bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of nitric oxide in oxygen transport in rat liver sinusoids during endotoxemia
Authors: Huang T , Nishida T , Kamike W , Kosaka H , Seiyama A , Morimoto Y , Tanaka S , Obunai S , Takei Y , Shiga T , Matsuda H
Published: July 2004

To evaluate the role of nitric oxide (NO) in hepatic microcirculation and liver injury during endotoxemia, we studied O2 transport in the hepatic microcirculation of endotoxin-infused rats. Rats were continuously infused with Escherichia coli lipopolysaccharide (LPS) (0.8 mg/kg/h) for 7 hours. LPS increased the plasma levels of NO2- + NO3- and aspartate transaminase (AST), and decreased the bile flow rate and hepatic adenosine triphosphate (ATP) level. Hepatic microcirculation was evaluated by two methods: reflectance spectrophotometry showed a decrease in the oxygenation of hemoglobin (Hb) in the liver, and dual-spot microspectroscopy indicated that LPS administration decreased blood velocity, the oxygenation of Hb, and O2 release from sinusoids to hepatocytes. The observed decreases in the O2 transport parameters were prominent in pericentral sinusoids. All of these phenomena were further aggravated by the administration of N(w)- nitro-L-arginine methyl ester (L-NAME) (5 mg/kg/h) plus LPS, and by aminoguanidine (AMG) (5 mg/kg/h) plus LPS, and these could be reversed by the concomitant administration of L-arginine (L-Arg) (100 mg/kg/h). These results suggest that deterioration of hepatic oxygen transport and liver function induced by endotoxin can be ameliorated by NO.
Effect of side chain length on biotransformation, hepatic transport, and choleretic properties of chenodeoxycholyl homologues in the rodent: Studies with dinorchenodeoxycholic acid, norchenodeoxycholic acid, and chenodeoxycholic acid
Authors: Yeh H , Schteingart C D , Hagey L R , Ton-Nu H , Bolder U , Gavrilkina M A , Steinbach J H , Hofmann A F
Published: July 2004

To assess the effect of side chain length on the metabolism and physiological effects of homologues of chenodeoxycholic acid (CDCA), dinorCDCA, the C22 homologue, was synthesized and its hepatic biotransformation, transport kinetics, and choleretic properties were defined in rat and hamster biliary fistula and in isolated perfused rat liver. Results were compared with those of norCDCA, the C23 homologue, and of CDCA, the natural C24 homologue. In the rat, dinorCDCA was secreted mostly in unconjugated form (the majority as dinor-α- muricholic acid); the remainder was glucuronidated. In the hamster, glucuronidation was greater, and the unconjugated fraction contained equal parts of dinorCDCA and 5β-hydroxy-dinorCDCA. NorCDCA was glucuronidated extensively (70%, rat; 40%, hamster). CDCA, in contrast, was efficiently amidated with taurine or glycine. In the perfused liver, the initial uptake rate of all three homologues was identical; later, regurgitation and/or cholehepatic shunting of dinorCDCA and norCDCA, but not of CDCA, occurred. In rats and hamsters with biliary fistulas, dinorCDCA and norCDCA, but not CDCA, induced a bicarbonate-rich hypercholeresis of canalicular origin. Hypercholeresis was not induced by the taurine conjugate of dinorCDCA. Hepatobiliary retention of both dinorCDCA and norCDCA occurred, consistent with efficient ductular absorption (calculated to be 94%) and cholehepatic cycling of the unmetabolized bile acids. It is concluded that dinorCDCA, as norCDCA, is inefficiently amidated, is metabolized as a xenobiotic, and induces hypercholeresis. DinorCDCA is the first dihydroxy bile acid to be identified that is secreted largely in unconjugated form in bile.
Hepatic mitochondrial proliferation in rats with secondary biliary cirrhosis: Time course and mechanisms
Authors: Forestier M , Solioz M , Isbeki F , Talos C , Reichen J , Krahenbuhl S
Published: July 2004

It is well known that the hepatic mitochondrial protein content is increased in rats 4 weeks after bile duct ligation. In the present study, we measured the time course of this increase and assessed the levels of selected mitochondrial messenger RNA (mRNA) species and the rate of mitochondrial protein synthesis by isolated mitochondria. Three days after surgery, the mitochondrial protein content was not significantly different between bile duct-ligated (BDL) and control rats, averaging 1,140 ± 220 mg/liver in BDL and 1,260 ± 50 mg/liver in sham-operated control rats. However, in comparison with control rats, it was increased in BDL rats by 35% at 7 days, by 81% at 14 days, and by 27% at 28 days after surgery. In vitro mitochondrial protein synthesis, which was assessed as the fractional incorporation of [35S]-methionine into mitochondrial protein, was not different between BDL and control rats at 3 days after surgery, but was decreased in BDL rats by 63% at 7 days, by 55% at 14 days, and by 36% at 28 days after surgery. Northern blot analysis revealed an increase in the mRNA levels of adenosine triphosphate (ATP)ase subunit 6 and apocytochrome b in BDL rats at day 7, but no significant differences between BDL and control rats in mitochondrial mRNA and ribosomal RNA species 14 and 28 days after surgery. These results show that the hepatic mitochondrial protein content rises early after surgery in BDL rats, but this rise cannot be ascribed to elevated rates of mitochondrial protein synthesis. Thus, increased synthesis of nuclearly encoded mitochondrial proteins and/or decreased degradation of mitochondrial proteins appear likely mechanisms that lead to the observed increase in the hepatic mitochondrial protein content in BDL rats.
Assessment of hepatic vitamin E status in adult patients with liver disease
Authors: Nagita A , Ando M
Published: July 2004

