bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterozygosity for hereditary hemochromatosis is associated with more fibrosis in chronic hepatitis C
Authors: Smith Belinda C. , Grove Jane , Guzail Muna A. , Day Christopher P. , Daly Ann K. , Burt Alastair D. , Bassendine Margaret F.
Published: July 2004

Hepatic iron has been associated with more aggressive liver disease in chronic viral hepatitis. We evaluated whether the recently described C282Y mutation of the hemochromatosis gene, designated HFE (responsible for at least 83% of hereditary hemochromatosis), was associated with more advanced liver disease in chronic hepatitis C. One hundred thirty-seven patients with biopsy-proven chronic hepatitis C were studied and liver biopsies scored for necroinflammation (grade 0-18) and fibrosis (stage 0-6). Genomic DNA was amplified by polymerase chain reaction and the C282Y mutation identified by restriction with RsaI and electrophoretic separation of restriction fragments. Ten (7.3%) patients had the C282Y mutation. No C282Y homozygous patients were identified. Age, sex distribution, and estimated weekly alcohol consumption were not significantly different between those with and without the mutation. Serum ferritin was higher in the heterozygotes (mean, 339 μg/L) compared with homozygous wild types (153 μg/L; P = .0005). In the majority of patients, liver iron was graded 0 out of 4, but hepatocyte iron staining was more commonly present in heterozygotes compared with homozygous normals (30% compared with 4% [P = .02]). Liver disease was more advanced in those with the mutant allele (mean fibrosis stage: 3.6, compared with wild type: 1.5 [P = .01]). Cirrhosis was found more often in those with the mutation (40%) compared with those without (8.7%) (P = .01; odds ratio: 7.6 [1.9-31.2]). There was no significant difference in inflammation scores between heterozygotes and wild type (mean, 5.4 compared with 4.1). Hepatitis C virus (HCV)-RNA titers were measured by branched DNA assay (HCV RNA 2.0-Chiron), and there was no difference between heterozygous and homozygous normal patients. Thus, despite relatively minor increases in iron stores, individuals who are heterozygous for hemochromatosis appear to develop more fibrosis in chronic hepatitis C. Venesection may be useful therapy in this subgroup.
Hepatitis D viremia following orthotopic liver transplantation involves a typical HDV virion with a hepatitis B surface antigen envelope
Authors: Smedile Antonina , Casey John L. , Cote Paul J. , Durazzo Marilena , Lavezzo Bruna , Purcell Robert H. , Rizzetto Mario , Gerin John L.
Published: July 2004

Patients receiving orthotopic liver transplantation (OLT) because of type D hepatitis frequently exhibit what appears to be an autonomous, or “isolated,” hepatitis D virus (HDV) infection following the transplantation, with no evidence of hepatitis B virus (HBV) in the graft or in the serum. These observations have led to the hypothesis that HBV might not always be required for HDV infection, or that HDV could exist as a latent infection until rescued by HBV. Alternatively, an apparently autonomous HDV infection could be explained by coinfection of a small number of hepatocytes with both viruses following transplantation, with a very low level of HBV expression that supports low-level HDV propagation. Our results are consistent with the latter hypothesis. Sensitive polymerase chain reaction (PCR)-based analysis of HBV and HDV viremia in transplantation patients with HDV infection previously characterized as isolated showed that HDV viremia was not independent of HBV viremia. Additional analyses, including PCR amplification, buoyant density analysis in a CsCl gradient, and immunoprecipitation with monoclonal hepatitis B surface antigen antibodies (anti-HBs), indicated that the posttransplant HDV particle is typical: it contains full-length HDV RNA and an envelope of hepatitis B surface antigen (HBsAg) and is not different from that found during the acute and chronic stages of HDV superinfection or coinfection. Moreover, an experimental test of the first hypothesis in chimpanzees did not support the idea that HDV can persist for several weeks as an isolated, latent infection that can be rescued subsequently by HBV. The data indicate, therefore, that latent HDV infection is not a factor in OLT recipients. We conclude that the HDV virion in the posttransplantation setting is typical, and that HDV viremia following OLT requires the helper function of HBV infection.
Molecular basis for persistent hepatitis B virus infection in the liver after clearance of serum hepatitis B surface antigen
Authors: Mason Andrew L. , Xu Lizhe , Guo Linsheng , Kuhns Mary , Perrillo Robert P.
Published: July 2004

Hepatitis B virus (HBV) DNA has been detected by polymerase chain reaction (PCR) in the liver of patients with resolved chronic HBV infection and sustained clearance of hepatitis B surface antigen (HBsAg) from serum. However, it is unknown whether the virus is transcriptionally active at this time or if the covalently closed circular (CCC) replicative intermediate of HBV DNA can still be detected. Therefore, hepatic nucleic acid extracts from seven patients who had cleared serum HBsAg were assessed by (PCR) for either reverse-transcribed HBV RNA, or an intact direct repeat region of the HBV genome indicative of the CCC replicative intermediate of HBV DNA. HBV transcripts were detected in four of seven patients in the study group, whereas an intact direct repeat region of the HBV genome was detected in three. Evidence for viral transcription and replication was more frequently detected in patients who had recently cleared serum HBsAg, but HBV RNA was also detected in one patient 5 years after HBsAg clearance, and an intact direct repeat region of HBV DNA was detected in another subject at nearly 4 years after resolution of disease. Therefore, hepatic HBV transcription may be associated with replicative intermediates of persistent HBV DNA in patients who have cleared HBsAg from serum, suggesting that, on occasion, HBV may not be in a latent state but undergoing low-level replication.
Chronic cholestasis in rats induces anhedonia and a loss of social interest
Authors: Swain Mark G. , Le Tai
Published: July 2004

