bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbon tetrachloride-induced hepatic injury is associated with global DNA hypomethylation and homocysteinemia: Effect of S -adenosylmethionine treatment
Authors: Varela-Moreiras Gregorio , Alonso-Aperte Elena , Rubio Mireia , Gasso Marta , Deulofeu Ramón , Alvarez Luis , Caballería Juan , Rodés Juan , Mato José M.
Published: March 2007

Carbon tetrachloride (CCl4) administration to rats produces hepatic cirrhosis and supplementation with S-adenosylmethionine (SAM) can partially prevent CCl4-induced liver injury. These effects are thought to be caused by oxidative stress and the subsequent formation of free radicals, but the mechanism whereby this occurs and the accurate nature of the mechanisms by which SAM exerts its protective action are not well understood. The effect of short-term administration of CCl4 on hepatic DNA methylation and on SAM and S-adenosylhomocysteine (SAH) were assessed. CCl4 administration to rats for 3 weeks resulted in hypomethylation of liver DNA, determined by comparing the extent to which DNA from livers of control or treated animals could be methylated in vitro using [3H-methyl] SAM as methyl donor. This CCl4 effect on DNA methylation was corrected by the administration of SAM (10 mg/kg/d, intramuscularly), with values of methyl groups incorporation comparable with those observed in the control animals. Hepatic SAM was decreased by CCl4 (65.3 ± 5.27 vs. 102.2 ± 4.89 nmol/g; P < .05) and SAH was increased (69.5 ± 14.6 vs. 29.4 ± 3.83 nmol/g; P < .05). This led to a marked reduction of the SAM/SAH ratio (the methylation ratio) from 3.47 in control rats to 0.94 in CCl4-treated animals (P < .05). SAM treatment partially prevented (P < .05) the reduction of the ratio SAM/SAH induced by CCl4. CCl4 also induced a marked elevation of serum homocysteine levels (more than 20-fold; P < .001), which was partially prevented by SAM administration. A decrease in serum methionine concentration was also observed (20.87 ± 1.76 vs. 31.25 ± 2.37 μmol/L; P < .05) in response to CCl4, whereas cystathionine levels remained unchanged. Hepatic folate was reduced by CCl4 (11.2 ± 2.1 vs. 17.6 ± 1.8 μg/g; P < .05) and SAM treatment prevented this effect (P < .05). Hepatic glutathione (reduced form) and the activity of the enzyme SAM synthetase, which have been found to be reduced after the administration of CCl4 to rats for longer periods, were unchanged after treatment with the hepatotoxic agent for only 3 weeks. It is proposed that CCl4 disrupts the distribution of homocysteine between remethylation and its degradation via the transsulphuration pathway and that SAM, by resetting the methylation ratio, restores this equilibrium. (Hepatology 1995; 22:1310-1315.).
Does interferon therapy for chronic hepatitis B reduce the risks of developing cirrhosis and hepatocellular carcinoma?
Authors: Lok Anna S. F.
Published: March 2007

Objective:

To estimate the cost-effectiveness of interferon-α2B for the treatment of patients with chronic hepatitis B infection who are positive for hepatitis B e antigen (HBeAg).

Design:

Meta-analysis of nine randomized controlled trials and cost-effectiveness analysis, projecting the clinical and economic outcomes expected from changes in serologic markers of hepatitis B viral replication.

Data Sources:

MEDLINE search, expert panel opinion, hospital cost data, and adjusted physician charges.

Patients:

552 patients with confirmed chronic hepatitis B infection who were positive for HBeAg.

Intervention:

Interferon-α2b.

Measurements:

Lifetime incidence of cirrhosis and hepatocellular carcinoma; life expectancy; quality-adjusted life expectancy; and costs and marginal cost-effectiveness ratios from a societal perspective.

Results:

Interferon-α2b increases the likelihood of becoming negative for HBeAg from 9.1% to 45.6% (difference, 36.5%; 95% CI, 23.7% to 49.2%) and of becoming negative for hepatitis B surface antigen from 1.7% to 7.7% (difference, 6.0%; CI, 2.8% to 9.3%) in the first year. For a 35-year-old person with chronic hepatitis B who is HBeAg positive, our analysis suggests that interferono; α2b will increase life expectancy by 3.1 years or 3.4 quality-adjusted life-years and will decrease projected lifetime costs, even if future savings are discounted; thus, interferon-α2b is the dominant strategy. Even with the model biased strongly in favor of standard care, the marginal cost-effectiveness ratio of interferon did not exceed $12 000 per life-year gained.

Conclusions:

Interferon-α2b should prolong life and lower costs for patients with chronic hepatitis B who are HBeAg positive.
In vitro modeling of liver membrane copper transport
Authors: Schilsky Michael L.
Published: March 2007

The process of hepatobiliary copper (Cu) secretion is still poorly understood Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting Ptype ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 μM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated K
m of 8.6 μM and a V
max of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT. (J. Clin. Invest. 1995.95:412-416.) Key words: copper · bile secretion · P-type ATPase · vesicle transport · Wilson disease. (Reproduced from The Journal of Clinical Investigation, 1995, vol. 95, pp. 412-416 by copyright permission of The Society for Clinical Investigation.).
Efficacy of interferon alfa therapy in chronic hepatitis C patients depends primarily on hepatitis C virus RNA level
Authors: Yamada Gotaro , Takatani Masahiro , Kishi Fumitoshi , Takahashi Michiko , Doi Toshihiko , Tsuji Takao , Shin Sadahito , Tanno Masashi , Urdea Mickey S. , Kolberg Janice A.
Published: March 2007