No published reports compare hepatic α-tocopherol (adjusted for hepatic lipid content) with indicators of blood α-tocopherol in adult patients with various liver diseases. α-Tocopherol was simultaneously measured in liver biopsy tissues and blood from 66 subjects (9 comparison patients hospitalized for biliary tract surgery, 13 with chronic persistent hepatitis, 9 with chronic aggressive hepatitis, 10 with acute hepatitis, 10 with cirrhosis, 7 with both cirrhosis and hepatic cell carcinoma, and 8 with fatty liver). Hepatic, erythrocyte, and plasma α-tocopherol concentrations were measured, as were hepatic and serum lipids. The ratios of α-tocopherol to total lipid concentrations (Toc/TL ratios) in plasma and liver were calculated. In both comparison patients and patients with chronic persistent hepatitis and fatty liver, hepatic α-tocopherol concentrations were strongly correlated with hepatic triglyceride and total lipid concentrations (r = .72, P < .001; and r = .75, P < .001, respectively); the relationships (slopes) when hepatic α-tocopherol concentrations were compared with hepatic triglyceride and total lipid concentrations were similar in these patients and in all subjects. No strong correlations were found between hepatic and blood α-tocopherol parameters in all subjects. These results suggest that hepatic α-tocopherol is present at similar concentrations in triglycerides as well as total cholesterol and phospholipids and that neither plasma Toc/TL ratios nor erythrocyte α-tocopherol concentrations are useful indicators of hepatic vitamin E status. The hepatic Toc/TL ratio may be useful to assess total hepatic vitamin E status.
Quantitative graphical description of portocentral gradients in hepatic gene expression by image analysis
Authors: Lamers W H , Geerts W J , Jonker A , Verbeek F J , Wagenaar G T , Moorman A F
Published: July 2004

The liver consists of numerous repeating, randomly oriented, more or less cylindrical units, the lobules. Although enzyme-histochemical or microbiochemical assays accurately reflect zonal differences in lobular enzyme content, their results cannot be directly compared to biochemical assays. This is because section-based assays typically sample along a linear portocentral column of cells, even though periportal regions contribute substantially more to hepatic volume than pericentral regions. We have developed a time-efficient approach that depends on image analysis to determine the prevalence of hepatocytes (pixels) with a defined cellular concentration of a particular gene product (absorbance), and that generates a graph with the average absorbance per hepatocyte on the ordinate and the percentage of hepatocytes with absorbances in each of a predetermined range of absorbances incrementally summed on the abscissa. The direction of the gradient is read directly from the section. The gradient is a graphical representation of the two-dimensional distribution pattern of the gene product between the portal tracts and the central veins. The total surface area underneath the resulting graph represents the integrated absorbance and is equivalent to the outcome of a biochemical assay. The typical linear portocentral gradient can be derived from that representing the two-dimensional distribution if we assume that liver lobules are uniformly cylindrical or prismatic. The analysis, therefore, yields a quantitative description of the relation between the enzymatic phenotype of hepatocytes and their position on a normalized portocentral radius. We have used the procedure to compare portocentral gradients of different enzymes in the same liver and of the same enzyme in different livers. In addition, bipolar portocentral gradients of the same enzyme in the same liver were analyzed.
Hepatitis G virus co-infection does not alter the course of recurrent hepatitis C virus infection in liver transplantation recipients
Authors: Cotler S J , Gretch D R , Bronner M P , Tateyama H , Emond M J , dela Rosa C , Perkins J D , Carithers R L
Published: July 2004

Although hepatitis G virus infection (HGV) is usually asymptomatic, it has been associated with mild hepatic injury. Whether hepatitis G co-infection alters the natural history of other viral hepatitis infections remains to be determined. In the present study, we investigated whether hepatitis G impacts on the time to recurrent hepatitis or on the time to progression to fibrosis in hepatitis C-infected patients who undergo liver transplantation. Forty-five liver transplantation recipients with persistent hepatitis C viremia by polymerase chain reaction (PCR) were evaluated. Stored sera obtained before and after liver transplantation was tested for HGV RNA by reverse transcriptase (RT)-PCR using primers to the 5′ region of the HGV genome. A median of eight serial liver biopsy specimens were reviewed per patient. The prevalence of HGV co-infection was 21% before transplantation and 22% following transplantation. During a median follow-up of 29 months, 78% (35/45) of patients with hepatitis C viremia developed histological features of recurrent hepatitis. Fifty-one percent (23/45) progressed to fibrous portal expansion and 16% (7/45) developed bridging fibrosis. Comparisons of patients with and without hepatitis G co-infection following transplantation showed no significant difference in time to recurrent hepatitis, fibrous portal expansion, bridging fibrosis, or of allograft or patient survival. In conclusion, hepatitis G co-infection does not seem to impact on the time to recurrent hepatitis C or progression of hepatitis C-related histological injury after liver transplantation.
Functional assessment of proliferating hepatocytes stimulated by hepatic stimulatory substance in ascorbic acid biosynthetic enzyme-deficient rats
Authors: Nakazawa F , Sawa M , Jiang B , Onodera K , Kasai S , Mito M
Published: July 2004