Central fatigue commonly occurs in patients with primary biliary cirrhosis (PBC) and correlates closely with depression, and cholestatic rats exhibit central fatigue. Therefore, we undertook a series of experiments in both rats with cholestasis caused by bile duct resection (BDR) and sham-resected controls (15 days after surgery) to determine if experimental cholestasis is associated with symptoms of depression that can be modeled in rats, namely anhedonia (loss of pleasure) and the loss of social interest. BDR rats exhibited significant anhedonia compared with sham controls as indicated by a loss in their preference for consuming a saccharin solution, a highly desirable drink for rats. Furthermore, social interest was examined by determining the time BDR or sham rats spent investigating a juvenile rat in an open-field apparatus compared with the time spent on nonsocial behaviors. BDR rats exhibited significantly reduced time spent in social investigation and significantly more time in nonsocial behaviors than did sham rats. Major depression in humans is often associated with elevated circulating glucocorticoid levels and impaired glucocorticoid feedback. Therefore, we measured these parameters in BDR and sham rats and found a striking elevation in circulating glucocorticoid levels in BDR compared with sham animals. However, elevated circulating glucocorticoid levels in BDR rats suppressed normally in response to exogenous dexamethasone, indicating intact glucocorticoid feedback control at the pituitary level in BDR rats. In summary, we have identified behaviors in cholestatic rats that are consistent with those seen in depression.
The diagnostic and predictive value of ascites nitric oxide levels in patients with spontaneous bacterial peritonitis
Authors: Garcia-Tsao Guadalupe , Angulo Paul , Garcia Juan Carlos , Groszmann Roberto J. , Cadelina Gregory W.
Published: July 2004

Nitric oxide (NO) is a messenger molecule involved in pathogen suppression. Cirrhosis is characterized by an increased risk for infections, including spontaneous bacterial peritonitis (SBP). The role of NO in the infections that develop in cirrhosis has not been clearly established. The aim of this study was to investigate the utility of measuring ascites NO in the diagnosis of SBP and/or in determining the predisposition of cirrhotic patients to develop this infection. Nitric oxide metabolites (nitrites + nitrates [NOx]) were measured by chemiluminescence in 105 ascites samples obtained from 87 cirrhotic patients and in 87 simultaneously obtained serum samples. Ascites NO levels were not significantly different among ascites from patients with SBP (n = 39; median, 48 μmol/L), patients with sterile ascites (n = 54; median, 42 μmol/L), and samples obtained after patients with SBP had been treated (n = 12; median, 62 μmol/L). No differences in ascites NO levels were observed between culture-positive and culture-negative peritonitis. Among 50 patients with sterile ascites on initial paracentesis, 7 patients developed peritonitis during follow-up; no differences in baseline NO levels were observed between patients who developed peritonitis (median, 46 μmol/L) and those who did not (median, 41 μmol/L). Among patients with SBP, mortality was significantly higher in those with NO levels >60 μmol/L. A very significant direct correlation was found between ascites and serum NO levels (r
2 = .86). In conclusion, ascites NO levels in cirrhotic patients are not useful either to diagnose or to determine predisposition to SBP. Rather, ascites NO levels reflect serum levels, are higher in cirrhotic patients with more severe liver disease, and may be a useful prognostic marker.
Development of pseudointima and stenosis after transjugular intrahepatic portasystemic shunts: Characterization of cell phenotype and function
Authors: Sanyal Arun J. , Contos Melissa J. , Yager Dorn , Zhu Ya-nan , Willey Amy , Graham Martin F.
Published: July 2004

The clinical utility of transjugular intrahepatic portasystemic shunts (TIPS) is frequently complicated by the ingrowth of tissue into the stent lumen, causing stent stenosis. These studies were undertaken to define the cellular and matrix components of the pseudointima, define the phenotype and function of the mesenchymal cells in the pseudointima and maintain them in culture, and to study the differences between stenotic and nonstenosed stents. A total of 35 stents were evaluated. TIPS pseudointima were examined histologically, by immunohistochemistry and in situ hybridization to determine the cellular and connective tissue constituents. Mesenchymal cells were grown from tissue within the TIPS and around it, and their phenotype was studied and compared with control smooth muscle cells and fibroblasts. Masson’s trichrome staining of histological sections demonstrated that TIPS tissue was composed of collagen and palisades of mesenchymal cells and was lined by an endothelium. Immunostaining demonstrated strong and uniform α-smooth muscle staining in TIPS mesenchymal cells and peri-TIPS cells. Type I procollagen mRNA expression was demonstrated in mesenchymal cells in and around the stent by in situ hybridization. TIPS mesenchymal cells secreted less radiolabeled fibronectin, and far more type III, relative to type I, collagen compared with peri-TIPS cells. TIPS cells also expressed high levels of type III procollagen mRNA compared with peri-TIPS cells. There was no difference between stenotic stents and nonstenosed stents with respect to clinical features, time from stenting, gross morphology, histology, presence of bile fistulae, and cell phenotype. However, smooth muscle cells (SMC) from stenotic stents demonstrated both greater cell proliferation and collagen I and III secretion compared with those from nonstenosed stents. These data demonstrate that TIPS stenosis results from an accumulation of collagen and proliferation of SMC within the stent lumen.
Subclinical hepatic encephalopathy impairs daily functioning
Authors: Groeneweg Michael , Quero Juan C. , De Bruijn Ilone , Hartmann Ieneke J.C. , Essink-bot Marie-louise , Hop Wim C.J , Schalm Solko W.
Published: July 2004

