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Volume 40, Issue 3 (September 2004)
Liver Biology and Pathobiology
TNF- regulates mouse fetal hepatic maturation induced by oncostatin M and extracellular matrices (p 527-536)
Akihide Kamiya, Frank J. Gonzalez
Fetal hepatic maturation consists of multisteps and is regulated by several cytokines and cell-cell or cell-matrices interactions. In the mid-to-late fetal stage, hepatocytes have few metabolic functions associated with adult liver homeostasis. Cultured fetal hepatocytes acquire the expression of several mature liver-specific genes through stimulation with hepatic maturation factor oncostatin M (OSM) and matrigel. Tumor necrosis factor- (TNF ) regulates fetal hepatic maturation stimulated by OSM and matrigel. TNF suppressed expression of mature liver-specific genes such as tyrosine aminotransferase and apolipoproteins. In addition, the expression of hematopoietic cytokines and cyclin A2, repressed by OSM and matrigel, is induced by TNF in the fetal hepatic cultures coincident with cell division. TNF inhibited the induction of hepatocyte nuclear factor 4 induced by OSM and matrigel, suggesting that down-regulation of hepatocyte nuclear factor 4 expression is involved in the mechanism of suppression of hepatic maturation by TNF . Interestingly, TNF is expressed in the prenatal and postnatal liver but not in adult liver, whereas TNFR1, a TNF receptor, is expressed in both fetal and adult livers. In conclusion , TNF is a suppressive factor of hepatic maturation. The balance between hepatic maturation factor (OSM and extracellular matrices) and TNF is important for liver development. (HEPATOLOGY 2004;40:527-536.) 
Rapamycin-sensitive induction of eukaryotic initiation factor 4F in regenerating mouse liver (p 537-544)
Melissa M. Goggin, Christopher J. Nelsen, Scot R. Kimball, Leonard S. Jefferson, Simon J. Morley, Jeffrey H. Albrecht
Following acute injuries that diminish functional liver mass, the remaining hepatocytes substantially increase overall protein synthesis to meet increased metabolic demands and to allow for compensatory liver growth. Previous studies have not clearly defined the mechanisms that promote protein synthesis in the regenerating liver. In the current study, we examined the regulation of key proteins involved in translation initiation following 70% partial hepatectomy (PH) in mice. PH promoted the assembly of eukaryotic initiation factor (eIF) 4F complexes consisting of eIF4E, eIF4G, eIF4A1, and poly-A binding protein. eIF4F complex formation after PH occurred without detectable changes in eIF4E-binding protein 1 (4E-BP1) phosphorylation or its binding eIF4E. The amount of serine 1108-phosphorylated eIF4G (but not Ser209-phosphorylated eIF4E) was induced following PH. These effects were antagonized by treatment with rapamycin, indicating that target of rapamycin (TOR) activity is required for eIF4F assembly in the regenerating liver. Rapamycin inhibited the induction of cyclin D1, a known eIF4F-sensitive gene, at the level of protein expression but not messenger RNA (mRNA) expression. In conclusion , increased translation initiation mediated by the mRNA cap-binding complex eIF4F contributes to the induction of protein synthesis during compensatory liver growth. Further study of factors that regulate translation initiation may provide insight into mechanisms that govern metabolic homeostasis and regeneration in response to liver injury. (HEPATOLOGY 2004;40:537-544.)
Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling (p 545-554)
Salman R. Khetani, Greg Szulgit, Jo A. Del Rio, Carrolee Barlow, Sangeeta N. Bhatia
Cocultivation of primary hepatocytes with a plethora of nonparenchymal cells (from within and outside the liver) has been shown to support hepatic functions in vitro . Despite significant investigation into this phenomenon, the molecular mechanism underlying epithelial-nonparenchymal interactions in hepatocyte cocultures remains poorly understood. In this study, we present a functional genomic approach utilizing gene expression profiling to isolate molecular mediators potentially involved in induction of liver-specific functions by nonparenchymal cells. Specifically, primary rat hepatocytes were cocultivated with closely related murine fibroblast cell types (3T3-J2, NIH-3T3, mouse embryonic fibroblasts) to allow their classification as high, medium, or low inducers of hepatic functions. These functional responses were correlated with fibroblast gene expression profiles obtained using Affymetrix GeneChips. Microarray data analysis provided us with 17 functionally characterized candidate genes in the cell communication category (cell surface, extracellular matrix, secreted factors) that may be involved in induction of hepatic functions. Further analysis using various databases ( i.e., PubMed, GenBank) facilitated prioritization of the candidates for functional characterization. We experimentally validated the potential role of two candidates in our coculture model. The cell surface protein, neural cadherin (N-cadherin), was localized to hepatocyte-fibroblast junctions, while adsorbed decorin up-regulated hepatic functions in pure cultures as well as cocultures with low-inducing fibroblasts. In the future, identifying mediators of hepatocyte differentiation may have implications for both fundamental hepatology and cell-based therapies ( e.g. , bioartificial liver devices). In conclusion , the functional genomic approach presented in this study may be utilized to investigate mechanisms of cell-cell interaction in a variety of tissues and disease states. (H EPATOLOGY 2004;40:545-554.)
Selective priming to Toll-like receptor 4 (TLR4), not TLR2, ligands by P. acnes involves up-regulation of MD-2 in mice (p 555-564)
Laszlo Romics Jr., Angela Dolganiuc, Karen Kodys, Yvonne Drechsler, Shilpa Oak, Arumugam Velayudham, Pranoti Mandrekar, Gyongyi Szabo
Lipopolysaccharide (LPS) triggers cytokine production through Toll-like receptor 4 (TLR4), which shares downstream signaling pathways with TLR2. We investigated the roles of TLR2 and TLR4 in Propionibacterium acnes (P. acnes )-primed, LPS-induced liver damage using selective TLR ligands. Stock LPS induced interleukin 8 in both TLR4- and TLR2-expressing human embryonic kidney (HEK) 293 cells. Purified LPS (TLR4 ligand) activated HEK/TLR4 cells, while peptidoglycan and lipoteichoic acid (TLR2 ligands) activated HEK/TLR2 cells, respectively. In mice, P. acnes priming resulted in increased liver messenger RNA (mRNA) and serum levels of tumor necrosis factor , interleukin 12, and interferon (IFN- ) by both stock LPS and purified LPS challenges compared with nonprimed controls. In contrast , P. acnes failed to sensitize to TLR2 ligands (peptidoglycan + lipoteichoic acid). In the liver, P. acnes -priming was associated with up-regulation of TLR4 and MD-2 proteins, and subsequent LPS challenge further increased MD-2 and CD14 mRNA levels. The lack of sensitization to TLR2 ligands by P. acnes correlated with no increase in hepatic TLR1 or TLR6 mRNA. In vitro ,P. acnes pretreatment desensitized RAW macrophages to a secondary stimulation via both TLR2 and TLR4. However, IFN- could selectively prevent desensitization to TLR4 but not to TLR2 ligands. Furthermore, P. acnes induced production of IFN- in vivo as well as in isolated splenocytes. In vitro ,P. acnes -primed Hepa 1-6 hepatocytes but not RAW macrophages produced increased MD-2 and CD14 mRNA levels after an LPS challenge. In conclusion ,P. acnes priming to selective TLR4-mediated liver injury is associated with up-regulation of TLR4 and MD-2 and is likely to involve IFN- and prevent TLR4 desensitization by P. acnes . (HEPATOLOGY 2004;40:555-564.)
