transcytosis across the epithelial wall of thyroid follicles. Circulating Tg is a potential ligand for the asialoglycoprotein receptor of hepatocytes. In this report we show, however, that clearance of circulating Tg occurred exclusively by endocytosis in liver macrophages, whereas hepatocytes did not participate in this process. The biological significance of this Tg uptake by the macrophages might consist in an increase of thyroid hormones in close proximity to the macrophages, thereby affecting the hepatocyte metabolism. To test this hypothesis, co-cultures of hepatocytes and macrophages were incubated with Tg, which resulted in the release of thyroid hormones and in a significant increase in the activity of lipogenesis and of hepatocellular key enzymes of the hexose monophosphate shunt. This effect of Tg could be mimicked by equivalent amounts of T3 or T4 exclusively in the co-cultures. When hepatocytes were incubated with thyroid hormones in the absence of macrophages, no or only little effect was observed, indicating that the interaction of macrophages and hepatocytes was a prerequisite for the stimulation of the hepatocellular metabolism. We conclude that the paracrine effect on HepG2 cells results from the degradation of Tg in J774 cells. Apparently, this process is not confined to the release of thyroid hormones, but it requires the interaction of both cell types, possibly mediated by an additional, as yet unknown stimulus.
Protective action of hepatocyte growth factor for acute liver injury caused by D-galactosamine in transgenic mice
Authors: Okano J , Shiota G , Kawasaki H
Published: July 2004

Hepatocyte growth factor (HGF) has been reported to be a potent mitogen for hepatocytes in vivo and in vitro. Recent reports have shown that HGF has cytoprotective actions in acute liver injury models, but its mechanisms remain to be resolved. In the present study, we investigated whether HGF could work as an anti-hepatitis agent for acute liver injury caused by D-galactosamine (D-GalN) using transgenic (TG) mice expressing HGF in hepatocytes, compared with wild-type (WT) mice of their siblings. After administration of 3 g/kg body weight of D-GalN, elevated serum transaminase levels and severe liver damage that were observed in WT mice were significantly improved in TG mice. In TG mice, the percentage of proliferating cell nuclear antigen (PCNA)-positive hepatocytes was high at 0 hours after D-GalN treatment, and increased at 24 hours. The percentage of PCNA-positive cells in WT mice was very low at 0 hours and 24 hours after treatment, but increased at 48 hours. To clarify the mechanisms via which HGF acts, we measured hepatic HGF and prostaglandin E2 (PGE2) contents after D-GalN administration by an enzyme immunoassay. Total hepatic HGF contents, which were composed of murine (endogeneous) and human (derived from transgene) HGF, in TG mice were higher than those in WT mice at 0, 12, and 24 hours after administration of 3 g/kg body weight of D-GalN. Hepatic PGE2 contents in TG mice were also significantly higher than those in WT mice. Hepatic PGE2 contents had a positive correlation with total HGF contents at both 12 hours and 24 hours after the treatment. Moreover, an administration of 0.5 μg of anti-HGF antibody into the portal vein suppressed hepatic PGE2 contents of mice, compared with saline- injected mice in acute liver injury. Anti-PGE2 antibody administration caused more severe liver damage than saline solution. A survival rate of TG mice that were given 6 g/kg body weight of D-GalN-a lethal dose of D-GalN-was improved compared with WT mice. These results provided direct evidences that HGF worked as an anti-hepatitis agent against acute liver injury caused by D-GalN, and that this action might be exerted through accelerated hepatic PGE2 production.
Bile acid-independent bile flow is differently regulated by glucagon and secretin in humans after orthotopic liver transplantation
Authors: Lenzen R , Elster J , Behrend C , Hampel K , Bechstein W , Neuhaus P
Published: July 2004

