bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired responsiveness to angiotensin II in experimental cirrhosis: Role of nitric oxide
Authors: Castro Anna , Jiménez Wladimiro , Clária Joan , Ros Josefa , Martínez Josep Maria , Bosch Marta , Arroyo Vicente , Piulats Jaume , River Francisca , Rodés Joan
Published: March 2007

Impaired vascular responsiveness to angiotensin II is a common feature in human cirrhosis with ascites. The aim of this study was to investigate whether vascular reactivity to angiotensin II is also decreased in rats with carbon tetrachloride-induced cirrhosis and ascites and to assess the role of endogenous nitric oxide in this abnormality. Increasing doses of angiotensin II (from 31 to 500 ng · kg−1 · min−1) induced significantly smaller increases in total peripheral resistance in conscious cirrhotic rats with ascites (n = 8) than in control animals (n = 9) at each dose tested. A reduced response to angiotensin II was also observed in vitro in aortic rings of rats with cirrhosis and ascites compared with that in control aortic rings (maximal response: 104 ± 16 mg vs. 204 ± 18 mg; p < 0.001). This in vitro hyporesponsiveness to angiotensin II in aortic rings of cirrhotic rats with ascites was reversed on endothelium denudation or nitric oxide synthesis inhibition with Nω-nitro-L-arginine but was not influenced by cyclooxygenase inhibition with indomethacin. In conclusion, this study shows reduced vascular reactivity to angiotensin II in carbon tetrachloride-induced cirrhosis with ascites and indicates that this abnormality is mediated by nitric oxide. (Hepatology 1993;18:367-372).
Increased blood levels of methyl tert-butyl ether but not of ethyl propionate during instillation with contact gallstone dissolution agents in the pig
Authors: Esch Oliver , Schteingart Claudio D. , Pappert Dirk , Kirby Diane , Streich Rita , Hofman Alan F.
Published: March 2007

We performed experiments in anesthetized piglets with two cholesterol gallstone solvents, methyl tertbutyl ether and ethyl propionate, to determine whether blood levels of either solvent would increase during gallbladder instillation of these solvents under conditions simulating gallstone dissolution. The solvent was oscillated rapidly in and out of the gallbladder with a computer-controlled syringe pump; intraluminal pressure was set below the leakage pressure, and oscillating volume was set below the leakage volume to decrease loss of solvent into the intestine. Blood levels were measured with gas chromatography. Six piglets received methyl tert-butyl ether, and six piglets received ethyl propionate. During 2 hr of instillation with methyl tert-butyl ether, blood levels increased steadily to concentrations averaging 0.3 ml/L blood at 2 hr; during a 6-hr period of instillation, blood levels rose to above 0.4 ml/L blood. Replacement of methyl tert-butyl ether with saline solution in the gallbladder caused blood levels to decline gradually; plasma levels decreased by half in 90 min. In contrast, when ethyl propionate was infused for 2 or 6 hr, blood levels remained below the detection limit, probably because of high first-pass hepatic extraction. We conclude that, under conditions simulating those likely present in patients undergoing contact dissolution of gallbladder stones, the two solvents differ: Ethyl propionate is removed so rapidly from blood that its levels remain undetectable, whereas methyl tert-butyl ether levels in blood (and, presumably, peripheral tissues) increase continuously. If the absorption from the gallbladder and subsequent metabolism of ethyl propionate and methyl tert-butyl ether in human beings are similar to those of the piglet, side effects attributable to increased blood levels will be fewer if ethyl propionate is used than if methyl tert-butyl ether is used for contact dissolution of cholesterol gallstones. (Hepatology 1993;18:373-379).
Nitric oxide mediates lipopolysaccharide-induced alteration of mitochondrial function in cultured hepatocytes and isolated perfused liver
Authors: Kurose Iwao , Kato Shinzo , Ishii Hiromasa , Fukumura Dai , Miura Soichiro , Suematsu Makoto , Tsuchiy Masaharu
Published: March 2007

The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization-sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 νg/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 μg/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate-labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule. These results suggest that nitric oxide mediates the lipopolysaccharide-activated, Kupffer cell-induced inhibition of mitochondrial electron transport in hepatocytes. (Hepatology 1993;18:380-388).
Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation
Authors: Chesné Christophe , Guyomard Claire , Fautrel Alain , Poullain Marie-gwenaëlle , Frémond Benjamin , de Jong Hendrik , Guillouz André
Published: March 2007

