bluuebunny/crossref_metadata_2025 huggingface dataset:
HBV-DNA sequences in tumor and nontumor tissue in a patient with the fibrolamellar variant of hepatocellular carcinoma
Authors: Davison Fergus D. , Fagan Elizabeth A. , Portmann Bernard , Williams Roger
Published: March 2007

One patient with the fibrolamellar variant of hepatocellular carcinoma was found to be seropositive for HBsAg and anti-HBe. DNA from tumor and nontumor areas of the liver was examined by molecular hybridization for hepatitis B virus DNA sequences. Undigested DNA from the tumor gave a high-molecular-weight smear, and restriction-enzyme analysis indicated a single instance of integration. Nontumor liver tissue was analyzed from three separate areas. Hepatitis B virus DNA was detected in two of these; restriction-enzyme digestion suggested they contained different sites of viral integration.

As with the typical hepatitis B virus—related hepatocellular carcinoma, analysis of hepatitis B virus DNA from nontumorous liver yielded a different pattern of high-molecular-weight bands, indicating that the virus genome had integrated at different chromosomal locations than that seen in the tumor. The finding of integrated hepatitis B virus DNA, especially in tumorous but also in nontumorous liver, would be consistent with an oncogenic role for hepatitis B virus in certain instances of fibrolamellar tumors and in the more typical hepatitis B virus—related hepatocellular carcinoma. (HEPATOLOGY 1990;12:676-679).
Transport of chenodeoxycholic acid and its 3-α- and 7-α-sulfates by isolated perfused rat liver
Authors: Gärtner Ulrich , Goeser Tobias , Stiehl Adolf , Raedsch Richard , Wolkoff Allan W.
Published: March 2007

In patients with cholestasis, levels of sulfated bile acids rise. Sulfate esters of chenodeoxycholic acid are the most abundant of these bile acid sulfates. These compounds are taken up by the liver and excreted into bile, although their plasma clearance and biliary excretion are reduced compared with that of unsulfated bile acids. It is not clear whether this is due to differences in intrinsic hepatic uptake or biliary excretion. In the present study, single-pass transport kinetics of chenodeoxycholic acid 3-α-sulfate, chenodeoxycholic acid 7-α-sulfate and unsulfated chenodeoxycholic acid were quantified in isolated perfused rat liver. Influx of the 7-α- and 3-α-sulfated derivatives was 57% and 20% that of chenodeoxycholic acid, respectively. These three compounds bound to albumin equally well, indicating that this was not a factor in their differential uptake. Although single-pass extraction of each of these compounds differed, biliary excretion of material taken up by the liver was identical. There was no difference in bile flow or biliary excretion rate, regardless of which bile acid sulfate was tested. These results indicate that the low plasma elimination of sulfated bile acids previously observed by others can be explained by low hepatic influx. The diminished transport into liver resulting from sulfation could lead to enhanced elimination of bile acids by the kidney. (HEPATOLOGY 1990;12:738-742).
Chronic ethanol administration impairs receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes†
Authors: Dalke Douglas D. , Sorrell Michael F. , Casey Carol A. , Tuma Dean J.
Published: March 2007

The effects of chronic ethanol administration on the receptor-mediated endocytosis of epidermal growth factor were studied in isolated rat hepatocytes. In initial experiments, it was demonstrated that significantly less ligand was bound by hepatocytes isolated from rats fed an ethanol liquid diet for 5 to 7 wk than by cells isolated from chow-fed or pair-fed controls. Reduced binding was shown to be primarily caused by a decreased number of surface receptors rather than by changes in receptor affinity. When hepatocytes were incubated at 37° C in the presence of a large saturating concentration of epidermal growth factor (80 nmol/L), intracellular levels of the ligand were significantly lower in cells from the ethanol-fed animals. However, no effect on degradation of the ligand was observed under those conditions. A defect in the initial stages of receptor-ligand internalization was also indicated because less surface-bound ligand was internalized and subsequently degraded in cells from the ethanoltreated rats. When the endocytosis of a lower, more physiological concentration of the growth factor (0.5 nmol/L) was studied, both the uptake of ligand and its degradation were markedly impaired in hepatocytes from the ethanol-fed animals. These results indicate that chronic ethanol administration impairs the receptor-mediated endocytosis of epidermal growth factor by the liver. The major impairment appears to be a reduction of cell surface receptors; however, other steps of the endocytotic pathway also appear to be affected. These altered steps include defective receptor-ligand internalization and changes in intracellular processing of the ligand leading to decreased degradation. (HEPATOLOGY 1990;12:1085-1091).
Zonal heterogeneity of the effects of chronic ethanol feeding on hepatic fatty acid metabolism
Authors: Guzman Manuel , Castro José
Published: March 2007

Periportal and perivenous hepatocytes were isolated from rats fed a high-fat, ethanol-containing diet to investigate the acinar heterogeneity of the effects of prolonged ethanol administration on lipid metabolism. Chronic feeding of ethanol caused a rather selective accumulation of triacylglycerols in the perivenous zone of the liver. In control animals the rate of lipogenesis and the activity of acetyl-CoA carboxylase were higher in perivenous than in periportal hepatocytes, whereas the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase I were higher in periportal than in perivenous cells; however, no zonation was evident for very-low-density-lipoprotein-lipid secretion. Prolonged ethanol administration abolished the zonal asymmetry of the lipogenic process and inverted the acinar distribution of the fatty acid—oxidative process (i.e., in ethanol-fed animals the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase I were higher in perivenous than in periportal hepatocytes). Moreover, chronic feeding of ethanol led to a marked and selective inhibition of very-low-density-lipoproteintriacylglycerol secretion by the perivenous zone of the liver. Nevertheless, no zonal differences were observed between control and ethanol-fed animals with respect to the effects of acute doses of ethanol and acetaldehyde on lipid metabolism. In conclusion, our results show that chronic ethanol intake produces important alterations in the acinar distribution of the different fatty acid—metabolizing pathways. (HEPATOLOGY 1990; 12:1098-1105).
Fatty liver hepatitis (steatohepatitis) and obesity: An autopsy study with analysis of risk factors
Authors: Wanless Ian R. , Lentz John S.
Published: March 2007