The functional ability of hepatic stimulatory substance (HSS)-stimulated proliferating hepatocytes was investigated by intrasplenic and/or intraportal transplantation in ascorbic acid (AsA) biosynthetic enzyme-deficient (ODS-od/od) rats that die of osteogenic disorders unless there is AsA supplementation. HSS was extracted from regenerating porcine livers. Hepatocytes isolated from the livers of congeneic ODS-+/+ rats that are capable of synthesizing AsA were transplanted into the spleen (Sp-HTx) and/or the portal vein (Pv-HTx) of ODS-od/od rats. The recipients were divided into eight groups as follows: HSS-untreated groups [group Ia, sham-operated, HTx(-); group IIa, Sp-HTx; group IIIa, Pv-HTx; and group IVa, Sp- and Pv-HTx], HSS-treated groups [group Ib, HSS only; group IIb, Sp-HTx + HSS; group IIIb, Pv-HTx + HSS; and group IVb, Sp- and Pv-HTx + HSS]. The recipients were given a diet and water containing AsA for 6 weeks after HTx, and AsA supplementation was then halted. The average bromodeoxyuridine (BrdU) labeling index (LI) and hepatocyte-occupied ratio in the spleen (H/S ratio) of HSS-treated rats were significantly higher than those of HSS-untreated rats. All the rats in HSS-untreated groups and group Ib died by 8 weeks after the cessation of AsA. In HSS-treated groups IIb, IIIb, and IVb, the survival rates were 60%, 50%, and 80%, respectively, at 16 weeks after HTx. The average serum AsA level of the surviving rats in groups IIb, IIIb, and IVb was significantly higher than that in HSS-untreated groups. These results indicate that HSS treatment induced rapid proliferation of transplanted hepatocytes in the spleen and the portal vein, and that these proliferating hepatocytes synthesized AsA and improved the survival rate of ODS-od/od rats.
The prediction of risk of recurrence and time to recurrence of hepatocellular carcinoma after orthotopic liver transplantation: A pilot study
Authors: Marsh J W , Dvorchik I , Subotin M , Balan V , Rakela J , Popechitelev E P , Subbotin V , Casavilla A , Carr B I , Fung J J , Iwatsuki S
Published: July 2004

Orthotopic liver transplantation (OLTx) in the presence of hepatocellular carcinoma (HCC) has been complicated by high recurrence rates. The ability to determine the risk and timing of HCC recurrence on an individual basis would greatly aid in the candidate selection process resulting in a more efficient use of donated organs and allow the individualization and better evaluation of adjuvant chemotherapy. The 214 patients who underwent OLTx in the presence of HCC were analyzed. From the 178 patients who survived more than 150 days, 71 (40%) have suffered HCC recurrence. Based on five risk factors, that is, gender, tumor number, lobar tumor distribution, tumor size, grade of vascular invasion, artificial neural network models predicting the likelihood of HCC recurrence within 1, 2, and 3 consecutive years after transplantation were developed. Based on model predictions, those combinations of risk factors that should/should not lead to recurrence were generated, allowing stratification of patients into the following three groups: 1) patients who should not suffer HCC recurrence and who should not need adjuvant therapy, 2) patients who will suffer recurrence and for whom postoperative chemotherapy significantly prolonged survival (but did not prevent recurrence), and 3) patients who may or may not suffer HCC recurrence and whose recurrence may be prevented by adjuvant chemotherapy. The outcome of OLTx for patients with HCC can be prognosticated based on a number of clinical variables. If verified through multicenter trials, these models could be made available to transplantation programs performing OLTx in the presence of HCC.
Hepatic expression of inducible nitric oxide synthase transcripts in chronic hepatitis C virus infection: Relation to hepatic viral load and liver injury
Authors: Mihm S , Fayyazi A , Ramadori G
Published: July 2004

Hepatitis C virus (HCV) causes acute and often also chronic liver disease. Hepatocellular injury might result from both a host response directed to inhibit viral spread and from processes initiated by the virus itself. To study mechanisms involved in hepatocellular injury, liver tissue from chronically HCV-infected patients was analyzed for the expression of the inducible isoform of nitric oxide synthase (iNOS) and for interferon γ (IFN-γ) by a quantitative, competitive reverse-transcription-polymerase chain reaction (RT-PCR) technique. Moreover, hepatic viral load was determined by two independent techniques, and liver tissue was evaluated histopathologically in detail. Liver tissue from HCV-infected patients was shown to express elevated levels of iNOS transcripts compared with non-HCV-infected patients. Increased iNOS transcript expression, however, was not accompanied by significantly elevated serum nitrite plus nitrate (NOx) concentration, although some of the chronic HCV-infected patients reached markedly higher serum NOx levels than the control group or healthy individuals, respectively. Hepatic iNOS expression was found to be positively correlated to hepatic HCV-RNA content on the one hand, and weakly to hepatic IFN-γ expression, previously shown to be solely associated with hepatic necro-inflammatory activity among the histopathological parameters studied, on the other hand. In contrast to IFN-γ transcript expression, neither hepatic iNOS expression nor hepatic HCV-RNA content were related to hepatic inflammatory activity. The presented data are compatible with the assumption that HCV might be able to stimulate iNOS expression both directly and indirectly via its capacity to induce IFN-γ.
Histological findings in chronic hepatitis C with autoimmune features
Authors: Czaja A J , Carpenter H A
Published: July 2004

