bluuebunny/crossref_metadata_2025 huggingface dataset:
Humoral inhibitor of rat hepatocyte DNA synthesis from patients with fulminant liver failure†
Authors: Yamada Haruki , Toda Gotaro , Yoshiba Makoto , Hashimoto Naoaki , Ikeda Yusei , Mitsui Hiroshi , Kurokawa Kiyoshi , Sugata Fumio , Hughes Robin D. , Williams Roger
Published: March 2007

Sera and ultrafiltrates (relative molecular mass<10,000 Da) from patients with fulminant liver failure inhibit hepatocyte DNA synthesis in vivo. In this study the effects of ultrafiltrates from pooled sera from fulminant liver failure patients in the United Kingdom and plasma ultrafiltrates from fulminant liver failure patients in Japan have been investigated in primary cultured rat hepatocytes, with incubation for up to 72 hr. Both types of ultrafiltrate inhibited the incorporation of [3H]thymidine into acid-precipitable material and reduced the cell labeling index as determined on autoradiography in hepatocytes stimulated by epidermal growth factor and insulin compared with normal sera/plasma ultrafiltrates. The inhibitory effects observed were dose dependent, reversible when the fulminant liver failure ultrafiltrate was removed and were not associated with increased release of lactate dehydrogenase or suppression of protein synthesis as assessed on the basis of the incorporation of [3H]leucine. The effects appeared to be specific for hepatocytes; in preliminary experiments DNA synthesis was not inhibited in cultured fibroblasts (NIH 3T3 cells). These experiments are further evidence of the presence of an inhibitory factor of relative molecular mass less than 10,000 Da in the blood of patients with fulminant liver failure. (Hepatology 1994;19:1133-1140.)
Acute hepatic and renal toxicity from low doses of acetaminophen in the absence of alcohol abuse or malnutrition: Evidence for increased susceptibility to drug toxicity due to cardiopulmonary and renal insufficiency
Authors: Bonkovsky Herbert L. , Kane Robert E. , Jones Dean P. , Galinsky Raymond E. , Banner Barbara
Published: March 2007

A 67-yr-old man with chronic cardiopulmonary disease exhibited severe hepatic and moderately severe renal injury after short-term ingestion of therapeutic doses of acetaminophen (1 to 3 gm/day for 3 days). Drug metabolism and other studies, performed 5 mo after recovery from the acute insult, indicated that the patient had decreased rates of hepatic metabolism of acetaminophen to its primary, nontoxic metabolites and decreased kidney function that was compromised further by acetaminophen ingestion. He also had abnormally low concentrations of hepatic and plasma reduced glutathione. Alcohol abuse and malnutrition could not be implicated in the pathogenesis of injury; rather it appeared that advancing age with chronic renal, cardiac and pulmonary insufficiency contributed to acetaminophen toxicity in this patient. (Hepatology 1994;19:1141-1148.)
The canadian multicenter double-blind randomized controlled trial of ursodeoxycholic acid in primary biliary cirrhosis
Authors: Heathcote Jenny E. , Cauch-Dudek Karen , Walker Valery , Bailey Robert J. , Blendis Laurence M. , Ghent Cameron N. , Michieletti Pina , Minuk Gerald Y. , Pappas Chris S. , Scully Linda J. , Steinbrecher Urs P. , Sutherland Lloyd R. , Williams Noel C. , Witt-Sullivan Helga , Worobetz Lawrence J. , Milner Ruth A. , Wanless Ian R.
Published: March 2007

Ursodeoxycholic acid, a dihydroxyl bile acid normally present in human beings in minimal amounts, becomes incorporated into the bile salt pool when taken orally. In cholestasis, bile acids are retained in the liver and are hepatotoxic. Ursodeoxycholic acid is the least-known hepatotoxic bile acid, has choleretic properties and is reported to benefit patients with chronic cholestasis. In a nationwide Canadian controlled trial, 222 patients with primary biliary cirrhosis were treated with ursodeoxycholic acid (14 mg/kg/body wt/day) or placebo for 24 mo. Only patients with a diagnosis confirmed by liver biopsy and serum positive for antimitochondrial antibodies were enrolled; 88% were symptomatic on entry. The primary outcome measure was percent change in total serum bilirubin from baseline to final follow-up. Treated patients (111) and controls (111) were comparable with regard to age, gender, biochemical parameters and liver histological condition. Although treatment was not associated with any improvement in symptoms, ursodeoxycholic acid therapy caused the bilirubin to fall significantly within the first 3 mo of therapy (p<0.001). Significant falls in serum alkaline phosphatase, aminotransferases, cholesterol and IgM levels were also noted in the treated group. Improvement in some histological features was observed but there was no difference between the groups in the number of patients who reached the endpoints of death or liver transplantation. Ursodeoxycholic acid, given to patients with primary biliary cirrhosis, leads to an improvement in serum markers of cholestasis. A larger sample size is needed to determine whether ursodeoxycholic acid therapy has a beneficial effect on the survival of patients with primary biliary cirrhosis. (Hepatology 1994;19:1149-1156.)
Ultrastructural location of human hepatocyte growth factor in human liver
Authors: Sakaguchi Hiroki , Seki Shuichi , Tsubouchi Hirohito , Daikuhara Yasushi , Niitani Yoshiyuki , Kobayashi Kenzo
Published: March 2007

