bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction between heat shock and interleukin 6 stimulation in the acute-phase response of human hepatoma (HepG2) cells
Authors: Karlsson Jens O. , Yarmush Martin L. , Toner Mehmet
Published: July 2004

Two characteristic elements of the acute-phase response are an altered pattern of circulating hepatic proteins and fever. Whereas a fever-induced heat shock response could affect expression of acute-phase proteins in the liver, the effects of a modest temperature increase on protein secretion in interleukin-6 (IL-6)-stimulated HepG2 cells were investigated. The response of HepG2 cells to IL-6 stimulation was significantly affected by heat treatment at 40°C. Albumin secretion rates, which were reduced by a factor of 2 in response to either heat shock or IL-6 stimulation alone, were down-regulated by a factor of 4 when IL-6 was administered simultaneously with a continuous 40°C heat shock. IL-6-induced fibrinogen up-regulation was significantly reduced by heat treatment (P < .01), and secretion rates were indistinguishable from control levels after 2 days (P > .10). Unexpectedly, heat shock at 40°C induced a fivefold up-regulation of haptoglobin production in the absence of IL-6. Simultaneous heat shock and IL-6 stimulation caused a synergistic enhancement of haptoglobin expression, with secretion rates increasing up to 30-fold compared with unstimulated control cells. For all three proteins, the interaction between temperature and IL-6 concentration was statistically significant (P < .001). Heat treatment resulted in significant alterations of both the kinetics and sensitivity of IL-6-induced protein synthesis, suggesting a major modification of the mechanism of acute-phase protein regulation at 40°C. In summary, the data show that heat shock can significantly modulate the pattern of acute-phase protein expression and that fever may be an important regulatory factor in the acute-phase response.
Efficacy of anti-intercellular adhesion molecule-1 immunotherapy on immune responses to allogeneic hepatocytes in mice
Authors: Bumgardner Ginny L. , Li Jiashun , Heininger Marie , Orosz Charles G.
Published: July 2004

Adhesion molecules appear to play important roles in vascularized organ allograft rejection, because antibodies directed against them are effective in prolonging survival of vascularized organ allografts in rodents. However, the efficacy of these agents for cellular allografts is unknown. The current studies were undertaken to determine the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on host immune responses to purified hepatocytes. Host mice (C3H, H-2k) grafted with hepatocytes in sponge matrix allografts (HC-SMA) received IgG isotype control, anti-ICAM-1, or anti-VCAM-1 monoclonal antibody (mAb) on days 0 through 9 after grafting. Twelve to 14 days later, host cells infiltrating the HC-SMA were assessed for the development of allospecific cytolytic T cells (allo-CTLs). Treatment with anti-ICAM-1 or anti-VCAM-1 mAb resulted in significantly decreased recruitment of host cells into HC-SMA (P < .035). However, only anti-ICAM-1 mAb resulted in abrogation of development of allo-CTLs in HC-SMA (P = .001). C3H (H-2k) hosts grafted with allogeneic hepatocytes from control C57BL/6 (H-2b) or ICAM-1 knockout [H-2b] mice elicited the development of allo-CTLs in HC-SMA (P = not significant). Furthermore, there was no difference in the development of allo-CTLs in HC-SMA of control hosts [C57BL/6, H-2b] compared with ICAM-1 knockout hosts (H-2b) (P = not significant). Treatment with anti-ICAM-1 mAb had no effect on the development of allo-CTLs in ICAM-1 knockout (H-2b) hosts bearing HC-SMA. The immunosuppressive effect of host treatment with anti-ICAM-1 mAb does not appear to be a consequence of simple blockage of donor hepatocyte or host immune cell expression of ICAM-1, but suggests a potential inhibitory effect on host immune cell activation or function, as well as an effect on recruitment of host cells to the allograft.
The hepatitis B virus X protein up-regulates tumor necrosis factor α gene expression in hepatocytes
Authors: Lara-Pezzi Enrique , Majano Pedro L. , Gómez-Gonzalo Marta , García-Monzón Carmelo , Moreno-Otero Ricardo , Levrero Massimo , López-Cabrera Manuel
Published: July 2004

Human hepatocytes infected by hepatitis B virus (HBV) produce the proinflammatory cytokine, tumor necrosis factor α (TNF-α). In this study, we explored the mechanism of induction of TNF-α synthesis by HBV. We found that the stable HBV-transfected hepatoma cell line, 2.2.15, expressed high-molecular-weight (HMW) TNF-α mRNAs, which were absent in the parent HepG2 cells. Treatment of 2.2.15 cells with interferon alfa (IFN-α) and/or interleukin-1β (IL-1β) reduced both viral gene transcription and TNF-α mRNA expression. Transient or stable transfection of hepatocyte-derived cell lines with HBV X protein (HBx) expression vectors induced the production of biologically active TNF-α. In these cells, the HBx-induced TNF-α was detected both as cell-associated and soluble forms. Luciferase gene-expression assays showed that the TNF-α gene promoter contained target sequences for HBx trans-activation within the proximal region of the promoter. These results indicate that the hepatocyte TNF-α synthesis induced by HBV is transcriptionally up-regulated by HBx. Thus, HBx may have a role in the induction of the intrahepatic inflammatory processes that take place during acute and chronic hepatitis B.
Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor κB independently of IκB degradation
Authors: Zwacka Ralf M. , Zhang Yulong , Zhou Weihong , Halldorson Jeff , Engelhardt John F.
Published: July 2004

