bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycine and Uridine Prevent D –Galactosamine Hepatotoxicity in the Rat: Role of Kupffer Cells
Authors: Stachlewitz Robert F. , Seabra Vitor , Bradford Blair , Bradham Cynthia A. , Rusyn Ivan , Germolec Dori , Thurman Ronald G.
Published: July 2004

Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in d–galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of d–galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from d–galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of d–galactosamine(500 mg/kg, intraperitoneally). d–galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl3prevented these effects. d–galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor–α (TNF–α) prevented hepatotoxicity caused by d–galactosamine. Moreover, apoptosis in hepatocytes caused by d–galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF–α antiserum, and uridine. Thus, it was hypothesized that TNF–α from Kupffer cells causes apoptosis after d–galactosamine administration in the rat. Indeed, increases in TNF–α messenger RNA (mRNA) were detected as early as 2.5 hours after d–galactosamine treatment. Previous work proposed that uridine blocks d–galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca2+]i and release of TNF–α caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by d–galactosamine treatment in vivo. These data support the hypothesis that uridine prevents d–galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF–α release from Kupffer cells thereby blocking apoptosis that occurs early after d–galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after d–galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.
Dna Hypermethylation At the D17s5 Locus and Reduced Hic–1 Mrna Expression Are Associated With Hepatocarcinogenesis
Authors: Kanai Yae , Hui Ai-Min , Sun Lin , Ushijima Saori , Sakamoto Michiie , Tsuda Hitoshi , Hirohashi Setsuo
Published: July 2004

To examine the significance of aberrant DNA methylation in hepatocarcinogenesis, the DNA methylation status at the D17S5 locus and mRNA expression of a candidate tumor suppressor gene, HIC–1 (hypermethylated–in–cancer), which was identified at the D17S5 locus, in primary hepatocellular carcinomas (HCCs) and their corresponding noncancerous liver tissues were assessed. DNA hypermethylation at the D17S5 locus was detected in 44% of the noncancerous liver tissues showing chronic hepatitis or cirrhosis, which are widely considered to be precancerous conditions, but was not observed in noncancerous liver tissues showing no remarkable histological findings. The incidence of DNA hypermethylation at this locus was significantly higher in HCCs (90%) than noncancerous liver tissues (P < .001). Loss ofheterozygosity at the D17S5 locus, which was preceded by DNA hypermethylation at the same locus, was detected in 54% of HCCs. The HIC–1 mRNA expression level of noncancerous liver tissues showing chronic hepatitis or cirrhosis was significantly lower than that of noncancerous liver tissues showing no remarkable histological findings (P < .01), and that of HCCs was even lower than that of noncancerous liver tissues (P < .05). Poorly differentiated HCCs showed lower expression levels than well– to moderately differentiated HCCs. Mutation of the p53 gene may be involved in HIC–1 inactivation. Moreover, wild–type p53 did not overcome DNA hypermethylation at the D17S5 locus to activate HIC–1 in HCCs. These data suggest that aberrant DNA methylation at this locus and reduced HIC–1 mRNA expression participate in hepatocarcinogenesis during both early developmental stages and malignant progression of HCCs.
Glucocorticoid Receptor Down–Regulates C–Jun Amino Terminal Kinases Induced by Tumor Necrosis Factor α in Fetal Rat Hepatocyte Primary Cultures
Authors: Ventura Juan-José , Roncero César , Fabregat Isabel , Benito Manuel
Published: July 2004

The effect of dexamethasone on Jun N–terminal kinase (JNK) activity was assayed by using fetal hepatocytes in primary culture. The addition of tumor necrosis factor α (TNF–α) caused an increase in JNK in a dose– and time–dependent manner. We show that activation of JNK by this extracellular signal is inhibited by dexamethasone in a dose–dependent fashion. This inhibitory effect was observed in cells treated for 10 minutes with dexamethasone in the presence of protein phosphatase inhibitors such as orthovanadate or okadaic acid, or in cells previously treated with actinomycin D. Glucocorticoid receptor (GR) can be precipitated with the fusion protein, GST-c–Jun (1–79), bound to agarose beads. However, the inhibitory effect of glucocorticoids on JNK activity was also observed using ATF–2 as substrate. In addition, dexamethasone inhibits JNK phosphorylation induced by TNF–α. Finally, we show that GR can also be phosphorylated in tyrosine residues in response to TNF–α and epidermal growth factor (EGF) upon ligand–binding. Our results suggest that the anti–inflammatory effect of glucocorticoids on the inflammatory pathways induced by TNF–α can be explained, at least in part, by modulating JNK activity through a direct protein–protein interaction; the JNK phosphorylation and tyrosine–phosphorylation state of GR may be regulatory steps also involved in that effect.
Epi–Illumination Fluorescent Light Microscopy for The In Vivo Study of Rat Hepatic Microvascular Response to Cryothermia
Authors: Schüder Gerhard , Vollmar Brigitte , Richter Sven , Pistorius Georg , Fehringer Michael , Feifel Gernot , Menger Michael D.
Published: July 2004

