bluuebunny/crossref_metadata_2025 huggingface dataset:
Gap Junction–Mediated Intercellular Communication in A Long–Term Primary Mouse Hepatocyte Culture System
Authors: Stoehr Stephanie A. , Isom Harriet C.
Published: October 2003

Gap junction–mediated intercellular communication (GJIC) is critical for maintaining integral cellular processes including differentiation and growth control. The disruption of GJIC has been correlated with aberrant function in many cell types, including hepatocytes in vivo; therefore it is imperative that cellular model systems support intercellular communication to simulate normal cellular functions. Functional GJIC has been shown in long–term primary rat hepatocyte cultures, which have been implemented widely to study various aspects of hepatocellular function; however, the onset of transgenic technology in murine species has necessitated the development of a primary mouse hepatocyte system. In this report, we analyze GJIC in a dimethylsulfoxide (DMSO)–containing long–term primary mouse hepatocyte culture system. The cells retain morphologic and biochemical characteristics of differentiated hepatocytes through day 30 post plating, including liver–specific gene expression. We further show that connexin32 and connexin26 expression and gap junction plaque formation increase over time in culture concomitant with an increase in GJIC between adjoining primary mouse hepatocytes. In conclusion, the findings described in this study make it possible to maintain differentiated primary mouse hepatocytes that also show GJIC in long–term culture for 30 days. In addition, this system has the potential to be extended to study primary mouse hepatocytes isolated from genetically engineered mice.
Intracellular Accumulation of Piga–R and Regulators of Transcytotic Trafficking in Cholestatic Rat Hepatocytes
Authors: Larkin Janet M. , Coleman Hope , Espinosa Angelica , Levenson Amy , Park Mee Soon , Woo Bonnie , Zervoudakis Alice , Tinh Vu
Published: October 2003

Bile duct ligation (BDL) impairs basolateral–to–apical transcytosis in hepatocytes, causing accumulation of transcytotic carriers for the polymeric IgA receptor (pIgA–R) and redistribution of secretory component (SC) from bile to blood. To gain insight into the mechanisms regulating transcytosis and the pathophysiology of cholestasis, we investigated nascent protein trafficking in control and BDL livers using cell fractionation in the context of in vivo pulse–chase experiments and immunoblot analysis. Control and cholestatic hepatocytes trafficked [35S]–labeled serum proteins and the pIgA–R along the secretory pathway with identical kinetics. However, BDL impaired transcytosis, causing (1) accumulation of the pIgA–R, rab3D, rab11a, and other candidate regulators of apical–directed secretion in a crude vesicle carrier fraction (CVCF) enriched in transcytotic carriers; (2) slow delivery of [35S]–labeled SC to bile; and (3) paracellular reflux of SC from bile to blood. In conclusion, these data indicate that the secretory and transcytotic pathways remain polarized in cholestatic hepatocytes and suggest that the pIgA–R traffics through postendosomal rab3D–, rab11a–, and syntaxin 2–associated compartments, implicating these proteins in the regulation of transcytosis.
Patients With Primary Biliary Cirrhosis React Against A Ubiquitous Xenobiotic–Metabolizing Bacterium
Authors: Selmi Carlo , Balkwill David L. , Invernizzi Pietro , Ansari Aftab A. , Coppel Ross L. , Podda Mauro , Leung Patrick S. , Kenny Thomas P. , Van De Water Judy , Nantz Michael H. , Kurth Mark J. , Gershwin Eric M.
Published: October 2003

Infectious and environmental agents have been proposed as immunologic triggers for primary biliary cirrhosis (PBC). Recently, a ubiquitous organism that metabolizes organic compounds and estrogens, Novosphingobium aromaticivorans, has been defined. Importantly, 2 bacterial proteins have homology with the E2 component of the pyruvate dehydrogenase complex (PDC–E2). Sera from 97 patients with PBC, 46 first–degree relatives, 10 spouses, and 195 controls were studied for reactivity against N. aromaticivorans and Escherichia coli. The reactivity was defined by absorption, affinity purification, and using monoclonal antibodies to PDC–E2. Stool samples from 20 patients with PBC and 34 controls were analyzed by polymerase chain reaction (PCR) for the presence of N. aromaticivorans. Sera from 100% of anti–PDC–E2 positive (77/77), 33% of anti–BCOADC E2 positive (1/3), and 12% of antimitochondrial antibody (AMA) negative patients with PBC (2/17) reacted with titers up to 10-6 against two known lipoylated bacterial proteins (47 and 50 kd) from N. aromaticivorans, including patients with early disease. This titer was approximately 100– to 1,000–fold higher than against E. coli and verified by absorption, use of affinity–purified sera, and monoclonal antibody reagents. Moreover, 78 of 80 AMA–positive and 5 of 17 AMA–negative patients with PBC had antibodies against 3 other N. aromaticivorans proteins. In contrast, 0 of 195 control sera reacted against N. aromaticivorans. Approximately 25% of patients and controls had N. aromaticivorans in their fecal specimens. In conclusion, based on protein homology, capacity to metabolize xenobiotics as well as modulate estrogens, its presence in feces, and specific immunologic response, we propose that N. aromaticivorans is a candidate for the induction of PBC.
Systemic, Renal, and Hepatic Hemodynamic Derangement in Cirrhotic Patients With Spontaneous Bacterial Peritonitis
Authors: Ruiz-del-Arbol Luis , Urman Jesús , Fernández Javier , González Mónica , Navasa Miguel , Monescillo Alberto , Albillos Agustín , Jiménez Wladimiro , Arroyo Vicente
Published: October 2003