Human hepatocyte growth factor has been purified from the plasma of patients with fulminant liver failure, but where this factor is produced in organs or cells of subjects with liver diseases is unknown. Therefore, we used a monoclonal antibody to human hepatocyte growth factor to stain cells in three normal and 29 diseased liver tissues by immunohistochemical techniques. By light microscopy, the immunostained cells seemed to be polymorphonuclear leukocytes because of their segmented nuclei. Some biliary epithelial cells also were stained. Electron microscopy confirmed that the immunostained cells with segmented nuclei were polymorphonuclear leukocytes and that the stained grains were on the membranes of rough endoplasmic reticulum, around specific or azurophilic granules and in the cell sap. Stained grains in the biliary epithelial cells were found sporadically on the inside and outside of the membranes of rough endoplasmic reticulum near the nuclei. Human hepatocyte growth factor is now known to be the same protein as scatter factor and tumor cytotoxic factor, both of which are produced by human fibroblasts in culture, but our results suggest that polymorphonuclear leukocytes in diseased livers are one cellular source of circulating human hepatocyte growth factor. The immunostaining properties of biliary epithelial cells in diseased livers also suggest that the cells produce and secrete human hepatocyte growth factor. (Hepatology 1994;19:1157-1163.)
Inhibition of procoagulant activity of human monocytes by chenodeoxycholic acid: Involvement of protein kinase C
Authors: Podevin Philippe , Calmus Yvon , Robert Annie , Poupon Raoul , Cherqui Gisele
Published: March 2007

Endogenous bile acids such as chenodeoxycholic acid have been shown to display a suppressive effect in vitro on mononuclear cell activation. We investigated the signal transduction pathway involved in the effect of chenodeoxycholic acid on monocyte procoagulant activity, a model of monocyte activation. Chenodeoxycholic acid (25 to 250 μmol/L) had a concentration-dependent inhibitory effect on procoagulant activity expressed by endotoxin-stimulated mononuclear cells, with half-maximal and maximal inhibition occurring at concentrations of 100 and 250 μmol/L, respectively. The inhibitory effect of chenodeoxycholic acid was (a) closely mimicked by 4β-phorbol 12β-myristate 13α-acetate (PMA), a protein kinase C activator, but not by forskolin or dibutyryl cyclic AMP, two activators of the protein kinase A-dependent pathway; (b) prevented by staurosporine, a potent protein kinase C inhibitor; (c) partially abolished in protein kinase C-depleted cells; and (d) observed in conditions under which chenodeoxycholic acid, like PMA, significantly increased (41%) protein kinase C activity, as assessed by phosphorylation of exogenous (histone III-S) and endogenous (37-kD protein) substrates. In conclusion, our results (a) provide clear evidence of a marked inhibitory effect of chenodeoxycholic acid on monocyte activation, suggesting a potential role of primary endogenous bile acids in the immune defect associated with cholestasis; and (b) indicate that the inhibition of monocyte activation by chenodeoxycholic acid is mediated by way of protein kinase C activation (Hepatology 1994;19:1164-1170.)
Human hepatoma cells synthesize and secrete lysozyme: Modulation by cytokines
Authors: Köbsel Norbert , Ramadori Giuliano
Published: March 2007

Plasma lysozyme levels are elevated in several different pathological conditions. In our study we show that well differentiated human hepatoma cells Hep3B and HepG2 are active synthesis sites of lysozyme and that this synthesis can be modulated by acute phase mediators. The production and modulation of lysozyme synthesis was studied by means of Northern-blot analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a specific bioassay after treatment of the cells with interleukin-1β, interleukin-6 and tumor necrosis factor-α. Hep3B and HepG2 cells constitutively synthesize high amounts of lysozyme. Lysozyme synthesis and secretion were found to be augmented by interleukin-1β and tumor necrosis factor-α in both cell lines. Interleukin-6 caused an increase in lysozyme production in Hep3B but a decrease in the HepG2 cells. As expected, the synthesis of albumin was decreased in both cell lines. Furthermore we demonstrated that HepG2 and Hep3B cells produce a biologically active form of the enzyme as measured by a specific bioassay. The results demonstrate that lysozyme is constitutively synthesized by Hep3B and HepG2 hepatoma cell lines and that lysozyme synthesis is modulated by acute-phase mediators. Well differentiated human hepatoma cells may respond differently to different cytokines. (Hepatology 1994;19:1182-1188.)
Direct regulation of bile secretion by prostaglandins in perfused rat liver
Authors: Beckh Karlheinz , Kneip Susanne , Arnold Rudolf
Published: March 2007

Prostaglandins have been postulated to act as mediators of intercellular communication between liver cell populations in the regulation of liver carbohydrate metabolism. Their role in the regulation of bile secretion is rather unclear. The action of prostaglandins on bile flow and bile acid secretion was studied in the in situ perfused rat liver. Infusion of prostaglandin F2α resulted in reduction of bile flow and bile acid secretion. In addition, portal flow was diminished and glucose output was increased. The parameters were altered in a dose-dependent manner. The effects of
Role of intracellular calcium in superoxide-induced hepatocyte injury
Authors: Murata Masaru , Monden Morito , Umeshita Koji , Nakano Hiroshi , Kanai Toshio , Gotoh Mitsukazu , Mori Takesada
Published: March 2007