To clarify the viral factors that may predict the therapeutic effect of interferon (IFN) in chronic hepatitis C (CHC) patients, we investigated the quantitative serum hepatitis C virus (HCV) RNA level, genotype, and liver biopsy histological features in 60 patients who were treated with 360 × 106 U of natural IFN-α for 36 to 48 weeks and for more than 12 months after therapy. A branched DNA (bDNA) assay was used to measure HCV RNA levels. All responders, defined as those individuals with normal alanine transaminase (ALT) levels at 48 weeks after therapy, had less than 2 × 106 HCV RNA Eq/mL before administration of IFN. Of 39 patients with RNA levels (less than 2 × 106 Eq/mL) 23 (59.0%) were responders. The genotype was determined for each patient using type-specific polymerase chain reaction (PCR) primers. There was a significant difference in rate of response between subtype 1b and subtypes 2a and 2b (P < .0002); however, all responders had less than 2 × 106 Eq/mL independent of genotype. In a multivariate analysis, RNA level was the most statistically significant factor affecting response to IFN. Although disease severity, as defined by histological features, was not statistically correlated with nonresponse, patients that responded to IFN tended to have less severe disease. (Hepatology 1995; 22:1351-1354).
Hepatitis B virus precore/core variation and interferon therapy
Authors: Fattovich Giovanna , McIntyre Graham , Thursz Mark , Colman Kathryn , Giuliano Giustina , Alberti Alfredo , Thomas Howard C. , Carman William F.
Published: March 2007

Precore/core genes from hepatitis B e antigen (HBeAg)-positive and antibody to HBeAg (anti-HBe) positive individuals with active hepatitis have been analyzed to search for correlations with response to interferon before and after treatment. Pretreatment, no precore stop codon mutants were detected, even at the 3% level, in HBeAg-positive responders or nonresponders. In anti-HBe-positive patients, precore mutants did not influence response. No significant core amino acid variability was observed in HBeAg-positive patients, irrespective of interferon response. However, anti-HBe-positive cases had multiple core protein substitutions, mostly in B- and T-helper cell epitopes, but responders had fewer (P = .02 for responders versus nonresponders and reactivators). None of four responders, three of seven reactivators, and three of three nonresponders had mutations within the major T-helper epitope from aa50 to aa69 (P = .03). Precore mutants appeared in eight of nine natural seroconverters compared with 3 of 10 interferon-induced anti-HBe seroconverters (P = .01). Those in whom precore wild-type remained after treatment often tested negative in the last available sample using polymerase chain reaction (PCR), whereas emergence of mutants led to ongoing viremia in all cases. In anti-HBe-positive cases, precore sequences remained stable during therapy, except for 2 cases in whom a precore mutant appeared accompanied by reactivation. In the core protein, anti-HBe-positive cases selected a mean of 3.5, 1.6, and 1.7 amino acid substitutions in responders, nonresponders, and reactivators respectively (P = NS). In conclusion, core but not precore sequence before therapy may predict response. Appearance of precore mutants during therapy usually predicts failure to clear virus but substitution in core does not influence outcome. (Hepatology 1995; 22:1355-1362).
T-cell-mediated autologous hepatocytotoxicity in patients with chronic hepatitis C virus infection
Authors: Liaw Yun-Fan , Lee Ching-Song , Tsai Sun-Lung , Liaw Bo-Wen , Chen Tse-Ching , Sheen I-Shyan , Chu Chia-Ming
Published: March 2007

Virus-specific cytotoxic T lymphocytes (CTLs) have been suggested to be responsible for the liver injuries in patients with hepatitis C virus (HCV) infection. However, there has been no report of direct evidence to substantiate this hypothesis. In this study, we performed in vitro autologous hepatocytotoxicity assay in 45 patients to examine a possible role of CTLs to HCV-in-fected liver cells. The data were correlated with histology activity index of liver biopsy specimens. Lymphocyte subsets and hepatocyte expression of human major histocompatibility complex antigens class I and class II (HLA-I and HLA-II) were also evaluated. The immunohistochemical study showed more prominent HLA-I expression than HLA-II on hepatocytes (mean score ± SEM: 2.34 ± 0.11 vs. 0.42 ± 0.08; P < .01). The lymphocyte subset analysis showed that CD8+ T cells were dominant in the lobular areas showing spotty necrosis, whereas CD4+ T cells were prominent in the portal and periportal areas (P < .01). Most patients had a significant T cell-mediated cytotoxicity to hepatocytes as compared with non-T cells (percentage cytotoxicity ± SEM: 46.4 ± 2.3 vs. 13.8 ± 2.7; P < .001). T cell-mediated hepatocytotoxicity had a linear correlation with HAI (P < .05). The T cell-mediated cytotoxicity could be blocked by anti-CD8 (43.7% vs. 9.5%, P < .05) or by anti-HLA-I (43.7% vs. 18.5%, P < .05) but not by anti-CD4 or anti-HLA-II monoclonal antibodies. These findings strongly suggest that HLA-I-restricted, CD8+ T cell-mediated hepatocytotoxicity is an important pathogenetic mechanism in patients with chronic HCV infection. (Hepatology 1995; 22:1368-1373).
Efficacy and safety of interferon alfa therapy in chronic hepatitis C with autoantibodies to liver-kidney microsomes
Authors: Todros Luca , Saracco Giorgio , Durazzo Marilena , Abate Maria Lorena , Touscoz Giovanni , Scaglione Luca , Verme Giorgio , Rizzetto Mario
Published: March 2007

