bluuebunny/crossref_metadata_2025 huggingface dataset:
Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion
Authors: Andreola Fausto , Calvisi Diego F. , Elizondo Guillermo , Jakowlew Sonia B. , Mariano Jennifer , Gonzalez Frank J. , De Luca Luigi M.
Published: January 2004

Aryl hydrocarbon receptor (AHR)-null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, tissue transglutaminase type II (TGaseII) activity, transforming growth factorβ (TGFβ) overexpression, and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR-null mice a vitamin A- deficient diet with the purpose to reverse the phenotype. Liver retinoid content decreased sharply within the first few weeks on the retinoid-deficient diet. Analysis of TGFβ1, TGFβ2, and TGFβ3 expression revealed a reduction to control levels in the AHR -/- mice accompanied by parallel changes in TGaseII protein levels. In addition, we observed an increase in the TGFβ receptors, TGFβ RI and TGFβ RII, as well as in Smad4, and their reduction to wild-type mouse liver levels in AHR -/- mice fed the retinoid-deficient diet. Reduction of peroxisomal proliferator-activated receptor γ (PPARγ) messenger RNA (mRNA) and protein levels in AHR -/- mice was consistent with the presence of hepatic stellate cell (HSC) activation and liver fibrosis. Vitamin A deficiency normalized PPARγ expression in AHR -/- mice. In conclusion, livers from AHR -/- mice fed the vitamin A-deficient diet showed a decrease in collagen deposition, consistent with the absence of liver fibrosis. (Hepatology 2004;39:157-166.)
Prediction of survival after liver retransplantation for late graft failure based on preoperative prognostic scores
Authors: Yao Francis Y. , Saab Sammy , Bass Nathan M. , Hirose Ryutaro , Ly David , Terrault Norah , Lazar Ann A. , Bacchetti Peter , Ascher Nancy L. , Roberts John P.
Published: January 2004

The current policy for determining priority for organ allocation is based on the model for end stage liver disease (MELD). We hypothesize that severity of graft dysfunction assessed by either the MELD score or the Child-Turcotte-Pugh (CTP) score correlates with mortality after liver retransplantation (re-OLT). To test this hypothesis, we analyzed the outcome of 40 consecutive patients who received re-OLT more than 90 days after primary orthotopic liver transplantation (OLT). The Kaplan-Meier 1-year and 5-year survival rates after re-OLT were 69% and 62%, respectively. The area under the curve (AUC) values generated by the receiver operating characteristics (ROC) curves were 0.82 (CI 0.70-0.94) and 0.68 (CI 0.49-0.86), respectively (P = .11), for the CTP and MELD models in predicting 1-year mortality after re-OLT. The 1-year and 5-year survival rates for patients with CTP scores less than 10 were 100% versus 50% and 40%, respectively, for CTP scores of at least 10 (P = .0006). Patients with MELD scores less than or equal to 25 had 1-year and 5-year survival rates of 89% and 79%, respectively, versus 53% and 47%, respectively, for MELD scores greater than 25 (P = .038). Other mortality predictors include hepatic encephalopathy, intensive care unit (ICU) stay, recurrent hepatitis C virus (HCV) infection, and creatinine level of 2 mg/dL or higher. Analysis of an independent cohort of 49 patients showed a trend for a correlation between CTP and MELD scores with 1-year mortality, with AUC of 0.59 and 0.57, in respective ROC curves. In conclusion, our results suggest that severity of graft failure based on CTP and MELD scores may be associated with worse outcome after re-OLT and provide a cautionary note for the “sickest first” policy of organ allocation. (Hepatology 2004;39:230-238.)
Pediatric fulminant hepatic failure in endemic areas of hepatitis B infection: 15 years after universal hepatitis B vaccination
Authors: Chen Huey-Ling , Chang Chee-Jen , Kong Man-Shan , Huang Fu-Chen , Lee Hung-Chang , Lin Chieh-Chung , Liu Ching-Chuan , Lee I-Hsien , Wu Tzee-Chung , Wu Shu-Fen , Ni Yen-Hsuan , Hsu Hong-Yuan , Chen Ding-Shinn , Chang Mei-Hwei
Published: January 2004

To investigate the role of hepatitis B virus (HBV) infection in pediatric fulminant hepatic failure (FHF) after the launch of universal HBV vaccination, the authors analyzed the data from patients with FHF collected from a nationwide collaborative study group. Children aged 1 month to 15 years who were diagnosed with FHF (62 males and 33 females) between 1985-1999 were included. HBV infection (hepatitis B surface antigen [HBsAg] and/or immunoglobulin M hepatitis B core antibody [IgM anti-HBc] seropositive) accounted for 46% (43 of 95 cases) of all the cases of FHF. The average annual incidence of FHF in the time period 1985-1999 was 0.053/100,000 in the group of patients ages 1-15 years and 1.29/100,000 in those patients age < 1 year. Approximately 61% (58 of 95 cases) of all FHF cases were infants. The percentage of HBV infection was found to be higher in infants (57%) compared with children ages 1-15 years (27%) (P = 0.004). The incidence rate ratio of those patients age < 1 year to those ages 1-15 years was 54.2 for HBV-positive FHF and 15.2 for HBV-negative FHF. Maternal HBsAg was found to be positive in 97% of the infants with HBV-positive FHF, and hepatitis B e antigen (HBeAg) was found to be negative in 84% of these infants. Approximately 74% of all HBV-positive FHF patients and 81% of the infantile HBV-positive patients had been vaccinated. In conclusion, within the first 15 years of universal vaccination, HBV was found to rarely cause FHF in children age > 1 year but remained a significant cause of FHF in infants. HBV-positive FHF was prone to develop in infants born to HBeAg-negative, HBsAg-carrier mothers; these infants had not received hepatitis B immunoglobulin according to the vaccination program in place. (Hepatology 2004;39:58-63.)
A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis† ‡
Authors: Promrat Kittichai , Lutchman Glen , Uwaifo Gabriel I. , Freedman Renee J. , Soza Alejandro , Heller Theo , Doo Edward , Ghany Marc , Premkumar Ahalya , Park Yoon , Liang Jake T. , Yanovski Jack A. , Kleiner David E. , Hoofnagle Jay H.
Published: January 2004

Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease for which there is no known effective therapy. A proportion of patients with NASH progress to advanced fibrosis and cirrhosis. NASH is considered one of the clinical features of the metabolic syndrome in which insulin resistance plays a central role. This prospective study evaluates the role of insulin-sensitizing agent in treatment of NASH. Eighteen nondiabetic patients with biopsy-proven NASH were treated with pioglitazone (30 mg daily) for 48 weeks. Tests of insulin sensitivity and body composition as well as liver biopsies were performed before and at the end of treatment. By 48 weeks, serum alanine aminotransferase values fell to normal in 72% of patients. Hepatic fat content and size as determined by magnetic resonance imaging decreased, and glucose and free fatty acid sensitivity to insulin were uniformly improved. Histological features of steatosis, cellular injury, parenchymal inflammation, Mallory bodies, and fibrosis were significantly improved from baseline (all P < 0.05). Using strict criteria, histological improvement occurred in two-thirds of patients. Pioglitazone was well tolerated; the main side effects were weight gain (averaging 4%) and an increase in total body adiposity. In conclusion, these results indicate that treatment with an insulin-sensitizing agent can lead to improvement in biochemical and histological features of NASH and support the role of insulin resistance in the pathogenesis of this disease. The long-term safety and benefits of pioglitazone require further study. (Hepatology 2004;39:188-196.)
Health-related quality of life in active injecting drug users with and without chronic hepatitis C virus infection
Authors: Dalgard Olav , Egeland Atle , Skaug Kjell , Vilimas Kostas , Steen Tore
Published: January 2004

This study assessed the effect of chronic hepatitis C virus (HCV) infection on the health-related quality of life (HRQOL) of injecting drug users, comparing the HRQOL of injecting drug users with and without chronic HCV infection. The study included 199 injecting drug users of more than 18 years of age who participated in a needle exchange program. Blood samples were tested for the presence of HCV RNA in serum with a polymerase chain reaction method. HRQOL was measured using the questionnaire SF-36, measuring HRQOL over the last 4 wk. The HCV RNA test was positive in 102 (51%) and negative in 97 (49%) subjects. The HRQOL scores of actively injecting drug users were markedly reduced compared to the population norm. However, we did not find poorer HRQOL in injecting drug users with chronic HCV infection than in injecting drug users without HCV infection. HCV RNA positive injecting drug users who were aware of the infection had lower HRQOL scores than those unaware of the infection in 4 of the 8 SF-36 dimensions (general health, physical functioning, physical role, and vitality). HCV RNA negative subjects, who believed they were infected, scored worse in one dimension (general health) compared to those who did not believe they were infected. In conclusion, chronic HCV infection per se did not negatively affect the HRQOL of active injecting drug users. Those who thought they were infected had a lower HRQOL scores than those who believed they were not infected. (Hepatology 2004;39:74-80.)
Differential modulation of rat hepatic stellate phenotype by natural and synthetic retinoids
Authors: Hellemans Karine , Verbuyst Peggy , Quartier Erik , Schuit Frans , Rombouts Krista , Chandraratna Ross A.S. , Schuppan Detlef , Geerts Albert
Published: January 2004

Activation of hepatic stellate cells (HSC) is a central event in the pathogenesis of liver fibrosis during chronic liver injury. We examined the expression of retinoic acid (RAR) and retinoid X receptors (RXR) during HSC activation and evaluated the influence of natural and synthetic retinoic acids (RA) on the phenotype of culture-activated HSC. The expression of the major RAR/RXR subtypes and isoforms was analyzed by Northern hybridization. Presence of functional receptor proteins was established by gel shift analysis. Retinoic acids, RAR, and RXR selective agonists and an RAR antagonist were used to evaluate the effects of retinoid signalling on matrix synthesis by Northern blotting and immunoprecipitation, and on cell proliferation by BrdU incorporation. The 9-cisRA and synthetic RXR agonists reduced HSC proliferation and synthesis of collagen I and fibronectin. All-trans RA and RAR agonists both reduced the synthesis of collagen I, collagen III, and fibronectin, but showed a different effect on cell proliferation. Synthetic RAR agonists did not affect HSC proliferation, indicating that ATRA inhibits cell growth independent of its interaction with RARs. In contrast, RAR specific antagonists enhance HSC proliferation and demonstrate that RARs control proliferation in a negative way. In conclusion, natural RAs and synthetic RAR or RXR specific ligands exert differential effects on activated HSC. Our observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or to animals subjected to fibrogenic stimuli. (Hepatology 2004;39:97-108.)
Viral features of lamivudine resistant hepatitis B genotypes A and D
Authors: Zöllner Bernhard , Petersen Jörg , Puchhammer-Stöckl Elisabeth , Kletzmayr Josef , Sterneck Martina , Fischer Lutz , Schröter Matthias , Laufs Rainer , Feucht Heinz-Hubert
Published: January 2004

Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo. Forty-one patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P = .006) and rt180M mutants (81% in group A vs 40% in group D, P = .015) prevailed in group A. The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P < .001). Additional resistance associated mutations were detected exclusively in group D (P = .004). In a multivariate analysis, HBV genotype (P = .039) and pretreatment serum HBV DNA (P = .001) were independently associated with emerging rt204I or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log10 6.99 copies/ml; range 3-9) compared with group D (mean log10 6.1 copies/ml; range 3.3-8; P = .04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up. In conclusion, the mutational pattern during selection of lamivudine resistant HBV strains differs between genotypes A and D. This may have consequences for a salvage regimen initiated for treatment of lamivudine resistant HBV. (Hepatology 2004;39:42-50.)
Over-expression of the ribosomal protein L36a gene is associated with cellular proliferation in hepatocellular carcinoma
Authors: Kim Jong-Hyun , You Kyung-Ran , Kim In Hee , Cho Baik-Hwan , Kim Chan-Young , Kim Dae-Ghon
Published: January 2004

Using messenger RNA (mRNA) differential display, we identified a single complementary DNA (cDNA) fragment (HG23T1) that was over-expressed in a hepatocellular carcinoma (HCC) specimen. We cloned the full-length HG23T1 gene by the rapid amplification of cDNA end (RACE) polymerase chain reaction (PCR) method. It perfectly matched the gene encoding human ribosomal protein L36a (RPL36A also referred to as RPL44). RPL36A mRNA was preferentially over-expressed in 34 of 40 HCC cases (85%, P < .001) and in all of 8 HCC cell lines. Ectopically over-expressed L36a ribosomal protein localized in the nucleoli of cells, and this localization seemed to be controlled by the N-terminal or the internal tetrapeptide consensus with its adjacent N-terminal domain. Over-expression of L36a led to enhanced colony formation and cell proliferation, which may have resulted from rapid cell cycling, and an antisense cDNA effectively reversed these alterations. In conclusion, RPL36A plays a role in tumor cell proliferation and may be a potential target for anticancer therapy of HCC. (Hepatology 2004;39:129-138.)
Identification of HBV DNA sequences that are predictive of response to lamivudine therapy
Authors: Ciancio Alessia , Smedile Antonina , Rizzetto Mario , Lagget Marco , Gerin John , Korba Brent
Published: January 2004

Numerous studies have shown that resistance to long-term lamivudine therapy occurs in as many as ⅔ of hepatitis B virus (HBV) chronic carriers. Additional studies have shown that reversion of HBV mutations in the precore/core promoter region conferring an HBeAg-negative phenotype/genotype can occur in up to 30% of lamivudine-treated patients. In this study, sequences of the HBV polymerase and precore/core coding regions in 26 HBV-infected patients (24 with HBeAg-negative virus infection, 25 genotype D, 1 genotype A) treated for 27 to 53 months with lamivudine were analyzed to determine the relationship between pretreatment HBV DNA sequence patterns and long-term treatment response, and the effect of therapy on the status of HBV precore mutations. Reversions of precore mutations A1762T/G1764A and G1896A were observed in 29% and 25% of patients, respectively, but none became HBeAg-positive. These data are consistent with previously published reversion frequencies for 2 other groups of lamivudine-treated patients. Two naturally-occurring DNA polymorphisms at aa91 and aa256 of the HBV polymerase were observed in the pretreatment serum samples, which correlated with extended treatment failure. In conclusion, reversion of mutations conferring an HBeAg-negative phenotype occur relatively frequently under lamivudine therapy. Furthermore, at least in HBeAg-negative patients infected predominately with HBV genotype D, specific viral DNA sequences which are present before therapy appear to be useful as predictors of long-term response to lamivudine treatment. (Hepatology 2004;39:64-73.)
Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease
Authors: Stewart Stephen F. , Vidali Matteo , Day Christopher P. , Albano Emanuele , Jones David E.J.
Published: January 2004

