bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular localization of endothelin-1 and increased production in liver injury in the rat: Potential for autocrine and paracrine effects on stellate cells
Authors: Rockey Don C. , Fouassier Laura , Chung John J. , Carayon Alain , Vallée Patrick , Rey Colette , Housset Chantal
Published: July 2004

Endothelin (ET) peptides have been implicated in the pathogenesis of several biological processes within the liver. ET levels are elevated in the circulation of patients with cirrhosis, and recent data suggest that ET may be overproduced in the liver itself in this condition. The aims of the current study were to elucidate the cellular source and expression of endothelin-1 (ET-1) in normal and injured liver, and to investigate its biological effects on stellate cells, the primary target of ETs in the liver. In normal hepatic cells, preproET-1 messenger RNA (mRNA) was detected in only nonparenchymal cells, predominantly in sinusoidal endothelial cells. After biliary fibrosis and early cirrhosis induced by bile duct ligation, preproET-1 mRNA and immunoreactive ET levels increased with progressive injury in whole liver extracts, as well as in isolated stellate and endothelial cell fractions. Eight days after bile duct ligation, the relative increase in preproET-1 mRNA was 1.6- and 7.6-fold above normal in sinusoidal endothelial and stellate cells, respectively. Additionally, immunoreactive ET peptide levels increased by 60% ± 27% over basal values in sinusoidal endothelial cells and 98% ± 40% in stellate cells. Cultured stellate cells responded dramatically to exogenous ET-1 by the spreading and up-regulation of smooth muscle α actin expression. Furthermore, in early culture before cellular activation, ET-1 (10 nmol/L) caused over a twofold increase in [3H]thymidine incorporation, while activated cells (i.e., those cultured for >1 week) exposed to ET-1 exhibited up to a fivefold decrease in [3H]thymidine incorporation. The data indicate that not only is ET-1 overproduced by both sinusoidal endothelial and stellate cells during liver injury, but that it also has potent effects on features of stellate cell activation. We conclude that autocrine and paracrine production of ET-1 is prominent and is likely to be important in the pathogenesis of hepatic diseases.
The portal lobule in rat liver fibrosis: A re-evaluation of the liver unit
Authors: Bhunchet Ekapot , Wake Kenjiro
Published: July 2004

We re-evaluated three schemes of liver organization: the classic lobule, the portal lobule, and Rappaport’s liver acinus. The lobular angioarchitecture of normal rat liver and the three-dimensional structure of pseudolubules found in rat livers with fibrosis induced by swine serum were compared with the classic lobule of the pig. Normal and fibrotic rat livers and pig livers were perfused, injected with either India ink or 0.75% OsO4 through the portal and/or hepatic vein, and immersionfixed. Whole lobes and hand-cut thick sections were made transparent with a solution of benzyl benzoate and methyl salicylate. The angioarchitecture of normal rat liver differs from pig liver. In the former, terminal portal branches and central veins interdigitate, and in the latter, numerous terminal portal branches that arise from interlobular portal veins establish a vascular basket surrounding one central vein and forming classic lobule. The structure of liver acinus is never found in the pig liver. The terminal portal branch, together with the terminal hepatic artery and bile duct, are present inside each pseudolobule of fibrotic rat livers. Blood from the terminal portal branch flows through inlet venules into radiating sinusoids, and, at the periphery converges into newly formed septal and angular outlet venules; these venules terminate in fibrotic central veins located at each corner. Pseudolobules are not rugby ball-like as Rappaport’s liver acini are but are polyhedron in shape. The rat pseudolobules are comparable with the portal lobule; its structure and microcirculation are the reverse of the pig classic lobule. Rat pseudolobules are different from liver acini, as shown by the following: 1) their three-dimensional shape is different; and 2) they have a reverse relationship to classic lobules while acini are defined to subdivide classic lobules. In normal and fibrotic rat livers, the liver unit is the portal lobule with a terminal portal branch as the axial branch and central veins at the periphery. The co-existence of liver acini and classic lobules is doubtful.
Involvement of intrasinusoidal hemostasis in the development of concanavalin a-induced hepatic injury in mice
Authors: Miyazawa Yuko , Tsutsui Hiroko , Mizuhara Hidekazu , Fujiwara Hiromi , Kaneda Kenji
Published: July 2004

Concanavalin A (Con A) stimulation induces T-cell activation-associated hepatic injury. This study is designed to show the involvement of microcirculatory disturbance in the pathogenesis. Con A administration led to prominent intrasinusoidal hemostasis, which consisted of erythrocyte agglutination, lymphocyte/neutrophil sticking to endothelial cells, and platelet aggregation and degranulation, resulting in a marked decrease in the intrahepatic blood flow and elevation of portal perfusion pressure. After hemostasis, confluent hepatic necrosis occurred within the congested area of liver parenchyma. Reduction in the extent of hemostasis by the treatment with heparin (thrombin inhibitor) or cyproheptadine (serotonin inhibitor) decreased hepatic injury. Pretreatment with either anti-tumor necrosis factor alfa (TNF-α) or anti-interferon gamma (IFN-γ) monoclonal antibody (MAb) moderately decreased hemostasis and hepatic injury, whereas combined use of two MAbs almost perfectly protected mice from these disorders. Complete obliteration of hemostasis and hepatic injury was also accomplished by the pretreatment with FK506 which suppressed TNF-α and IFN-γ production. Intrasinusoidal accumulation of leukocytes and platelets was, however, not blocked by FK506, indicating that Con A activities other than the stimulation of cytokine production are responsible for this event. The administration of anti-CD3 MAb, a T-cell stimulant without agglutination activities, which elevated plasma cytokine levels in a comparable degree without inducing prominent leukocyte infiltration, did not induce hepatic congestion and injury. These findings indicate that the agglutination activities of Con A and T-cell activation mediated TNF-α/IFN-γ production are both required for the induction of intrasinusoidal hemostasis, which is indispensable for the development of hepatic injury.
Chemokine involvement in hepatic ischemia/reperfusion injury in mice: Roles for macrophage inflammatory protein-2 and kupffer cells
Authors: Lentsch Alex B. , Yoshidome Hiroyuki , Cheadle William G. , Miller Frederick N. , Edwards Michael J.
Published: July 2004

