bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphoblastoid interferon alfa with or without steroid pretreatment in children with chronic hepatitis B: A multicenter controlled trial
Authors: Gregorio G V , Jara P , Hierro L , Diaz C , de la Vega A , Vegnente A , Iorio R , Bortolotti F , Crivellaro C , Zancan L , Daniels H , Portmann B , Mieli-Vergani G
Published: November 2004

The comparative efficacy of prednisolone followed by interferon alfa (IFN-α) versus IFN-α alone in enhancing the rate of antibody to hepatitis B e antigen (anti-HBe) seroconversion has not been evaluated in a large cohort of white children. To determine this, a multicenter-controlled trial was conducted in 95 hepatitis B virus (HBV)-DNA/hepatitis B e antigen (HBeAg)-positive children (median age, 9 years [range, 2-16 years]; 56 boys; 84 [89 %] white), all having inflammatory changes on liver biopsy. Patients were randomized to receive either prednisolone followed by IFN-α (n = 34); placebo followed by IFN-α (n = 30); or no treatment (n = 31). The prednisolone/placebo was given on a double-blind basis. Lymphoblastoid IFN-α was given at 5 MU/m(2) three times a week for 12 weeks. Baseline clinical, biochemical, and histological features were similar for the three groups. The majority (85 %) had a baseline aspartate aminotransferase (AST) level ≤ 100 IU/L. On follow-up between 12 and 18 months (median, 15 months) after treatment, the loss of HBeAg with anti-HBe seroconversion was more common in patients pretreated with steroids (12 of 34 [35 %]) or placebo [12 of 30 (40 %)] as against controls (4 of 31 [13 %], P< .05). Factors predictive of anti-HBe seroconversion were baseline HBV-DNA concentration of ≤ 1,000 pg/mL and a greater degree of portal tract inflammation on pretrial biopsy. Our results show that in white children treatment with IFN-α, at the dose and duration used in this study, improves the rate of anti-HBe seroconversion. Steroid priming does not potentiate the effect of IFN-α.
Comparison of glyceraldehyde-3-phosphate dehydrogenase and 28S-ribosomal RNA gene expression in human hepatocellular carcinoma
Authors: Gong Y , Cui L , Minuk G Y
Published: November 2004

The gene responsible for transcribing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is commonly used as a reporter gene to estimate the amount of RNA present in Northern analyses. However, recent data suggest that GAPDH gene expression may vary with the extent of cell proliferation and differentiation. 28S-ribosomal RNA (28S-rRNA) has also been employed to normalize Northern blots prepared with total RNA. In the present study, we compared the expression of GAPDH messenger RNA (mRNA) with 28S-rRNA by Northern blot analyses in human hepatocellular carcinoma tissues (HCC) and adjacent non-HCC tissues from eight patients with chronic viral hepatitis-induced cirrhosis and normal liver tissue from eight healthy control subjects. The results of the study revealed that GAPDH mRNA levels in HCC were significantly higher (14×-16×) than those in adjacent non-HCC and normal liver tissues. Conversely, 28S-rRNA levels did not vary among HCC, adjacent non-HCC, and normal liver tissues. We also demonstrated that the 28S-RNA signal was proportional to the amount of RNA loaded. These findings indicate that 28S-rRNA, rather than GAPDH mRNA, should be used as RNA loading controls for Northern blot analyses involving HCC and nontumor tissues. The findings also raise the possibility that GAPDH mRNA gene expression might serve as a diagnostic indicator for human HCC.
Interleukin 1 β markedly stimulates nitric oxide formation in the absence of other cytokines or lipopolysaccharide in primary cultured rat hepatocytes but not in Kupffer cells
Authors: Kitade H , Sakitani K , Inoue K , Masu Y , Kawada N , Hiramatsu Y , Kamiyama Y , Okumura T , Ito S
Published: November 2004

To investigate whether a single inflammatory cytokine could stimulate nitric oxide formation in the absence of other cytokines or lipopolysaccharide (LPS), NO was measured by the redox chemiluminescence method in primary cultured rat hepatocytes and in rat Kupffer cells. Interleukin (IL) 1 beta, but neither IL-6, tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), nor LPS stimulated NO formation in a dose-dependent manner and induced half-maximal effects at 30 pmol/L. Maximal stimulation was achieved at 12 to 16 hours after the addition of 1 nmol/L of IL-1 beta, and was 50- to 60-fold above basal levels in rat hepatocytes. The combined effect of these cytokines with LPS or IFN-gamma on NO formation was also examined. Neither LPS nor IFN-gamma affected the IL-1 beta-induced NO formation. TNF-alpha, however, stimulated IL-1 beta-induced NO formation, while IL-6 inhibited it, although independently these cytokines had no effect on NO formation. None of the cytokines tested stimulated NO formation in cultured rat Kupffer cells. In hepatocytes, the NO formation induced by IL-l beta was blocked by both the NO synthase (NOS) inhibitor NG-monomethyl-l-arginine (l-NMMA) and by IL-1 receptor antagonist (IL-1ra). Furthermore, IL-1 beta markedly increased NOS activity, and this increase in activity was accompanied by the expression of inducible NOS (iNOS) messenger RNA (mRNA). This study clearly demonstrated that IL-1 beta markedly stimulates NO formation in hepatocytes, in the absence of other cytokines or LPS.
Persistent sex differences in growth control of early rat liver lesions are programmed during promotion in the resistant hepatocyte model
Authors: Liao D , Porsch-Hallstrom I , Gustafsson J A , Blanck A
Published: November 2004