Cryopreserved hepatocytes from various animal species and human beings were tested for their ability to survive and function in primary culture. The freeze/thaw protocol primarily designed for rat hepatocytes was used with slight modifications for the cells of all other species; it consisted of suspending parenchymal cells in the Leibovitz L15 medium containing 10% fetal calf serum and 10% to 16% dimethyl sulfoxide. After transient storage at 4° C cell suspensions were transferred to −20° C and then to −70° C before being plunged in liquid nitrogen. Hepatocytes were stored for a few weeks to 4 yr. Prolonged storage did not augment loss of cell viability and function. Cell viability after thawing was estimated by the trypan blue exclusion test, and attachment efficiency to plastic was estimated by measuring intracellular lactate dehydrogenase content. Similar values were obtained for most species tested; after cryopreservation cell viability and attachment were decreased by 10% to 25% and by 40% to 50%, respectively. A lower attachment rate was found with dog hepatocytes. Total cytochrome P-450 and protein synthesis were compared in fresh and cryopreserved cells from four species after 4, 24, 48 or 72 hr of culture. Similar values were found in both cells after 24 or 48 hr of culture. In addition, drug-metabolizing activities were measured in human hepatocytes from five donors. In most cases phenacetin deethylation activity was decreased whereas procainamide N-acetylation and paracetamol sulfoconjugation and glucuronidation were increased in cryopreserved cells. These results show that a simple and reproducible freeze/thaw protocol can be used to cryopreserve hepatocytes from various species including human beings and that after thawing a large fraction of the cells is still viable and capable of expressing various functions in vitro.
Effect of propylthiouracil on the ethanol-induced increase in liver oxygen consumption in awake rats
Authors: Carmichael Frederick J. , Orrego Hector , Saldivia Victor , Israel Yedy
Published: March 2007

It has been postulated that the beneficial effects of the antithyroid drug propylthiouracil in the treatment of alcoholic liver disease depend primarily on the action of propylthiouracil in suppressing the increase in hepatic oxygen consumption induced by ethanol. The evidence for this effect of propylthiouracil is derived from studies in which liver oxygen consumption has been determined in in vitro preparations. In our study the effects of ethanol and propylthiouracil on liver oxygen consumption were assessed in vivo in an unrestrained and unanesthetized rat model, where liver blood flow and hepatic vein and portal vein oxygen content can be measured. Data show that the liver oxygen consumption increased in rats treated with ethanol-containing liquid diets for 4 to 6 wk, both on withdrawal of alcohol (30%, p < 0.01), and after readministration of ethanol (50%, p < 0.01). Single-dose ethanol administration increased portal tributary blood flow without affecting hepatic arterial blood flow in both controls and rats withdrawn from long-term ethanol treatment. Long-term ethanol administration per se had no effect on portal tributary blood flow; however, hepatic arterial blood flow was increased by 38% (p < 0.01). Treatment with propylthiouracil for 5 days resulted in complete suppression of the increase in liver oxygen consumption induced by long-term ethanol administration. Propyithiouracil treatment also attenuated the increase in portal tributary blood flow after the administration of a single dose of ethanol. These determinations were made 24 hr after the last dose of propylthiouracil. In conclusion, use of a new in vivo model with unrestrained and unanesthetized rats has confirmed that long-term administration of ethanol increases liver oxygen consumption. For the first time, it has been shown in vivo that propylthiouracil abolishes the ethanol-induced increase in liver oxygen consumption that follows long-term ethanol administration. (Hepatology 1993;18:415-421).
Mechanism of ionomycin-induced intracellular alkalinization of rat hepatocytes
Authors: Anwer Sawkat M.
Published: March 2007

Calcium ionophores such as ionomycin and A23187 are often used to determine the role of intracellular Ca+ + in cellular processes. Ionomycin but not Ca+ +-mobilizing agonists increases basal intracellular pH in hepatocytes. To explain this difference in effects of agents that increase intracellular Ca+ + concentration, the mechanism of ionomycin-induced increases in basal intracellular pH in isolated rat hepatocytes was studied. Changes in intracellular pH and intracellular Ca+ + concentration were measured with the fluorescent probes BCECF (2′,7′-bis-2-[carboxyethyl ester]-5[6]carboxyfluorescein) and quin-2, respectively. Ionomycin produced dose-dependent increases in intracellular pH and intracellular Ca+ + concentration, with the increase in intracellular Ca+ + concentration preceded by the increase in intracellular pH. Ionomycin-induced increases in intracellular pH were not affected by I mmol/L amiloride, 100 μmol/L diisothiocyanostilbene disulfonate or removal of extracellular Na+, indicating that the effect is not mediated by Na+/H+ exchange, Cl−/HCO3
− exchange or Na+/HCO3
− cotransport. Ionomycin failed to increase intracellular pH or intracellular Ca+ + concentration in the absence of extracellular Ca+ +, and both intracellular pH and intracellular Ca+ + concentration increased promptly when extracellular Ca+ + was reintroduced. Ionomycin-induced increases in intracellular Ca+ + concentration but not intracellular pH were smaller in hepatocytes loaded with the Ca+ + buffering agent MAPTA. Thapsigargin increased intracellular Ca+ + concentration but failed to increase intracellular pH. Thus the effect of ionomycin is independent of the effect of ionomycin on intracellular Ca+ + concentration and dependent on extracellular intracellular Ca+ + concentration. Experimental conditions that produce cell depolarization did not increase basal intracellular pH but lowered ionomycininduced increases in intracellular pH by 25% without affecting increases in intracellular Ca+ + concentration. Taken together, these results indicate that the increase in basal intracellular pH may primarily be due to ionomycin-mediated electroneutral Ca+ +/2H+ exchange across the hepatocyte plasma membrane. Because the effect of ionomycin is not mediated by Na+/H+ exchange, the activity of this exchanger under basal conditions is not regulated by intracellular Ca+ +. These results also suggest that the pharmacological effects of ionomycin in hepatocytes are mediated by changes in intracellular pH in addition to or independent of changes in intracellular Ca+ + concentration. (Hepatology 1993;18:433-439).
Intercellular immune adhesion molecules in human liver transplants: Overview on expression patterns of leukocyte receptor and ligand molecules
Authors: Steinhoff Gustav , Behrend Matthias , Schrader Bettina , Pichlmayr Rudolf
Published: March 2007