Hepatic injury induced by ischemia and reperfusion is an important clinical problem after liver resection or transplantation. Neutrophils are known to mediate the organ injury, but the precise mechanisms leading to hepatic neutrophil recruitment are undefined. Two CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and KC, are potently chemotactic for neutrophils in vitro and have been reported to be involved in neutrophil-dependent inflammatory tissue injury. The objective of the present study was to determine the roles of MIP-2 and KC in the induction of hepatic ischemia/reperfusion injury. C57BL/6 mice were subjected to 90 minutes of partial hepatic ischemia followed by reperfusion. Hepatic injury was associated with neutrophil sequestration, edema, and elevated serum levels of hepatic transaminases. The expression of MIP-2 messenger RNA (mRNA) was induced within 3 hours after reperfusion, before any detectable increase in neutrophil accumulation, and was also increased to a greater extent in the ischemic lobe after 9 hours of reperfusion. These data suggest that MIP-2 may be involved in the initial recruitment of neutrophils to the ischemic lobe. In contrast, KC mRNA expression was not increased after 3 hours of reperfusion but after 9 hours increased equivalently in both ischemic and non-ischemic lobes, suggesting a more generalized role in neutrophil recruitment. Neutralization of MIP-2 or KC resulted in significant decreases in hepatic neutrophil accumulation, edema, and hepatocellular injury. These data suggest that the local expression of MIP-2 and KC are important mediators involved in neutrophil-dependent hepatic injury induced by ischemia and reperfusion in mice.
Strain difference in the induction of T-cell activation-associated, interferon gamma-dependent hepatic injury in mice
Authors: Mizuhara Hidekazu , Kuno Masako , Seki Nobuo , Yu Wen-Gong , Yamaoka Makiko , Yamashita Masakatsu , Ogawa Toshikazu , Kaneda Kenji , Fujii Takashi , Senoh Hachiro , Fujiwara Hiromi
Published: July 2004

A single intravenous injection of concanavalin A (Con A) induces T-cell activation-associated inflammatory injury selectively in the liver. This study investigated the strain difference in the development of Con A-induced hepatic injury. Normal C57BL/6 and BALB/c spleen cells produced comparable levels of T-cell-derived lymphokines (interferon gamma [IFN-γ], tumor necrosis factor α [TNF-α], and interleukin-2 [IL-2]) following in vitro stimulation with Con A. A single intravenous injection of Con A to C57BL/6 mice induced the plasma levels of TNF-α and IL-2 comparable with or slightly higher than those observed in BALB/c mice, whereas the same treatment resulted in an apparently lower level of IFN-γ production in C57BL/6 mice. RNA from livers of Con A-treated C57BL/6 mice exhibited lower levels of IFN-γ mRNA than RNA of BALB/c livers. Unexpectedly, a dramatic difference in the severity of hepatic injury was observed between C57BL/6 and BALB/c. Namely, the peak alanine transaminase (ALT) level was more than 15,000 U/L and inducible as early as 8 hours after injection of 0.2 mg Con A per mouse in the C57BL/6 strain, whereas the peak was approximately 3,000 U/L and induced as late as 24 hours after Con A injection in the BALB/c strain. The increase in plasma ALT levels was limited to less than 10% by injection of anti-IFN-γ monoclonal antibody (mAb) in both strains. The C57BL/6 strain inducing lower levels of IFN-γ exhibited higher IFN-γ responsiveness as exemplified by the intrahepatic expression of an IFN-γ-inducible gene, an inducible type of nitric oxide (NO) synthase (iNOS). These results indicate that, while IFN-γ produced in vivo by activated T cells induces hepatic injury, there exists a striking strain difference in the induction of IFN-γ-dependent hepatic injury.
Taurohyodeoxycholic acid protects against taurochenodeoxycholic acid-induced cholestasis in the rat
Authors: Roda Aldo , Piazza Francesco , Baraldini Mario , Speroni Ester , Guerra Maria Clelia , Cerré Carolina , Forti Giorgio Cantelli
Published: July 2004

