bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatic lobar differences in progression of chronic liver disease: Correlation of asialoglycoprotein scintigraphy and hepatic functional reserve
Authors: Matsuzaki S , Onda M , Tajiri T , Kim D Y
Published: July 2004

We studied the hepatic functional reserve in the lobes of the liver in 28 patients with chronic liver disease and 13 controls using single photon emission computed tomography (SPECT) imaging with a radiolabeled asialoglycoprotein analog, Technetium-99m-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (Tc-99m GSA). Counts of Tc-99m GSA radioactivity in the liver on SPECT images significantly correlated (P < .0001) with the serum albumin level (r = .612), log (serum cholinesterase activity) (r = .618), serum bilirubin level (r = .628), prothrombin time (r = .715), hepaplastin test (r = .637), and indocyanine green retention rate at 15 minutes (r = .771), making it possible to estimate the distribution of functional reserve in the liver based on counts. Using the intact hepatocyte theory, we estimated the number of viable hepatocytes based on the counts. With progression of hepatic functional degeneration, counts per unit hepatic volume decreased (ρ = .779, P < .0001), and left lobe to right lobe ratio of this parameter increased (ρ = .491, P = .0019) significantly. These findings suggest that the reduction of hepatic functional reserve per unit hepatic volume and numerical density of the hepatocytes, and the proliferation of fibrosis in patients with chronic liver disease is slower in the left lobe than in the right. We discuss a possible biological basis for these apparent lobar differences and for hepatic morphological changes seen in cirrhosis.
Liver function, cerebral blood flow autoregulation, and hepatic encephalopathy in fulminant hepatic failure
Authors: Strauss G , Hansen B A , Kirkegaard P , Rasmussen A , Hjortrup A , Larsen F S
Published: July 2004

In acute liver failure, massive hepatic necrosis may result in impaired regulation of cerebral blood flow (CBF), development of encephalopathy, and cerebral edema. In 10 consecutive patients with fulminant hepatic failure (FHF), CBF autoregulation was found to be absent, as transcranial Doppler mean flow velocity (Vmean) in the middle cerebral artery increased from 49 (27-59) to 69 (49-92) cm/s (P < .05) during a 30- (28-34) mm Hg rise in mean arterial pressure (MAP). In 7 patients, restoration of CBF autoregulation was shown within 48 (24-120) hours after spontaneous hepatic recovery or liver transplantation, before complete alleviation of hepatic encephalopathy (HE). The extraordinarily rapid restoration of CBF autoregulation in patients with FHF following re-establishment of liver function is unique compared with other conditions affecting the CBF autoregulation, indicating a close connection between liver function and regulation of cerebral circulation. Because CBF autoregulation was restored after initial alleviation of HE, it does not appear to be of major pathophysiological importance in the mediation of HE.
Sinusoidal endothelium release of hydrogen peroxide enhances very late antigen-4-mediated melanoma cell adherence and tumor cytotoxicity during interleukin-1 promotion of hepatic melanoma metastasis in mice
Authors: Anasagasti M J , Alvarez A , Martin J J , Mendoza L , Vidal-Vanaclocha F
Published: July 2004

The hepatic sinusoidal endothelium (HSE) releases large amounts of reactive oxygen species (ROS) in response to endotoxins and interleukin-1 (IL-1). Such pro-inflammatory mediators have been shown to promote hepatic metastasis. We have investigated the involvement of ROS released by IL-1-stimulated HSE in this promoting effect. Recombinant human interleukin-1β (rHuIL-1β) (5 μg/kg) was intravenously injected into C57BL/6J mice, and the hepatic metastasizing ability of B16 melanoma cells following intrasplenic injection was studied in the presence of ROS scavengers. rHuIL-1β-promoted hepatic metastases were significantly (P < .01) reduced by catalase (1 mg/kg) and enhanced by recombinant human superoxide dismutase (rHuSOD) (5 mg/kg). rHuIL-1β-stimulated HSE-conditioned medium (HSE-CM) significantly (P < .01) enhanced B16 melanoma cell adhesion to HSE compared with unstimulated HSE-CM, which in turn also significantly (P < .01) increased with melanoma cell adherence compared with basal medium. The addition of catalase completely abrogated proadhesive effects induced by rHuIL-1β-stimulated HSE-CM with respect to unstimulated HSE-CM, but did not affect the proadhesive effects induced by unstimulated HSE-CM over basal medium. The rat monoclonal antibody to mouse vascular cell adhesion molecule-1 (VCAM-1) significantly (P < .01) inhibited the enhanced melanoma cell adherence effects of both unstimulated and rHuIL-1β-stimulated HSE-CM, indicating that adherence was very late antigen-4 (VLA-4)-mediated. Not surprisingly, the percentage of VLA-4 expressing B16 melanoma cells significantly (P < .05) increased in response to unstimulated (21% of controls) and rHuIL-1β-stimulated (32% of controls) HSE-CM. Catalase addition abrogated these effects of rHuIL-1β-stimulated-HSE-CM. Melanoma cell damage was observed from the second hour of adhesion to HSE and significantly (P < .01) increased when the cells adhered to rHuIL-1β-stimulated HSE. This increase was abrogated by catalase. Cytolysis of the HSE was not observed during melanoma cell adhesion. Neither was the enhancement of B16 melanoma hydrogen peroxide production observed in response to rHuIL-1β. Thus, the effects of IL-1 in the liver may consist of a balance between the prometastatic effect of enhanced adherence to the HSE and the antimetastatic effect of H2O2-mediated cytotoxicity. Our results suggest that the enhancement of H2O2 production by the rHuIL-1β-stimulated HSE may contribute to the hepatic metastasis progression of ROS-resistant melanoma cells. Results in vitro indicate that this progression is associated with a H2O2-mediated increase in melanoma cell adhesion to HSE.
Expression of urokinase-type plasminogen activator receptor in hepatocellular carcinoma
Authors: Morita Y , Hayashi Y , Wang Y , Kanamaru T , Suzuki S , Kawasaki K , Ohta K , Yamamoto M , Saitoh Y , Itoh H , Doe W F
Published: July 2004