Recently it has become clear that the inflammatory response of immune cells to target cells and extracellular matrix is regulated by several receptor-ligand molecules. Three main classes of molecules mediating intercellular adhesion and activation processes have been identified: the integrin, immunoglobulin and selectin families. This study surveys the expression of adhesion molecules on resident and infiltrating cells in human liver grafts. The patterns of cellular expression and inducibility in different pathological conditions of the graft are described. Our results show organ-specific regulation of the different adhesion molecules during alloreactive reactions and other types of inflammatory reactions. No rejection-specific patterns were detected on comparison with reperfusion damage or infectious transplant inflammation. Major differences were noted in the composition of the portal tract and sinusoid with regard to endothelial and parenchymal cell expression of cell-cell and cell-matrix adhesion molecules. Intravascular and interstitial differences in the expression patterns of leukocyte adhesion receptors support a concept of stepwise expression. The implications for the appearance of inflammatory reactions in human liver in immunosuppressive and therapeutic interventions are discussed. (Hepatology 1993;18:440-453).
Hepatitis B vaccine: Should we give it a shot?
Authors: Dickinson Chris J. , Olson Alan
Published: March 2007

Objective:

To determine clinical and economic consequences of alternative vaccination strategies for preventing hepatitis B virus infection (HBV).

Methods:

Decision analysis was used to evaluate costs, outcomes, and cost-effectiveness of three HBV management strategies (“no vaccination,” “universal vaccination,” and “screen and vaccinate”) in four populations (newborns, 10-year-old adolescents, a high-risk adult population, and the general adult U.S. population). Information on HBV incidence and prevalence, clinical course, and management of acute illness and chronic sequelae was obtained from the literature and a panel of experts. Actual payments (costs) were obtained from Blue Cross/Blue Shield and local pharmacies. Incremental cost-effectiveness was calculated from the perspective of the payer of medical care and subjected to sensitivity analysis.

Results:

Vaccination (with or without screening) prevents more disease at somewhat increased cost than no vaccination for the neonatal, adolescent, and adult populations. Vaccination (with or without screening) is a dominant strategy in adult high-risk populations (lower cost and greater benefit than no vaccination). Optimal cost-effectiveness, with nonmonetary benefits not discounted, results if all pregnant women are screened for active HBV infection, and HBV vaccine and hepatitis B immune globulin are administered to babies born to mothers with positive screening tests. Then HBV vaccine is administered to all children at age 10 and again 10 years later (incremental cost-per-year-of-life-saved relative to the “no vaccination” strategy is $375). A strategy of universal newborn vaccination alone leads to an incremental cost-per-year-of-life saved of $3332. If adolescents are vaccinated at age 10, incremental cost-per-year-of-life saved is $13,938; for the general adult population, the incremental cost-per-year-of-life saved of universal vaccination is $54,524. Discounting benefits will increase cost-per-year-of-life saved 7 to 12 times for all strategies.

Conclusions:

HBV vaccine is most cost-effective when a strategy of screening newborns is combined with routine administration to 10-year-old children. The means to achieve substantial improvements in the health of the public in a cost-effective fashion are now available and should be pursued aggressively.
Human leukocyte antigen class I-independent pathways may contribute to hepatitis B virus-induced liver disease after liver transplantation
Authors: Missale Gabriele , Brems John J. , Takiff Howard , Pockros Paul J. , Chisari Francis V.
Published: March 2007

The proliferative response of peripheral blood lymphocytes to the HBcAg was compared with serological, molecular and immunohistochemical parameters of hepatitis B virus infection and with biochemical and histological parameters of liver disease in a patient who received a completely human leukocyte antigen class I-mismatched liver allograft for fulminant hepatitis. The proliferative response increased progressively after transplantation, as hepatitis B virus infection became reestablished in the hepatic allograft. Strikingly, the HBcAg-specific T cells suddenly disappeared from the peripheral blood immediately before the acute onset of a severe necroinflammatory liver disease in which more than 80% of the hepatocytes expressed HBcAg. These observations are compatible with the hypothesis that human leukocyte antigen class I-independent hepatitis B virus-specific T cells might play a previously unsuspected role in the pathogenesis of hepatitis B virus-induced liver disease. (HEPATOLOGY 1993;18:491-496.)
B-cell epitopes on the hepatitis C virus nucleocapsid protein determined by human monospecific antibodies
Authors: Akatsuka Toshitaka , Donets Mickhail , Scaglione Luca , Ching Wei-Mei , Shih Wai-Kuo J. , Di Bisceglie Adrian M. , Feinstone Stephen M.
Published: March 2007