The effects of gonadal hormones on several parameters associated with sex-differentiated promotion in the resistant hepatocyte (RH) model were studied. Male and female rats were initiated with diethylnitrosamine and promoted with 2-acetylaminofluorene (2-AAF) and partial hepatectomy [correction of hepatecomy] (PH). Before promotion, some female rats were ovariectomized, with or without receiving subcutaneous testosterone implants. Rats were killed either at the time of cessation of 2-AAF treatment or 2 weeks later. Ovariectomy decreased the messenger RNA (mRNA) expression of the female-specific cytochrome P450 2C12 (CYP2C12) at the time of PH, but did not increase the male-specific CYP2C11. Testosterone treatment further decreased CYP2C12 and induced CYP2C11 to the level in male liver. Hepatic foci positive for the placental form of glutathione-S-transferase (GST-P) were larger in male than in female rats. Ovariectomy did not affect the size of foci, whereas testosterone treatment increased the size to the male level. At the time of cessation of 2-AAF treatment, the labeling index, determined as cells staining for proliferating cell nuclear antigen, was higher in foci of males and testosterone-treated females than in foci from females with or without ovariectomy, whereas the labeling index in the surrounding hepatocytes was lower in males and testosterone-treated females. Two weeks later, the sex differences in labeling index were still present in foci, but no differences were observed in the surrounding hepatocytes. An elevated c-myc expression was observed in nodules isolated 3 weeks after PH from males and testosterone-treated females, but not in nodules from intact females. In conclusion, ovarian hormones did not affect promotion in the RH-model, whereas testosterone administration to ovariectomized females masculinized growth hormone-regulated hepatic parameters and response to promotion. The persistent sex differentiation in focal proliferation and c-myc expression indicated that a hormone-regulated change in growth control was introduced during 2-AAF/PH treatment.
Hepatic mucosal mast cell hyperplasia in rats with secondary biliary cirrhosis
Authors: Rioux K P , Sharkey K A , Wallace J L , Swain M G
Published: November 2004

Mast cells have been shown to play a role in many chronic inflammatory and fibrotic disorders. However, their possible contribution to the pathological changes that occur in liver cirrhosis is unknown. To explore this, we examined whether changes in hepatic mast cell number and mediator content were associated with fibrotic changes in experimental biliary cirrhosis. Rats were studied 7, 14, or 21 days after bile duct resection (BDR). Hepatic mast cells were identified by histochemical and immunohistochemical stains. Rat mast cell protease II (RMCP-II), a marker of mast cell degranulation, was measured in liver by enzyme-linked immunosorbent assay. Hepatic collagen deposition was assessed by Sirius Red F3BA staining. In day 21 BDR rats, there was a one- to twofold increase (P < .001) in the number of hepatic mast cells, but this was not observed in day 7 or 14 BDR rats. Mild fibrotic changes were noted in BDR rat livers as early as 7 days after induction of cholestasis. Significant expansion and organization of fibrous tissue had occurred in day 14 BDR rats which progressed to bridging fibrosis by day 21. Liver RMCP-II levels were decreased by 50 percent (P < .05) and mast cell degranulation was apparent as shown by histamine immunostaining. These results suggest that hepatic mast cell hyperplasia and degranulation occur during prolonged cholestasis in the rat. Although these changes do not correlate with the onset of hepatic fibrosis, they do occur at a time during which there is significant deposition and organization extracellular matrix elements. Hepatic mast cells, by releasing profibrogenic mediators, may contribute to fibrotic changes in biliary cirrhosis.
Ultracentrifugation systematically overestimates vesicular cholesterol levels in bile
Authors: Yuet P K , Blankschtein D , Donovan J M
Published: November 2004

To accurately determine the cholesterol (Ch) distribution between mixed micelles and vesicles in lithogenic bile, both ultracentrifugation and gel chromatography with the correct intermixed micellar/vesicular bile salt concentration (IMC) have been proposed. We have systematically compared both separation techniques with physiological model biles to ascertain their quantitative separation ability. After determination of optimal ultra-centrifugation conditions in systems containing only micelles or vesicles, Ch-supersaturated model biles [3-10 g/dL, 10 mol percent Ch, taurocholate (TC)]/([TC + egg yolk phosphatidylcholine (EYPC)] = 0.6 and 0.7) were adjusted to a density of 1.03 g/mL, and ultracentrifuged at 42,000 rpm and 37 degrees C for 13 hours. Identical model biles were subjected to gel chromatography with the correct IMC, either directly or after remixing and incubation at 37 degrees C after ultracentrifugation. By ultracentrifugation, 31 percent +/- 2 percent (TC/(TC + EYPC) = 0.6) and 40 percent +/- 5 percent (TC/(TC + EYPC) = 0.7) of total Ch were found in vesicles (Ch/EYPC molar ratios = 1.0 and 1.3, respectively). However, by gel chromatography, only 19 percent +/- 2 percent (Ch/EYPC = 1.0) and 22 percent +/- 2 percent (Ch/EYPC = 1.5) of total Ch were found in the corresponding biles. Gel chromatography of biles (TC/(TC + EYPC) = 0.7) ultracentrifuged for various durations showed a progressive increase in vesicular Ch to 41 percent after 13 hours. On incubation for 11.5 hours after ultracentrifugation, vesicular Ch decreased to 31 percent, thus approaching the initial (gel chromatography) value. Quasielastic light scattering also demonstrated formation of vesicles in ultracentrifuged Ch-unsaturated model bile (cholesterol saturation index (CSI) approximately 0.97). As compared with gel chromatography, ultracentrifugation systematically elevates vesicular Ch, possibly because of induced shifts in lipids between lipid aggregates caused by variation in local bile salt concentration. Because ultracentrifugation can alter the phases present in bile, gel chromatography with the correct IMC more accurately represents the distribution of Ch in biliary lipid aggregates.
Non–Neoplastic Liver Disease Associated With Chronic Ground Squirrel Hepatitis Virus Infection
Authors: Cullen J M , Marion P L
Published: November 2004