Serum antibodies reactive with neo-antigens generated during ethanol metabolism have been identified in patients with alcoholic liver disease (ALD), although their role in the pathogenesis of disease remains unclear. In this study, we characterized peripheral blood mononuclear cell (PBMC) T-cell and antibody responses to human serum albumin (HAS) adducted with acetaldehyde under reducing conditions (AcA-HSA) or with malondialdehyde (MDA-HSA) in patients with advanced ALD (AALD, n = 28), heavy drinkers with no liver disease (NALD, n = 14), and mild/moderate drinking controls (n = 22). Peak proliferative responses of PBMC were assessed in vitro by tritiated thymidine incorporation after the addition of optimized concentrations of antigen or OKT3. Antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). MDA-HSA induced PBMC T-cell proliferation was significantly higher in ALD than in NALD or control patients. Moreover, 10 of 28 (36%) of ALD patients had significant T-cell proliferative responses to MDA-HSA compared to 0 of 14 (0%, P = .02) of the NALD group and 2 of 22 (9%, P < .05) of controls. No significant difference in PBMC T-cell response to Aca-HSA was seen between subject groups. Patients with positive cellular responses to MDA had higher serum anti-MDA antibody titers than those not exhibiting a positive cellular response (P < .005). In conclusion, the pattern of cellular and humoral responses to MDA adducts suggests that the development of these responses may be a susceptibility factor for the development of advanced alcoholic liver disease. The apparent importance of T-cell responses to MDA adducts suggests that oxidative stress may represent an important stimulus for the development of cellular immune responses associated with advanced ALD. (Hepatology 2004;39:197-203.)
Spontaneous cholecysto- and hepatolithiasis in Mdr2 −/− mice: A model for low phospholipid-associated cholelithiasis†
Authors: Lammert Frank , Wang David Q.-H. , Hillebrandt Sonja , Geier Andreas , Fickert Peter , Trauner Michael , Matern Siegfried , Paigen Beverly , Carey Martin C.
Published: January 2004

Previously, we identified needle-like and filamentous, putatively “anhydrous” cholesterol crystallization in vitro at very low phospholipid concentrations in model and native biles. Our aim now was to address whether spontaneous gallstone formation occurs in Mdr2 (Abcb4) knockout mice that are characterized by phospholipid-deficient bile. Biliary phenotypes and cholesterol crystallization sequences in fresh gallbladder biles and non-fixed liver sections were determined by direct and polarizing light microscopy. The physical chemical nature and composition of crystals and stones were determined by sucrose density centrifugation and before mass and infrared spectroscopy. Gallbladder biles of Mdr2
−/− mice precipitate needle-like cholesterol crystals at 12 weeks of age on chow. After 15 weeks, more than 50% of Mdr2
−/− mice develop gallbladder stones, with female mice displaying a markedly higher gallstone-susceptibility. Although gallbladder biles of Mdr2
−/− mice contain only traces (≤ 1.1 mM) of phospholipid and cholesterol, they become supersaturated with cholesterol and plot in the left 2-phase zone of the ternary phase diagram, consistent with “anhydrous” cholesterol crystallization. Furthermore, more than 40% of adult female Mdr2
−/− mice show intra- and extrahepatic bile duct stones. In conclusion, spontaneous gallstone formation is a new consistent feature of the Mdr2
−/− phenotype. The Mdr2
−/− mouse is therefore a model for low phospholipid-associated cholelithiasis recently described in humans with a dysfunctional mutation in the orthologous ABCB4 gene. The mouse model supports the concept that this gene is a monogenic risk factor for cholesterol gallstones and a target for novel therapeutic strategies. (Hepatology 2004;39:117-128.)
Relative contribution of iron burden, HFE mutations, and insulin resistance to fibrosis in nonalcoholic fatty liver
Authors: Bugianesi Elisabetta , Manzini Paola , D'Antico Sergio , Vanni Ester , Longo Filomena , Leone Nicola , Massarenti Paola , Piga Antonio , Marchesini Giulio , Rizzetto Mario
Published: January 2004

The mechanism(s) determining the progression from fatty liver to steatohepatitis is currently unknown. Our goal was to define the relative impact of iron overload, genetic mutations of HFE, and insulin resistance on the severity of liver fibrosis in a population of subjects with nonalcoholic fatty liver disease (NAFLD) who had low prevalence of obesity and no overt symptoms of diabetes. In a cohort of 263 prospectively enrolled patients with NAFLD, 7.4% of patients had signs of peripheral iron overload and 9% had signs of hepatic iron overload, but 21.1% had hyperferritinemia. The prevalence of C282Y and H63D HFE mutations was similar to the general population and mutations were not associated with iron overload. Although subjects were on average only moderately overweight, insulin sensitivity, measured both in the fasting state and in response to oral glucose, was lower. Univariate analysis demonstrated that the presence of severe fibrosis was independently associated with older age, female sex, overweight, aspartate/alanine aminotransferase ratio, serum ferritin level, fasting glucose and insulin levels, decreased insulin sensitivity, and with histologic features (degree of necroinflammation and steatosis). After adjustment for body mass index (BMI), age, sex, and degree of steatosis, ferritin levels (odds ratio [OR] = 1.77; 95% CI = 1.21- 2.58; P = .0032) and the oral glucose insulin sensitivity (OR = 0.53; CI = 0.33-0.87; P = .0113) were independent predictors of severe fibrosis. In conclusion, the current study indicates that insulin resistance is a major, independent risk factor for advanced fibrosis in patients with NAFLD. Increased ferritin levels are markers of severe histologic damage, but not of iron overload. Iron burden and HFE mutations do not contribute significantly to hepatic fibrosis in the majority of patients with NAFLD. (Hepatology 2004;39:179-187.)
Cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis†
Authors: Xu Lizhe , Sakalian Michael , Shen Zhiwei , Loss George , Neuberger James , Mason Andrew
Published: January 2004