For many inherited and acquired hepatic diseases, liver transplantation is the only possible therapeutic strategy. Ischemia/reperfusion (I/R) damage to donor tissue is thought to be one component that may play a role in the decline of posttransplant tissue function and ultimately rejection. The transcription factors, AP-1 and nuclear factor κB (NF-κB), play important roles in the acute cellular responses to tissue damage, as well as the inflammatory phase following I/R. We have found that the DNA binding activity of AP-1 was dramatically increased following warm ischemia at 1 to 3 hours postreperfusion. Induced DNA binding activity was composed of predominately c-Jun and JunD hetero- and homodimers as determined by electrophoretic mobility supershift assays. This increase in AP-1 activity occurred in the absence of significant changes in the steady-state protein levels of c-Jun and JunB. Maximal activation of Jun amino-terminal kinase (JNK) occurred within the 25 to 30 minutes postreperfusion, just before the peak in AP-1 DNA binding. These findings suggest that phosphorylation may play an important role in regulating AP-1 transcriptional complexes. Furthermore, JunD protein levels slightly increased at 3 hours postreperfusion, concordant with changes in AP-1 DNA binding activity. The activation of NF-κB at 1 hour postreperfusion was independent of proteolytic degradation of IκB-α or IκB-β. This activation of NF-κB DNA binding activity in the nucleus was preceded by an increase in tyrosine phosphorylation of IκB-α. These studies suggest that JNK, IκB tyrosine kinase, and JunD are potential targets for therapeutic intervention during liver I/R injury.
Vasopressin-induced disruption of actin cytoskeletal organization and canalicular function in isolated rat hepatocyte couplets: Possible involvement of protein kinase C
Authors: Roma Marcelo G. , Stone Vicki , Shaw Robert , Coleman Roger
Published: July 2004

The effect of vasopressin (VP) on canalicular function and hepatocellular morphology, with particular regard to actin cytoskeletal organization and the concomitant plasma membrane bleb formation, was studied in isolated rat hepatocyte couplets. VP induced the concentration-dependent formation of multiple plasma membrane blebs as well as simultaneous impairment in both canalicular vacuolar accumulation (cVA) and retention (cVR) of the fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), which evaluate couplet secretory function and tight-junction integrity, respectively. These effects were mimicked by the protein kinase C (PKC) activator, phorbol dibutyrate (PDB), but not by the protein kinase A (PKA) activator, dibutyryl-cAMP. VP-induced bleb formation and canalicular dysfunction were fully prevented by the protein kinase inhibitor, H-7, but not by the PKA inhibitor, KT5720, further suggesting a specific role of PKC. VP-induced alterations were also prevented by pretreatment with the Ca2+-buffering agent, BAPTA/AM, but not with the calmodulin-dependent protein kinase II antagonist, calmidazolium. Neither the Ca2+-activated neutral protease inhibitor, leupeptin, nor the antioxidants, α-tocopherol or deferoxamine, were able to prevent either VP-induced plasma membrane blebbing or canalicular dysfunction. The Ca2+-ionophore, A23187, mimicked the VP-induced alterations, but its harmful effects were completely prevented by H-7. Bleb formation induced by VP and PDB was accompanied by an extensive redistribution of filamentous actin from the pericanalicular area to the cell body, and this effect was fully prevented by H-7. These results suggest that VP-induced canalicular and cytoskeletal dysfunction is mediated by PKC and that classical (Ca2+-dependent) PKC appear to be involved because intracellular Ca2+ is required for VP to induce its harmful effects.
Nitric oxide inactivates rat hepatic methionine adenosyltransferase in vivo by S -nitrosylation
Authors: Ruiz Félix , Corrales Fernando J. , Miqueo Carmen , Mato José M.
Published: July 2004

We investigated the mechanism of nitric oxide (NO) action on hepatic methionine adenosyltransferase (MAT) activity using S-nitrosoglutathione (GSNO) as NO donor. Hepatic MAT plays an essential role in the metabolism of methionine, converting this amino acid into S-adenosylmethionine. Hepatic MAT exists in two oligomeric states: as a tetramer (MAT I) and as a dimer (MAT III) of the same subunit. This subunit contains 10 cysteine residues. In MAT I, S-nitrosylation of 1 thiol residue per subunit was associated with a marked inactivation of the enzyme (about 70%) that was reversed by glutathione (GSH). In MAT III, S-nitrosylation of 3 thiol residues per subunit led to a similar inactivation of the enzyme, which was also reversed by GSH. Incubation of isolated rat hepatocytes with S-nitrosoglutathione monoethyl ester (EGSNO), a NO donor permeable through the cellular membrane, induced a dose-dependent inactivation of MAT that was reversed by removing the NO donor from the cell suspension. MAT, purified from isolated rat hepatocytes, contained S-nitrosothiol groups and the addition of increasing concentrations of EGSNO to the hepatocyte suspension led to a progressive S-nitrosylation of the enzyme. Removal of the NO donor from the incubation media resulted in loss of most NO groups associated to the enzyme. Finally, induction in rats of the production of NO, by the administration of bacterial lipopolysaccharide (LPS), induced a fivefold increase in the S-nitrosylation of hepatic MAT, which led to a marked inactivation of the enzyme. Thus, the activity of liver MAT appears to be regulated in vivo by S-nitrosylation
Neither intestinal sequestration of bile acids nor common bile duct ligation modulate the expression and function of the rat ileal bile acid transporter
Authors: Arrese Marco , Trauner Michael , Sacchiero Robert J. , Crossman Michael W. , Shneider Benjamin L.
Published: July 2004