It is well known that a urokinase-type plasminogen activator receptor (uPAR) is a key protein in the plasminogen activation system, which plays a proteolytically important role in the invasion and metastasis of various cancer cells. To assess the expression of uPAR in hepatocellular carcinoma (HCC), we analyzed the expression of uPAR messenger RNA (mRNA) and the protein in 31 pair-samples of solitary HCC and nontumorous liver tissues from the same patients. Fifteen samples exhibited no histological potential of recurrence, such as portal involvement or intrahepatic metastasis (group A), and 16 samples exhibited such histological features (group B). Seventy-one percent of the cases showed uPAR signals, and these signals were mainly localized at the cytoplasm of the tumor cells and tended to be at the front of invasive foci. 87.5% of the cases in group B showed uPAR signals against 53.3% of the cases in group A (P < .05). The rate of recurrence in the uPAR positive/negative cases in group A was 75.0% and 14.3%, respectively (P < .05). In non-neoplastic cases, e.g., chronic active hepatitis and cirrhosis, weak uPAR mRNA and protein signals were detected in hepatocytes neighboring the portal tracts, suggesting that this protein plays some role in such cases. The present study indicates that uPAR plays an important role at least in its initial stage in invasion and metastasis of HCC, and that uPAR expression can be a candidate predictor of these factors.
Expression of hepatocyte growth factor and its receptor c- met proto-oncogene in hepatocellular carcinoma
Authors: Ueki T , Fujimoto J , Suzuki T , Yamamoto H , Okamoto E
Published: July 2004

The c-met proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), a potent mitogen and motogen for epithelial cells. Because of its profound effects on cell growth and motility, HGF may be important in the development of cancer metastases in hepatocellular carcinoma (HCC). In this study, we examined HGF concentration and expression of the c-met-proto-oncogene product (c-met) in 62 patients with HCC to determine the relationship between the level of expression and clinicopathological features, and patient outcome following hepatectomy. Western blotting was used to examine the c-met expression, and HGF concentration in tumors was measured using an enzyme-linked immunosorbent assay. c-met was found to be overexpressed in HCC compared with nontumorous liver tissue (P < .01), and correlated with an increased incidence of intrahepatic metastases (P = .039). Patients were divided into two groups, low c-met HCC and high c-met HCC. Patients with high c-met HCC had a significantly shorter 5-year survival than patients with low c-met HCC (33.5% vs. 80.3%, respectively; P < .05). However, there was no correlation between HGF concentration in the tumor tissue and clinicopathological factors and patient survival. These results indicate that expression of c-met played an important role in tumor growth and metastases in patients who underwent hepatectomy for HCC.
Target gene modulation in hepatocellular carcinomas by decreased DNA-binding of p53 mutations
Authors: Kubicka S , Trautwein C , Niehof M , Manns M
Published: July 2004

The crystallographic structure of the p53 core domain showed that most of the p53 mutations found in human tumors are located in conserved regions of the p53 DNA-binding domain. The aim of our study was to investigate the effect on DNA-binding and transactivation of three p53 mutations frequently found in hepatocellular carcinomas (HCC). Two of these mutations are located near the DNA-binding surface and are induced by aflatoxin B1 (249ser) and oxiradicals (249met). In contrast, mutation 220cys is not associated with a specific carcinogen in HCCs and is located outside the DNA binding structures of p53. Cotransfection experiments in two HCC cell lines, with mutated or deleted P53 genes, showed that all three mutations did not enhance reporter gene activity (RGC-CAT), in contrast to wt p53. However, in hepatoma cell lines all three mutations did suppress the p53 wildtype (wt) transactivation in a dose-dependent fashion. DNA-binding was monitored by gel shift assays using the consensus-, Waf-, and RGC-p53 binding sites. All three p53 mutations did decrease DNA-binding versus all binding sites included. Interestingly although all mutations showed the same DNA-binding and transactivation properties, differences in the ectopic expression in different hepatoma cells were observed. Therefore our results indicate that p53 mutations in HCC found in the DNA-binding domain and outside the conserved DNA-binding structures modulate target gene expression by decreasing sequence specific DNA-binding in a dominant negative fashion. The cellular environment may contribute to an additional selection advantage of some mutations.
Kupffer cell depletion abolishes induction of interleukin-10 and permits sustained overexpression of tumor necrosis factor alpha messenger RNA in the regenerating rat liver
Authors: Rai R M , Loffreda S , Karp C L , Yang S , Lin H , Diehl A M
Published: July 2004

