bluuebunny/crossref_metadata_2025 huggingface dataset:
Hetero-oligomeric tagging diminishes non-specific aggregation of target proteins fused with Anthozoa fluorescent proteins
Authors: BULINA Maria E. , VERKHUSHA Vladislav V. , STAROVEROV Dmitry B. , CHUDAKOV Dmitry M. , LUKYANOV Konstantin A.
Published: March 2003

The tendency for tetramerization is the main disadvantage in the green fluorescent protein homologues from Anthozoa species. We report a universal method called hetero-oligomeric tagging, which diminishes troublesome consequences of tetramerization of Anthozoa-derived fluorescent proteins (FP) in intracellular protein labelling. This approach is based on the co-expression of the FP-tagged protein of interest together with an excess of free non-fluorescent FP mutant. The resulting FP heterotetramers contain only a single target polypeptide and, therefore, can be considered pseudo-monomeric. Feasibility of the method has been demonstrated with a red FP fused with cytoplasmic β-actin or tubulin-binding protein Tau34. In addition, heterotetramers appeared to be a unique model for biophysical characterization of Anthozoa FPs in pseudo-monomeric state.
A randomized 4-arm multicenter study of interferon alfa-2b plus ribavirin in the treatment of patients with chronic hepatitis C relapsing after interferon monotherapy
Authors: Saracco Giorgio , Olivero Alda , Ciancio Alessia , Carenzi Silvia , Smedile Antonina , Cariti Giuseppe , Andreoni Massimo , Orsi Pier Giulio , Biglino Alberto , Tabone Marco , Roffi Luigi , Croce Guido , Manca Aldo , Tappero Gianfranco , Ciccone Giovannino , Rizzetto Mario
Published: March 2003

To determine whether a higher dosage of interferon (IFN) and/or a prolonged time of administration may improve the efficacy of combination therapy, we conducted a 4-arm randomized trial on patients with chronic hepatitis C relapsing after 1 or more previous treatment courses with IFN monotherapy. Group A (n = 70) received 3 MU IFN alfa-2b 3 times per week plus ribavirin 1,000 mg/d for 12 months; group B (n = 70) received 5 MU 3 times per week plus ribavirin for 12 months; group C (n = 82) received 3 MU 3 times per week plus ribavirin for 6 months, and group D (n = 73) received 5 MU 3 times per week plus ribavirin for 6 months. The primary end point was the clearance of viremia at the end of 6-month follow-up: test results for hepatitis C virus (HCV)-RNA were negative in 54% of group A, 56% of group B, 40% of group C, and 49% of group D patients (P = NS). Among patients with genotype 1 and 4, the sustained response was significantly higher in groups A and B than in group C (45%, 49% vs. 22%, P = .03; group D = 33%, P = NS). In patients with genotype 2 and 3, the sustained virologic response was not affected by the different regimens (group A = 69%, group B = 68%, group C = 62%, group D = 71%, P = NS). In conclusion, duration of therapy rather than IFN dosage is more important in increasing the sustained virologic rate among HCV-positive patients with genotype 1 and 4 relapsing after IFN monotherapy; patients with genotypes 2 and 3 can be effectively retreated with a 6-month course of combination therapy, avoiding unnecessary side effects and waste of resources.
Hepatitis C in adults and adolescents with hemophilia: A randomized, controlled trial of interferon alfa-2b and ribavirin
Authors: Fried Michael W. , Peter Joy , Hoots Keith , Gaglio Paul J. , Talbut Donald , Davis Charleton P. , Key Nigel S. , White Gilbert C. , Lindblad Lauren , Rickles Frederick R. , Abshire Thomas C.
Published: March 2003

Adolescents and adults with inherited disorders of coagulation have one of the highest prevalence rates of hepatitis C among known risk groups. Few data are available on the use of combination therapy with interferon and ribavirin in this population. Patients 13 years of age and older who were positive for hepatitis C virus (HCV) RNA by polymerase chain reaction and negative for human immunodeficiency virus were randomized to receive interferon alfa-2b (3 million units 3 times a week) plus ribavirin (1,000 mg/day) or interferon alfa-2b alone for 48 weeks with 24 weeks of posttreatment follow-up. Patients started on interferon alone who remained positive for HCV RNA at week 12 crossed over to treatment with interferon plus ribavirin. A total of 113 patients were treated. Thirty-seven patients were younger than 18 years. At the end of treatment, 18 of 56 (32%) treated with interferon plus ribavirin and 6 of 57 (11%) treated with interferon alone were negative for HCV RNA (P = .005). Sustained virologic response in the combination arm was 29% (16 of 56) compared with 7% (4 of 57) for those started on interferon alone (P = .027). Among adolescents younger than 18 years who were treated with combination therapy, 10 of 17 (59%) had sustained response compared with 6 of 39 (15%) of adult patients on the same regimen (P = .001). In conclusion, in this U.S. multicenter, randomized trial of therapy for HCV in patients with inherited bleeding disorders, sustained virologic response rate was significantly improved for patients treated with interferon and ribavirin compared with those started on interferon alone. Adolescents treated with combination therapy had a significantly higher sustained response than adults did on the same regimen.
Predictive value of ALT levels for histologic findings in chronic hepatitis C: A European collaborative study
Authors: Pradat Pierre , Alberti Alfredo , Poynard Thierry , Esteban Juan-Ignacio , Weiland Ola , Marcellin Patrick , Badalamenti Salvatore , Trépo Christian
Published: March 2003