The present study characterizes recovery of bile secretion after orthotopic liver transplantation (OLT) in humans with special regard to hormonal regulation of bile acid-independent bile flow by glucagon and secretin. Sixty-seven patients with an uncomplicated postoperative course were studied during the first 3 weeks after OLT to determine normalization of bile flow. A group of 7 and 10 patients, respectively, underwent a biliary stimulation test by either glucagon at days 7, 14, and 21 after OLT or by secretin at days 2, 10, and 21 after OLT. Secretin tests were similarly performed in patients with acute severe rejection during the first 10 days after OLT, while glucagon tests were performed in patients with acute allograft rejection occurring 2 weeks after OLT. Furthermore, hormone effects were studied in nontransplanted patients after cholecystectomy with indwelling biliary T tube. After OLT, bile secretory function recovered and stabilized within 14 days after surgery by reconstitution of both bile acid-dependent and -independent bile flow. Two weeks after OLT, bile secretion was comparable with nontransplanted patients after cholecystectomy. Glucagon and secretin stimulated bile acid-independent bile flow in transplanted and nontransplanted patients significantly, yet secretin choleresis, unlike glucagon choleresis, had already occurred during the first days after OLT and was unaffected by acute allograft rejection. These results allow the speculation that, in humans, glucagon and secretin exert their choleretic activity by different mechanisms and/or at different anatomical sites in the liver. Assuming that secretin acts at the bile duct cells, its secretory capacity was not altered by the transplantation procedure and during moderate or severe rejection episodes, as opposed to glucagon choleresis, which most likely originates in the hepatocytes and requires an entirely reconstituted canalicular transport system after OLT.
Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts
Authors: Attavar P , Budhai L , Kim B , Roy-Chowdhury N , Roy-Chowdhury J , Davidson A
Published: July 2004

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells.
Long-term longitudinal study of intrahepatic hepatitis C virus replication after liver transplantation
Authors: di Martino V , Saurini F , Samuel D , Gigou M , Dussaix E , Reynes M , Bismuth H , Feray C
Published: July 2004

Recurrence of hepatitis C after liver transplantation is common and can lead to severe liver diseases. Although immunosuppression and high levels of viremia suggest a direct pathogenicity of hepatitis C virus (HCV), the relations between viral replication and long-term histological course are still unknown. Thirty-three patients with a mean histological follow-up of 3.5 years (3 months - 8.6 years) were analyzed. Nineteen patients were infected by genotype 1b. Liver HCV RNA was determined in parallel with the quantitation of an internal control (28S ribosomal RNA) by competitive polymerase chain reaction (PCR). Lobular hepatitis (LH) and chronic active hepatitis (CAH) occurred in 27 and 19 patients, respectively. Levels of liver HCV RNA determined in 84 biopsies were higher in cases of LH than in the other patterns (82 ± 123 vs. 19 ± 38; P < .01) and were unrelated to the genotype. Progression from LH to CAH was associated with a highly significant decrease of liver HCV RNA (P = .006), which was not observed in patients with stable histology. Among patients with CAH, those infected by genotype 1b had more severe liver damage and lower levels of liver HCV RNA than others (P = .04). Multivariate analysis showed that high levels of liver HCV RNA at the time of the first posttransplantation biopsy was an independent predictor of CAH (P = .01). After liver transplantation, the progression to CAH together with a decrease of liver HCV RNA suggests that a host's response is involved in the long-term viral pathogenicity. This response may be stronger and liver disease more severe in patients with high levels of replication at the time of LH and in those infected by genotype 1b.
The effects of low dietary levels of polyunsaturates on alcohol-induced liver disease in Rhesus monkeys
Authors: Pawlosky R J , Flynn B M , Salem N
Published: July 2004

Rhesus monkeys that were maintained on a diet containing low, yet adequate, amounts of vitamins C and E and in which linoleate and linolenate represented 1.4% and 0.08% of the total caloric intake, respectively, developed liver fibrosis after consuming alcohol (mean, 2.6 g kg-1 d-1) over a period of 3 years. In the liver, several polyunsaturated fatty acids including 18:2n6, 20:4n6, and 22:6n3 decreased compared with dietary controls, and similar findings were also observed in plasma lipoproteins and erythrocytes. The amount of alcohol consumed correlated positively with plasma lipid peroxidation products, 4-hydroxynonenal (4-HNE) and 8-isoprostane F2α, and negatively with 20:4n6 and 22:6n3 levels. These findings imply that alcoholics who also have a marginal intake of essential fatty acids and antioxidants in their diets may be at an increased risk of developing liver disease.
Clinical, histological, and virological features of hepatitis C virus carriers with persistently normal or abnormal alanine transaminase levels
Authors: Puoti C , Magrini A , Stati T , Rigato P , Montagnese F , Rossi P , Aldegheri L , Resta S
Published: July 2004

