bluuebunny/crossref_metadata_2025 huggingface dataset:
Zonal and regional differences identified from precision mapping of vitamin a-storing lipid droplets of the hepatic stellate cells in pig liver: A novel concept of addressing the intralobular area of heterogeneity
Authors: Zou Zhenzhen , Ekataksin Wichai , Wake Kenjiro
Published: July 2004

Knowledge of hepatic heterogeneity has been strikingly increased, while an accurate means for addressing intralobular positions is still lacking. We examined pig liver preparations of the gold impregnation method for vitamin A-storing lipid droplets in hepatic stellate cells. Droplet morphometry was performed under oil immersion, and the calculated volumes plotted on computerized maps. The heterogeneous results were assessed with five concentric zones and five radial regions; the latter were determined based on midseptum visualized by portal injection. Zonation and regionation thus subdivided lobules into 5-zone/5-region (5Z/5R) compartmentalization. Distribution of values exhibited a distinct zonal gradient, heightened at peripheral zones 1 and 2, decreased over intermediate zone 3 toward centrilobular zones 4 and 5; peak was always found at zone 2. Within a single zone, variations were obvious, forming a regional gradient. Values were significantly higher at periportal than midseptal regions. Digitized mapping showed that low values filled up centrilobular zones, whereas high values concentrated in periportal regions. Along the periphery, inlet venules were quantified, revealing an occurrence rate of 60% at periportal, and 5% at midseptal regions, closely compatible with the regional gradient of vitamin A-storing capacity. The interweaving between zonal and regional gradients results in a vitamin A-low territory, a compound area composed of centrilobular zones plus extensions into midseptal regions. Because the results could account for physiological and pathological events, we regard the 5Z/5R compartmentalization a model worth routine adoption for a precise description of any morphofunctionally demonstrable heterogeneity of the liver lobules.
Factors associated with serum alanine transaminase activity in healthy subjects: Consequences for the definition of normal values, for selection of blood donors, and for patients with chronic hepatitis C
Authors: Piton Annie , Poynard Thierry , Imbert-Bismut Françoise , Khalil Lina , Delattre Jacques , Pelissier Elisabeth , Sansonetti Nicole , Opolon Pierre
Published: July 2004

In clinical research, the definition of the upper limit of normal (ULN) alanine transaminase (ALT) is never detailed. However, such a definition can vary and may have practical consequences. Our aim was to assess factors associated with serum ALT activity in apparently healthy subjects and then to apply seven different definitions of ULN in three different populations so as to assess the prevalence of subjects with normal ALT among blood donors and among hepatitis C patients before (normal ALT hepatitis C patients) and after treatment (interferon [IFN] responders). ALT measurements were performed in the same laboratory using the same technique; 1,033 donors were prospectively investigated, 186 patients with hepatitis C never treated and 40 patients treated with 3 MU three times per week of IFN-α for at least 6 months. The seven definitions (D) of ULN were: D1: 95th percentile of ALT; D2: 95th percentile after separating males and females; D3: males and females separately, ULN = 10 (mean of log10 ALT + 1.96 SD); D4: ULN = 45 IU/L given by the manufacturer; D5: mean + 1 SD after exclusion of the 5% extreme values; D6: 95th percentile after separating subjects with body mass index (BMI) under or equal to the median (23); and D7: 95th percentile after separating subjects according to BMI and sex. BMI and male sex were independently associated (P < .0001; logistic regression) with ALT, without an association with alcohol. The range of ULN varied from 26 IU/L in females (D5) to 66 IU/L in males with BMI >23 (D7). Depending on the definition, the prevalence of blood donors with normal ALT varied from 82% to 96%, i.e., a range of 14%; that of hepatitis C patients with normal ALT varied from 16% to 27%, i.e., a range of 11%; the prevalence of IFN responders varied from 25% to 42%, i.e., a range of 17%. Definitions of normal ALT values should be adjusted for sex and BMI to reduce artificial heterogeneity in blood donor selection and in hepatitis C clinical studies
Loss of butyrate-induced apoptosis in human hepatoma cell lines HCC-M and HCC-T having substantial Bcl-2 expression
Authors: Saito Hidetsugu , Ebinuma Hirotoshi , Takahashi Masahiko , Kaneko Fumihiko , Wakabayashi Kanji , Nakamura Mitsuyasu , Ishii Hiromasa
Published: July 2004

We have demonstrated that sodium butyrate induces differentiation in human hepatoma cells; however, recent studies have shown that this agent causes apoptosis in some types of cancer cells. In this study, we examined whether sodium butyrate causes apoptosis in the human hepatoma cell lines, HCC-M and HCC-T. The growth of human hepatoma cells was dose-dependently reduced by sodium butyrate. Flow cytometric analysis showed cell-cycle arrest at the G1 phase in the sodium butyrate-treated cells. Apoptotic change was never found in treated cells at concentration levels of less than 5 mmol/L. Sodium butyrate decreased p53 expression and increased p21WAF-1 expression in HCC-T and HCC-M cells having the wild-type p53 gene. Western blot analysis showed that Bcl-2 was expressed in the HCC-T and HCC-M cells, and its expression was increased after exposure to sodium butyrate. Antisense oligodeoxynucleotide against bcl-2 easily caused apoptosis. These results indicate that sodium butyrate hardly induces apoptotic change in the human hepatoma cell lines, HCC-T and HCC-M, with the increase of Bcl-2 expression. Cell-cycle arrest in the G1 phase caused by sodium butyrate was suggested to be induced by the increase in p21WAF-1 expression, but this change did not link with the p53 increase.
Characterization of the IGF axis components in isolated rat hepatic stellate cells
Authors: Scharf Jens-Gerd , Knittel Thomas , Dombrowski Frank , Müller Lars , Saile Bernhard , Braulke Thomas , Hartmann Heinz , Ramadori Giuliano
Published: July 2004