Subclinical hepatic encephalopathy (SHE) is assumed to have a negative effect on patients’ daily functioning; therefore, treatment is recommended. However, no studies have been performed that document the clinical relevance of SHE. We performed a study in which the prevalence of SHE was determined in 179 outpatients with cirrhosis using two psychometric tests (Number Connection Test Part A [NCT-A] and the Digit Symbol Test [DGT]) and automated analysis of the electroencephalogram (EEG). SHE was defined by the presence of at least one abnormal psychometric test and/or abnormal slowing of the EEG. The influence of cirrhosis and SHE on patients’ daily functioning was assessed using the Sickness Impact Profile (SIP) questionnaire. The distribution of SIP scores of the patients with cirrhosis differed from the reference scores of the general population. Patients with cirrhosis and SHE (n = 48) reported significantly more impairment in all 12 scales of the SIP, in the psychosocial subscore, the physical subscore, as well as in the total SIP score, compared with cirrhotic patients without SHE (n = 131). Multivariate analysis taking into account severity of liver disease (Child-Pugh score), presence of varices, and alcoholic etiology, showed that SHE independently was related to a diminished total SIP score. The reproducibility of the SIP was high when the test was repeated after a 3-month period. We conclude that SHE implies impaired daily functioning and warrants attempts at treatment.
Sorbitol as a test substance for measurement of liver plasma flow in humans
Authors: Keiding Susanne , Engsted Eva , Ott Peter
Published: July 2004

The objective of this study was to evaluate whole-body removal kinetics of sorbitol, the use of extrarenal sorbitol clearance to estimate hepatic plasma flow in humans, and to compare measurements of liver flow by Fick’s principle using either indocyanine green (ICG) or sorbitol. A sorbitol bolus (5 mmol/kg) was given intravenously to 6 controls for determination of sorbitol elimination capacity (SEC)and distribution volume, Vd
sorb
. Sorbitol infusion (287 μmol/min) was given to 17 liver patients and 11 controls. Extrarenal sorbitol clearance (0Vx
sorb
) was calculated as infusion rate (corrected for renal excretion and accumulation in Vd
sorb
) divided by arterial concentration. Liver flow(QICG
) was calculated from the ICG infusion and arterial and hepatic venous ICG concentrations by Fick’s principle. Average SEC was 73 μmol/min/kg, Vd
sorb
was 0.16 L plasma per kilogram, and in vivo
Vd
sorb
was 3 mmol/L. Renal sorbitol excretion rate was 0.03 to 0.31 of infusion rate. Extrahepatic extrarenal removal was not significantly different from zero but varied considerably. Hepatic extraction fraction of sorbitol,(Esorb
), measured by liver vein catheterization, was 0.35 to 1.04(median, 0.86)in cirrhotic patients and 0.90 to 0.98 (0.86) in controls.The requirements for using Clx
sorb
as an estimate of QICG
was not violated by the data in controls, Clx
sorb
/QICG
0.70 to 1.55 [median, 1.08]), whereas there was a systematic underestimation in cirrhotic patients (0.72-1.08 [0.85]). Liver flow calculated by Fick’s principle using either sorbitol or ICG agreed well. Esorb
>E
ICG in each individual except one. Curvilinear relationship between Esorb
and EICG
was in agreement with different kinetic parameters for sorbitol and ICG, and did not require additional assumption of intrahepatic shunts.
Interleukin-8 and hIRH (SDF1-α/PBSF) mRNA expression and histological activity index in patients with chronic hepatitis C
Authors: Shimoda Katsuhiro , Begum Nasim Ara , Shibuta Kenji , Mori Masaki , Bonkovsky Herbert L. , Banner Barbara F. , Barnard Graham F.
Published: July 2004

Recombinant human intercrine reduced in hepatomas (hIRH)/stromal cell-derived factor 1 (SDF1-α)/pre-B-cell growth-stimulating factor (PBSF), a new chemokine, exhibits an in vitro chemotaxis to neutrophils and a mixed in vivo chemotactic activity to neutrophils, lymphocytes, and monocytes in a rat intradermal injection model. We have investigated the messenger RNA (mRNA) expression of interleukin-8 (IL-8) and hIRH, in chronic hepatitis C of differing severity. Levels of expression of IL-8 and hIRH mRNA obtained from 37 human liver biopsy samples were measured by reverse-transcription and semiquantitative polymerase chain reaction (RT-PCR) amplification. We examined the correlation between mRNA expression and components of the histological activity index (HAI). Patients with HAI ≥ 8 had a significantly higher corrected IL-8 mRNA expression ratio (0.24 ± 0.13 [mean ± SD]; n = 20) than those with HAI ≤ 7 (0.05 ± 0.03; n = 17; P< .0001). Additionally, IL-8 mRNA expression was strongly associated with the severity of portal inflammation (PI) (high PI vs. low PI, 0.22 ± 0.14 vs. 0.05 ± 0.04; P < .0001) and with the presence of bile duct lesions (0.29 ± 0.15 vs. 0.11 ± 0.1; P < .01). In contrast, hIRH mRNA expression was not associated with the total HAI, any components of the HAI, or bile duct inflammation or injury. These results suggest that hIRH, although having the -CXC-, alpha chemokine motif, and exhibiting in vivo and in vitro inflammatory activity as does IL-8, plays a different role from IL-8 in hepatic inflammation and injury. IL-8 expression is directly associated with inflammation in patients with chronic hepatitis C, while hIRH expression does not correlate with histopathological severity of inflammation.
Protein phosphatase inhibition in normal and keratin 8/18 assembly-incompetent mouse strains supports a functional role of keratin intermediate filaments in preserving hepatocyte integrity
Authors: Toivola Diana M. , Omary Bishr M. , Ku Nam-on , Peltola Olli , Baribault Hélène , Eriksson John E.
Published: July 2004