The aim of this retrospective study was to determine the predictive value of alanine aminotransferase (ALT) levels for histologic findings in patients with chronic hepatitis C virus (HCV) infection. Data on 864 HCV RNA-positive patients were collected. ALT values were obtained at the time of biopsy (before treatment), and normal ALT values were defined as normal values obtained at serial evaluations during a 6-month period. Histologic results were scored using the METAVIR system. Among all patients, 99% of those with elevated ALT levels had a score of at least F1 (positive predictive value [PPV], 99%) and 88% had a score greater than A1F1. Among patients with persistently normal ALT values, 65% had a score of at least F1 (negative predictive value [NPV], 35%) and 26% had a score greater than A1F1. The receiver operating characteristics analysis indicates that the ALT threshold for the best compromise sensitivity-specificity was about 2.25 times the upper limit of normal (ULN). In conclusion, almost all HCV RNA-positive patients with elevated ALT levels have some degree of fibrosis. However, an important proportion of patients with persistently normal ALT levels also show some histologic signs of fibrosis; the degree of fibrosis is usually mild but is sometimes more marked, and in rare cases cirrhosis may be present. In this subset of patients, the indication of liver biopsy and the potential benefit of therapy need to be further evaluated. These results suggest the need to revisit the algorithm for liver biopsy practice.
Uroporphyria in the uroporphyrinogen decarboxylase-deficient mouse: Interplay with siderosis and polychlorinated biphenyl exposure
Authors: Franklin Michael R. , Phillips John D. , Kushner James P.
Published: March 2003

Several methods have been used to develop rodent models with the hepatic manifestations of porphyria cutanea tarda (PCT). Acute iron administration or mutations of the hemochromatosis gene (Hfe) have been used to generate hepatic siderosis, a nearly uniform finding in PCT. Heterozygosity for a null mutation at the uroporphyrinogen decarboxylase (Uro-D+/−) locus has been developed to mimic familial PCT in humans. This study examines the interplay of these 2 genetic risk factors and their influence, alone and combined with polychlorinated-biphenyl exposure. Neither an Hfe-null mutation nor iron-dextran administration alone or in combination with polychlorinated biphenyl exposure was porphyrinogenic in a 3-week model using mice wild-type at the Uro-D locus. Homozygosity for an Hfe-null mutation significantly elevated hepatic iron but not to the extent seen with parenteral iron-dextran administration. Homozygosity for an Hfe-null mutation but not iron-dextran administration was porphyrinogenic in animals heterozygous for the Uro-D mutation. Polychlorinated biphenyls were also porphyrinogenic in these animals. Uroporphyria in Uro-D+/− animals was exacerbated by combinations of the homozygous Hfe-null mutation and polychlorinated biphenyls and iron-dextran and polychlorinated biphenyls. In all cases in which uroporphyria developed, a greater degree of experimental uroporphyria was seen in female animals. All elevated hepatic uroporphyrin concentrations were accompanied by depressed uroporphyrinogen decarboxylase activity and the presence of a factor in cytosol that inhibits recombinant human uroporphyrinogen decarboxylase. In conclusion, the expression of the uroporphyric phenotype, dependent on the susceptibility imparted by a genetic mutation, provides a uniquely facile model for dissecting the molecular pathogenesis of the disease.
Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse
Authors: Yoshiji Hitoshi , Kuriyama Shigeki , Yoshii Junichi , Ikenaka Yasuhide , Noguchi Ryuichi , Nakatani Toshiya , Tsujinoue Hirohisa , Yanase Koji , Namisaki Tadashi , Imazu Hiroo , Fukui Hiroshi
Published: March 2003

It has been suggested that the tissue inhibitor of metalloproteinases-1 (TIMP-1) is involved in spontaneous resolution of liver fibrosis. The aim of this study was to investigate whether TIMP-1 altered spontaneous resolution of liver fibrosis in conjunction with matrix metalloproteinases (MMP) inhibition and hepatic stellate cell (HSC) activation. The livers of liver-targeted TIMP-1 transgenic (TIMP-Tg) and control hybrid (Cont) mice were harvested at 0, 3, 7, and 28 days following spontaneous recovery from CCl4-induced liver fibrosis. The extent of fibrosis resolution, MMP expression, α-smooth-muscle actin (α-SMA) positive cells, and procollagen-(I) messenger RNA (mRNA) in the liver were assessed at the respective periods in both groups. We also examined the effect of TIMP-1 on HSC apoptosis. The TIMP-Tg mice showed significantly attenuated resolution of spontaneous liver fibrosis compared with the Cont mice. The hydroxyproline content, number of α-SMA positive cells, and procollagen-(I) mRNA rapidly decreased with time in the Cont mice, whereas these markers were little changed in TIMP-Tg mice. The level of the active form of metalloproteinases-2 (MMP-2) in the TIMP-Tg mice was less than that in the Cont mice. TIMP-1 markedly decreased the nonparenchyma apoptotic cells in the liver fibrosis resolution model, and it also inhibited HSC apoptosis associated with suppression of caspase-3 activity in vitro. In conclusion, TIMP-1 significantly attenuated spontaneous resolution of liver fibrosis by the combination of a net reduction of the MMP activity and suppression of apoptosis in HSC.
Terlipressin therapy with and without albumin for patients with hepatorenal syndrome: Results of a prospective, nonrandomized study
Authors: Ortega Rolando , Ginès Pere , Uriz Juan , Cárdenas Andrés , Calahorra Blas , De Las Heras Dara , Guevara Mónica , Bataller Ramón , Jiménez Wladimiro , Arroyo Vicente , Rodés Juan
Published: March 2003