The insulin-like growth factors I and II (IGF-I, -II) are circulating peptides known to participate in the regulation of metabolism, growth, and cellular differentiation. In the present study, “early cultured” (days 2-3 of culture) and “culture-activated” (days 6-7 of culture) rat hepatic stellate cells (HSCs) were analyzed for expression of individual components of the IGF axis. Northern blot analysis of IGF-I messenger RNA (mRNA) revealed transcripts of 7.5, 4, 2, and 1.0 to 1.5 kb in culture-activated HSCs, while early cultured HSCs did not express IGF-I mRNA. In culture-activated HSCs, an IGF-I secretion of 8.3 ± 2.5 ng/106 cells per 24 hours was determined radioimmunologically. In media from early cultured HSCs, IGF-I was not detectable. The IGF-I receptor (IGF-I-R) mRNA expression was threefold higher in early cultured HSCs than in culture-activated HSCs. By immunohistochemistry, a decrease of IGF-I-R expression of HSCs in vivo following CCl4-induced liver damage was noted as well. IGF binding proteins (IGFBPs) were detected in conditioned media from HSCs by 125I-IGF-I ligand blotting at apparent molecular masses of 24 and 41 to 45 kd that were immunologically identified as IGFBP-4 and -3, respectively. Synthesis of these IGFBPs increased with time of culture. At neutral pH, no IGFBP proteolysis was observed in conditioned media of early cultured and culture-activated HSCs, whereas at acidic pH, protease activities against IGFBP-3 and -4 were detectable. IGFBP protease activities were completely abolished by inhibitors of aspartyl and cysteine proteases. Addition of 100 nmol/L IGF-I stimulated cell proliferation of early cultured HSCs 5.6 ± 1.1- and 4.6 ± 0.2-fold as measured by [3H]thymidine and 5-bromo-2′-deoxyuridine incorporation, respectively. In culture-activated HSCs, proliferation was increased 1.2 ± 0.1-fold in the presence of 100 nmol/L IGF-I in both proliferation assays. It can be concluded that due to a higher expression of the IGF-I-R and lower levels of IGFBPs, early cultured HSCs are more susceptible to the mitogenic actions of IGFs than the culture-activated HSCs. The present data suggest a role for the IGF axis components in the initiation rather than the perpetuation of HSC proliferation during hepatic fibrogenesis
Inhibition of NFκB in activated rat hepatic stellate cells by proteasome inhibitors and an IκB super-repressor
Authors: Hellerbrand Claus , Jobin Christian , Iimuro Yuji , Licato Laura , Sartor Balfour R. , Brenner David A.
Published: July 2004

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFκB activity in activated but not in quiescent HSCs with subsequent expression of NFκB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IκB super-repressor on the cytokine mediated activation of NFκB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1β or tumor necrosis factor α (TNFα) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IκB super-repressor (Ad5IκB) or β-galactosidase (Ad5LacZ) as a control. NFκB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IκB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IκB, and the Ad5IκB, which provides an exogenous nondegradable IκB, block the stimulation of NFκB activity by TNFα and IL-1β in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFκB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IκB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFκB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IκB in activated HSCs. Inhibition of IκBα degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
Hepatic venous hemoglobin oxygen saturation predicts regenerative status of remnant liver after partial hepatectomy in rats
Authors: Yoshioka Shigeru , Miyazaki Masaru , Shimizu Hiroaki , Ito Hiroshi , Nakagawa Koji , Ambiru Satoshi , Nakajima Nobuyuki , Fukuda Yasuichiro
Published: July 2004

This study was designed to evaluate the use of hepatic venous hemoglobin oxygen saturation (Shvo
2) as an indicator of hepatic oxygen supply-demand relation and also regenerative status of the liver after partial hepatectomy in rats. We assessed the Shvo
2 levels for 7 days, as well as hepatic hemodynamics, oxygen consumption, DNA synthesis and energy charge of the remnant liver for 3 days after 50% hepatectomy or sham operation. Total hepatic oxygen consumption (HVo
2) per liver weight, hepatic oxygen extraction ratio (Ho
2ER), and DNA synthesis were significantly elevated at days 1 and 3 after hepatectomy, compared with the preoperative levels. Meanwhile, significantly decreased Shvo
2 levels were observed at days 1 and 3, and the Shvo2 levels were significantly correlated with the HVo
2. Furthermore, the decreased Shvo
2 levels were synchronized with the increased DNA synthesis in the remnant liver. Energy charge levels were also significantly decreased at day 1 after hepatectomy. These results suggest that the regenerating liver demands an increased amount of oxygen for mitochondrial oxidative phosphorylation to restore hepatic energy charge. In conclusion, the Shvo
2 after hepatectomy may reflect oxygen metabolic status in the remnant liver and could be useful for estimating liver regeneration.
Efficacy of thymosin α1 in patients with chronic hepatitis B: A randomized, controlled trial
Authors: Chien Rong-Nan , Liaw Yun-Fan , Chen Tse-Ching , Yeh Chau-Ting , Sheen I.-Shyan
Published: July 2004