Steatohepatitis (fatty liver hepatitis), histologically identical to alcoholic disease, occurs in some obese patients after jejunoileal bypass. A similar lesion occurs rarely in obese patients without bypass surgery, but the risk factors are poorly understood. Hepatic steatosis, steatohepatitis and fibrosis were sought in 351 apparently nonalcoholic patients at autopsy and various risk factors were evaluated. Incidence of steatosis and steatohepatitis correlated with the degree of obesity. Steatohepatitis was found in 18.5% of markedly obese patients and 2.7% of lean patients. Additional risk factors for steatohepatitis were type II diabetes, weight loss in the preterminal period shortly before death and intravenous glucose therapy in the last week of life. Severe fibrosis was found in 13.8% of markedly obese patients and in 6.6% of lean patients; this difference was largely explained by the higher prevalence of diabetes in obese groups.

The risk factors defined in this study are known to be associated with abnormalities of free fatty acid metabolism. Obesity, type II diabetes and intravenous glucose therapy are associated with hyperinsulinemia, which may inhibit fatty acid oxidation. Obesity and weight loss increase the presentation of fatty acids to the liver. Similar metabolic changes may occur in obese patients after jejunoileal bypass surgery. Thus this study supports the hypothesis that fatty acids have a role in the hepatocellular necrosis found in some obese individuals. (HEPATOLOGY 1990;12:1106-1110).
Impaired lipopolysaccharide-inducible tumor necrosis factor production in vitro by peripheral blood monocytes of patients with viral hepatitis
Authors: Müller Christian , Zielinski Christoph C.
Published: March 2007

We investigated lipopolysaccharide-induced tumor necrosis factor production in vitro by peripheral blood monocytes from patients with various liver diseases. Tumor necrosis factor production was found to be significantly reduced in patients with chronic hepatitis B (n = 17; 135 ± 30 pg tumor necrosis factor/ml; mean ± S.E.M.) and patients with chronic non-A, non-B hepatitis (n = 15; 212 ± 22 pg tumor necrosis factor/ml) compared with healthy control individuals (n = 47; 411 ± 40 pg tumor necrosis factor/ml; p < 0.0005 and p < 0.01, respectively). This reduced tumor necrosis factor production was not only seen with an optimal stimulating concentration of lipopolysaccharide (100 ng/ml) but also with suboptimal concentrations (0.1 ng/ml). In contrast to patients with chronic viral hepatitis, monocytes from patients with alcohol-induced cirrhosis (n = 26; 444 ± 49 pg tumor necrosis factor/ml), primary biliary cirrhosis (n = 7; 412 ± 81 pg tumor necrosis factor/ml) and alcohol-induced fatty liver changes (n = 5; 401 ± 62 pg tumor necrosis factor/ml) produced normal amounts of tumor necrosis factor when stimulated with an optimal concentration of lipopolysaccharide. Lipopolysaccharide (0.1 ng lipopolysaccharide/ml)— stimulated peripheral blood monocytes of patients with chronic hepatitis B (n = 15; 102 ± 32 pg/ml) or non-A, non-B hepatitis (n = 13; 97 ± 16 pg/ml) could not be induced to produce more tumor necrosis factor either when prestimulated with γ-interferon (170 ± 45 pg/ml and 149 ± 32 pg/ml, respectively), a lymphokine known to activate monocytes, or with the cyclooxygenase inhibitor indomethacin to reduce the suppressive effect of prostaglandin E2 (148 ± 40 pg/ml and 153 ± 45 pg/ml, respectively). In contrast, patients with alcoholic cirrhosis (n = 11; 178 ± 31 ng tumor necrosis factor/ml) showed significant increase of tumor necrosis factor production by lipopolysaccharide-stimulated monocytes when prestimulated with γ-interferon (n = 11; 395 ± 80 pg tumor necrosis factor/ml; p < 0.025) or indomethacin (n = 11; 393 ± 82 pg tumor necrosis factor/ml; p < 0.05). A significant reduction in lipopolysaccharide-induced tumor necrosis factor production by peripheral blood monocytes was observed in acute hepatitis B but not in acute hepatitis A or non-A, non-B hepatitis. These investigations suggest that lipopolysaccharide-induced tumor necrosis factor production by peripheral blood monocytes is impaired in patients with chronic viral hepatitis and acute hepatitis B. (HEPATOLOGY 1990;12:1118-1124).
Human hepatic regenerative stimulator substance: Partial purification and biological characterization of hepatic stimulator substance from human fetal liver cells
Authors: Zhiqiang Yao , Weisong Yang , Wenbin Zhang , Yong Chen , Fengyi Yang
Published: March 2007