Patients with primary biliary cirrhosis (PBC) have both serologic and tissue evidence of infection. A recently identified human betaretrovirus was originally cloned from the biliary epithelium cDNA library of a patient with PBC. By conducting a BLASTN search, the initial partial pol gene fragment was found to have 95% to 97% nucleotide homology with mouse mammary tumor virus (MMTV) and with retrovirus sequences derived from human breast cancer samples. Using an anti-p27CA MMTV antibody, viral proteins were detected in the perihepatic lymph nodes but not in liver tissue samples from patients with PBC, suggesting a higher viral burden in lymphoid tissue. Therefore, in the current study, we used lymph node DNA to clone the proviral genome of the human betaretrovirus from two patients with PBC using a polymerase chain reaction (PCR) walking methodology with conserved primers complementary to MMTV. The human betaretrovirus genome contains five potential open reading frames (ORF) for Gag, protease (Pro), polymerase (Pol), envelope (Env), and superantigen (Sag) proteins that are collinear with their counterparts in MMTV. Alignment studies performed with characterized MMTV and human breast cancer betaretrovirus amino acid sequences revealed a 93% to 99% identity with the p27 capsid proteins, a 93% to 97% identity with the betaretrovirus envelope proteins, and a 76% to 85% identity with the more variable superantigen proteins. Phylogenetic analysis of known betaretrovirus superantigen proteins showed that the human and murine sequences did not cluster as two distinct species. In conclusion, human betaretrovirus nucleic acid sequences have been cloned from patients with PBC. They share marked homology with MMTV and human breast cancer-derived retrovirus sequences. (Hepatology 2004;39:151-156.)
High prevalence of occult hepatitis B in Baltimore injection drug users
Authors: Torbenson Michael , Kannangai Rajesh , Astemborski Jacquie , Strathdee Steffanie A. , Vlahov David , Thomas David L.
Published: January 2004

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in a serum or liver in the absence of hepatitis B surface antigen (HBsAg). The prevalence and clinical correlates of occult hepatitis B remain incompletely defined. A cross-sectional study was performed to determine the prevalence of occult hepatitis B in a high-risk cohort composed of 188 injection drug users in Baltimore, Maryland. All individuals had chronic hepatitis C viral infections confirmed by RNA detection and liver biopsy. Serologic assays for HBsAg and core antibody (HBcAb) were performed. Serum HBV DNA was detected using the COBAS HBV AMPLICOR monitor assay (lower limit of detection, 200 HBV copies per milliliter) and a semi-nested polymerase chain reaction (PCR) assay (lower limit of detection, 15 HBV copies per milliliter). Although almost all individuals (96%) were anti-HBC positive, only 8 of 188 (4%) were HBsAg positive. Occult hepatitis B was not identified using the COBAS assay, but was found in 81 of 180 (45%) of individuals using semi-nested PCR. Of the 8 HBsAg positive individuals, HBV DNA was found in 1/8 using the COBAS assay and 6/8 using the nested PCR assay. Overall, liver disease was mild, with a median serum alanine aminotransferase (ALT) of 38 IU/L, median activity grade of 3/18, and median fibrosis stage of 1/6. No association was found between the serum AST (aspartate aminotransferase), activity grade, or stage of liver disease and the presence of occult hepatitis B. Serum ALT levels were slightly higher in patients without occult hepatitis B (46 vs. 35 IU/L), and the median years since first injection drug use was somewhat longer in those without occult hepatitis B (24 vs. 20 years). In conclusion, although further research is needed to assess its clinical significance, there is a high prevalence of occult HBV infection in this cohort of HCV-infected injection drug users. (Hepatology 2004;39:51-57.)
Vascular involvement of the liver in Turner's syndrome
Authors: Roulot Dominique , Degott Claude , Chazouillères Olivier , Oberti Frédéric , Calès Paul , Carbonell Nicolas , Benferhat Said , Bresson-Hadni Solange , Valla Dominique
Published: January 2004

Unexplained liver test abnormalities are frequent in patients with Turner's syndrome. This cohort study was performed to clarify the histopathologic features, causes, and long-term outcome of liver involvement in these patients. Thirty patients with persistently abnormal liver test results were followed-up for 8.8 ± 5.2 years. Liver specimens were available in 27 patients. Marked architectural changes were present in 10 patients, including nodular regenerative hyperplasia in six, multiple focal nodular hyperplasia in two, and cirrhosis in two patients. These changes frequently were associated with obliterative portal venopathy lesions and with aortic malformations. There was mild to moderate portal fibrosis in 15 of the 17 other patients, inflammatory infiltrates in nine patients, and nonalcoholic fatty liver disease in 11 patients. Bile duct alterations resembling small duct sclerosing cholangitis were observed in 21 patients (with or without architectural changes). There was no viral, alcoholic, autoimmune, or drug-induced liver damage. Portal hypertension was observed in four patients with marked architectural changes, including three in whom refractory ascites or recurrent variceal bleeding developed, one of whom underwent transplantation. None of the patients without marked architectural changes experienced progressive or decompensated liver disease. There was no evidence of liver toxicity from estrogen replacement therapy. In conclusion, the main causes of liver involvement in Turner's syndrome are vascular disorders, probably of a congenital origin, and nonalcoholic fatty liver disease. In patients with vascular disorders, severe liver disease requiring liver transplantation may develop. Estrogen therapy does not appear to be pathogenically implicated. (Hepatology 2004;39:239-247.)
The Gambia Liver Cancer Study: Infection with hepatitis B and C and the risk of hepatocellular carcinoma in West Africa
Authors: Kirk Gregory D. , Lesi Olufunmilayo A. , Mendy Maimuna , Akano Aliu O. , Sam Omar , Goedert James J. , Hainaut Pierre , Hall Andrew J. , Whittle Hilton , Montesano Ruggero
Published: January 2004