The regulatory responses of bile acid (BA) transport in the terminal ileum to perturbations in BA homeostasis are complex, and conflicting results have been reported by different investigators. These studies were designed to examine the response of this system to a reduction in ileal bile salt concentrations at both a functional and molecular level. Common bile duct ligation (BDL) or feeding of a novel bile acid-binding compound, GT31-104HB, for 7 days were used to reduce ileal apical membrane bile salt flux. Apical bile acid transport function was assessed by examining sodium-dependent uptake of [3H]-taurocholate (TC) into brush border membrane vesicles (BBMV). Expression of the apical sodium-dependent bile acid transporter (ASBT) and the ileal lipid-binding protein (ILBP) were assessed by Western blotting with quantitation using [125I]-labeled secondary antibody and a phosphorimager. Neither common BDL nor intestinal sequestration of BA led to a change in ileal bile acid transport function or the expression of the ASBT or the ILBP. These results indicate that a reduction in presentation of bile salts to the apical surface of the terminal ileum does not modulate the expression of the genes involved in their transport.
Gender-related differences in bile acid and sterol metabolism in outbred CD-1 mice fed low- and high-cholesterol diets
Authors: Turley Stephen D. , Schwarz Margrit , Spady David K. , Dietschy John M.
Published: July 2004

These studies were undertaken to determine whether in young adult outbred CD-1 mice there were any gender-related differences in basal bile acid metabolism that might be important in determining how males and females in this species responded to a dietary cholesterol challenge. When fed a plain cereal-based rodent diet without added cholesterol, 3-month-old females, compared with age-matched males, manifested a significantly larger bile acid pool (89.1 vs. 54.1 μmol/100 g body weight), a higher rate of fecal bile acid excretion (13.6 vs. 8.5 μmol/d/100 g body weight), a more efficient level of intestinal cholesterol absorption (41.1% vs. 25.3%), and a lower rate of hepatic sterol synthesis (338 vs. 847 nmol/h/g). Similar results were found in C57BL/6 and 129Sv inbred mice. In matching groups of CD-1 mice fed a diet containing 1% cholesterol for 21 days, hepatic cholesterol levels increased much more in the females (from 2.4 to 9.1 mg/g) than in the males (from 2.1 to 5.2 mg/g). This occurred even though the level of stimulation of cholesterol 7α-hydroxylase activity in the females (79%) exceeded that in the males (55%), as did the magnitude of the increase in fecal bile acid excretion (females: 262% vs. males: 218%). However, in both sexes, bile acid pool size expanded only modestly and by a comparable degree (females: 19% vs. males: 26%) so that in the cholesterol-fed groups, the pool remained substantially larger in the females than in the males (102.3 vs. 67.6 μmol/100 g body weight). Together, these data demonstrate that while male and female CD-1 mice do not differ qualitatively in the way cholesterol feeding changes their bile acid metabolism, the inherently larger bile acid pool in the female likely facilitates the delivery of significantly more dietary cholesterol to the liver than is the case in males, thereby resulting in higher steady-state hepatic cholesterol levels.
α3β1-integrin as a critical mediator of the hepatic differentiation response to the extracellular matrix
Authors: Lora José M. , Rowader Kathleen E. , Soares Lorena , Giancotti Filippo , Zaret Kenneth S.
Published: July 2004

The extracellular matrix (ECM) promotes the differentiation of many cell types, and ECM remodeling in the liver has been implicated in embryonic development, tissue injury, and oncogenesis. Integrins are heterodimeric ECM receptors that play critical roles in transducing the composition of the ECM in the cell environment. We previously showed that mouse H2.35 cells, a conditionally transformed, liver-derived cell line, assume a more differentiated hepatocyte morphology and enhanced liver-specific gene expression when the cells are cultured on gelatinous ECM substrata. Here we show that H2.35 cells express relatively high levels of α3β1-integrins, similar to that previously shown for immature hepatocytes, transformed hepatocytes, and biliary cells. However, the cell morphological responses that depend on α3β1-integrin have not been defined. We found that transfecting H2.35 cells with antisense RNA construct directed to α3-subunit messenger RNA perturbs the initial cell attachment to laminin and collagen, and strongly inhibits cell morphological, proliferative, and gene expression responses to a collagen gel substratum. In situhybridization to mouse embryo tissues demonstrates the presence of α3-subunit messenger RNAs in newly formed hepatocytes. We suggest that α3β1-integrins are important for immature and transformed hepatocytes to respond morphologically to the extracellular matrix
Hepatic iron overload in patients with chronic viral hepatitis: Role of HFE gene mutations
Authors: Piperno Alberto , Vergani Anna , Malosio Ida , Parma Laura , Fossati Laura , Ricci Alessandra , Bovo Giorgio , Boari Giuseppe , Mancia Giuseppe
Published: July 2004