The function and regulation of keratin 8 (K8) and 18 (K18), intermediate filament (IF) proteins of the liver, are not fully understood. We employed the liver damage induced by microcystin-LR (MC-LR), a liver-specific inhibitor of type-1 and type-2A protein phosphatases, in normal and in keratin assembly-incompetent mouse strains as a model to elucidate the roles of IF phosphorylation in situ. The mouse strains used were wild-type (wt) mice and mice with abnormal filament assembly, caused by a targeted null mutation of the K8 gene or caused by expression of a point-mutated dominant negative human K18. In vivo
32P-labeled wt mice, subsequently injected with a lethal dose of MC-LR, showed hyperphosphorylation, disassembly, and reorganization of K8/K18, in particular K18, indicating high phosphate turnover on liver keratins in situ. At lethal doses, the keratin assembly-incompetent mice displayed liver lesions faster than wt mice, as indicated histopathologically and by liver-specific plasma enzyme elevations. The histological changes included centrilobular hemorrhage in all mouse strains. The assembly-incompetent mice showed a marked vacuolization of periportal hepatocytes. Indistinguishable MC-LR-induced reorganization of microfilaments was observed in all mice, indicating that this effect on microfilaments is not dependent on the presence of functional K8/K18 networks. At sublethal doses of MC-LR, all animals had the same potential to recover from the liver damage. Our study shows that K8/K18 filament assembly is regulated in vivo by serine phosphorylation. The absence or occurrence of defective K8/K18 filaments render animals more prone to liver damage, which supports the previously suggested roles of keratin IFs in maintenance of structural integrity.
Effects of diosgenin, A plant-derived steroid, on bile secretion and hepatocellular cholestasis induced by estrogens in the rat
Authors: Accatino Luigi , Pizarro Margarita , Solís Nancy , Koenig Cecilia S.
Published: July 2004

Increased biliary secretion of cholesterol and lipid vesicles (unilamellae and multilamellae) induced by diosgenin (D), a plant-derived steroid, has cytoprotective effects in the rat liver subjected to obstructive cholestasis. In this study, our aims were to investigate the following: 1) the effects of D on the bile secretory process and on the cholestasis induced by estradiol-17β -(β -d -glucuronide) (E17G) or 17 α-ethynylestradiol (E) administration; 2) whether the potentially protective effects of D are related to D-induced increase of biliary cholesterol and lipid lamellae; and 3) whether D has other effects capable of modifying specific bile secretory processes or preventing the cholestatic effects of estrogens. Rats were fed a standard ground chow (control group) or chow containing D for 6 days. E17G was administered i.v. to control and D-fed rats and bile flow, bile salt output, and alkaline phosphatase excretion were examined. 17α-E was administered from days 4 to 6 to rats fed standard chow or chow plus D for 6 days and different functional parameters of the bile secretory process as well as the ultrastructure of hepatocytes and histochemistry of alkaline phosphatase and Mg2+-adenosine triphosphatase (ATPase) were examined. D-treatment markedly increased cholesterol and lamellar structures in bile and attenuated the acute cholestatic effects of E17G. D-feeding prevented the decrease of taurocholate maximum secretory rate and the increase of biliary alkaline phosphatase and Ca2+,Mg2+-EctoATPase (EctoATPase) excretion, as well as the increase of cholesterol/phospholipids ratio, alkaline phosphatase activity, and EctoATPase content in canalicular plasma membranes induced by E. D-feeding did not prevent E-induced decrease of basal bile flow, bile salt, cholesterol, and phospholipid secretory rates nor the decrease of Na+,K+-ATPase activity and Na+-taurocholate cotransporting polypeptide (Ntcp) content in isolated sinusoidal membranes. Cholestatic alterations of canalicular domain were apparent in E-treated rats. D administration was also associated with changes of ultraestructure and histochemistry of hepatocytes. E-induced alterations in ultrastructure and acinar distribution and intensity of histochemical reaction of both enzymes were partially prevented by D-feeding. We conclude that D administration, in addition to inducing a marked increase of biliary cholesterol and lipid lamellar structures output, was associated to changes in hepatocyte morphology and plasma membrane composition, enzymes activity, and histochemistry. D-feeding attenuated the acute cholestatic effects of E17G. D-induced increase of bile cholesterol and lipid lamellae content was not apparent when D-fed rats received E. Despite this fact, D administration prevented some cholestatic effects of E, probably through different metabolic effects and/or direct membrane effects, not related to increased lipid lamellae excretion.
Insulin resistance in cirrhosis: Prolonged reduction of hyperinsulinemia normalizes insulin sensitivity
Authors: Petrides Alexander S. , Stanley Timmye , Matthews Dwight E. , Vogt Christoph , Bush Andrew J. , Lambeth Helen
Published: July 2004