Interferon alfa (IFN) therapy is efficacious in chronic viral hepatitides. It may, however, cause adverse immunologic reactions in patients with concomitant autoimmune phenomena. A minority of patients with chronic type C hepatitis have antibodies against liver and kidney microsomes (anti-LKM) in serum. We therefore carried out this study to find out whether IFN is safe and efficacious also in this subgroup. We treated 92 consecutive cases of chronic hepatitis C with IFN. Twelve patients had anti-LKM, and the remaining 80 tested negative to the anti-LKM. The hepatitis C virus (HCV) infection was diagnosed on the basis of positive anti-HCV and HCV-RNA tests. We compared the clinical and virological results of the therapy and the side effects found in the two groups. We found that the response to therapy and the outcome after 1 year of follow-up were similar. Treatment was discontinued in one anti-LKM-positive patient because of a drastic increase in ALT levels at the fourth month of therapy. No untoward effect was observed in the other cases. Hepatitis C patients with anti-LKM may be exposed to an increased risk of an adverse hepatitic reaction while being treated with IFN. However, we found that the extent of the risk was minimal compared with the expected benefits of the therapy. IFN is therefore recommended as the first therapy to choose in these patients. They must, however, be monitored more closely for possible liver dysfunction than the ordinary hepatitis C patient. (Hepatology 1995; 22:1374-1378).
The significance of spontaneous hepatitis B e antigen seroconversion in childhood: With special emphasis on the clearance of hepatitis B e antigen before 3 years of age
Authors: Chang Mei-Hwei , Hsu Hong-Yuan , Hsu Hey-Chi , Ni Yen-Hsuan , Chen Juei-San , Chen Ding-Shinn
Published: March 2007

To investigate the significance of spontaneous hepatitis B e antigen (HBeAg) seroconversion during childhood, 415 hepatitis B surface antigen (HBsAg) carrier children (ages 0 to 15 years) were prospectively followed for 7.1 ± 2.9 years. Hepatitis B virus (HBV) markers and liver function profiles of each child were tested at least once every 6 months. Among them, 50 were initially anti-HBe positive and 140 seroconverted from HBeAg to anti-HBe during follow-up. Before HBeAg seroconversion, jaundice occurred in 9 and alanine transaminase (ALT) activities elevated in 99 of the 140 seroconverters. Serum ALT returned to normal in all patients within 1 to 5 years of seroconversion. Six had reelevated ALT later after seroconversion. Only 7 (9.7%) of the 72 carrier infants seroconverted before 3 years of age. The peak ALT levels in five of them exceeded 100 IU/L, and two had jaundice before HBeAg seroconversion. One of the early seroconverters developed hepatocellular carcinoma (HCC) at 11 years of age, although his liver function profiles remained normal after HBeAg seroconversion. Liver biopsy specimens from 30 children during the anti-HBe-positive stage showed inactive cirrhosis in 2 (including one with HCC), chronic hepatitis with marked fibrosis in 1, mild activity and moderate fibrosis in 2, mild activity and mild fibrosis in 9, and minimal histologic changes in the remaining 16. Although most will achieve a normalization of ALT and inactive liver histologic changes, the seroconversion of HBsAg carrier children from HBeAg to anti-HBe is not necessarily an indicator of favorable prognosis; a small proportion of children will develop cirrhosis or even HCC. (Hepatology 1995; 22: 1387-1392).
Integration of hepatitis B virus and alteration of the 1p36 region found in cancerous tissue of primary hepatocellular carcinoma with viral replication evidenced only in noncancerous, cirrhotic tissue
Authors: Simon Daniela , Carr Brian I.
Published: March 2007

We have studied the genetic profile of the host genome and hepatitis B virus (HBV) in HBV-associated primary hepatocellular carcinoma (HCC). Comparative analyses of HCC cell line Hep 40 and the original biopsy specimens showed the episomal and replicating form of HBV only in the biopsy specimen from nontumor (NT) cirrhotic liver tissue, where a molecular change in the 1p36 region was detected (NT tissue showed a normal 46XY karyotype). In contrast, only integrated HBV was detected in HCC tumor (T) tissue and Hep 40 cells. Two HBV integration sites were identical in HCC tissue and the hyperdiploid Hep 40 cell line, where genetic alteration in the 1p36 region was identified. These data indicate that viral replication is ongoing only in NT cirrhotic-hyperplastic, chromosomally normal tissue with evidence for genetic instability. Only the tumor cell with altered genotype has virus integrated. (Hepatology 1995; 22:1393-1398).
Primary sclerosing cholangitis and ulcerative colitis: Evidence for increased neoplastic potential
Authors: Broomé Ulrika , Löfberg Robert , Veress Béla , Eriksson Ljusk Siw
Published: March 2007