Tumor necrosis factor α (TNF), initiates a cytokine cascade that promotes hepatocyte proliferation after 70% partial hepatectomy (PH) but the mechanisms regulating TNF production after PH are unknown. We previously reported that gadolinium chloride (GdCl), an agent that depletes the liver of phagocytically active Kupffer cells, enhances hepatic expression of TNF messenger RNA (mRNA) and promotes liver regeneration after subsequent PH. This suggests that GdCl interferes with Kupffer cell mechanisms that normally constrain TNF production after PH. To evaluate this, the pre- and post-PH expression of TNF, TNF-inducible cytokines (interleukin [IL]-1, IL-6) and cytokines (transforming growth factor [TGF]β1 and IL-10) that down-regulate TNF were compared in controls and GdCl-treated rats. In controls, TNF, IL-1, IL-6, and IL-10 increase within 3 hours after PH, whereas TGF-β1 is induced much later (> 24 hours after PH). GdCl causes sustained overexpression of TNF mRNA and transient overexpression of circulating TNF protein after PH; both TNF-inducible cytokines are also relatively overexpressed. Cytokines that down-regulate TNF are effected differentially by GdCl. Regenerative induction of IL-10 is abolished but TGF-β1 induction is unaltered. Because IL-10 is known to shorten the half-life of TNF mRNA, these results suggest that Kupffer cell production of IL-10 is an important mechanism that down-regulates TNF production during liver regeneration.
Lack of specificity of albumin-mRNA-positive cells as a marker of circulating hepatoma cells
Authors: Muller C , Petermann D , Pfeffel F , Oesterreicher C , Fugger R
Published: July 2004

The aim of the study was to assess the specificity of albumin-messenger RNA (mRNA)-positive cells in peripheral blood as an indicator for circulating malignant hepatocytes. Albumin-mRNA-positive cells in the peripheral blood mononuclear cell (PMNC) fraction were detected by reverse-transcription polymerase chain reaction (RT-PCR). Albumin-mRNA-positive cells in PMNC were found in 12 of 19 (63%) patients with hepatocellular carcinoma, but also in 22 of 25 (88%) patients with chronic hepatitis without evidence for hepatoma, and in 18 of 19 (94%) of patients with acute viral hepatitis. In addition, 8 of 28 (28%) of healthy control individuals had also albumin-mRNA-positive cells in peripheral blood. PMNC known to be spontaneously negative for albumin-mRNA could be induced in vitro to transcribe albumin-mRNA after activation with a variety of substances such as interleukin-1 (IL-1) plus concanavalin A (Con A), IL-2, bacterial lipopolysaccharide, platelet derived growth factor, α-fibroblast growth factor, or hepatocyte growth factor. These results show that the majority of patients with acute and chronic liver disease without evidence for hepatocellular carcinoma has albumin-mRNA-positive cells in their PMNC fraction indicating the nonspecificity of that parameter for the presence of circulating malignant hepatocytes. In addition, in vitro experiments suggest that PMNC are capable of transcribing mRNA for albumin at a low level after activation. In vivo-activated PMNC are likely to be the source of positivity in healthy controls, patients with nonmalignant acute and chronic liver disease, and patients with hepatocellular carcinoma.
The role of insulin-like growth factor II in the malignant transformation of rat liver oval cells
Authors: Zhang N , Siegel K , Odenthal M , Becker R , Oesch F , Dienes H P , Schirmacher P , Steinberg P
Published: July 2004

Oval cells are small nonparenchymal epithelial cells that first appear in the periportal areas of the liver and thereafter invade the whole parenchyma when mice or rats are exposed to a variety of chemical carcinogens. In the present study we have analyzed the expression of insulin-like growth factor II (IGF II) in the recently established oval cell line OC/CDE 22 and its malignantly transformed counterpart (the M22 cells) and the biological consequences of the constitutive expression of IGF II in oval cells. OC/CDE 22 cells do not express the above-mentioned growth factor, whereas the M22 cells do and addition of a neutralizing anti-IGF II antibody to M22 cells resulted in an almost complete proliferation stop. The presence of type 1 as well as type 2 insulin-like growth factor receptors in OC/CDE 22 and M22 cells was revealed by Northern blotting; however, only neutralizing antibodies directed against the type 1 IGF receptor were able to inhibit the proliferation of the cultured oval cells. Finally, transfection of an IGF II complementary DNA (cDNA) into OC/CDE 22 cells resulted in the release of active IGF II into the extracellular medium but not in the concomitant malignant transformation of the cells. Taken together these results show that: (1) upon transformation oval cells start producing IGF II and (2) IGF II acts on oval cells as a pure mitogen (without being per se oncogenic) via an autocrine loop involving the activation of the type 1 IGF receptor.
Inherent increase of apoptosis in liver tumors: Implications for carcinogenesis and tumor regression
Authors: Grasl-Kraupp B , Ruttkay-Nedecky B , Mullauer L , Taper H , Huber W , Bursch W , Schulte-Hermann R
Published: July 2004