To elucidate the hepatic microvascular response to cryothermia, we studied the liver microcirculation of Sprague–Dawley rats after one and two 4–minute freeze–thaw cycles using intravital fluorescence microscopy. Irrespective of the number of freeze–thaw cycles applied, the nature of hepatic microvascular injury was characterized by complete stasis of sinusoidal blood flow within the central part of the cryolesions and heterogeneous sinusoidal perfusion in a critically perfused border zone located at the periphery of the lesions. Analysis over time (2 hours) revealed a successive shutdown of sinusoidal perfusion within this critically perfused border zone, which was caused by intravascularly lodging cell aggregates, blocking the lumen of individual sinusoids. The aggregates consisted of parenchymal cells and cell fragments, but did not include leukocytes or platelets. Strikingly, microvascular perfusion failure was associated with Ito cell disintegration and marked dilation of sinusoids (15.6 ± 0.8 μm vs. 8.8 ± 0.8 μm; P < .05). This excludes sinusoidal constriction as the cause of nutritive perfusion failure, and may indicate dysfunction of Ito cell-regulated vasomotor control by cryothermia. However, because circulating cell aggregates were frequently observed plugging individual microvessels, dilation of sinusoids may just be the result of passive distension caused by outflow blockade. Analysis of hepatic tissue at 8 weeks after cryothermia did not reveal regeneration and microvascular remodeling, but loss of hepatic tissue, which corresponded well with the tissue area presenting with sinusoidal perfusion failure during the initial observation period after cryothermia. The fact that there was no recovery of sinusoidal perfusion over the initial 2–hour observation period, but loss of tissue after 8 weeks, supports the view that cryothermia induces injury not only by direct low–temperature-mediated action, but also through ischemia caused by irreversible deterioration of the microcirculation.
Transforming Growth Factor β1 Induces the Expression of α1(I) Procollagen Mrna by A Hydrogen Peroxide–C/Ebpβ–Dependent Mechanism in Rat Hepatic Stellate Cells
Authors: García-Trevijano Elena R. , Iraburu María J. , Fontana Luis , Domínguez-Rosales José A. , Auster Anitra , Covarrubias-Pinedo Amador , Rojkind Marcos
Published: July 2004

Oxidative stress plays a key role in liver fibrosis. Both inflammatory cells and activated Kupffer cells produce H2O2, an oxidant involved in the activation of hepatic stellate cells (HSC). Increased production of reactive oxygen intermediates (ROIs) in fibrotic livers is associated in part with the up–regulation of transforming growth factor β (TGF–β), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF–β induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H2O2 is a mediator of TGF–β–elicited α1(I) collagen gene (col1a1) up–regulation. We demonstrated that TGF–β induces the accumulation of H2O2, and that this oxidant is, in turn, directly involved in up–regulating the expression of the col1a1 gene. While the addition of H2O2 to HSC induced the expression of α1(I) procollagen mRNA, catalase, an H2O2 enzyme scavenger, abrogated TGF–β–mediated col1a1 gene up–regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped a cis–acting element (-370 to -344) essential for TGF–β responsiveness. We further showed that TGF–β induced the activation and binding of a C/EBPβ–containing transcriptional complex to this sequence, an effect that was also mimicked by the addition of H2O2. Taken together, these data demonstrate a direct connection between TGF–β–mediated accumulation of H2O2 and the up–regulation of col1a1 gene in HSC.
Cryptogenic Cirrhosis: Clinical Characterization and Risk Factors for Underlying Disease
Authors: Caldwell Stephen H. , Oelsner David H. , Iezzoni Julia C. , Hespenheide Elizabeth E. , Battle Emily H. , Driscoll Carolyn J.
Published: July 2004

We characterized 70 consecutive patients with cryptogenic cirrhosis to assess major risks for liver disease. Each patient was reevaluated for past alcohol exposure, scored by the International Autoimmune Hepatitis (IAH) score and assessed for viral hepatitis risks and risks for nonalcoholic steatohepatitis (NASH). The results were compared with 50 consecutive NASH patients, 39 nonalcoholic patients age 50 and over with cirrhosis from hepatitis C, and 33 consecutive patients with cirrhosis caused by primary biliary cirrhosis (PBC). Among the cryptogenic group, 49 (70%) were female, and the mean age was 63 ± 11 years. Although ascites and variceal bleeding were common, almost one half lacked major signs of complicated portal hypertension. A history of Type 2 diabetes mellitus and/or obesity was present in 51 (73%). Nineteen (27%) patients had a history of blood transfusions antedating the diagnosis of cirrhosis. No clinical or histological features distinguished this group from the other patients, and 14 (74%) of these had a history of obesity and/or diabetes. Nineteen of the remaining nontransfused patients had indeterminant IAH scores but were histologically and biochemically indistinguishable from the others. Twelve of these (63%) also had a history of obesity and/or diabetes. Both diabetes and obesity were significantly more common in the cryptogenic cirrhotic patients compared with the cirrhotic patients with PBC or hepatitis C. In contrast, the prevalence of obesity and diabetes was similar to the NASH patients who were, on average, a decade younger. Although there is some diversity that indicates more than one cause, our findings suggest that NASH plays an under–recognized role in many patients with cryptogenic cirrhosis, most of whom are older, type 2 diabetic and obese females.
Long–Term Response to Hepatitis B Vaccination and Response to Booster in Children Born to Mothers With Hepatitis B E Antigen
Authors: Huang Li-Min , Chiang Bor-Luen , Lee Chin-Yun , Lee Ping-Ing , Chi Wei-Kuang , Chang Mei-Hwei
Published: July 2004