To evaluate the histological findings in patients with chronic hepatitis C and autoimmune features, liver tissue specimens from 60 patients were graded under code for individual features and composite patterns that denoted autoimmune, viral, combined autoimmune and viral, and nondiscriminative changes. Portal, interface, and acinar hepatitis in any combination with plasma cell infiltration connoted an autoimmune pattern that was associated with higher serum levels of γ-globulin (2.4 ± 0.2 g/dL vs. 1.7 ± 0.1 g/dL; P = .0003) and immunoglobulin G (2,211 ± 227 mg/dL vs. 1,508 ± 83 mg/dL; P = .001) than patients with other patterns. Patients with the autoimmune pattern also had a greater frequency of cirrhosis (43% vs. 8%; P = .003), higher mean Knodell score (13.2 ± 0.9 vs. 6.8 ± 0.9; P < .0001), and a greater occurrence of high-titer smooth muscle antibodies (SMA) (13% vs. 0%; P = .05) than patients with other histological findings. HLA DR3 also occurred more frequently in these individuals than in other patients (48% vs. 15%; P = .01) and normal subjects (43% vs. 16%; P = .01). Patients with nondiscriminative patterns and interface hepatitis had clinical findings similar to those with autoimmune patterns, except for a lower mean serum level of γ-globulin. We conclude that the composite histological pattern that resembles autoimmune hepatitis is associated with greater immunoreactivity, inflammatory activity, and disease severity than other patterns. Interface hepatitis may be the most important histological finding associated with these clinical manifestations.
Enlargement of perihepatic lymph nodes in relation to liver histology and viremia in patients with chronic hepatitis C
Authors: Dietrich C F , Lee J , Herrmann G , Teuber G , Roth W K , Caspary W F , Zeuzem S
Published: July 2004

Inflammatory processes in organs frequently lead to hyperplasia of regional lymph nodes. In the present study, we investigated whether lymph node enlargement within the hepatoduodenal ligament may reflect the inflammatory activity within the liver of patients chronically infected with the hepatitis C virus (HCV). In 114 patients with chronic hepatitis C and 49 healthy controls, the total lymph node volume within the hepatoduodenal ligament was prospectively investigated by ultrasound. In patients with chronic hepatitis C, a liver biopsy was taken at the same occasion, and specimens were semiquantitatively evaluated by the histological activity index (HAI). Hepatitis C viremia was assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Genotyping was performed by a reverse hybridization assay. In 104 of 114 patients (91.2%) and in 45 of 49 healthy controls (91.8%), adequate visualization of the region of the hepatoduodenal ligament was achieved by ultrasound. Lymph nodes were detected in all patients with chronic hepatitis C and in 33 of 45 controls. The mean perihepatic lymph node volume in healthy controls (2.2 ± 1.8 mL) was lower than in HCV-infected patients with mild to moderate inflammatory activity, severe inflammatory activity, and patients with cirrhosis (5.8 ± 2.2 mL, 18.1 ± 10.4 mL, and 22.8 ± 18.8 mL, respectively). In patients with HCV-RNA levels of less than 106 copies/mL, the total lymph node volume was 5.8 ± 1.6 mL and was significantly increased in patients with higher viremia (20.3 ± 13.8 mL; P < 10 < −6). No correlation was found between the total lymph node volume within the hepatoduodenal ligament, HCV genotypes, and liver function test results. In conclusion, enlargement of perihepatic lymph nodes in patients with chronic hepatitis C is predictive for the presence of severe inflammatory activity. The mechanism of portal lymphadenopathy in patients with chronic hepatitis is unknown but appears to be related to viral replication within the liver and the immune-mediated inflammatory response of the host.
Tolerance and efficacy of oral ribavirin treatment of chronic hepatitis C: A multicenter trial
Authors: Bodenheimer H C , Lindsay K L , Davis G L , Lewis J H , Thung S N , Seeff L B
Published: July 2004

Hepatitis C is a common cause of chronic liver disease that may progress to cirrhosis. We conducted a multicenter double-blind placebo-controlled trial of ribavirin 600 mg given orally twice daily for 36 weeks with follow-up off therapy for an additional 16 weeks. Fifty-nine patients with compensated chronic hepatitis C were entered. Efficacy was measured at the end of therapy and after follow-up by normalization of alanine aminotransferase (ALT), improvement in liver histology, reduction in hepatitis C virus (HCV) RNA level and improvement of symptoms. Among the ribavirin recipients, 12 of 29 (41.4%) had normal ALT values at 36 weeks compared with only 1 of 30 (3.3%) placebo recipients (P < .001). No patient maintained a normal ALT when therapy was stopped. No significant decrease in level of HCV RNA was observed during the study. Histological improvement among subjects who normalized ALT (-1.67 Knodell index) was significantly greater than that in other treated patients (+0.33 Knodell index; P < .05). Fatigue improved in 19.2% of ribavirin-treated subjects and in 8.3% of placebo recipients whereas no worsening of fatigue was reported by ribavirin recipients compared with 16.7% of controls. This difference in fatigue was significant at weeks 36 and 52 (P < .05; .02, respectively). Adverse events were generally comparable between treatment groups except for a reversible hemolytic anemia experienced by ribavirin recipients. Chest pain was noted in four patients on ribavirin. Ribavirin was well tolerated and improved aminotransferase values and reduced fatigue in patients with hepatitis C viral infection while treatment was being administered. Because this action was produced without change in viral level, the mechanism of action of this agent requires further investigation.
Distribution of hepatitis G viremia and antibody response to recombinant proteins with special regard to risk factors in 709 patients
Authors: Feucht H , Zollner B , Polywka S , Knodler B , Schroter M , Nolte H , Laufs R
Published: July 2004