We examined 95 ground squirrels to compare the histological appearance of liver sections from animals that were chronically infected with ground squirrel hepatitis virus (GSHV) (n = 29), uninfected (n = 42), or had recovered from infection (n = 24). We studied the effects of long–term infection because these animals had been infected with GSHV for up to 10 years. Chronic infection generally produced a mild, persistent hepatitis characterized by light lymphocytic and plasmacytic portal infiltrates with occasional individual necrotic hepatocytes and small aggregates of Kupffer cells or mononuclear inflammatory cells in the parenchyma. In a few of the portal tracts from each of the more inflamed livers (grade 2), the inflammatory infiltrate penetrated the limiting plate and extended into the adjacent parenchyma. Hepatitis (grades 1 or 2) was detected more often in chronically infected animals (17 of 29) than in recovered (4 of 24) or uninfected ground squirrels (7 of 42). Fibrosis was generally not increased, but fine strands of collagen extended from the portal tracts and central veins into the parenchyma of about one quarter of the infected and recovered animals. Cytoplasmic pigment accumulation and variation in the size of hepatocyte nuclei appeared to be related to aging, not infection. Serum levels of aspartate and alanine transaminases (AST and ALT) were mildly elevated in samples from seven infected animals compared with seven control animals. Despite many years of chronic infection, liver injury was similar to that reported in previous studies on animals infected for shorter intervals, indicating that liver injury is not progressive in GSHV–infected ground squirrels.
The Hepatocyte Growth Factor Regulates the Synthesis of Acute–Phase Proteins in Human Hepatocytes: Divergent Effect on Interleukin–6–Stimulated Genes
Authors: Guillen M I , Gomez-Lechon M J , Nakamura T , Castell J V
Published: November 2004

Our study addressed the role of the human hepatocyte growth factor (HGF), a potent mitogen for mature rat and human hepatocytes, in the regulation of specific hepatic genes. The experimental evidence obtained in primary cultured human hepatocytes indicates that HGF regulates the synthesis of plasma proteins in a dose–response fashion. It stimulates the synthesis of the negative acute–phase proteins albumin, transferrin, and fibronectin, decreases that of alpha1–antichymotrypsin (ACT) and haptoglobin, and stimulates that of alpha2–macroglobulin (AMG), which in man is insensitive to inflammatory mediators. HGF had no effect on C–reactive protein (CRP) synthesis. These effects differ from those elicited by interleukin–6 (IL–6). The effects of HGF on fibrinogen and alpha1–antitrypsin were, however, similar to those induced by IL–6. The effects of HGF were also observed at the messenger RNA (mRNA) level. Time–course induction experiments showed that the effects of HGF on protein synthesis were delayed by about 48 to 72 hours, in contrast with the 12–hour lag found after IL–6 stimulation. Although the presence of glucocorticoids was not absolutely necessary for HGF to affect plasma protein synthesis, it moderately extended the effects. In pulse–chase experiments, it was found that the action of HGF was not due to an alteration of the rate of secretion of the proteins. The effects of HGF on the synthesis of albumin, transferrin, fibronectin, alpha1–antichymotrypsin, and haptoglobin could be counteracted by the simultaneous presence of IL–6 in the incubation media. A clear additive effect was observed only in the case of fibrinogen. No interaction was observed in the cases of CRP and AMG. The results of this study indicate that the effects of HGF on human hepatocytes may not simply be limited to its mitogenic activity, but that it also regulates hepatic–specific genes and antagonizes, in part, the action of IL–6.
High Plasma Levels of Von Willebrand Factor As A Marker of Endothelial Perturbation in Cirrhosis: Relationship to Endotoxemia
Authors: Ferro D , Quintarelli C , Lattuada A , Leo R , Alessandroni M , Mannucci P M , Violi F
Published: November 2004

The aim of this study was to evaluate whether there is endothelial dysfunction in patients with cirrhosis and to detect the mechanism that may account for it. We measured plasma levels of von Willebrand factor (vWF), a marker of endothelial perturbation, and endotoxin, which releases vWF from endothelial cells

in vitro

, in 32 patients (18 men, 14 women, aged 39–70 years) with cirrhosis classified as mild (class A, n = 10), moderate (class B, n = 16), or severe (class C, n = 6) according to Child–Pugh's classification. vWF antigen (

P

< .0001) and endotoxemia (ρ < .0001) progressively increased from A to class C; but the increase of vWF antigen was not strictly related to liver failure, as shown by the lack of correlation between vWF and several indexes of liver protein synthesis. Analysis of the vWF subunit showed no sign of proteolytic fragmentation of the molecule. Multimeric analysis indicated intact vWF multimeric structure. In all patients, there was a strong correlation between vWF antigen and endotoxemia (rho = .92;

P

= .0001). In 20 selected patients, vWF antigen and endotoxemia were measured before and after 7 days of standard therapy (n = 10) or standard therapy plus nonabsorbable antibiotics. There was a significant decrease of vWF antigen (