Thymosin α1 (Tα) is an immune modifier that has been shown in a pilot study to be effective for chronic hepatitis B; this requires confirmation. Ninety-eight patients with clinicopathologically proven chronic hepatitis B were randomly allocated to 3 groups: 1) group A received a 26-week course of Tα with a 1.6-mg subcutaneous injection two times a week (T6 group); 2) group B received the same regimen as group A, but Tα therapy extended for 52 weeks (T12 group); and 3) group C served as a control group and was followed up for 18 months without specific treatment (T0 group). The three groups were comparable in clinicohistological features at entry. The complete virological response rate (clearance of serum hepatitis B virus [HBV] DNA and hepatitis B e antigen [HBeAg]) was higher in group A (40.6%) and group B (26.5%) than in group C (9.4%) (group A vs. group C: P = .004; group B vs. group C: P = .068) when assessed 18 months after entry, although complete response rates among these three groups were similar when first assessed at the end of therapy. There was a trend for complete virological response to increase or accumulate gradually after the end of Tα therapy. None of the responders lost hepatitis B surface antigen. Blinded histological assessment showed a significant improvement in treated patients, particularly in lobular necroinflammation and scores excluding fibrosis. No significant side effects were observed. These results suggest that a 26-week course of Tα therapy is effective and safe in patients with chronic hepatitis B.
Immunoreactivity of porcine heart dihydrolipoamide acetyl- and succinyl-transferases (PDC-E2, OGDC-E2) with primary biliary cirrhosis sera: Characterization of the autoantigenic region and effects of enzymatic delipoylation and relipoylation
Authors: Koike Kichiko , Ishibashi Hiromi , Koike Masahiko
Published: July 2004

Analysis of the primary structure of the lipoyl domain of the dihydrolipoamide acetyltransferase (PDC-E2) component of the porcine pyruvate dehydrogenase complex (PDC) reveals a high degree of homology with M2 antigen and human PDC-E2. The porcine PDC-E2 and the dihydrolipoamide succinyltransferase (OGDC-E2) component of the porcine 2-oxoglutarate dehydrogenase complex (OGDC) were identified as mitochondrial autoantigen with sera from patients with primary biliary cirrhosis (PBC). Immunodominant regions (autoepitopes) on the porcine-PDC-E2 component have been mapped to two regions around Lys-46 (outer lipoyl domain) and Lys-173 (inner lipoyl domain), which contained covalently bound lipoic acid prosthetic group. When these lipoyl domains were cleaved at Asp-45 or Asp-172 with endoproteinase Asp-N, the autoantigenicities of the two domains completely disappeared; this suggested the requirement of Asp-45 or Asp-172 residues for the immunoreaction with PBC sera. In addition, a single 14-amino acid epitope peptide histidine-substituted at Asp-172 did not exhibit competitive inhibition of autoantigen binding. Fragmentation of lipoyl domain of the porcine PDC-E2 by limited proteolysis and BrCN-cleavage after alkylation resulted in rapid loss of autoantigenicity. Enzymatic delipoylation and relipoylation of the complexed and free PDC-E2 and OGDC-E2 components did not influence immunoreactivity with PBC sera.
Detection of reovirus RNA in hepatobiliary tissues from patients with extrahepatic biliary atresia and choledochal cysts
Authors: Tyler Kenneth L. , Sokol Ronald J. , Oberhaus Stephanie M. , Le Mysan , Karrer Frederick M. , Narkewicz Michael R. , Tyson Weslie R. , Murphy James R. , Low Robert , Brown William R.
Published: July 2004

Extrahepatic biliary atresia (EHBA) and choledochal cysts (CDC) are important causes of obstructive jaundice in pediatric patients. Viruses in general, and reoviruses in particular, have long been considered as possible etiologic agents responsible for inciting the inflammatory process that leads to these infantile obstructive cholangiopathies. In an effort to determine whether reovirus infection is associated with these disorders, we used a sensitive and specific reverse-transcriptase polymerase chain reaction (RT-PCR) technique designed to amplify a portion of the reovirus L1 gene segment from extracts of liver and/or biliary tissues. These tissues were obtained at the time of liver biopsy or surgical procedures from 23 patients with EHBA, 9 patients with CDC, and 33 patients with other hepatobiliary diseases. Hepatic and biliary tissues obtained at autopsy from 17 patients who died without known liver or biliary disease were also analyzed. Reovirus RNA was detected in hepatic and/or biliary tissues from 55% of patients with EHBA and 78% of patients with CDC. Reovirus RNA was found also in extracts of hepatic and/or biliary tissue from 21% of patients with other hepatobiliary diseases and in 12% of autopsy cases. The prevalence of reovirus RNA in tissues from patients with EHBA and CDC was significantly greater than that in patients with other hepatobiliary diseases (χ2P= .012 EHBA vs. OTHER,P= .001 CDC vs. OTHER), or AUTOPSY cases (χ2P= .006 EHBA vs. AUTOPSY,P< .001 CDC vs. AUTOPSY).
Immunohistochemical demonstration of β-naphthoflavone-inducible cytochrome P450 1A1/1A2 in rat intrahepatic biliary epithelial cells
Authors: Shen Jinshan , Moy Jadine A. , Green Mitchell D. , Guengerich Peter F. , Baron Jeffrey
Published: July 2004