The role of iNOS in alcohol-dependent hepatotoxicity and mitochondrial dysfunction in mice (p 565-573)
Aparna Venkatraman, Sruti Shiva, Amanda Wigley, Elena Ulasova, David Chhieng, Shannon M. Bailey, Victor M. Darley-Usmar
Nitric oxide (NO) is now known to control both mitochondrial respiration and organelle biogenesis. Under conditions of ethanol-dependent hepatic dysfunction, steatosis is increased, and this is associated with increased expression of inducible nitric oxide synthase (iNOS). We have previously shown that after chronic exposure to ethanol, the sensitivity of mitochondrial respiration to inhibition by NO is enhanced, and we have proposed that this contributes to ethanol-dependent hypoxia. This study examines the role of iNOS in controlling the NO-dependent modification of mitochondrial function. Mitochondria were isolated from the livers of both wild-type (WT) and iNOS knockout (iNOS -/- ) mice that were fed an isocaloric ethanol-containing diet for a period of 5 weeks. All animals that consumed ethanol showed some evidence of fatty liver; however, this was to a lesser extent in the iNOS -/- mice compared to controls. At this early stage in ethanol-dependent hepatic dysfunction, infiltration of inflammatory cells and the formation of nitrated proteins was also decreased in response to ethanol feeding in the iNOS -/- animals. Mitochondria isolated from wild-type ethanol-fed mice showed a significant decrease in respiratory control ratio and an increased sensitivity to NO-dependent inhibition of respiration relative to their pair-fed controls. In contrast, liver mitochondria isolated from iNOS -/- mice fed ethanol showed no change in the sensitivity to NO-dependent inhibition of respiration. In conclusion , the hepatic response to chronic alcohol-dependent cytotoxicity involves a change in mitochondrial function dependent on the induction of iNOS. (HEPATOLOGY 2004;40:565-573.)
Peroxynitrite alters the catalytic activity of rodent liver proteasome in vitro and in vivo (p 574-582)
Natalia A. Osna, James Haorah, Viatcheslav M. Krutik, Terrence M. Donohue Jr.
The proteasome is an important multicatalytic enzyme complex that degrades misfolded and oxidized proteins, signal transduction factors, and antigenic peptides for presentation. We investigated the in vitro effects of peroxynitrite (PN) on the peptidase activity of both crude 20S and 26S and purified 20S proteasome preparations from rat liver as well as proteasome activity in Hep G2 cells and in mouse liver. Crude and purified proteasome preparations were exposed to PN or to the PN donor, 3-morpholinosydnonimine hydrochloride (SIN-1), and then assayed for chymotrypsin-like activity. For in vivo experiments, mice were treated with molsidomine, which is metabolized to SIN-1 in liver. PN and SIN-1 dose-dependently modulated the chymotrypsin-like activity of the 20S proteasome: lower concentrations enhanced proteasome activity, and higher concentrations caused its decline. The NO donor S-nitroso-N-acetylpenicillamine (SNAP), at all concentrations, suppressed 20S proteasome activity. We observed similar results when liver soluble fractions (S-100) were treated with PN, SIN-1, or SNAP, except that enzyme activity in S-100 fractions was less sensitive than the purified enzymes to these agents. Treatment of Hep G2 cells with 0.01 or 0.1 mmol/L SIN-1 stimulated in situ proteasome activity in these cells, while 1 mmol/L SIN-1 suppressed it. SNAP treatment did not affect proteasome activity in Hep G2 cells. Mice treated with molsidomine had enhanced liver proteasome activity 6 hours after treatment, but after 24 hours enzyme activity declined below control levels. In conclusion , PN dose-dependently modulated proteasome activity, regulating protein degradation by the proteasome in liver cells. (HEPATOLOGY 2004;40:574-582.)