P

< .02) concomitantly with the decrease of endotoxemia (

P

< .006) in patients taking nonabsorbable antibiotics. Human umbilical vein endothelial cells incubated in vitro with 125 to 500 pg/mL endotoxin released vWF antigen into the medium dose dependently. These results demonstrate that there is endothelial perturbation in cirrhosis and that endotoxemia may play a key role in its occurrence.
Coagulation Factor V Levels As A Prognostic Indicator in Fulminant Hepatic Failure
Authors: Izumi S , Langley P G , Wendon J , Ellis A J , Pernambuco R , Hughes R D , Williams R
Published: November 2004

Data reported by Bernuau et al. have strongly supported the measurement of coagulation factor V as the best prognostic indicator in fulminant hepatic failure (FHF) and as the test on which selection for urgent liver transplantation should be made. In this study, we have measured plasma factor V in 110 patients with FHF, in grades I–IV coma, in 88 of whom the etiology was acetaminophen overdose. On admission, patients who did not survive had significantly lower factor V levels (median, 5%; range, 1–27; n = 49), compared with those who did (median, 10%; range, 2–70;

P

< .001). In the 81 patients with acetaminophen–induced FHF who did not receive a transplant, there was no cutoff level of factor V that clearly separated the patients. On statistical analysis, a positive predictive value (the mortality in patients predicted to have a poor prognosis) of 0.49 was calculated for factor V <20% and 0.57 for factor V < 10%. If the prognostic criteria included deep coma (grades III and IV) as well as factor V <20%, a positive predictive value of 0.73 was calculated. This compared with a value of 0.92 for the well–established King's prognostic criteria based on pH, and a combination of international normalized ratio (INR), renal failure, and coma. In the 17 mixed, nonacetaminophen group of patients who did not receive a liver graft, the positive predictive value was 0.85 for a factor V level <20% and 1.00 for factor V <10%, compared with 0.93 for the King's criteria for that etiologic group. This study demonstrates that the predictive accuracy of plasma factor V level is much less effective than the well–validated King's criteria in the selection of patients with acetaminophen–induced FHF needing liver grafting, although it may be useful in patients with FHF due to other causes.
Comparison of Glial Fibrillary Acidic Protein and Desmin Staining in Normal and Ccl4–Induced Fibrotic Rat Livers
Authors: Niki T , De Bleser P J , Xu G , Van der Berg K , Wisse E , Geerts A
Published: November 2004

Fat–storing cells are the major producers of extracellular matrix in the liver. A good immunocytochemical marker is, however, still lacking for this cell type. Desmin, frequently used by most investigators, fails to stain many pericentral fat–storing cells in normal rat liver. The aim of the present study is to evaluate glial fibrillary acidic protein (GFAP) as an alternative marker of fat–storing cells. In normal rat liver, immunostaining of GFAP revealed numerous fat–storing cells with characteristic cytoplasmic extensions. Unlike desmin, which was preferentially expressed in periportal fat–storing cells, GFAP–positive fat–storing cells were distributed more evenly in the lobules. In a narrow periportal zone, however, GFAP–positive cells were occasionally absent. Dual GFAP/desmin staining revealed colocalization of these markers, but fat–storing cells positive only for GFAP or desmin were also present. Chronic carbon tetrachloride exposure induced a spatial change in the expression of GFAP and desmin. At 3 weeks, accumulation of GFAP/desmin double–positive cells was observed in developing fibrotic septa. At 8 weeks, the GFAP positivity in the septa persisted but became weak, while desmin expression became stronger. In contrast, the expression of GFAP within the lobule was gradually decreased as fibrosis progressed. We conclude that GFAP is expressed by a subpopulation of fat–storing cells, which differs partially from the population that expresses desmin. Because in normal rat liver desmin– negative fat–storing cells can be identified by GFAP staining and vice versa, dual GFAP/desmin staining allows more complete identification of fat–storing cells. In chronically injured liver, GFAP may not be as useful as in normal rat liver. The coexpression of GFAP/desmin in developing septa and the subsequent downregulation of GFAP in an advanced stage of fibrosis may reflect different stages of fat–storing cell activation. Further investigation is required to determine the functional significance of alteration of GFAP expression in fat–storing cells.
Influence of Continuous Interleukin–2 Administration Via the Portal Vein on Liver Regeneration Following Partial Hepatectomy in Rats
Authors: Wadamori K , Oka M , Tokuda N , Fujikura Y , Hazama S , Fukumoto T , Suzuki T
Published: November 2004

We have reported the efficacy of intraarterial–combined immunochemotherapy including interleukin–2 (IL–2) for unresectable hepatocellular carcinoma (HCC). To further test this therapy for prevention of intrahepatic recurrence after hepatectomy, the influence of IL–2 on liver regeneration was examined using mitotic index (MI) and the bromodeoxyuridine (BrdU) labeling index (LI) in 70% hepatectomized Donryu rats. In addition, gap junction appearance, which may change during liver regeneration, was analyzed using a monoclonal antibody (HAM8). Serum albumin, alanine transaminase, and total bilirubin (TB) levels were also evaluated. IL–2 (45,000 Japanese reference units [JRU]/d) or saline was administered continuously via the portal vein immediately after hepatectomy using an infusion pump. We also examined the influence of IL–2 on liver regeneration after hepatectomy with splenectomy. No difference in the weight of the liver, serum albumin, alanine transaminase, or TB was observed in any groups at 1, 2, or 4 days after hepatectomy. Neither IL–2 nor splenectomy influenced MI and BrdU LI at all three points. Gap junctions began to disappear after hepatectomy and reached a minimum on day 2 in all groups. Four days after hepatectomy, the density of the reappearing gap junctions was markedly lower in groups treated with IL–2 than in those receiving saline with or without splenectomy. However, the density returned to close to preoperative levels 6 days after hepatectomy in all groups. Continuous portal infusion of IL–2 transiently disturbed gap junction reappearance during liver regeneration. However, no other parameters of liver regeneration or liver functions differed. These results suggest that the liver regeneration after partial hepatectomy may be suppressed by the administration of IL–2, even though the suppression may not be harmful for overall recovery of the resected liver. However, it seems that hepatic IL–2 administration can be performed without serious complications after hepatectomy.
Decreased Ureagenesis From Alanine, But Not From Ammonia and Glutamine, in the Perfused Rat Liver After Partial Hepatectomy
Authors: Moriyama M , Makiyama I , Shiota M , Uesugi K , Kannan Y , Ohta M , Kimura K , Sugano T
Published: November 2004