Although intrahepatic biliary epithelial cells are targets for certain hepatotoxic chemicals, including some procarcinogens, their ability to monooxygenate, and thereby bioactivate and inactivate xenobiotics, remains to be established. Thus, the present study was undertaken to immunohistochemically determine if cytochrome P450 (CYP) 1A1/1A2 is present and can be induced within these non- parenchymal liver cells. Immunoperoxidase and immunofluorescent staining for CYP1A1/1A2 was detected within intrahepatic biliary epithelial cells as well as hepatocytes of control rats and was markedly enhanced in both cell types by β-naphthoflavone (BNF). Color confocal laser microscopic analyses of dual immunofluorescent staining for CYP1A1/1A2 and cytokeratins 6 and 9 (56 and 64 kd, respectively) provided unequivocal evidence for the presence and induction of CYP1A1/1A2 within intrahepatic bile duct epithelia. Moreover, microdensitometric analyses of immunoperoxidase staining intensities for CYP1A1/1A2 revealed that intrahepatic biliary epithelial cells of control rats contain 44%, 56%, and 58% as much CYP1A1/1A2 as do centrilobular, midzonal, and periportal hepatocytes, respectively. These analyses further revealed that BNF increased the content of CYP1A1/1A2 in biliary epithelial cells by approximately 120%, while CYP1A1/1A2 levels in centrilobular, midzonal, and periportal hepatocytes were increased by 82%, 159%, and 160%, respectively. The results of this study represent the first in situ demonstration that mammalian intrahepatic biliary epithelial cells contain a CYP isoform, and further that CYP1A1/1A2 can be induced in these cells by BNF. These findings therefore indicate that intrahepatic biliary epithelial cells can oxidatively metabolize xenobiotics in situ and that their ability to bioactivate and inactivate xenobiotics can be significantly enhanced by CYP1A1/1A2 induction.
Aly/Aly mice: A unique model of biliary disease
Authors: Tsuneyama Koichi , Kono Naoko , Hoso Masahiro , Sugahara Hiroyuki , Yoshida Kazuharu , Katayanagi Kazuyoshi , Gershwin Eric M. , Saito Katsuhiko , Nakanuma Yasuni
Published: July 2004

An autosomal recessive murine mutation, coined “aly/aly” or “alymphoplasia,” was recently reported. Homozygotes for aly are defective in both humoral and cell-mediated immune function and have diffuse lymphoid cell infiltration of various tissues, particularly around the conduit ducts of the pancreas and salivary glands. In pilot studies in our laboratories, aly/aly mice were found to have peculiar biliary tract lesions, which were analyzed histologically and immunohistochemically in the present study. The livers of aly/aly mice older than 8 weeks consistently showed a variable lymphoid cell infiltration with lymph follicle formation in portal tracts; intrahepatic biliary epithelial cells showed various types of damage including pseudopyloric gland metaplasia and proliferative changes. In addition, the extrahepatic bile duct and intrahepatic large bile duct were found to contain an acidophilic substance in their epithelial cytoplasm. In the lumen and occasionally in the cytoplasm of these bile ducts, acidophilic crystals were also seen. Ultrastructurally, the intracytoplasmic acidophilic substances consisted of membrane-bound intracytoplasmic inclusions with homogeneous electron density, likely derived from rough endoplasmic reticulum (ER). Immunohistochemically, the cytoplasmic acidophilic substances were simultaneously positive for cystatin C, gastrin, serotonin, and somatostatin. In contrast, the acidophilic crystals did not react with any of these antibodies. These findings suggest that the intracytoplasmic acidophilic substances may contain a precursor of the peptide hormones, possibly because of defective secretion or intracellular transport. We believe that the aly/aly mouse is a useful model for the analysis of biliary metabolic events, and for studies of the interaction of the immune system and biliary destruction.
Cholesterol crystallization in human gallbladder bile: Relation to gallstone number, bile composition, and apolipoprotein E4 isoform
Authors: Van Erpecum Karel J. , Van Berge-Henegouwen Gerard P. , Eckhardt Erik R.M. , Portincasa Piero , Van De Heijning Bert J.M. , Dallinga-Thie Geesje M. , Groen Albert K.
Published: July 2004