The prevention of the hepatotoxic effects produced by intravenous infusion of taurochenodeoxycholic acid (TCDCA) by coinfusion with taurohyodeoxycholic acid (THDCA) was evaluated in bile fistula rats; the hepatoprotective effects of the latter were also compared with those of tauroursodeoxycholic acid (TUDCA). Rats infused with TCDCA at a dose of 8 μmol/min/kg showed reduced bile flow and calcium secretion, as well as increased biliary release of alkaline phosphatase (AP) and lactate dehydrogenase (LDH). This was associated with a very low biliary secretion rate of TCDCA (≈1 μmol/min/kg). Simultaneous infusion of THDCA or TUDCA at the same dose preserved bile flow and almost totally abolished the pathological leakage of the two enzymes into bile. The effect was slightly more potent for THDCA. The maximum secretion rate of TCDCA increased to the highest value (8 μmol/min/kg) when coinfused with either of the two hepatoprotective bile acids (BA), which were efficiently and completely secreted in the bile, without metabolism. Calcium output was also restored and phospholipid (PL) secretion increased with respect to the control saline infusion. This increase was higher in the THDCA study. These data show that THDCA is highly effective in the prevention of hepatotoxicity induced by intravenous infusion of TCDCA by facilitating its biliary secretion and reducing its hepatic residence time; this was associated with selective stimulation of PL biliary secretion.
Magnetic resonance microscopy and histopathology: Comparative approach of bromobenzene-induced hepatotoxicity in the rat
Authors: Delnomdedieu Marielle , Hedlund Laurence W. , Maronpot Robert R. , Johnson Allan G.
Published: July 2004

The development of magnetic resonance (MR) microscopy has provided new approaches to histology and histopathology. Recent work has shown the promise of increased sensitivity in animal models of chemically induced hepatotoxicity. However, the field is so new that there is little experience to relate changes seen in MR micrographs to the more traditional optical images stained with hematoxylin and eosin. This work compares the sensitivity and reproducibility of MR microscopy with conventional histopathology in detecting bromobenzene-induced hepatotoxicity in the rat. A time-course study was undertaken to provide a range of histopathologies. Specimens were studied at 24, 48, 72, and 96 hours after exposure to 10% of the median lethal dose of bromobenzene. Using 4 animals per group (a total of 32 rats) added statistical significance to the study and defined a range of interanimal variability over 96 hours. This work shows that MR microscopy, besides being nondestructive and three-dimensional, is at least as sensitive as conventional hematoxylin-eosin staining in detecting bromobenzene-induced centrilobular lesions and recovery of the hepatocellular architecture in the rat. This study further suggests that, as we begin to understand the underlying mechanisms of contrast in MR histology, MR may, in fact, supply even higher specificity than more traditional studies: variations were observed in MR images of treated livers at a given time point that could be not be differentiated based on the grading of necrosis and inflammation on hematoxylin-eosin-stained sections.
The beneficial effects of ciprofloxacin on survival and hepatic regenerative activity in a rat model of fulminant hepatic failure
Authors: Kaita Kelly D. , Assy Nimar , Gauthier Tony , Zhang Manna , Meyers Adrienne F. , Minuk Gerald Y.
Published: July 2004

Recently, we reported that ciprofloxacin, an antimicrobial agent with γ-aminobutyric acid (GABAA) receptor antagonist properties, significantly increases hepatic regenerative activity in animal models of alcohol-induced liver disease and cirrhosis. In the present study, we documented the effects of ciprofloxacin on survival and hepatic regeneration in a d -galactosamine (d -gal)-induced model of acute hepatic injury in rats. One hundred nineteen adult, male Sprague-Dawley rats (n = 19-20/group) were treated with intraperitoneal d -gal (total dose: 2.5 g/kg), followed by gastric gavage with either saline, ciprofloxacin (10, 50, or 100 mg/kg), norfloxacin (250 mg/kg), or intraperitoneal putrescine (300 μmol/kg), a potent hepatic growth promoter. Mortality rates were then documented over a 4-day period. An additional 45 rats (n = 15/group) received a sublethal dose of d -gal (1.0 g/kg), followed by gastric gavage with either saline or ciprofloxacin (100 mg/kg), or intraperitoneal putrescine (300 μmol/kg). In these rats, hepatic regenerative activity was documented at 12, 24, and 60 hours post-d -gal by 3H-thymidine incorporation into hepatic DNA and proliferating cell nuclear antigen (PCNA) staining. In the survival study, a dose-response effect of ciprofloxacin on survival was observed (ciprofloxacin: 10 mg/kg, 10%; 50 mg/kg, 26%; and 100 mg/kg, 35%) with the results in the 100-mg/kg-treated group being significant when compared with the 5% survival rate in saline-treated controls (P < .05). Survival figures in the norfloxacin- and putrescine-treated groups were not significantly improved (15% and 25%, respectively). In the regeneration study, compared with the d -gal + saline-treated control group, DNA synthesis rates at 60 hours were increased in the d -gal + ciprofloxacin and d -gal + putrescine groups (10.2 ± 3.3 vs. 18.2 ± 5.1 and 18.8 ± 6.8 × 103 dpm/mg DNA respectively; P < .05). The results of PCNA staining also supported enhanced hepatic regeneration in the ciprofloxacin-treated group at 60 hours (saline, 13.4 ± 3.7; ciprofloxacin, 47.4 ± 7.3; and putrescine, 8.4% ± 2.8% hepatocytes staining positive). Ciprofloxacin at a dose of 100 mg/kg significantly improves survival and hepatic regenerative activity in this animal model of acute hepatic injury.
Impaired activity of the bile canalicular organic anion transporter (Mrp2/cmoat) is not the main cause of ethinylestradiol-induced cholestasis in the rat
Authors: Koopen Nynke R. , Wolters Henk , Havinga Rick , Vonk Roel J. , Jansen Peter L. , Müller Michael , Kuipers Folkert
Published: July 2004