Hepatitis B (HB) vaccine provides an uncertain duration of protection and the optimal timing of booster vaccine remains unclear. This study examined the immune response at 10 years of 118 children who had developed protective anti–HB surface (anti–HBs) levels after a primary series of HB immunizations in infancy. All of the children were born to hepatitis B e Antigen (HBeAg)–positive hepatitis B surface antigen (HBsAg) carrier mothers. HB markers in all subjects and cellular immune response in some were determined. A booster was given to all subjects after the collection of samples and another blood sample was collected 4 weeks later. The results showed that a total of 39 (33%) of the children were seronegative for anti–HBs. T–cell proliferative response to HBsAg was noted in 47% of children. On HBsAg stimulation, leukocyte samples from a significantly higher proportion of subjects produced cytokines (81% of T cells produced interleukin–2 [IL–2] and 100% produced IL–5). The booster dose of HB vaccine induced the production of a protective level of anti–HBs (≥10 mIU/mL) in all subjects. Cellular immunity was augmented with a positive rate of 58%, 90%, and 100% for HBsAg–induced T–cell proliferation, IL–2 production, and IL–5 production, respectively. Although 14 (11.9%) of the subjects were HB core antibody positive at 10 years of age, no new HBsAg carrier was detected. The results of this study show that protection afforded by HB vaccination persisted to the age of 10 years in all vaccinees. Immunologic memory was detected in all subjects including those who had lost their anti–HBs seropositivity. These results suggest that no booster vaccination is needed before 10 years of age. The most sensitive marker of immunologic memory is IL–5 production of T cells.
Role of Intercellular Adhesion Molecule–1 and Lymphocyte Function-Associated Antigen–1 During Nonsuppurative Destructive Cholangitis in A Mouse Graft–Versus–Host Disease Model
Authors: Howell Charles D. , Li Jian , Chen Weiran
Published: July 2004

Intercellular adhesion molecule–1(ICAM–1) is expressed abnormally on the bile duct epithelium during the course of primary biliary cirrhosis (PBC), but the importance of ICAM–1 and its lymphocyte function–associated antigen–1 (LFA–1) receptor during the course of nonsuppurative destructive cholangitis (NSDC) has not been defined. To address this question, we defined the relationship between ICAM–1 on the intrahepatic bile duct epithelium and the evolution of NSDC lesions in a mouse graft–versus–host disease (GVHD) model. We also determined the effects of anti-ICAM–1 and anti-LFA–1 treatments on NSDC, intrahepatic lymphokine production, and the homing of lymphocytes to the livers of GVHD mice. ICAM–1 was initially detected on the bile duct epithelium and portal vein endothelium on day 7 of GVHD. There was a significant positive correlation between the intensity of ICAM–1 staining and histological bile duct damage (r = .58, P < .05) between day 3 and 28. Treatment with anti-ICAM–1 (but not anti-LFA–1) decreased both the mean grades of portal inflammation (P = .003) and NSDC (P = .002) lesions compared with control immunoglobulin G (IgG) treatments. Combined treatment with anti-ICAM–1 and anti-LFA–1 caused a further decrease in the amount of portal inflammation and bile duct damage compared with anti-ICAM–1, alone (P = .02). Anti-ICAM–1 treatment also decreased both the percentage of T cells and the production of interleukin–2 (IL–2) and IL–12 in the liver (P < .01), but had no effect on IL–4, IL–10, and interferon gamma. Neither anti-ICAM–1 nor anti-LFA–1 prevented lymphocytes from homing to the liver. These results indicate that both ICAM–1 and LFA–1 are important to the pathogenesis of NSDC
In Vitro Evidence for the Presence of Hematopoietic Stem Cells in the Adult Human Liver
Authors: Crosbie Orla M. , Reynolds Maura , McEntee Gerry , Traynor Oscar , Hegarty John E. , O'Farrelly Cliona
Published: July 2004

Previous studies have identified novel lymphoid phenotypes in the adult human liver and provided evidence to suggest that lymphoid differentiation can occur locally in this organ. The aim of this study was to examine the adult human liver for the presence of hematopoietic stem cells that may provide the necessary precursor population for local hematopoietic and lymphoid differentiation. Hepatic mononuclear cells (HMNC) were extracted from normal adult liver biopsy specimens using a combination of mechanical disruption and enzymatic digestion. The stem cell marker CD34 was found on 0.81% to 2.35% of isolated HMNCs by flow cytometry. CD34+ HMNCs were positively selected using magnetically labeled beads, and the enriched population was further examined for surface markers characteristically expressed by immature hematopoietic cells and early progenitors. CD45 was expressed by 49% (±23%) of CD34+ HMNCs, indicating their hematopoietic origin. CD38, one of the first markers to be expressed by developing progenitor cells was found on 50% (±22%) of CD34+ HMNCs indicating the presence of both pluripotent stem cells and committed precursors. The majority (90%) of CD34+ HMNCs coexpressed the activation marker human leukocyte antigen DR, consistent with actively cycling cells. Functional maturation of these hepatic progenitors was shown by the detection of multilineage hematopoietic colony formation after tissue culture. Erythroid (BFU–E), granuloctye–monocyte (CFU–GM), and mixed colonies (CFU–GEMM) were detected after culture of unseparated HMNCs and the enriched CD34+ HMNC population; 14.3 ± 13.2 (mean ± SD) BFU–E, 3.1 ± 3.1 CFU–GM, and 0.4 ± 0.9 CFU–GEMM per 1 × 105 unseparated HMNCs and 16.0 ± 9.5 BFU–E and 1.7 ± 0.9 CFU–GM were identified per 2.4 × 103 CD34+ HMNCs plated. The detection of surface markers characteristic of immature hematopoietic cells and colony formation in tissue culture provides evidence for the presence of hematopoietic stem cells and early progenitor cells in the adult human liver. This would suggest that the adult human liver continues to contribute to hematopoiesis and may be an important site for the differentiation of lymphohematopoietic cells involved in disease states, such as autoimmune hepatitis and graft rejection after liver transplantation
Autoepitope Mapping and Reactivity of Autoantibodies to the Dihydrolipoamide Dehydrogenase–Binding Protein (E3bp) and the Glycine Cleavage Proteins in Primary Biliary Cirrhosis
Authors: Dubel Laurence , Tanaka Atsushi , Leung Patrick S. , Van de Water Judy , Coppel Ross , Roche Thomas , Johanet Catherine , Motokawa Yutaro , Ansari Aftab , Gershwin Eric M.
Published: July 2004