The mechanisms of hepatocyte injury caused by exogenous superoxide were investigated with the use of cultured rat hepatocytes. Cell viability, cytosolic free calcium concentration and cell surface structure were observed. Superoxide was produced by adding hypoxanthine and xanthine oxidase to the buffer. Cytosolic free calcium concentration was calculated by means of ratio imaging of fura 2 fluorescence with multiparameter digitized microscopy. In the buffer containing 1.27 mmol/L of calcium, lactate dehydrogenase release into the buffer began to increase at 1 hr and reached a plateau in 5 hr. Eighteen minutes after the addition of hypoxanthine and xanthine oxidase, small blebs were recognized on the cell surface with a scanning electron microscope; then a gradual rise in cytosolic free calcium concentration was observed. Thirty minutes after exposure to superoxide, large blebs were recognized with a phase-contrast microscope, when cytosolic free calcium concentration had risen to about 700 nmol/L. Depriving the buffer of calcium (< 10 μmol/L) significantly suppressed bleb formation and cell death, and cytosolic free calcium concentration was found to remain around the basal level (200 nmol/L). When ethylene glycol-bis (β-amino-ethyl ether)-N,N,N′,N′-tetraacetic acid was added to the buffer, bleb formation and cell death were suppressed more completely, and cytosolic free calcium concentration decreased. Superoxide dismutase combined with catalase or nifedipine allowed the hepatocytes to maintain their viability and suppressed cytosolic free calcium concentration elevation. Calpeptin, a Ca2+-dependent neutral protease inhibitor, did not affect the rise in cytosolic free calcium concentration but prevented cell injury. We concluded that cell injury caused by superoxide is initiated by influx of calcium and that the resultant activation of Ca2+-dependent protease may play an important role in bleb formation and cell death. (Hepatology 1994;19:1223-1228.)
Reversal of behavioral changes in rats subjected to portacaval shunt with oral neomycin therapy
Authors: Conjeevaram Hari S. , Nagle Alexander , Katz Andrew , Kaminsky-Russ Kristine , McCullough Arthur J. , Mullen Kevin D.
Published: March 2007

The portacaval shunt rat is often used as a model of human portal-systemic encephalopathy, but its relevance to human portal-systemic encephalopathy remains uncertain. Specifically, it has not been demonstrated that the behavioral changes seen in this model respond to measures known to improve portal-systemic encephalopathy in human subjects. Accordingly, the aim of this study was to establish whether neomycin (an effective treatment for portal-systemic encephalopathy in human beings) added to the drinking water of rats subjected to portacaval shunt reversed or ameliorated the reduction in spontaneous motor activity, which represents a measure of encephalopathy in this animal model. A randomized, placebocontrolled crossover design was used, with each animal serving as its own control. After establishment of baseline activities, 12 rats with portacaval shunt and 12 sham-operated rats were divided into two equal groups: Group A animals received neomycin for 1 wk; this was followed by 1 wk off neomycin; in group B rats, the sequence was reversed. Spontaneous intake of neomycin for 7 days at doses comparable to human usage (0.1 to 0.2 gm/kg/day) was associated with a significant increase in spontaneous motor activity in rats subjected to portacaval shunt (26.4% in group A, 66.3% in group B; p<0.01 for each protocol) with no significant effect in sham-operated animals. Withdrawal of neomycin resulted in reversal of this effect in group A rats subjected to portacaval shunt. Similar significant improvements for exploratory activity as measured on the basis of nose-hole pokes was also seen in rats subjected to portacaval shunt and given neomycin. These neomycin-associated changes in the motor activity of rats subjected to portacaval shunt could not be attributed to any effect of neomycin on serum ammonia level. These findings support the concept that the rat subjected to portacaval shunt is a valid model of human portal-systemic encephalopathy that can be used to explore the pathophysiology of this syndrome. (Hepatology 1994;19:1245-1250.)
The fate of intravenously injected endotoxin in normal rats and in rats with liver failure
Authors: Nakao Akimasa , Taki Shigemi , Yasui Motoshi , Kimura Yasunori , Nonami Toshiaki , Harada Akio , Takagi Hiroshi
Published: March 2007

The elimination of endotoxin from the blood was studied in rats with D-galactosamine-induced liver failure and in normal controls after intravenous injection of various doses of endotoxin. Endotoxin was found to localize in liver tissue by immunohistochemical staining with factor C, which is derived from amebocyte lysate of the horseshoe crab and which reacts specifically with endotoxin. Before injection, the blood endotoxin concentrations were normal both in control rats and in rats with liver failure. The blood concentrations of endotoxin were significantly higher after intravenous injection of endotoxin in the D-galactosamine-induced liver failure group (p<0.05) and decreased much more slowly (p<0.05). Endotoxin concentrations were also significantly higher after in vitro incubation with plasma from rats with liver failure (p<0.05). After intravenous injection of endotoxin (1 mg/kg), endothelial and Kupffer cells in the liver sinusoids were positively stained for endotoxin in the control group, but not stained or faintly stained in the liver failure group. Endotoxemia in liver failure thus results from reduced inactivation of endotoxin in plasma and from impaired hepatic clearance. (Hepatology 1994;19:1251-1256.)
Impaired function of pancreatic islets from rats with portal hypertension resulting from cirrhosis and partial portal vein ligation
Authors: Gomis Ramon , Fernández-Alvarez Josefa , Pizcueta Pilar , Fernández Mercedes , Casamitjana Roser , Bosch Jaume , Rodés Joan
Published: March 2007

Increased circulating insulin and glucagon levels are a common observation in patients with cirrhosis, as well as in portal hypertensive models. Hyperinsulinemia and hyperglucagonemia may be caused either by increased beta- and alpha-cell secretion or by defective hepatic clearance of these hormones. To elucidate whether an abnormal endocrine pancreatic function might contribute to the hyperinsulinism or to the hyperglucagonism observed in chronic portal hypertension, insulin and glucagon secretion were measured in vitro in isolated pancreatic islets from rats with partial portal vein ligated and rats with cirrhosis caused by carbon tetrachloride poisoning. Both rats with partial portal vein ligation and rats with cirrhosis caused by carbon tetrachloride poisoning exhibited hyperinsulinism and hyperglucagonism as compared with control rats. Isolated pancreatic islets from both experimental portal hypertensive models showed an impaired insulin secretion after glucose stimulation. On the contrary, glucagon secretion was significantly increased, and there was a markedly enhanced response to arginine. This increased in vitro glucagon production could not be corrected, even in the presence of high glucose concentrations in the incubation medium. Therefore our data show that although hyperglucagonism in rats with partial portal vein ligation and in rats with cirrhosis caused by carbon tetrachloride poisoning is promoted by an enhanced alpha-cell secretion, hyperinsulinism is associated with impaired beta-cell secretion. (Hepatology 1994;19:1257-1261.)
Lipopolysaccharide-neutralizing antibody reduces hepatocyte injury from acute hepatotoxin administration
Authors: Czaja Mark J. , Xu Jun , Ju Yue , Alt Elaine , Schmiedeberg Phyllis
Published: March 2007