Spontaneous bacterial peritonitis (SBP) is frequently associated with renal failure. This study assessed if systemic and hepatic hemodynamics are also affected by this condition. Standard laboratory tests, tumor necrosis factor α (TNF–α) in plasma and ascitic fluid, plasma renin activity (PRA) and norepinephrine (NE), and systemic and hepatic hemodynamics were determined in 23 patients with SBP at diagnosis and after resolution of infection. Eight patients developed renal failure during treatment. At diagnosis of infection, patients developing renal failure showed significantly higher values of TNF–α, blood urea nitrogen (BUN), PRA and NE, peripheral vascular resistance, and hepatic venous pressure gradient (HVPG) and lower cardiac output than patients not developing renal failure. During treatment, a significant reduction in cardiac output and arterial pressure and increase in PRA and NE, HVPG, and Child–Pugh score were observed in the first group but not in the second. Peripheral vascular resistance remained unmodified in both groups. Changes in PRA and NE correlated inversely with changes in arterial pressure and directly with changes in BUN, Child–Pugh score, and HVPG. Five patients in the renal failure group developed encephalopathy, and 6 died. In the group without renal failure, none of the patients developed encephalopathy or expired. In conclusion, patients with SBP frequently develop a rapidly progressive impairment in systemic hemodynamics, leading to severe renal and hepatic failure, aggravation of portal hypertension, encephalopathy, and death. This occurs despite rapid resolution of infection and is associated with an extremely poor prognosis.
Establishment, Characterization, and Long–Term Maintenance of Cultures of Human Fetal Hepatocytes
Authors: Lázaro Catherine A. , Croager Emma J. , Mitchell Claudia , Campbell Jean S. , Yu Changpu , Foraker Jessica , Rhim Jonathan A. , Yeoh George C.T. , Fausto Nelson
Published: October 2003

Cultured human hepatocytes have broad research and clinical applications; however, the difficulties in culturing rodent and human hepatocytes are well known. These problems include the rapid loss of the hepatocytic phenotype in primary culture and the limited replicating capacity of the cultured cells. We describe the establishment of serum–free primary cultures of human fetal hepatocytes (HFHs) that retain hepatocytic morphology and gene expression patterns for several months and maintain sufficient proliferative activity to permit subculturing for at least 2 passages. Initially, HFH cultures contained 2 main cell types that morphologically resembled large and small hepatocytes. The fetal hepatocytes expressed α–fetoprotein (AFP), cytokeratin (CK) 19, albumin, and other hepatic proteins. Treatment of the cultures with oncostatin M (OSM) increased cell size and enhanced cell differentiation and formation of bile canaliculi, probably through an effect on hepatocyte nuclear factor (HNF) 4α. Approximately 1 month after plating, multiple clusters of very small cells became apparent in the cultures. These cells had very few organelles and are referred to as blast–like cells. Flow cytometric analysis of these cells showed that they express oval cell/stem cell markers such as CD90 (Thy–1), CD34, and OV–6 but do not stain with antibodies to β2–microglobulin. HFH cultures maintained for 9 to 12 months produced grossly visible organoids containing ductular structures that stained for CK18, CK19, and AFP. In conclusion, HFH cultures, which might contain a population of hepatic stem cells, constitute an excellent tool for a variety of studies with human hepatocytes, including the mechanisms of viral infection.
Cyclosporin A Suppresses Replication of Hepatitis C Virus Genome in Cultured Hepatocytes
Authors: Watashi Koichi , Hijikata Makoto , Hosaka Masahiro , Yamaji Masashi , Shimotohno Kunitada
Published: October 2003

Persistent infection of hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Searching for a substance with anti–HCV potential, we examined the effects of a variety of compounds on HCV replication using a HCV subgenomic replicon cell culture system. Consequently, the immunosuppressant cyclosporin A (CsA) was found to have a suppressive effect on the HCV replicon RNA level and HCV protein expression in these cells. CsA also inhibited multiplication of the HCV genome in a cultured human hepatocyte cell line infected with HCV using HCV–positive plasma. This anti–HCV activity of CsA appeared to be independent of its immunosuppressive function. In conclusion, our results suggest that CsA may represent a new approach for the development of anti–HCV therapy.
Alcohol–Induced Liver Injury in Mice Lacking Cu, Zn–Superoxide Dismutase
Authors: Kessova Irina G. , Ho Ye-Shih , Thung Swan , Cederbaum Arthur I.
Published: October 2003