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial antibodies (AMA) directed primarily against the E2 subunits of the pyruvate dehydrogenase complex, the branched chain 2–oxo–acid dehydrogenase complex, the 2–oxoglutarate dehydrogenase complex, as well as the dihydrolipoamide dehydrogenase–binding protein (E3BP) of pyruvate dehydrogenase complex. The autoantibody response to each E2 subunit is directed to the lipoic acid binding domain. However, hitherto, the epitope recognized by autoantibodies to E3BP has not been mapped. In this study, we have taken advantage of the recently available full–length human E3BP complementary DNA (cDNA) to map this epitope. In addition, another lipoic binding protein, the H–protein of the glycine cleavage complex, was also studied as a potential autoantigen recognized by AMA. Firstly, the sequence corresponding to the lipoic domain of E3BP (E3BP–LD) was amplified by polymerase chain reaction and recombinant protein and then purified. Immunoreactivity of 45 PBC sera (and 52 control sera) against the purified recombinant E3BP–LD was analyzed by enzyme–linked immunosorbent assay (ELISA) and immunoblotting. Secondly, reactivity of PBC sera was similarly analyzed by immunoblotting against H–protein. It is interesting that preabsorption of patient sera with the lipoic acid binding domain of E3BP completely removed all reactivity with the entire protein by immunoblotting analysis, suggesting that autoantibodies to E3BP are directed solely to its lipoic acid binding domain. Fifty–three percent of PBC sera reacted with E3BP–LD, with the majority of the response being of the immunoglobulin G (IgG) isotype (95%). Surprisingly, there was little IgM response to the E3BP–LD suggesting that the immune response was secondary because of determinant spreading. In contrast, H–protein does not appear to possess (or expose) autoepitopes recognized by PBC sera. This observation is consistent with structural data on this moiety.
Characterization of the Human Multidrug Resistance Protein Isoform Mrp3 Localized to the Basolateral Hepatocyte Membrane
Authors: König Jörg , Rost Daniel , Cui Yunhai , Keppler Dietrich
Published: July 2004

Several members of the multidrug resistance protein (MRP) family are expressed in the liver. Adenosine triphosphate (ATP)-dependent transport of glutathione and glucuronoside conjugates across the hepatocyte canalicular membrane is mediated by the apical MRP isoform, MRP2 (APMRP), also known as canalicular multispecific organic anion transporter (cMOAT). We have cloned an additional MRP isoform, MRP3, from human liver and localized it to the basolateral membrane domain of hepatocytes. Basolateral MRP (BLMRP) is composed of 1,527 amino acids and encoded by 4,581 base pairs of complementary DNA. Northern blotting of various human tissues indicated an expression of MRP3 in the liver, colon, pancreas, and, at a lower level, in the kidney. The amino acid identity of MRP3 with MRP1 and MRP2 is 58% and 48%, respectively. These three isoforms, encoded by genes on different chromosomes, have a similar predicted topology of transmembrane segments and ATP–binding domains. Antibodies raised against two peptide sequences of MRP3 that are not shared by other MRP family members detected recombinant MRP3 expressed in polarized MDCK cells. Both antibodies served to localize MRP3 to the basolateral membrane of hepatocytes. Double–label immunofluorescence microscopy confirmed that MRP3 was not detectable in the canalicular membrane domain. A particularly strong expression of the MRP3 protein was observed in the basolateral hepatocyte membrane of two patients with Dubin–Johnson syndrome who are deficient in MRP2. These results indicate that the basolateral MRP isoform, MRP3, may be upregulated when the canalicular secretion of anionic conjugates by MRP2 is impaired
Infections Caused By Escherichia Coli Resistant to Norfloxacin in Hospitalized Cirrhotic Patients
Authors: Ortiz Jordi , Vila Maria Carme , Soriano Germán , Miñana Josep , Gana Jordi , Mirelis Beatriz , Novella Maria Teresa , Coll Susana , Sàbat Miriam , Andreu Montserrat , Prats Guillem , Solà Ricard , Guarner Carlos
Published: July 2004