This study was aimed to evaluate demographic, clinical, histological, and virological characteristics of 46 hepatitis C virus (HCV) carriers with persistently normal alanine transaminase (ALT) levels and to compare the results with those obtained in a group of 52 HCV-RNA-positive patients with elevated ALT levels. Subjects with normal ALT were more often females (P < .001), were more likely to be asymptomatic (P < .001), and have a lower incidence of risk factors for HCV transmission (P < .01). All patients with normal ALT had significant histological liver damage. The mean grading and staging did not differ between patients with normal and those with raised ALT concentrations. Moderate to severe hepatitis was more frequently found among subjects with normal than with elevated ALT. HCV genotype 2a was far more common in subjects with normal (43%) than with abnormal ALT levels (6%; P < .002), genotype 1b being more frequent in these latter (50% vs. 17%; P < .001). Patients with normal ALT levels had similar serum HCV-RNA titers than subjects with raised ALT. Neither HCV genotype distribution nor viral load correlated with the severity of liver damage. We conclude that significant liver disease may occur irrespective of clinical symptoms, ALT levels, HCV genotypes, and viral load.
Enhanced apoptosis relates to bile duct loss in primary biliary cirrhosis
Authors: Harada K , Ozaki S , Gershwin M E , Nakanuma Y
Published: July 2004

Primary biliary cirrhosis (PBC) is characterized by an immune-mediated destruction of intrahepatic small bile ducts. Apoptosis, a unique pattern of cell death, has been suggested to be responsible for the biliary destruction in PBC. To address this issue, we attempted to detect the apoptosis of biliary epithelial cells by in situ nick-end labeling and by the expression of apoptosis-related proteins using immunohistochemistry in patients with various hepatobiliary diseases, including PBC. The data was noteworthy for several reasons. First, apoptosis was occasionally detected on biliary cells in all liver specimens; however, the positive rate was high in PBC and relatively low in other livers. Strong expression of CD95 was frequently observed in the epithelial cells of the injured bile ducts of PBC, which accompanied high intensity CD95 ligand-expressing mononuclear cells. Perforin and granzyme B immunoreactivities were occasionally found on the bile ducts in control liver diseases as well as PBC, but granzyme B-positive biliary cells were prominent in PBC. In contrast, Lewis Y expression, as detected using BM-1 antibody, was consistently present in the injured bile ducts of PBC. These data suggest that apoptosis, via the perforin/granzyme B pathway, may be associated with the degrading fraction of cell cycle regulation in the small-sized biliary tree under physiological and pathological liver conditions. Moreover, enhanced apoptosis, mediated by CD95/CD95 ligand interaction, may contribute to the bile duct injury and loss observed in PBC.
Infection complicating percutaneous liver biopsy in liver transplant recipients
Authors: Larson A M , Chan G C , Wartelle C F , McVicar J P , Carithers R L , Hamill G M , Kowdley K V
Published: July 2004

There is controversy about the frequency of and risk factors for infectious complications of percutaneous liver biopsy in liver transplant recipients. The aim of this study was to identify the incidence and nature of complications associated with liver biopsy after orthotopic liver transplantation (OLT), with particular emphasis on infection. The medical records of all patients undergoing OLT between January 1990 and August 1994 were reviewed retrospectively to identify complications requiring hospitalization within one week of percutaneous liver biopsy. The nature and severity of complications were recorded and possible risk factors for infectious complications were examined. One hundred ninety-eight patients underwent 1,136 percutaneous liver biopsies. There were eleven complications (0.96%), including as follows: 7 infections, 3 bleeding episodes, and 1 vasovagal reaction. Infections after percutaneous liver biopsy included fever and bacteremia (n = 6), and fever without bacteremia (n = 1). All infections developed only in patients with underlying biliary tract abnormalities; the frequency of infection was higher (9.8%) in patients with choledochojejunostomy when compared with those with choledochocholedochostomy (1.4%). Bacteremia was more likely caused by skin flora in patients with choledochocholedochostomy (CDC) and by enteric bacteria in patients with choledochojejunostomy (CDJ). All infections were treated successfully with parenteral antibiotics. We conclude that biliary tract abnormalities are the primary risk factors for infection after percutaneous liver biopsy, although the risk is higher in patients with CDJ than with CDC. These data support the use of antibiotic prophylaxis before percutaneous liver biopsy in OLT recipients with biliary tract abnormalities.
Clinical efficacy of lactulose in cirrhotic patients with and without subclinical hepatic encephalopathy
Authors: Watanabe A , Sakai T , Sato S , Imai F , Ohto M , Arakawa Y , Toda G , Kobayashi K , Muto Y , Tsujii T , Kawasaki H , Okita K , Tanikawa K , Fujiyama S , Shimada S
Published: July 2004