We quantitatively assessed rates of cell replication and of apoptosis during the development and regression of liver cancer. In rats, apoptotic activity gradually increased from normal liver to putative preneoplastic foci (PPF), to hepatocellular adenoma (HCA), and to hepatocellular carcinoma (HCC). At all stages, rates of cell replication were higher than of apoptosis, allowing a preferential net gain of (pre)neoplastic cells. As in rats, in human HCC, birth and death rates were increased manifold, indicating a species-independent phenomenon. Implications of the increasing cell turnover were studied in rats using the administration and withdrawal of nafenopin (NAF), a liver mitogen and nongenotoxic carcinogen. Prolonged NAF treatment enhanced cell number in normal liver by 25%, while PPF and liver tumors were amplified at least 100-fold. After stopping NAF treatment, cell replication ceased, while cell elimination by apoptosis was increased in normal and (pre)neoplastic liver. HCA and HCC showed the most pronounced shifts from replication toward apoptosis. As a result, 5 weeks after halting NAF, 20% of cells in normal liver, but about 85% of (pre)neoplastic lesions including HCC, were eliminated. The implications of these findings include that nongenotoxic carcinogens can act as survival factors even for malignant cells. Furthermore, tumor cells not only exhibit excessive proliferation, but also undergo apoptosis at rates that far exceed those in normal tissue. Therefore, inhibition of cell death by the survival activity of nongenotoxic carcinogens results in selective growth of (pre)neoplastic lesions. On the other hand, blockade of survival effects leads to excessive apoptosis in (pre)neoplasia and seems promising as a therapeutic concept for the selective elimination of (liver) cancer.
Roles of tyrosine kinases in the regulation of nitric oxide synthesis in murine liver cells: Modulation of NF-κB activity by tyrosine kinases
Authors: Lee B , Kang H , Pyun K , Choi I
Published: July 2004

Nitric oxide (NO) synthesis is upregulated during chronic hepatic inflammation. The present study characterized the mechanisms involved in the induction of NO production and inducible NO synthase (iNOS) messenger RNA (mRNA) expression in murine embryonic liver cell line, BNL CL.2 cells. No production by BNL CL.2 cells was induced by interferon-r (IFN-r) plus lipopolysaccharide (LPS). However, other inflammatory cytokines such as interleukin (IL)-β, tumor necrosis factor α (TNF-α), and IL-6 had no additional effects on it. The stimulatory effects of IFN-r and LPS were time- and dose-dependent. NO secretion was inhibited by treatment with inducible NOS inhibitors such as NG-monomethyl L-arginine, N
G-amino-L-arginine, and diphenylene iodonium. iNOS mRNA was induced 3 hours after IFN-r plus LPS treatment, and iNOS expression was maximal in the presence of IFN-r and LPS. The protein tyrosine kinase inhibitors such as genistein and tyrphostin reduced IFN-r plus LPS-induced iNOS mRNA expression and NO production. In contrast, the inhibitors of protein kinase C, protein kinase A, and protein phosphatases did not affect iNOS expression induced by IFN-r plus LPS. In addition, iNOS mRNA expression was completely blocked by treatment with tyrphostin. However, mRNA expression of an early response gene, JunB, and constitutively expressed genes β-actin and GAPDH were not inhibited by tyrphostin. Furthermore, tyrphostin inhibited the promoter activation of iNOS gene induced by IFN-γ plus LPS, and it also suppressed IFN-γ plus LPS-induced nuclear factor-κ B-binding activity but not AP-1-binding activity. These results suggest that NO production and iNOS mRNA expression in this cell line is dependent on protein tyrosine kinases but does not require protein kinase C, protein kinase A, or protein phosphatases.
Chronic enteral ethanol treatment causes hypoxia in rat liver tissue in vivo
Authors: Arteel G E , Iimuro Y , Yin M , Raleigh J A , Thurman R G
Published: July 2004

It is known that ethanol increases oxygen consumption in in vitro liver models, which could lead to hypoxia. Although it was shown recently that one large dose of ethanol caused hypoxia in rat liver in vivo, whether ethanol produces hypoxia in a clinically relevant chronic model remains unclear. In the present study, therefore, the effect of chronic ethanol on hypoxia was investigated in vivo using the 2-nitroimidazole hypoxia marker, pimonidazole. Male Wistar rats (300-325 g) were exposed to enteral ethanol continuously for 4 weeks. In this model, rats develop steatosis, inflammation, and necrosis characteristic of early stages of clinical alcoholic liver disease in humans. One hour before they were killed, rats were injected with pimonidazole (120 mg/kg intravenously), and livers were surgically isolated, removed, and fixed. Protein-bound pimonidazole adducts were identified on formalin-fixed, paraffin-embedded tissue with immunohistochemistry. Ethanol administration for 4 weeks significantly increased serum aspartate transaminase levels and hepatic pathology scores for steatosis, inflammation, and necrosis, as expected. Ethanol treatment significantly increased both the extent and number of cells that stained positive for pimonidazole compared with control animals given an enteral diet without ethanol. Quantitative image-analysis of pimonidazole binding showed that 4 weeks of ethanol administration nearly doubled the pimonidazole-positive area in tissue. Ethanol also increased pimonidazole binding significantly at 7 days, long before inflammation and necrosis could be detected. These results indicate that chronic ethanol causes hypoxia at the cellular level in rat liver in vivo and lend support to the hypothesis that hypoxia is involved in mechanisms of early alcoholic liver injury.
Bile acids with differing hydrophilic-hydrophobic properties do not influence cytokine production by human monocytes and murine Kupffer cells
Authors: Bergamini A , Dini L , Baiocchi L , Cappannoli L , Falasca L , Bolacchi F , Capozzi M , Faggioli E , Nistri A , Salanitro A , Ventura L , Rocchi G , Angelico M
Published: July 2004