Current support or replacement therapies for fulminant acute hepatic failure are frequently very disappointing. In this study, human hepatic stimulator substance—a liver-specific growth factor—was partially purified from human fetal liver cells and characterized by its biological effects. Almost 70-fold protein content was purified with an approximately 80-fold increase in specific growth stimulator activity. Human hepatic stimulator substance proved to be heatstable, protase-sensitive, organ-specific and species-nonspecific. Human hepatic stimulator substance produced a two- to threefold increase of 3H-thymidine incorporation into hepatic DNA when injected intraperitoneally into growing weanling mice (nonhepatectomized) or regenerating rats (34% hepatectomy). The effects of hHSS in reversing the lethality of D-galactosamine (1.6 gm/kg body weight)—induced hepatic necrosis in rats were further evaluated. A survival rate of 4% (n = 24), 41% (n = 12, p < 0.05), 33% (n = 12, p < 0.05), 31% (n = 13, p < 0.05) and 18% (n = 11, p > 0.05) was observed when the rats were injected with 4 ml of saline intraperitoneally, 4 ml of human intact fetal hepatocytes (2.4 × 108) intraperitoneally, 4 ml of human hepatic stimulator substance intraperitoneally, 2 ml of twofold concentrated human hepatic stimulator substance intravenously and 1 ml of fourfold human hepatic stimulator substance intramuscularly, respectively, 20 hr after poisoning. These results suggested that human hepatic stimulator substance could successfully improve survival in rats with chemically induced acute liver failure by hepatocyte proliferation and might represent a significant potential for the management of clinical fulminant hepatic failure (HEPATOLOGY 1990;12:1144-1151).
Hepatic vascular disease and portal hypertension in polycythemia vera and agnogenic myeloid metaplasia: A clinicopathological study of 145 patients examined at autopsy†
Authors: Wanless Ian R. , Peterson Powers , Das Asha , Boitnott John K. , Moore William G. , Bernier Vincent
Published: March 2007

The pathogenesis of portal hypertension arising in patients with myeloproliferative disorders has been difficult to understand because liver biopsy findings often show minimal changes. It has been suggested that increased splenic blood flow, hepatic infiltration with hematopoietic cells or sinusoidal fibrosis may be important. We have reviewed the autopsy findings and clinical histories of 97 patients with polycythemia vera and 48 patients with agnogenic myeloid metaplasia collected from three institutions and from the Polycythemia Vera Study Group. Cirrhosis was present in seven patients, one of whom had bleeding varices. Esophageal varices were present clinically in 10 patients without cirrhosis (seven polycythemia and three agnogenic myeloid metaplasia). All of these patients had lesions in small or medium-sized portal veins and four had stenosis of the extrahepatic portal vein with histology compatible with organized thrombi. Nodular regenerative hyperplasia occurred in 14.6% and correlated closely with the presence of portal vein lesions. Thirty patients had > 500 ml of ascites, seven of these patients also had varices and six of them had hepatic vein thrombosis. Ascites also correlated with hepatic vein disease confined to small intrahepatic branches. No correlation was seen between hepatic hematopoietic infiltration and signs of portal hypertension.

We conclude that esophageal varices are common and are almost always associated with portal vein lesions visible by light microscopy. These portal vein lesions, and the secondary effects of nodular regenerative hyperplasia and portal hypertension, are most likely a result of portal vein thrombosis in patients with myeloproliferative disorders. (HEPATOLOGY 1990;12:1166-1174).
Acute-phase response of human hepatocytes: Regulation of acute-phase protein synthesis by interleukin-6
Authors: Castell José V. , Gómez-lechón Maria José , David Martina , Fabra Ricardo , Trullenque Ramón , Heinrich Peter C.
Published: March 2007

Human hepatocytes in primary culture were used as a model system to investigate the mechanism(s) involved in the induction of the acute-phase response in human liver. Hepatocytes were incubated with increasing amounts of recombinant human interleukin1β;, recombinant interleukin-6 and tumor necrosis factor-α. Synthesis of C-reactive protein was studied at the mRNA and protein levels. Only recombinant interleukin-6 was capable of inducing C-reactive protein—mRNA and C-reactive protein—protein synthesis. Also, fibrinogen and α−1-antitrypsin synthesis measured by immunoprecipitation with specific antisera increased in a dose-dependent, time-dependent manner, whereas albumin synthesis decreased to about 50% of controls. Maximal effects were observed at 100 to 300 units of recombinant interleukin-6/ml culture medium after 20 hr of incubation. Although the synthetic glucocorticoid dexamethasone slightly modulated the effect of recombinant interleukin-6, it was not an absolute requirement for the induction of acute-phase protein synthesis in human hepatocytes. In pulse-chase experiments it was shown that the time course of the disappearance of the acute-phase proteins from the cells and their appearance in the medium is not influenced by recombinant interleukin-6. This finding suggests that recombinant interleukin-6 exerts its regulatory effect on acute-phase protein synthesis at the pretranslational level. (HEPATOLOGY 1990;12:1179-1186).
Effect of chronic administration of ursodeoxycholic acid on the ileal absorption of endogenous bile acids in man
Authors: Marteau Philippe , Chazouiléres Olivier , Myara Anne , Jian Raymond , Rambaud Jean-Claude , Poupon Raoul
Published: March 2007

The effect of long-term administration of ursodeoxycholic acid on the ileal absorption of endogenous bile acids was determined using the tauro 23 (75Se) selena-25 homotaurocholic acid test in a controlled double-blind study involving healthy subjects (n = 15). Subjects received placebo or 13 to 15 mg/kg/day ursodeoxycholic acid for 5 wk. In the placebo group (n = 7) there was no change in the composition of serum bile acids or in the mean percentage of retention of tauro 23(75Se) selena-25 homotaurocholic acid (36.1% ± 6.0% vs. 38.7% ± 6.7%). In contrast, in the ursodeoxycholic acid group, serum ursodeoxycholic acid conjugates increased and the percentage of retention of tauro 23 (75Se) selena-25 homotaurocholic acid fell from 45.8% ± 6.8% to 20.5% ± 5.7% (p < 0.01). We conclude that ursodeoxycholic acid administration reduces ileal absorption of endogenous bile acids. These findings provide a rational explanation for the changes in the composition of the bile acid pool during ursodeoxycholic acid therapy and could have important therapeutic implications. (HEPATOLOGY 1990;12:1206-1208).
Bile acids exert negative feedback control on bile acid synthesis in cultured pig hepatocytes by suppression of cholesterol 7α-hydroxylase activity
Authors: Kwekkeboom Jaap , Princen Hans M. G. , van Voorthuizen Eline M. , Kempen Herman Jan M.
Published: March 2007