A new virus named hepatitis G virus (HGV) has been detected recently. Until now, no assays for the detection of antibodies against different HGV proteins have been commercially available. Therefore, a strip immunoblot assay has been established to investigate seroreactivity against recombinant structural (core) and nonstructural proteins (NS3 and NS4) of HGV produced in Escherichia coli. Seropositivity for HGV was evaluated and concordanced with HGV polymerase chain reaction (PCR) results in 709 subjects. These individuals were classified into a nonrisk or a risk group, on the basis of infection with human immunodeficiency virus (HIV) or hepatitis C virus (HCV) or frequent parenteral exposure, including hemophilia, intravenous drug addiction, receipt of blood transfusion, or hemodialysis. The nonrisk group consisted of 257 healthy blood donors with normal alanine transaminase (ALT) levels (ALT < 30 U/L) and 154 patients with suspected non-A-E hepatitis (ALT > 45 U/L). In the group of healthy blood donors, 1.9% (5 of 257) had detectable HGV viremia and 15.9% (41 of 257) showed antibody response to HGV. In the collective of patients with suspected non-A-E hepatitis, results from 1.9% of patients (3 of 154) were positive by HGV PCR, and 15.6% of patients (24 of 154) showed seropositivity against the recombinant HGV proteins. In six groups of patients (n = 298) with different risk factors, the prevalence of both HGV viremia (V) and serological reactivity (SR) was higher compared with that of the nonrisk group: V, 6.80%-35.2%; serological reactivity (SR), 25.4%-52.9%. The following conclusions can be derived from our data. HGV infection is widespread in the general population. The prevalence of antibodies against HGV or detectable HGV viremia is higher in patients with risk factors for parenteral viral transmission than in those without risk factors. The majority of HGV infections (70.2%) is self-limiting and not persistent in our collective of patients. We found no correlation between HGV viremia and clinical or biochemical signs of hepatitis in individuals without risk factors for acquiring parenterally transmitted agents.
Pilot study of the combination of interferon alfa and ribavirin as therapy of recurrent hepatitis C after liver transplantation
Authors: Bizollon T , Palazzo U , Ducerf C , Chevallier M , Elliott M , Baulieux J , Pouyet M , Trepo C
Published: July 2004

Recurrent hepatitis C virus (HCV) in liver transplant patients is a major cause of graft loss, liver failure, and need for retransplantation. The results available to date with the use of interferon alfa (IFN-α) in the treatment of recurrent HCV in liver transplant patients have been disappointing. The aim of this study was to evaluate the efficacy and clinical utility of post-transplant combination therapy with IFNα2b (3 million units 3 times weekly) and oral ribavirin (1,200 mg/d) for a duration of 6 months, followed by maintenance with ribavirin alone for an additional 6 months. Twenty-one liver transplant recipients with recurrent hepatitis C infection (HCV- RNA-positive; active hepatitis without rejection on biopsy) were enrolled in this study. Pretreatment serum alanine transaminase (ALT) levels were at least two times the upper limit of normal. Before treatment, all patients were HCV-RNA-positive and mean HCV-RNA titers were 125 million genome-equivalents/mL. Mean pretreatment histological score was 6.3 ± 2. After 6 months of combination therapy, all 21 patients had normal ALTs. Ten patients (48%) cleared HCV-RNA from their serum, as assessed by polymerase chain reaction (PCR), and HCV-RNA levels decreased significantly in the others (P = .0001). Improvement in histological score was seen in all patients after combination therapy (P = .0013). During maintenance ribavirin monotherapy, ALT remained normal in all but 1 of the 18 patients who tolerated therapy. HCV-RNA reappeared in 5 patients, but HCV-RNA levels did not return to pretreatment levels (P = .0004). Comparison of pretreatment and postribavirin monotherapy liver biopsies revealed improvement in all but 1 of the 18 patients who tolerated ribavirin (P = .0002). Side effects were restricted to anemia, which necessitated cessation of ribavirin therapy in 3 patients. No patient experienced graft rejection during the study period. These results are significantly better than those reported with IFN-α monotherapy. Most importantly, there was a complete absence of graft rejection. These results suggest that the combination of IFN-α and ribavirin is effective in reducing HCV-RNA levels and ameliorating hepatocellular injury in recurrent HCV after liver transplantation, and that maintenance therapy with ribavirin monotherapy can maintain the biochemical and histological response.
A new rat bile ductular epithelial cell culture model characterized by the appearance of polarized bile ducts in vitro
Authors: Sirica A E , Gainey T W
Published: July 2004

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or α-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2′deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.
The role of granulomatous phlebitis and thrombosis in the pathogenesis of cirrhosis and portal hypertension in sarcoidosis
Authors: Moreno-Merlo F , Wanless I R , Shimamatsu K , Sherman M , Greig P , Chiasson D
Published: July 2004