Four monospecific antibodies against the hepatitis C virus nucleocapsid protein, which was expressed by recombinant baculovirus, were obtained by Epstein-Barr virus transformation of B cells from three patients with chronic hepatitis C virus infection. One of these antibodies was IgG and the other three were IgM. Their specificities were characterized initially by enzyme-linked immunosorbent assay and immunoblotting against hepatitis C virus proteins expressed by six recombinant baculoviruses with different hepatitis C virus sequence insertions. These specificities were confirmed, and their epitopes were more precisely determined with a series of overlapping decapeptides made by solid-phase pin technology. Two antibodies (1F4 and 2G6) reacted with the same peptides located near the amino(N)-terminus of nucleocapsid protein (amino acids 33-50). The third antibody (3B5) recognized the peptide consisting of amino acids 133-142, and the fourth antibody (3B9) was mapped to the carboxy(C)-terminus and reacted with a peptide consisting of amino acids 165-174. This epitope has not previously been reported. Two antibodies, 1F4 and 3B9, which are specific to the N-terminus and C-terminus of nucleocapsid protein, respectively, have been stably produced for more than 6 mo and are being subcloned to establish monoclonality. These antibodies should be useful reagents for the study of hepatitis C virus. (HEPATOLOGY 1993;18:503-510.)
Secretion of soluble chemotactic factors, including interleukin-6: A mechanism for the recruitment of cd8-positive t lymphocytes to human liver allografts during rejection
Authors: Hathaway Mark , Burnett David , Elias Elwyn , Adams David H.
Published: March 2007

In 35 patients receiving first liver transplants we assessed the chemotactic responsiveness of peripheral blood lymphocytes to bile, the subset composition of the responding population and the ability of peripheral blood lymphocytes to produce chemotactic factors. In 13 patients in whom acute rejection developed, lymphocyte chemotaxis in vitro was significantly greater in bile sampled 1 or 2 days after transplantation and before episodes of acute rejection than in bile sampled when rejection was clinically apparent or after steroid therapy during stable graft function. The chemotactic factors present showed preferential activity for CD8-positive T cells. Bile sampled during the same post-transplant periods from 11 patients who did not exhibit rejection showed significantly less chemotactic activity. In vitro cultured peripheral blood lymphocytes from patients in whom rejection developed produced lymphocyte chemotactic factors that induced a similar pattern of chemotactic responsiveness with preferential activity for CD8-positive T cells. Separate culture of purified CD4-positive and CD8-positive T cells obtained from patients in whom rejection developed showed that CD4-positive cells produced the factor(s). Analysis of the subset responses of normal peripheral blood lymphocytes to a variety of chemotactic cytokines showed interleukin-6 to have specificity similar to that observed with chemotactic bile, mixed peripheral blood lymphocyte culture supernatants and supernatants of CD4-positive T cells. Chemotactic activity was reduced by 45% to 90% in five chemotactic bile samples and three peripheral blood lymphocyte culture supernatants by treatment with affinity purified interleukin-6 monoclonal antibody. These data suggest that recruitment of CD8-positive T lymphocytes in response to chemotactic factors secreted by CD4-positive cells may be important in human liver allograft rejection. (HEPATOLOGY 1993;18:511-518.)
Refractory ascites in cirrhosis: Roles of volume expansion and plasma atrial natriuretic factor level elevation
Authors: Wong Florence , Tobe Sheldon , Legault Louis , Logan Alexander G. , Skorecki Karl , Blendis Laurence M.
Published: March 2007

Cirrhotic patients with ascites refractory to diuretics also have blunted response to marked elevations of plasma atrial natriuretic factor levels alone or to moderate intravascular volume expansion by head-out water immersion. However, these patients usually undergo natriuresis after peritoneovenous shunting. To dissect the factors responsible for this response, we studied the effects on separate days of moderate intravascular volume expansion and highly elevated plasma atrial natriuretic factor levels (head-out water immersion and atrial natriuretic factor infusion) or marked volume expansion and moderate plasma atrial natriuretic factor level elevation (head-out water immersion and albumin infusion) in 13 alcoholic cirrhotic patients with massive ascites. Three of these patients, who responded to initial head-out water immersion with a negative sodium balance, served as controls. Unresponsiveness to head-out water immersion was confirmed in the remaining 10 patients on both days on the basis of blunted natriuretic response (urinary sodium excretion < 0.8 mmol/hr after 2 hr). In contrast, these 10 refractory patients were able to achieve negative sodium balance with both combinations. Mean urinary sodium excretion increased from a baseline level of 0.13 ± 0.10 mmol/hr to a peak level of 2.29 ± 0.61 mmol/hr after head-out water immersion and atrial natriuretic factor infusion and from 0.10 ± 0.3 mmol/hr to 1.61 ± 0.62 mmol/hr after head-out water immersion and albumin infusion. Both maneuvers were associated with suppression of plasma renin activity and serum aldosterone levels. With head-out water immersion and atrial natriuretic factor infusion, we noted a significant increase in 5′ cyclic GMP levels, a second messenger of atrial natriuretic factor, indicating possible activation of atrial natriuretic factor receptors at the inner medullary collecting ducts. In contrast, with head-out water immersion and albumin infusion no such increase in levels occurred, indicating that the increase in urinary sodium excretion was mainly due to increased delivery of sodium to the cortical distal nephron, as indicated by a disproportionate increase in urinary potassium excretion. In conclusion, massive (as opposed to moderate) volume expansion or greatly elevated levels of plasma atrial natriuretic factor associated with moderate volume expansion can improve blunted atrial natriuretic factor responsiveness in cirrhotic patients with refractory ascites. This appears to be achieved by way of a marked increase in distal delivery of filtrate in the kidney, with or without activation of distal atrial natriuretic factor receptors in the inner medullary collecting ducts. (HEPATOLOGY 1993;18:519-528.)
Visualization of the uptake and processing of oxidized low-density lipoproteins in human and rat liver
Authors: Esbach Sebastiaan , Pieters Moniek N. , Van Der Boom Johannes , Schouten Donald , Van Der Heyde Niels M. , Roholl Paul J. M. , Brouwer Adriaan , Van Berkel Theo J. C. , Knook Dick L.
Published: March 2007