Insulin resistance is present in nearly all patients with cirrhosis, but its etiology remains unknown. Chronic hyperinsulinemia has been suspected as a potential candidate, and we therefore tested the hypothesis that, in cirrhosis, prolonged reduction of the hyperinsulinemia restores insulin sensitivity. Whole-body insulin sensitivity (euglycemic insulin-clamp technique), glucose turnover (6,6-2H2-glucose isotope dilution), glucose oxidation (indirect calorimetry), non-oxidative glucose disposal, and fractional glycogen synthase activity in muscle (biopsies) were measured in eight clinically stable patients with cirrhosis before and at the end of a 4-day continuous subcutaneous infusion of the somatostatin-analogue octreotide (200 μg/24 h) designed to continuously reduce plasma insulin levels. Baseline data were compared with results obtained in healthy individuals matched for sex, age, and weight (n = 8). During the baseline (pre-octreotide) study, patients demonstrated a significant decrease in insulin-mediated glucose uptake compared with controls (5.75 ± 0.21 vs. 7.98 ± 0.84 mg/kg/min;P < .03), which was entirely accounted for by an impairment in non-oxidative glucose disposal (P < .04). Four-day infusion of octreotide to cirrhotic patients: 1) reduced postabsorptive and meal-stimulated plasma insulin levels by ≈35% to 45% without significantly affecting glucose tolerance; 2) did not significantly alter plasma free fatty acids (FFA), growth hormone, and glucagon levels in the postabsorptive state and during the meal test; 3) normalized insulin-mediated whole-body glucose disposal (7.63 ± 0.72 mg/kg/min post-octreotide; P = not significant vs. control). Restoration of insulin-mediated glucose utilization was entirely caused by normalization of non-oxidative glucose disposal; 4) was associated with a considerably more pronounced stimulation by insulin of the fractional glycogen synthase in muscle compared with pre-octreotide results (increment above baseline pre: 0.035 ± 0.010 vs. post: 0.060 ± 0.023 nmol/min/mg protein; P < .04). Fractional glycogen activity significantly correlated with non-oxidative glucose disposal during insulin infusion (r = .69; P < .03). Prolonged reduction of hyperinsulinemia for 96 hours in cirrhotic patients normalizes insulin-mediated glucose uptake and glycogen synthesis in muscle. We conclude that chronic hyperinsulinemia causes insulin resistance in cirrhosis.
The polarized hepatic human/rat hybrid WIF 12-1 and WIF-B cells communicate efficiently in vitro via connexin 32-constituted gap junctions
Authors: Chaumontet Catherine , Mazzoleni Giovanna , Decaens Catherine , Bex Valérie , Cassio Doris , Martel Paule
Published: July 2004

Gap junction intercellular communication (GJIC) plays an essential role in the control of growth, differentiation, and functions of different tissues. The expression of connexins (Cxs), the structural proteins of gap junctions, is developmentally regulated and tissue-specific In vivo hepatocytes express Cx32 and Cx26. Most currently available in vitro hepatic cell systems express Cx43 instead of the expected Cxs. This work analyzes the GJIC competence and Cx expression of the highly differentiated and polarized hepatoma-derived hybrid cell lines, WIF 12-1 and WIF-B. It shows (using two dye transfer assays) that both lines communicate efficiently and that the acquisition of GJIC competence precedes the formation of bile canaliculi. Interestingly, these cells communicate via Cx32 expression, whereas Cx26 and Cx43 are not expressed, as demonstrated by Western and Northern blotting, immunocytochemistry, and confocal microscopy. The human fibroblast WI38 parent communicates via Cx43, whereas the rat hepatoma parent Fao and the subclone WIF 12-1 TGδ, that has lost the human X chromosome, do not communicate, the expression of Cx32 being restricted to the mRNA in these two lines. The GJIC competence of WIF cells could thus result from the activation of the human X chromosome-linked Cx32 gene.
Antiviral activity and toxicity of fialuridine in the woodchuck model of hepatitis B virus infection
Authors: Tennant Bud C. , Baldwin Betty H. , Graham Lou Ann , Ascenzi Mary A. , Hornbuckle William E. , Rowland Peter H. , Tochkov Ilia A. , Yeager Amy E. , Erb Hollis N. , Colacino Joseph M. , Lopez Carlos , Engelhardt Jeffery A. , Bowsher Ronald R. , Richardson Frank C. , Lewis William , Cote Paul J. , Korba Brent E. , Gerin John L.
Published: July 2004

Woodchucks were used to study the antiviral activity and toxicity of fialuridine (FIAU; 1,-2′deoxy-2′fluoro-1-β-d-arabinofuranosyl-5-iodo-uracil). In an initial experiment, groups of six chronic woodchuck hepatitis virus (WHV) carrier woodchucks received daily doses of FIAU by intraperitoneal injection for 4 weeks. At 0.3 mg/kg/d, the antiviral effect was equivocal, but at 1.5 mg/kg/d, FIAU had significant antiviral activity. No evidence of drug toxicity was observed during the 4-week period of treatment or during posttreatment follow-up. In a second experiment, groups of nine WHV carriers or uninfected woodchucks were given 1.5 mg/kg/d of FIAU orally for 12 weeks, and the results compared with placebo-treated controls. After 4 weeks, the serum WHV-DNA concentration in the FIAU-treated carrier group was two to three logs lower than that in the placebo-treated group. After 12 weeks of FIAU treatment, serum WHV DNA was not detectable by conventional dot-blot analysis, hepatic WHV-DNA replicative intermediates (RI) had decreased 100-fold, and hepatic expression of WHV core antigen was remarkably decreased. No evidence of toxicity was observed after 4 weeks, but, after 6 to 7 weeks, food intake decreased and, after 8 weeks, the mean body weights of woodchucks treated with FIAU were significantly lower than controls. Anorexia, weight loss, muscle wasting, and lethargy became progressively severe, and all FIAU-treated woodchucks died or were euthanized 78 to 111 days after treatment began. Hepatic insufficiency (hyperbilirubinemia, decreased serum fibrinogen, elevated prothrombin time), lactic acidosis, and hepatic steatosis were characteristic findings in the final stages of FIAU toxicity in woodchucks. The syndrome of delayed toxicity in woodchucks was similar to that observed previously in humans treated with FIAU, suggesting that the woodchuck should be valuable in future investigations of the molecular mechanisms of FIAU toxicity in vivo and for preclinical toxicological evaluation of other nucleoside analogs before use in patients.
Cytokine and hepatitis B virus DNA Co-immunizations enhance cellular and humoral immune responses to the middle but not to the large hepatitis B virus surface antigen in mice
Authors: Geissler Michael , Schirmbeck Reinhold , Reimann Jörg , Blum Hubert E. , Wands Jack R.
Published: July 2004