Role of the renin-angiotensin system in hepatic ischemia reperfusion injury in rats (p 583-589)
Luping Guo, Katharine S. Richardson, Lindsay M. Tucker, Mark A. Doll, David W. Hein, Gavin E. Arteel
It has been shown that the renin-angiotensin system (RAS) plays key roles in the development of fibrosis in numerous organs, including the liver. Other studies have suggested that the RAS also may play roles in diseases of chronic inflammation. However, whether the RAS also can mediate acute inflammation in liver is unclear. The purpose of this study therefore was to determine the effect of the RAS inhibitors captopril and losartan on acute liver damage and inflammation caused by hepatic ischemia and subsequent reperfusion. Accordingly, male rats were subjected to 1 hour of hepatic ischemia (70%) followed by reperfusion; animals were killed 3, 8, or 24 hours after reperfusion. The effect of captopril or losartan (100 or 5 mg/kg intragastrically, respectively) was compared with that of vehicle (saline). The expression of angiotensinogen in liver increased fivefold 3 hours after reperfusion. Indices of liver damage and inflammation ( e.g. , alanine aminotransferase levels, pathological features, tumor necrosis factor- levels, and intercellular adhesion molecule-1 expression) all were significantly elevated in vehicle-treated animals after hepatic ischemia and subsequent reperfusion. Ischemia and reperfusion also caused an increase in the accumulation of protein adducts of 4-hydroxynonenal, an index of oxidative stress. Captopril or losartan treatment showed profound protective effects under these conditions, significantly blunting the increase in all these parameters caused by ischemia and reperfusion. In conclusion , RAS inhibitors prevent acute liver injury in a model of inflammation caused by ischemia and reperfusion. These data further suggest that the RAS may play a key role in mediating such responses in the liver and suggest a novel role for this system. (HEPATOLOGY 2004;40:583-589.)
In vitro differentiation of rat liver derived stem cells results in sensitization to TNF -mediated apoptosis (p 590-599)
Aránzazu Sánchez, Valentina M. Factor, Luis A. Espinoza, Insa S. Schroeder, Snorri S. Thorgeirsson
Hepatic stem cells are activated after liver damage and have a critical role in tissue homeostasis and repair. Characterization of molecular and cellular events accompanying the expansion and differentiation of liver stem cells is essential for understanding the basic biology of stem cells and for facilitating clinical application of the stem cells. We assessed whether in vitro differentiation of putative hepatic progenitor (rat liver epithelial [RLE]) cells toward hepatocytic lineage affects the response to TNF -mediated cytotoxicity, a common determinant of liver injury. The data show that 50% of differentiated cells underwent apoptosis after 6 hours of TNF treatment whereas control RLE cells were resistant. Both cell types displayed mitochondrial depolarization and release of cytochrome c but the TNF treatment resulted in activation of caspases 9 and 3 and the execution of apoptosis only in differentiated RLE cells. Apoptotic death was associated with increased ROS production and depletion of glutathione. Antioxidants completely prevented both glutathione depletion and apoptosis induced by TNF in differentiated RLE cells. Conversely, glutathione-depleting agents sensitized control RLE cells to TNF induced apoptosis. In conclusion , efficient antioxidant defense system involving glutathione renders hepatic progenitor cells resistant to TNF -mediated apoptosis and acquisition of sensitivity to death stimuli is an implicit feature of the differentiation process. (HEPATOLOGY 2004;40:590-599.)