Feedback regulation of bile acid synthesis by its end products was studied in cultured hepatocytes of young weaned pigs. We previously showed that conversion of exogenous [14C] cholesterol into bile acids was suppressed by addition of bile acids to the culture medium. In the present study, the effects of bile acids on bile acid mass production and cholesterol 7α-hydroxylase activity were examined. Mass production of bile acids was strongly inhibited by addition of taurocholic acid (50 and 100 μmol/L) to the culture medium. The inhibitory action was exerted specifically on activity of cholesterol 7α-hydroxylase because conversion of [14C] 7α-hydroxycholesterol to bile acids by pig hepatocytes was not affected. Suppression of cholesterol 7α-hydroxylase activity after incubation of the hepatocytes with taurocholic acid was concentration- and time-dependent. Maximum suppression (−80%) was achieved after a 20 to 30 hr incubation of hepatocytes with 100 μmol/L of this bile acid. Decline of enzyme activity caused by 100 μmol/L taurocholic acid followed first-order kinetics with a half-life of 10 hr. Taurocholic acid had no direct effect on cholesterol 7α-hydroxylase activity in homogenates of hepatocytes as assessed by addition of the bile acid to the assay mixture.

The effects of several other bile acids in a concentration of 100 μmol/L on cholesterol 7α-hydroxylase activity were examined in 48 hr incubations. Glycochenodeoxycholic and glycohyodeoxycholic acids, which are the major bile acids in pig bile, their unconjugated forms and also deoxycholic and cholic acid pronouncedly inhibited activity of the enzyme. In contrast, hyocholic acid almost failed to inhibit, whereas ursodeoxycholic acid was a weak inhibitor.

It is concluded that bile acids, in physiological concentrations (i.e., as observed in portal blood), inhibit bile acid synthesis in cultured pig hepatocytes by suppression of cholesterol 7α-hydroxylase activity through a direct effect on the hepatocoyte. (HEPATOLOGY 1990;12:1209-1215).
Mechanism of cholate uptake: NA+-cotransport, but not cholate/OH− exchange
Authors: Anwer Sawkat M.
Published: March 2007

The mechanism(s) and driving force(s) for hepatocellular uptake of the unconjugated bile acid cholate were investigated in isolated basolateral (sinusoidal) rat liver plasma membrane (blLPM) vesicles and in protein free liposomes. In blLPM vesicles both an inwardly directed Na+ gradient and a transmembrane pH difference (8.0 in/6.0 out) stimulated cholate uptake 2-3-fold above equilibrium uptake values (overshoot). While Na+ gradient driven cholate uptake could be inhibited by the anion transport inhibitor 4,4′-diisothiocyanato-2,2′-disulfonic acid stilbene (DIDS), the pH gradient dependent portion of cholate uptake was insensitive to DIDS, but could be inhibited by furosemide. Furthermore, initial rates (1-s values) of the pH gradient stimulated cholate uptake were linear with increasing substrate concentrations (no saturability). In liposomes a similar inside alkaline pH gradient also induced a transient DIDS insensitive/furosemide inhibitable intravesicular accumulation (approx. 2-fold) of cholate (overshoot). These findings confirm that hepatocellular uptake of cholate occurs in part via the common Na+/bile acid cotransport system. In addition, the data strongly indicate that in isolated membrane vesicles pH gradient driven cholate uptake represents nonionic diffusion rather than a carrier mediated process (Blitzer, B.L., Terzakis, C. and Scott, K.A. (1986) J. Biol. Chem. 261, 12042-12046). Since in the perfused liver DIDS inhibited uptake of both cholate and taurocholate to a similar extent, DIDS-insensitive pH gradient dependent membrane diffusion appears to be of minor significance for cholate uptake in the intact organ.
Effect of lactulose on the metabolism of short-chain fatty acids
Authors: Uribe Misael , Campollo Octavio , Cote Christiane
Published: March 2007

Short-chain (C2·C5) fatty acids account for 60%-70% of the anions in the colon. Acetate (C2) is nontoxic in contrast to C(3)4·C5 fatty acids (propionate, butyrate, isobutyrate, valerate, and isovalerate), which induce coma in animals and may be important in the pathogenesis of hepatic coma in humans. An in-vitro fecal incubation system was used to map out short-chain fatty acid production in the presence of lactulose, amino acids, albumin, or blood. Albumin and blood increased production of all C2·C5 fatty acids. In contrast, lactulose was converted to acetate only and increased fecal acidity. The degradation of amino acids, albumin, and blood to short-chain fatty acids was completely inhibited by 10-25 mM lactulose. This was caused mainly by the acidifying effect of lactulose. pH-independent inhibition of blood and amino acid degradation to short-chain fatty acids required concentrations of lactulose exceeding 50-100 mM. Thus, the effect of lactulose in the treatment of hepatic coma may be related to its rapid fermentation into organic acids at rates exceeding colonic buffering capacity. This probably reduces formation of toxic fatty acids and ammonia from amino acids, polypeptides, and blood in the colon.
Alcohol content of various beverages: All booze is created equal
Authors: Conn Harold O.
Published: March 2007

After consuming comparable amounts of ethanol, women have higher blood ethanol concentrations than men, even with allowance for differences in size, and are more susceptible to alcoholic liver disease. Recently, we documented significant “first pass metabolism” of ethanol due to its oxidation by gastric tissue. We report a study of the possible contribution of this metabolism to the sex-related difference in blood alcohol concentrations in 20 men and 23 women. Six in each group were alcoholics.