Seventy-five cirrhotic patients with hyperammonemia in the past or at the time of the study were randomly divided into two groups (treated with lactulose or nontreated) in 14 hospitals in Japan. Thirty-six cirrhotic patients were diagnosed as having subclinical hepatic encephalopathy (SHE), and 39 were diagnosed as non-SHE. SHE was diagnosed when the results of all three of the quantitative psychometric tests used (number connection test, and symbol digit and block design tests of the Wechsler adult intelligence scale [revised]) were abnormal as compared with age-matched normal values. The mean number of abnormal psychometric test results and the prevalence of SHE were used for a quantitative evaluation of the efficacy of the lactulose treatment. Twenty-two of the SHE patients were treated with lactulose (45 mL/d) for 8 weeks, and the other 14 SHE patients did not receive lactulose. In the SHE patients administered lactulose, the results of the quantitative psychometric evaluation were significantly improved at 4 and 8 weeks after the beginning of the lactulose administration. The SHE had disappeared in 10 (50%) of the 20 treated patients at week 8, but it persisted in 11 (85%) of the untreated 13 patients. We concluded that lactulose treatment in cirrhotic patients with SHE is effective with respect to psychometric tests.
Large cell change (liver cell dysplasia) and hepatocellular carcinoma in cirrhosis: Matched case-control study, pathological analysis, and pathogenetic hypothesis
Authors: Lee R G , Tsamandas A C , Demetris A J
Published: July 2004

Large cell change (LCC), characterized by cellular enlargement, nuclear pleomorphism and hyperchromasia, and multinucleation of hepatocytes, is a common lesion in cirrhotic livers, but its nature, significance, and pathogenesis remain uncertain. Therefore, we assessed the prognostic value of LCC as a marker of subsequent hepatocellular carcinoma (HCC) through a case-control study that compared pretransplant liver biopsy specimens from 37 cirrhotic liver transplant recipients with HCC to specimens from a control group of recipients without HCC, matched for sex, age (±5 years), and cause of cirrhosis. LCC was identified in 16 (43%) of the study and 7 (19%) of the control group biopsy specimens. By matched-pair analysis, LCC conveyed a moderately increased risk of later HCC with an estimated odds ratio of 3.3 (95% CI, 1.2-15; P = .038). However, a pathology review of 45 HCCs showed adjoining LCC in only 12 (27%) and did not suggest a morphological transition or a histogenetic association between the two lesions. LCC hepatocytes displayed a low proliferative rate by Ki-67 or proliferating cell nuclear antigen immunostaining (labeling indices of 0.27 and 0.73) but showed a greater degree of apoptosis than normal hepatocytes (labeling indices of 1.9 and 0.23; P = .03) To reconcile these findings, we propose that LCC derives from derangements in the hepatocyte's normal process of polyploidization. Such derangements, possibly caused by chronic inflammation-induced DNA damage, could yield a population of enlarged liver cells with nuclear atypia and pleomorphism, frequent binuclearity, and minimal proliferation. According to this hypothesis, LCC would be a habitual feature of cirrhosis and a regular accompaniment of HCC but would not represent a direct malignant precursor.
Quantitative liver function tests as surrogate markers for end-points in controlled clinical trials: A retrospective feasibility study
Authors: Lotterer E , Hogel J , Gaus W , Fleig W E , Bircher J
Published: July 2004