In vivo regulation of murine CYP7A1 by HNF-6: A novel mechanism for diminished CYP7A1 expression in biliary obstruction (p 600-608)
Minhua Wang, Yongjun Tan, Robert H. Costa, Ai-Xuan L. Holterman
Disruption of the enterohepatic bile acid circulation during biliary tract obstruction leads to profound perturbation of the cholesterol and bile acid metabolic pathways. Several families of nuclear receptor proteins have been shown to modulate this critical process by regulating hepatic cholesterol catabolism and bile acid synthesis through the transcriptional control of cholesterol 7- hydroxylase (CYP7A1). Hepatocyte nuclear factor (HNF) 6 (also known as OC-1) is a member of the ONECUT family of transcription factors that activate numerous hepatic target genes essential to liver function. We have previously shown that hepatic expression of mouse HNF-6 messenger RNA (mRNA) and protein significantly decrease following bile duct ligation. Because CYP7A1 contains potential HNF-6 binding sites in its promoter region, we tested the hypothesis that HNF-6 transcriptionally regulates CYP7A1. Following bile duct ligation, we demonstrated that diminished HNF-6 mRNA levels correlate with a reduction in CYP7A1 mRNA expression. Increasing hepatic levels of HNF-6 either by infection with recombinant adenovirus vector expressing HNF-6 cDNA by growth hormone treatment leads to an induction of CYP7A1 mRNA. To directly evaluate if HNF-6 is a transcriptional activator for CYP7A1, we used deletional and mutational analyses of CYP7A1 promoter sequences and defined sequences -206/-194 to be critical for CYP7A1 transcriptional stimulation by HNF-6 in cotransfection assays. In conclusion , the HNF-6 protein is a component of the complex network of hepatic transcription factors that regulates the expression of hepatic genes essential for bile acid homeostasis and cholesterol/lipid metabolism in normal and pathological conditions. (HEPATOLOGY 2004;40:600-608.)
Proteomic signature corresponding to alpha fetoprotein expression in liver cancer cells (p 609-617)
Hideki Yokoo, Tadashi Kondo, Kazuyasu Fujii, Tesshi Yamada, Satoru Todo, Setsuo Hirohashi
Alpha fetoprotein (AFP) has been implicated in the development of hepatocellular carcinoma and is considered to be a diagnostic and prognostic tumor marker. Because elevated expression of AFP is associated with many characteristics of hepatocellular carcinoma tissues, we hypothesized that multiple proteins may function in a coordinated manner with AFP. To identify such proteins, we performed global protein expression analysis, namely a proteomic study. The protein expression profiles of 9 AFP-producing liver cancer cell lines (JHH-5, HuH-1, PLC/PRL/5, Hep3B, HT-17, JHH-7, HuH-7, HepG2, Li-7) and 7 nonproducing liver cancer cell lines (HLE, JHH-6, Sk-Hep-1, JHH-4, HLF, RBE, SSP-25) were generated by fluorescence 2-dimensional difference gel electrophoresis. In fluorescence 2-dimensional difference gel electrophoresis, proteins are labeled with fluorescent dyes before electrophoresis for more accurate quantitative expression analysis. We identified 11 protein spots that distinguished AFP-producing cell lines from nonproducing cell lines by multivariate studies. The spots showed consistent alterations in amount in AFP-producing cell lines (6 up-regulated and 5 down-regulated). An additional 5 liver cancer cell lines (KIM-1, KYN-2, KYN-3, PH5-CH, PH5-T) also were correctly grouped with respect to their AFP production on the basis of the intensity of the 11 protein spots. The proteins corresponding to the 11 selected spots were identified by mass spectrometry and were categorized into 4 groups based on their known role in apoptosis, glucose metabolism, cytoskeletal organization, or translation. In conclusion , we found a novel association of AFP with other proteins. Their interaction should provide insight into the biology of AFP-producing hepatocellular carcinoma cells. (HEPATOLOGY 2004;40:609-617.)
Negative regulation of hepatocellular carcinoma cell growth by signal regulatory protein 1 (p 618-628)
He-Xin Yan, Hong-Yang Wang, Rui Zhang, Lei Chen, Bao-An Li, Shu-Qin Liu, Hui-Fang Cao, Xiu-Hua Qiu, Yun-Feng Shan, Zhong-Hua Yan, Hong-Ping Wu, Ye-Xiong Tan, Meng-Chao Wu
Signal regulatory protein (SIRP) 1 is a member of the SIRP family that undergoes tyrosine phosphorylation and binds SHP-2 tyrosine phosphatase in response to various mitogens. The expression levels of SIRP 1 were decreased in HCC tissues, compared with the matched normal tissues. Exogenous expression of wild type SIRP 1, but not of a mutant SIRP 1 lacking the tyrosine phosphorylation sites, in SIRP 1-negative Huh7 human HCC cells resulted in suppression of tumor cell growth both in vitro and in vivo . Treatment of Huh7 transfectants with EGF or HGF induced tyrosine phosphorylation of SIRP 1 and its association with SHP-2, which were accompanied by reduced ERK1 activation. Expression of SIRP 1 significantly suppressed activation of NF- B and also sensitized Huh7 cells to TNF or cisplatin-induced cell death. In addition, SIRP 1-transfected Huh7 cells displayed reduced cell migration and cell spreading in a fashion that was dependent on SIRP 1/SHP-2 complex formation. In conclusion , a negative regulatory effect of SIRP 1 on hepatocarcinogenesis is exerted, at least in part, through inhibition of ERK and NF- B pathways. (HEPATOLOGY 2004;40:618-628.)