Because alcoholic liver disease has been linked to oxidative stress, we investigated the effect of a compromised antioxidant defense system, Cu, Zn–superoxide dismutase (Sod1) deficiency, on alcohol–induced liver injury. C57BL/129SV wild–type (Sod1
+/+) and Sod1 knockout (Sod1
-/-) mice were fed dextrose or ethanol (10% of total calories) liquid diets for 3 weeks. Histologic evaluation of liver specimens of Sod1
-/- mice fed ethanol showed the development of liver injury ranging from mild to extensive centrilobular necrosis and inflammation. Sod1
+/+ mice fed ethanol showed mild steatosis; both Sod1
+/+ and Sod1
-/- mice fed the dextrose diet had normal histology. Alanine transaminase levels were significantly elevated only in Sod1
-/- mice fed ethanol. Cytochrome P450 2E1 (CYP2e1) activity was elevated about 2–fold by ethanol in Sod1
+/+ and Sod1
-/- mice. Ethanol consumption increased levels of protein carbonyls and lipid peroxidation aldehydic products in the liver of Sod1
-/- mice. Hepatic adenosine triphosphate (ATP) content was reduced dramatically in Sod1
-/- mice fed ethanol in association with a decrease in the mitochondrial reduced glutathione (GSH) level and activity of MnSOD. Immunohistochemical determination of 3–nitrotyrosine (3NT) residues in liver sections of the Sod1 knockout mice treated with ethanol showed a significant increase of 3NT staining in the centrilobular areas. In conclusion, a rather moderate ethanol consumption promoted oxidative stress in Sod1
-/- mice, with increased formation of peroxynitrite, protein carbonyls, and lipid peroxidation and decreased mitochondrial GSH and MnSOD. We speculate that the increased oxidative stress causes mitochondrial damage and reduction of ATP content, leading to alcoholic liver injury. This model may be useful in further mechanistic studies on alcohol–induced liver injury.
Hepatitis B Virus X Protein Regulates Transactivation Activity and Protein Stability of the Cancer–Amplified Transcription Coactivator Asc–2
Authors: Kong Hee Jeong , Park Min Jung , Hong SunHwa , Yu Hyun Jung , Lee Young Chul , Choi Young Hyun , Cheong JaeHun
Published: October 2003

Hepatitis B virus X protein (HBx) is a transcriptional coactivator that plays a significant role in the regulation of genes involved in inflammation and cell survival. A recently identified cellular coactivator, activating signal cointegrator 2 (ASC–2), is enriched in liver cancer cells and associates with many transcription factors that are active in hepatocytes. The tissue colocalization of these 2 proteins, in view of their similar regulatory functions, led us to examine whether HBx and ASC–2 cooperate in transcriptional activation of gene expression. Glutathione S–transferase (GST) pull–down assays and mammalian 2–hybrid analysis show that the transactivation domain of HBx interacts with the C–terminal domain of ASC–2. In fact, these 2 proteins associated in a ternary complex that included the transcriptional activator retinoid X receptor (RXR). Mechanistically, on expression of HBx, the half–life of the ASC–2 coactivator is observed to increase in concordance with the observed increase in ASC–2–dependent coactivation of transcription. In conclusion, these results show that HBx stabilizes the cellular coactivator ASC–2 through direct protein–protein interaction, affecting the regulation of genes actively transcribed in liver cancer cells.
Exploring the Biological Basis of Hepatitis B E Antigen in Hepatitis B Virus Infection
Authors: Milich David , Liang Jake T.
Published: October 2003

The function of the hepatitis B e antigen (HBeAg) is largely unknown because it is not required for viral assembly, replication, or infection. In this report we chronicle clinical and experimental studies in an attempt to understand the role of HBeAg in natural infection. These studies largely have focused on clinical–pathologic features of HBeAg–negative variants in acute and chronic HBV infection, mutational analysis in animal models of hepadnavirus infection, and the use of transgenic murine models. The clinical and experimental data suggest that serum HBeAg may serve an immunoregulatory role in natural infection. To the contrary, cytosolic HBeAg serves as a target for the inflammatory immune response. These dual roles of the HBeAg and its ability to activate or tolerize T cells show the complexity of the interactions between the HBeAg and the host during HBV infection.
Ptk2 And Eif3s3 Genes May Be Amplification Targets At 8Q23–Q24 and Are Associated With Large Hepatocellular Carcinomas
Authors: Okamoto Hiroyuki , Yasui Kohichiroh , Zhao Chen , Arii Shigeki , Inazawa Johji
Published: October 2003

We investigated 39 primary hepatocellular carcinomas (HCCs) for aberrations in DNA copy number, using comparative genomic hybridization (CGH). Gain of DNA at 8q was common in these tumors; high–level gains, indicative of gene amplification, occurred most frequently at 8q23–q24. Gains of 8q correlated with large (>5 cm) tumor size. To identify targets of the amplification events involving 8q, we determined expression levels of 14 candidate genes within that region in a total of 41 HCCs by means of real–time quantitative reverse–transcription polymerase chain reactions (RT–PCR). Significant correlation was found between elevated levels of expression and increases in copy number for PTK2 (located at 8q24.3) and EIF3S3 (at 8q23.3), but for none of the other candidates, which included MYC (8q24.1). Southern blot analyses confirmed that PTK2 and EIF3S3 were amplified, respectively, in 5 (19%) and 7 (26%) of the 27 tumors examined in accordance with expression patterns, an indication that expression of PTK2 and EIF3S3 was probably up–regulated by the amplification mechanism. When we analyzed potential relationships between elevated expression of PTK2 and EIF3S3 and clinicopathologic parameters, high expression of the 2 transcripts was significantly associated with large (>5 cm) tumor size and with hepatitis B virus (HBV) infection. In conclusion, PTK2 and EIF3S3, which, respectively, encode focal adhesion kinase and the p40 subunit of the eukaryotic initiation factor 3, were probable targets within the amplification at 8q23–q24 and may be involved in progression of HCC.
Determinants for Sustained Hbeag Response to Lamivudine Therapy
Authors: Chien Rong-Nan , Yeh Chau-Ting , Tsai Sun-Lung , Chu Chia-Ming , Liaw Yun-Fan
Published: October 2003