Mild to moderate hepatic iron overload is frequent in patients with chronic viral hepatitis (CH). We evaluated the role of hemochromatosis (HFE) gene mutations and other acquired factors in the development of iron overload in these patients. We studied 110 patients with chronic B or C viral hepatitis (31 women, 79 men), including 20 with cirrhosis, and 139 controls. Hepatic iron was evaluated by semiquantitative analysis in all the patients, and hepatic iron concentration (HIC) was determined in 97 of them (26 women, 71 men). C282Y and H63D mutations were sought in all the subjects by a polymerase chain reaction-restriction assay. The frequency of HFE genotypes and alleles did not differ in patients and controls. No relation was detected between hepatic iron stores and HFE gene mutations in women. In men, all C282Y heterozygotes had iron overload, and the H63D mutation was significantly more frequent in patients with more marked hepatic siderosis than in those with mild or no siderosis (P = .0039) and in controls (P = .0008). Heavy alcohol intake and hepatic cirrhosis were also associated with increased hepatic iron stores in the men. In the 71 men in whom HIC was measured, multiple regression analysis showed that this variable was related independently only to alcohol intake and HFE gene mutations. We suggest that in patients with CH, iron accumulates in the liver as the result of an interplay between genetic and acquired factors, and that increased liver iron stores may influence progression toward liver fibrosis.
High titers of antibodies inhibiting the binding of envelope to human cells correlate with natural resolution of chronic hepatitis C
Authors: Ishii Koji , Rosa Domenico , Watanabe Yushiro , Katayama Tohru , Harada Hideharu , Wyatt Colby , Kiyosawa Kendo , Aizaki Hideki , Matsuura Yoshiharu , Houghton Michael , Abrignani Sergio , Miyamura Tatsuo
Published: July 2004

Most cases of hepatitis C virus (HCV) infection result in chronic disease; however, a very small fraction of patients naturally clear the virus and resolve chronic hepatitis. In an attempt to correlate immune response with chronic disease resolution, we compared the antibody response in patients with different outcomes of the infection. Antibody responses to HCV structural proteins were assessed in 34 patients originally diagnosed with acute hepatitis. Five cases resolved acute infection, 22 developed chronic hepatitis, and 7 naturally resolved chronic hepatitis C. To estimate HCV neutralizing antibodies we used the neutralization of binding (NOB) assay, which evaluates inhibition of the envelope-2 protein binding to human cells. Enzyme-linked immunosorbent assay was used for the quantitative assessment of serum antibodies. The presence of HCV RNA was ascertained by reverse transcription-polymerase chain reaction. In 6 of 7 patients naturally recovered from chronic hepatitis C, the emergence and the persistence (for more than 3 months) of high serum titers (>1/600) of NOB antibodies coincided with virus clearance and clinical resolution of hepatitis. NOB antibody activity was observed in only 2 of 5 patients recovered from acute hepatitis C. Chronic patients who did not show any resolution during the course of the study developed low or no NOB antibodies. Because of the correlation between prolonged high NOB titers and natural resolution of chronic hepatitis C, vaccination or passive immunization aimed at high titers of NOB antibodies may be valuable new therapeutic approaches for chronic hepatitis C.
Ultrastructural analysis of hepatitis B virus in HepG2-transfected cells with special emphasis on subviral filament morphogenesis
Authors: Roingeard Philippe , Sureau Camille
Published: July 2004

The intracellular accumulation of empty hepatitis B virus (HBV) particles of filamentous shape leads to a direct cytopathic effect in so-called ground-glass hepatocytes. The aim of this study was to investigate how these filaments can be structurally formed at the cellular level. By electron microscopy, we reexamined the HBV-producer HepG2T-14 cells, which have been described as producing a substantial amount of empty HBV filaments compared with the other forms of HBV particles. Examination of ultrathin sections of HepG2T14 cells revealed the presence of HBV virions and filaments at the periphery of extremely large intracellular cisternae, probably related to a pre-Golgi compartment. Very long filaments appeared to be formed by a tubular budding of a long portion of the cisterna membrane. This phenomenon may be identical to that observed in the hepatocytes of HBV chronic carriers, in which the inability of the infected cell to export long HBV filamentous particles through the cellular secretion pathway seems to be at the origin of a direct cytopathic effect.
Immunological Cross–Reactivity to Multiple Autoantigens in Patients With Liver Kidney Microsomal Type 1 Autoimmune Hepatitis
Authors: Choudhuri Kaushik , Gregorio Germana V. , Mieli-Vergani Giorgina , Vergani Diego
Published: July 2004