Ureagenesis from ammonia, alanine, and glutamine in the liver after partial hepatectomy (PH) was determined by using the liver–perfusion system. The maximum rate of ureagenesis from ammonium chloride (10 mmol/L) in hepatectomized (HX) rats at 24 hours after surgery was obtained in the presence of ornithine, lactate, and pyruvate, and it was almost identical to that in sham–operated (SO) rats. The rate of urea production from glutamine (1 mmol/L or 10 mmol/L) in HX rats was significantly lower than that of SO rats with a concomitant decrease in hepatic glutaminase activities. However, the rate of urea synthesis from glutamine (1 mmol/L) in the presence of added ammonia (0.5 mmol/L) was accelerated approximately 10–fold, and the significant difference in the rate of urea formation between HX and SO rats was abolished. This result indicates that there is enough glutaminase to generate ammonia from glutamine in the liver of HX rats. The rate of urea production from alanine (1 mmol/L or 10 mmol/L) in HX rats was significantly decreased at 24 hours following surgery, while that of SO rats was increased. The decreased formation of urea from alanine was not seen at 72 and 120 hours after the operation. These results suggest that during the proliferation phase of liver regeneration, a reduction of ureagenesis from alanine facilitates the remnant liver to make nonessential amino acids such as aspartate. This metabolic alteration might be related to the proliferation of liver cells.
Critical Involvement of Interferon γ in the Pathogenesis of T–Cell Activation–Associated Hepatitis and Regulatory Mechanisms of Interleukin–6 for the Manifestations of Hepatitis
Authors: Mizuhara H , Uno M , Seki N , Yamashita M , Yamaoka M , Ogawa T , Kaneda K , Fujii T , Senoh H , Fujiwara H
Published: November 2004

A single intravenous injection of concanavalin A (Con A) induces T–cell activation and an acute hepatitis in mice. This study investigated the role of interferon γ (IFN–γ) in the pathogenesis of this hepatitis model. Striking increases in the plasma levels of various cytokines, including tumor necrosis factor (TNF), interleukin–2 (IL–2), and IFN–γ, were detected before the increase in plasma aminotransferase levels induced by Con A injection. TNF levels peaked within 2 hours, whereas IFN–γ levels peaked at 6 hours after Con A injection. In contrast to a sharp peak of TNF levels, high IFN–γ levels were detected for a more prolonged period. Passive immunization with anti–IFN–γ monoclonal antibody (MAb) conferred a dose–dependent protection against liver injury in this model. This protection was observed when anti–IFN–γ MAb was administered at least 30 minutes before Con A injection but not when given 1 hour after Con A injection. The protection from Con A–induced hepatitis was also induced by administration of rIL–6 before Con A injection. rIL–6 treatment induced significant albeit incomplete inhibition of IFN–γ and TNF production, whereas this regimen did not affect IL–2 production. Despite striking protective effects of rIL–6 or anti–IFN–γ MAb, comparable levels of cellular (both T cell and polymorphonuclear cell) infiltration were detected in liver sections from animals untreated, or treated with either rIL–6 or anti–IFN–γ MAb. Moreover, electron microscopic examination showed that infiltrating T cells exhibited a blastoid appearance in all groups. These results indicate that IFN–γ plays a critical role in the development of Con A–induced acute hepatitis and suggest that IL–6 administration can regulate the manifestation of hepatitis through mechanisms including the reduced production of inflammatory cytokines such as IFN–γ.
Evidence That Interference With Binding to Hepatic Cytosol Binders Can Inhibit Bile Acid Excretion in Rats
Authors: Takikawa H , Sugiyama Y , Fernandez-Checa J C , Kuhlenkamp J , Ookhtens M , Kaplowitz N
Published: November 2004

We previously identified that Y′ bile acid binders (3α– hydroxysteroid dehydrogenases) interact with bile acids in intact rat hepatocytes using [3β–3H, C24–14C]bile acids and that indomethacin, a competitive inhibitor of 3α–hydroxysteroid dehydrogenase, inhibits 3H–loss from the C3–position of bile acids as well as inhibits hepatic bile acid removal and excretion. To study the kinetics of these inhibitory effects, glycocholate transport was studied in the absence and presence of indomethacin in the single–pass perfused rat liver. Indomethacin decreased net hepatic glycocholate uptake in the perfused liver, which was confirmed in isolated hepatocytes and basolateral liver plasma membrane vesicles. However, indomethacin markedly increased the sinusoidal efflux and decreased the biliary excretion of glycocholate in the perfused liver. These observations indicate that the effect of indomethacin to delay biliary glycocholate excretion is related to either intracellular or canalicular glycocholate transport. The latter possibility seemed unlikely because indomethacin did not inhibit electrogenic or adenosine triphosphate (ATP)–dependent glycocholate uptake by canalicular liver plasma membrane vesicles. Thus, the current data support an important role for binding of bile acids to cytosolic proteins in overall hepatic transport and suggest that specific interference with cytosolic binding can interfere with the excretion of bile acids.
Involvement of Platelet–Activating Factor in Cytokine Production and Neutrophil Activation After Hepatic Ischemia–Reperfusion
Authors: Serizawa A , Nakamura S , Suzuki S , Baba S , Nakano M
Published: November 2004