Primary sclerosing cholangitis (PSC) is a biliary destructive disease mostly affecting patients with ulcerative colitis (UC). PSC has been suggested to be an independent risk factor for the development of colorectal malignancy in UC. Patients with PSC also have an increased risk of developing cholangiocarcinoma. This study aimed at assessing the cumulative risk of colorectal neoplasia in PSC and UC, and also to determine risk factors for the development of cholangiocarcinoma. Fifty-eight PSC patients were included. Forty PSC patients having extensive UC were each matched to two control patients of the same age, with extensive colitis and a comparable duration of the colitis, but without PSC. All UC patients had been under colonoscopic surveillance with multiple biopsies. Among the 40 PSC patients with UC, 16 developed colonic dysplasia or carcinoma, versus 10 in the control group (P < .001). The absolute cumulative risk of developing colorectal dysplasia/carcinoma in the PSC/UC groups was 9%, 31%, and 50% after 10,20, and 25 years of disease duration. In the group with UC only, the corresponding risk was 2%, 5%, and 10%, respectively (P < .001). Ten patients with PSC developed cholangiocarcinoma, all but one having UC. In the control group, no cholangiocarcinoma occurred. Patients with PSC and UC with colorectal neoplasia developed cholangiocarcinoma significantly more often compared with patients with UC and PSC without colonic dysplasia/carcinoma (P < .02). This study demonstrates not only that patients with PSC and UC have a significantly higher risk of developing colorectal neoplasia compared with patients having UC only, but also that patients with PSC and UC having colorectal neoplasia are more prone to develop cholangiocarcinoma. (Hepatology 1995; 22:1404-1408).
An investigation of whether polymorphisms of cytochrome P4502E1 are genetic markers of susceptibility to alcoholic end-stage organ damage in a chinese population
Authors: Chao You-Chen , Young Tong-Ho , Chang Wei-Kuo , Tang Hung-Shang , Hsu Chung-Te
Published: March 2007

The human cytochrome P4502E1 gene (P4502E1), coding for an ethanol-inducible nitrosamine-metabolizing P-450, is involved in the metabolism of ethanol and many known carcinogens. Recently, restriction fragment length polymorphisms (RFLps) within the P4502E1 have been suggested as genetic markers of susceptibility to alcohol-induced liver disease but the previous studies disagree whether alcoholics with c1 or c2 allele are more susceptible to alcohol-induced liver disease. Using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, we determined the RsaI and PstI polymorphism of P4502E1 in 77 Chinese alcoholic patients (54 with alcohol-induced cirrhosis and 23 with acute alcohol-induced pancreatitis) and 164 nonalcoholics and compared them with previously published data. The PCR-RFLPs showed three P4502E1 genotypes: type A, homozygote c1/c1; type B, heterozygote c1/c2; and type C, homozygote c2/c2. The RsaI and PstI polymorphism of P4502E1 were completely linked in both Chinese alcoholics and nonalcoholic controls. The rare allele (c2) occurs at similar frequency of 0.232 and 0.234 (P > .05) in nonalcoholic controls and alcoholics, respectively. The genotype distributions of P4502E1 between Chinese alcoholics and nonalcoholics are not sig nificantly different. The genotype and allele frequencies of P4502E1 for Chinese are significantly different from those of Swedes, European-Americans, and African-Americans, respectively (P < .0001), but very similar to Japanese (P > .05). In conclusion, ethnic variations exist between Asians and Caucasians and between Asians and African-Americans. No allelic variants at loci associated with RsaI PstI RFLPs result in phenotypes displaying greater susceptibility to alcohol-induced cirrhosis or alcoholism in Chinese populations, which contradicts previous reports from Japanese groups. (HEPATOLOGY 1995;22:1409-1414).
Systemic hypotension and diuresis by l-arginine in cirrhotic patients with ascites: Role of nitric oxide
Authors: Tajiri Kazuo , Miyakawa Happei , Izumi Namiki , Marumo Fumiaki , Sato Chifumi
Published: March 2007

To investigate the role of nitric oxide in renal function and hemodynamics in cirrhotic patients with ascites, l-arginine (30 g in 300 mL of distilled water), a substrate for nitric oxide synthase, was infused into six cirrhotic patients with ascites, and the effects were compared with those of saline infusion. Healthy controls (n = 5) were also studied under the same conditions. In the patients, l-arginine infusion significantly decreased systolic and diastolic blood pressures while markedly increasing urinary flow and urinary sodium excretion; no significant changes were seen with saline infusion. In controls, only diastolic blood pressure was decreased by l-arginine infusion, whereas urinary flow and urinary sodium excretion were increased by both l-arginine and saline infusion. In both groups, a similar increase of plasma atrial natriuretic factor (ANF) was seen with l-arginine and saline infusions; endotheline and cate-cholamines were not affected by either infusion. In both groups, plasma levels of vasopressin were increased by l-arginine infusion. In the cirrhotic patients, urinary excretions of cyclic guanosine monophosphate (cGMP) and nitrates/nitrites (NOx) were significantly increased by l-arginine infusion, whereas no significant changes were seen with saline infusion. In controls, only the excretion of cGMP was increased by l-arginine infusion. In summary, l-arginine infusion induces diuresis and natriuresis accompanied by increased excretions of cGMP and NOx in cirrhotic patients with ascites. This differs from the response in controls, where the increase in urinary sodium excretion is not accompanied by an increase in markers of increased nitric oxide synthesis. (Hepatology 1995; 22:1430-1435).
Differential display and integrin alpha 6 messenger RNA overexpression in hepatocellular carcinoma
Authors: Begum Nasim Ara , Mori Masaki , Matsumata Takashi , Takenaka Kenji , Sugimachi Keizo , Barnard Graham F.
Published: March 2007