Sarcoidosis often involves the liver with mild elevation of serum enzymes and granulomas histologically. Rarely, chronic cholestasis, portal hypertension, cirrhosis, or nodular hyperplasia may be found. The pathogenesis of the portal hypertension and of the cirrhosis are not understood, in part because large samples of tissue have seldom been described. We describe the clinical and anatomic findings of four patients with sarcoid liver disease in whom the whole livers were available for examination. One patient had cirrhosis, one had diffuse nodular hyperplasia, and two had small regions of parenchymal fibrosis. The first two of these had a history of variceal bleeding and healed portal vein thrombosis. One had chronic cholestasis without cirrhosis. We suggest that the cirrhosis and focal fibrosis were caused by ischemia secondary to primary granulomatous phlebitis of portal and hepatic veins. The portal hypertension in two patients was likely secondary to portal vein thrombosis, because cirrhosis was absent at the onset of variceal bleeding.
Serum autoantibodies in chronic hepatitis C: Comparison with autoimmune hepatitis and impact on the disease profile
Authors: Cassani F , Cataleta M , Valentini P , Muratori P , Giostra F , Francesconi R , Muratori L , Lenzi M , Bianchi G , Zauli D , Bianchi F B
Published: July 2004

Antibodies to nuclei (ANA), smooth muscle (SMA), and liver/kidney microsomes type 1 (anti-LKM1) may occur in chronic hepatitis C. Distinct subspecificities, including ANA with the homogeneous pattern (ANA-H) and SMA with antiactin specificity (SMA-AA), are found in autoimmune hepatitis (AIH). This study was performed to characterize the hepatitis C virus (HCV)-associated autoantibodies and to evaluate their influence on the profile of the disease. Two hundred ninety consecutive patients with chronic hepatitis C and 35 control cases with AIH were screened for autoantibodies by indirect immunofluorescence (IFL) at 1:40 serum dilution. The ANA pattern was defined by IFL on HEp-2 cells and the SMA-AA identified by the presence of at least two of the following elements: 1) SMA(T) or SMA(G) pattern by IFL on kidney sections; 2) XR1 precipitating system by counterimmunoelectrophoresis; or 3) typical pattern by IFL on liver sections from phalloidin-intoxicated rats. ANA, SMA, and anti-LKM1 occurred in 9%, 20%, and 6% of chronic hepatitis C cases, respectively. The overall prevalence of autoantibodies was 30% (87 of 290). Compared with AIH, HCV-associated ANA and SMA exhibited ANA-H and SMA-AA at a lower prevalence (38% vs. 71%, P = .04 and 8% vs. 87%, P < .000001, respectively) and had a lower median titer (1:80 vs. 1:320, P < .001 and 1:40 vs. 1:320, P < .000001, respectively). The concomitant positivity for ANA-H and SMA-AA was detected in none of the HCV cases, but in 46% of AIH sera (P < .000001). Two parameters were independently associated with the autoantibodies in chronic hepatitis C: high alanine transaminase (ALT) serum levels (F = 14.04) and female gender (F = 5.03). At the univariate analysis, patients with autoantibodies had a more severe portal-periportal necroinflammation (median Scheuer's score: 2.05 vs. 1.64, P = .003). The presence of autoantibodies did not influence the response to interferon (IFN). In chronic hepatitis C, serum autoantibodies are common, but their subspecificities are distinct from those occurring in AIH. Whereas the absence of ANA-H and/or SMA-AA does not exclude AIH, the characterization of ANA and SMA may help to discriminate between the two conditions. As compared with the seronegative counterpart, autoantibody-positive chronic hepatitis C is more common in females and exhibits a more severe biochemical and histological activity. The response to IFN therapy, however, is similar.
Antibodies to single-stranded and double-stranded DNA in antinuclear antibody-positive type 1-autoimmune hepatitis
Authors: Czaja A J , Morshed S A , Parveen S , Nishioka M
Published: July 2004

To determine the significance of antibodies to single-stranded (anti-ssDNA) and double-stranded DNA (anti-dsDNA) in antinuclear antibody (ANA)-positive type 1 autoimmune hepatitis, sera from 53 patients were tested by enzyme immunosorbent assay (ELISA) and indirect immunofluorescence using the Crithidia luciliae substrate. Anti-dsDNA were detected in 18 patients (34%) by ELISA and 12 patients (23%) by the Crithidia-based assay. Twenty patients with anti-dsDNA by either assay (38%) had higher serum levels of immunoglobulin G (3971 ± 270 mg/dL vs. 3201 ± 247 mg/dL, P = .05) than seronegative patients. They also had human leukocyte antigen (HLA) DR4 more commonly than other patients (83% vs. 41%, P = .006) and normal subjects (83% vs. 30%, P = .00007). In contrast to patients seropositive by the Crithidia-based assay, those seropositive by ELISA failed corticosteroid therapy more commonly (24% vs. 3%, P = .04). Anti-ssDNA were found in 45 patients (85%) and they did not distinguish patients with different clinical features or outcomes. We conclude that anti-dsDNA are common in ANA-positive type 1 autoimmune hepatitis. HLA DR4 is associated with their production, and seropositivity by ELISA characterizes patients who have a poorer immediate response to corticosteroid treatment. Anti-ssDNA are common but they do not have important clinical implications.
Ki-67 expression during rat liver regeneration after partial hepatectomy
Authors: Gerlach C , Sakkab D Y , Scholzen T , Daβler R , Alison M R , Gerdes J
Published: July 2004