Although platelet–activating factor (PAF) is implicated as an important mediator in the pathogenesis of hepatic ischemia–reperfusion (IR) injury, the precise mechanism of its action has not been studied. We examined the hypothesis that PAF may influence neutrophils by promoting the production of tumor necrosis factor α (TNF–α) and cytokine–induced neutrophil chemoattractant (CINC), a member of the interleukin–8 (IL–8) family, and may be associated with liver and lung injury during the early phase of reperfusion after total hepatic ischemia. Rats pretreated with a specific PAF receptor antagonist exhibited suppression of the increase in plasma TNF–α and CINC levels, as well as the priming of peripheral neutrophils for superoxide production after reperfusion when compared with animals pretreated with physiological saline. These effects resulted in a reduction of plasma liver enzymes and of hepatic and pulmonary neutrophil sequestration, as well as an increased survival rate. There was a strong correlation between the time course of CINC release and hepatic or pulmonary neutrophil sequestration. We concluded that PAF activates neutrophils, either directly or by promoting the production of TNF–α and CINC, and is involved in hepatic IR injury.
Biliary Atresia: Current Concepts and Research Directions. Summary of A Symposium
Authors: Balistreri W F , Grand R , Hoofnagle J H , Suchy F J , Ryckman F C , Perlmutter D H , Sokol R J
Published: November 2004

Biliary atresia (BA) is the end result of a destructive, inflammatory process that affects intra– and extrahepatic bile ducts, leading to fibrosis and obliteration of the biliary tract with the development of biliary cirrhosis. It is the commonest cause of chronic cholestasis in infants and children, and therefore is the most frequent indication for liver transplantation in this age group. The disease occurs worldwide, affecting an estimated 1 in 8,000 to 12,000 live births. At present, there is no specific therapy for BA; however, sequential surgical therapy begins with creation of a hepatoportoenterostomy (HPE); in those with end–stage liver disease, liver transplantation is indicated. Since most candidates are young children of small size, there is a shortage of size–matched donors for liver transplantation. At present, an increased awareness to ensure early diagnosis and development of methods to prevent progressive fibrosis are needed. These considerations are dependent on detailed studies of the pathogenesis of BA. Recent studies have focused on normal and altered bile duct morphogenesis and the role of various factors (infectious or toxic agents and metabolic insults) in isolation or in combination with a genetic or immunologic susceptibility in the etiology of BA.
Differences in the Mechanisms of Uptake and Endocytosis of Small and Large Chylomicron Remnants by Rat Liver
Authors: Windler E , Greeve J , Robenek H , Rinninger F , Greten H , Jäckle S
Published: November 2004

Initial binding and subsequent endocytosis of small and large chylomicron remnants by rat liver were compared. Small and large chylomicrons were obtained from mesenteric lymph of glucose– or fat–fed rats, respectively. The low–density lipoprotein (LDL) receptor was up– and down–regulated as shown by LDL receptor messenger RNA (mRNA). The rate of removal of small chylomicron remnants by isolated perfused rat livers followed closely the activity of the LDL receptor. When mRNA was undetectable, the uptake was as low as that of lymphatic small chylomicrons. In contrast, the uptake of large chylomicron remnants into perfused rat livers was unaffected by changes of the LDL–receptor activity, but significantly reduced after livers were flushed with heparin or heparinase. Large chylomicron remnants were cleared from plasma much faster than small chylomicron remnants, but were more slowly internalized into hepatocytes. Both, small and large chylomicron remnants entered the pathway of receptor–mediated endocytosis as shown by electron microscopy and analysis of isolated endosomes. Yet, large chylomicron remnants were taken up into the compartment of uncoupling of receptors and ligands and multivesicular bodies at a much slower rate. This was independent of the activity of the LDL receptor and the heparin–releasable binding site. From these findings it is concluded that large chylomicron remnants initially bind rapidly to surface components other than the LDL receptor, one of which may be hepatic lipase. Yet, the consecutive internalization is slow. In contrast, small chylomicron remnants are removed at a slower rate from plasma, binding predominantly to the LDL receptor, but are more readily taken up into endosomes.
Salmonella Hepatitis: Analysis of 27 Cases and Comparison With Acute Viral Hepatitis
Authors: El-Newihi H M , Alamy M E , Reynolds T B
Published: November 2004

The liver is commonly involved in patients with typhoid fever. However, severe hepatic derangement simulating acute viral hepatitis is rare. Our aim was to characterize the clinical picture, biochemical features, and prognosis of Salmonella hepatitis. Retrospective case–control analysis of medical records included 27 patients with Salmonella hepatitis and 27 inpatients with acute viral hepatitis from 1973 to 1993. Travel history, clinical picture, a standard battery of 18 biochemical tests, complete blood counts, disease complications, duration of hospital admission, and final outcome were analyzed. Eleven patients with Salmonella hepatitis (40%) travelled abroad within 1 month of illness. A greater proportion of Salmonella hepatitis patients developed fever > 104 degrees (44% vs. 4%, respectively;