To test the hypothesis that impaired activity of the bile canalicular organic anion transporting system mrp2 (cmoat) is a key event in the etiology of 17α-ethinylestradiol (EE)-induced intrahepatic cholestasis in rats, EE (5 mg/kg subcutaneously daily) was administered to male normal Wistar (NW) and mrp2-deficient Groningen Yellow/Transport-deficient Wistar (GY/TR−) rats. Elevated plasma bilirubin levels in GY/TR− rats increased upon EE-treatment from 65 ± 8.4 μmol/L to 183 ± 22.7 μmol/L within 3 days, whereas bilirubin levels remained unaffected in NW rats. Biliary bilirubin secretion was 1.5-fold increased in NW rats but remained unaltered in GY/TR− rats. Plasma bile salt concentrations remained unchanged in both strains, although hepatic levels of the sinusoidal Na+-taurocholate cotransporting protein (ntcp) were markedly reduced. Biliary secretion of endogenous bile salt was not affected in either strain. A clear reduction of mrp2 levels in liver plasma membranes of NW rats was found after 3 days of treatment. The bile salt-independent fraction of bile flow (BSIF) was reduced from 2.6 to 2.0 μL/min/100 g body weight in NW rats with a concomitant 62% reduction of biliary glutathione secretion. The absence of mrp2 and biliary glutathione in GY/TR− rats did not prevent induction of EE-cholestasis; a similar absolute reduction of BSIF, i.e., from 1.1 to 0.6 μL/min/100 g body weight, was found in these animals. EE treatment caused a reduction of the maximal biliary secretory rate (SRM) of the mrp2 substrate, dibromosulphthalein (DBSP), from 1,040 to 695 nmol/min/100 g body weight (−38%) in NW rats and from 615 to 327 nmol/min/100 g body weight (−46%) in GY/TR− rats. These results demonstrate that inhibition of mrp2 activity and/or biliary glutathione secretion is not the main cause of EE-induced cholestasis in rats. The data indicate that alternative pathways exist for the biliary secretion of bilirubin and related organic anions that are also affected by EE.
Extrahepatic deposition and cytotoxicity of lithocholic acid: Studies in two hamster models of hepatic failure and in cultured human fibroblasts
Authors: Ceryak Susan , Bouscarel Bernard , Malavolti Mauro , Fromm Hans
Published: July 2004

Effects of bile acids on tissues outside of the enterohepatic circulation may be of major pathophysiological significance under conditions of elevated serum bile acid concentrations, such as in hepatobiliary disease. Two hamster models of hepatic failure, namely functional hepatectomy (HepX), and 2-day bile duct ligation (BDL), as well as cultured human fibroblasts, were used to study the comparative tissue uptake, distribution, and cytotoxicity of lithocholic acid (LCA) in relation to various experimental conditions, such as binding of LCA to low-density lipoprotein (LDL) or albumin as protein carriers. Fifteen minutes after iv infusion of [24-14C]LCA, the majority of LCA in sham-operated control animals was recovered in liver, bile, and small intestine. After hepatectomy, a significant increase in LCA was found in blood, muscle, heart, brain, adrenals, and thymus. In bile duct-ligated animals, significantly more LCA was associated with blood and skin, and a greater than twofold increase in LCA was observed in the colon. In the hepatectomized model, the administration of LCA bound to LDL resulted in a significantly higher uptake in the kidneys and skin. The comparative time- and concentration-dependent uptake of [14C]LCA, [14C]chenodeoxycholic acid (CDCA), and [14C]cholic acid (CA) in cultured human fibroblasts was nonsaturable and remained a function of concentration. Initial rates of uptake were significantly increased by approximately tenfold, with decreasing hydroxylation of the respective bile acid. After 1 hour of exposure of fibroblasts to LCA, there was a significant, dose-dependent decrease in mitochondrial dehydrogenase activity from 18% to 34% of the control, at LCA concentrations ranging from 1 to 20 μmol/L. At a respective concentration of 100 and 700 μmol/L, CDCA caused a 35% and 99% inhibition of mitochondrial dehydrogenase activity. None of the bile acids tested, with the exception of 700 μmol/L CDCA, caused a significant release of cytosolic lactate dehydrogenase into the medium. In conclusion, we show that bile acids selectively accumulate in nonhepatic tissues under two conditions of impaired liver function. Furthermore, the extrahepatic tissue distribution of bile acids during cholestasis may be affected by serum lipoprotein composition. At a respective concentration of 1 and 100 μmol/L, LCA and CDCA induced mitochondrial damage in human fibroblasts, after just 1 hour of exposure. Therefore, enhanced extrahepatic uptake of hydrophobic bile acids during liver dysfunction, or disorders of lipoprotein metabolism, may have important implications for bile-acid induced cytotoxic effects in tissues of the systemic circulation.
Enhanced expression of hepatic acyl-coenzyme A synthetase and microsomal triglyceride transfer protein messenger RNAs in the obese and hypertriglyceridemic rat with visceral fat accumulation
Authors: Kuriyama Hiroshi , Yamashita Shizuya , Shimomura Iichiro , Funahashi Tohru , Ishigami Masato , Aragane Katsumi , Miyaoka Kouji , Nakamura Tadashi , Takemura Kaoru , Man Zhiwei , Toide Kiyotaka , Nakayama Natsuki , Fukuda Yoji , Lin Marie C. , Wetterau John R. , Matsuzawa Yuji
Published: July 2004