We describe two patients with liver kidney microsomal antibody type 1 (LKM1)–positive autoimmune hepatitis (AIH) with associated endocrinopathies. The first patient had insulin–dependent diabetes (IDDM), and the second patient had Addison's disease and hypoparathyroidism, and is also positive for islet cell antibodies, without overt diabetes. To account for the existence of multiple endocrinopathy in these patients, we investigated whether there is sequence similarity between the target of LKM1 antibodies, cytochrome P4502D6 (CYP2D6), and other human proteins, and if so, whether this structural similarity produces a detectable cross–reactive immune response. Our database search identified two proteins, carboxypeptidase H, an autoantigen in insulin–dependent diabetes, and 21–hydroxylase, the major autoantigen in Addison's disease, that share sequence similarity to the second major LKM1 epitope on CYP2D6. We tested the reactivity of sera from these patients to the homologous regions of the three autoantigens using an enzyme–linked immunosorbent assay (ELISA). The cut–off for positivity was established by testing sera from 22 healthy children. To determine the significance of reactivity to the peptide homologues of the three autoantigens, we investigated 16 additional patients with LKM1 AIH and 20 children with chronic hepatitis B virus infection as pathological controls. We found that reactivity to the second major epitope of CYP2D6 is significantly associated with reactivity to the homologous regions of carboxypeptidase H (CPH) and 21–hydroxylase (21–OHase) in patients with LKM1 AIH, and that this simultaneous recognition is cross–reactive. We suggest that a cross–reactive immune response between homologous autoantigens may contribute to the development of multiple endocrinopathies in LKM1 AIH.
Proposal of A New Nomenclature for Budd–Chiari Syndrome: Hepatic Vein Thrombosis Versus Thrombosis of the Inferior Vena Cava At Its Hepatic Portion
Authors: Okuda Kunio , Kage Masayoshi , Shrestha Santosh M.
Published: July 2004

Budd–Chiari syndrome (BCS) was initially defined as a symptomatic occlusion of the hepatic veins, but subsequent reports on various obliterative changes that occur in the hepatic portion of the inferior vena cava (IVC) and hepatic vein orifices have resulted in a broadened and ambiguous definition. Membranous obstruction of the inferior vena cava has been regarded by many as a congenital vascular malformation, but its relation to the classical BCS has remained obscure. With modern imaging and recent histological study of new cases, membranous obstruction of the IVC is now considered to be a sequela to thrombosis. How to classify various forms of occlusion and stenosis of the IVC and hepatic vein ostia is a major challenge. In this review, we emphasize that primary hepatic vein thrombosis (classical Budd–Chiari) and an obliterative disease predominantly affecting the hepatic portion of the IVC, both of which account for most patients with venous outflow block, are clinically quite different. In the West, the former is more common than the latter, which constitutes the vast majority of cases of outflow block in developing countries such as Nepal, South Africa, China, and India. The latter is frequently complicated by hepatocellular carcinoma (HCC), and primary hepatic vein thrombosis is not. The major cause of thrombosis is a hypercoagulable state in hepatic vein thrombosis, but more of the latter cases are idiopathic. The clinical presentation of the latter is milder, and onset is frequently inapparent, whereas the former is more severe, sometimes causing acute hepatic failure. Markedly enlarged subcutaneous veins over the body trunk characterize the latter. We propose that these two disorders be clinically distinguished with a suggested term “obliterative hepato–cavopathy” for the latter against classical BCS.
Reduction of Renal Cortical Blood Flow Assessed by Doppler in Cirrhotic Patients With Refractory Ascites
Authors: Rivolta Roberto , Maggi Alessandra , Cazzaniga Massimo , Castagnone Daniela , Panzeri Anna , Solenghi Daniela , Lorenzano Elettra , di Palo Filippo Quarto , Salerno Francesco
Published: July 2004

The usefulness in cirrhotic patients of hemodynamic measurements by Doppler ultrasonography (US) is still not defined. We investigated the relationships between Doppler measurements and the severity of ascites. Portal blood flow velocity and volume, and hepatic and renal arterial resistance indexes (RI) were measured in 57 cirrhotic patients (19 without ascites, 28 with responsive ascites, and 10 with refractory ascites) and 15 healthy controls. The renal arterial RI were obtained for the main renal artery, interlobar vessels, and cortical vessels. Cirrhotic patients had decreased portal blood flow and an increased congestion index (CI). Only the CI was correlated to the severity of ascites, showing that it is also a reliable measure of the severity of portal hypertension in patients with ascites. The hepatic and renal artery RI were increased in cirrhotic patients, and the two values were correlated (r = .68; P = .00001). The RI of renal interlobar and cortical vessels were higher in patients with refractory ascites than in patients without ascites (P < .02 and P < .009), and correlated with sodium excretion rate (r = -.45; P < .003), the renin–aldosterone system, and creatinine clearance (r = -.62; P < .0002). The RI decreased from the hilum of the kidney to the outer parenchyma in healthy subjects and patients with responsive ascites, but this difference disappeared in patients with refractory ascites. This indicates that the degree of renal vasoconstriction varies in different areas according to the severity of the ascites. Cortical vessels are involved mainly in patients with refractory ascites, suggesting that the intrarenal blood flow distribution in cirrhosis tends to preserve the cortical area and that severe cortical ischemia is a feature of refractory ascites.
Fibrosis Accelerates the Development of Enzyme–Altered Lesions in the Rat Liver
Authors: Sakaida Isao , Hironaka Koji , Uchida Koichi , Suzuki Chieko , Kayano Kozo , Okita Kiwamu
Published: July 2004