Bile acids have been proposed to exert immunological effects of potential pathogenic or therapeutic relevance, yet the experimental evidence remains preliminary. We reexamined the effects of a variety of bile salts with differing hydrophilic-hydrophobic properties on the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF α) from monocytes and Kupffer cells. Monocytes from healthy human donors and Kupffer cells from 5-week-old mice were incubated for up to 18 hours with or without varying concentrations of bile salts and lipopolysaccharide (LPS). Monocyte viability was ≥ 95% with up to 250 μmol/L sodium ursodeoxycholate and ≤ 90% with 200 μmol/L chenodeoxycholate, decreasing sharply at higher concentrations. Kupffer cells were more vulnerable, particularly to chenodeoxycholate (viabilities of 25% and 0% at concentrations of 100 μmol/L and 200 μmol/L, respectively). In monocytes incubated in the presence of 20% fetal calf serum, neither ursodeoxycholate and chenodeoxycholate, nor a variety of other unconjugated and conjugated bile acids, tested up to their maximal noncytotoxic concentrations, influenced the IL-6 and TNF alpha production, at any level of LPS stimulation. Similar to monocytes, incubation of murine Kupffer cells with ursodeoxycholate and chenodeoxycholate did not influence cytokine release. In contrast, the addition of 10 nmol/L dexamethasone to monocytes significantly decreased TNF-alpha and IL-6 release (69 ± 11% and 48 ± 15%, respectively). When monocytes were incubated with 200 μmol/L chenodeoxycholate in the presence of lower concentrations of fetal calf serum (10% and 5%, respectively) a significant inhibition of cytokine release was observed, whereas incubation with ursodeoxycholate did not cause any effect. Flow cytometry using fluoresceinated LPS showed that chenodeoxycholate does not interact with the CD14 receptor, thus excluding the possibility of an interference with the LPS uptake by monocytes. Incubation with [14C]-chenodeoxycholate showed that the intracellular bile acid uptake was inversely related to the concentration of fetal calf serum, being negligible (< 3 fmol/cell) at the highest level. In conclusion, bile acids with widely different hydrophobicities are incapable of influencing the release of IL-6 and TNF alpha by monocytes and Kupffer cells, provided they are studied at noncytotoxic concentrations and in the presence of physiological amounts of proteins.
Protective effect of preconditioning on the injury associated to hepatic ischemia-reperfusion in the rat: Role of nitric oxide and adenosine
Authors: Peralta C , Hotter G , Closa D , Gelpí E , Bulbena O , Rosello-Catafau J
Published: July 2004

The effects of ischemic preconditioning on rat liver integrity, as well as the implication of nitric oxide (NO) and adenosine in this process, has been evaluated. Preconditioning before ischemia-reperfusion prevented the increases in alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) levels, but did not modify blood flow. Adenosine or NO administration previous to hepatic ischemia-reperfusion simulated the effect of preconditioning, whereas inhibition of adenosine or NO synthesis abolished the protective effect of hepatic preconditioning. Nevertheless, inhibition of adenosine and simultaneous administration of NO in preconditioned animals offered similar results to those found in the preconditioned group, indicating that, in the absence of adenosine, NO is able to maintain the preconditioning benefits. It is suggested that, in preconditioning, adenosine stimulates NO production to protect against the injury associated with ischemia-reperfusion.
Hepatoprotective effect of the endothelin receptor antagonist TAK-044 against ischemia-reperfusion injury in the canine liver
Authors: Kitayama Y , Yamanaka N , Kawamura E , Kuroda N , Okamoto E
Published: July 2004

The present study was designed to investigate if TAK-044, a novel endothelin (ET) ETA/ETB receptor antagonist, inhibits ischemia- reperfusion liver injury. The initial study showed the presence of both ETA and ETB receptors in canine hepatic membrane fractions using the specific binding assay of labeled ET-1 with ET isomers and TAK-044. The nonselective ETA/ETB receptor antagonist TAK-044 inhibited the specific binding of ET-1 to the receptors in a concentration-dependent manner. In subsequent studies using a canine 70% partial liver ischemic model (60 minutes), we found that an intravenous injection of TAK-044 (3 mg/kg) before ischemia significantly inhibited the release of serum liver enzymes (aspartate transaminase, alanine transaminase, mitochondrial glutamic oxaloacetic transaminase, and an increase of indocyanine green retention rate after reperfusion, compared with the control group. Elevation of the portal venous pressure was also suppressed significantly during the portal triad occlusion, and a rapid restoration of oxygen pressure in the liver tissue after reperfusion was observed in the TAK-044-treated group. Morphometric analysis revealed that the hepatocyte swelling and sinusoidal contraction 1 hour after reperfusion were significantly less severe in the treated group than in the control group. The sludging of erythrocytes in the sinusoidal lumens was also minimal in the treated group. In conclusion, the significant suppression of hepatic microcirculatory disturbance and tissue injury after ischemia-reperfusion were shown in the TAK-044-treated group. This finding indicates that the pretreatment of TAK-044 is useful as a hepatoprotective agent against ischemia-reperfusion injury, which is otherwise produced by a pathway involving ET-1.
Galactosamine-induced fulminant hepatic necrosis in unanesthetized canines
Authors: Diaz-Buxo J A , Blumenthal S , Hayes D , Gores P , Gordon B
Published: July 2004