The interaction of oxidized human low-density lipoproteins with human and rat liver was analyzed by light and electron microscopy. At the light microscopic level oxidized low-density lipoprotein was visualized by the fluorescent dye 1,1′ dioctadecyl 3,3,3′,3′ tetramethyl indocarbocyanine perchlorate, whereas at the electron microscopic level, an indirect immunolabeling procedure was used that detected the apoprotein B of the oxidized low-density lipoprotein. In rats, oxidized low-density lipoprotein was administered intravenously, and uptake by human liver was studied by perfusion of tissue blocks. Both in human and in rat liver, fluorescently labeled oxidized low-density lipoprotein was mainly found to become concentrated in Kupffer cells and, to a lesser extent, in endothelial cells. In both species the cell association of fluorescently labeled oxidized low-density lipoprotein could be inhibited by preadministration of polyinosinic acid, indicating a scavenger receptor-mediated process. At the electron microscopic level, oxidized low-density lipoprotein was found to bind mainly to areas of the plasma membrane of the Kupffer cells without clathrin coating, although binding to coated regions was also noticed. Internalization of the ligand occurred through coated vesicle formation and through membrane folding of interacting lamellipodia and wormlike structures. No indication for phagocytosis of aggregated oxidized low-density lipoprotein particles was noticed. After internalization, the immunoreactive oxidized low-density lipoprotein was detected in relatively electron-lucent endosomes and, subsequently, in lysosomes. Endothelial cells internalized oxidized lowdensity lipoprotein solely through coated pits, after which the particles were transferred through endosomes into lysosomes. The endosomes often contained tubular extensions, which were devoid of immunolabel. In human Kupffer and endothelial liver cells, essentially the same organelles were demonstrated to be involved in the internalization and processing of oxidized low-density lipoprotein as in the rat. Our morphological results confirm earlier biochemical data on the relative involvement of the various liver cell types in the uptake of oxidized low-density lipoprotein in rats, and the relevance of these data for the human situation is indicated. The uptake process, coupled to oxidized low-density lipoprotein recognition by Kupffer cells, as presently analyzed, indicates that both rat and human Kupffer cells are equipped with a similar removal system to protect the body against the occurrence of the atherogenic oxidized low-density lipoprotein particles in the blood. (HEPATOLOGY 1993;18:537-545.)
Establishment and immunological characterization of cultured human gallbladder epithelial cells
Authors: Auth Marcus K. H. , Keitzer Raymond A. , Scholz Martin , Blaheta Roman A. , Hottenrott Christoph E. , Herrmann Günther , Encke Albrecht , Markus Bernd H.
Published: March 2007

Biliary epithelial cells are a primary site of damage in liver allograft rejection and in immunologically mediated diseases such as primary biliary cirrhosis. Human leukocyte antigens and adhesion molecules on the biliary epithelium are associated with T-lymphocytic binding, recognition and destruction. To investigate relevant cellular immunological mechanisms under standard conditions, we have established anin vitromodel using human gallbladder epithelial cells. Although not directly affected in these aberrations, gallbladder epithelial cells are excellent objects for immunological investigations. More than 108highly purified cells were isolated and cultured longer than 6 wk in confluent monolayers. Cell growth was routinely established on uncoated plastic culture dishes, and serum-free media could be applied for immunological experiments. Cell characterization was performed by means of specific monoclonal antibodies typical for biliary epithelial cells. Cytokeratins 1 through 8, 18 and 19 and human epithelial cell antibody 125 always showed strong positive staining. Antigen patterns were examined before and after treatment with interferon-γ by use of immunohistochemical staining methods. Untreated human gallbladder epithelial cells expressed human leukocyte class I antigens but few or no class II antigens. After stimulation with interferon-γ induction of human leukocyte antigen-DR, -DP and -DQ was detected. In addition, intercellular adhesion molecule 1 was induced on these gallbladder epithelial cells. Therefore an immunological competence similar to that of biliary epithelial cells could be demonstrated.In vitrocell cultures of gallbladder epithelial cells offer a promising tool for subsequent investigations concerning intrahepatic biliary epithelial cells and their interactions with cells of the immune system. (HEPATOLOGY 1993;18:546-555.)
Plasma endopeptidase 24.11 (enkephalinase) activity is markedly increased in cholestatic liver disease
Authors: Swain Mark G. , Vergalla John , Jones Anthony E.
Published: March 2007