Endogenous lipopolysaccharide has been implicated as a cofactor in the hepatocellular injury and death resulting from toxic liver injury. To prevent this lipopolysaccharide-induced injury and to further understand the mechanism of this effect, an anti-lipopolysaccharide antibody was administered to rats in which toxic hepatocellular injury was induced. Rats were given the hepatotoxin galactosamine together with an isotypic control antibody B55 or the anti-lipopolysaccharide antibody E5. E5 treatment resulted in reductions of serum AST levels of 43% at 36 hr (p<0.02) and 60% at 48 hr (NS) after galactosamine administration. These decreases in AST values were accompanied by diminished histological evidence of injury and inflammation. In carbon tetrachloride-induced liver injury, E5 similarly reduced serum AST levels at 36 and 48 hr by 47% (p<0.04) and 54% (p<0.03), respectively. E5 treatment was equally effective in reducing AST levels 48 hr after administration of carbon tetrachloride, whether the initial dose of antibody was given 1 hr before or 3 or 6 hr after the administration of this toxin. To understand the mechanism of this E5 effect, the activation of the toxic cytokine tumor necrosis factor-α and the chemotactic cytokine monocyte chemoattractant protein 1 was examined by Northern-blot analysis of RNA from rat livers after galactosamine-induced injury and treatment with B55 or E5. Despite E5’s efficacy in reducing hepatocellular damage, E5 treatment did not affect the timing or magnitude of tumor necrosis factor-α or monocyte chemoattractant protein 1 activation during galactosamine-induced injury. These data suggest that antibody neutralization of lipopolysaccharide may be an effective therapy for toxic liver injury and that the mechanism of this effect is not through the prevention of cytokine expression. (Hepatology 1994;19:1282-1289.)
Hepatocellular Na+/H+ exchange is activated early, transiently and at a posttranscriptional level during rat liver regeneration
Authors: Dällenbach Andreas , Marti Ulrich , Renner Eberhard L.
Published: March 2007

In hepatocytes in vitro, Na+/H+ exchange, an important regulator of intracellular pH, is activated by epidermal growth factor, but its activity during liver regeneration in vivo is unknown. We therefore compared activity and regulation of Na+/H+ exchange in hepatocytes isolated after two-thirds partial hepatectomy or sham surgery, respectively, by measuring intracellular pH (fluorimetry) and steady state Na+/H+ exchange mRNA levels (Northern blotting). Resting intracellular pH increased from 7.06 ± 0.02 to 7.12 ± 0.02 (p<0.05) 2 hr but not 20 hr after partial hepatectomy. Na+/H+ exchange-mediated rates of intracellular pH recovery from an acid load increased from 0.075 ± 0.018 to 0.151 ± 0.018 pH units/min (p<0.05) 2 hr but not 20 hr after partial hepatectomy. Because intracellular buffering capacity was not affected, this reflects increased Na+/H+ exchange activity. The inverse relationship between Na+/H+ exchange activity and intracellular pH was shifted by about 0.1 pH units toward more alkaline pH values 2 hr but not 20 hr after partial hepatectomy, whereas steady-state Na+/H+ exchange mRNA levels remained unchanged. In conclusion, hepatocellular Na+/H+ exchange is activated early, transiently and at a posttranscriptional level during liver regeneration induced in the rat by partial hepatectomy. (Hepatology 1994;19:1290-1301.)
Distinguishing primary and metastatic carcinomas of the liver: Immunohistochemical updates and reminders
Authors: Theise Neil O.
Published: March 2007

Distinguishing primary hepatocellular carcinoma (HCC) from metastatic carcinomas to the liver is often difficult, if not impossible, particularly in needle biopsy and fine-needle aspiration specimens. In an attempt to identify a specific immunohistochemical profile that would distinguish HCC from metastatic carcinomas, we studied 56 HCCs, 8 cholangio-carcinomas, and 24 metastatic adenocarcinomas with monoclonal antibodies to alpha-fetoprotein (AFP), keratin (AE1, AE3, and CAM5.2), Leu-M1, human milk fat globule (HMFG-2), tumor-associated glycoprotein-72 (B72.3), epithelial specific membrane antigen (Ber-EP4), and BCA-225 (CU-18). Both monoclonal and polyclonal (mCEA and pCEA) antibodies to carcinoembryonic antigen also were used. Metastatic adenocarcinomas were often positive for CU-18 (71%), Leu-M1 (75%), B72.3 (50%), HMFG-2 (67%), Ber-EP4 (83%) and mCEA (71%). Using these antibodies, the frequency of positivity for HCC was 9%, 16%, 11%, 20%, 36%, and 11%, respectively. CU-18 was the only monoclonal antibody in which there was a significant difference in positive rates between HCC and metastatic adenocarcinomas. Most HCCs (71%) revealed a bile canalicular staining pattern with pCEA. Because this staining pattern was absent in metastatic carcinomas, pCEA appears to be useful in confirming a diagnosis of HCC, AE1, AE3 and CAM5.2 antibodies were not useful in distinguishing HCC from metastatic carcinomas. Each cholangiocarcinoma shared a staining profile similar to that of metastatic carcinomas.