Quantitative liver function tests such as the determination of galactose elimination capacity (GEC) or the aminopyrine breath test (ABT) may have the potential to serve as refined entry criteria and surrogate markers for end-points in controlled clinical trials. The magnitude of a statistically detectable difference in test results and the period of observation required to document such a difference must be known to properly design such trials. Therefore, we explored retrospectively the time course of changes in GEC and ABT and their reproducibility from a cohort of patients with alcoholic cirrhosis followed for 12 to 42 months, with a median of 34 months. In 15 patients who stopped drinking, GEC improved significantly by 0.64 mg/min/kg within 1 year (mean; 95% confidence interval [CI]: 0.42; 0.86). In contrast, it deteriorated by 0.53 mg/min/kg within 1 year (95% CI: 0.32; 0.74) in another 17 patients who continued to drink (P < .01). The residual standard deviation of the changes in GEC with respect to the patients' initial values was 0.43 mg/min/kg (95% CI: 0.32; 0.52). In addition, ABT improved significantly by 0.14% dose · kg/mmol CO2 (95% CI: 0.09; 0.18) in the abstinent group, and deteriorated by 0.09% dose · kg/mmol CO2 (95% CI: 0.06; 0.13) in the nonabstinent group (P < .01). The residual standard deviation in the above sense for ABT was 0.08% dose · kg/mmol CO2 (95% CI: 0.06; 0.10). These data indicate that clinical trials with a sample size of n = 20 in each group must achieve absolute differences (ADs) in GEC of 0.6 mg/min/kg and of 0.7 mg/min/kg to reach statistical significance at the 5% and 1% level, respectively. In the present study, a period of 11 and 12 months was necessary to observe such differences. The corresponding results for the ABT are 0.11% dose · kg/mmol CO2 (9 months of follow- up; 5% level) and 0.13% dose · kg/mmol CO2 (11 months of observation; 1% level), respectively. Provided that patients with liver diseases treated with drugs are similar to the abstinent and nonabstinent patients with alcoholic liver disease investigated in this study, such numbers could serve for the planning of controlled clinical trials, in which the control group is likely to deteriorate and the treated group is expected to improve. Trials based on GEC or ABT would require only 37 or 30 patient years of observation compared with a median of 444 patient years (range, 50-2,100 patient years) reported for various published controlled clinical trials using survival analysis.
Coexpression of C- myc and transforming growth factor alfa in the liver promotes early replicative senescence and diminishes regenerative capacity after partial hepatectomy in transgenic mice
Authors: Factor V M , Jensen M R , Thorgeirsson S S
Published: July 2004

We have recently shown that overexpression of c-myc and transforming growth factor α (TGF-α) in the liver of double-transgenic mice results in severe DNA damage, aberrant hepatic growth, and development of tumors at a much younger age than that observed in c-myc single- transgenic mice. We now report that double-transgenic TGF-α/c-myc hepatocytes rapidly lose their ability to proliferate upon mitogenic stimulation following partial hepatectomy (PH). At 4 weeks of age, the overall rate of bromodeoxyuridine (BrdU) incorporation following PH was comparable in c-myc and TGF-α/c-myc livers and exceeded that seen in wild-type (WT) mice. However, by 10 weeks of age, c-myc single- transgenic hepatocytes showed proliferative advantages over the WT cells, whereas TGF-α/c-myc double-transgenic hepatocytes had a decreased capacity to proliferate upon mitogenic stimulation. This decreased proliferative response was accompanied by a reduction in the total fraction of proliferating hepatocytes, as well as by a decline in the induction of cyclin A, cyclin B, and cdc2 gene expression. These data show that constitutive coexpression of c-myc and TGF-α accelerates age-related loss in the regenerative potential following PH, and suggest that early replicative senescence of differentiated hepatocytes may have a role in providing a selective growth advantage to initiated cell populations in this model.
Exposure of primary rat hepatocytes in long-term DMSO culture to selected transition metals induces hepatocyte proliferation and formation of duct-like structures
Authors: Cable E E , Isom H C
Published: July 2004