Endopeptidase 24.11 (enkephalinase), an enzyme known to be present in plasma and liver, is capable of metabolizing a substantial number of bioactive peptides. We measured plasma endopeptidase 24.11 activity in normal subjects and in patients with chronic hepatocellular disease or chronic cholestatic liver disease. The mean level of plasma endopeptidase 24.11 activity was 13 times higher in cholestatic patients than in controls or patients with hepatocellular disease (p < 0.01). Plasma endopeptidase 24.11 activity in patients correlated closely with traditional serum markers of cholestasis, including levels of alkaline phosphatase, γ-glutamyltranspeptidase and aminopeptidase (p < 0.01 for all). However, plasma endopeptidase 24.11 activity correlated poorly with serum ALT aminotransferase level (p = NS), indicating that it is not a marker of hepatocellular disease. The lack of overlap between plasma endopeptidase 24.11 activity in cholestatic patients and noncholestatic liver disease controls suggests that this enzyme activity is a useful biochemical marker of cholestasis. In addition, because of the broad spectrum of peptides metabolized by endopeptidase 24.11, increased plasma endopeptidase 24.11 activity may contribute to the pathophysiology of the syndrome of cholestasis. (HEPATOLOGY 1993;18:556-558.)
The humoral immune response to p53 in patients with hepatocellular carcinoma is specific for malignancy and independent of the α-fetoprotein status
Authors: Volkmann Martin , Müller Martina , Hofmann Walter J. , Meyer Martina , Hagelstein Jens , Räth Ulrich , Kommerell Burkhard , Zentgraf Hanswalter , Galle Peter R.
Published: March 2007

Recently, p53 gene aberrations have been recognized as a relevant factor in hepatocarcinogenesis, in tumors from both high-risk and low-risk areas. Because p53 gene mutations typically result in increased p53 levels in tumor cells, this cellular protein might become immunogenic during tumor development. To test this hypothesis, we have analyzed sera from 80 European patients with hepatocellular carcinoma for the presence of p53 antibodies. For this purpose we developed an immunoblot assay using recombinant p53 as antigen. Sixty-seven sera from patients with different acute and chronic liver diseases were used as controls. In addition, serum α-fetoprotein assays were performed. Circulating antibodies against p53 were found in 25% (20 of 80) of the sera from patients with hepatocellular carcinoma but not in various nonmalignant liver diseases The association of p53 antibodies with malignancy was highly significant (p < 0.00003). In 73.8% (59 of 80) of the hepatocellular carcinoma sera the α-fetoprotein levels were elevated. Among the 21 α-fetoprotein-negative hepatocellular carcinoma sera, 5 were found to contain p53 antibodies (23.8%). In conclusion, an antibody response against p53 developed in a significant proportion of patients with hepatocellular carcinoma but not in those with nonmalignant liver diseases. Serological testing for p53 antibodies gives the opportunity to identify a subgroup of patients with hepatocellular carcinoma not detected by conventional tests for serum α-fetoprotein. (HEPATOLOGY 1993;18:559-565.)
Mutation at codon 249 of p53 gene in a human hepatoblastoma
Authors: Kar Siddhartha , Jaffe Ron , Carr Brian I.
Published: March 2007

G-to-T transversion at codon 249 of the p53 gene has been shown to be specifically associated with human hepatocellular carcinomas, particularly that subset associated with exposure to the chemical hepatocarcinogen aflatoxin B1. We surveyed 47 North American adult hepatocellular carcinomas and three childhood liver tumors for codon 249 mutation. We report here a case of childhood hepatoblastoma in a patient, without known exposure to aflatoxin B1 or hepatitis B or C virus, whose tumor had a mutation at codon 249 involving G-to-T transversion. (HEPATOLOGY 1993;18:566-569.)
Increased plasma interleukin-8 concentrations in alcoholic hepatitis
Authors: Hill Daniel L. B. , Marsano Luis S. , McClain Craig J.
Published: March 2007