Liver Failure and Liver Disease
Randomized controlled study of TIPS versus paracentesis plus albumin in cirrhosis with severe ascites (p 629-635)
Francesco Salerno, Manuela Merli, Oliviero Riggio, Massimo Cazzaniga, Valentina Valeriano, Massimo Pozzi, Antonio Nicolini, Filippo Salvatori, GIST
The transjugular intrahepatic portosystemic shunt (TIPS) has been shown to be effective in the control of refractory or recidivant ascites. However, the effect of TIPS on survival as compared with that of large-volume paracentesis plus albumin is uncertain. A multicenter, prospective, clinical trial was performed in 66 patients with cirrhosis and refractory or recidivant ascites (16 Child-Turcotte-Pugh class B and 50 Child-Turcotte-Pugh class C) randomly assigned to treatment with TIPS (n = 33) or with large-volume paracentesis plus human albumin (n = 33). The primary endpoint was survival without liver transplantation. Secondary endpoints were treatment failure, rehospitalization, and occurrence of complications. Thirteen patients treated with TIPS and 20 patients treated with paracentesis died during the study period, 4 patients in each group underwent liver transplantation. The probability of survival without transplantation was 77% at 1 year and 59% at 2 years in the TIPS group as compared with 52% and 29% in the paracentesis group ( P= .021). In a multivariate analysis, treatment with paracentesis and higher MELD score showed to independently predict death. Treatment failure was more frequent in patients assigned to paracentesis, whereas severe episodes of hepatic encephalopathy occurred more frequently in patients assigned to TIPS. The number and duration of rehospitalizations were similar in the two groups. In conclusion , compared to large-volume paracentesis plus albumin, TIPS improves survival without liver transplantation in patients with refractory or recidivant ascites. (HEPATOLOGY 2004;40:629-635.)
Serum from patients with fulminant hepatic failure causes hepatocyte detachment and apoptosis by a 1-integrin pathway (p 636-645)
Philip N. Newsome, John Tsiaoussis, Steven Masson, Robert Buttery, Cameron Livingston, Ian Ansell, James A. Ross, Tariq Sethi, Peter C. Hayes, John N. Plevris
Hepatocyte transplantation is restricted by the impaired ability of hepatocytes to engraft and survive in the damaged liver. Understanding the mechanisms that control this process will permit the development of strategies to improve engraftment. We studied changes in liver matrix during acute injury and delineated the mechanisms that perturb the successful adhesion and engraftment of hepatocytes. Collagen IV expression was increased in sinusoidal endothelium and portal tracts of fulminant hepatic failure explants, whereas there were minimal changes in the expression of fibronectin, tenascin, and laminin. Using an in vitro model of cellular adhesion, hepatocytes were cultured on collagen-coated plates and exposed to serum from patients with liver injury to ascertain their subsequent adhesion and survival. There was a rapid, temporally progressive decrease in the adhesive properties of hepatocytes exposed to such serum that occurred within 4 hours of exposure. Loss of activity of the 1-integrin receptor, which controls adhesion to collagen, was seen to precede this loss of adhesive ability. Addition of the 1-integrin activating antibody (TS2/16) to cells cultured with liver injury serum significantly increased their adhesion to collagen, and prevented significant apoptosis. In conclusion , we have identified an important mechanism that underpins the failure of infused hepatocytes to engraft and survive in liver injury. Pretreating cells with an activating antibody can improve their engraftment and survival, indicating that serum from patients with liver injury exerts a defined nontoxic biological effect. This finding has important implications in the future of cellular transplantation for liver and other organ diseases. (HEPATOLOGY 2004;40:636-645.)