Selective intestinal decontamination with norfloxacin is useful to prevent bacterial infections in several groups of cirrhotic patients at high risk of infection. However, the emergence of infections caused by Escherichia coli resistant to quinolones has recently been observed in cirrhotic patients undergoing prophylactic norfloxacin. Our aim is to determine the characteristics of the infections caused by E. coli resistant to norfloxacin in hospitalized cirrhotic patients. One hundred and six infections caused by E. coli in 99 hospitalized cirrhotic patients were analyzed and distributed into two groups: group I (n = 67), infections caused by E. coli sensitive to norfloxacin, and group II (n = 39), infections caused by E. coli resistant to norfloxacin. The clinical and analytical characteristics at diagnosis of the infection were similar in both groups. Previous prophylaxis with norfloxacin was more frequent in group II (15/67, 22.4% vs. 32/39, 82%, P < .0001), as a result of a higher number of patients submitted to continuous long–term prophylaxis in this group, whereas previous short–term prophylaxis was similar in both groups. Infections were more frequently nosocomial–acquired in group II than in group I (17/67, 25.3% vs. 20/39, 51.2%, P = .01). The type of infections was similar in both groups: urinary tract infections 38 in group I and 24 in group II, spontaneous bacterial peritonitis 8 and 2, spontaneous bacteremia 4 and 4, and bacterascites 1 and 0, respectively (pNS). Mortality during hospitalization was similar in the two groups (4/67, 5.9% vs. 5/39, 12.8%, pNS). None of the E. coli resistant to norfloxacin were also resistant to cefotaxime and only one of them was resistant to amoxicillin–clavulanic acid. Prophylaxis with norfloxacin, usually continuous long–term prophylaxis, favors the development of infections caused by norfloxacin–resistant E. coli. Long–term antibiotic prophylaxis should therefore be restricted to highly selected groups of cirrhotic patients at high–risk of infection. Infections caused by E. coli resistant to norfloxacin show a severity similar to those caused by sensitive E. coli. No significant associated resistance between norfloxacin and the antibiotics most frequently used in the treatment of bacterial infections in cirrhotic patients has been observed.
Ischemia–Reperfusion Injury in Rat Fatty Liver: Role of Nutritional Status
Authors: Caraceni Paolo , Nardo Bruno , Domenicali Marco , Turi Paola , Vici Manuela , Simoncini Mara , De Maria Nicola , Trevisani Franco , Van Thiel David H. , Derenzini Massimo , Cavallari Antonino , Bernardi Mauro
Published: July 2004

Fatty livers are more sensitive to the deleterious effects of ischemia–reperfusion than normal livers. Nutritional status greatly modulates this injury in normal livers, but its role in the specific setting of fatty liver is unknown. This study aimed to determine the effect of nutritional status on warm ischemia–reperfusion injury in rat fatty livers. Fed and fasted rats with normal or fatty liver induced by a choline deficient diet underwent 1 hour of lobar ischemia and reperfusion. Rat survival was determined for 7 days. Serum transaminases, liver histology and cell ultrastructure were assessed before and after ischemia, and at 30 minutes, 2 hours, 8 hours, and 24 hours after reperfusion. Survival was also determined in fatty fasted rats supplemented with glucose before surgery. The preischemic hepatic glycogen was measured in all groups. Whereas survival was similar in fasted and fed rats with normal liver (90% vs. 100%), fasting dramatically reduced survival in rats with fatty liver (14% vs. 64%, P < .01). Accordingly, fasting and fatty degeneration had a synergistic effect in exacerbating liver injury. Mitochondrial damage was a predominant feature of ultrastructural hepatocyte injury in fasted fatty livers. Glucose supplementation partially prevented the fasting–induced depletion of glycogen and improved the 7–day rat survival to 45%. These data indicate that rat fatty livers exposed to normothermic ischemia–reperfusion injury are much more sensitive to fasting than histologically normal livers. Because glucose supplementation improves both the hepatic glycogen stores and the rat survival, a nutritional repletion procedure may be part of a treatment strategy aimed to prevent ischemia–reperfusion injury in fatty livers
Cost Analysis for the Prevention of Variceal Rebleeding: A Comparison Between Transjugular Intrahepatic Portosystemic Shunt and Endoscopic Sclerotherapy in A Selected Group of Italian Cirrhotic Patients
Authors: Meddi Patrizia , Merli Manuela , Lionetti Raffaella , De Santis Adriano , Valeriano Valentina , Masini Andrea , Rossi Plinio , Salvatori Filippo , Salerno Francesco , de Franchis Roberto , Capocaccia Livio , Riggio Oliviero
Published: July 2004

The aim of the present study was to compare the cumulative cost of the first 18–month period in a selected group of Italian cirrhotic patients treated with transjugular intrahepatic portosystemic shunt (TIPS) versus endoscopic sclerotherapy (ES) to prevent variceal rebleeding. Thirty–eight patients enrolled in a controlled trial were considered (18 TIPS and 20 sclerotherapy). The number of days spent in the hospital for the initial treatment and during the follow–up period were defined as the costs of hospitalization. ES sessions, TIPS procedures, angioplasty or addition of a second stent to maintain the shunt patency, were defined as the costs of therapeutic procedures. The two groups were comparable for age, sex, and Child–Pugh score. During the observation period 4 patients died in the TIPS group, and 2 died and 1 was transplanted in the sclerotherapy group. The rebleeding rate was significantly higher in the sclerotherapy group. Despite this, the number of days spent in the hospital was similar in the two groups. This was because of a higher number of hospital admissions for the treatment of hepatic encephalopathy and shunt insufficiency in the TIPS group. The therapeutic procedures were more expensive for TIPS. Consequently, the cumulative cost was higher for patients treated with TIPS than for those treated with sclerotherapy. The extra cost was because of the initial higher cost of the procedure and the difference was still maintained at the end of the 18–month follow–up. When the cumulative costs were expressed per month free of rebleeding, the disadvantage of TIPS disappeared. In conclusion, a program of prevention of variceal rebleeding with TIPS, despite the longer interval free of rebleeding, is not a cost–saving strategy in comparison with sclerotherapy
A Mechanistic Model for the Development and Maintenance of Portocentral Gradients in Gene Expression in the Liver
Authors: Christoffels Vincent M. , Sassi Habib , Ruijter Jan M. , Moorman Antoon F. , Grange Thierry , Lamers Wouter H.
Published: July 2004