Our aim was to isolate potentially important differentially expressed gene products from paired human hepatocellular carcinoma (HCC) and normal liver samples using the differential messenger RNA (mRNA) display technique. Total RNA samples were reverse transcribed with anchoring oligonucleotide primers and then amplified by the polymerase chain reaction (PCR) with additional upstream random primers. Differentially expressed complementary DNA (cDNA) products were subsequently used as probes in Northern blot analysis. One such cDNA product, present in tumor but absent in normal displays, showed identity with the adhesion molecule integrin alpha 6. In Northern blots of 16 HCC pairs, the ˜5.5 kb signal of integrin alpha 6 mRNA was overexpressed in seven tumors, with a weak signal in the normal livers. For those patients with versus without integrin alpha 6 mRNA overexpression: (1) grade III (or IV) histology was noted in seven of seven versus three of nine tumors, respectively (P = .03); (2) tumor recurrence or death (at mean follow-up of 18 months) was noted in six of seven versus three of eight patients, respectively (P = .17). Similar results were obtained using semiquantitative PCR co-amplification with glyceraldehyde 3-phosphate dehydrogenase as a control; ±50% of the tumors had stronger integrin alpha 6 bands than their paired normals. Both A and B variants of integrin alpha 6 mRNA were detectable in the tumor and normal liver samples. The B variant was more pronounced than the A variant by 8.9-fold in the tumors (n = 10) compared with threefold in the normal livers (n = 10), suggesting that the overexpression of integrin alpha 6 may be more reflective of abnormalities of B variant levels than of A variant levels. Important genes whose expression correlates with significant patient variables may be isolated by differential display; based on this small series there is a trend for integrin alpha 6 mRNA to be overexpressed in high-grade HCC and to predict a tendency toward a poorer outcome. (Hepatology 1995; 22:1447-1455).
Hepatic sinusoidal endothelial cells can store and metabolize serum immunoglobulin
Authors: Iwamura Shinichi , Enzan Hideaki , Saibara Toshiji , Onishi Saburo , Yamamoto Yasutake
Published: March 2007

Sinusoidal inclusion-containing endothelial cells in the liver were investigated with particular interest in their capacity of metabolizing immunoglobulin. Formalin-fixed deparaffinized liver specimens were used for immunohistochemistry, and pronase digestion was proved to be effective for antigen retrieval of immunoglobulin. The inclusions in sinusoidal endothelial cells were strongly immunostained with anti-immunoglobulin (Ig)G, IgA, and IgM antibodies in predigested sections. The complements were not identified immunohistochemically in the inclusions even after pronase treatment. Two women with autoimmune liver disease, who initially represented high levels of serum γ globulin and abundant inclusion-containing endothelial cells, were studied. The subsequent biopsy after effective corticosteroid therapy demonstrated significant histological improvement as well as the disappearance of inclusion-containing endothelial cells (ICECs). During and after treatment, their serum γ globulin levels were drastically reduced. In conclusion, the hepatic sinusoidal endothelial cells can take up serum immunoglobulin, probably through a receptor-mediated pathway, and its excessive storage results in the formation of cytoplasmic inclusions that are easily recognized by a light microscope. The stored immunoglobulin may be degraded in the cytoplasm, and the inclusions would disappear in association with the reduction of sinusoidal γ globulin content. In other words, the intralobular density of inclusion-containing endothelial cells is a morphological predictor for the serum γ globulin level. (Hepatology 1995; 22:1456-1461).
Serum carbohydrate-deficient transferrin: Mechanism of increase after chronic alcohol intake
Authors: Xin Yan , Lasker Jerome M. , Lieber Charles S.
Published: March 2007

Carbohydrate-deficient transferrin (CDT) is now considered to be the most sensitive and specific biological marker of alcohol abuse. However, the mechanism by which chronic alcohol consumption causes an elevation of CDT levels in serum is still not understood. Therefore, we fed eight pairs of male rats a nutritionally adequate liquid diet containing either alcohol (36% of energy) or isocaloric dextrose (control) for 4 weeks, after which blood and liver samples were obtained. Serum CDT content in alcohol-treated rats increased by 45% (P < .05) as assessed with an isoelectric focusing/Western blot method (IEF/WB) thereby mimicking the clinical condition. In particulate fractions (plasma membranes) prepared from the liver samples, the activity of sialidase was increased 1.62-fold (P < .05) in ethanol-fed animals compared with their corresponding controls. In contrast, in rats fed ethanol, the activities of sialyltransferase (ST), galactosyltransferase (GT), and N-acetylglu-cosamine transferase (N-AGT), which are glycosyltransferases involved in transferrin carbohydrate side chain synthesis, were diminished by 24% and 40% (P < .05), 23% and 51% (P < .05, .001), and 20% and 26% (P < .05) in total liver homogenates and Golgi fraction (GF) 1, respectively, when expressed as units/100 g body weight. These enzymes were also significantly less active in hepatic GFs 2 and 3. The depression of the transferase activities in ethanol-fed rats appeared to be due, at least in part, to enzyme inactivation by acetaldehyde, whereas ethanol itself was without effect. Similar results were obtained in humans; five alcohol abusers were found to exhibit a 23% decrease in hepatic sialyltransferase and a 41% increase in sialidase activities, respectively, when compared with three nondrinking subjects. In conclusion, the increased activity of sialidase in liver particulate fractions, combined with the decrease in transferrin glycosyltransferases in GFs, mediated by ethanol (or acetaldehyde derived from ethanol) most likely accounts for the enhanced serum CDT in response to chronic ethanol consumption in rats as well as in humans. (Hepatology 1995; 22:1462-1468).
Impaired biliary excretion and whole body elimination of methylmercury in rats with a congenital defect in biliary glutathione excretion
Authors: Ballatori Nazzareno , Gatmaitan Zenaida , Truong Anh T.
Published: March 2007