Vasopressin analogues associated with albumin improve renal function in hepatorenal syndrome (HRS). The current study was aimed at assessing the efficacy of the treatment, predictive factors of response, recurrence of HRS, and survival after therapy. Twenty-one consecutive patients with HRS (16 with type 1 HRS, 5 with type 2 HRS) received terlipressin (0.5-2 mg/4 hours intravenously) until complete response was achieved (serum creatinine level < 1.5 mg/dL) or for 15 days; 13 patients received intravenous albumin together with terlipressin. Twelve of the 21 patients (57%) showed complete response. Albumin administration was the only predictive factor of complete response (77% in patients receiving terlipressin and albumin vs. 25% in those receiving terlipressin alone, P = .03). Treatment with terlipressin and albumin was associated with a remarkable decrease in serum creatinine level, increase in arterial pressure, and suppression of the renin-aldosterone system. By contrast, no significant changes in these parameters were found in patients treated with terlipressin alone. Only 1 patient showed ischemic adverse effects. Recurrence of HRS occurred in 17% of patients with complete response. The occurrence of complete response was associated with an improved survival. In conclusion, terlipressin therapy reverses HRS in a high proportion of patients. Recurrence rate after treatment withdrawal is uncommon. Albumin appears to improve markedly the beneficial effects of terlipressin.
The significance of human jagged 1 mutations detected in severe cases of extrahepatic biliary atresia
Authors: Kohsaka Takao , Yuan Zeng-rong , Guo Shu-xia , Tagawa Manabu , Nakamura Akio , Nakano Miwako , Kawasasaki Hideo , Inomata Yukihiro , Tanaka Koichi , Miyauchi Jun
Published: March 2003

Mutations of human jagged 1 (JAG1) gene are responsible for Alagille Syndrome (AGS), whose 2 main symptoms are intrahepatic bile duct hypoplasia and pulmonary stenosis. We examined the JAG1 mutation in extrahepatic biliary atresia (EHBA), which is similar in phenotype to AGS, although a different pathogenesis is suggested. In 102 cases of EHBA, 9 missense mutations were detected, including 2 intrafamilial expressions in the propositus and an aunt of one family. These mutations were all missense and sporadic except for those of this particular family. The JAG1 gene mutations were generally found in severely ill patients subjected to liver transplantation at less than 5 years of age. None of the 9 cases of EHBA revealed any of the 5 major symptoms of AGS nor any identical pathological findings after 3 years of follow-up. Our cases were clearly separated from AGS by pathological findings and clinical features, and could be diagnosed as EHBA and not as atypical AGS. The increase of interleukin 8 (IL-8) production induced by tumor necrosis factor α (TNF-α) in Huh 7 cells was suppressed by the coexistence of JAG1 protein. We examined the different influences between wild-type cells and the 3 kinds of mutants detected in EHBA on Huh 7 cells and found that 2 of 3 mutants showed about half of the repressed activity compared with that of wild type. In conclusion, these results suggest that the JAG1 gene abnormality may be an aggravating factor in EHBA.
p53 gene and Wnt signaling in benign neoplasms: β-catenin mutations in hepatic adenoma but not in focal nodular hyperplasia
Authors: Chen Ya-Wen , Jeng Yung-Ming , Yeh Shiou-Hwei , Chen Pei-Jer
Published: March 2003

Hepatocellular adenoma (HA) and focal nodular hyperplasia (FNH) are 2 rare, benign liver neoplasms that often are discovered incidentally. To date, few genetic changes have been found in these 2 benign lesions. However, the 2 pathways of p53 and Wnt signaling, which may be the most common molecular targets involved in liver tumorgenesis, were studied in HA and FNH. Ten HAs and 11 FNHs were analyzed for loss of heterozygosity (LOH) and sequencing analysis of mutation hot spots in exons 5 to 8 of the p53 gene. No LOH or mutant sequences were identified, indicating that p53 was not associated with these benign lesions. Genes in the Wnt signaling pathway, including β-catenin, axin, and adenomatous polyposis coli (APC), also were studied. Polymerase chain reaction (PCR) amplification and direct sequencing of all samples of HA and FNH displayed no mutations in exon 3 of the β-catenin gene. However, 3 HAs (30%) contained interstitial deletions from exon 3 to exon 4. Truncated forms of β-catenin detected by Western blot and immunohistochemical analyses showed they had accumulated in the cytoplasm and nuclei. However, for the axin and APC genes, no genetic changes, including allelic loss, interstitial deletions and point mutations, were detected in any of the HAs and FNHs. In conclusion, β-catenin, which participates in the Wnt signaling pathway, might play a more important role in the formation of HA than in that of FNH, but p53 is not associated with the development of either HA or FNH.
Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model
Authors: Forns Xavier , Ampurdanès Sergi , Llovet Josep M. , Aponte John , Quintó Llorenç , Martínez-Bauer Eva , Bruguera Miquel , Sánchez-Tapias Jose Maria , Rodés Juan
Published: March 2003