Patients with alcoholic hepatitis often have hepatic polymorphonuclear leukocyte infiltration and neutrophilia. Interleukin-8 is a cytokine that stimulates neutrophil chemotaxis and release of lysosomal enzymes. It is made by several types of cells, including fibroblasts, Kupffer cells and hepatocytes. In this study, serial plasma interleukin-8 concentrations were measured with enzyme-linked immunosorbent assay in 40 consecutive patients with moderate-to-severe alcoholic hepatitis over a 6-mo period. Two control groups included 10 patients without clinically important liver disease admitted for treatment of alcohol dependence and 12 healthy male volunteers. The mean plasma interleukin-8 level on admission was markedly increased: 695 ± 146 pg/ml in the alcoholic hepatitis patients. The alcohol-dependent control group and the normal volunteer controls had mean interleukin-8 concentrations of 106 ± 28 pg/ml and 10 ± 5 pg/ml, respectively. Initially increased interleukin-8 levels in alcoholic hepatitis patients decreased to a meals of 182 ± 42 pg/ml over the first month; levels had decreased further to 124 ± 79 pg/ml after 6 mo. Increased interleukin-8 concentrations in patients with alcoholic hepatitis suggest a role for interleukin-8 in the neutrophilia and hepatic polymorphonuclear leukocyte infiltration of alcoholic hepatitis. (HEPATOLOGY 1993;18:576-580.)
Spatial and temporal patterns of gene expression for the proteoglycans biglycan and decorin and for transforming growth factor-β1 revealed by in situ hybridization during experimentally induced liver fibrosis in the rat
Authors: Krull Nora B. , Zimmermann Thomas , Gressner Axel M.
Published: March 2007

Expression of the proteoglycans biglycan and decorin and of transforming growth factor-α1 at various stages of liver fibrosis induced experimentally in rats by oral administration of thioacetamide was examined. Using in situ hybridization combined with immunocytochemical staining for cell-type characteristic markers, we demonstrate spatial and temporal expression patterns specific for each of the genes. Biglycan gene expression levels coincided tightly with the activity and extent of fibrosis, fat-storing cells and their transformed form, the myofibroblast-like cells, being the major contributors. Decorin messenger RNA was detectable only after the transition to the chronic inflammatory stage in nonparenchymal cells of periportal fields and, transiently, in the forming septa. In the cirrhotic stage, expression was detected solely in periportal fields with enhanced bile duct proliferation. Transforming growth factor-β1 expression was undetectable in normal liver. During the subacute inflammatory stage, a hepatocyte subpopulation expressing low levels of transforming growth factor-β1 occurred at the limiting plate. With the progression of fibrosis, transforming growth factor-β1 expression levels increased considerably but remained restricted to the mesenchymal cells of the fibrotic septa. (HEPATOLOGY 1993;18:581-589.)
Effect of chronic iron overload on procollagen gene expression
Authors: Roberts Faye D. , Charalambous Paul , Fletcher Linda , Powell Lawrie W. , Halliday June W.
Published: March 2007

The pathogenesis of hepatic fibrosis and cirrhosis in genetic hemochromatosis may involve a direct effect of excess iron on collagen synthesis in the liver. To investigate this theory, we measured procollagen messenger RNA levels (types I, III and IV) in the livers of rats in which we produced chronic parenchymal iron overload by feeding them dietary carbonyl iron (2.5%, wt/wt) for up to 18 mo. This feeding resulted in predominantly parenchymal iron deposition in a periportal distribution similar to that seen in genetic hemochromatosis. Increased amounts of collagen fibrils were observed in iron-loaded livers on electron microscopy; all iron-loaded livers showed some periportal fibrosis. Although very high hepatic iron concentrations (range = 340 to 1,100 μmol/gm dry wt) were achieved in the carbonyl iron-loaded rats, we saw no consistent difference between steady-state messenger RNA levels for procollagens types I, III and IV in control and iron-loaded livers examined at five different time points up to 18 mo. Messenger RNA levels of the cytokine transforming growth factor-β1, which has been implicated as having a role in the production of extracellular matrix proteins, were also measured. No significant differences were observed between ironloaded and control livers. These results suggest that excess parenchymal iron does not have a direct effect on the expression of the procollagens or transforming growth factor-β1 genes in iron-loaded livers and that factors other than, or in addition to, iron are necessary for fibrosis to occur. (HEPATOLOGY 1993;18:590-595.)
Role of prostacyclin in hemodynamic alterations in conscious rats with extrahepatic or intrahepatic portal hypertension
Authors: Oberti Frederic , Sogni Philippe , Cailmail Stephane , Moreau Richard , Pipy Bernard , Lebrec Didier
Published: March 2007

Although prostaglandins are thought to be involved in the hyperdynamic circulation of portal hypertension, the role of this substance has not been elucidated. Dose-response curves, the hemodynamic effects of prostacyclin (20 μg/kg) and its inhibitor indomethacin and measurements of plasma and urinary levels of 6-keto-prostaglandin F1αwere compared in three groups of six rats each: normal, with portal vein stenosis and with secondary biliary cirrhosis. Plasma and urinary levels of 6-keto-prostaglandin F1αwere higher in rats with portal vein stenosis and cirrhotic rats than in normal rats. Dose-response curves showed similar maximal decreases in arterial pressure in the three groups, whereas the maximal increase in portal pressure was less marked in cirrhotic rats than in normal rats and rats with portal vein stenosis. In normal rats, prostacyclin increased cardiac output by 21% and portal pressure by 41%. Similar increases were observed in rats with portal vein stenosis. In contrast, prostacyclin did not affect cardiac output and portal pressure in cirrhotic rats. Indomethacin induced a more marked vasoconstrictive effect in normal rats than in cirrhotic rats. This study shows that prostacyclin plays a role in the hemodynamic alterations in portal hypertension. Moreover, the hyporeactivity observed in cirrhotic rats suggests that prostacyclin plays a major role in the circulatory changes of portal hypertension due to chronic liver disease. (HEPATOLOGY 1993;18:621-627)
Involvement of nitric oxide and prostaglandins in gastric mucosal hyperemia of portal-hypertensive anesthetized rats
Authors: Casadevall María , Panés Julián , Piqué Josep M. , Marroni Norma , Bosch Jaume , Whittle Brendan J. R.
Published: March 2007