Injection of pig serum into rats twice a week for 8 weeks induced stellate cell activation resulting in liver fibrosis without parenchymal cell injury. Administration of a choline deficient L–amino acid defined (CDAA) diet for 6 weeks with or without pig serum pretreatment led to the development of preneoplastic lesions that were positive for the placental form of glutathione S–transferase (GSTP). Pig serum pretreatment induced more activated stellate cells in the livers of rats subsequently fed a CDAA diet for 6 weeks compared with rats fed the CDAA diet alone. Activated stellate cells were detected as α smooth muscle actin (αSMA)–positive cells and by the expression of αSMA messenger RNA. These cells caused severe fibrosis as assessed by the hepatic hydroxyproline content. Pre–existing fibrosis induced by the activation of stellate cells with pig serum pretreatment increased hepatic malondialdehyde (MDA) level in parallel with GSTP–positive lesions. These results indicate that pre–existing fibrosis with the activated stellate cells accelerates the development of preneoplastic lesions in a CDAA diet model.
Expression and Regulation of Leukotriene–Synthesis Enzymes in Rat Liver Cells
Authors: Shimada Kazuo , Navarro Javier , Goeger Douglas E. , Mustafa Shamimunisa B. , Weigel Paul H. , Weinman Steven A.
Published: July 2004

The liver plays a major role in metabolism and elimination of leukotrienes (LT). It produces cysteinyl leukotrienes (cLT), and cLT have been implicated in hepatocellular toxicity in several models of lipopolysaccharide (LPS)–associated liver injury. However, the liver cell types responsible for cLT production are poorly defined, and the expression of the LT–synthesis enzymes, 5–lipoxygenase (5–LO) and LTC4 synthase (LTC4–S), in liver cells has never been demonstrated. The aim of the present study was to examine the ability of rat liver cells to produce cLT by determining whether hepatocytes, Kupffer cells, and sinusoidal endothelial cells express mRNA and enzyme activities of the LT–synthesis enzymes and whether expression is altered by LPS. 5–LO mRNA was expressed in whole liver, and expression was enhanced by LPS. Cell fractionation studies demonstrated that expression was present in Kupffer cells and sinusoidal endothelial cells, but not in hepatocytes. LTC4–S mRNA was detected in whole liver, hepatocytes, and sinusoidal endothelial cells, but not in Kupffer cells. Semiquantitative reverse–transcriptase polymerase chain reaction (RT–PCR) showed that LPS increased LTC4–S expression in hepatocytes by a factor of 3 (n = 3; P < .03). LTC4–S enzyme activity in the microsomal fraction of hepatocytes was also increased from 0.52 ± 0.13 to 1.90 ± 0.66 nmol · mg protein-1 · 5 min-1 (n = 6; P < .015) after LPS treatment. These results indicate that hepatocytes do not possess the ability for de novo synthesis of cLT from arachidonic acid, but they may actively participate in cLT production by conjugation of LTA4 with glutathione to produce LTC4. LPS enhances LTC4–S expression in hepatocytes. This intrinsic cLT production may contribute to hepatocellular injury during inflammation.
Discontinuous Total Parenteral Nutrition Prevents Postischemic Mitochondrial Dysfunction in Rat Liver
Authors: Morikawa Nobuyuki , Suematsu Makoto , Kyokane Takanori , Goda Nobuhito , Kumamoto Yusuke , Okitsu Taro , Ishimura Yuzuru , Kitajima Masaki
Published: July 2004

Although discontinuous total parenteral nutrition (d–TPN) has recently been favored for clinical use over continuous total parenteral nutrition (c–TPN) to ameliorate liver dysfunction, mechanisms for the protection against postoperative liver dysfunction remain unknown. This study aimed to examine differences in mitochondrial function in d–TPN– and c–TPN-pretreated livers during ischemia–reperfusion. Rat livers pretreated with d–TPN or c–TPN were perfused with Krebs–Ringer buffer and were exposed to 25% low–flow hypoxia followed by reperfusion. Intrahepatic mitochondrial membrane potential (△Ψ) and cell viability were assessed by dual–color digital microfluorography using rhodamine 123 (Rh123) and propidium iodide (PI), respectively. In response to hypoxia, livers pretreated with c–TPN, d–TPN, and an ordinary chow diet exhibited a significant △Ψ reduction among the entire lobules. Upon reperfusion, the regional △Ψ values further decreased in the c–TPN liver, whereas those in the d–TPN-treated or chow–treated livers displayed a rapid recovery toward the control levels. The severity of cell injury did not differ among the groups, showing that the reperfusion–induced △Ψ drop in the c–TPN-pretreated liver is not a consequence of cell injury. Differences in the △Ψ drop among the groups appear to occur irrespective of those in the glycogen storage, because the livers undergoing d–TPN display a marked △Ψ recovery even when reperfused at the end of a fasted state. These results indicate that c–TPN, but not d–TPN, jeopardizes mitochondrial re–energization and suggest that a circadian pattern of the TPN serves as a potentially beneficial strategy to reduce the risk of postischemic mitochondrial dysfunction in the liver.
Macrophage–Induced Inhibition of Nitric Oxide Production in Primary Rat Hepatocyte Cultures Via Prostaglandin E2 Release
Authors: Griffon Bénédicte , Cillard Josiane , Chevanne Martine , Morel Isabelle , Cillard Pierre , Sergent Odile
Published: July 2004

Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN–γ) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages. In cocultured hepatocytes incubated with LPS and IFN–γ, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage–number-dependent. Macrophage–conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins. Ultrafiltration of macrophage–conditioned medium through a 500–d membrane to rule out higher–molecular–weight molecules, such as anti–inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophages did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by N
G–monomethyl–l–arginine (l–NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E2 (PGE2) release by macrophages was concomitantly observed. Moreover, inhibition of PGE2 production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE2 release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.
The Modified Dipeptide, Enalapril, An Angiotensin–Converting Enzyme Inhibitor, Is Transported by the Rat Liver Organic Anion Transport Protein
Authors: Pang Sandy K. , Wang Pi Jun , Chung Alfred Y. , Wolkoff Allan W.
Published: July 2004

Oatp1, the organic anion transport polypeptide, is an integral membrane protein cloned from rat liver that mediates the uptake of various organic anions such as bromosulfophthalein (BSP) and taurocholate (TCA). Recent studies by others revealed that the thrombin inhibitor, CRC 220, a modified dipeptide, was transported by oatp1. The present study was designed to examine whether another modified peptide, enalapril, an angiotensin–converting enzyme inhibitor, was also a substrate. Transport was studied with enalapril (1 to 800 μmol/L, with [3H]enalapril) in a HeLa cell line stably transfected with oatp1–cDNA under the regulation of a Zn2+–inducible promoter. Noninduced transfected cells (without zinc) that did not express oatp1 failed to take up enalapril. In contrast, cells expressing oatp1 transported enalapril, estrone sulfate (E1S), taurolithocholic acid sulfate (TLCAS), and the glutathione conjugate of BSP (BSPGSH). Uptake of enalapril by oatp1 at 37°C was substantially higher than that at 4°C. The rate at 37°C (uptake rates for induced – noninduced, transfected cells) was linear over 5 minutes and was concentration–dependent, characterized by a K
m of 214 ± 67 μmol/L and a V
max of 0.51 ± 0.15 nmol/min/mg protein. Enalapril uptake was inhibited competitively by BSP (at 1, 5, 10, and 50 μmol/L) and TCA (at 5, 25, and 100 μmol/L) with inhibition constants (K
i) of 2 and 32 μmol/L, respectively. The metabolite enalaprilat was, however, not transported by oatp1. That oatp1 is not a general transporter of anionic compounds was further shown by the lack of transport of harmol sulfate, benzoate, and hippurate. These observations attest to the role of oatp1 as a specific transporter for at least two classes of pharmacologically important peptides.
Copper Resistant Human Hepatoblastoma Mutant Cell Lines Without Metallothionein Induction Overexpress Atp7b
Authors: Schilsky Michael L. , Stockert Richard J. , Kesner Amanda , Gorla Giridhar R. , Gagliardi Gregg S. , Terada Kunihiko , Miura Naoyuki , Czaja Mark J.
Published: July 2004

Mutant human hepatoblastoma cell lines resistant to copper toxicity were isolated from mutagenized HuH7. Two copper resistant cell lines (CuR), CuR 23 and CuR 27, had reduced basal expression of metallothionein (MT) messenger RNA (mRNA) and exhibited minimal or no increase in resistance to cadmium or zinc toxicity. Copper uptake, efflux of newly transported copper, glutathione content, and efflux rate were comparable with HuH7, whereas holoceruloplasmin synthesis and secretion were slightly decreased. Subcellular distribution of copper at steady–state showed an increase in organelle and membrane fractions with a reduction in cytosol. Expression of ATP7B mRNA was fivefold increased, and ATP7B protein approximately threefold increased in both CuR 23 and 27. Another cell line, CuR 41, showed increased basal expression of MT and ATP7B mRNA but not ATP7B protein, and resistance to cadmium and zinc toxicity. Copper uptake in CuR 41 was comparable with HuH7, but initial rates of efflux of copper and glutathione were reduced. The synthesis of holoceruloplasmin but not ceruloplasmin peptide was markedly diminished in CuR 41. Subcellular distribution of copper showed an increase in cytosolic and decreased organelle and membrane–associated copper. These data suggest that cellular resistance to copper toxicity was achieved in two independent cell lines without MT induction and that the induction of ATP7B may lead to the enhanced intracellular sequestration of copper by organelles.
Defective Nonoxidative Leucine Degradation and Endogenous Leucine Flux in Cirrhosis During An Amino Acid Infusion
Authors: McCullough Arthur J. , Mullen Kevin D. , Kalhan Satish C.
Published: July 2004