Liver biopsy is required for staging hepatic fibrosis in patients with chronic hepatitis C, but it is an expensive procedure with occasional complications and poor patient acceptance. This cohort study was designed to assess the accuracy of a noninvasive method aimed to discriminate between patients with and without significant liver fibrosis (stages 2-4 versus 0-1). Clinically relevant variables were analyzed in a cohort of 476 consecutive untreated patients (estimation group, 351 patients; validation group, 125 patients) with chronic hepatitis C who underwent a liver biopsy. Multivariate analysis identified age, gamma glutamyl transpeptidase (GGT), cholesterol, platelet count, and prothrombin time as independent predictors of fibrosis. We constructed a model and a score system combining age, GGT, cholesterol, and platelet count that proved useful to identify patients without significant hepatic fibrosis. The area under the ROC curve was 0.86 for the estimation group and 0.81 for the validation group. Using the best cutoff score (less than 4.2), presence of significant fibrosis (F2 to F4) could be excluded with high accuracy (negative predictive value of 96%) in 125 (36%) of 351 patients. Similarly, it could be excluded with the same certainty in 49 (39%) of the 125 patients of the validation group. Only 2 patients with liver fibrosis stage 2 were incorrectly classified. In conclusion, a combination of easily accessible variables accurately predicts the absence of significant fibrosis and might render liver biopsy unnecessary in more than one third of patients with chronic hepatitis C.
Preemptive lamivudine therapy based on HBV DNA level in HBsAg-positive kidney allograft recipients
Authors: Chan Tak Mao , Fang Guo Xiang , Tang Colin S. O. , Cheng Ignatius K. P. , Lai Kar Neng , Ho Stephen K. N.
Published: March 2003

Hepatitis B surface antigen (HBsAg)-positive kidney transplant recipients have increased liver-related mortality. The impact of lamivudine treatment on patient survival, the optimal time to start treatment, and the feasibility of discontinuing treatment have not been determined. This study examined these issues with a novel management protocol. Serum hepatitis B virus (HBV) DNA levels were measured serially in HBsAg-positive kidney transplant recipients, and lamivudine was administered preemptively to patients with increasing HBV DNA levels with or without elevation of aminotransferase levels. Outcomes of patients who underwent transplantation before or after institution of this preemptive management strategy (in January 1996) were compared. Eleven de novo patients (91.7%) who underwent transplantation between 1996 and 2000 and 15 existing patients (39.5%) who underwent transplantation between 1983 and 1995 received preemptive lamivudine therapy for 32.6 ± 13.3 months. The treatment criteria were met by de novo patients at 8.4 ± 6.2 months (range, 1-18 months) after transplantation. Suppression of HBV DNA and normalization of aminotransferase levels were achieved in all treated patients, and 21.4% had hepatitis B e antigen (HBeAg) seroconversion. The survival of preemptively managed de novo transplant patients was similar to that of HBsAg-negative controls, whereas HBsAg-positive patients who underwent transplantation before January 1996 had inferior survival (relative risk of death, 9.7 [P < .001]; relative risk of liver-related mortality, 68.0 [P < .0001]). Eleven patients (40.7%) developed lamivudine resistance. Discontinuation of lamivudine was attempted in 12 low-risk patients after stabilization and was successful in 5 (41.7%). In conclusion, preemptive lamivudine therapy based on serial HBV DNA levels and clinical monitoring improved the survival of HBsAg-positive renal allograft recipients. Treatment can be discontinued safely in selected patients after stabilization to minimize the selection of drug-resistant HBV mutants.
Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma
Authors: Price Julie A. , Kovach Stephen J. , Johnson Timothy , Koniaris Leonidas G. , Cahill Paul A. , Sitzmann James V. , McKillop Iain H.
Published: March 2003

Hepatocyte growth factor-scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P < .05). Despite activation of a MAPK pathway and increased proto-oncogene expression, IGF-I failed to significantly affect cell mitogenesis. In contrast, HGF significantly inhibited cell mitogenesis in HCC lines (68.4% ± 9.4% vs. control, n = 4, P < .05). Pretreatment of HCC cells with IGF-I (60 minutes) led to significant HGF-SF stimulation of total cell mitogenesis dependent on both IGF-I and HGF-SF dose (194% ± 8% increase vs. control, n = 4, P < .05). In conclusion, tumor burden is important in altering intrahepatic growth factor synthesis. Signal cooperation between multiple cytokine pathways is an important factor in the progression of HCC.
Loss of cyclin D1 does not inhibit the proliferative response of mouse liver to mitogenic stimuli
Authors: Ledda-Columbano Giovanna M. , Pibiri Monica , Concas Danilo , Cossu Costanza , Tripodi Marco , Columbano Amedeo
Published: March 2003

Cyclin D1 is considered to play a critical role in the progression from G1 to S phase of the cell cycle, and its overexpression is seen in many human tumors. However, previous studies in cell lines have shown that cyclin D1 is not sufficient to trigger cell replication. To directly test the role of cyclin D1 in the progression of the cell cycle, we have examined the proliferative response of hepatocytes to the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in mice with homozygous disruption of the cyclin D1 gene. We found that 24 hours after administration of TCPOBOP, the number of bromodeoxyuridine (BrdU)-positive hepatocytes was significantly reduced in cyclin D1−/− (labeling index was 1.9% in knockout mice vs. 9.7% of heterozygous mice); however, no difference in the number of proliferating hepatocytes was found 36 or 72 hours after treatment (labeling index was 16% and 43% in cyclin D1−/− mice vs. 20% and 41% of heterozygous mice), indicating that lack of cyclin D1 may transiently delay entry into S phase but is not sufficient to inhibit the response of hepatocytes to mitogenic stimuli. The results also show that although there was no difference in hepatic protein levels of cyclin D2 and D3 between untreated cyclin D1−/− and cyclin D1+/− mice, messenger RNA (mRNA) and protein levels of cyclin E were much higher in the former. In conclusion, our results show that cyclin D1 is not essential for liver development and hepatocyte proliferation induced by mitogenic stimuli and suggest that overexpression of cyclin E may compensate for the lack of cyclin D1. (Hepatology 2002;36:1098-1105.)
A pilot study of interferon alfa and ribavirin combination in liver transplant recipients with recurrent hepatitis C
Authors: Shakil Obaid A. , McGuire Brendon , Crippin Jeff , Teperman Lewis , Demetris Jake A. , Conjeevaram Hari , Gish Robert , Kwo Paul , Balan Vijayan , Wright Teresa L. , Brass Clifford , Rakela Jorge
Published: March 2003