Patients with multiple cholesterol gallstones are at increased risk of recurrence after nonsurgical therapy, possibly because of fast biliary cholesterol crystallization. Serum apolipoprotein E4 (apo E4) is a risk factor for primary cholesterol gallstone formation as well as recurrence. We examined potential effects of stone number and apolipoprotein E genotype on crystallization and on various crystallization-influencing factors in gallbladder biles of 36 cholesterol stone patients (25 multiple stones: 10 carrying the ε4 allele). Biliary cholesterol saturation, bile salt composition or concentrations of total protein, immunoglobulin (Ig)A, IgG, α1-acid glycoprotein, haptoglobin, or mucin—all crystallization promoters—did not differ between multiple and solitary stone patients, apparently not explaining different speed of crystallization (crystal observation time 3.5 ± 0.6 days vs. 12.7 ± 2.4 days, respectively; P = .0003). In contrast, biliary aminopeptidase-N activities (2,607 ± 592 mU/mL vs. 947 ± 185 mU/mL; P = .04) were higher and IgM levels (179 ± 39 vs. 65 ± 8 mg/L; P = .09) tended to be higher in the case of multiple stones. Although patients carrying the ε4 allele had similar stone numbers and crystallization as patients without the ε4 allele, their cholesterol saturation index (CSI) was lower (1.08 ± 0.09 vs. 1.54 ± 0.13; P = .01), whereas total protein and bile salt concentrations tended to be higher with preferential taurine-conjugation. In conclusion, fast cholesterol crystallization is associated with multiple stones but not with apolipoprotein E4. Whereas fast crystallization may contribute to high recurrence rates after nonsurgical therapy in case of multiple gallstones, the mechanism for increased risk of gallstone formation in patients carrying the ε4 allele remains unknown.
Early cyclosporine monotherapy in liver transplantation: A 5-year follow-up of a prospective, randomized trial
Authors: Belli Luca S. , de Carlis Luciano , Rondinara Gianfranco , Alberti Alberto B. , Bellati Giorgio , De Gasperi Andrea , Forti Domenico , Idèo G.
Published: July 2004

Maintenance of adequate immunosuppression and avoidance of side-effects are the goals of long-term management of all organ-transplanted patients. We here report the final results of a prospective, randomized trial comparing early cyclosporine monotherapy versus double-drug therapy (cyclosporine and steroids) in adult liver transplantation patients. One hundred four patients were randomized 3 months after transplantation either to continue (Group I = 50 patients) or to stop steroids (Group II = 54 patients). Patients on a double-drug regimen were maintained long term on methylprednisolone at a dose of 0.1 mg/kg/d. Target cyclosporine trough levels were between 150 and 250 ng/mL in both groups. Our main points of interest were the prevalence of acute and chronic rejections and steroid-related side-effects in the two groups of patients. Mean follow-up was 41 ± 16 months (range, 4-68 months). Patient actuarial survival 2 and 5 years after randomization was similar in the two groups (82% vs. 83% and 82% vs. 77%). The prevalence of acute rejections after randomization was, respectively, 8% and 4%. A single episode of chronic rejection was observed only in a patient on long-term steroid therapy. Side-effects of steroid therapy were less frequent in patients weaned off steroids, and when considering hypertension and diabetes, the differences between the two groups were statistically significant. Early cyclosporine monotherapy is a safe undertaking in liver transplantation because it allows a significant reduction of steroid-related side-effects without increasing the risk of acute and chronic rejection. After 5 years, patient survival was similar in patients with or without steroids.
Potassium channels participate in gastric mucosal protection in rats with partial portal vein ligation
Authors: Li Michael K.K. , Tsui C. P. , Sung Joseph J.Y. , Scremin Oscar U. , Leung Felix W.
Published: July 2004

Glybenclamide, an adenosine triphosphate-dependent potassium (K+
ATP) channel blocker, lowered portal pressure and attenuated the hyperdynamic splanchnic circulation in rats with partial portal vein ligation (PPVL). The purpose of this report was to confirm these observations and to test the hypothesis that glybenclamide could reduce acidified ethanol-induced gastric mucosal injury in rats with PPVL. Gastric mucosal blood flow (hydrogen gas clearance), systemic blood pressure, and portal pressure were monitored in rats with PPVL or sham operation (SO). Intravenous glybenclamide (20 mg/kg) or vehicle was administered, followed by intragastric acidified ethanol (0.15 N HCl and 15% ethanol). The area of gastric mucosal lesions was assessed by image analysis. In contrast to published findings, there was no significant elevation of portal pressure after glybenclamide administration in rats with PPVL. Glybenclamide did not alter the gastric mucosal hyperemia in these rats. Glybenclamide significantly increased mucosal injury. The data are consistent with the hypothesis that K+
ATP channels play a role in protecting the gastric mucosa in rats with PPVL.
Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: Possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver
Authors: El-Assal Osama N. , Yamanoi Akira , Soda Yukiko , Yamaguchi Minekazu , Igarashi Masahiko , Yamamoto Akitaka , Nabika Toru , Nagasue Naofumi
Published: July 2004