The liver plays a central role in lipoprotein metabolism. In particular, very-low density lipoprotein (VLDL) is assembled in the hepatocytes and secreted into the blood circulation. The VLDL is then catabolized to low-density lipoprotein by lipoprotein lipase and hepatic triglyceride lipase. Obese subjects, especially those with visceral fat accumulation, are frequently associated with hyperlipidemia, non-insulin-dependent diabetes mellitus (NIDDM), and hypertension. The mechanism of hyperlipidemia in visceral fat obesity has not yet been elucidated. Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of NIDDM, characterized by obesity with visceral fat accumulation, hyperlipidemia, and late-onset insulin resistance. To elucidate the mechanism of hyperlipidemia observed in OLETF rats, we focused on the production of VLDL by the liver and investigated hepatic messenger RNA (mRNA) levels of microsomal triglyceride transfer protein (MTP), acyl-coenzyme A synthetase (ACS), and apolipoprotein B (apo B), which play important roles in VLDL synthesis and secretion. In 6-week-old OLETF rats, in which insulin resistance had not been manifested, visceral fat weight was already higher and portal free fatty acid (FFA) and VLDL-triglyceride levels were elevated compared with the control rats. Hepatic ACS activity and mRNA levels, and MTP mRNA levels were also increased in OLETF rats, whereas apo B mRNA levels were similar; these results suggest that the enhanced expression of both ACS and MTP genes associated with visceral fat accumulation before developing insulin resistance may be involved in the pathogenesis of hyperlipidemia in obese animal models with NIDDM.
Compatible organic osmolytes in rat liver sinusoidal endothelial cells
Authors: Weik Christian , Warskulat Ulrich , Bode Johannes , Peters-Regehr Thorsten , Häussinger Dieter
Published: July 2004

Compatible organic osmolytes, such as betaine and taurine are involved in the regulation of Kupffer cell (KC) function, but nothing is known about osmolytes in liver endothelial cells. This was investigated here by studying the effect of aniso-osmotic exposure of rat liver sinusoidal endothelial cells (SEC) on osmolyte transport and the messenger RNA (mRNA) levels for the transport systems for betaine (BGT1), taurine (TAUT), and myo-inositol (SMIT). Compared with normo-osmotic exposure (305 mosmol/L), hyperosmotic exposure (405 mosmol/L) of SEC led to an increase in the mRNA levels for these transport systems and simultaneously to a stimulation of betaine, taurine, and myo-inositol uptake, which led to an increase of cell volume. Conversely, hypo-osmotic exposure decreased osmolyte uptake. When hyperosmotically pre-exposed SEC were loaded with betaine, taurine, or myo-inositol, hypo-osmotic stress stimulated the efflux of these osmolytes from the cells. Studies on osmolyte tissue levels revealed that taurine was an important compatible organic osmolyte under normo-osmotic conditions and predominantly released following hypo-osmotic stress. Conversely, following hyperosmotic exposure, the increase in cellular betaine and myo-inositol exceeded that of taurine. In lipopolysaccharide (LPS)-treated SEC, hyperosmotic exposure markedly raised the mRNA levels for cyclo-oxygenase-2 (COX-2), but not for inducible nitric oxide synthase (iNOS). The increase of COX-2 mRNA levels was counteracted by betaine and taurine and, to a lesser extent, by myo-inositol. The findings indicate that SEC use taurine, betaine, and myo-inositol as compatible organic osmolytes.
Hepatic uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients: The influence of virus C infection
Authors: Moran Maria Jose , Fontanellas Antonio , Brudieux Eric , Hombrados Isabelle , de Ledinghen Victor , Couzigou Patrice , de Verneuil Hubert , De Salamanca Rafael Enriquez
Published: July 2004

Porphyria cutanea tarda (PCT) is caused by a decreased activity of the hepatic enzyme uroporphyrinogen decarboxylase (URO-D). This deficiency causes overproduction, hepatic deposition, and increased excretion of uroporphyrin. Iron overload and hepatic viral infections are considered aggravating factors of the disease. Two forms of PCT have been described, as follows: a familial one with an inherited decrease of URO-D activity in all tissues and a sporadic one with a decreased activity of URO-D restricted to the liver. To assess whether the hepatic URO-D returns to normal during a remission of the disease, this activity was measured in liver biopsy samples in 24 sporadic PCT patients. The hepatic and urinary porphyrin concentrations were also measured. Viral status and histopathological findings were analyzed to assess their involvement in PCT. Six patients treated by phlebotomy to reduce hepatic iron and who were considered to be in clinical remission, characterized by a disappearance of cutaneous lesions, showed higher hepatic URO-D activities and lower hepatic porphyrin concentrations than did patients with overt PCT. The medians of these variables, however, did not achieve normal values. The hepatic URO-D activity showed a significant inverse relationship with both hepatic porphyrins and urinary uroporphyrin excretion. Hepatic URO-D activity was not reduced by hepatitis C virus (HCV) infection and liver damage. We conclude that the achievement of remission in PCT largely depends on the transient normalization of hepatic URO-D activity. A small increase in hepatic coproporphyrin in nonporphyric patients could reflect hepatic injury/iron/alcohol-induced oxidative stress oxidizing the accumulated heme precursors rather than a direct effect on hepatic URO-D enzyme.
Expression of α-fetoprotein and albumin genes in human hepatocellular carcinomas: Limitations in the application of the genes for targeting human hepatocellular carcinoma in gene therapy
Authors: Ohguchi Souhei , Nakatsukasa Harushige , Higashi Toshihiro , Ashida Kozo , Nouso Kazuhiro , Ishizaki Masahiko , Hino Naoki , Kobayashi Yoshiyuki , Uematsu Shuji , Tsuji Takao
Published: July 2004