Although interferon alfa (IFN-α) and ribavirin are widely used in the treatment of hepatitis C, their role in the transplant recipient is unclear. We conducted a pilot study to determine the efficacy and safety of this therapy in transplant recipients with recurrent hepatitis C. Patients at least 6 months posttransplantation were treated with IFN-α 3 million units 3 times a week subcutaneously and ribavirin 800 mg daily by mouth for 48 weeks followed by ribavirin monotherapy for 24 weeks. The primary end point was sustained virologic response, and secondary end points included biochemical, virologic, and histologic responses at the end of combination treatment. Thirty-eight patients initiated therapy but 16 withdrew due to adverse effects, including 2 with myocardial infarction. Median age was 50 years; 74% were men, and 91% had genotype 1. The median interval between transplantation and enrollment was 23 months. On an intention-to-treat basis, 7 patients (18%) had a biochemical and 5 (13%) had a virologic response at the end of combination treatment. Inflammatory activity did not change, but fibrosis worsened in virologic nonresponders. Ribavirin maintenance caused a further decrease in serum alanine aminotransferase levels, but hepatitis C virus (HCV) RNA levels increased. Only 2 of the 38 patients (5%) had a sustained virologic response. Several patients required treatment with erythropoietin for anemia. In conclusion, IFN-α and ribavirin are effective in a small proportion of liver allograft recipients with recurrent hepatitis C. Adverse effects occur commonly, requiring dose reductions and treatment withdrawal.
Bone metabolism in advanced cholestatic liver disease: Analysis by bone histomorphometry
Authors: Guichelaar Maureen M. J. , Malinchoc Michael , Sibonga Jean , Clarke Bart L. , Hay Eileen J.
Published: March 2003

Despite the clinical importance of cholestatic osteopenia, little is known about its pathophysiologic mechanism. By tetracycline-labeled histomorphometric analysis of bone biopsies taken at the time of liver transplantation, we prospectively evaluated bone resorption and formation in 50 consecutive patients with advanced primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Histomorphometric analysis confirmed low bone volume parameters, consistent with the mean T-score of the lumbar spine of −1.9 by dual energy x-ray absorptiometry. Dynamic (bone formation rates, adjusted apposition rates) and static (osteoid markers, osteoblast number) parameters of bone formation were decreased in cholestatic patients with no abnormalities in mineralization. Increased osteoclast numbers and increased eroded surface areas suggested increased bone resorption, and this was supported in female patients by increased trabecular separation and decreased trabecular number. Male cholestatic patients, however, did not have significant increases in resorption parameters, although they were as osteopenic as female patients and had low bone formation markers. Bone histomorphometric changes were similar in PBC and PSC, suggesting an etiologic effect of chronic cholestasis rather than the individual diseases. Cancellous bone volume and osteoid markers correlated with bone mineral density measurements but no correlations were found between histomorphometric parameters and biochemical markers of bone metabolism. In conclusion, cholestatic osteopenia appears to result from a combination of decreased bone formation and increased resorption, especially in female patients, but the relative importance of these two abnormalities and their actual etiology remain to be elucidated.
Region selective alterations of soluble guanylate cyclase content and modulation in brain of cirrhotic patients
Authors: Corbalán Regina , Chatauret Nicolas , Behrends Sönke , Butterworth Roger F. , Felipo Vicente
Published: March 2003

Modulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) is altered in brain from experimental animals with hyperammonemia with or without liver failure. The aim of this work was to assess the content and modulation of sGC in brain in chronic liver failure in humans. Expression of the α-1, α-2, and β-1 subunits of sGC was measured by immunoblotting in autopsied frontal cortex and cerebellum from cirrhotic patients and controls. The contents of α-1 and α-2 subunits of guanylate cyclase was increased both in cortex and cerebellum, whereas the β-1 subunit was not affected. Addition of the NO-generating agent S-nitroso-N-acetyl-penicillamine (SNAP) to homogenates of frontal cortex from controls increased the activity of sGC 87-fold, whereas, in homogenates from cirrhotic patients, the increase was significantly higher (183-fold). In contrast, in cerebellum, activation of guanylate cyclase by NO was significantly lower in patients (156-fold) than in controls (248-fold). A similar regional difference was found in rats with portacaval anastomosis. In conclusion, these findings show that the NO-guanylate cyclase signal transduction pathway is strongly altered in brain in patients with chronic liver failure and that the effects are different in different brain areas. Given that activation of sGC by NO in brain is involved in the modulation of important cerebral processes such as intercellular communication, learning and memory, and the sleep-wake cycle, these changes could be implicated in the pathogenesis of hepatic encephalopathy in these patients.
Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong, China
Authors: Ming Lihua , Thorgeirsson Snorri S. , Gail Mitchell H. , Lu Peixin , Harris Curtis C. , Wang Nengjin , Shao Yongfu , Wu Zhiyuan , Liu Guoting , Wang Xiaohong , Sun Zongtang
Published: March 2003