There are inconsistent data on the durability of hepatitis B e antigen (HBeAg) seroconversion after lamivudine is discontinued. The aim of this study was to examine the determinants for sustained HBeAg response to lamivudine therapy. Both host and viral factors as well as the drug factor were compared between 43 patients with sustained HBeAg response and 39 patients whose response was not sustained. All of them received a mean period of 16 months (range, 3–55 months) lamivudine therapy and had achieved complete response (HBeAg seroconversion plus HBV DNA seroclearance by hybrid capture assay and normal alanine aminotransferase [ALT]) and were followed–up for a mean period of 44 months (range, 12–88 months). Stepwise logistic regression model was used to estimate the sustained response on the presence of the following variables: age; gender; pretherapy ALT; total bilirubin and HBV DNA levels; time to HBeAg seroconversion; additional lamivudine treatment after HBeAg seroconversion; total duration of treatment; hepatitis activity index scores; periportal, intralobular, and portal inflammation and fibrosis scores; scores excluding fibrosis; status of precore mutation; basal core promoter mutation; and genotype. The results showed that genotype (OR, 5.922; 95% CI, 1.611–21.768; P = .007), age (OR, 0.943; 95% CI, 0.891–0.997; P = .040), and additional treatment (OR, 1.097; 95% CI, 1.028–1.171; P = .005) were independent factors to sustained HBeAg response. Further categorical analysis disclosed that patients with genotype B, age ≤36 years, and additional lamivudine treatment over 8 months have higher sustained response. In conclusion, HBV genotype, age, and additional treatment are the major determinants for the sustained HBeAg response to lamivudine therapy.
Membrane Cholesterol But Not Putative Receptors Mediates Anandamide–Induced Hepatocyte Apoptosis
Authors: Biswas Kamal Krishna , Sarker Krishna Pada , Abeyama Kazuhiro , Kawahara Ko-ichi , Iino Satoshi , Otsubo Yasuharu , Saigo Kazuhiko , Izumi Hiroyuki , Hashiguchi Teruto , Yamakuchi Munekazu , Yamaji Kazuyo , Endo Ryujin , Suzuki Kazuyuki , Imaizumi Hitoshi , Maruyama Ikuro
Published: October 2003

The endogenous cannabinoid anandamide, a lipid mediator, induces various physiologic events such as vascular relaxation, inhibition of gap–junctions formation, tumor proliferation, neurologic analgesia, and apoptosis. Although increased concentration of anandamide in plasma has been implicated in pathophysiologic states including endotoxin–induced hypotension, the effects of anandamide on hepatocytes still remain unclear. In this study, we present evidence that plasma anandamide concentration is highly increased in severe hepatitis and cirrhosis patients. In addition, concentrations of anandamide within the pathophysiologic range potently induced apoptosis of hepatoma cell line (Hep G2) and primary hepatocytes, suggesting a possible link between increased anandamide level and hepatocyte damage. Anandamide–induced cell death was preceded by G0/G1 cell–cycle arrest, activation of proapoptotic signaling (i.e., p38 MAPK and JNK), and inhibition of antiapoptotic signaling (i.e., PKB/Akt) pathways. Moreover, anandamide increased susceptibility to oxidative stress–induced hepatocyte damage. In this context, methyl–β–cyclodextrin (MCD), a membrane cholesterol depletor, or mevastatin, an HMG–CoA reductase inhibitor, or N–acetyl cysteine, an antioxidant, potently inhibited the anandamide–induced proapoptotic events and cell death, whereas putative cannabinoid receptor antagonists did not exhibit an inhibitory effect on anandamide–induced cell death. Furthermore, binding assay using polymyxin beads revealed that anandamide could interact with cholesterol. In conclusion, our data suggest that cholesterol present in the cell membrane determines the fate of hepatocytes exposed to anandamide, possibly functioning as an anandamide receptor.
Inhibition of Proteasome Function Leads to Nf–κB–Independent Il–8 Expression in Human Hepatocytes
Authors: Joshi-Barve Swati , Barve Shirish S. , Butt Waseem , Klein Jon , McClain Craig J.
Published: October 2003