We previously showed that primary rat hepatocytes plated on a rat-tail collagen coated dish and fed a chemically-defined medium supplemented with 2% dimethylsulfoxide (DMSO) can be maintained in a well-differentiated, non-replicating state for periods of several months. In this study, we show that the addition of copper, iron, and zinc to the DMSO-containing chemically defined medium induced DNA synthesis and cell replication during the first two months in culture without loss of hepatic differentiation. DNA synthesis occurred throughout the hepatocyte population without regard to cellular size. No changes were observed in properties indicative of well-differentiated hepatocytes, including cellular morphology, ultrastructure, albumin, or cytokeratin-8 expression. During the third month in culture, after the hepatocytes had become confluent, pseudoduct structures became apparent. Examination of cells lining the ducts by immunohistochemistry showed that these cells lost the ability to express albumin and stained more intensely for cytokeratin 19 and laminin. The ultrastructure of the cells lining the ducts was altered and became more characteristic of bile duct cells. Immunoelectron microscopy revealed that connexin 43, a marker of bile-duct proliferation, was expressed in the duct-like cells. We conclude that under these specific nutritive conditions, primary rat hepatocytes proliferate and, with time, begin to form duct-like structures with altered gene expression and ultrastructural properties.
Human hepatic myofibroblasts increase invasiveness of hepatocellular carcinoma cells: Evidence for a role of hepatocyte growth factor
Authors: Neaud V , Faouzi S , Guirouilh J , Le Bail B , Balabaud C , Bioulac-Sage P , Rosenbaum J
Published: July 2004

The stroma of hepatocellular carcinomas (HCC) is infiltrated with myofibroblasts (MFs). Preliminary in vivo data have suggested that liver MF express hepatocyte growth factor (HGF), a cytokine that has been implicated in several tumor models. Our aim was to investigate the role of MF and HGF in HCC. Cultured liver MF expressed HGF messenger RNA (mRNA) and secreted HGF in their medium, as shown by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Addition of MF-conditioned medium to the HepG2 HCC cell line induced cell scattering. This was associated with a decrease in cell proliferation. MF also increased about 100-fold the ability of HepG2 to invade Matrigel. Increased invasiveness was also shown for HuH7 cells, but no scattering was observed and cell proliferation was stimulated. All the effects of MF on both tumor cell types were blocked by addition of an antibody to HGF and they all could be reproduced by adding recombinant HGF to the tumor cells. RT-PCR and Western blot analysis confirmed that both tumor cell lines expressed c-met, the receptor for HGF. The effects of MF-conditioned medium were not reproduced by acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), or platelet-derived growth factor (PDGF-BB). Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that HGF was expressed in human HCC. Our data show that human liver MF act on HCC cells to increase their invasiveness and suggest that MF-derived HGF could be involved in the pathogenesis of HCC.
Presence of distinct AP-1 dimers in normal and transformed rat hepatocytes under basal conditions and after epidermal growth factor stimulation
Authors: Nadori F , Lardeux B , Rahmani M , Bringuier A , Durand-Schneider A , Bernuau D
Published: July 2004

Activation of the transcriptional regulator AP-1, a dimeric complex formed of various combinations of Fos and Jun proteins, is a key step in the cellular response to mitogens. Because different dimers are believed to display different regulatory functions, we hypothesized that transformed cells that lack normal growth constraints might display AP-1 dimers that are different from those of normal cells. We therefore compared in primary and transformed rat hepatocytes (1) the composition of AP-1 dimers under basal conditions and (2) AP-1 induction by epidermal growth factor (EGF). Under basal conditions, AP-1 contained predominantly Jun homodimers in both cell types. However, whereas normal hepatocytes contained only JunD, both JunD and JunB were present in the AP-1 complex of 7777 cells. EGF treatment triggered almost identical programs of fos and jun gene activation at the messenger RNA (mRNA) level in both cell types, with an early accumulation of c-fos, c-jun, and junB mRNAs, but no change in junD mRNA levels. In both cell types, c-Fos and Fra-1 proteins increased after EGF treatment, but differences in the induction of Jun proteins were noted, with an increase of c-Jun in hepatocytes and an increase of JunB in 7777 cells. In both cell types, activation of AP-1 DNA binding activity by EGF was accompanied by the recruitment of Fra-1 into AP-1, detected earlier in 7777 cells than in hepatocytes, and with the transient appearance of c-Fos in 7777 cells only. Finally, EGF activated AP-1-dependent transcription in 7777 cells but not in hepatocytes. These data indicate important differences in the functional activity of AP-1 in transformed hepatocytes.
Altered expression of mitogen-activated protein kinases in a rat model of experimental hepatocellular carcinoma
Authors: McKillop I H , Schmidt C M , Cahill P A , Sitzmann J V
Published: July 2004