The first pass metabolism was determined on the basis of the difference in areas under the curves of blood alcohol concentrations after intravenous and oral administration of ethanol (0.3 g per kilogram of body weight). Alcohol dehydrogenase activity was also measured in endoscopic gastric biopsies. In nonalcoholic subjects, the first pass metabolism and gastric alcohol dehydrogenase activity of the women were 23 and 59 percent, respectively, of those in the men, and there was a significant correlation (r8 = 0.659) between first pass metabolism and gastric mucosal alcohol dehydrogenase activity. In the alcoholic men, the first pass metabolism and gastric alcohol dehydrogenase activity were about half those in the nonalcoholic men; in the alcoholic women, the gastric mucosal alcohol dehydrogenase activity was even lower than in the alcoholic men, and first-pass metabolism was virtually abolished.

We conclude that the increased bioavailability of ethanol resulting from decreased gastric oxidation of ethanol may contribute to the enhanced vulnerability of women to acute and chronic complications of alcoholism.
Early and frequent detection of HBxAg and/or anti-HBx in hepatitis B virus infection
Authors: Vitvitski-Trépo Ludmila , Kay Alan , Pichoud Christian , Chevallier Philippe , de Dinechin Stéphane , Shamoon Blanche-Marie , Mandart Elisabeth , Trépo Christian , Galibert Francis
Published: March 2007

To clarify the significance of the X gene of hepatitis B virus, we have tested for anti-HBx in the serum and HBxAg in the liver at different stages of the natural history of hepatitis B virus infection. Sera were screened by enzyme-linked immunosorbent assay and positive results confirmed by immunoblot. Purified recombinant MS2 Pol-HBx fusion protein was used as target for both assays.

Among serial sera of patients with nonfulminant acute hepatitis, 24 of 64 patients (37.5%) were positive for anti-HBx. In fulminant cases, 15 of 36 patients (42%) had anti-HBx. In chronic hepatitis patients with high rates of hepatitis B virus replication, we found a significantly (p < 0.01) higher prevalence of anti-HBx, 14 of 25 patients (56%), than in those with low replication, 14 of 66 patients (21%), or among asymptomatic HBsAg carrier blood donors (20 of 126 = 16%) without detectable hepatitis B virus replication (p < 0.0001). The highest prevalence of anti-HBx was found in HBsAg carriers with cirrhosis (41 of 54 patients = 76%) and/or with hepatocellular carcinoma (18 of 33 patients = 54%).

The findings suggest that anti-HBx appears as a common and early marker of hepatitis B virus infection, transient in self-limited hepatitis but persisting with progression to chronicity. In chronic hepatitis, the prevalence of anti-HBx correlated with the intensity and duration of hepatitis B virus replication but neither with the severity of the liver disease nor with malignant transformation per se.

HBxAg was also frequently detected by immunoperoxidase staining in paraffin liver sections of 60 chronic HBsAg carriers with liver disease. Its localization overlapped that of HBcAg. A discrepancy between the detection of HBxAg in liver and that of anti-HBx in serum was observed in chronic hepatitis cases, which was not found in patients with more advanced forms of liver disease. (HEPATOLOGY 1990;12:1278-1283).
Association of autoimmune hepatitis with HLA-Bw54 and DR4 in Japanese patients
Authors: Seki Takeshi , Kiyosawa Kendo , Inoko Hidetoshi , Ota Masao
Published: March 2007

Human leukocyte antigen-D region—related alleles (human leukocyte antigen DR and DQ) and human leukocyte antigen class I alleles were typed serologically in 31 Japanese patients with autoimmune hepatitis. These patients had increased serum levels of AST and IgG, high titers of autoantibodies, no history of blood transfusion and were negative for HBsAg and antibodies to HBc. Three hundred eighty-six healthy subjects and 30 patients with cryptogenic chronic hepatitis served as control groups. The frequency of DR4 was significantly higher in autoimmune hepatitis patients (90.3%) than in healthy subjects (38.6%) and in cryptogenic chronic hepatitis patients (30%). The frequency of Bw54 was significantly higher in autoimmune hepatitis patients (45.2%) than in healthy subjects (10.9%). The risk to DR4-positive subjects for autoimmune hepatitis was 14.8 relative to healthy subjects. Two of 31 patients (6.5%) with autoimmune hepatitis were positive for antibody to hepatitis C virus; both clearly satisfied criteria for autoimmune hepatitis and both had Bw54 and DR4. This study revealed a highly significant association of autoimmune hepatitis with human leukocyte antigen Bw54 and DR4 in Japanese patients. Among the DR4-positive patients with autoimmune hepatitis, no significant differences were seen between those positive or negative for Bw54 with regard to clinical or laboratory data, relapse of disease or efficacy of prednisolone. Thus human leukocyte antigen class II alleles contribute to susceptibility and resistance to autoimmune hepatitis in Japanese patients, with distinct racial differences from those in white patients. (HEPATOLOGY 1990;12:1300-1304).
A quantitative analysis of T lymphocyte populations in human liver allografts undergoing rejection: The use of monoclonal antibodies and double immunolabeling
Authors: McCaughan Geoffrey W. , Davies Stewart J. , Waugh Jenny A. , Bishop Alex G. , Hall Bruce M. , Gallagher Neil D. , Thompson John F. , Sheil Ross A. G. , Painter Dorothy M.
Published: March 2007