Breakdown of cellular proteins is a highly regulated process, and the ubiquitin–proteasome pathway is the major proteolytic system in the cell. It regulates the levels of numerous proteins that control gene expression and cell division, as well as responses to stress and inflammation. Recent studies have reported abnormalities in proteasome function in alcoholic liver disease (ALD). Moreover, a direct relation has been reported between impaired proteasome function and oxidative stress in experimental models of ALD. Neutrophil infiltration is a hallmark of ALD, and activated neutrophils are thought to play a role in the pathology of ALD. As a potent neutrophil chemoattractant and activator, interleukin 8 (IL–8) likely plays a key mechanistic role in many forms of liver injury. In this study, we evaluated the effects of inhibition of proteasome function on expression and release of IL–8 by human fetal hepatocytes and hepatoma cells. Our data demonstrate that inhibition of proteasome function in hepatocytes leads to apoptotic cell death. Decreased hepatocyte survival coincides with enhanced expression of IL–8, both at the protein and the messenger RNA (mRNA) levels. This increase in IL–8 is independent of nuclear factor κB (NF–κB) activation and is associated with an increase in c–Jun N–terminal kinase (JNK) and activator protein–1 (AP–1) activity. In conclusion, hepatocytes dying because of inhibition of proteasome function produce massive quantities of the proinflammatory chemokine IL–8, possibly resulting in neutrophil infiltration, increased inflammation, and liver injury.
Role of Vα 14 Nkt Cells in the Development of Impaired Liver Regeneration In Vivo
Authors: Ito Hiroyasu , Ando Kazuki , Nakayama Toshinori , Taniguchi Masaru , Ezaki Takayuki , Saito Kuniaki , Takemura Masao , Sekikawa Kenji , Imawari Michio , Seishima Mitsuru , Moriwaki Hisataka
Published: October 2003

Although we have previously demonstrated that IL–12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL–12 or by α–galactosylceramide (α–GalCer) on murine liver regeneration using Vα 14 NKT knockout (Jα 281-/-) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jα 281+/+ but not in Jα 281-/- mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) γ and tumor necrosis factor α (TNF–α) messenger RNA (mRNA) after stimulation with both factors in Jα 281+/+ mice. Either anti–IFN–γ or TNF–α antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF–α injection similarly caused injury in hepatectomized livers of both Jα 281+/+ and Jα 281-/- mice; indeed, adoptively transferred TNF–α+/+ NKT cells enhanced liver injury after hepatectomy in TNF–α knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF–α synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.
Kupffer Cell Engulfment of Apoptotic Bodies Stimulates Death Ligand and Cytokine Expression
Authors: Canbay Ali , Feldstein Ariel E. , Higuchi Hajime , Werneburg Nate , Grambihler Annette , Bronk Steve F. , Gores Gregory J.
Published: October 2003

Hepatocyte apoptosis by death receptors, hepatic inflammation, and fibrosis are prominent features of liver diseases. However, the link between these processes remains unclear. Our aim was to ascertain whether engulfment of apoptotic bodies by Kupffer cells promotes hepatic inflammation and fibrosis. Isolated murine Kupffer cells efficiently engulfed apoptotic bodies generated from UV–treated mouse hepatocytes. Engulfment of the apoptotic bodies, but not latex beads, stimulated Kupffer cell generation of death ligands, including Fas ligand, and tumor necrosis factor α (TNF–α). Both apoptotic body phagocytosis and death ligand generation were attenuated by gadolinium chloride, a Kupffer cell toxicant. Kupffer cells isolated from 3–day bile duct–ligated (BDL) mice were phenotypically similar to apoptotic body–“fed” Kupffer cells with enhanced death ligand expression; inhibition of hepatocyte apoptosis with a caspase inhibitor prevented this Kupffer cell activation. Consistent with a role for Kupffer cells in liver inflammation and fibrosis, gadolinium chloride attenuated neutrophil infiltration and markers for stellate cell activation. In conclusion, these findings support a model of cholestatic liver injury where Kupffer cell engulfment of apoptotic bodies promotes inflammation and fibrogenesis.
Adjuvant Intra–Arterial Injection of Iodine–131-Labeled Lipiodol After Resection of Hepatocellular Carcinoma
Authors: Boucher Eveline , Corbinais Stéphane , Rolland Yan , Bourguet Patrick , Guyader Dominique , Boudjema Karim , Meunier Bernard , Raoul Jean-Luc
Published: October 2003

The high rate of recurrence after surgical resection of hepatocellular carcinoma (HCC) is a major therapeutic challenge. Postoperative injection of 131–iodine–labeled lipiodol (131I–Lip) into the hepatic artery has been proposed as adjuvant treatment (Lau et al.). We analyzed 2 retrospective series of matched patients treated in our unit before and after addition of 131I–Lip adjuvant therapy to our standard surgical strategy. Thirty–eight patients who had undergone surgical resection of HCC after January 1999 were given adjuvant intra–arterial injection of 131I–Lip after surgery. These patients were matched with 38 other patients who had undergone surgical resection only between January 1997 and January 1999. The frequency of recurrences, disease–free rates, and overall survival rates were compared. The 2 groups were similar for clinical, biologic, or histologic parameters studied and Cancer Liver Italian Program scores. There were 15 recurrences in the group without adjuvant treatment and 9 in the group with 131I–Lip adjuvant treatment. The 1–, 2–, and 3–year disease–free survival rates (±95% confidence interval) were different (P < .02): 94.7% ± 3.6%, 83.7% ± 6.1%, and 68.4% ± 9.7%, respectively, in the 131I–Lip group versus 73.7% ± 7.1%, 54.3% ± 8.2%, and 41.5% ± 10.5% in the surgery group. The 1–, 2–, and 3–year survival rates (±95% confidence interval) also were different (P < .02): 94.7% ± 3.6%, 91.7% ± 4.6%, and 91.7% ± 4.6%, respectively, in the 131I–Lip group versus 94.7% ± 3.6%, 71.3% ± 7.8%, and 49.9% ± 10% in the surgery group. In conclusion, this retrospective analysis supports the promising contribution of postoperative injection of 131I–Lip after resection of HCC. A randomized study including more patients would be necessary to confirm its contribution to therapeutic management.
A Pilot Study of Therapeutic Vaccination With Envelope Protein E1 in 35 Patients With Chronic Hepatitis C
Authors: Nevens Frederik , Roskams Tania , Van Vlierberghe Hans , Horsmans Yves , Sprengers Dirk , Elewaut Ann , Desmet Valeer , Leroux-Roels Geert , Quinaux Emmanuel , Depla Erik , Dincq Stephanie , Stichele Christine Vander , Maertens Geert , Hulstaert Frank
Published: October 2003