We assessed the separate and combined effects of hepatitis B virus (HBV), hepatitis C virus (HCV), and aflatoxin in causing hepatocellular carcinoma (HCC) in Qidong, China. A consecutive series of 181 pathologic-diagnosed HCC cases were studied for hepatitis B surface antigen (HBsAg), anti-HBc, HBV X gene sequence, anti-HCV, the 249ser-p53 mutation, and chronic hepatitis pathology. Each of the 181 incident HCC cases had markers for HBV infection and hepatitis pathology; only 6 of 119 cases were coinfected with HCV. The 249ser-p53 mutation was found in 54% (97/181) of HCC cases and in all 7 cases with tissue for analysis from the hepatitis cohort but in none of 42 matched cases from Beijing. The estimated cumulative dose of aflatoxin B1 in these 7 cases ranged from 0.13 to 0.49 mg/kg. Follow-up data through 13.25 years on a cohort of 145 men with chronic HBV hepatitis showed that the relative risk from aflatoxin exposure was 3.5 (1.5-8.1). A similar relative risk was found using 249ser-p53 mutation as a marker for aflatoxin exposure. In conclusion, HBV hepatitis is ubiquitous in Qidong HCC cases, whereas HCV contributes little to its risk. The 249ser-p53 mutation appears to result from coexposure to aflatoxin and HBV infection. Even modest levels of aflatoxin exposure tripled the risk of HCC in HBV-infected men.
Trafficking and functional defects by mutations of the ATP-binding domains in MRP2 in patients with Dubin-Johnson syndrome
Authors: Hashimoto Kenkichi , Uchiumi Takeshi , Konno Toshikazu , Ebihara Takuya , Nakamura Takanori , Wada Morimasa , Sakisaka Shotaro , Maniwa Fumio , Amachi Teruo , Ueda Kazumitsu , Kuwano Michihiko
Published: March 2003

Dubin-Johnson syndrome (DJS) is a hereditary disease characterized by hyperbilirubinemia. We investigated the consequences of 2 missense mutations, R768W and Q1382R, of nucleotide-binding domains (NBDs) of the multidrug resistance protein 2 (MRP2; ABCC2) that were previously identified in patients with DJS. Pulse chase analysis revealed that the precursor form of the wild-type and Q1382R MRP2 were converted to the mature form, which is resistant to endoglycosidase H (Endo H) in about 60 minutes. However, the precursor form of the R768W MRP2, which is sensitive to endoglycosidase H, was degraded within 120 minutes and did not mature to the fully glycosylated form. Proteasome inhibitors inhibited the degradation of the precursor form of the R768W MRP2. Unlike the R768W MRP2, the Q1382R MRP2 was mainly localized on the apical membrane in the wild-type form. However, efflux of glutathione monochlorobimane (GS-MCLB) and ATP-dependent leukotriene C4 (LTC4) uptake into plasma membrane vesicles from cells expressing the Q1382R MRP2 were markedly reduced, suggesting that the Q1382R MRP2 on the apical membrane was nonfunctional. Vanadate-induced nucleotide trapping with 8-azido-[α-32P]ATP in the wild-type MRP2 was stimulated by estradiol glucuronide (E217βG) in a concentration-dependent manner but that in the Q1382R MRP2 was not. In conclusion, the R768W mutation causes deficient maturation and impaired sorting, and the Q1382R mutation does not affect maturation or sorting but impairs the substrate-induced ATP hydrolysis.
Synergistic induction of apoptosis by acyclic retinoid and interferon-β in human hepatocellular carcinoma cells
Authors: Obora Akihiro , Shiratori Yoshimune , Okuno Masataka , Adachi Seiji , Takano Yukihiko , Matsushima-Nishiwaki Rie , Yasuda Ichiro , Yamada Yasuhiro , Akita Kuniharu , Sano Tetsuro , Shimada Jun , Kojima Soichi , Okano Yukio , Friedman Scott L. , Moriwaki Hisataka
Published: March 2003

Acyclic retinoid, a synthetic retinoid analog, as well as interferon alfa (IFN-α) and IFN-β induce apoptosis in hepatocellular carcinoma (HCC) cells and are used clinically in the prevention of HCC. Here, we show that acyclic retinoid acts synergistically with IFNs in suppressing the growth and inducing apoptosis (as characterized by DNA fragmentation and chromatin condensation) in 5 human HCC cell lines (JHH7, HuH7, PLC/PRF/5, HLE, and HLF). This synergism was only observed when cells were pretreated with the acyclic retinoid, whereas natural retinoic acids (all-trans and 9-cis retinoic acid) were ineffective. This promotion may be due to up-regulation of type 1 IFN receptor (IFNR) expression by the retinoid. Accordingly, incubation with antitype 1 IFNR antibody abolished the synergy. Enhanced IFNR expression was accompanied by increased expression and DNA-binding activity of STAT1, an intracellular signal transducing molecule of IFNR, and increased induction of 2’, 5’-oligoadenyl-5’-triphosphate synthetase, which is a target gene of STAT1. Acyclic retinoid did not have any effects on the growth of normal human hepatocytes (Hc) probably because of a lack of IFNR and STAT1 up-regulation. In conclusion, these results provide a rationale for combined biochemoprevention of HCC using acyclic retinoid and IFN-β.
Hepatitis C virus genotype 3 is cytopathic to hepatocytes: Reversal of hepatic steatosis after sustained therapeutic response
Authors: Kumar Dinesh , Farrell Geoffrey C. , Fung Caroline , George Jacob
Published: March 2003