The metabolic fate of leucine's first and second carbon may be different depending on the tissue in which leucine is metabolized, as well as the prevailing hormonal milieu of that tissue. However, previous studies of leucine kinetics in humans have used only leucine labeled (as tracer) at the first carbon position. Because cirrhosis is associated with factors (such as insulin resistance and altered fuel substrate utilization) that may influence how leucine is degraded, the kinetics of leucine's first and second carbon using a simultaneous infusion of [1–14C] leucine and [2–13C] leucine were studied in the postabsorptive state and during an amino acid infusion in 6 stable cirrhotic patients and 6 matched controls. The data were normalized for different body compartments that were quantified from the dilution of H2 [180] and bromide. The body cell mass, but not body weight or fat–free body mass, was decreased in cirrhosis (P < .001). In response to the amino acid infusion, total leucine appearance from proteolysis and leucine's incorporation into protein increased significantly in both groups, but were higher in cirrhotic patients. Endogenous protein breakdown decreased in normals but remained unchanged in cirrhosis. These alterations in leucine metabolism became more prominent when data were expressed based on the body cell mass rather than on body weight. The oxidation of leucine's first carbon (C1) was decreased in cirrhosis, but the oxidation of leucine's second carbon (C2) did not differ between groups during both the postabsorptive period and the amino acid infusion, while nonoxidative leucine degradation [the difference between the oxidation of leucine's (C1) and (C2)] was also decreased in cirrhosis. In addition, there was a positive correlation between nonoxidative leucine degradation (which represents leucine incorporation into fat), and the respiratory quotient obtained from indirect calorimetry (r = .87; P < .001). These data suggest that the extent of leucine carbon oxidation is dependent on whether fat or carbohydrate is the prevailing fuel substrate. In addition, cirrhotic patients have decreased nonoxidative leucine degradation and are unable to suppress endogenous protein breakdown normally in response to amino acid administration. These abnormalities may contribute to the diminished fat stores and body cell mass commonly observed in cirrhosis.
Intrasplenic Transplantation of Allogeneic Hepatocytes Prolongs Survival in Anhepatic Rats
Authors: Arkadopoulos Nikolaos , Lilja Helene , Suh Kyung Suk , Demetriou Achilles A. , Rozga Jacek
Published: July 2004

To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single–stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor β1 (TGF–β1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 × 107 cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham–transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 ± 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF–β1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham–transplanted anhepatic controls (34.1 ± 8.5 vs. 15.5 ± 4.8 hrs, P < .01). Additionally, at 12 hours post–hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF–β1 levels when compared with sham–transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF–β1 levels in rats rendered anhepatic. (Hepatology 1998;28:1365–1370.)
Transport of Residual Endocytosed Products Into Terminal Lysosomes Occurs Slowly in Rat Liver Endothelial Cells
Authors: Hellevik Turid , Martinez Inigo , Olsen Randi , Toh Ban-Hock , Webster Paul , Smedsrød Bård
Published: July 2004

Receptor–mediated endocytosis of circulating collagen is a major physiological scavenger function of the liver endothelial cell and an important catabolic event in the complete turnover of this abundant connective tissue protein. In the present study, transport of collagen through the endocytic pathway was investigated in cultured liver endothelial cells. Collagen conjugated to fluorescein isothiocyanate, to allow detection of the ligand by fluorescence and immunoelectron microscopy, was found sequentially in three different organelles that compose the basic degradative endocytic pathway of eukaryotic cells: early endosomes, late endosomes, and terminal lysosomes. Early endosomes were identified as vesicles positive for early endosome antigen 1 (EEA1). Late endosomes were distinguished as structures positive for the late endosomal/lysosomal marker rat lysosomal membrane glycoprotein 120, but negative for EEA1 and lysosomally targeted BSA-gold. Lysosomes were defined by their content of BSA-gold, injected 24 hours before isolation of cells. Coated pits and coated vesicles mediated an extremely rapid internalization. Shortly after internalization and during the first 20 minutes, ligand was found in early endosomes. From 20 minutes on, ligand started to appear in late endosomes (23%), and by 2 hours the transfer was largely complete (82.5%). Only 2.5% of ligand was transferred to the lysosomes after 2 hours, and this number slowly increased to 21% and 53% after 6 and 16 hours, respectively. We conclude that 1) EEA1 is a useful marker for tracing early events of endocytosis in liver endothelial cells; 2) in contrast to the rapid internalization, transit of internalized ligand through early sorting endosomes generally takes from 20 minutes to 2 hours; and 3) exit from the late endosomes is very slow, requiring several hours.
Functional Analysis of Hbv Genomes From Patients With Fulminant Hepatitis
Authors: Sterneck Martina , Kalinina Tatjana , Günther Stephan , Fischer Lutz , Santantonio Teresa , Greten Heiner , Will Hans
Published: July 2004

Two previous case reports suggest that hepatitis B virus (HBV) core promoter variants with a high replication competence contribute to the pathogenesis of fulminant hepatitis B (FHB). We recently found in HBV genomes from patients with FHB an accumulation of mutations within the core promoter region. Therefore, the aim of this study was to investigate the phenotype of these HBV variants. Replication competence and expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) of viral genomes from seven patients with FHB and one patient with fulminant recurrent hepatitis after liver transplantation were analyzed by transfection experiments in human hepatoma cells. Compared with wild–type virus, the HBV variants from the seven patients with FHB produced similar or slightly lower levels of intracellular replicative intermediates and extracellular viral particles. In contrast, the HBV genomes from the patient with fulminant recurrent hepatitis synthesized and secreted significantly more HBV DNA. All genomes tested expressed similar or even higher levels of HBeAg compared with wild–type virus, except for those from four patients with a precore stop codon mutation in the respective dominant viral populations. The level of HBsAg produced by all variant genomes was similar or reduced compared with wild–type virus. These data indicate that in some cases HBV variants with enhanced replication competence and/or a defect in HBeAg expression may contribute to the development of FHB. However, neither phenotype is an essential prerequisite; thus, an additional role of other viral or host factors in the pathogenesis of FHB is suggested.