New treatments are needed for chronic hepatitis C patients in whom viral clearance cannot be achieved. Thirty–five chronic hepatitis C patients (genotype 1) were randomized to receive 20 μg of recombinant HCV E1 (E1) (n = 26) or placebo (n = 9) intramuscularly at weeks 0, 4, 8, 12, and 24. Thirty–four then received open–label E1 vaccine at weeks 50, 53, 56, 59, 62, and 65. Twenty–four patients (12 men, 12 women; mean age, 52 y; 18 interferon–based treatment failures; mean baseline alanine aminotransferase [ALT] level, 118 IU/L) underwent a biopsy before and after 2 courses of E1, 17 months later. Liver histology was scored by 2 blinded pathologists according to the Ishak and Metavir systems. Postinjection reactions were similar to placebo (alum only). Nine of 24 patients (38%) had improvement of 2 points or more, 10 (41%) remained stable, and 5 (21%) showed worsening in total Ishak score. Nine patients (38%) improved both on Ishak and Metavir fibrosis scores. Plasma HCV–RNA levels remained unchanged, whereas ALT levels showed a trend toward a decrease during treatment. All but 3 patients developed a significantde novoE1–specific T–cell response. The increase in anti–E1 antibody levels correlated with the decrease in total Ishak score and with the relative decreases in both Ishak fibrosis score and ALT level (allP≤ .01). In conclusion, E1 therapeutic vaccination is well tolerated and the observed effects warrant further study.
Validity of the 13C–Caffeine Breath Test As A Noninvasive, Quantitative Test of Liver Function
Authors: Park Gordon Jung-Hyuk , Katelaris Peter Harry , Jones Brian D. , Seow Francis , Le Couteur David George , Ngu Meng Chong
Published: October 2003

The properties of caffeine render it an ideal substrate for a quantitative test of liver function. The aim of this study was to determine whether the caffeine breath test (CBT) using orally administered 13C–caffeine correlates reliably with plasma caffeine clearance and reflects varying degrees of liver dysfunction. The CBT was performed in 25 healthy controls; 20 subjects with noncirrhotic, chronic hepatitis B or C; and 20 subjects with cirrhosis. Plasma caffeine clearance was assayed simultaneously with the CBT in a cohort of these subjects. Over a broad range of caffeine clearances, the CBT exhibited a highly significant correlation with plasma clearance (r = 0.85, P < .001). Cirrhotic patients were characterized by significantly reduced CBT values (1.15 ± 0.75 Δ‰ mg-1) compared with controls (2.23 ± 0.76; P = .001) and hepatitic patients (1.83 ± 1.05; P = .04). There was a significant inverse relationship between the CBT and Child–Pugh score (r = -.74, P = .002). The intraclass correlation coefficient between repeated CBTs in 20 subjects with normal and cirrhotic livers was 0.89. Although smoking was associated with an 86% to 141% increase in CBT in all groups, the CBT was able to distinguish control, hepatitic, and cirrhotic smokers (5.36 ± 0.82, 3.63 ± 1.21, and 2.14 ± 1.14, respectively, P = .001). Multivariate analysis revealed that only smoking (P < .001) and disease state (P = .001) were significant predictors of the CBT. In conclusion, the 13C–CBT represents a valid indicator of plasma caffeine clearance and correlates reproducibly with hepatic dysfunction.
Quantitative T1 Mapping of Hepatic Encephalopathy Using Magnetic Resonance Imaging
Authors: Shah Nadim Joni , Neeb Heiko , Zaitsev Maxim , Steinhoff Sven , Kircheis Gerald , Amunts Katrin , Häussinger Dieter , Zilles Karl
Published: October 2003

Changes are shown in the spin–lattice (T1) relaxation time caused by the putative deposition of manganese in various brain regions of hepatic encephalopathy (HE) patients using a novel and fast magnetic resonance imaging (MRI) method for quantitative relaxation time mapping. A new method, T1 mapping with partial inversion recovery (TAPIR), was used to obtain a series of T1–weighted images to produce T1 maps. Imaging of 15 control subjects and 11 patients was performed on a 1.5T MRI scanner. The measurement time per patient with this technique, including adjustments, was ˜5 minutes. Regions of interest in the globus pallidus, the caudate nucleus, the posterior and anterior limbs of the internal capsule, the putamen, the frontal and occipital white matter, the white matter of the corona radiata, the occipital visual and frontal cortices, and the thalamus were interactively defined in the left hemisphere and analyzed with respect to their T1 values. T1 changes in the brains of HE patients can be determined quantitatively with TAPIR in short, clinically relevant measurement times. Significant correlations between the change in T1 and HE severity have been shown in the globus pallidus, the caudate nucleus, and the posterior limb of the internal capsule. No significant correlation of T1 with grade of HE was found in the putamen, frontal white matter, white matter of the corona radiata, white matter in the occipital lobe, the anterior limb of the internal capsule, visual cortex, thalamus, or frontal cortex. In conclusion, these measurements show that T1 mapping is feasible in short, clinically relevant acquisition times.
Characterization of Hcv–Specific Patr Class Ii Restricted Cd4+ T Cell Responses in An Acutely Infected Chimpanzee
Authors: Woollard David J. , Grakoui Arash , Shoukry Naglaa H. , Murthy Krishna K. , Campbell Katherine J. , Walker Christopher M.
Published: October 2003