As in other tumors, the assessment of microvessel density (MVD) in hepatocellular carcinoma (HCC) may be essential to perform an effective anti-angiogenic therapy for this tumor. The relationship between vascular endothelial growth factor (VEGF) and MVD of HCC as well as the surrounding liver remains to be elucidated. In 71 patients who had undergone curative hepatic resection for HCC, MVD and VEGF expressions were evaluated for HCC and the liver by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and/or immunostaining. The intensity and extent of VEGF immunoreactivity were evaluated using a computer image analyzer-cell analysis system (CAS). Angiographic data were re-evaluated and compared with MVD in 50 tumors. Tumoral MVD was significantly correlated with tumor capsule formation (t test, P = .0016). Small HCCs (≤2 cm) had a significantly lower MVD compared with moderate-sized HCCs (2-5 cm) (t test, P = .016), and the MVD of large HCCs was relatively lower than that of moderate tumors. Tumor vascularity on angiography was not correlated with the MVD. Neither VEGF mRNA levels nor protein expression in HCC were correlated with the tumoral MVD or any histopathological features of the tumor. However, cirrhotic livers had significantly higher MVD and VEGF expressions compared with noncirrhotic livers (t test, P = .0015 and P = .047, respectively). Only the MVD of tumor was significantly correlated with intrahepatic recurrence (t test, P = .0048) and disease-free survival (DFS) rates (log rank test, P = .0035). Moreover, the MVD was an independent predictor for DFS by multivariate analysis (χ2 test, P = .03). In conclusion, the MVD in HCC may be involved in the dismal prognosis of this tumor, and VEGF may be associated with the angiogenic process of the cirrhotic liver, but not with the angiogenesis of HCC.
Liver transplantation for small hepatocellular carcinoma: The tumor-node-metastasis classification does not have prognostic power
Authors: Llovet Josep M. , Bruix Jordi , Fuster Josep , Castells Antoni , Garcia-Valdecasas Juan Carlos , Grande Lluís , França Alex , Brú Concepció , Navasa Miquel , Ayuso Maria del , Solé Manel , Real Maria Isabel , Vilana Ramon , Rimola Antoni , Visa Josep , Rodés Joan
Published: July 2004

Tumoral recurrence rate and survival of patients with hepatocellular carcinoma (HCC) treated by orthotopic liver transplantation (OLT) depend on tumor stage. Thereby, from the beginning of our program, we selected only patients with solitary tumors ≤5 cm without vascular invasion, thus avoiding the use of the tumor-node-metastasis (TNM) staging system as a selection tool. The present study reports the results obtained in 58 consecutive patients (52 ± 8 years, 47 males) with HCC (7 incidentals) transplanted between 1989 and 1995. Transplantation was indicated because of tumor diagnosis in 40 cases and advanced liver failure in 18. Mean tumor size at staging was 28.2 ± 12.1 mm. No adjuvant treatment was applied during the waiting period (58.9 ± 45.1 days). The pathological tumor-node-metastasis (pTNM) classification allocated 15 patients to stage I, 19 to stage II, 11 to stage IIIA, and 13 to stage IVA showing preoperative understaging in 43% of the cases with known tumor. After a median follow up of 31 months, only two patients have shown tumor recurrence and fifteen have died, the 1-, 3-, and 5-year survival being 84%, 74%, and 74%. All HCV+ patients remain infected and 94% showed significant liver disease (6 cirrhosis). Six patients have had a second transplant. In conclusion, the application of restrictive criteria not following the TNM staging system prompts excellent results for liver transplantation in patients with HCC, both in terms of survival and disease recurrence, even without applying adjuvant treatment; however, the survival data should be tempered by the appearance of complications that may worsen the long-term prognosis.
Transarterial embolization versus symptomatic treatment in patients with advanced hepatocellular carcinoma: Results of a randomized, controlled trial in a single institution
Authors: Bruix Jordi , Llovet Josep M. , Castells Antoni , Montañá Xavier , Brú Concepció , Ayuso Maria Del Carmen , Vilana Ramon , Rodés Joan
Published: July 2004

This randomized, controlled trial assessed the effect of transarterial embolization (TAE) (without associated chemotherapy) on the survival of patients with nonsurgical hepatocellular carcinoma (HCC). Eighty consecutive patients were randomized to treatment with embolization (Group A, n = 40), or to symptomatic treatment (Group B, n = 40), there being no differences between both groups regarding the degree of liver function impairment and tumor stage. Eighty-two percent of the patients presented a self-limited postembolization syndrome, without treatment-related mortality. Fifty-five percent of the treated cases exhibited a partial response, which resulted in a lower probability of tumor progression during follow-up (57% vs. 77% at 1 year; P < .005). However, after a median follow-up of 24 months (30 deaths in each group), there are no differences in survival (Group A: 49% and 13%; Group B: 50% and 27%, at 2 and 4 years, respectively; P = .72). The absence of differences was maintained even when dividing patients according to Child-Pugh’s grade, Okuda stage, or performance status test (PST). Furthermore, there were no differences in the probability of complications or in the need of hospital admissions. In conclusion, TAE has a marked antitumoral effect associated to a slower growth of the tumor, but it does not improve the survival of patients with nonsurgical HCC.
Complement-mediated cytotoxicity and inhibition of ligand binding to hepatocytes by woodchuck hepatitis virus-induced autoantibodies to asialoglycoprotein receptor
Authors: Diao Jingyu , Churchill Norma D. , Michalak Tomasz I.
Published: July 2004