P

< .0001), and had relative bradycardia (42% vs. 4%, respectively;

P

< .002) than viral hepatitis patients. Salmonella hepatitis was associated with lower peak serum alanine transaminase (ALT), aspartate transaminase, and higher peak serum alkaline phosphatase (296 vs. 3,234 U/L, 535 vs. 2,844 U/L, and 500 vs. 228 U/dL, respectively;

P

< .0001, <.0003, and <.004). The admission ALT/lactic dehydrogenase (LDH) ratio, when levels of both enzymes were expressed as multiples of upper limit of normal value for each, was significantly lower in Salmonella hepatitis. All Salmonella hepatitis cases had a ratio < 4, and all viral hepatitis cases had a ratio > 5,

P

< .0001. Left shift of white blood cells was more common in Salmonella hepatitis (83% vs. 37%;

P

< .004). Patients with Salmonella hepatitis had a longer hospitalization (14.8 vs. 6.5 days, respectively;

P

< .0001). All 54 patients survived their illness. The clinical picture of Salmonella hepatitis is frequently indistinguishable from viral hepatitis. The admission ALT/LDH ratio is the best discriminator between both entities. Other clues that raise the possibility of Salmonella hepatitis include high fever, relative bradycardia, and left shift of WBCs. Despite long hospitalization, Salmonella hepatitis responds to proper antibiotic therapy and has an excellent prognosis.
Abnormal Expression of Muc1 Apomucin and Mature Muc1 Mucin in Biliary Epithelial Cells in Various Cystic Liver Diseases
Authors: Sasaki M , Nakanuma Y
Published: November 2004

MUC1 apomucin is proposed as an “oncofetal” antigen in the biliary system. We surveyed the biliary expression of MUC1 apomucin and mature MUC1 mucin (highly–glycosylated MUC1 apomucin) in hepatic cysts, biliary microhamartomas, and intrahepatic bile ducts in various cystic liver diseases and clarified the relationship between the expression of these mucins and cystogenesis in the liver. The expression of MUC1 apomucin and mature MUC1 mucin were detected immunohistochemically using the monoclonal antibodies DF3 and SM3, and HMFG1, respectively. We surveyed six livers with adult–type polycystic disease (APCD), six with multiple hepatic cysts without polycystic kidney, 11 with solitary hepatic cysts, and 11 “histologically normal” livers. Biliary microhamartomas were frequently found in the former three. MUC1 apomucin was frequently expressed at the luminar surface of the epithelial cells lining cysts and also in biliary microhamartomas, independent of the type of cystic disease. MUC1 apomucin detected by both monoclonal antibodies (MAbs) DF3 and SM3 was also frequently expressed in the intrahepatic bile ducts from polycystic diseases, particularly the adult–type, whereas such expression was rare in the intrahepatic bile ducts in normal livers. Mature MUC1 mucin was expressed on the luminal surface of the epithelial cells lining cysts in approximately half of hepatic cysts studied, but it was infrequently expressed in biliary microhamartomas and almost absent in the intrahepatic bile ducts. The biliary epithelial cells lining hepatic cysts, biliary microhamartomas, the apparently normal intrahepatic bile ducts in APCD, multiple hepatic cysts, and to a lesser degree, solitary hepatic cysts, express abnormal levels MUC1 apomucin. This implies that biliary epithelial cells expressing MUC1 apomucin are related to the whole cystogenetic process in these diseases. In contrast, the expression of mature MUC1 mucin may be related to the late cystogenetic process in these diseases.
Ultrasonographic Determination of Ascitic Volume
Authors: Inadomi J , Cello J P , Koch J
Published: November 2004

The purpose of this study was to develop a method by which ascitic volume can be calculated using transcorporeal ultrasonography, and to determine the accuracy of this method by comparison with the volume of distribution of a radiolabeled tracer (indicator dilution technique [IDT]). Subjects with ascites confirmed by ultrasonography were recruited from the San Francisco General Hospital Gastroenterology and Liver Clinics. With subjects in the prone position on their hands and knees, ultrasonographic measurements were obtained along the ventral surface of the abdomen. The greatest vertical depth of ascitic fluid was recorded, and the abdominal circumference was measured from this point. The ascitic fluid volume was modeled as a segment of a sphere. IDT was performed as the reference method by injecting 99mTc–labeled macroalbumin into the peritoneal cavity and determining the volume of distribution of the indicator. Nine patients were evaluated. The median volume of ascites measured by the IDT was 11.2 L (range, 1.5–17.0 L). The median volume calculated by the ultrasonographic method was 10.3 L (range, 1.2–18.0 L). The correlation coefficient between the ultrasonographic and IDT was 0.96 (

P

< .001). Our technique accurately determines the volume of ascites using simple ultrasonographic measurements.
Bile Ductule Formation in Fetal, Neonatal, and Infant Livers Compared With Extrahepatic Biliary Atresia
Authors: Cocjin J , Rosenthal P , Buslon V , Luk L , Barajas L , Geller S A , Ruebner B , French S
Published: November 2004