On the basis of cross-sectional studies, it has been proposed that hepatic steatosis is a cytopathic effect of hepatitis C virus (HCV) genotype 3 but not genotype 1 infections. We tested this hypothesis by examining whether antiviral treatment altered hepatic steatosis in chronic hepatitis C. In 28 patients with genotype 1 and 34 with genotype 3 HCV, we determined the severity of steatosis in pre- and posttreatment liver biopsies using computer-assisted morphometric image analysis as well as conventional semiquantitative scoring. Before treatment, hepatic steatosis was present in 16 (57%) patients infected with HCV genotype 1 and 21 (62%) of those with genotype 3. Sustained viral response (SVR) was achieved in 9 (32%) patients with genotype 1 and 22 (65%) with genotype 3. In neither group were there significant changes in body weight or alcohol consumption between pre- and posttreatment biopsies. In patients with HCV genotype 1, there was no change in hepatic steatosis after treatment, irrespective of the treatment response. Among those infected with genotype 3, SVR significantly reduced steatosis (
P
< .001), but there was no change in steatosis among those without a SVR. By logistic regression analysis, SVR was the only variable predictive of improvement in hepatic steatosis (OR = 36, 95% CI = 2.7-481, P = .007). In conclusion, these data provide strong support for a direct causal association between HCV genotype 3 infection and hepatic steatosis.
Hypoxia is an inducer of vasodilator agents in peritoneal macrophages of cirrhotic patients
Authors: Cejudo-Martín Pilar , Morales-Ruiz Manuel , Ros Josefa , Navasa Miguel , Fernández-Varo Guillermo , Fuster Josep , Rivera Francisca , Arroyo Vicente , Rodés Juan , Jiménez Wladimiro
Published: March 2003

The aim of the investigation was to assess whether hypoxia induces the production of endogenous vasoactive peptides in macrophages of cirrhotic patients with ascites because low tissue oxygenation is a relatively frequent event in these patients. Peritoneal macrophages were isolated from ascites, seeded on well plates, and cultured at different times under hypoxic (5% O2) or normoxic conditions (21% O2). Then, accumulation of vasoactive peptides sensitive to hypoxia including endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and adrenomedullin (ADM) was measured. Only VEGF and ADM were constitutively secreted, and hypoxia further stimulated the release of these vasodilator peptides. In concordance, increased messenger RNA (mRNA) levels of VEGF and ADM were found at culturing macrophages in hypoxia. This characteristic response was not observed in circulating monocytes of either cirrhotic patients or healthy subjects. Next the expression of the transcription factor, hypoxia inducible factor 1 (HIF-1), was analyzed. Expression of HIF-1α and HIF-1β messengers and HIF-1β protein subunit remained unchanged regardless of O2 tension, whereas HIF-1α protein subunit was overexpressed under hypoxic conditions. Moreover, conditioned medium from macrophages cultured under hypoxic conditions promoted a larger nitric oxide (NO) release in endothelial cells than that of normoxic macrophages. In conclusion, these data indicate that hypoxia induces the synthesis of VEGF and ADM in macrophages of cirrhotic patients, likely through HIF-1-enhanced transcriptional activity. These data suggest that a local reduction in O2 tension could enhance the synthesis of macrophage-derived vasodilators, thus aggravating the circulatory disturbance of these patients.
Specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in SCID mice
Authors: Wolschek Markus F. , Thallinger Christiane , Kursa Malgorzata , Rössler Vanessa , Allen Matthew , Lichtenberger Cornelia , Kircheis Ralf , Lucas Trevor , Willheim Martin , Reinisch Walter , Gangl Alfred , Wagner Ernst , Jansen Burkhard
Published: March 2003

Systemic tumor-targeted gene delivery is attracting increasing attention as a promising alternative to conventional therapeutical strategies. To be considered as a viable option, however, the respective transgene has to be administered with high tumor specificity. Here, we describe novel polyethylenimine (PEI)-based DNA complexes, shielded by covalent attachment of polyethylene glycol (PEG), that make use of epidermal growth factor (EGF) as a ligand for targeting gene delivery to EGF receptor-expressing human hepatocellular carcinoma (HCC) cells.
In vitro
transfection of luciferase reporter DNA resulted in high levels of gene expression in the human HCC cell lines Huh-7 and HepG2. An excess of free EGF during transfection clearly reduced expression levels, indicating a specific EGF receptor-mediated uptake of the DNA particles. Following intravenous injection into human HCC xenograft-bearing SCID mice, luciferase expression was predominantly found in the tumor, with levels up to 2 logs higher than in the liver, which was the highest expressing major organ. Histologic investigation showed reporter gene expression (β-galactosidase) localized to tumor cells. Assessing DNA distribution within the tumor by immunofluorescence microscopy, rhodamine-labelled transgene DNA was found to be mainly associated with HCC cells. In the liver, DNA was taken up almost exclusively by Kupffer cells and, as indicated by the low expression, subsequently degraded. In conclusion, we have shown that intravenous injection of PEGylated EGF-containing DNA/PEI complexes allows for highly specific expression of a transgene in human HCC tumors.
Pharmacodynamic effects of a nonpeptide antidiuretic hormone V2 antagonist in cirrhotic patients with ascites
Authors: Guyader Dominique , Patat Alain , Ellis-Grosse Evelyn J. , Orczyk Gayle P.
Published: March 2003