The aim of this study was to quantitate T-cell populations infiltrating portal tracts, bile ducts and hepatic lobules in 82 biopsy specimens from 25 patients after orthotopic liver transplantation. Biopsy specimens taken immediately after revascularization of the graft were used as controls. Patients studied include 18 with initial rejection episodes, 11 with unresolved rejection, five with vanishing bile duct syndrome and eight patients with other forms of liver injury. Quantitation was done in a blinded fashion for the first 20 biopsy specimens. A double immunolabeling technique was used to simultaneously immunolabel bile duct structures (with anti-major histocompatibility complex class II or antikeratins) and lymphoid populations (with anti-CD2, anti-CD4 or anti-CD8). This facilitated the accurate quantitation of intraepithelial lymphocytes within bile ducts. This technique also enabled simultaneous detection of CD4 and CD8 antigens on lymphocytes in portal tracts. The predominant lymphocyte subtype within biliary epithelium during acute and chronic rejection was of the CD2+/CD8+ phenotype. CD8+/CD4+ ratio in bile ducts was approximately 5:1 in acute, unresolved and chronic rejection. In vanishing bile duct syndrome, double immunolabeling enabled the detection of destroyed interlobular bile duct remnants that were not apparent on routine hematoxylin and eosin staining. Attached to some of these structures were CD8+ lymphocytes. Lobular CD8+ cells were not prominent in acute rejection but increased significantly in biopsy specimens from patients with unresolved and chronic rejection. In chronic rejection, a selective increase was seen in these CD8+ cells in centrizonal regions. In contrast, portal tract T-cell numbers decreased in nonresolving and chronic rejection biopsy specimens compared with acute rejection specimens. CD8+ and CD4+ T cells were present in approximately equal numbers in such infiltrates. It is concluded that in the evolution from acute to chronic rejection a selective accumulation of CD8+/CD2+ cells occurs in the biliary epithelium and hepatic lobules. The role of these cells in the actual pathogenetic process of hepatic allograft rejection remains to be established. (HEPATOLOGY 1990;12:1305-1313).
Site-directed mutagenesis of lysine within the immunodominant autoepitope of PDC-E2
Authors: Leung Patrick S. C. , Iwayama Takashi , Coppel Ross L. , Gershwin Eric M.
Published: March 2007

The major autoantigens of PBC have been identified as the four closely related mitochondrial enzymes PDC-E2, BCKD-E2 OGDC-E2 and protein X. A major structural similarity of these enzymes is the presence of one or more lipoyl domains. The immunodominant epitope of each autoantigen has either been postulated or been demonstrated to be located within the lipoate binding region. However, it is not clear whether the binding of lipoic acid to the epitope is necessary for autoantibody recognition. To address this issue we have constructed by oligonucleotide site-directed mutatagenesis three mutants in the lipoyl domain of human PDC-E2. Because lipoic acid is covalently bound to the ξ-amino group of the lysine residue of PDC-E2, the mutants were designed to replace the lysine residue in the lipoyl domain with glutamine, a negatively charged amino acid; histidine, a positively charged amino acid; and tyrosine, an aromatic amino acid. Binding reactivity of sera from patients with PBC were analyzed by enzyme-linked immunosorbent assay, immunoblotting and specific absorption against each of the three mutants and control clones. All data were compared with parallel studies with a control recombinant clone, the liver-specific F alloantigen. We believe the recognition of the lipoyl domain is a reflection of the surface-exposed, hydrophilic and relatively mobile nature of this region of the autoantigen. Further studies on direct assay for the presence of lipoic acid will be needed to clarify these issues. (HEPATOLOGY 1990;12:1321-1328).
Decreased toxicity of polymorphonuclear neutrophils toward hepatocytes isolated from rats with acute inflammatory reaction
Authors: Mavier Philippe , Rosenbaum Jean , Preaux Anne-Marie , Mallat Ariane , Dhumeaux Daniel
Published: March 2007

We have recently demonstrated that polymorphonuclear neutrophils were toxic to hepatocytes through a protease-mediated mechanism. Since synthesis of antiproteases is markedly increased during acute inflammatory reaction, the aim of this work was to investigate the toxicity of neutrophils against normal vs. inflammatory rat hepatocytes. Acute inflammatory reaction was induced by subcutaneous injection of turpentine 24 hr before the experiments. Hepatocytes from normal and turpentine-treated rats were isolated by collagenase digestion. They were incubated with human neutrophils stimulated by 1 mg/ml opsonized zymosan. Cytotoxicity was quantified by the percentage of alanine aminotransferase activity released by hepatocytes in culture medium after an 18-hr incubation period. By comparison to normal hepatocytes, inflammatory hepatocytes were more resistant to the toxicity of neutrophils. At a neutrophil/hepatocyte ratio of 20:1, the alanine aminotransferase activity releases were 53.7% ± 5.4% (mean ± 1 S.E.) and 27.4% ± 4.8% for normal and inflammatory hepatocytes, respectively. Similarly, inflammatory hepatocytes were found to be less sensitive than normal hepatocytes to the toxic effect of purified neutrophil cathepsin G. In contrast, both types of hepatocytes exhibited the same sensitivity to H2O2 generated by a system consisting of glucose and glucose oxidase. Two arguments suggested that the resistance of inflammatory hepatocytes to protease toxicity was explained by an increased production of antiproteases by these cells: (a) when tested against cathepsin G and porcine pancreatic elastase activities, the protease inhibitory capacity of conditioned medium from inflammatory hepatocytes was higher than that of conditioned medium from normal hepatocytes; (b) conditioned medium from inflammatory hepatocytes markedly reduced the toxicity of stimulated neutrophils as that of cathepsin G. These results show that, during an acute inflammatory reaction, the increased synthesis of antiproteases by hepatocytes may inhibit the proteasemediated toxicity of neutrophils. In the clinical disorders in which a pathogenetic role of neutrophils has been suggested, the accompanying inflammatory reaction might thus have a beneficial effect by reducing neutrophil-mediated tissue injury. (HEPATOLOGY 1990;12:1337-1341).
Restoration of liver function in gunn rats without immunosuppression using transplanted microencapsulated hepatocytes
Authors: Dixit Vivek , Darvasi Ruth , Arthur Marika , Brezina Maria , Lewin Klaus , Gitnick Gary
Published: March 2007