For an approach of gene therapy for hepatocellular carcinoma (HCC), transcriptional regulatory sequence (TRS) of either α-fetoprotein (AFP) or albumin has been used for targeting cancer cells. To examine the feasibility of using TRSs of these genes for possible gene therapy of HCCs, the cellular distribution of AFP and albumin gene transcripts was studied in 25 cases of surgically removed human HCCs. AFP gene expression was observed in HCC nodules of 13 cases. The expression in HCC was heterogeneous, and the distribution of the transcripts was mostly sparse and spotty. The higher the serum AFP levels, the larger population of the AFP-expressing HCC cells tended to reflect. In noncancerous liver, a slight AFP expression was found by Northern blot analysis, but the transcripts were not detected in the liver sections. In contrast, albumin expression was found in all HCCs as well as in noncancerous hepatocytes. In HCC, the transcripts for albumin were distributed in cancer cells, and the expression varied with nodules. There were more albumin-expressing cancer cells than the AFP-expressing cells. Albumin expression was retained even in poorly differentiated HCC, although the intensity of the signal was not as strong as in more-differentiated HCCs. Metastatic HCC nodules revealed transcripts for both AFP and albumin genes, and those were clearly recognized in the lung tissue. These results suggest that, for gene therapy for HCCs, neither AFP nor albumin are ideal options for targeting HCC cells. AFP-TRS may be used as a transcriptional regulator in selected cases in which AFP gene expression is observed in the cancer nodules. The serum AFP level appears to be an important indicator in selecting cases. Albumin-TRS in conjunction with retroviral vector might be used in limited cases such as HCCs with no AFP expression. However, careful consideration must be taken, because albumin is constitutively expressed in normal hepatocytes, and AFP-expressing nonmalignant progenitor cells possibly exist.
Pancreastatin receptor is coupled to a guanosine triphosphate-binding protein of the Gg/11α family in rat liver membranes
Authors: Santos-Álvarez José , González-Yanes Carmen , Sánchez-Margalet Víctor
Published: July 2004

Pancreastatin (PST), a recently discovered regulatory peptide derived from chromogranin A, has been shown to have a glycogenolytic effect in the hepatocyte that is mediated by increasing intracellular calcium. Our previous studies on pancreastatin signaling suggested that PST receptor is coupled to some G proteins in the plasma membrane of the hepatocyte. The nature of this interaction was investigated using antisera against Gq/11α by different approaches. Indirect evidence of a pertussis toxin (PT)-insensitive G protein of the family of Gq/11α was obtained by measuring high-affinity guanosine triphosphatase (GTPase) activity in soluble rat liver membranes. PST increased GTPase activity in a dose-dependent manner. This effect was only slightly inhibited by PT pretreatment of the membranes, whereas anti-Gq/11α antisera blocked most of the PST-stimulated GTPase activity. The selective association of the PST receptor with this G protein was further studied by the coelution in wheat germ agglutinin semipurification of the receptor and by immunoprecipitation of the G protein-PST receptor complexes using G-protein-specific antisera. A G protein of the family of Gq/11α was found to be associated with the semipurified PST receptor. Moreover, anti-Gq/11α antisera immunoprecipitated most PST-binding activity (95%), bringing down most of the specific G protein, whereas anti-Gi1,2α and -Go,i3α failed to immunoprecipitate the PST-binding activity. Finally, the coupling of the PST receptor with the effector phospholipase C was disrupted by blocking with Gq/11α antisera, suggesting that a G protein of the family of Gq/11α is a signal mediator from PST receptors to phospholipase C activation in rat liver membranes.
Bile acid synthesis in primary cultures of rat and human hepatocytes
Authors: Ellis Ewa , Goodwin Bryan , Abrahamsson Anna , Liddle Chris , Mode Agneta , Rudling Mats , Björkhem Ingemar , Einarsson Curt
Published: July 2004

The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7α-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 ± 24 ng/mL (67% cholic acid) on day 1 to 55 ± 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, α-muricholic acid, and β-muricholic acid were formed. Treatment with triidothyronine (T3) or dexamethasone alone had no significant effect on bile acid production. A combination of T3 and dexamethasone significantly increased the total bile acid production on day 4 (224 ± 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12α-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7α-hydroxylase activity of 3.3 ± 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T3 had an activity of 16.4 ± 3.6 pmol/mg protein/min. The cholesterol 7α-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 ± 0.9 cpm/μg total nucleic acids (tNAs). T3- or dexamethasone-treated cells did not differ from control cells, whereas the combination of T3 and dexamethasone increased the mRNA levels to 20.6 ± 2.8 cpm/μg tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 ± 79 ng/mL on day 2 to 1490 ± 594 ng/mL on day 6 and then declined slightly. Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7α-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T3or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3- to 5-fold higher than that in human liver in vivo. It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo, this represents a useful model for further studies of the synthesis and regulation of bile acids.
Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†
Authors: Aizaki Hideki , Aoki Yoichiro , Harada Takashi , Ishii Koji , Suzuki Tetsuro , Nagamori Seishi , Toda Gotaro , Matsuura Yoshiharu , Miyamura Tatsuo
Published: July 2004