Biliary excretion of methylmercury, a major route of elimination of this toxic compound, was less than 2% of control in Eisai hyperbilirubinemic (EHBR) rats, a mutant Sprague-Dawley strain with a defect in biliary excretion of a variety of organic anions, including glutathione S-conjugates and reduced glutathione (GSH). Biliary GSH excretion in EHBR rats was also <2% of controls, confirming previous findings. Impaired biliary methylmercury and GSH excretion was not explained by decreased hepatic content of these compounds. Indeed, hepatic methylmercury and GSH concentrations in EHBR rats were actually double those of controls. To assess the significance of the impaired biliary excretion in the whole body elimination of the toxicant, 203Hg excretion was measured over a 17-day period after intraperitoneal administration of either 0.5 or 5 μmol/kg of 203Hg-methylmercury chloride. The results for the two doses were similar. Methylmercury was eliminated by a first order process; however, the biological half-life was significantly longer in the EHBR rats, 46 to 54 days versus 18 to 22 days. Fecal excretion was the main route of elimination in both control and mutant animals. At necropsy (17 days), 16% to 25% of the 203Hg dose was recovered in the liver of the EHBR rats, whereas livers of control animals contained less than 2% of the administered dose. These findings demonstrate that biliary excretion of methylmercury is markedly impaired in EHBR rats and is associated with a low biliary GSH excretion, providing support for the hypothesis that methylmercury is normally transported across the canalicular membrane by a GSH-dependent mechanism, and presumably as a GSH mercaptide (CH3Hg-SG). This study also provides a direct measure of the contribution of biliary excretion to the whole-body elimination of methylmercury. The biological half-life of the metal was 2.5 times longer in the mutant animals, indicating a critical role for biliary secretion in methylmercury detoxification. (Hepatology 1995; 22:1469-1473).
Transforming growth factorβ1 increases the number of apoptotic bodies and decreases intracellular pH in isolated periportal and perivenular rat hepatocytes
Authors: Benedetti Antonio , di Sario Antonio , Baroni Gianluca Svegliati , Jezequel Anne Marie
Published: March 2007

Transforming growth factor beta 1 (TGFβ1) is involved in promoting cell death by apoptosis in the liver, whereas the activation of Na+/H+ exchanger has been related to cell proliferation. The aim of this study was to gain information on the effects of TGFβ1 on intracellular pH and Na+H+ exchange activity in isolated periportal (PP) and perivenular (PV) rat hepatocytes using the pH-sensitive dye BCECF in a perfused subconfluent hepatocyte monolayer. Steady-state intracellular pH (pHi) in a bicarbonate-free solution (HEPES) were 7.17 ± 0.031 in PP and 7.15 ± 0.041 in PV cells. Treatment with TGFβ1 (120 pmol/L) for 7 hours increased the number of apoptotic bodies by 25% and 38%, and decreased steady-state pHi to 7.11 ± 0.018 (P = .05) and to 7.07 ± 0.021 (P ± .02), respectively, in PP and PV hepatocytes. In HEPES, cells recovered from an acid load, extruding protons at a rate (JH) of 4.85 ± 1.01 mmol/L/min in PP cells and of 4.91 ± 0.99 mmol/L/min in PV hepatocytes. This recovery appeared amiloride inhibitable (1 mmol/L). Culture with TGFβ1 for 7 hours induced (in HEPES) a decrease of pHi recovery rate from an acid load more in PV (by 46%) than in PP hepatocytes (by 35%, P ± .05). Acute administration of epidermal growth factor (EGF) (10 to 100 ng/mL) induced an increase in Na+/H+ exchange activity by 32% and 27%, respectively, in PP and PV cells compared with controls. In contrast, in cells cultured for 7 hours with 120 pmol/L TGFβ1, the acute administration of EGF slightly increased Na+/H+ exchange activity (by 18%, P ± .05) only in PP cells. This study demonstrates that pHi and Na+/H+ exchange activity are decreased by TGFβ1, which increases the number of apoptotic bodies in periportal and perivenular rat hepatocyte primary cultures. (Hepatology 1995; 22:1488-1498).
Endotoxin and interleukin-1 related hepatic inflammatory response promotes liver failure after partial hepatectomy
Authors: Boermeester Marja A. , Straatsburg Irene H. , Houdijk Alexander P. J. , Meyer Catharina , Frederiks Wilma M. , Wesdorp Robert I. C. , van Noorden Cornelis J. F. , van Leeuwen Paul A. M.
Published: March 2007

Impairment of various functions of the liver and concomitantly increased levels of parameters of liver damage, a clinical entity termed liver failure, is commonly seen after partial hepatectomy. We investigated in a rat model whether damage of the remnant liver was due to local inflammatory responses, and related to endotoxin or interleukin-1 (IL-1). To address this question, the effects of partial hepatectomy on infiltration of immunocompetent cells and expression of major histocompatibility complex (MHC) class II antigen of macrophages in the remnant liver was studied using immunohistochemical techniques. Specific intervention with recombinant N-terminal bactericidal/permeability-increasing protein (rBPI23) to neutralize endotoxin and with IL-1 receptor antagonist (IL-1ra) to block IL-1 activity was used to examine the respective roles of endotoxin and IL-1. After partial hepatectomy, we found an influx of neutrophils, an increased expression of MHC class II antigens, and morphologic changes of Kupffer cells consistent with activation. These inflammatory events coincided with increased serum levels of markers of liver damage (aspartate aminotransferase, alanine aminotransferase, ammonia). Both neutralization of endotoxin and blocking of IL-1 activity reduced hepatic inflammation and reduced serum levels of aminotransferases and ammonia. In addition, liver cell proliferation as assessed by staining for proliferating cell nuclear antigen (PCNA) expression was significantly enhanced when either endotoxin or IL-1 effects were blocked. Thus, our results suggest that local hepatic inflammatory responses inhibit liver cell proliferation and promote liver failure, presumably by affecting the functional capacity of the remnant liver. (Hepatology 1995; 22:1499-1506).
Molecular dissection of the mitogenic effect of hepatocytes on cultured hepatic stellate cells
Authors: Gressner Axel M. , Lahme Birgit , Brenzel Arnfried
Published: March 2007