A large animal model of fulminant hepatic necrosis is necessary to test the efficacy of artificial liver support systems. A recent model was developed using D-galactosamine in anesthetized dogs. Because of the difficulties encountered with prolonged anesthesia, a similar protocol was used in 10 unanesthetized dogs. Intravenous infusions of D-galactosamine (1.0 to 1.5 gm/kg) did not result in uniform death of all animals at 72 hours or development of hypoglycemia. Severe hepatic necrosis was observed in all animals, but residual hepatocyte viability was evident in some. All animals developed severe consumption coagulopathy with histological evidence of disseminated intravascular coagulation (DIC) in four. A clinical picture characteristic of endotoxic shock was observed in most animals as a terminal event. The presence of endotoxin was confirmed in all dogs tested after 12 hours (7/10). The differences observed between this model and the anesthetized model are probably because of the toxic synergism between halothane and D-galactosamine. Neither model seems satisfactory for the testing of artificial liver support systems. The halothane model requires extremely long periods of anesthesia and mechanical ventilation. The present model does not cause uniform or universal death of the animals within 3 days. Foremost, both models result in DIC and endotoxic shock, neither of which is likely to respond to bioartificial liver support or treatment with conventional dialysis membranes.
Ceramide induces hepatocyte cell death through disruption of mitochondrial function in the rat
Authors: Arora A S , Jones B J , Patel T C , Bronk S F , Gores G J
Published: July 2004

Although ceramide signaling pathways have been implicated in cell death, neither their role in hepatocellular death nor the cellular mechanisms mediating ceramide-induced cell death are known. The mitochondrial membrane permeability transition (MMPT) has been proposed as a common final pathway in cell death. Thus the aims of our study were to determine if ceramides cause hepatocellular death by necrosis and not apoptosis as confirmed by morphology and the absence of internucleosomal DNA cleavage. Ceramide-mediated hepatocyte necrosis was acyl chain-length, concentration, and time-dependent. Ceramides induced cell necrosis was associated with adenosine triphosphate (ATP) depletion and mitochondrial depolarization suggesting that ceramides caused mitochondrial dysfunction. In isolated mitochondria, ceramides induced the cyclosporine A-sensitive MMPT in an acyl chain-length and concentration dependent manner. Ceramide toxicity was specific as the less potent dihydro form did not induce cell necrosis, significant ATP depletion, mitochondrial depolarization nor the MMPT. In conclusion, ceramide induced cell death is acyl-chain length dependent and mediated by the MMPT. These data show for the first time that ceramide acts as a mediator of hepatocyte necrosis by causing mitochondrial failure.
Effects of growth hormone and insulin-like growth factor-I singly and in combination on in vivo capacity of urea synthesis, gene expression of urea cycle enzymes, and organ nitrogen contents in rats
Authors: Grøfte T , Wolthers T , Jensen S A , Møller N , Jørgensen J O , Tygstrup N , Ørskov H , Vilstrup H
Published: July 2004

Improvement of nitrogen balance is desirable in patients with acute or chronic illness. Both growth hormone (GH) and insulin-like growth factor-I (IGF-I) are promising anabolic agents, and their combined administration has been shown to reverse catabolism more efficiently than each of the peptides alone. This is believed to be mediated primarily through increased peripheral protein synthesis, whereas little attention has focused on a possible participation of amino acid metabolism in the liver. Four groups of rats were given: 1) placebo; 2) GH (200 μg/d); 3) IGF-I (300 μg/d); and 4) both GH and IGF-I. After 3 days, the maximum capacity of urea-nitrogen synthesis was determined by saturating infusion of alanine (n = 8 in each group), together with measurements of liver messenger RNA (mRNA) levels for urea cycle enzymes (n = 5 in each group) and N-contents of muscles, heart, and kidney. Basal plasma alpha-amino acid concentrations were similar in all groups. The capacity of urea-N synthesis [μmol/(min x 100 g body weight)] was reduced in a stepwise manner (placebo: 8.25 ± 1.2; GH treatment: 6.52 ± 0.8; IGF-I treatment: 5.5 ± 0.6; and GH/IGF-I: 4.22 ± 1.6 [P < .001 by ANOVA]), each step being lower than the former. Serum IGF-I increased stepwise from placebo (699 ± 40 to 1,579 ± 96 μg/L in the combined GH/IGF-I group), and was correlated negatively with the capacity of urea-nitrogen synthesis (P < .01). mRNA levels for urea cycle enzymes in the liver decreased after GH and IGF-I treatment, and the effect was more pronounced after the combined treatment in which the rate-limiting enzyme, argininosuccinate synthetase, was halved. Nitrogen contents of organs increased after both GH and IGF-I treatment, and even more so after the combination treatment, reaching an increase of 30% (P < .05). Data suggest that GH and IGF-I singly and, even more so in combination, additively inhibit urea synthesis. This is supposed to favor protein buildup in organs. We speculate that this inhibitory effect on the capacity of urea synthesis is caused by a decreased translation rate of the urea cycle enzymes caused by GH and IGF-I's down-regulatory effect on urea cycle enzyme gene transcription. The findings may indicate a novel mechanism of the protein anabolic action of GH and IGF-I.
Na+-dependent and -independent Cl−/HCO3 − exchange mediate cellular HCO3 − transport in cultured human intrahepatic bile duct cells
Authors: Strazzabosco M , Joplin R , Zsembery A , Wallace L , Spirli C , Fabris L , Granato A , Rossanese A , Poci C , Neuberger J M , Okolicsanyi L , Crepaldi G
Published: July 2004