We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3′ end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.
Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective
Authors: Melegari Margherita , Scaglioni Pier Paolo , Wands Jack R.
Published: July 2004

Hepatitis B virus (HBV) variant strains may develop during therapy for chronic infection with the nucleoside analog 2′,3′-dideoxy-3′-thiacytidine (3TC). HBV mutants result from isoleucine (I) or valine (V) substitutions in the methionine (M) of the YMDD motif in the viral reverse-transcriptase catalytic domain. In addition, other mutations in the reverse-transcriptase “B domain” involving either a phenylalanine (F)-to-leucine (L) at amino acid 501 (F501L) or an L-to-M substitution at amino acid 515 (L515M) have been observed during 3TC and Famciclovir therapy as well. To determine the biologic consequences of these mutations on viral replication, variant viral genomes were constructed and transiently transfected into hepatocellular carcinoma (HCC) and HEK 293 human embryo kidney-derived cell lines. In transiently transfected HCC cells, the viruses bearing the YI/VDD or F501L mutations had greatly impaired replication as compared to wild-type virus, whereas the virus carrying the L515M substitution showed the least defect. Double mutants with the L515M substitution showed intermediate defect between the YI/VDD or F501L and the L515M single-mutant strains. In contrast, when transfected into HEK 293 cells, the viruses bearing the YI/VDD or L515M mutation replicated as wild-type. However, under conditions of deoxynucleotide depletion produced by hydroxyurea treatment of HEK 293 cells, all mutants but not the wild-type virus exhibited a reduced replication phenotype similar to that observed in HCC cells. In both HCC and HEK 293 cells, the mutant viruses carrying the F501L substitution showed a decreased pregenomic RNA encapsidation level, suggesting that the defect in HBV DNA synthesis occurs at the RNA packaging level. These findings show that 3TC and Famciclovir selected mutations alter the properties of the HBV reverse transcriptase, resulting in impaired viral replication within the cell.
Cholesterol metabolism and serum and biliary noncholesterol sterols in gallstone patients during simvastatin and ursodeoxycholic acid treatments†
Authors: Miettinen Tatu E. , Kiviluoto Tuula , Taavitsainen Matti , Vuoristo Matti , Miettinen Tatu A.
Published: July 2004

Effects of long-term high-dose ursodeoxycholic acid (UDCA) and simvastatin treatments on cholesterol metabolism and biliary lipid compositions were compared in patients with cholesterol gallstones. Absorption and synthesis of cholesterol, serum and biliary noncholesterol sterols and lipids were determined in 14 patients randomized to UDCA (23-25 mg/kg/d) or simvastatin (40 mg/d) for 1 year. Simvastatin reduced serum low-density lipoprotein cholesterol by 55%, and UDCA, by 9%. Cholesterol absorption was decreased (35%) by UDCA, but nonsignificantly increased by simvastatin (P< .05 for difference of changes caused by the two drugs). Whole-body synthesis and biliary output of cholesterol were both significantly decreased only by UDCA. In addition, UDCA inconsistently increased the proportions of serum and biliary precursor sterols of cholesterol, known to reflect cholesterol synthesis, but did not affect their biliary secretions. Simvastatin, however, dramatically reduced serum and also biliary cholesterol precursor sterol proportions and their biliary secretions and increased proportions of serum and biliary plant sterols and cholestanol, known to reflect cholesterol absorption, but had no effect on their biliary secretion. Only UDCA significantly decreased the molar percentage of cholesterol, the lithogenic index, and the cholesterol/phospholipid (CH/PL) ratio in bile, whereas both treatments inconsistently decreased the vesicular CH/PL ratio (P < .07 in both groups). It is concluded that both drugs decreased serum cholesterol and inhibited cholesterol synthesis, but had a differing influence on precursor sterols and the absorption of cholesterol. UDCA had more beneficial effects than simvastatin on the antilithogenic properties of bile.
Time dependent changes in the concentration and type of bacterial sequences found in cholesterol gallstones
Authors: Swidsinski Alexander , Khilkin Michael , Pahlig Hartmut , Swidsinski Sonja , Priem Friedrich
Published: July 2004