The mitogen-activated protein kinase (MAPK) cascade acts as a focal point for signal transduction following activation of both G-protein-linked and tyrosine kinase growth factor receptors. A common intermediate between both of these diverse receptor subtypes includes the small guanosine triphosphate (GTP)-binding protein, p21ras. Point mutations of p21ras have been identified in various tumor types and lead to constitutive activation of this protein and subsequent activation of downstream pathways including the MAPK cascade. Using an in vivo model of hepatocellular carcinoma (HCC), we investigated the abundance and function of individual components of the MAPK cascade and the presence of specific p21ras mutations in this model. Expression of components of the MAPK cascade were determined in tumor and adjacent, non-neoplastic liver specimens by Western blot analysis and functional activity confirmed by substrate phosphorylation assays. Mutations in p21ras were analyzed using an enzyme-linked immunosorbent assay. In tumor, extracellular regulated kinases (ERKs) ERK1, ERK2, and mitogen-activated ERK-regulated kinase-1 (MEK1) were elevated by three-to fourfold as compared with adjacent nontumorigenic normal liver. In contrast, MEK2 was elevated by only 28%. Substrate phosphorylation and detection of phosphorylated ERK1/2 proteins showed increased functional activity of these proteins of the same magnitude as that observed for protein expression. Mutations in p21ras were not detected in this experimental model of HCC. We conclude that HCC is associated with marked changes in expression and function of components of the MAPK cascade independent of common p21ras mutations.
Chromosomal aberrations in hepatocellular carcinomas: Relationship with pathological features
Authors: Zimmermann U , Feneux D , Mathey G , Gayral F , Franco D , Bedossa P
Published: July 2004

Fluorescence in situ hybridization performed on tissue sections can reveal chromosomal abnormalities related to histopathological features. This technique was performed on serial frozen sections from seven normal livers and 29 hepatocellular carcinomas (HCCs) using pericentromeric repeat-specific probes for chromosomes 1, 4, 6, 7, 8, 16, and 17. For each HCC and each probe, the percentage of cells showing one, two, or more than two signals was counted and compared with the distribution in the normal liver. According to these results, HCCs were categorized as monosomic, disomic, or polysomic (more than two signals) for the chromosome tested. These data were compared with the main histopathological characteristics of HCC. Chromosome gains were very common, preferentially affecting chromosome 1 (23 of 27 cases, 85%), chromosome 16 (16 of 27 cases, 59%), chromosome 7 (16 of 29 cases, 55%), chromosome 6 (15 of 29 cases, 52%) and chromosome 8 (14 of 29 cases, 48%). Monosomy was seen more rarely, affecting preferentially chromosome 16 (19%), chromosome 17 (14%), and chromosome 4 (10%). A significant correlation was observed between aneusomy of chromosome 4 and tumor size (P < .05) or the presence of vascular embolism (P < .05). In conclusion, chromosomal gains are frequent genetic events in human HCC. A significant association between a gain in chromosome 4 and large tumor size or vascular embolism suggests that this genetic abnormality is a late event in liver carcinogenesis.
Role of Kupffer cells in gut ischemia/reperfusion-induced hepatic microvascular dysfunction in mice
Authors: Horie Y , Wolf R , Russell J , Shanley T P , Granger D N
Published: July 2004

Kupffer cells (KCs) have been implicated in the leukocyte recruitment and microvascular dysfunction associated with liver inflammation. The overall objective of this study was to assess the role of KCs in the leukocyte adhesion and oxidative stress elicited in the liver by gut ischemia/reperfusion (I/R). The accumulation of rhodamine-6G-labeled leukocytes and the number of nonperfused sinusoids (NPS) were monitored (by intravital microscopy) in mouse liver for 1 hour after a 15-minute period of normothermic intestinal ischemia. Autofluorescence of pyridine nucleotide [NAD(P)H] was measured as an index of mitochondrial O2 consumption and redox status. Leukostasis, as well as increases in NPS and NAD(P)H autofluorescence (indicating hypoxia), were observed in the liver at 60 minutes after gut I/R. Pretreatment with gadolinium chloride (GdCl3), which reduces KC function, attenuated the liver leukostasis and NPS elicited by gut I/R. The platelet activating factor (PAF) antagonist, WEB2086, and a tumor necrosis factor (TNF)-α-specific antibody were also effective in attenuating the gut I/R-induced leukostasis and NAD(P)H autofluorescence. The findings of this study suggest that KCs play an important role in mediating the leukocyte recruitment, impaired sinusoidal perfusion, and tissue hypoxia elicited in the liver after gut I/R. These KC-mediated responses appear to involve the participation of both PAF and TNF-α.
Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat
Authors: Iimuro Y , Gallucci R M , Luster M I , Kono H , Thurman R G
Published: July 2004