Resolution of hepatitis C virus (HCV) infection is associated with strong and sustained virus–specific CD4+ T cell responses. In this study, we investigated the evolution of functional T cell responses during acute infection of a chimpanzee and the longevity of these lymphocytes in blood and liver after resolution of infection. Viremia increased through the first 3 weeks of infection and then remained stable until the onset of T cell responses at weeks 6 and 8 postinfection. CD4+ T cells targeting nonstructural HCV proteins were detected in proliferation assays by week 6 postinfection, but they failed to produce interferon γ (IFN–γ). HCV–specific CD4+ and CD8+ T cells with the ability to produce IFN–γ appeared at week 8 when a rapid 10–fold reduction in plasma viremia was first observed. This cytokine response persisted through to week 24 when infection apparently resolved. T cell lines targeting 3 CD4+ T cell epitopes and 1 CD8+ T cell epitope were derived from liver and their Patr major histocompatibility complex (MHC) restriction elements were identified. In retrospective studies performed on cryopreserved peripheral blood mononuclear cells (PBMCs) collected at various timepoints after infection, the onset of an IFN–γ response measured against the class II restricted epitopes correlated with viral clearance. In conclusion, the characterization of the HCV epitopes and MHC class II restriction elements described here will facilitate a detailed comparison of CD4+ T cell function in animals with resolved and persistent infections.
Adenovirus–Mediated Expression of Cu/Zn– or Mn–Superoxide Dismutase Protects Against Cyp2e1–Dependent Toxicity
Authors: Pérez María José , Cederbaum Arthur I.
Published: October 2003

CYP2E1 induction by ethanol is one mechanism by which ethanol creates oxidative stress in the liver. The superoxide dismutases (SODs) are an important antioxidant enzyme defense system against reactive oxygen species (ROS). To investigate the protective role of SOD against CYP2E1–dependent toxicity, a transfected HepG2 cell line overexpressing CYP2E1 (E47 cells) was infected with adenoviral vectors containing Cu/Zn–SOD complementary DNA (cDNA) (Ad.SOD1) and Mn–SOD cDNA (Ad.SOD2). Forty–eight hours after infection, intracellular levels and activity of Cu/Zn–SOD and Mn–SOD were increased about 2– and 3–fold, respectively. Localization of the overexpressed Cu/Zn–SOD in the cytosol and Mn–SOD in the mitochondria was confirmed by assaying the levels and activity of SOD in the corresponding isolated fractions. Arachidonic acid (AA) plus iron–induced cell death was partially prevented in both Ad.SOD1– and Ad.SOD2–infected E47 cells. Overexpression of Cu/Zn–SOD and Mn–SOD also partially protected E47 cells from the increase in reactive oxygen production and lipid peroxidation and the loss of mitochondrial membrane potential induced by AA and iron. Infection with Cu/Zn–SOD and Mn–SOD also protected the E47 cells against AA toxicity or buthionine sulfoximine (BSO)–dependent toxicity. CYP2E1 levels and catalytic activity were not altered by overexpression of Cu/Zn–SOD or Mn–SOD. Cu/Zn–SOD in the cytosol and Mn–SOD in mitochondria each are capable of protecting HepG2 cells expressing CYP2E1 against cytotoxicity induced by pro–oxidants. In conclusion, these enzymes may be useful in the prevention or improvement of liver injury produced by agents known to be metabolized by CYP2E1 to reactive intermediates and to cause oxidative stress.
Adenovirus–Mediated Overexpression of Follistatin Enlarges Intact Liver of Adult Rats
Authors: Takabe Kazuaki , Wang Lili , Leal Angela M.O. , MacConell Leigh A. , Wiater Ezra , Tomiya Tomoaki , Ohno Akihiko , Verma Inder M. , Vale Wylie
Published: October 2003

Under normal physiologic conditions, liver size is under strict regulatory control. Activin, a member of the transforming growth factor β (TGF–β) superfamily, is expressed in the intact adult liver and is an inhibitor of hepatocyte growth. However, the exact role played by endogenous activin in maintaining the size of a normal adult liver has yet to be completely examinedin vivo. Here, we report the development of an adenoviral vector (AdexCAFS288) that expressed human follistatin–288, which binds to activin and neutralizes its biologic activities. AdexCAGFP, a control virus, expressed green fluorescent protein. AdexCAFS288 effectively expressed follistatin–288, as measured both in HepG2 cell lysate and conditioned medium and blocked activin signaling and its biologic functionsin vitro. Intraperitoneal injection of AdexCAFS288in vivoresulted in significant liver growth (146% of control) in intact liver of adult male rats 12 days following treatment without significant dysfunctions. The increase in liver size was attributed to increased hepatocyte proliferation, as monitored by the mitotic index. Furthermore, there was a significant correlation between serum follistatin levels and liver weight. In conclusion, our results suggest that activin plays a critical role in maintaining optimal liver size and implicates the endogenous activin system as a therapeutic target in the treatment of liver disease.
Deficiency in Virion Secretion and Decreased Stability of the Hepatitis B Virus Immune Escape Mutant G145r
Authors: Kalinina Tatyana , Iwanski Alicja , Will Hans , Sterneck Martina
Published: October 2003