Hepatocyte paraffin 1 is a monoclonal antibody that has been developed specifically to react with hepatocytes in routine formalin-fixed and paraffin-embedded surgical pathology tissues. It results in a distinct, granular cytoplasmic staining of hepatocytes but fails to react with bile ducts and nonparenchymal liver cells. The antibody decorates a majority of hepatocellular carcinomas, including fibrolamellar variants. It fails to react with a wide variety of other adult malignancies, with the exception of focal staining in a few gastrointestinal malignancies, including a sub-population of gastric carcinomas.
Rat liver epithelial cells: When it comes to differentiation, there’s no place like home
Authors: Hixson Douglas C. , Faris Ronald A.
Published: March 2007

Following intrahepatic transplantation in adult syngeneic Fischer 344 rats, diploid cultured rat liver epithelial cells (WB-F344), modified to carry the Eschericia coli β-galactosidase reporter gene and/or the fluorescent membrane dye PKH26-GL, integrate into hepatic plates and acquire the size and nuclear structure of mature hepatocytes. Additionally, of two aneuploid, neoplastically transformed derivatives of WB-F344 cells, both of which produce aggressively growing tumors when transplanted subcutaneously, cells of one line (GN6TF) do not produce tumors in the liver but integrate into hepatic plates and morphologically differentiate. The other transformed line (GP7TB) retains tumorigenicity in the liver, but cells in the intrahepatic tumors are more differentiated morphologically than are tumors at subcutaneous sites. These results suggest that WB-F344 cells are stemlike cells for hepatocytes and that the hepatic microenvironment induces them to incorporate into hepatic plates and differentiate. Our results also suggest that the hepatic microenvironment regulates the differentiation of some neoplastically transformed hepatic stemlike cells, thereby eliminating or reducing their tumorigenic potential.
Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: Definition of optimal conditions for specimen collection and clinical application in interferon-treated patients
Authors: Davis Gary L. , Lau Johnson Y.-N. , Urdea Mickie S. , Neuwald Paul D. , Wilber Judith C. , Lindsay Karen , Perrillo Robert P. , Albrecht Janice
Published: March 2007

To determine the optimal conditions for preparation of serum specimens for quantitative hepatitis C virus RNA determination, patient samples were processed such that differences in time from clot formation to centrifugation, centrifugation to separation of serum and collection of serum until freezing could be independently assessed. The effects of multiple cycles of freezing and thawing were also determined. There was progressive and significant loss of hepatitis C virus RNA activity when the time from the formation of the clot until centrifugation was longer than 2 hr. This reduction reached 32% after 6 hr and 49% after 24 hr. If centrifugation was performed immediately after formation of the clot, loss of hepatitis C virus RNA activity was reduced to less than 10% even though the serum remained unseparated from the clot for up to 6 hr. Centrifugation of blood through a paraffin plug (serum separator tube) prevented loss of hepatitis C virus RNA activity for up to 24 hr. There was no loss of hepatitis C virus RNA activity with up to three freeze-thaw cycles. When patient specimens were prepared under these optimal conditions, the sensitivity of the quantitative branched DNA signal amplification assay in patients with hepatitis C virus infection was 83% and the specificity in patients with liver disease was 100%. Fluctuations in hepatitis C virus RNA levels were shown to correlate with biochemical changes observed in patients treated with recombinant inter-feron-α2b. These data demonstrate that improper or inconsistent methods of serum preparation may result in falsely low and unreliable levels of hepatitis C virus RNA. Clinical applications of quantitative hepatitis C virus RNA assays will only be possible when sera are properly prepared. (Hepatology 1994;19:1337-1341.)
Significance of specific antibody assay for genotyping of hepatitis C virus
Authors: Tanaka Takeshi , Tsukiyama-Kohara Kyoko , Yamaguchi Kenjiro , Yagi Shintaro , Tanaka Satoshi , Hasegawa Akira , Ohta Yohsuke , Hattori Nobu , Kohara Michinori
Published: March 2007

Group I and II hepatitis C virus genotypes were determined by a newly developed serological genotyping assay. This assay detected antibodies against group-specific recombinant proteins in the putative NS4 protein region (amino acid no. 1676-1760) by an enzyme-linked immunosorbent assay. This region of the hepatitis C virus peptide has many group-specific amino acids; fewer than 50% of these amino acids are identical between groups I and II. Genotypes determined by the serological genotyping assay were compared with those determined by a method in which the polymerase chain reaction was used in 91 chronic hepatitis patients. The group-specific polymerase chain reaction was performed within the genome region corresponding to the putative NS5 protein, where the group II hepatitis C virus genome is 57 nucleotides longer than that of group I. Among 91 chronic hepatitis C patients who had positive results in the second-generation hepatitis C virus antibody (core and NS3 region) assay, hepatitis C virus RNA was detected in 80 patients by polymerase chain reaction in the 5′ untranslated region and in 78 patients by this group-specific polymerase chain reaction. As a result, in 76 of 91 patients (84%) genotypes determined by the serological genotyping assay showed complete agreement with those determined by the group-specific polymerase chain reaction, and none of the patients revealed a group opposite to that of hepatitis C virus genotype. The detection rate of the serological genotyping assay (89 of 91; 98%) was even higher than that of the polymerase chain reaction assay (78 of 91; 86%). Thus, serological genotyping assay is specific and sensitive for the determination of hepatitis C virus genotypes, and this enzyme-linked immunosorbent assay may be useful for epidemiological studies of hepatitis C virus genotypes. (Hepatology 1994;19:1347-1353.)
Hepatitis C virus replication and antibody responses toward specific hepatitis C virus proteins
Authors: Yuki Nobukazu , Hayashi Norio , Kasahara Akinori , Hagiwara Hideki , Ohkawa Kazuyoshi , Fusamoto Hideyuki , Kamada Takenobu
Published: March 2007