This study investigates the effects of inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME), the inhibition of prostaglandin synthesis with indomethacin and the combined effects on gastric mucosal hyperemia of ketamine-anesthetized rats with portal hypertension induced by partial portal vein ligation. The hydrogen gas-clearance technique was used for measurement of gastric mucosal blood flow. Blood pressure increased with L-NAME administration in a similar manner in portal-hypertensive and sham-operated rats. Low doses of L-NAME (1 and 3 mg/kg, intravenously) caused a significant and dosedependent reduction in gastric mucosal blood flow in portal-hypertensive rats but had no effect on shamoperated animals. With a higher dose of L-NAME (13 mg/kg, intravenously), a significant decrease in gastric mucosal blood flow was observed in both portal-hypertensive and sham-operated rats. Indomethacin pretreatment (5 mg/kg, subcutaneously) caused a significant decrease in basal gastric mucosal blood flow of portal-hypertensive rats but did not modify this parameter in sham-operated animals. In sham-operated rats pretreated with indomethacin, the lower dose of L-NAME (3 mg/kg) did not significantly modify basal gastric mucosal blood flow. Likewise, pretreatment with indomethacin in sham-operated rats did not augment the significant reduction in gastric mucosal blood flow produced by the higher dose of L-NAME. In portal-hypertensive rats the significant dose-dependent reduction in gastric mucosal blood flow induced by L-NAME (3 and 13 mg/kg) was not significantly altered by pretreatment with indomethacin. Portal pressure was higher in portal-hypertensive than in sham-operated rats, and no significant differences were observed in this parameter between portal-hypertensive animals treated with different doses of L-NAME. These results indicate that both nitric oxide and prostaglandins may be involved in the gastric mucosal hyperemia of portal-hypertensive rats. However, no synergistic interactions between these two endogenous vasodilators could be observed in this experimental model. (HEPATOLOGY 1993;18:628-634.)
Effect of ethanol on rat fetal hepatocytes: Studies on cell replication, lipid peroxidation and glutathione
Authors: Devi Bheemappa G. , Henderson George I. , Frosto Teri A. , Schenker Steven
Published: March 2007

Studies have shown that ethanol at moderate concentrations inhibits epidermal growth factor-dependent replication of fetal rat hepatocytes in culture. This may account for the growth/development impairment associated with fetal alcohol syndrome and decreased liver regeneration in alcoholic liver disease. In this study, we further define the mechanism(s) of the negative impact of ethanol on fetal rat hepatocytes and provide evidence that ethanol-induced injury to these cells is associated with membrane damage caused by lipid peroxidation, altered cell glutathione homeostasis and deranged mitochondrial structure and function. Exposure of fetal rat hepatocyte replication to ethanol (2 mg/ml) promptly resulted in blockade of replication, as indicated by a 40% reduction in DNA synthesis (p < 0.05). Assessment of cell injury on the basis of lactate dehydrogenase and ALT leakage indicated a statistically significant but not appreciable effect, whereas 51Cr leakage was more substantially increased (p < 0.05). Within 6 hr of ethanol exposure, superoxide radical levels increased more than twofold (p < 0.05). We noted a 56% increase in levels of diene conjugates, a 131% increase in malonaldehyde concentration and a 66% increase in fluorescent products of lipid peroxidation (all p < 0.05). Glutathione levels were decreased to 47% below control values (p < 0.05). Electron microscopic studies illustrated a slight disruption of mitochondrial structure (enlargement of mitochondria and dilation of cristae). This disruption was accompanied by mitochondrial swelling (increased permeability), altered mitochondrial membrane potential (a 16% decrease in rhodamine uptake), a 28% decrease in succinate dehydrogenase activity and a 30% decrease in cellular ATP level (p < 0.05). Pretreatment of fetal rat hepatocytes with 0.1 mmol/L N-acetylcysteine or S-adenosylmethionine for 24 hr prevented the ethanol-induced reduction of ATP and glutathione levels, essentially restored cell replication, ameliorated 51Cr leakage and decreased malonal-dehyde and diene conjugate levels to 41% to 65% and 25% above control values, respectively. Pretreatment with 0.1 mmol/L vitamin E fully normalized malonal-dehyde and diene conjugate levels and 51Cr leakage but failed to improve ATP levels or to increase significantly cell replication and glutathione levels. Concomitant administration of glutathione precursors with ethanol, rather than pretreatment, did not alter the impaired cell replication. Thus our data underscore the importance of cellular glutathione and ATP in preventing ethanol-induced decreases in fetal cell replication and suggest that alleviation of cellular lipid peroxidation alone is not sufficient to prevent this abnormality in fetal rat hepatocyte function. (HEPATOLOGY 1993;18:648-659.)