Hepatocellular carcinoma (HCC) is the most common cancer in The Gambia. Hepatitis B virus (HBV) infection is endemic, with 15% to 20% of the population being chronic carriers, whereas hepatitis C virus (HCV) prevalence is low. We recruited 216 incident cases of HCC and 408 controls from three sites. HBV carriage was present in 61% (129/211) of HCC patients and 16% (64/402) of controls, whereas 19% (36/191) of HCC patients were HCV seropositive compared with 3% (11/382) of controls. HCC patients with HCV were notably older and were more likely to be female than those with HBV. Increased HCC risk was strongly associated with chronic HBV (odds ratio, 16.7; 95% CI, 9.7-28.7), HCV (16.7; 6.9-40.1), and dual infection (35.3; 3.9-323). We interpret the additive nature of risk with coinfection as representative of HBV and HCV acting primarily through shared steps in the multistage process of hepatocarcinogenesis. HCV infection was not observed among younger participants, suggesting a possible cohort effect. Reasons for the striking age and gender differences in HCC associated with HBV compared with HCV are unclear, but transmission patterns and age at exposure may be factors. In conclusion, in a standardized evaluation of well-characterized study participants from The Gambia, most cases of HCC are attributable to HBV (57%), but HCV adds a significant fraction (20%), especially among older patients and females. If HCV transmission is not perpetuated in future cohorts, focusing available resources on HB vaccination efforts could greatly ameliorate a major cause of cancer death in sub-Saharan Africa. (Hepatology 2004;39:211-219.).
Prevalence and predictors of esophageal varices in patients with primary sclerosing cholangitis
Authors: Zein Claudia O. , Lindor Keith D. , Angulo Paul
Published: January 2004

Patients with primary sclerosing cholangitis (PSC) may develop and bleed from esophageal varices. However, the exact prevalence of esophageal varices in patients with PSC remains unknown and potential predictors of esophageal varices in this population have not been identified. Our aim was to determine the prevalence of esophageal varices in patients with PSC and the variables that predict their presence. Data were collected on 283 patients with PSC treated for the first time at the Mayo Clinic (Rochester, MN) during 8 consecutive years. Thirty-six percent (102 of 283) of patients had esophageal varices including 56% (57 of 102) with moderate/large varices. After excluding 28 patients with a history of variceal bleeding, data on 183 patients were analyzed to identify independent predictors of esophageal varices and of moderate/large size varices. Platelet count, albumin level, and advanced histologic disease were independent predictors of esophageal varices (area under the receiver operator characteristic [ROC] curve = 0.88). After controlling for the presence of advanced histologic stage and albumin levels, the odds ratios (OR) of platelet count less than 150 × 103/dL for the presence of esophageal varices was 6.3 (95% CI: 2.6-15.8). The diagnostic accuracy of these results was corroborated by cross-validation of the data in an independent set of 72 patients with PSC (area under the ROC = 0.90). In conclusion, in patients with PSC, noninvasive markers of portal hypertension and of advanced liver disease predict the presence of esophageal varices. Our results suggest a clinically applicable and useful approach to identify patients with PSC who are more likely to benefit from endoscopic screening for esophageal varices. (Hepatology 2004;39:204-210.)
Long-term outcome (35 years) of hepatitis C after acquisition of infection through mini transfusions of blood given at birth
Authors: Casiraghi Maria Antonietta , De Paschale Massimo , Romanò Luisa , Biffi Renato , Assi Agnese , Binelli Giorgio , Zanetti Alessandro Remo
Published: January 2004

Long-term follow up studies of hepatitis C virus (HCV) infection rarely exceed 20-25 yr. We studied the outcome of HCV infection in 35-yr-old adults infected at birth (1968) through mini transfusions of blood. A retrospective-prospective study was carried out. The cohort included 31 individuals who were given mini blood transfusions (21-30 ml) collected from a donor subsequently revealed to be HCV infected. At enrollment (1998), 18 of 31 (58.1%) recipients had anti-HCV antibody and 16 (88.9%) of them were HCV-RNA positive. All viremic recipients and the infectious donor had the same genotype 1b. Sequence analysis of E1/E2 and NS5b regions, coupled with phylogenetic analysis, indicated that HCV isolates from donor/recipients were linked. Eleven of the 16 viremic recipients gave consent to liver biopsy. Nine had no fibrosis or mild portal fibrosis and 2 had either discrete (Ishak's staging 3) or marked (Ishak's staging 4) fibrosis. During the prospective follow-up period (1998-2003), 2 patients were given therapy, one of whom achieved sustained clinical and virologic response. A second biopsy, performed in 5 patients at a 5 yr interval, revealed no substantial modifications in 4 cases and progression from absence of fibrosis to mild portal fibrosis in the fifth. In conclusion, taking into account the limited study sample, these findings suggest that HCV infection acquired early in life shows a slow progression and mild outcome during the first 35 yr of infection. (Hepatology 2004;39:90-96.)
MUC4 is a novel prognostic factor of intrahepatic cholangiocarcinoma-mass forming type
Authors: Shibahara Hiroaki , Tamada Shugo , Higashi Michiyo , Goto Masamichi , Batra Surinder K. , Hollingsworth Michael A. , Imai Kohzoh , Yonezawa Suguru
Published: January 2004