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3
− and Cl−. Mechanisms responsible for transepithelial H+/HCO3
−secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, γ-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2′7′-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3
−, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na+-dependent and amiloride-inhibitable. No Na+-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3
−, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl-depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl−-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na+-dependent and Na+-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3
−exchange activity was not prevented by the Cl-channel inhibitor 5′-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na+-dependent Cl-/HCO3
−exchange) and an acid loader (Cl-/HCO3
−exchange), whereas no evidence was found for cAMP activated H+-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl−/HCO3
−exchange, whereas Na+:HCO3
− cotransport is not active in the physiological range of pHi. Stimulation of Na+-independent Cl−/HCO3
−exchanger by cAMP does not require activation of Cl−conductances. These mechanisms may underlay hormone-regulated biliary HCO3
−secretion in the human biliary tree.
Measurement of transferrin receptor kinetics in the baboon liver using dynamic positron emission tomography imaging and [18F]holo-transferrin
Authors: Aloj L , Carson R E , Lang L , Herscovitch P , Eckelman W C
Published: July 2004

We have evaluated the use of [18F]holo-transferrin ([18F]Tf) and positron emission tomography (PET) to measure in vivo Tf receptor expression and recycling using the baboon liver as a model. [18F]Tf was intravenously injected in three baboons and dynamic PET was performed over the region containing liver and spleen. In two of the three baboons, [18F]albumin ([18F]Alb), labeled with the same technique, was administered 3 hours later. Time activity curves (TACs) were obtained from liver and spleen for both tracers. TACs for [18F]Tf over the liver were fit to a pharmacokinetic model including vascular radioactivity and an extravascular tissue compartment corresponding to transferrin uptake and release. [18F]Alb data provided an independent estimate of plasma volume. Kinetic analysis showed the presence of a tissue compartment for [18F]Tf that rapidly reaches equilibrium (half time 7-10 minutes). In this organ, the measured rates for Tf turnover obtained with quantitative PET are similar to previously published data using cell culture systems. A model for [18F]Tf in the spleen was not statistically improved by adding a tissue compartment. These data and the pharmacokinetic modeling provide in vivo evidence of a high flux equilibrium binding compartment in the liver, consistent with Tf internalization and recycling.
Reversal of cyclosporine-inhibited low-density lipoprotein receptor activity in HepG2 cells by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors
Authors: Rayyes O A , Wallmark A , Florén C
Published: July 2004

Previously we have shown that cyclosporine inhibits low-density lipoprotein (LDL) catabolism in HepG2 cells. This inhibition mainly occurs through reduced LDL-receptor activity. 3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors up-regulate LDL receptor activity with a subsequent increase in LDL uptake and degradation. In this study, in HepG2 cells, we investigated the effects of HMG-CoA reductase inhibitors on cellular LDL catabolism in the presence of cyclosporine. Different concentrations of cyclosporine and HMG-CoA reductase inhibitors, which were within the range of therapeutic concentrations used in humans, were added to the culture medium and the cellular LDL receptor activity was then measured. The results show that HMG-CoA reductase inhibitors reverse the down-regulatory effect of cyclosporine on LDL receptor activity, thus further supporting our previous findings and also providing a rationale for the already established treatment in cyclosporine-induced hypercholesterolemia with HMG-CoA reductase inhibitors.
Biochemical and molecular identification of distinct forms of alkaline phosphodiesterase I expressed on the apical and basolateral plasma membrane surfaces of rat hepatocytes
Authors: Scott L J , Delautier D , Meerson N R , Trugnan G , Goding J W , Maurice M
Published: July 2004

We have identified B10, a plasma membrane protein previously defined by a monoclonal antibody, as an alkaline phosphodiesterase I (APDE) expressed in the plasma membrane of rat hepatocytes and enterocytes, with a restricted apical distribution. B10 complementary DNA (cDNA) was cloned from a rat intestinal library screened with a polyclonal antibody directed to the hepatic protein. Two distinct B10 clones with an open reading frame of 2,625 bp were obtained that differed only by 12 bases in the coding region. One B10 clone had a single base difference with gp130RB13-6 cDNA, which was recently cloned in rat fetal brain. B10/gp130RB13-6 had 50% identity at the amino acid level with the plasma cell antigen PC-1, an APDE cloned in the mouse and in human. Anti-B10 antibodies immunoprecipitated 34% of the APDE activity in liver plasma membranes and over 95% of the APDE activity in intestinal cells. Most of the remaining activity in hepatocytes (44%) could be immunoprecipitated by antibodies directed to PC-1. APDE activity immunoprecipitated with anti-B10 antibodies was found in the apical rat liver plasma membrane fractions on a sucrose gradient whereas most of the remaining APDE activity was associated with the basolateral fractions, which contained PC-1. By immunofluorescence, B10 was localized to the apical surfaces of hepatocytes and enterocytes whereas PC-1 was present on the basolateral surfaces of hepatocytes. B10/gp130RB13-6 and rat PC-1 are a unique example of distinct molecules having similar enzymatic activity but different apical/basolateral location, and possibly different functions.
Posttransplantation chronic hepatitis in fulminant hepatic failure
Authors: Mohamed R , Hubscher S G , Mirza D F , Gunson B K , Mutimer D J
Published: July 2004