Hepadnavirus invasion in woodchucks has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediate clearance of desialylated serum proteins. We evaluated the possible pathogenetic importance of anti-ASGPR triggered by woodchuck hepatitis virus (WHV), using anti-ASGPR-reactive serum immunoglobulins (Igs) from five animals with different stages of WHV hepatitis or self-limited WHV infection and isolated woodchuck hepatocytes or HepG2 cells as targets. The results revealed that WHV-induced anti-ASGPR can specifically inhibit asialoglycoprotein recognition by both homologous and heterologous liver cells, as tested in an asialofetuin (ASFN)-binding radioassay. However, the extent of the interference significantly varied (from 85% inhibition to none) for anti-ASGPR with similar titer from different animals, indicating a high degree of heterogeneity in the ASGPR epitope specificity and in the potential biological effects of these autoantibodies. The WHV-triggered anti-ASGPR also induced complement-mediated hepatocytolysis in a microculture tetrazolium (MTT) assay, which ranged from 8.9% ± 0.3% to 33.6% ± 3.6% (mean ± SD) for different animals and target cell numbers. This cytopathic effect was strictly ASGPR-specific, complement-dependent, and was not related to the anti-ASGPR ability to inhibit ligand-hepatocyte binding. Our findings indicate that among pathways by which anti-ASGPR autoimmunity could cause liver damage, hepadnavirus-induced anti-ASGPR might impair hepatocytes by both disrupting clearance of desialylated proteins and activation of the complement-mediated cytolysis. These cytopathic effects might contribute to the pathogenesis, aggravate severity, and prolong recovery from liver injury in viral hepatitis.
Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases
Authors: Jones Richard A. , Johnson Victoria L. , Buck Neil R. , Dobrota Miloslav , Hinton Richard H. , Chow Sek C. , Kass George E.
Published: July 2004

The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-dl -Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L -phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L -lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L -norleucinal, Ac-Leu-Leu-L -methional, and trans-epoxysuccinyl-L -leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3α) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.
Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation
Authors: Gao Wenshi , Bentley Rex C. , Madden John F. , Clavien Pierre-Alain
Published: July 2004

In livers excised for transplantation, sinusoidal endothelium appears especially vulnerable to injury during organ preservation in the cold and subsequent reperfusion. The degree of endothelial cell injury correlates with functional impairment of the graft following transplantation. The mechanism of injury remains obscure, but endothelial cell damage has been described as coagulative necrosis secondary to irreversible physico-chemical damage. We investigated whether endothelial cell death is caused by apoptosis rather than by necrosis. Tissue from rat livers stored for varying periods in cold (1°C) Euro-Collins solution and then reperfused for 1 hour at 37°C were studied for evidence of apoptosis by detection of DNA fragmentation using the in situ terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay, DNA gel electrophoresis, and by transmission electron microscopy (EM). DNA fragmentation of the type characteristic of apoptosis was identified in 49.7% ± 2.2% of sinusoidal lining cells after 8 hours of ischemia + reperfusion (viable graft) vs. 70.7% ± 4.3% after 16 hours + reperfusion (nonviable graft) (P < .001). No such fragmentation was observed after cold preservation without reperfusion or in unpreserved, reperfused livers. EM demonstrated changes characteristic of apoptosis exclusively in endothelial cells. The study suggests that the apoptosis of sinusoidal endothelial cells is a pivotal mechanism of preservation injury in liver transplantation.
Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in north america†
Authors: Bonkovsky Herbert L. , Poh-Fitzpatrick Maureen , Pimstone Neville , Obando Jorge , Di Bisceglie Adrian , Tattrie Christine , Tortorelli Kristina , LeClair Paula , Mercurio Mary G. , Lambrecht Richard W.
Published: July 2004

In some, but not all countries, porphyria cutanea tarda (PCT) has been associated with chronic infection with the hepatitis C virus (HCV). Recently, PCT has also been associated with mutations in the HFE gene that are associated with HLA-linked hereditary hemochromatosis. Until now, few studies of these associations have been reported from North America. The aims of this study were: 1) to assess the prevalence of HCV infection and HFE mutations in North American patients with PCT; 2) to compare demographic and laboratory features between those who are HCV-positive and HCV-negative; and 3) to study urinary porphyrin excretions in American HCV-positive patients without clinically manifest PCT. Clinical and laboratory data, including tests for HCV and urinary porphyrins, were collected from 70 unselected patients with typical PCT. Urinary porphyrins were also measured in 110 non-PCT patients with chronic hepatitis C. Mutational analyses of the HFE gene were performed in 26 PCT patients. Thirty-nine of 70 (56%) of the PCT patients had evidence of HCV infection. Thirty-two of 39 PCT patients with HCV were men, all of whom used alcohol. In contrast, 22 of 31 PCT patients without HCV infection were women, 12 of whom had taken estrogens. The HCV-positive group was more likely to have used illicit intravenous drugs (45% vs. 0%; P = 0.01), to have had several (>4) sex partners (48% vs. 13%; P = 0.005), and less likely to have no known risk factors for HCV infection (33% vs. 78%; P = 0.004). Total urinary porphyrin excretion was the same in the two groups, but those with HCV infection had a significantly lower percentage of uroporphyrin and higher percentages of hepta-and hexa-carboxy porphyrins in urine. Sixteen of 110 (15%) HCV-positive subjects without PCT had increased urinary porphyrins, but, unlike PCT, these were mainly coproporphyrin. Forty-two percent of PCT patients carried the C282Y mutation of HFE (15% homozygous), and another 31% carried the H63D mutation (8% homozygous). Thus, 73% of PCT patients had one of these mutations. The prevalence of HCV infection (56%) and mutations in the HFE gene (73%) are high among North American patients with PCT. Alcohol and estrogen use are important additional risk factors. All PCT patients should be tested for HCV infection and for HFE gene mutations. Although HCV infection is a trigger for PCT, preclinical PCT is rare in chronic HCV hepatitis C in the United States.
Identification and characterization of mutations in hepatitis B virus resistant to lamivudine
Authors: Allen Marchelle I. , Deslauriers Manon , Andrews Webster C. , Tipples Graham A. , Walters Kathie-Anne , Tyrrell David L. , Brown Nathaniel , Condreay Lynn D.
Published: July 2004