Water retention and dilutional hyponatremia, mainly attributable to an impairment of free water excretion and increased vasopressin activity, are well-documented complications in cirrhotic patients with ascites. VPA-985 is a selective, nonpeptide, orally active, vasopressin-2-receptor antagonist. The aim of this study was to determine the pharmacodynamics, safety, and pharmacokinetics of ascending single doses (25, 50, 100, 200, and 300 mg) in cirrhotic patients with ascites in a randomized, double-blind, placebo-controlled trial. Each dose level was studied in 5 patients (4 active and 1 placebo). After an overnight fast and fluid restriction (continued for 4 hours after dose administration), all patients were given placebo on baseline day and an oral suspension of VPA or placebo on the following day. VPA produced a significant dose-related increase in daily urine output (1,454 ± 858 mL to 4,568 ± 4,385 mL with VPA 300 mg) and a dose-related decrease in urine osmolality. The free water clearance reached greater than 3 mL/min for doses 100 mg or greater. Simultaneously, significant increases in serum osmolality, sodium, and vasopressin levels were found. There was a significant increase in sodium urine excretion. VPA was rapidly absorbed and maximum serum concentrations were achieved within 1 hour after administration. Elimination half-life ranged from 9.0 hours after 100 mg to 22.6 hours after 200 mg. In conclusion, VPA induced a dose-related aquaretic response, suggesting a therapeutic potential in managing water retention in patients with liver cirrhosis with ascites.
Recombinant alfa-interferon plus ribavirin therapy in children and adolescents with chronic hepatitis C
Authors: Wirth Stefan , Lang Thomas , Gehring Stephan , Gerner Patrick
Published: March 2003

Treatment with alfa-interferon alone yielded poor results in children with chronic hepatitis C and was not generally recommended. Owing to limited experience with combination therapy in children, the aim of the study was to evaluate the efficacy and tolerability of alfa-interferon 2b in combination with ribavirin in these patients with different routes of viral transmission. In an uncontrolled pilot study, 41 children and adolescents ranging from 3 to 16 years were treated with alfa-interferon at a dose of 3 or 5 MU/m2 3 times weekly in combination with oral ribavirin (15 mg/kg/d) for 12 months. The mode of infection was unknown in 4, parenterally transmitted in 16, and vertically transmitted in 21 children. Forty patients completed the study. Eleven children, who remained hepatitis C virus (HCV)-RNA positive 6 months after the beginning, discontinued therapy. One boy stopped treatment because of side effects. At the end of treatment 25 patients were HCV-RNA negative (61%). All individuals remained HCV-RNA negative during the 6-month follow-up period. Nine of 15 children with parenteral (56.3%), 14 of 21 with vertical (66.6%), and 2 of 4 with unknown route of infection responded. Side effects included minor clinical signs such as fever, flu-like symptoms, anorexia, and more severe signs (21.4%) such as the development of thyroid autoantibodies and impairment of thyroid function. In conclusion, combination of alfa-interferon with ribavirin seems to be an important advance in the treatment of chronic hepatitis C in children and adolescents. This also is true for both vertically infected patients and for individuals with normal transaminase levels before therapy.
Remethylation and transsulfuration of methionine in cirrhosis: Studies with l-[2H3-methyl-1-13C]methionine
Authors: Russmann Stefan , Junker Edith , Lauterburg Bernhard H.
Published: March 2003

Disturbances of the methionine cycle may result in liver injury. Patients with alcohol-induced liver disease often exhibit hypermethioninemia and a delayed clearance (CL) of methionine, but the extent to which transsulfuration and remethylation pathways of the cyclic methionine metabolism are affected is unknown. Methionine turnover was determined in 7 healthy volunteers and 6 patients with alcohol-induced cirrhosis after oral administration of 2 mg/kg [2H3-methyl-1-13C]methionine, which permitted us to follow transsulfuration by its decarboxylation to 13CO2 and remethylation by replacement of the labeled methyl group by an unlabeled one. Basal plasma concentrations of endogenous methionine (50 ± 5 vs. 25 ± 2 μmol/L, mean ± SEM, P < .001) were significantly higher in patients with cirrhosis and its CL was significantly decreased (774 ± 103 vs. 2,050 ± 141 mL/min, P < .001). Methionine turnover amounted to 42 ± 4 vs. 27 ± 3 μmol/kg/h (P < .05) in controls and patients with cirrhosis, respectively. The fraction of administered methionine undergoing remethylation was lower in patients with cirrhosis (7.6 ± 1.5 vs. 14.1 ± 1.1%, P < .005). However, because of the larger pool of circulating methionine, the total flux of methionine through the remethylation pathway was similar in both groups. A significantly lower fraction of the administered dose appeared in the form of 13CO2 in breath in patients with cirrhosis (2.2 ± 0.4 vs. 11.0 ± 0.8%, P < .001). In conclusion, the data indicate that the liver with cirrhosis compensates for a decreased activity of remethylating enzymes by operating at higher concentrations of methionine. In contrast, transsulfuration is impaired in patients with alcohol-induced cirrhosis such that an assessment of transsulfuration by a simple breath test may provide a clinically useful estimate of hepatic function.