In the liver, genes are expressed along a portocentral gradient. Based on their adaptive behavior, a gradient versus compartment type, and a dynamic versus stable type of gradient have been recognized. To understand at least in principle the development and maintenance of these gradients in gene expression in relation to the limited number of signal gradients, we propose a simple and testable model. The model uses portocentral gradients of signal molecules as input, while the output depends on two gene–specific variables, viz., the affinity of the gene for its regulatory factors and the degree of cooperativity that determines the response in the signal–transduction pathways. As a preliminary validity test for its performance, the model was tested on control and hormonally induced expression patterns of phosphoenolpyruvate carboxykinase (PCK), carbamoylphosphate synthetase I (CPS), and glutamine synthetase (GS). Affinity was found to determine the overall steepness of the gradient, whereas cooperativity causes these gradients to steepen locally, as is necessary for a compartment–like expression pattern. Interaction between two or more different signal gradients is necessary to ensure a stable expression pattern under different conditions. The diversity in sequence and arrangement of related DNA–response elements of genes appears to account for the gene–specific shape of the portocentral gradients in expression. The feasibility of testing the function of hepatocyte–specific DNA–response units in vivo is demonstrated by integrating such units into a ubiquitously active promoter/enhancer and analyzing the pattern of expression of these constructs in transgenic mice
Preoperative Portal Vein Embolization: An Audit of 84 Patients
Authors: Imamura Hiroshi , Shimada Ryo , Kubota Mitsuru , Matsuyama Yutaka , Nakayama Ataru , Miyagawa Shin-ichi , Makuuchi Masatoshi , Kawasaki Seiji
Published: July 2004

Preoperative portal vein embolization (PVE) was performed in 84 patients before extensive liver resection for various diseases. By the criteria of liver volumetric determination, some patients were candidates for PVE, whereas others were not, even though the same surgical procedure, such as extended right lobectomy (ERL), was scheduled. PVE using gelatin sponge powder induced hypertrophy in the nonembolized lobe (0%–171%; median, 30%) and proportional atrophy in the embolized lobe in 2 weeks without eliciting any major inflammatory or necrotic reaction, as evidenced histologically and by the minimal elevations in the serum aspartate transaminase (AST) and alanine transaminase (ALT) values. Alterations in the total bilirubin level and prothrombin time were also insignificant and transient, indicating that hepatocyte functions were not impaired by PVE. Not all patients who undergo PVE proceed with the scheduled hepatic resection procedure, so it is a great advantage that gelatin sponge causes minimal damage compared with other embolizing materials such as cyanoacrylate and absolute ethanol, which have been reported to induce an inflammatory reaction or histological alteration. Our multiple regression analysis showed that three factors, diabetes mellitus, a high total bilirubin level at the time of PVE, and being male, each reduced the extent of hypertrophy in the nonembolized lobe (r
2 = .30). By contrast, cholestasis appeared to accelerate the process of atrophy in the embolized lobe (r
2 = .16). In conclusion, PVE by gelatin sponge powder is a safe and effective preoperative maneuver that induces hypertrophy of the section of the liver that will remain after partial hepatectomy
Vascular Endothelial Growth Factor Production in Peritoneal Macrophages of Cirrhotic Patients: Regulation by Cytokines and Bacterial Lipopolysaccharide
Authors: Pérez-Ruiz Manuel , Ros Josefa , Morales-Ruiz Manuel , Navasa Miguel , Colmenero Jordi , Ruiz-del-Arbol Luis , Cejudo Pilar , Clària Joan , Rivera Francisca , Arroyo Vicente , Rodés Juan , Jiménez Wladimiro
Published: July 2004

Vascular endothelial growth factor (VEGF) is an angiogenic peptide with vascular permeability and relaxing properties. This study assessed whether peritoneal macrophages of cirrhotic patients can be up–regulated to produce VEGF under proper stimulatory conditions. Macrophages were isolated from ascites. VEGF protein secretion and mRNA expression were measured in basal conditions and after stimulation with lipopolysaccharide (LPS), tumor necrosis factor α (TNF–α), and interleukin–1 (IL–1). These substances induced a time– and dose–dependent increase in both VEGF production and transcript expression. Assays with actinomycin D showed that VEGF mRNA induction is secondary to both higher VEGF gene transcription and mRNA stability. Ascites and plasma concentration of VEGF was also measured in cirrhotic patients with (n = 15) and without (n = 10) spontaneous bacterial peritonitis (SBP). Plasma values did not differ between both groups of patients. However, ascites VEGF levels were higher in SBP patients than in noninfected cirrhotic patients (710 ± 183 vs. 94 ± 15 pg/mL; P < .025). These results indicate that cytokines and LPS markedly increase VEGF protein secretion and mRNA expression in macrophages of cirrhotic patients, and suggest that this substance could be an important mediator of the pronounced arterial vasodilation frequently occurring in SBP patients
Low–Dose Midazolam Sedation: An Option for Patients Undergoing Serial Hepatic Venous Pressure Measurements
Authors: Steinlauf Adam F. , Garcia-Tsao Guadalupe , Zakko Maram F. , Dickey Kevin , Gupta Tarun , Groszmann Roberto J.
Published: July 2004