Genetic immunization is a potentially useful strategy to prevent or treat hepatitis B virus (HBV) infection. We have previously shown that HBV envelope proteins are highly immunogenic using this technique. The large envelope protein (LHBs), however, induced significantly weaker humoral and cellular immune responses when compared with the middle envelope protein (MHBs). We studied the effect of co-immunizations with cytokine DNA expression constructs encoding for interleukin (IL)-2 and (GM-CSF) on the immunogenicity of LHBs at the B-and T-cell level. Co-immunizations of mice with plasmids encoding for MHBs and IL-2 or GM-CSF increased anti-HBs responses, helper T-cell proliferative activity, and cytotoxic T lymphocyte (CTL) killing. In contrast, co-immunizations of plasmids encoding for LHBs and IL-2 or GM-CSF had no effect on humoral and cellular immune responses. LHBs did not inhibit the production or secretion of IL-2 and GM-CSF. In addition, IL-2, tumor necrosis factor alfa (TNF-α), and interferon gamma (IFN-γ) had no suppressive effect on HBV envelope protein expression in vitro. Based on these data, MHBs, but not LHBs, genetic immunization can be augmented by IL-2 or GM-CSF cytokines.
Hepatitis C virus persistence in human hematopoietic cells injected into SCID mice
Authors: Bronowicki Jean-Pierre , Loriot Marie-Anne , Thiers Valérie , Grignon Yves , Zignego Anna Linda , Bréchot Christian
Published: July 2004

The issue of infection of peripheral blood mononuclear cells (PBMC) by the hepatitis C virus (HCV) has potentially important implications, but is still debated. We have used the severe combined immunodeficiency (SCID) mouse model to test for the persistence of HCV in PBMC. Hematopoietic cells isolated from 14 subjects infected by HCV were inoculated intraperitoneally into SCID mice. Serum and blood cell samples from these mice were obtained with a mean follow-up of 8 weeks. As controls, human fibroblasts and sheep PBMC, preincubated with a human HCV-positive serum, were inoculated concomitantly into mice and analyzed. HCV-RNA positive strands were detected in 7 of 26 serum samples and 8 of 26 cell fractions from SCID mice inoculated with HCV-positive PBMC, after 8 weeks of follow-up. In contrast, no HCV RNA was detectable in the 10 control mice. HCV-RNA negative strands were detected in only 2 of 10 tested samples from 2 mice, and both positive mice had been inoculated with PBMC from HCV-positive subjects with malignant hematopoietic syndrome. Our study offers strong evidence for the persistence of HCV infection in mononuclear cells. Our results are also consistent with a low rate of HCV multiplication. This SCID mouse model might therefore be useful in analyzing the mechanisms of HCV persistence in mononuclear cells.
Effects of long-term ursodeoxycholate administration on expression levels of secretory low-molecular-weight phospholipases A and mucin genes in gallbladders and biliary composition in patients with multiple cholesterol stones
Authors: Kano Masahito , Shoda Junichi , Irimura Tatsuro , Ueda Tetsuya , Iwasaki Ryu , Urasaki Tetsuya , Kawauchi Yasushi , Asano Toru , Matsuzaki Yasushi , Tanaka Naomi
Published: July 2004

Group IIA phospholipase A2 (PLA2), a secretory low-molecular-weight PLA2, may play a critical role in the process of gallbladder mucosal inflammation in multiple cholesterol stones, which in turn may produce biliary pronucleating proteins as well as mucin. On the other hand, ursodeoxycholate (UDC) decreases biliary levels of various pronucleating proteins, possibly because of its membrane-protective effects on the inflamed gallbladder mucosa. To elucidate that beneficial effect of UDC, the expression levels of low-molecular-weight PLA2s, group IIA PLA2 (PLA2-IIA), and group V PLA2 (PLA2-V), and mucin core polypeptide genes in the gallbladders were studied for UDC-treated patients and untreated patients with multiple cholesterol stones. Furthermore, the results were correlated with alterations in biliary composition. With long-term administration of UDC, the PLA2-IIA protein mass (2.7 ± 0.5 vs. 5.0 ± 0.4 ng/mg · protein [mean ± SEM]; P < .01) and steady-state mRNA level, as well as the PLA2-V mRNA level, were significantly decreased in the gallbladders, where the prostaglandin E2 (PGE2) level was concomitantly decreased (190.7 ± 27.9 vs. 393.6 ± 55.3 pg/mg · protein; P < .01). In the gallbladder bile, the immunoradiometrically determined PLA2-IIA levels were significantly decreased in the UDC-treated patients (43 ± 4 ng/dL; P < .01) in comparison with untreated patients (78 ± 6 ng/dL). Significant decreases were similarly found for total protein, mucin, and free arachidonate concentrations, as well as nucleation activity in the bile. The degree of the changes was found to be rather small in solitary stones. In contrast to the decreased mucin concentration, however, there were no significant changes in the expression levels of mucin core polypeptide genes (MUC1-MUC6) between the UDC-treated and untreated patients. Long-term UDC administration was observed to lower the increased PLA2-IIA protein mass and mRNA level, as well as the PLA2-V mRNA level, in the gallbladders of patients with multiple cholesterol stones, which in turn may be of therapeutic importance in improving the gallbladder mucosal inflammation. Effects of UDC on secretory low-molecular-weight PLA2s as inflammatory mediators may relate to the reported efficacy of UDC treatment in cholesterol gallstone disease.
The normal adult human liver biopsy: A quantitative reference standard
Authors: Crawford Aleta R. , Lin Xi-Zhang , Crawford James M.
Published: July 2004