We assessed the correlation between hepatitis C virus replication and antibody responses toward hepatitis C virus core (C22-3), NS3 (C33C), NS4 (5-1-1 and C100-3) and NS5 proteins in 59 virus carriers. The concentration of serum hepatitis C virus RNA was determined by a competitive reverse transcription-polymerase chain reaction assay. All 50 patients with high viremic levels of ≥ 106 copies/mL had antibodies to C22-3 and C33C. Antibodies to 5-1-1, C100-3 and NS5 proteins were detected less frequently (p<0.01) in 72% (36 of 50), 78% (39 of 50) and 84% (32 of 38) of such patients, respectively. As for the nine patients with low viremic levels of<106 copies/mL, antibodies to C22-3, C33C, 5-1-1 and NS5 proteins were detected in only one patient (11%), which was significantly less than the frequency for highly viremic patients (p<0.01). Antibody to C100-3 was also found less frequently in only four patients (44%) (p<0.05). Thus, only four (44%) of the nine low viremic patients tested positive for any antibody compared with all 50 highly viremic patients (p<0.01). These results indicate that highly viremic carriers can be detected by the presence of hepatitis C virus antibodies, but a considerable proportion of low viremic carriers may not show any serological evidence of hepatitis C virus infection. (Hepatology 1994;19:1360-1365.)
Detection of precore hepatitis B virus mutants in asymptomatic HBsAg-positive family members
Authors: Akarca Ulus Salih , Greene Sheila , Lok Anna Suk Fong
Published: March 2007

Precore hepatitis B virus mutants have been detected mainly in HBeAg-negative patients with active liver disease. We previously reported two novel mutations: M1 (C-to-T change at nucleotide 1856 [pro-ser at codon 15]) and M3 (G-to-A change at nucleotide 1898 [gly-ser at codon 29]) in addition to two well-described mutations: M2 (G-to-A change at nucleotide 1896 [trp-stop at codon 28]); and M4 (G-to-A change at nucleotide 1899 [gly-asp at codon 29]) in Chinese patients. The aims of this study were to determine (a) the prevalence of precore HBV mutations in asymptomatic carriers and (b) whether family members share the same mutated sequence as the index patients. Fifty-three index patients and 89 HBsAg-positive family members were studied by means of direct sequencing of polymerase chain reaction-amplified hepatitis B virus DNA. M0, a conserved mutation (T-to-C at nucleotide 1858, codon 15), was detected in 81% and 12% family members of index patients with and without M0, respectively (p<0.0001). The clustering of M0 indicates that most subjects were infected through intrafamilial transmission. M1 was detected in all the family members of patients with M1 but in none of the family members of patients with wild-type or M2 sequences (p<0.0001). M2 was detected in 25%, 0% and 15% of family members of patients with M2, M1 and WT sequences, respectively (p=0.19). M3 was detected in five and M4 in four family members. M1 was equally distributed among HBeAg-positive and HBeAg-negative family members, 19.5% vs. 9% (p=0.34), whereas M2 was detected more frequently in HBeAg-negative family members: 45.5% vs. 4.5% in HBeAg-positive family members (p<0.0001). Ten (77%) of 13 family members with M2 and all 15 family members with M1 had normal serum aminotransferase levels. The family members with M2 were significantly older than those with wild-type or M1 sequences (mean ages, respectively, 37.9 ± 5,23 ± 1.4 and 24.1 ± 3yr;p=0.0005). In addition, M2 was more frequently detected in family members who were older than the index patients. Longitudinal studies documented progression from wild-type sequence to M2 in some family members, but progression from wild-type to M1 or M1 to M2 was not observed. Our data showed that precore HBV mutants can be detected in 33% asymptomatic carriers. M1 appears to be present at the onset of infection, whereas M2 emerges (from wild-type but not M1) during the course of infection. Initiation of infection with M2 only seems to be rare. (Hepatology 1994;19:1366-1370.)
Distribution of pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PDC-E2) and another mitochondrial marker in salivary gland and biliary epithelium from patients with primary biliary cirrhosis
Authors: Joplin Ruth E. , Johnson Gerald D. , Matthews John B. , Hamburger John , Lindsay Gordon J. , Hubscher Stefan G. , Strain Alastair J. , Neuberger James M.
Published: March 2007