The activation of proliferation of rat liver hepatic stellate cells (HSC) in cooperation with hepatocytes (PC) was studied using a coculture system and cell-conditioned media, respectively. The proliferation of HSC was followed by incorporation of [3H] thymidine and BrdU into DNA and by DNA content per culture. Strong stimulation of HSC proliferation was noticed under reduced fetal calf serum (FCS) conditions (0.2%) during a 48-hour coculture with PC, rat hepatoma, human hepatoma, and transforming growth factor (TGF)-α-transgenic mouse PC, respectively. The extent of stimulation was frequently higher than that observed by the addition of 10% FCS. Transformed HSC (myofibroblasts) could also be stimulated by cocultured PC, but the magnitude of activation was lower than that of (untransformed) HSC. Using radioreceptor assays, we could demonstrate significant concentrations of insulinlike growth factor (IGF)-1 (300 ng/106 cells × 48 hours) and quite lower concentrations of bFGF and TGF-α in the hepatocyte-conditioned media (PCcM), whereas IGF-2 was not detectable. With anti-IGF-1 neutralizing antibody, the stimulatory activity of PCcM could be reduced by approximately 50%. PCcM, which mimics the effects of cocuitures and supports strongly the action of exogenous IGF-1 on HSC proliferation, leaving that of other cytokines (TGF-α, IL-1 α, bFGF, aFGF, TNF-α), added either separately or in various combinations, uninfluenced. The latter cytokines were without significant effects on HSC proliferation. The mitogenic activity of cytokine combinations containing IGF-1 could be enhanced severalfold by limiting amounts of PCcM. Maximum stimulation of cell proliferation of 40-fold above control cultures was reached by IGF-1 in combination with TGF-α and bFGF in presence of diluted PCcM, which is approximately 6-fold higher than in the absence of PCcM. [125I] IGF-1 added to PCcM was bound by more than 90% to carrier proteins. The results confirm in cocultures strong mitogenic activation of HSC by PC. It is suggested that IGF-1 and respective IGF-binding proteins are of great importance in the mitogenic signal transfer between hepatocytes and hepatic stellate cells. (Hepatology 1995; 22:1507-1518).
Dynamic measurements of the acute and chronic effects of lysosomotropic agents on hepatocyte lysosomal pH using flow cytometry
Authors: Myers Brent M. , Tietz Pamela S. , Tarara James E. , Larusso Nicholas F.
Published: March 2007

Previous investigators measuring the pH of lysosomes have used digitized video microscopy (DVM) in freshly isolated or cultured cells. Although useful, this technique is time consuming, requires the use of an image analysis system, and is limited by the fact that measurements can be made in only a relatively small number of cells. The aim of this study was to develop and initially apply a technique using flow cytometry to make dynamic measurements of lysosomal pH in a large number of living hepatocytes. Rats were injected intraperitoneally with fluorescein isothiocyanate-dextran (FITC-Dex), a pH-sensitive fluorescent probe that is sequestered into lysosomes. Hepatocytes were isolated 16 hours after injection by collagenase perfusion. Lysosomal pH was measured in 20,000 hepatocytes per animal using flow cytometry with excitation at 488 nm and emission at 530 nm (pH sensitive) and 585 nm (pH insensitive). A standard curve of pH versus the 530/585 nm ratio was generated with FITC-Dex-loaded hepatocytes by equilibrating intralysosomal pH with extracellular pH using ionophores and metabolic inhibitors. The acute effects of chloroquine and methylamine were determined by exposing isolated hepatocytes to these lysosomotropic agents. The effect of chronic administration of chloroquine and Triton WR-1339 (Rutger Chemical, Inc., Irvington, NJ) on lysosomal pH was also measured. Intralysosomal pH was 4.67 + 0.02, nearly identical to the value 4.70 + 0.05 previously measured by us using DVM. Both chloroquine and methylamine caused both rapid (< 1 minute), major (0.5 to 2.0 pH units), and dose-dependent increases in lysosomal pH as well as changes in lysosome morphology. In contrast, although chronic chloroquine and Triton WR-1339 administration caused dramatic ultrastructural changes in lysosomal morphology, lysosomal pH was not increased by either of these agents suggesting that their pharmacological mechanisms are not mediated by their effects on lysosomal pH. These results show the utility and validity of using flow cytometry in living cells to quantify dynamic changes in the pH of lysosomes and show a dissociation of morphological and pH responses of hepatocyte lysosomes to chronic treatment with lysosomotropic agents. (Hepatology 1995; 22:1519-1526).
Specific targeting of human hepatocellular carcinoma cells by immunoliposomes in vitro
Authors: Moradpour Darius , Compagnon Béatrice , Wilson Byron E. , Nicolau Claude , Wands Jack R.
Published: March 2007