The cell of origin of intrahepatic bile ducts during fetal development remains a subject of controversy, although there has been recent evidence that they form from hepatocytes. However, the origin of neoductules and ducts in the setting of liver disease has not been extensively investigated in humans. Using anticytokeratins characteristic of hepatocytes and bile ducts, we repeated earlier studies of fetal development to compare ductule formation in normal developing and newborn livers with the ductules formed during extrahepatic biliary atresia. We utilized an antibody to proliferating cell nuclear antigen (PCNA) staining to determine which cells were in active DNA synthesis (S phase) during fetal development and liver disease progression. The results indicated that hepatocytes undergo a phenotypic switch (metaplasia) to form ductular cells during fetal development. There was no ductular cell replication in the fetal livers. In contrast, both bile ductular metaplasia and proliferation were observed in biliary atresia. Therefore, both a limiting plate phenotypic switch to ductules and replication of ductular cells play a role in the increase in the ductules seen in the progression to biliary cirrhosis. Bile ductular proliferation in biliary atresia, however, was less than that seen in hepatocytes, whereas the number of bile ductules increased and the relative proportion of hepatocytes diminished as the accompanying periductular fibrosis progressed to cirrhosis.
Inactivation of P16Ink4 in Hepatocellular Carcinoma
Authors: Hui A , Sakamoto M , Kanai Y , Ino Y , Gotoh M , Yokota J , Hirohashi S
Published: November 2004

We analyzed the p16INK4 status of 6 hepatocellular carcinoma (HCC) cell lines and 32 primary HCC tumors, including 9 early–stage tumors, to determine whether p16INK4 tumor–suppressor gene inactivation participates in hepatocarcinogenesis. p16INK4 was studied at its protein level through Western blotting, at its messenger RNA (mRNA) level through reverse–transcriptase polymerase chain reaction analysis (RT–PCR) and Northern blotting, and at its genomic level through Southern blotting and PCR–single–strand conformation polymorphism analysis. The p16 protein was absent from 3 of 6 cell lines (50%) and 11 of 32 primary tumors (34%), but present in noncancerous tissues, indicating that p16INK4 is involved in hepatocarcinogenesis. Furthermore, we suggest that the p16 protein loss may contribute to the following: (1) early–stage hepatocarcinogenesis, because it was observed in 22% of early stage tumors; and (2) tumor progression, because it occurred approximately twice as often in advanced rather than in early stage tumors (40%). It was striking that neither p16INK4 homozygous deletion and mutation nor loss of p16INK4 mRNA expression were observed in HCC cell lines and primary tumors, including those specimens from which the p16 protein was absent except the Li7HM cell line, in which p16INK4 mRNA was not detected. These results suggest that p16INK4 in HCC is inactivated predominantly by posttranscriptional regulation rather than by genomic aberrations and lack of transcription.
Mallory Body Induction in Drug–Primed Mouse Liver
Authors: Yuan Q X , Marceau N , French B A , Fu P , French S W
Published: November 2004

The aim of this study was to determine the various factors that are involved in the induction of Mallory body (MB) formation. A model was developed where MB formation was induced by refeeding either of the drugs griseofulvin or diethyl 1,4–dihydro–1,4,6–trimethyl–3,5– pyridinedicarboxylate (DDC). Mice were fed the drugs for 5 months, followed by withdrawal of the drugs for 1 month (drug–primed livers). The drugs were refed for 1,3,5,7, or 11 days. Early MBs first appeared as small, enlarged aggregates of filaments in the perinuclear or pericanalicular location on the third day of refeeding. Mature MBs appeared on the fifth day of refeeding. MBs reached maximum concentration on day 5 of refeeding. Western blots showed a progressive increase in the cytokeratin proteins (CK49 and CK55) and actin while refeeding the drugs. Liver cell regeneration, as indicated by the percent of proliferating cell nuclear antigen (PCNA)–positive nuclei, increased on the third day of refeeding. However, there was no correlation between the frequency of MBs and the percent of PCNA– positive nuclei. It is concluded that MB formation is not related to the liver cell regeneration response to injury but rather involves a separate regulation pathway. The MBs were heavily ubiquitinated and were associated with increased ubiquitin–protein conjugates as indicated by Western blotting, suggesting that ubiquitinization of cytokeratin protein are involved in the formation of MB aggregation.
Influence of Dietary Calcium Phosphate on the Disposition of Bilirubin in Rats With Unconjugated Hyperbilirubinemia
Authors: Van Der Veere C N , Schoemaker B , Bakker C , Van Der Meer R , Jansen P L , Elferink R P
Published: November 2004

The aim of this study was to test a possible form of therapy that could be used in the management of unconjugated hyperbilirubinemia. We hypothesized that unconjugated bilirubin (UCB) can permeate the intestinal wall and can thus be secreted with the feces. We have previously observed that UCB binds to amorphous calcium phosphate

in vitro

. Orally ingested amorphous calcium phosphate may act as a trapping agent for bilirubin in the intestine, thereby preventing back– diffusion across the intestinal wall. In this study, we tested whether feeding calcium phosphate leads to enhanced excretion of unconjugated bilirubin in Gunn rats. When a purified control diet was substituted by a high calcium phosphate diet, a decrease in bilirubin levels of 30% to 50% in male Gunn rats and of 23% in female rats was observed. The fecal output of bilirubin was more than doubled in Gunn rats in the first 3 days after the normal diet had been replaced by the high calcium– phosphate diet. The biological half–life of 3H–labeled bilirubin in blood was 89.8 +/– 17.2 hours in rats fed the purified control diet and 50.9 +/– 1.4 hours in rats fed the high calcium phosphate diet (

P

= .004). After 30 weeks, plasma bilirubin levels were still significantly lower in Gunn rats fed a high calcium phosphate diet. No differences were found in plasma concentrations of calcium, magnesium, phosphate, urea, and creatinine in both Gunn rats and Wistar rats on control or high calcium phosphate diets. This therapy might be useful in the management of Crigler–Najjar patients, for example, as an adjunct to phototherapy.