In assessing adult human liver histology, questions remain concerning the normal number of portal tracts and bile ducts in a liver biopsy. We therefore reviewed liver biopsies obtained with use of a percutaneous Menghini cutting needle (14G, internal diameter 1.6 mm), from 16 patients undergoing liver biopsy for screening procedures (age 49 ± 14 years, ±SD) and found to be normal by histological examination. The average aggregate length of the liver tissue was 1.8 ± 0.8 cm (area of 16.4 ± 10.7 mm2), representing 7 ± 3 tissue fragments. Portal triads containing at least one profile each of a portal vein, hepatic artery, and interlobular bile duct numbered 11 ± 6 per biopsy (range 3-23). Portal dyads, which did not contain one of these profiles, usually the portal vein, numbered 8 ± 5 (range 1-18). On a per-specimen basis, 38% of portal tracts did not contain a portal vein, 7% did not contain a bile duct, and 9% did not contain a hepatic artery. Because of multiplicity of profiles within portal tracts, however, the average number of profiles per portal tract was 6 ± 5 (range 2-35). Notably, on average there were 2.3 ± 2.2 interlobular bile ducts per portal tract, compared to 2.6 ± 2.3 hepatic arteries and 0.7 ± 0.7 portal veins. The average minimum external diameter of interlobular bile ducts was 13 ± 4 μm, of hepatic arteries 12 ± 5 μm, and of portal veins 35 ± 25 μm. Bile ducts greater than 30 μm in diameter were rare, only one each in two biopsies were observed. In contrast, probable canals of Hering were occasionally evident at the periphery of portal tracts (6 ± 6 per biopsy) and within the lobular parenchyma as strings of cuboidal cells (5 ± 5 per biopsy). We conclude that, although multiplicity of profiles is normal, portal dyads are almost as common as portal triads in normal peripheral liver tissue. On average, there are two interlobular bile ducts, two hepatic arteries, and one portal vein per portal tract, with 6 full portal triads per linear cm of tissue obtained by external Menghini biopsy technique with use of a 14G needle, equivalent to 0.8 ± 0.5 portal triads per mm2. By serving as a reference standard for adult human liver histology, these findings may assist in the histopathological assessment of liver biopsies, particularly those performed for disease conditions featuring loss of intrahepatic bile ducts.
Atypical antineutrophil cytoplasmic antibodies with perinuclear fluorescence in chronic inflammatory bowel diseases and hepatobiliary disorders colocalize with nuclear lamina proteins
Authors: Terjung Birgit , Herzog Volker , Worman Howard J. , Gestmann Ingo , Bauer Christoph , Sauerbruch Tilman , Spengler Ulrich
Published: July 2004

Antineutrophil cytoplasmic antibodies (ANCA) are frequently associated with chronic inflammatory bowel diseases (IBD) and hepatobiliary disorders. However, their target antigens have not been identified yet. Recently, we observed an atypical perinuclear ANCA fluorescence (p-ANCA) together with an intranuclear staining using ANCA-positive sera from patients with IBD and hepatobiliary disorders. This observation suggests that the target antigens are localized within the nucleus of neutrophilic granulocytes. To further investigate this hypothesis, we examined sera from patients with ulcerative colitis, primary sclerosing cholangitis, autoimmune hepatitis or systemic vasculitis on ethanol or formaldehyde-fixed neutrophils using confocal laser scanning microscopy and immunoelectron microscopy. Counterstaining with propidium iodide, a DNA-specific dye, showed that ANCA-positive sera in IBD and heptobiliary disorders react with intranuclear antigens at the nuclear periphery of the neutrophils. Double immunolabeling techniques revealed that nuclear lamina proteins, lamins A, C and B1, and lamin B receptor were colocalized with the antigen(s) recognized by atypical p-ANCA. No colocalization was observed with classical p-ANCA and antibodies against histones (H1-H4). Our study showed that atypical p-ANCA are antinuclear antibodies reactive with granulocyte-specific antigens present in the nuclear lamina.
Sequential versus concomitant administration of ribavirin and interferon alfa-n3 in patients with chronic hepatitis C not responding to interferon alone: Results of a randomized, controlled trial
Authors: Sostegni Raffaello , Ghisetti Valeria , Pittaluga Fabrizia , Marchiaro Giovanna , Rocca Giuseppe , Borghesio Elisabetta , Rizzetto Mario , Saracco Giorgio
Published: July 2004