Tumor necrosis factor (TNF)α, a pivotal cytokine involved in inflammation, is produced primarily by Kupffer cells in the liver. It has been shown that inactivation of Kupffer cells prevents alcohol-induced liver injury; therefore, the purpose of this study was to determine if neutralizing anti-TNF-α antibody is also effective. Male Wistar rats were exposed to ethanol (11 to 12 g · kg-1 · d-1) continuously for up to 4 weeks via intragastric feeding using an enteral feeding model. Before ethanol exposure, polyclonal anti-mouse TNF-α rabbit serum was injected (2.0 mg/kg intravenously). There were no significant differences in body weight, mean ethanol concentration, or cyclic patterns of ethanol in urine when ethanol-and ethanol plus antibody-treated groups were compared. Expression of TNF- α and macrophage inflammatory protein 2 (MIP-2) messenger RNA (mRNA), determined using reverse transcription-polymerase chain reaction, was three- to four-fold higher in livers of ethanol-treated rats than in those of rats fed an ethanol-free, high-fat control diet. In addition, MIP-2 levels were also elevated when detected by Northern blot analysis. Anti-TNF-α antibody did not affect expression of mRNA for interleukin (IL) 1α, IL-6, transforming growth factor β1, or TNF-α. However, MIP-2 mRNA expression, which is regulated by TNF-α, was decreased significantly by anti-TNF-α antibody treatment. Serum aspartate transaminase levels were elevated in ethanol-treated rats to 136 ± 12 IU/L after 4 weeks but only reached 90 ± 5 IU/L (P < .05) in rats treated with anti-TNF-α antibody. The hepatic inflammation and necrosis observed in ethanol-fed rats were attenuated significantly by antibody treatment, and steatosis was not. These results support the hypothesis that TNF-α plays an important role in inflammation and necrosis in alcohol-induced liver injury and that treatment with anti-TNF-α antibody may be therapeutically useful in this disease.
Dietary saturated fatty acids down-regulate cyclooxygenase-2 and tumor necrosis factor alfa and reverse fibrosis in alcohol-induced liver disease in the rat
Authors: Nanji A A , Zakim D , Rahemtulla A , Daly T , Miao L , Zhao S , Khwaja S , Tahan S R , Dannenberg A J
Published: July 2004

We investigated the potential of dietary saturated fatty acids to decrease endotoxemia and suppress expression of cyclooxygenase 2 (Cox-2) and tumor necrosis factor α (TNF-α) in established alcohol-induced liver injury. Six groups (five rats/group) of male Wistar rats were studied. Rats in group 1 were fed a fish oil-ethanol diet for 6 weeks. Rats in groups 2, 3, and 4 were fed fish oil and ethanol for 6 weeks. Ethanol administration was stopped at this time, and the rats were switched to isocaloric diets containing dextrose with fish oil (group 2), palm oil (group 3), or medium-chain triglycerides (group 4) as the source of fat for an additional 2 weeks. Rats in groups 5 and 6 were fed fish oil-ethanol and fish oil-dextrose, respectively, for 8 weeks. Liver samples were analyzed for histopathology, lipid peroxidation, and levels of messenger RNA (mRNA) for Cox-2 and TNF-α. Concentrations of endotoxin were determined in plasma. The most severe inflammation and fibrosis were detected in groups 1 and 5, as were the highest levels of endotoxin, lipid peroxidation, and mRNA for Cox-2 and TNF-α. After ethanol was discontinued, there was minimal histological improvement in group 2 but near normalization of the histology, including regression of fibrosis, in groups 3 and 4. Histological improvement was associated with decreased levels of endotoxin, lipid peroxidation, and reduced expression of Cox-2 and TNF-α. The data indicate that a diet enriched in saturated fatty acids (groups 3 and 4) effectively reverses alcohol-induced liver injury, including fibrosis. The therapeutic effects of saturated fatty acids may be explained, at least in part, by reduced endotoxemia and lipid peroxidation, which in turn result in decreased levels of TNF-α and Cox-2.