The hepatic venous pressure gradient (HVPG) is becoming increasingly used clinically. It is useful in the differential diagnosis of portal hypertension and provides a prognostic index in cirrhotic patients. Performance of serial measurements has been shown to be useful in guiding pharmacological therapy of portal hypertension and variceal hemorrhage. The technique is safe to perform; however, many patients are anxious and reluctant to undergo serial measurements. The effects of sedatives on portal pressure measurements have not yet been defined. The objective of this study was to evaluate the effects of midazolam on the HVPG. Twenty patients with compensated cirrhosis were included in this prospective, double–blind study. The HVPG was determined by subtracting the free hepatic venous pressure (FHVP) from the wedged hepatic venous pressure (WHVP). Patients were randomized to receive either placebo, 0.02 mg/kg midazolam, or 0.03 mg/kg midazolam, administered intravenously over 3 minutes. Immediately after drug administration and every 3 minutes thereafter, for a total of 30 or 40 minutes, measurements were repeated. Three hours later, patients were asked to state whether the sedative affected their state of comfort/relaxation. The effects of both doses of midazolam on HVPG did not differ significantly from those of placebo. Furthermore, neither dose of midazolam induced significant changes in HVPG as compared with baseline values. However, higher–dose midazolam (0.03 mg/kg) was associated with significant reductions in FHVP from baseline and a tendency for a reduction in WHVP. Both doses significantly increased patient comfort and relaxation during the test. Midazolam, used at a dose of 0.02 mg/kg, is effective in increasing patient comfort and relaxation during hepatic venous pressure measurements, without significantly affecting pressures (HVPG, WHVP, or FHVP). It is therefore an acceptable option for patients undergoing serial hepatic venous pressure measurements
No Association Between Hepatitis C and B–Cell Lymphoma
Authors: Collier Jane D. , Zanke Brent , Moore Malcolm , Kessler Garry , Krajden Mel , Shepherd Frances , Heathcote Jenny
Published: July 2004

Chronic viral infection has been implicated in the pathogenesis of B–cell lymphoma, and hepatitis C virus (HCV) infects lymphocytes. Chronic infection with HCV may result in B–cell proliferation. Individuals infected with hepatitis C are often co–infected with the RNA virus GB virus type C. Studies from Europe where hepatitis C infection is more common than in North America have shown a high prevalence of hepatitis C infection in patients with B–cell lymphoma. The aim of this study was to establish the prevalence of HCV and GBV–C infection in patients with B–cell lymphoma in an area of low HCV prevalence. One hundred patients with B–cell lymphoma (10 high grade, 46 intermediate grade, and 44 low grade) and 100 controls with nonhematological malignancies were studied. Serum was analyzed for HCV antibodies by third generation enzyme–linked immunosorbant assay, and HCV RNA and GBV–C RNA was analyzed by reverse transcriptase PCR. None of the controls or lymphoma patients had antibodies to HCV. HCV RNA was undetected in 60 out of 100 lymphoma patients tested. GBV–C RNA was detected in the serum of 5 out of 100 (5%) of lymphoma patients and in 3 out of 100 (3%) controls. Hepatitis C and GBV–C are, therefore, unlikely to play a major role in the pathogenesis of B–cell lymphoma in North America.
Lipopolysaccharide Induces Cholangiocyte Proliferation Via An Interleukin–6-Mediated Activation of P44/P42 Mitogen–Activated Protein Kinase
Authors: Park Joongwon , Gores Gregory J. , Patel Tushar
Published: July 2004

The biliary epithelium is exposed to mediators of inflammation such as bacterial endotoxin or lipopolysaccharide (LPS) in a variety of inflammatory conditions. These conditions are also characterized by cholangiocyte proliferation and a predisposition to malignancy. Furthermore, LPS can enhance the expression of interleukin–6 (IL–6), a known biliary mitogen. However, the effects of LPS on cholangiocyte proliferation or IL–6 secretion are unknown. Thus, our aims were to determine if LPS stimulates cholangiocyte proliferation by IL–6-dependent signaling pathways. H69 cells derived from normal human intrahepatic cholangiocytes proliferated in response to LPS. Cholangiocytes responded to LPS (and other inflammatory cytokines such as tumor necrosis factor α [TNF–α] and IL–1β) by increased secretion of IL–6, which had a mitogenic effect on H69 cells. Preincubation with anti-IL–6 neutralizing antibodies inhibited LPS–induced proliferation. Furthermore, cholangiocytes possessed the IL–6 receptor complex subunits and intact signaling mechanisms leading to activation of signal transducers and activators of transcription (STAT) factors. Although both p38 and p44/p42 mitogen–activated protein kinases (MAPKs) were constitutively present and active in cholangiocytes, IL–6 increased p44/p42, but not p38 MAPK activity. PD098059 inhibited activation of p44/p42 MAPK in cholangiocytes and completely blocked DNA synthesis in response to IL–6 or LPS. These studies identify a critical role for the p44/p42 MAPK in cholangiocyte proliferation and demonstrate that the proliferative response of cholangiocytes to inflammatory mediators such as LPS involves IL–6-mediated activation of the p44/p42 MAPK pathway
Determination of the Chelatable Iron Pool of Isolated Rat Hepatocytes by Digital Fluorescence Microscopy Using the Fluorescent Probe, Phen Green Sk
Authors: Petrat Frank , Rauen Ursula , de Groot Herbert
Published: July 2004