Previous studies in which quantitative immunofluorescence was used have shown that certain biliary epithelial cells in liver with primary biliary cirrhosis show increased levels of pyruvate dehydrogenase dihydrolipoamide acetyltransferase compared with controls. This study was designed to determine whether the increase in intensity of pyruvate dehydrogenase dihydrolipoamide acetyltransferase in biliary epithelial cells is accounted for by an increase in the number of mitochondria in the same cells. A double-antibody staining technique was used with antibodies specific for pyruvate dehydrogenase dihydrolipoamide acetyltransferase and another mitochondrial inner membrane marker, recognized by the mouse monoclonal antibody MCA151A. Distribution of the antigens was studied in sections of liver and salivary gland, an additional site that is frequently involved in primary biliary cirrhosis. Confocal microscopy was used to quantify the intensity of fluorescence resulting from binding of fluorochrome-labeled antibody. In both liver and salivary glands MCA151A binding was similar in normal and sections with primary biliary cirrhosis and corresponded to the predicted distribution of mitochondria in these tissues. In the liver staining was less intense in biliary epithelial cells than in hepatocytes. In salivary gland binding of both antibodies was predominantly localized to duct cells, with those forming striated ducts, known to be rich in mitochondria, being most intensely stained. There was high coincidence of the two antigens in salivary glands (p<0.01) and in biliary epithelial cells from normal liver (p=0.01). However, in liver with primary biliary cirrhosis, despite high coincidence between the antigens on hepatocytes, biliary epithelial cells showed high intensity of pyruvate dehydrogenase dihydrolipoamide acetyltransferase but not MCA151A. The results indicate that an increase in mitochondria does not account for high intensity of pyruvate dehydrogenase dihydrolipoamide acetyltransferase in biliary epithelial cells in liver with primary biliary cirrhosis. (Hepatology 1994;19:1375-1380).
Bile acid metabolism and biliary secretion in patients receiving orthotopic liver transplants: Differing effects of cyclosporine and FK 506†
Authors: McCashland Timothy M. , Donovan Jeremiah P. , Amelsberg Andree , Rossi Steven S. , Hofmann Alan F. , Shaw Byers W. , Quigley Eamonn M. M.
Published: March 2007

Bile acid metabolism and biliary secretion were characterized in the first 2 wk after orthotopic liver transplantation in 15 patients receiving cyclosporine and in five patients receiving FK 506. Analyses were performed on hepatic bile obtained by T-tube drainage; values obtained were compared with literature values for bile samples obtained in patients who had undergone cholecystectomy. Biliary bile acid output, which is equivalent to bile acid biosynthesis from cholesterol, was low (mean ± S.E.M.) and increased with time: day 1, 0.50 ± 0.1 mmol/day; day 3, 0.8 ± 0.1 mmol/day; and day 6, 1.6 ± 0.5 mmol/day. Chenodeoxycholic acid biosynthesis, as percent of total bile acid biosynthesis, was abnormally low in patients receiving cyclosporine (16.2 ± 1.1) but not in patients receiving FK 506 (38.2 ± 4.8) (p<0.005). Before the T-tube was clamped, the proportion of deoxycholic acid (a secondary bile acid formed by bacterial 7-dehydroxylation of cholic acid) was low in both groups: cyclosporine, 0.4 ± 0.1; FK 506, 4.8 ± 2.5 (p<0.01). The mean concentration of bile acids in hepatic bile between days 4 and 11 did not differ significantly between groups: cyclosporine, 7.7 ± 1.3 mmol/L; FK 506 4.3 ± 0.7 mmol/L (mean ± S.E.M.). (These values are similar to those reported for patients who have undergone cholecystectomy.) Bile acid-dependent bile flow, expressed as apparent choleretic activity (microliters of bile per micromole of bile acid output), was markedly elevated: in patients receiving cyclosporine the value was 129, and in patients receiving FK 506 the value was 220. (In patients who have undergone cholecystectomy, this value is less than 30.) It is concluded that canalicular secretion of bile acids is markedly decreased in the immediate postoperative period after liver transplantation, whereas bile flow is relatively unimpaired, possibly because of impaired biliary duct function. During this period, administration of cyclosporine but not FK 506 causes a selective reduction in chenodeoxycholic acid biosynthesis, probably attributable to its known inhibition of cholesterol 27-hydroxylase. (Hepatology 1994;19:1381-1389.)
Characterization of human hepatocyte lines derived from normal liver tissue
Authors: Roberts Eve A. , Letarte Michelle , Squire Jeremy , Yang Suyun
Published: March 2007

Four separate continuous lines of human hepatocytes (HH01, HH02, HH09, HH25) were developed from normal liver tissue by subjecting cocultures of human hepatocytes with rat liver epithelial cells in a highly enriched medium to frequent subculturing. The addition of conditioned medium from either the human hepatoma line Hep G2 or one of these stable human hepatocyte lines (HH09) appeared to facilitate establishment of line HH25. These human hepatocyte lines have been in continuous culture for 2 to 5 yr and, consist of approximately 95% human cells by analysis of cell surface antigens. Cytogenetic analysis also confirmed the human origin of these cells and showed clonal origin with abnormal ploidy. Cells in these human hepatocyte lines retain morphological features of hepatocytes by both light and electron microscopy. They also retain glucose-6-phosphatase activity and secrete proteins characteristic of hepatocytes, such as albumin, α-fetoprotein and transferrin. After incubation with 13 μmol/L dihenz(a,h) anthracene for 24 hr, each line had detectable activity of aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase. Thus, these human hepatocyte lines retain important differentiated characteristics of hepatocytes. Derived from normal liver tissue, they appear to be immortalized. They provide a new model system for studying human hepatocellular drug metabolism. These lines may also be useful for studying the regulation of synthesis of albumin, α-fetoprotein and other proteins in human hepatocytes, determining the effects of cytokines and growth factors and designing systems to effect gene transfer into human hepatocytes for the purpose of gene therapy. (Hepatology 1994;19:1390-1399.)
Cerebral blood flow and metabolism in fulminant liver failure
Authors: Wendon Julia A. , Harrison Phillip M. , Keays Richard , Williams Roger
Published: March 2007