Hepatitis B virus with a G145R mutation in the small surface protein is considered the quintessential immune escape mutant because it frequently is found in vaccinated individuals with breakthrough infections and liver transplant recipients under anti–hepatitis B surface antigen (HBsAg) immunoglobulin prophylaxis. Nowadays the prevalence of the variant progressively increases. However, because spread of a virus depends not only on immune pressure but also on the viral phenotype, we investigated the biologic properties of the G145R variant. The G145R mutation was introduced into wild–type (Wt) virus genome by in vitro mutagenesis. After transfection into human hepatoma cells, the DNA, RNA, and protein synthesis and viral secretion ability of the mutant were studied. Furthermore, cotransfection studies were performed with the G145R variant and a Wt virus S–protein expressing construct and vice versa. Production and stability of viral messenger RNAs (mRNAs), DNA, and proteins were not affected by the G145R mutation. In contrast, secretion of mutant virions was reduced significantly. Only 20% of virions were found in the medium of G145R variant–transfected cells compared with Wt virus. Furthermore, mutant virions were more sensitive to detergent treatment suggesting a diminished stability. In cotransfection studies, Wt virus S–protein rescued secretion of mutant virions, whereas mutant S–protein had a transdominant negative effect on secretion of Wt virus. Both mechanisms may support persistence of the defective mutant in a mixed population with Wt virus. In conclusion, the significant defect of the G145R mutant for secretion of infectious virions and the diminished stability of mutant virions may limit global spread of the mutant.
Talin1 is critical for force-dependent reinforcement of initial integrin–cytoskeleton bonds but not tyrosine kinase activation
Authors: Giannone Grégory , Jiang Guoying , Sutton Deborah H. , Critchley David R. , Sheetz Michael P.
Published: October 2003

Cells rapidly transduce forces exerted on extracellular matrix contacts into tyrosine kinase activation and recruitment of cytoskeletal proteins to reinforce integrin–cytoskeleton connections and initiate adhesion site formation. The relationship between these two processes has not been defined, particularly at the submicrometer level. Using talin1-deficient cells, it appears that talin1 is critical for building early mechanical linkages. Deletion of talin1 blocked laser tweezers, force-dependent reinforcement of submicrometer fibronectin-coated beads and early formation of adhesion sites in response to force, even though Src family kinases, focal adhesion kinase, and spreading were activated normally. Recruitment of vinculin and paxillin to sites of force application also required talin1. FilaminA had a secondary role in strengthening fibronectin–integrin–cytoskeleton connections and no role in stretch-dependent adhesion site assembly. Thus, force-dependent activation of tyrosine kinases is independent of early force-dependent structural changes that require talin1 as part of a critical scaffold.
Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells
Authors: Parisi Silvia , D'Andrea Daniela , Lago Carmine T. , Adamson Eileen D. , Persico M. Graziella , Minchiotti Gabriella
Published: October 2003

The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.
Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase
Authors: Jackson Antony P. , Flett Alexander , Smythe Carl , Hufton Lindsay , Wettey Frank R. , Smythe Elizabeth
Published: October 2003

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.
Integrin-linked kinase is required for laminin-2–induced oligodendrocyte cell spreading and CNS myelination
Authors: Chun Soo Jin , Rasband Matthew N. , Sidman Richard L. , Habib Amyn A. , Vartanian Timothy
Published: October 2003

Early steps in myelination in the central nervous system (CNS) include a specialized and extreme form of cell spreading in which oligodendrocytes extend large lamellae that spiral around axons to form myelin. Recent studies have demonstrated that laminin-2 (LN-2; α2β1γ1) stimulates oligodendrocytes to extend elaborate membrane sheets in vitro (cell spreading), mediated by integrin α6β1. Although a congenital LN-2 deficiency in humans is associated with CNS white matter changes, LN-2–deficient (dy/dy) mice have shown abnormalities primarily within the peripheral nervous system. Here, we demonstrate a critical role for LN-2 in CNS myelination by showing that dy/dy mice have quantitative and morphologic defects in CNS myelin. We have defined the molecular pathway through which LN-2 signals oligodendrocyte cell spreading by demonstrating requirements for phosphoinositide 3-kinase activity and integrin-linked kinase (ILK). Interaction of oligodendrocytes with LN-2 stimulates ILK activity. A dominant negative ILK inhibits LN-2–induced myelinlike membrane formation. A critical component of the myelination signaling cascade includes LN-2 and integrin signals through ILK.
Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome
Authors: Bilodeau Patricia S. , Winistorfer Stanley C. , Kearney William R. , Robertson Andrew D. , Piper Robert C.
Published: October 2003

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting “receptors,” the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.