The role of bacteria in gallstone formation could not be conclusively evaluated until bacterial presence or absence in a stone was consistently shown. Cultural bacteriologic investigations at the time of cholecystectomy, however, led to the assumption that cholesterol gallstones were free of bacteria. In this study, we used a culture independent, molecular genetic approach to detect, quantify, and identify bacteria in cholesterol gallstones from 100 patients at the time of cholecystectomy and 6 months following. Bacterial growth was recorded in the culture in 9 of 100 gallstones; bacterial DNA, however, was detected in 82 of 91 sterile gallstones. High concentrations corresponding to between 106 to 107 bacteria/g were detected in 11 stones and low concentrations of 105 bacteria/g were detected in 71 sterile stones. The infection in stones with a positive bacterial culture was characterized by the predominance of single bacterial sequence(s) of the bacteria cultured. A similar predominance, indicating a recent infection, was found in sterile gallstones with low DNA concentrations. A high diversity of non-repeating bacterial sequences, possibly arising from previous overlapping infections, was found in sterile gallstones with high concentrations of bacterial DNA. After 6 months concentrations of bacterial DNA fell significantly in all groups of gallstones. As bacterial DNA is quickly destroyed upon storage, but is nevertheless readily found in most gallstones at the time of cholecystectomy, there must be a mechanism by which it is replenished. One such mechanism is the frequently reoccurring, possibly self-terminating infection and another one is the permanent colonization of the gallstone with bacteria at low concentrations. Both can promote cholecystolithiasis.
Hepatic plasma flow estimated according to fick’s principle in patients with hepatic encephalopathy: Evaluation of indocyanine green and d-sorbitol as test substances
Authors: Clemmesen Jens Otto , Tygstrup Niels , Ott Peter
Published: July 2004

The magnitude of hepatic plasma flow in patients with liver failure and hepatic encephalopathy (HE) is unknown because a reliable flow estimate has not been available. The purpose of this study was to estimate hepatic plasma flow in patients with HE and to evaluate indocyanine green (ICG) and sorbitol as test compounds. Fourteen patients with acute liver failure (ALF) and nine patients with chronic liver failure (CLF), all with HE grade II or more, were studied. After hepatic vein catheterization, hepatic plasma flow was estimated by use of constant infusion, simultaneous arterial and hepatic vein concentration measurements, and calculated according to Fick’s principle. The hepatic extraction fraction of d-sorbitol 0.179 ± 0.144 (mean ± SD) was higher than the hepatic extraction fraction of ICG 0.054 ± 0.085 (P < .001). The low hepatic extraction fraction of ICG rendered this compound unfit for estimation of hepatic plasma flow in these patients. In contrast, by using d-sorbitol the hepatic plasma flow could be estimated in 21 of 23 patients with a median SD of 8.4% (range, 2.6% to 29%). The d-sorbitol estimated hepatic plasma flow was 1.2 ± 0.5 L/min (n = 12) in patients with ALF and 1.4 ± 0.9 L/min (n = 9) in patients with CLF. These values are higher than what has been reported in normal subjects and in patients with cirrhosis without HE. An elevated hepatic flow should increase oxygen delivery and may enhance the failing liver’s ability to remove substances from the blood. At the same time, hepatic first pass metabolism is reduced. We conclude that an elevated hepatic flow in these patients is of clinical importance.
Splenic impedance indices: A useful method to monitor patients after liver transplantation?
Authors: Bolognesi Massimo , Sacerdoti David , Bombonato Giancarlo , Angeli Paolo , Gerunda Giorgio , Merenda Roberto , Volpin Roberta , Iemmolo Rosamaria , Gatta Angelo
Published: July 2004

Splenic Doppler impedance indices are influenced in portal hypertensive patients, by the resistance of the portal system. The aim of the study was to verify the usefulness of these indices in evaluating the presence of a pathological increase in portal resistance in patients with complications after liver transplantation. Splenic impedance indices have been evaluated in 46 patients before orthotopic liver transplantation (OLT), and 2 days, 1, 4, 8, and 12 to 18 months after transplantation. The results showed that spleen size slowly decreased after liver transplantation. From a baseline longitudinal diameter value of 18.0 ± 3.6 cm (M ± SD), the decrease was by 0% ± 3%, 8% ± 8%, 13% ± 9%, 15% ± 11%, and 14% ± 11% at 2 days and 1, 4, 8, and 12 to 18 months after liver transplantation. Splenic impedance indices—resistance index = (peak systolic − end diastolic) / peak systolic velocity; pulsatility index = (peak systolic − end diastolic) / mean velocity—which were increased before liver transplantation, showed a rapid decrease to normal values: resistance index: from 0.62 ± 0.08 to 0.55 ± 0.08 after 2 days, and to 0.49 ± 0.09, 0.51 ± 0.10, 0.54 ± 0.10, 0.55 ± 0.11 after 1, 4, 8, 12-18 months; pulsatility index: from 0.96 ± 0.21 to 0.82 ± 0.17 after 2 days, and to 0.69 ± 0.19, 0.72 ± 0.21, 0.81 ± 0.26, 0.84 ± 0.26 after 1, 4, 8, and 12 to 18 months. Patients who had a good outcome, without any major complications, showed a clear and steady decrease in splenic impedance indices. On the contrary, patients who had complications affecting portal resistance (e.g., acute rejection, relapse of chronic hepatitis C virus-related hepatitis or cirrhosis, stenosis of portal anasthomosis, portal thrombosis), showed a lack of decrease, or, after an initial decrease, a subsequent re-increase in splenic impedance indices to pathological values. Splenic impedance indices measured in patients with complications were higher than those of patients without complications (P< .0004). Specificity and sensitivity of splenic impedance indices in the evaluation of the presence of complications increasing portal resistance were good. In conclusion, after OLT, splenic impedance indices could be useful aspecific parameters for identifying patients with complications that are able to affect or increase portal resistance.