Fulminant liver failure is a syndrome that is frequently complicated by cerebral edema and increased intracranial pressure. Cerebral blood flow has been reported as high in some studies but low in others. This study undertook to measure cerebral blood flow and cerebral metabolic rate for oxygen in 30 patients with fulminant liver failure in grade 4 encephalopathy and to assess these parameters after intervention with hyperventilation and infusions of mannitol, epoprostenol and acetylcysteine. Cerebral blood flow varied widely, from 14 to 71 ml/100 gm/min (normal range, 41 to 66), whereas the cerebral metabolic rate for oxygen was low in all patients, 0.16 to 2.03 ml/100 gm/min (normal range, 3.12 to 3.96). Twenty-one of the 30 patients had evidence of anaerobic metabolism with cerebral lactate production. Hyperventilation resulted in a significant decrease in both cerebral blood flow (median, 36 to 28 ml/100 gm/min) and cerebral metabolic rate for oxygen (median, 0.92 to 0.65 ml/100 gm/min); mannitol and acetylcysteine infusions resulted in significant increases in both of these parameters. Prostaglandin I2 infusion did not significantly alter cerebral blood flow, but there was a significant increase in cerebral metabolic rate for oxygen. The depressed cerebral metabolic rate for oxygen in patients with fulminant liver failure is inappropriate to metabolic requirements, as demonstrated by both cerebral lactate production and the increase in cerebral oxygen consumption after improvement in cerebral blood flow. Mannitol infusion should remain the main treatment of the cerebral complications of fulminant liver failure. Acetylcysteine has a beneficial effect that is not restricted to fulminant liver failure after acetaminophen overdose and has a role in the management of patients, regardless of cause of illness. The use of prostaglandin I2 appears to be safe and may be beneficial. Acute hyperventilation causes decreases in both cerebral blood flow and cerebral metabolic rate for oxygen and has the potential to worsen cerebral ischemia. (Hepatology 1994;19:1407-1413.)
Serum α-L-fucosidase activity and tumor size in hepatocellular carcinoma
Authors: Takahashi Hirofumi , Saibara Toshiji , Iwamura Shinichi , Tomita Akira , Maeda Takashi , Onishi Saburo , Yamamoto Yasutake , Enzan Hideaki
Published: March 2007

The serum level of α-L-fucosidase activity has been suggested as a useful marker in the diagnosis of hepatocellular carcinoma, although the precise mechanism behind the elevation of this parameter has not been determined. We found that the serum α-L-fucosidase activity level was significantly higher in 67 patients with hepatocellular carcinoma (695.1 ± 245.5 nmol/ml/hr) than in 47 patients with cirrhosis (389.1 ± 188.2 nmol/ml/hr; p<0.001) and in 54 controls (202.0 ± 104.6 nmol/ml/hr; p<0.001). However, α-L-fucosidase activity was not correlated with tumor size (r=0.134), whereas the α-fetoprotein level was correlated with tumor size (r=0.580, p<0.001). When 515.8 nmol/ml/hr was taken as the cutoff value (mean value in the controls plus 3 standard deviations), α-L-fucosidase activity was above the cutoff value in 12 of the 17 patients with a hepatocellular carcinoma less than 2 cm in diameter, in 28 of the 37 patients with a hepatocellular carcinoma less than 3 cm in diameter and in 52 of the 67 patients with hepatocellular carcinoma. In contrast, only 10 of the 47 patients with cirrhosis had levels above the cutoff value. These findings suggest that an increase in serum α-L-fucosidase activity in patients with cirrhosis may be a marker for detecting a hepatocellular carcinoma, especially a small tumor, because α-fetoprotein and des-γ-carboxy-prothrombin are less promising as tumor markers. (Hepatology 1994;19:1414-1417.)
Tissue inhibitor of metalloproteinase is increased in the serum of precirrhotic and cirrhotic alcoholic patients and can serve as a marker of fibrosis
Authors: Li Jianjun , Rosman Alan S. , Leo Maria A. , Nagai Yasuo , Lieber Charles S.
Published: March 2007

One of the contributory factors to the development of cirrhosis is a decrease in collagenase activity, which may be related to levels of inhibitors such as serum tissue inhibitor of metalloproteinase. We therefore measured serum tissue inhibitor of metalloproteinase and serum procollagen III peptides (another proposed marker of fibrosis) in 16 healthy controls and 44 alcoholic patients with biopsy-proved liver disease, namely steatosis without fibrosis (n=13), perivenular fibrosis (n=10), septal fibrosis or cirrhosis or both (n=15) and alcoholic hepatitis (n=6). In alcoholic patients, serum tissue inhibitor of metalloproteinase values strongly correlated with fibrosis (r

s
=0.70, p<0.001). Compared with values in controls (177 ± 12 ng/ml), serum tissue inhibitor of metalloproteinase was significantly elevated in perivenular fibrosis (330 ± 22 ng/ml, p<0.05), in septal fibrosis, cirrhosis or both (406 ± 29 ng/ml, p<0.001) and in alcoholic hepatitis (526 ± 143 ng/ml, p<0.001) but not in steatosis (204 ± 17 ng/ml). In contrast, procollagen III peptides were significantly increased only in the septal fibrosis-cirrhosis group but not in the perivenular fibrosis group. With the threshold defined as the upper value of the steatosis group (resulting in a specificity of 100%), we found that serum tissue inhibitor of metalloproteinase was elevated in 50% of patients with perivenular fibrosis, in 87% of subjects with extensive fibrosis (septal fibrosis, cirrhosis or both) and in 67% of individuals with alcoholic hepatitis. The overall sensitivity of serum tissue inhibitor of metalloproteinase for detecting either perivenular fibrosis or more extensive fibrosis was 71%. Receiver operating characteristic curve analysis revealed that serum tissue inhibitor of metalloproteinase was significantly better than procollagen III peptides in discriminating alcoholics with fibrosis from those with steatosis (area under the receiver operating characteristic curves, 0.958 ± 0.028 vs. 0.803 ± 0.066, p<0.05). In conclusion, serum tissue inhibitor of metalloproteinase is increased in early fibrotic states in alcoholic liver disease and is a useful marker of precirrhotic and cirrhotic states. (Hepatology 1994;19:1418-1423.)