The intracellular pool of chelatable iron is considered to be a decisive pathogenetic factor for various kinds of cell injury. We therefore set about establishing a method of detecting chelatable iron in isolated hepatocytes based on digital fluorescence microscopy. The fluorescence of hepatocytes loaded with the fluorescent metal indicators, phen green SK (PG SK), phen green FL (PG FL), calcein, or fluorescein desferrioxamine (FL–DFO), was quenched when iron was added to the cells in a membrane–permeable form. It increased when cellular chelatable iron available to the probe was experimentally decreased by an excess of various membrane–permeable transition metal chelators. The quenching by means of the ferrous ammonium sulfate + citrate complex and also the “dequenching” using 2,2′–dipyridyl (2,2′–DPD) were largest for PG. We therefore optimized the conditions for its use in hepatocytes and tested the influence of possible confounding factors. An ex situ calibration method was set up to determine the chelatable iron pool of cultured hepatocytes from the increase of PG SK fluorescence after the addition of excess 2,2′–DPD. Using this method, we found 9.8 ± 2.9 μmol/L (mean ± SEM; n = 18) chelatable iron in rat hepatocytes, which constituted 1.0% ± 0.3% of the total iron content of the cells as determined by atomic absorption spectroscopy. The concentration of chelatable iron in hepatocytes was higher than the one in K562 cells (4.0 ± 1.3 μmol/L; mean ± SEM; n = 8), which were used for comparison. This method allowed us to record time courses of iron uptake and of iron chelation by different chelators (e.g., deferoxamine, 1,10–phenanthroline) in single, intact cells
Characterization of the Effects of Hepatitis C Virus Nonstructural 5A Protein Expression in Human Cell Lines and on Interferon–Sensitive Virus Replication
Authors: Polyak Stephen J. , Paschal Denise M. , McArdle Susan , Gale Michael J. , Moradpour Darius , Gretch David R.
Published: July 2004

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. Biochemical studies have demonstrated that NS5A interacts in vitro with and inhibits the IFN–induced, RNA–dependent protein kinase, PKR, and that NS5A interacts with at least one other cellular kinase. The present study describes the establishment and characterization of various stable NS5A–expressing human cell lines, and the development of a cell culture-based assay for determining the inherent IFN resistance of clinical NS5A isolates. Human epithelioid (Hela) and osteosarcoma (U2–OS) cell lines were generated that express NS5A under tight regulation by the tetracycline–dependent promoter. Maximal expression of NS5A occurred at 48 hours following the removal of tetracycline from the culture medium. The half–life of NS5A in these cell lines was between 4 to 6 hours. NS5A protein expression was localized cytoplasmically, with a staining pattern consistent with the location of the Golgi apparatus and endoplasmic reticulum. In the majority of cell lines, no obvious phenotypic changes were observed. However, three genotype 1b NS5A-expressing osteosarcoma cell lines exhibited cytopathic effect and severely reduced proliferation as a result of high–level NS5A expression. Full–length NS5A protein isolated from a genotype 1b IFN-nonresponsive patient (NS5A–1b) was capable of rescuing encephalomyocardititis virus replication during IFN challenge up to 40–fold, whereas a full–length NS5A–1a and an interferon sensitivity determining region (ISDR) deletion mutant (NS5A–1a–△ISDR) isolated from a genotype 1a IFN-nonresponsive patient showed no rescue activity. The NS5A–1b and NS5A–1a proteins also rescued vesicular stomatitis virus replication during IFN treatment by two– to threefold. These data cummulatively suggest that NS5A expression alone can render cells partially resistant to the effects of IFN against IFN–sensitive viruses, and that in some systems, these effects may be independent of the putative ISDR. A scenario is discussed in which the NS5A protein may employ multiple strategies contributing to IFN resistance during HCV infection.
Human Leukocyte Antigen Class Ii and Iii Alleles and Severity of Hepatitis C Virus-Related Chronic Liver Disease
Authors: Asti Margherita , Martinetti Miryam , Zavaglia Claudo , Cuccia Maria Clara , Gusberti Laura , Tinelli Carmine , Cividini Agostino , Bruno Savino , Salvaneschi Laura , Ideo Gaetano , Mondelli Mario U. , Silini Enrico M.
Published: July 2004

Hepatitis C outcome is likely related both to viral factors and host's immune responses. We correlated the severity of liver disease with human leukocyte antigen (HLA) genes (C4A, C4B, TNFA, TNFB, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, TAP1, and TAP2) in three groups of subjects: 99 patients with chronic hepatitis, 41 asymptomatic carriers, and 179 uninfected controls. Patients with grade/stage 3 to 4 hepatitis significantly differentiated for their low frequency of alleles TNFB*1, DRB1*1104, and DRB3*03, which had a protective role, and high frequency of allele DRB1*1001, which was associated with disease severity. HLA–DRB*11 subtypes were differentially distributed: DRB1*1104 was most frequent in carriers, whereas DRB1*1101 was more frequent in patients. The TAP1C,2A haplotype was also underrepresented in patients with respect to controls. Finally, a decrease of heterozygous subjects was observed in patients with respect to carriers at the DQB1 locus. Multivariate analysis by correspondence analysis and multiple logistic regression indicated that age, sex, and hepatitis C virus (HCV) type were the strongest risk factors; however, some immunogenetic variables (TNFB*1, DRB1*1104, and DRB3*03) showed an independent contribution, especially in comparing patients with extreme manifestations of disease. The involvement of different genes in various HLA subregions suggests that anti–HCV responses are modulated by a complex gene interplay rather than by single alleles