Microencapsulation of cells within synthetic semipermeable membranes is a novel technique that enables the transplantation of cell cultures without the need for immunosuppression. We have previously shown that transplanted isolated encapsulated hepatocytes can provide sufficient short-term metabolic support to improve the survival of animals with galactosamine-induced fulminant hepatic failure. Here we have demonstrated the feasibility of isolated encapsulated hepatocyte transplantation in providing long-term metabolic liver support in Gunn rats. Gunn rats have a congenital inability to conjugate bilirubin and thus exhibit lifelong hyperbilirubinemia. We studied the feasibility of isolated encapsulated hepatocyte transplantation in restoring this specific liver function. Free hepatocytes, isolated from male Wistar rats, were microencapsulated with collagen within a trilayered sodium alginate-poly-L-lysine-sodium alginate membrane using techniques developed in our laboratory. A total of 45 Gunn rats underwent intraperitoneal transplantation with free hepatocytes (5 × 107), isolated encapsulated hepatocytes (5 × 107), control (empty) microcapsules or no transplant (untreated controls). Serum bilirubin levels were monitored daily for 10 days after transplantation, and subsequent weekly samples were obtained for up to 1 mo. Microcapsules were studied by light and electron microscopy 1 mo after transplantation. During the first week after transplantation, the mean maximum reduction in serum bilirubin levels for the isolated encapsulated hepatocytes, free hepatocytes and control microcapsule transplanted groups was 45.7%, 18.6% and 14.3%, respectively. For up to 1 mo thereafter the mean reduction in serum bilirubin levels in these respective groups was 34.8%, 13.5% and 3.3%. Light and electron microscopy of the isolated encapsulated hepatocytes revealed preservation of a number of hepatocytes containing numerous mitochondria, smooth and rough endoplasmic reticulum, Golgi bodies and glycogen. We conclude that isolated encapsulated hepatocyte transplantation was effective in significantly lowering serum bilirubin levels in the Gunn rat. Transplantation of isolated encapsulated hepatocytes is a unique approach to the restoration of at least one liver function in congenital liver deficiency. (HEPATOLOGY 1990;12:1342-1349).
Rapid decrease of cytochrome P-450IIE1 in primary hepatocyte culture and its maintenance by added 4-methylpyrazole
Authors: Wu De Feng , Clejan Liviu , Potter Barry , Cederbaum Arthur I.
Published: March 2007

Studies were conducted to evaluate the possible induction or the maintenance of cytochrome P-450IIE1 in primary hepatocyte cultures by the inducing agent 4-methylpyrazole. Hepatocytes were isolated from control (noninduced) rats and from rats treated in vivo with either pyrazole or 4-methylpyrazole to induce P-450IIE1. The content of P-450IIE1 was determined by Western blots with antipyrazole P-450 IgG, and catalytic activity was assessed by assays of dimethyl-nitrosamine demethylase activity. The treatment with 4-methylpyrazole in vivo increased the content of P-450IIE1 and dimethylnitrosamine demethylase activity sevenfold and fourfold, respectively. In cultures prepared from noninduced hepatocytes, P-450IIE1 levels fell to values of 76%, 65%, 31% and 1% of freshly isolated hepatocytes after 1, 3, 6 and 9 days in culture. A similar decrease in dimethylnitrosamine demethylase was observed during this time. In cultures prepared from induced hepatocytes, the decline in P-450IIE1 was more rapid as levels fell to 77%, 31%, 3% and 3% of initial values after 1, 3, 6 and 9 days in culture. Again, the fall in dimethylnitrosamine demethylase activity paralleled the decline in content of P-450IIE1 and was more rapid with the induced hepatocytes. With cultures prepared from noninduced or induced hepatocytes, the addition of 4-methylpyrazole in vitro did not increase the content of P-450IIE1 or the activity of dimethylnitrosamine demethylase over the initial values. However, 4-methylpyrazole appeared to stabilize the P-450IIE1 and to decrease its rate of decline in culture. In noninduced cultures, the percent remaining content of P-450IIE1 after 6 days was 31% in the absence of and 52% in the presence of 5 mol/L 4-methylpyrazole. In cultures from 4-methylpyrazole—induced hepatocytes, the percent remaining P-450IIE1 after 3 days was 31% in the absence of inducer and 59% with 4-methylpyrazole added in vitro. Similarly 4-methylpyrazole helped to prevent the rapid decline of dimethylnitrosamine demethylase activity in induced and noninduced cultures. Viability of the induced and noninduced cultures in the absence or presence of added 4-methylpyrazole was similar. Levels of mRNA for P-450IIE1 were similar for livers from control rats and from rats treated in vivo with 4-methylpyrazole. The mRNA levels rapidly declined in induced and noninduced cultures, and this decline, unlike the fall in P-450IIE1 or dimethylnitrosamine demethylase activity, could not be prevented by the addition of 4-methylpyrazole in vitro to the cultures. These results suggest that the already induced P-450IIE1 isozyme is more labile and subject to rapid decline in culture and that inducers such as 4-methylpyrazole appear to stabilize the P-450IIE1 and thus help to maintain this isozyme and associated catalytic activity in cultures prepared from noninduced and induced hepatocytes. (HEPATOLOGY 1990;12:1379-1389).
Attenuation of alcohol-induced hepatic fibrosis by polyunsaturated lecithin†
Authors: Lieber Charles S. , Decarli Leonore M. , Mak Ki M. , Kim Cho-Il , Leo Maria A.
Published: March 2007