Non-A, non-B or seronegative hepatitis is the leading indication for liver transplantation in patients with fulminant hepatic failure (FHF). We examined protocol annual review liver allograft biopsy specimens in consecutive adult patients transplanted for FHF in an attempt to determine the extent of the histological changes. One hundred eleven biopsy specimens from 41 patients transplanted for fulminant seronegative hepatitis and 34 from a comparison group of 16 patients transplanted for other causes of FHF (11 paracetamol overdose, 2 idiosyncratic drug reaction, 3 Wilson's disease) were available. Specimens were analyzed using standard proforma without knowledge of the original diagnosis. Chronic hepatitis was present in 29 patients (71%) transplanted for fulminant seronegative hepatitis (23 mild, 3 moderate, and 3 severe) compared with 5 patients (31%, all mild) transplanted for other causes of FHF. Twenty-five patients (61%) grafted for seronegative FHF had fibrosis (13 mild, 9 moderate, and 3 severe) in contrast to 4 fibrosis (25%) (all mild) in the comparison group. Excluding early allograft failure because of primary graft nonfunction or vascular complications, six patients with seronegative FHF required retransplantation (2 = chronic rejection; 1 = severe hepatitis with panacinar necrosis, resembling original liver; and 3 = chronic hepatitis with precirrhotic fibrosis and prominent cholestasis of unknown cause). One patient in the comparison group had a second graft (chronic rejection). Posttransplantation chronic hepatitis is more frequent and severe in patients transplanted for seronegative hepatitis. Graft survival may be adversely influenced by the development of chronic hepatitis, which may represent persistent or recurrent disease.
Persistent infiltration of CD45RC−CD4+ T cells, Th2-like effector cells, in prolonging hepatic allografts in rats pretreated with a donor-specific blood transfusion
Authors: Miyanari N , Yamaguchi Y , Matsuno K , Tominaga A , Goto M , Ichiguchi O , Mori K , Ogawa M
Published: July 2004

A single intravenous injection of freshly heparinized blood from a donor-specific blood transfusion (DST) seven days before transplantation significantly prolongs the subsequent survival of hepatic allografts from ACI(RT1a) to LEW(RT11) rats. We used W3/25 (anti-CD4) and OX22 (anti-CD45 RC: an isoform of leukocyte-common antigen [CD45R]) monoclonal antibodies to investigate the cellular identity of hepatic allograft infiltrates following transplantation. The number of CD4+ and CD45RC+ cells in untreated allografts increased equally over time by day seven. However, in DST-treated hepatic allografts, CD4+ and CD45RC+ cells also increased over time by day 14, but the increment in the number of CD4+ cells was significantly greater than that in CD45RC+ cells. While the number of CD4+ cells remained persistently elevated in the hepatic allografts of rats pretreated with DST, they did not initiate rejection. Fluorescence-activated cell sorter (FACS) analysis revealed that the accumulated CD4+ T cells could be divided into two subsets, CD45RC−CD4+ and CD45RC+ CD4+ T cells, and that the ratio of CD45RC−CD4+/CD45RC+ CD4+ T cells in the hepatic allografts of recipients pretreated with DST was significantly greater than that in untreated allografts. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated that CD45RC-CD4+ T cells expressed interleukin (IL)-4 and IL-10 messenger RNA (mRNA), but not IL-2 and interferon gamma (IFN-γ). The pattern of messenger RNA expression in hepatic allograft infiltrates from animals pretreated with DST provides compelling evidence for the selective in vivo preservation of T-helper (Th2)-specific cytokines in the rat system. Our studies show that CD45RC leukocyte-common antigen expression can define different populations of hepatic infiltrating CD4+ T cells. A persistent infiltration of CD45RC−CD4+ T cells, Th2-like effector cells, is characteristic of hepatic allografts with a prolonged survival in DST-pretreated rats.
Transgenic expression of hepatitis C virus structural proteins in the mouse
Authors: Kawamura T , Furusaka A , Koziel M J , Chung R T , Wang T C , Schmidt E V , Liang T J
Published: July 2004

Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the role of viral cytopathic effects remains unclear. To study the biosynthesis of HCV structural proteins and their pathogenic role, we constructed transgenic mice, expressing type 1b HCV structural proteins (core, E1, and E2) in liver tissues. Two liver-specific promoters were used. The mouse major urinary protein (MUP) promoter has been shown to be developmentally regulated with little or no expression in utero but high-level expression after birth. The albumin (Alb) promoter provides constitutive, high levels of transgenes in live. Expression of both HCV transgenes was detected in several lines by Northern blots, HCV-specific reverse transcriptase-polymerase chain reactions (RT-PCR), and Western immunoblotting. Alb HCV lines showed higher levels of HCV expression than the MUP HCV lines. Immunohistochemical analysis revealed a predominantly cytoplasmic presence of core protein with occasional nuclear staining, and both cytoplasmic and membrane expression of the E2 protein in the transgenic livers. In both transgenes, the highest levels of both antigens were seen in perivenular hepatocytes, suggesting potential processing specificity in those cells. At six months of age, the livers of all transgenic lineages remained histologically normal. We concluded that HCV structural proteins are not directly cytopathic in this animal model.