Cirrhosis and hepatocellular carcinoma occur as long-term complications of chronic hepatitis B virus (HBV) infection. Antiviral therapy is potentially a successful approach for the treatment of patients with HBV infection, which includes the nucleoside analog, lamivudine [(-)2′-deoxy-3′-thiacytidine, 3TC]. Although resistance to lamivudine therapy has been reported in several HBV-infected patients, the pattern of resistance-associated mutations in HBV has not been fully characterized. We report a DNA sequence database that includes a 500-base pair region of the HBV polymerase gene from 20 patients with clinical manifestations of lamivudine resistance. Analysis of the database reveals two patterns of amino acid substitutions in the tyrosine, methionine, aspartate, aspartate (YMDD) nucleotide-binding locus of the HBV polymerase. HBV DNA from the sera of patients in Group I exhibits a substitution of valine for methionine at residue 552, accompanied by a substitution of methionine for leucine at residue 528. Patients in Group II had only an isoleucine-for-methionine substitution at position 552. Reconstruction of these mutations in an HBV replication-competent plasmid was performed in a transient transfection cell assay to determine the function/relevance of these mutations to lamivudine resistance. Both Group I and Group II mutations resulted in a substantial decrease in sensitivity to lamivudine treatment (>10,000-fold shift in IC50 over wild-type [wt] IC50), strongly indicating that these mutations were involved in resistance to lamivudine. A hypothetical model of the HBV reverse transcriptase has been generated for further study of the role of these mutations in lamivudine resistance.
Dynamics of hypervariable region 1 variation in hepatitis C virus infection and correlation with clinical and virological features of liver disease
Authors: Brambilla Sabrina , Bellati Giorgio , Asti Margherita , Lisa Antonella , Candusso Maria Elena , D’Amico Maria , Grassi Gabriele , Giacca Mauro , Franchini Angelo , Bruno Savino , Ideo Gaetano , Mondelli Mario U. , Silini Enrico M.
Published: July 2004

Hepatitis C virus (HCV) infection is a dynamic process during which molecular variants are continously selected as the result of virus adaptation to the host. Understanding the nature of HCV genetic variation is central to current theories of pathogenesis and immune response. We prospectively studied hypervariable region 1 (HVR1) variation in the E2 gene of 36 hepatitis C patients, including 10 asymptomatic carriers, followed up for 1 to 2 years. Sequence changes in single and consecutive serum samples were assessed and correlated with clinical and virological parameters of liver disease. A region of the E1 gene was sequenced for comparison in 3 subjects. HVR1 heterogeneity at single time points widely varied in individual patients, did not increase cumulatively over the follow-up period, and did not correlate with HVR1 evolutionary rates. Conversely, the process of HVR1 sequence diversification, although differed considerably among patients, was stable over time and directly correlated with infections by HCV type 2, lower alanine aminotransferase (ALT) levels, and absence of cirrhosis. HCV carriers showed the highest HVR1 variation rates. Our findings indicate that HVR1 variation has an adaptive significance and is associated with favorable features of liver disease and suggest that prospective, rather than static, observations are required to model the process of HCV variation.
Ribavirin inhibits protein synthesis and cell proliferation induced by mitogenic factors in primary human and rat hepatocytes
Authors: Ilyin Gennady P. , Langouët Sophie , Rissel Maryvonne , Delcros Jean-Guy , Guillouzo Andre , Guguen-Guillouzo Christiane
Published: July 2004

Ribavirin, a guanosine analog, used in combination with interferon α (IFN-α) in the treatment of chronic hepatitis induced by hepatitis C virus (HCV) infection, has been shown to improve liver histology and to decrease transaminases even when administered alone. We analyzed the direct effects of ribavirin on the liver by using primary cultures of human and rat hepatocytes. Between 10 to 60 μmol/L, ribavirin was found to inhibit both the synthesis and secretion of whole proteins in a time- and dose-dependent fashion. Such an effect was confirmed by the measurement of albumin and haptoglobin secretion rates. [3H]-Thymidine incorporation was suppressed both in hepatocyte growth factor-stimulated human hepatocytes and in epidermal growth factor (EGF)-stimulated rat hepatocytes in the presence of ribavirin. The inhibitory effect on DNA synthesis was associated with a delayed progression to S phase of the cell cycle, as determined by flow cytometry and detection of cyclin A and cdc2 which are two proteins expressed during the S phase. The inhibition of DNA synthesis, caused by 50 μmol/L ribavirin, was completely restored by the addition of 80 μmol/L guanosine. These observations demonstrate that ribavirin at concentrations close to those found in plasma of treated patients can directly affect hepatic functions in vitro. Its effects could, however, be reduced in vivo by guanosine salvage supply.