The monoclonal antibody AF-20 was raised against the human hepatocellular carcinoma (HCC) cell line FOCUS and binds with high affinity to a rapidly internalized 180-kd homodimeric glycoprotein that is abundantly expressed on the surface of human HCC and other human cancer cell lines. Immunoliposomes were produced by covalently coupling AF-20 to liposomes containing carboxyfluorescein. Interaction of immunoliposomes with various HCC cell lines in uitro was quantitatively assessed by flow cytometry and qualitatively analyzed by fluorescence microscopy. Liposomes bearing an isotypematched nonrelevant monoclonal antibody (MAb) and cell lines not expressing AF-20 antigen served as controls. AF-20-immunoliposomes specifically bound to HCC and other human cancer cell lines expressing the AF-20 antigen and were rapidly internalized at 37°C. Interaction of AF-20-conjugated liposomes with these cell lines was between 5 and 200 times greater than that of unconjugated liposomes, whereas no difference was observed between control liposomes bearing a nonrelevant antibody and unconjugated liposomes. Specificity of liposome-target cell interaction was confirmed by competitive inhibition assays. Kinetic analysis showed rapid association of AF-20 immunoliposomes with target cells, with saturation conditions being reached after 60 minutes. We conclude that the MAb AF-20 directs highly efficient, specific, and rapid targeting of immunoliposomes to human HCC and other human cancer cell lines in vitro. This targeted liposomal delivery system represents a promising approach for the development of immunotargeted diagnosis and therapy strategies against HCC. (HEPATOLOGY 1995;22:1527-1537).
Vascular adhesion molecule-1 and intercellular adhesion molecule-1 expression on rat liver cells after lipopolysaccharide administration in vivo
Authors: van Oosten Marijke , van de Bilt Erika , de Vries Helga E. , van Berkel Theo J. C. , Kuiper Johan
Published: March 2007

During sepsis the infiltration of leukocytes plays a pivotal role in tissue damage. Induction of septic shock results in an early accumulation of polymorphonuclear leukocytes in the liver (after 3 hours), which is followed by an infiltration of mononuclear phagocytes (after 30 hours). Expression of adhesion molecules may contribute to the migration of leukocytes to the site of inflammation. Therefore, in the present study we determined the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) on hepatocytes, liver endothelial cells, and Kupffer cells after lipopolysaccharide (LPS) treatment of rats in vivo. Parenchymal cells showed no constitutive expression of VCAM-1 and the expression could not be upregulated by LPS treatment in vivo, whereas Kupffer and endothelial cells had a low basal expression of VCAM-1 and this expression was increased 40-fold by LPS treatment in vivo. All three cell types showed a basal expression of ICAM-1 and the expression on endothelial liver cells of untreated rats was two times higher than the expression on parenchymal and Kupffer cells. Stimulation with LPS increased the expression of ICAM-1 2.5 times for parenchymal cells and approximately 4 times for endothelial and Kupffer cells. It is concluded that the expression of adhesion molecules may contribute to the influx of leukocytes during septic shock and, therefore, play a role in tissue damage during septic shock. (Hepatology 1995; 22:1538-1546).
The effect of prostaglandin E1 on liver adenine nucleotides and cytoplasmic enzymes in a porcine model of normothermic hepatic ischemia
Authors: Helling Thomas S. , Wogahn Brent M. , Olson Steve A. , Evans Laurie S. , Reddy Bokka R. , Vanway Charles
Published: March 2007

The liver has been judged relatively resistant to ischemia, but prolonged inflow occlusion at normothermic conditions can produce evidence of reversible or irreversible hepatocellular damage. Cytoprotective agents have been used both experimentally and clinically to afford extended viability of hepatocytes under reduced perfusion. One agent, prostaglandin E1, has been described clinically as effective in sustaining liver function under ischemic conditions. We have sought to verify this observation in an experimental model using prolonged normothermic inflow occlusion. Twenty miniature pigs were anesthetized and subjected to subtotal normothermic hepatic inflow occlusion (portal vein, hepatic artery, choledochal vessels) to allow for sufficient splanchnic decompression. Half of the animals received pretreatment with prostaglandin E1 (alprostadil) 500 μg intravenously. Inflow occlusion was maintained for 2 hours followed by reperfusion and killing 24 hours later. As a measure of functional preservation, the tissue adenine nucleotides adenosine monophosphate, diphosphate, and triphosphate (AMP, ADP, ATP) were measured in ischemic liver by freeze-clamping and high-performance liquid chromatography during occlusion and after reperfusion. Cytosolic enzyme determinations (aspartate transaminase, alanine transaminase, lactate dehydrogenase) were also made before occlusion and after reperfusion. As a possible indicator of cellular injury, blood ionized Ca++ was measured before inflow occlusion and after reperfusion. Although no difference was found in levels of AMP and ADP between prostaglandin E1 and control animals, ATP levels rose significantly higher during recovery in prostaglandin E1 animals at 60 minutes and 24 hours after reperfusion (13.97 ± 1.29 and 13.60 ± 0.91 μmoles/gm dry weight prostaglandin E1 vs. 9.25 ± 0.97 and 9.80 ± 0.85 μmoles/g dry weight control, P < .01). However, energy charge (ATP + ½ ADP/ATP + ADP + AMP) showed no significant difference between prostaglandin E1 and control groups at any time measured. There also was no significant difference in cytosolic enzymes or blood ionized Ca++ levels between prostaglandin E1 and control animals. We conclude pretreatment with prostaglandin E1 facilitates recovery of ATP on reperfusion after normothermic hepatic ischemia. The mechanism for this phenomenon remains unclear but does not seem to involve transcellular Ca++ flux. More rapid recovery of ATP may allow for continued viability of marginally damaged hepatocytes. (Hepatology 1995; 22:1554-1559).