Complete surgical resection of the tumor is the sole approach to improve the cure rate of patients with intrahepatic cholangiocarcinoma-mass forming type (ICC-MF). Although patients are treated by curative resection, many of them show poor outcome. Mucin (MUC)4 expression has been implicated as a marker for diagnosis and progression of pancreatic adenocarcinomas, but there is no study of the relationship between MUC4 expression and patient's prognosis in ICC-MF. In the present study, we examined the expression profile of MUC4 in ICC-MF tissue from 27 patients using immunohistochemistry. MUC4 was expressed in the carcinoma tissues of 10 (37%) of the 27 ICC-MF tumors, whereas it was not expressed in normal liver tissue. Because MUC4 is an intramembrane ligand for receptor tyrosine kinase ErbB2 and is related with regulation of p27, we also compared the MUC4 expression with ErbB2 and p27 expressions in ICC-MFs. The patients with MUC4 and ErbB2 double positive expression showed a short survival period compared to non-expressing patients. MUC4 and p27 showed no relationship. The univariate analysis showed that tumor size, intrahepatic metastasis, lymph node metastasis, MUC4 expression, and MUC1 expression were statistically significant risk factors affecting the outcome of the patients with ICC-MF. The multivariate analysis demonstrated that MUC4 expression, as well as surgical margin, were statistically significant independent risk factors. In conclusion, the results suggest that expression of MUC4 in ICC-MF is a new independent factor for poor prognosis and is a useful marker to predict the outcome of the patients with ICC-MF. (Hepatology 2004;39:220-229.)
Structural biology of hepatitis C virus
Authors: Penin François , Dubuisson Jean , Rey Felix A. , Moradpour Darius , Pawlotsky Jean-Michel
Published: January 2004

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Studies of this virus have been hampered by the lack of a productive cell culture system; most information thus has been obtained from analysis of the HCV genome, heterologous expression systems, in vitro and in vivo models, and structural analyses. Structural analyses of HCV components provide an essential framework for understanding of the molecular mechanisms of HCV polyprotein processing, RNA replication, and virion assembly and may contribute to a better understanding of the pathogenesis of hepatitis C. Moreover, these analyses should allow the identification of novel targets for antiviral intervention and development of new strategies to prevent and combat viral hepatitis. This article reviews the current knowledge of HCV structural biology. (Hepatology 2004;39:5-19.)
AQP4 transfected into mouse cholangiocytes promotes water transport in biliary epithelia
Authors: Splinter Patrick L. , Masyuk Anatoliy I. , Marinelli Raul A. , LaRusso Nicholas F.
Published: January 2004

Rodent cholangiocytes express 6 of the 11 known channel proteins called aquaporins (AQPs) that are involved in transcellular water transport in mammals. However, clarifying the role of AQPs in mediating water transport in biliary epithelia has been limited in part because of the absence of physiologically relevant experimental models. In this study, we established a novel AQP4-transfected polarized mouse cholangiocyte cell line suitable for functional studies of transepithelial water transport, and, using this model, we define the importance of this AQP in water transport across biliary epithelia. Polarized normal mouse cholangiocytes (NMCs) lacking endogenous AQP4 were transfected stably with functional AQP4 or cotransfected with functional AQP4 and a transport-deficient AQP4 dominant negative mutant using a retroviral delivery system. In transfected NMCs, AQP4 is expressed on both the mRNA and protein levels and is localized at both the apical and basolateral membranes. In nontransfected NMCs, the transcellular water flow, P

f
, value was relatively high (i.e., 16.4 ± 3.2 μm/sec) and likely was a reflection of endogenous expression of AQP1 and AQP8. In NMCs transfected with AQP4, P

f
increased to 75.7 ± 1.4 μm/sec, that is, by 4.6-fold, indicating the contribution of AQP4 in channel-mediated water transport across MNCs monolayer. In cotransfected NMCs, AQP4 dominant negative reduced P

f
twofold; no changes in P

f
were observed in NMCs transfected with the empty vector. In conclusion, we developed a novel polarized mouse cholangiocyte monolayer model, allowing direct study of AQP4-mediated water transport by biliary epithelia and generated data providing additional support for the importance of AQP4 in cholangiocyte water transport. (Hepatology 2004;39:109-116.)
Commentary: Clinical diagnostic use of cystatin C
Authors: Massey Davis
Published: January 2004

Clinicians recognize and compensate for limitations in estimating the glomerular filtration rate (GFR) using serum creatinine (sCr) measurements by the use of timed collections and mathematical manipulations of sCr. These limitations stem from that fact that sCr is affected by nonrenal influences, including muscle mass and disease state. In addition, sCr may not be sensitive enough to detect minimal declines in GFR in those patient populations in which it is important to recognize early decline. This brief review describes the limitations of sCr, and examines the contribution that sCysC may be able to make in the early recognition of declining renal function. The physiology of CysC is presented, as are the results of clinical investigations that suggest sCysC is in many instances superior to sCr in the recognition of early decline in renal function. Certain exceptions to this are noted. J. Clin. Lab. Anal. 18:55–60, 2004. © 2004 Wiley‐Liss, Inc.
Gene transfer and genetic modification of embryonic stem cells by Cre‐ and Cre‐PR‐expressing MESV‐based retroviral vectors
Authors: Psarras Stelios , Karagianni Niki , Kellendonk Christoph , Tronche François , Cosset François‐Loic , Stocking Carol , Schirrmacher Volker , Boehmer Harald von , Khazaie Khashayarsha
Published: January 2004

Background

Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post‐translationally inducible Cre‐Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells.