Antibodies to the cell-cycle-associated Ki-67 protein have been widely used for more than a decade as markers of proliferative cells. The prototype antibody Ki-67 reacted only with snap-frozen human tissue, but a novel antibody, MIB-1, was able to detect the Ki-67 antigen in paraffin wax-embedded human tissue. The ability of MIB-5, a novel antibody reactive with the rat equivalent Ki-67 protein, to immunohistochemically detect cycling parenchymal and littoral cells in the regenerating rat liver is reported. Rats underwent a standard two-thirds partial hepatectomy (PH), and groups of three animals were killed at intervals for up to 192 hours after PH. DNA synthesis was monitored by flash labeling with bromodeoxyuridine, and the response was as expected with a significant upsurge in hepatocyte labeling at 16 to 17 hours after PH. On the other hand, MIB-5 labeled a relatively constant percentage of hepatocytes (4%-8%) during the first 16 hours after PH, before a large proportion became labeled, also at 17 hours. The temporal pattern of MIB-5 labeling was similar to that of bromodeoxyuridine labeling, although, as expected, MIB-5 indices were higher. Semiquantification of Ki-67 messenger RNA (mRNA) levels by reverse-transcription polymerase chain reaction showed modest (fourfold to fivefold) increases in abundance during the first 12 hours after PH, but then levels increased dramatically to be at least 15-fold those of intact liver at 36 hours after PH. Much higher than normal levels of Ki-67 mRNA persisted throughout the period of study and even at 96 hours after PH they were still ninefold greater than normal. This study has shown the usefulness of the MIB-5 antibody to monitor proliferation in the rat liver, and furthermore, the pattern of expression of both the mRNA and the protein suggest that the Ki-67 protein, with hitherto unknown function, is more abundant in DNA synthesis and mitosis than in the early or even very late first G1 phase.
Hepatitis B and C virus infection, alcohol drinking, and hepatocellular carcinoma: A case-control study in Italy
Authors: Donato F , Tagger A , Chiesa R , Ribero M L , Tomasoni V , Fasola M , Gelatti U , Portera G , Boffetta P , Nardi G
Published: July 2004

We performed a case-control study to assess the association of hepatocellular carcinoma (HCC) with hepatitis B virus (HBV) and hepatitis C virus (HCV) infection and alcohol drinking. We recruited as cases 172 subjects with an initial diagnosis of HCC, who were admitted to the two major hospitals in the province of Brescia, northern Italy, and 332 subjects, sex-, age-, and hospital-matched, who were admitted to the Departments of Ophthalmology, Dermatology, Urology, Cardiology, and Internal Medicine, as controls. Of the HCC cases, 23.8% were positive for HBsAg and 37.8% for HCV RNA; among the controls, 5.4% were positive for HBsAg and 4.8% for HCV RNA. History of heavy alcohol intake (>80 g of ethanol per day for at least 5 years) was found among 58.1% of the cases and among 36.4% of the controls. The relative risks (RRs) for HBsAg, HCV RNA positivity, and heavy alcohol intake were, respectively: 11.4 (95% confidence interval: 5.7-22.8), 23.2 (95% confidence interval: 11.8-45.7), and 4.6 (95% confidence interval: 2.7- 7.8). Positive interactions (synergisms) between both HBsAg positivity and HCV RNA positivity and heavy alcohol intake were found, suggesting more than additive effects of viral infections and alcohol drinking on the risk of HCC. Infection with HCV genotype 1b showed a higher risk than type 2 (RR = 2.9; 95% confidence interval: 0.9-10), suggesting a major role for the former type in causing HCC. On the basis of population attributable risks (AR), heavy alcohol intake seems to be the single most relevant cause of HCC in this area (AR: 45%), followed by HCV (AR: 36%), and HBV (AR: 22%) infection.
Different changes in expression and function of connexin 26 and connexin 32 during DNA synthesis and redifferentiation in primary rat hepatocytes using a DMSO culture system
Authors: Kojima T , Yamamoto M , Mochizuki C , Mitaka T , Sawada N , Mochizuki Y
Published: July 2004