Characteristic features of alcoholic liver injury include fibrosis and striking membrane alterations, with associated phospholipid changes. To offset some of these abnormalities, a 10-yr study was conducted in baboons: 12 animals (eight females, four males) were fed a liquid diet supplemented with polyunsaturated lecithin (4.1 mg/kcal) for up to 8 yr, with either ethanol (50% of total energy) or isocaloric carbohydrate. They were compared with another group of 18 baboons fed an equivalent amount of the same diet (with or without ethanol), but devoid of lecithin. In the two groups, comparable increases in lipids developed in the ethanol-fed animals, but striking differences in the degree of fibrosis were seen. Whereas at least septal fibrosis (with cirrhosis in two) and transformation of their lipocytes into transitional cells developed in seven of the nine baboons fed the regular diet with ethanol, septal fibrosis did not develop in any animals fed lecithin (p < 0.005). They did not progress beyond the stage of perivenular fibrosis (sometimes associated with pericellular and perisinusoidal fibrosis) and had a significantly lesser activation of lipocytes to transitional cells. Furthermore, when three of these animals were taken off lecithin, but continued on the same amount of the ethanol-containing diet, they rapidly (within 18 to 21 mo) progressed to cirrhosis, accompanied by an increased transformation of their lipocytes to transitional cells. These results indicate that some component of lecithin exerts a protective action against the fibrogenic effects of ethanol. Because we had previously found that choline, in amounts present in lecithin, has no comparable action, the polyunsaturated phospholipids themselves might be responsible for the protective effect. (HEPATOLOGY 1990;12:1390-1398).
Characterization and accumulation of ferritin in hepatocyte nuclei of mice with iron overload
Authors: Smith Andrew G. , Carthew Philip , Francis Jean E. , Edwards Richard E. , Dinsdale David
Published: March 2007

After a single subcutaneous dose of iron-dextran (600 mg of iron/kg), iron overload developed in C57BL/10ScSn mice. At 4, 24 and 78 wk liver nonheme iron concentrations were 67-, 42- and 21-fold higher than controls, respectively. Much of the iron was in macrophages, but hepatocytes were also strongly positive for Perls' stainable iron. One feature was the development of iron-positive nuclear inclusions in hepatocytes. After a delay of at least 8 wk when no stainable iron was evident, a maximum of 37% of periportal hepatocytes contained inclusions by 24 wk. Although this proportion remained constant for the remainder of the study, the size of the inclusions (which were not membrane-limited) increased to > 3 μm in diameter, occupying > 25% of the nuclear volume. The presence of iron in the inclusions was confirmed by energy dispersive x-ray microanalysis. Immunocytochemical studies showed that the iron was present as aggregates of ferritin. Quantitation of nonaggregated ferritin molecules by image analyses after electron microscopy demonstrated that within 4 wk ferritin levels in cytoplasm and nucleoplasm had greatly increased but that there was a concentration gradient of approximately one order of magnitude across the nuclear envelope.

These findings are consistent with the hypothesis that in iron-loaded mouse hepatocytes there is a slow passage of ferritin-molecules through the nuclear pores; the gradient is maintained by the continual aggregation of ferritin within the nucleus. Intranuclear ferritin may provide a source of iron for catalyzing hydroxyl radical formation in nuclei during some toxic, carcinogenic and aging processes. (HEPATOLOGY 1990;12:1399-1405).
Quantification of hepatic iron with CT and MRI: Practical considerations
Authors: Kier Ruben
Published: March 2007

The diagnostic efficacy of magnetic resonance (MR) and computed tomography (CT) for detection and quantification of hepatic iron was assessed in a series of patients under investigation for clinical or biochemical evidence of hepatic iron overload. Thirty patients underwent MR imaging (SE 30,60/1000 or SE 30,60/2000) at 0.5 Tesla with calculation of hepatic T2 and liver to paraspinous muscles signal intensity ratios. Twenty-nine patients also had measurement of hepatic attenuation on noncontrast CT images. Results of these imaging studies were correlated in all patients with quantitative iron determination from liver biopsy specimens. The best predictor of liver iron among parameters studied was the ratio of the signal intensities of liver and paraspinous muscle (L/M) on a SE 60/1000 sequence. Both MR using L/M ratios and CT were sensitive methods for detection of severe degrees of hepatic iron overload with 100% of patients with hepatic iron on biopsy > 600 μg/100 mg liver dry weight detected on the basis of L/M < 0.6 or CT attenuation > 70 Hounsfield units (HU). The MR parameter, however, was more specific than CT (100 vs 50%) and showed a higher degree of correlation with quantitated hepatic iron from biopsy. T2 measurements showed poor correlation with hepatic iron, due to difficulty in obtaining precise T2 measurements in vivo when the signal intensity is low. None of the parameters utilized was sensitive for detecting mild or moderate degrees of hepatic iron overload.

We conclude that MR and CT are sensitive techniques for noninvasive detection of severe hepatic iron overload, with MR providing greater specificity than CT. Lesser degrees of iron deposition, however, may go undetected by our current imaging techniques.