We conducted a three-arm, randomized trial in 96 patients with chronic hepatitis C who did not respond to interferon alfa to compare treatments. Group 1 (33 patients) received ribavirin alone (1,000 mg/daily for 6 months) followed by interferon alfa n-3 alone (3 MU thrice weekly for 6 months); group 2 (33 patients) received ribavirin plus interferon alfa n-3 for 6 months at the above doses; and group 3 (30 patients) received interferon alfa n-3 alone (3 MU thrice weekly for 6 months). At the end of treatment, 3 patients (10%) in group 1, 13 (41%) in group 2, and 5 (17%) in group 3 had normal alanine transaminase (ALT) levels (group 2 vs. groups 1 and 3,P= .008). After 6 months of follow-up, only 4 patients (12.5%) in group 2 still had normal ALT values (P= .03). At the end of therapy, hepatitis C virus (HCV) RNA was no longer detectable by polymerase chain reaction in 4 (13%), 9 (27%), and 2 (7%) patients, respectively, in groups 1, 2, and 3 (P= NS). Six months posttherapy, only 5 (15%) patients in group 2 were still HCV RNA negative (P= .02). At the time of follow-up liver biopsy, performed 6 months after the end of treatment, a significant improvement of the necroinflammatory scores was observed among group 2 patients (P= .01) but not in the other two groups. Side effects reflected the profile of each drug as monotherapy; mild hemolytic anemia was the most frequent side effect caused by ribavirin. In conclusion, concomitant administration of ribavirin and interferon alfa n-3 was significantly superior to the sequential schedule or interferon alfa n-3 monotherapy in inducing a sustained response in patients with chronic hepatitis C who had not responded to interferon alone. However, combination therapy at the dose and duration adopted in this study is capable of modifying the natural course of the disease in only a minority of these patients.
Cytokeratin expression is reduced in glycogenotic clear hepatocytes but increased in ground-glass cells in chronic human and woodchuck hepadnaviral infection†
Authors: Su Qin , Zerban Heide , Otto Gerd , Bannasch Peter
Published: July 2004

Hepatocytes of normal adult liver express cytokeratins (CKs) 8/18, but bile duct cells additionally contain CK7/19. We have previously demonstrated the frequent occurrence of foci of altered hepatocytes in association with hepatic tumors in humans and provided evidence for a preneoplastic nature of the focal lesions. In this study, we investigated the CK expression in both the preneoplastic lesions and extrafocal parenchyma. Sixty-seven explanted livers with cirrhosis or advanced fibrosis harboring preneoplastic focal lesions, with or without hepatitis B virus (HBV) infection, as well as 9 livers with HBV-associated fulminant hepatitis, were studied for the expression of CK7/8/14/18/19. Five livers from woodchucks infected with the woodchuck hepatitis virus (WHV) were also investigated. Glycogenotic clear hepatocytes were negative or weakly positive for CK8/18, while amphophilic hepatocytes were strongly positive for these CKs, the changes being associated with marked reduction and increase, respectively, of highly organized membranous components in their cytoplasm. This allows the distinct recognition of the clear-cell and clear-cell-dominant preneoplastic lesions in the human and woodchuck livers. In ground-glass hepatocytes expressing viral antigens, an unusual accumulation of CK8/18 was observed, but there was no evidence of preferential necrosis of ground-glass hepatocytes. Many CK7- and CK19-positive ductular (oval) cells were found in extrafocal liver tissue, but only rarely were they present within focal lesions.
Decreased survival in rat liver transplantation with extended cold preservation: Role of portal vein clamping time
Authors: Urata Koichi , Nguyen Bich , Brault Antoine , Lavoie Joël , Rocheleau Bernard , Huet Pierre-Michel
Published: July 2004

Primary liver graft dysfunction is currently related to cold ischemia-reperfusion injury, although a wide survival range has been reported using 24-hour preservation in cold University of Wisconsin (UW) solution. We hypothesized that the portal vein clamping time (PVCT) played a more important role than cold preservation injury in the postoperative outcome. Rat liver transplantation was performed using different clamping times after 24-hour cold ischemia in the UW solution. Survival rates, plasma tumor necrosis factor (TNF), and nitrate/nitrite levels were examined. Subsequently, the effect of clamping time was evaluated on hepatocyte and sinusoidal endothelial cell (SEC) function using isolated perfused livers. Survival rate was directly related to clamping time length. Marked increases in TNF and nitrate/nitrite levels were found after surgery, particularly after long clamping times. In perfusion studies, the SEC function was already markedly altered after preservation alone and was not further modified by transplantation. By contrast, the hepatocyte function was moderately altered after transplantation, irrespective of clamping times, even when rats operated with long clamping times were in terminal conditions. In rats, 24-hour preservation in cold UW solution is not a severely compromising condition leading to primary liver nonfunction. Long PVCTs are associated with an endotoxemia-like syndrome more related to a warm intestinal ischemia than to cold ischemia injury of the liver.
Noncirrhotic portal hypertension and nodular regenerative hyperplasia of the liver in dogs with mucopolysaccharidosis type I
Authors: McEntee Michael F. , Wright Kathy N. , Wanless Ian , DeNovo Robert , Schneider John F. , Shull Robert
Published: July 2004

Nodular regenerative hyperplasia (NRH) is an uncommon hepatic lesion often associated with noncirrhotic portal hypertension (PHT). We have noted that NRH and PHT are frequent occurrences in a colony of dogs with the genetic storage disease, mucopolysaccharidosis I (MPS-I). This observation provides the opportunity to study the histology and pathogenesis of NRH and noncirrhotic PHT in a new animal model. Thirteen of 32 dogs (41%) with MPS-I developed multiple portocaval shunts between 4 and 48 months of age that were grossly visible at necropsy. Seven of the 13 developed marked ascites, whereas all those without shunts and littermates (n = 24) heterozygous for the mutated α-l-iduronidase allele (carriers unaffected by the storage disease) did not. The large and medium-sized portal veins were widely patent without thrombosis or vascular malformations. Hepatic parenchymal fibrosis was absent or mild and did not correlate with shunt formation. All 32 livers had varying degrees of diffuse periportal hepatocellular hyperplasia with multifocal atrophy and compression of centrolobular cords (NRH) most prominent in dogs with shunts. Many small portal veins were reduced in diameter or absent, especially in animals with shunts. Noncirrhotic PHT and NRH appear to be related to the obliteration of small portal veins in these dogs, but the pathogenesis of this vascular change remains unknown.