In the present study, we determined in detail the changes of liver gap junctions, connexin 26 (Cx26), and connexin 32 (Cx32), during DNA synthesis and redifferentiation of hepatocytes in vitro. We used primary rat hepatocytes that expressed the liver gap junction proteins, which were cultured in the medium containing epidermal growth factor (EGF) with 2% dimethylsulfoxide (DMSO) and 10-7 mol/L glucagon (a DMSO culture system), as we previously reported. In the present cultures, almost confluent hepatocytes cultured in the medium containing EGF with 2% DMSO and 10-7 mol/L glucagon, underwent a nearly synchronous wave of DNA synthesis induced by the removal of 2% DMSO and 10-7 mol/L glucagon, and the addition of 10 mmol/L nicotinamide, after which the DNA synthesis was completely re-inhibited by the re-addition of 2% DMSO and 10-7 mol/L glucagon. During stimulation of DNA synthesis, both Cx26 and Cx32 messenger RNA (mRNAs) in hepatocytes transiently increased in the G1 phase and then markedly decreased before the onset of the S phase, while only Cx26 messenger RNA (mRNA) increased slightly in the S/M phase. Furthermore, before the onset of the S phase, a disappearance of both Cx26 and Cx32 immunoreactivities and gap junction plaques were observed. Gap junctional intercellular communication (GJIC), as measured by lucifer yellow, which indicated the function of Cx32, decreased markedly from before the onset of the S phase. GJIC measured by propidium iodide, which indicated the function of Cx26, decreased from before the onset of the S phase and then increased slightly in the S/M phase. During the re-inhibition after the stimulation of DNA synthesis, Cx32 mRNA, but not Cx26 mRNA, rapidly returned to the pretreatment control level. Cx32 immunoreactivity and gap junction plaques also recovered. However, the recovery of GJIC measured by lucifer yellow was later than that of Cx32 expression. These results indicated the different changes of expression and function of Cx26 and Cx32 in the hepatocytes during stimulation and re-inhibition of DNA synthesis. This culture system should be useful as a model in which to study liver gap junctions during hepatocyte growth and differentiation in vitro.
Excess iron induces hepatic oxidative stress and transforming growth factor β1 in genetic hemochromatosis
Authors: Houglum K , Ramm G A , Crawford D H , Witztum J L , Powell L W , Chojkier M
Published: July 2004

Genetic hemochromatosis (GH) is associated with excess iron deposition in hepatocytes, which results in progressive hepatic injury. The pathogenesis of hepatic injury in GH is poorly understood. In this study, we found enhanced oxidative stress in patients with GH, as evidenced by hepatic malondialdehyde (MDA)-protein adducts and by increased oxidatively modified serum proteins. MDA-lysine epitopes and oxidatively modified serum proteins, as well as immunoglobulin G autoantibodies against MDA-lysine epitopes, were increased in untreated GH patients and to a lesser extent in GH heterozygotes compared with normal individuals. These markers of ongoing oxidative stress decreased with phlebotomy treatment in GH patients. In addition, TGF-β1 colocalized with hepatic iron and MDA protein adducts in hepatocytes and sinusoidal cells of hepatic acinar zone 1 and normalized after iron removal. Our data suggest that iron overload increases both lipid peroxidation and TGF-β1 expression, which together could promote hepatic injury and fibrogenesis.
Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis
Authors: Ogata I , Auster A S , Matsui A , Greenwel P , Geerts A , D'Amico T , Fujiwara K , Kessler E , Rojkind M
Published: July 2004

Using a polyclonal antibody raised against a liver stellate cell (LSC) line derived from a rat CCl4-cirrhotic liver, we isolated 14 clones from a complementary DNA library prepared with total RNA extracted from the same cell line, with nucleotide sequences homologous to that of the type I procollagen C-proteinase enhancer protein (PCPE) gene. The longest PCPE insert of 1,530 base pairs contained an open reading frame coding for 468 amino acids. PCPE cDNA recognized by Northern blot a 1.7-kilobase messenger RNA (mRNA) in total RNA extracted from freshly isolated and early passaged LSC, LSC lines derived from normal (NFSC) and cirrhotic (CFSC) rat livers, and various LSC clones derived from CFSC. The expression of PCPE mRNA was increased threefold in CFSC compared with NFSC. PCPE mRNA was not detected in total rat liver, freshly isolated hepatocytes, or endothelial or Kupffer cells. However, the expression of PCPE mRNA was induced in fibrotic livers of rats treated with CCl4. PCPE mRNA expression in LSC was up-regulated by transforming growth factor β1 (TGF-β1) and down-regulated by tumor necrosis factor α (TNF-α), similar to the changes in α1 (1) procollagen mRNA induced by these cytokines. PCPE was not detectable in liver biomatrix proteins obtained from normal liver. However, PCPE was increased in liver biomatrix proteins from cirrhotic livers and was proportional to the amount of collagen. These data suggest that PCPE may play an important role in the processing of type I collagen during liver fibrogenesis, and that TGF-β1 and TNF-α regulate its expression.
Manipulation of glutathione stores in rat hepatic stellate cells does not alter collagen synthesis
Authors: Maher J J , Neuschwander-Tetri B A
Published: July 2004

Hepatic stellate cells are resistant to the fibrogenic effects of lipid hydroperoxides in primary culture. Recent studies from our laboratory suggest that antioxidants, particularly glutathione, play a role in this resistance. We have observed that glutathione accumulates rapidly in stellate cells during primary culture; in the current study, we investigated whether glutathione modulates stellate cell collagen synthesis. Stellate cells from normal rat liver were plated in primary culture and maintained for 7 days. From day 4 through day 7, the cells were treated with L-buthionine sulfoximine (BSO) to deplete glutathione stores. BSO profoundly diminished stellate cell glutathione but had no effect on morphology, viability, or basal levels of collagen synthesis and gene expression. When cultured stellate cells were incubated with the putative fibrogenic mediator 4-hydroxynonenal or iron/ascorbate, little or no increase in collagen synthesis occurred regardless of glutathione content. In contrast, iron/ascorbate induced collagen synthesis by cultured fibroblasts. The data indicate that stellate cells strongly resist oxidant- and lipid peroxide-induced collagen synthesis in primary culture. They demonstrate that the mechanism of this resistance does not involve glutathione.