Methods

To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses β‐galactosidase only upon Cre‐mediated recombination. This was used together with the ROSA26‐R ES cell Cre‐reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti‐Cre polyclonal antibody, and by monitoring the expression of β‐galactosidase.

Results

Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone‐inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre‐PR (fusion of Cre and progesterone receptor) or β‐galactosidase. Cre‐mediated recombination in more than 60% of ROSA26‐R ES cells was achieved when infected by a VSV‐G‐pseudotyped MESV retrovirus at MOI of 50.

Conclusions

The MESV‐based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells. Copyright © 2003 John Wiley & Sons, Ltd.
Sequence‐specific gene silencing in murine muscle induced by electroporation‐mediated transfer of short interfering RNA
Authors: Kishida Tsunao , Asada Hidetsugu , Gojo Satoshi , Ohashi Suzuyo , Shin‐Ya Masaharu , Yasutomi Kakei , Terauchi Ryu , Takahashi Kenji A. , Kubo Toshikazu , Imanishi Jiro , Mazda Osam
Published: January 2004

Background

Post‐genomic biomedical research requires efficient techniques for functional analyses of poorly characterized genes in living organisms. Sequence‐specific gene silencing in mammalian organs may provide valuable information on the physiological and pathological roles of predicted genes in mammalian systems. Here, we attempted targeted gene knockdown in vivo in murine skeletal muscle through the electroporation‐mediated transfer of short interfering RNA (siRNA).

Methods

siRNA duplexes corresponding to the firefly luciferase (Luc), green fluorescent protein (GFP), or glyceraldehyde‐3‐phosphate dehydrogenase (GAPD) genes were delivered by electroporation into the tibial muscle of normal or enhanced GFP (EGFP) transgenic mice. Plasmid vectors carrying the Luc, hRluc or β‐galactosidase (β‐gal) reporter genes were also delivered. The Luc and hRluc activities in the muscle lysates were assayed. The EGFP and GAPD expression was detected by fluorescence microscopic observation and RT‐PCR, respectively.

Results

When Luc‐specific siRNA was co‐delivered with the Luc expression vector into the tibial muscle, the reporter gene expression was markedly suppressed (less than 1% of the control level) for 5 days. As little as 0.05 µg of siRNA almost completely blocked the reporter gene expression from 10 µg of the plasmid. To examine whether siRNA can also suppress expression of an endogenous gene, transgenic mice carrying the EGFP gene received intramuscular transfection of a mixture of β‐gal plasmid and GFP‐specific siRNA. β‐Gal‐positive cells failed to express detectable levels of EGFP, while EGFP expression was not inhibited in control mice that received nonspecific siRNA. Expression of GAPD was also suppressed by the specific siRNA.

Conclusions

The present system may provide a useful means of phenotypic analysis of genetic information in mammalian organs for basic research as well as therapeutic molecular targeting in the post‐genomic era. Copyright © 2003 John Wiley & Sons, Ltd.
Intramuscular SP1017‐formulated DNA electrotransfer enhances transgene expression and distributes hHGF to different rat tissues
Authors: Riera Marta , Chillon Miguel , Aran Josep M. , Cruzado Josep M. , Torras Joan , Grinyó Josep M. , Fillat Cristina
Published: January 2004

Background

The systemic administration of a therapeutic protein is a common approach for the treatment of multiple disorders. Intramuscular (i.m.) injection of plasmids, followed by electroporation, has been shown to facilitate naked DNA gene transfer in skeletal muscle allowing proteins to be produced and secreted at therapeutically relevant levels.

Methods

Plasmid DNA, unformulated or formulated with the non‐ionic carrier SP1017, was injected at the rat tibialis anterior muscle followed by the application of electric pulses. Follow‐up of protein expression was measured.

Results

In our study we report that the non‐ionic carrier SP1017 significantly increases transgene expression in rat muscle after the i.m. injection of a formulated‐pCMVβ plasmid followed by electroporation. Such increased expression was observed after delivering square‐wave unipolar electric pulses at two different field strengths: low (110 V/cm) and high (175 V/cm). Moreover, elevated secreted placental alkaline phosphatase (SEAP) plasma levels were achieved with low‐voltage (110 V/cm) electroporation. Our results also show that human hepatocyte growth factor (hHGF) can be produced from rat muscle and delivered to blood circulation at a biologically active level after a single i.m. injection of an SP1017‐formulated plasmid (pCMV/hHGF) followed by electroporation. Tissue distribution studies mostly identified hHGF in the liver, but it was also found in the kidneys and lungs suggesting that here too the HGF could be of therapeutic benefit.

Conclusions

Our results indicate that SP1017 enhances the expression of electrotransferred genes. Such a delivery approach could prove an efficient method for the systemic production of therapeutic proteins. Copyright © 2003 John Wiley & Sons, Ltd.