Effects of contrast media on renal function in patients with cirrhosis: A prospective study (p 646-651)
Mònica Guevara, Glòria Fernández-Esparrach, Carlo Alessandria, Aldo Torre, Carlos Terra, Xavier Montañà, Carlos Piera, Maria Luisa Alvarez, Wladimiro Jiménez, Pere Ginès, Vicente Arroyo
Patients with cirrhosis are frequently submitted to radiological procedures that require the administration of contrast media. Contrast media is a well-known cause of renal failure, particularly in the presence of some predisposing conditions. However, it is not known whether cirrhosis constitutes a risk factor for contrast media-induced renal failure. The aim of this study was to assess the possible nephrotoxicity of contrast media in patients with cirrhosis. In a first protocol, renal function was evaluated with sensitive methods (glomerular filtration rate using iothalamate I 125 clearance and renal plasma flow using iodohippurate I 131 clearance) before and 48 hours after the administration of contrast media in 31 patients with cirrhosis (20 with ascites, 5 with renal failure). Solute-free water clearance, urine sodium, prostaglandins, and markers of tubular damage were also measured. The administration of contrast media was not associated with significant changes in renal function tests, neither in the whole group of patients nor in patients with ascites or renal failure. Urinary prostaglandin E2 and N-acetyl- -D-glucosaminidase increased significantly, but sodium and solute-free water excretion remained unchanged. In a second protocol, a different series of 60 patients with cirrhosis and renal failure were examined prospectively. No patient had renal failure due to contrast media. Only in 1 patient with septic shock was contrast media a possible contributing factor. In conclusion, the administration of contrast media is not associated with adverse effects on renal function in patients with cirrhosis. Cirrhosis does not appear to be a risk factor for the development of contrast media-induced nephrotoxicity. (HEPATOLOGY 2004;40:646-651.)
Improved survival after variceal bleeding in patients with cirrhosis over the past two decades (p 652-659)
Nicolas Carbonell, Arnaud Pauwels, Lawrence Serfaty, Olivier Fourdan, Victor George Lévy, Raoul Poupon
Over the past two decades, new treatment modalities have been introduced for the management of variceal bleeding. The aim of this retrospective study in a single center was to assess whether these treatments have improved the prognosis for cirrhotic patients with variceal bleeding. We reviewed the clinical records of all patients with cirrhosis admitted to our Liver Intensive Care Unit due to variceal bleeding during the years 1980, 1985, 1990, 1995, and 2000. Whereas balloon tamponade was still the first-line treatment in 1980, patients treated in 2000 received a vasoactive agent, an endoscopic treatment, and an antibiotic prophylaxis in, respectively, 90%, 100%, and 94% of cases. The in-hospital mortality rate steadily decreased over the study period: 42.6%, 29.9%, 25%, 16.2%, and 14.5% in 1980, 1985, 1990, 1995, and 2000, respectively ( P< .05). Mortality decreased from 9% in 1980 to 0% in 2000 in Child-Turcotte-Pugh class A patients, from 46% to 0% in class B patients, and from 70% to 32% in class C patients. This improved survival was associated with a decrease of rebleeding (from 47% in 1980 to 13% in 2000) and bacterial infection rates (from 38% to 14%). On multivariable analysis, endoscopic therapy and antibiotic prophylaxis were independent predictors of survival. In conclusion , in-hospital mortality of patients with cirrhosis and variceal bleeding decreased threefold over the past two decades, in concurrence with an early and combined use of pharmacological and endoscopic therapies and short-term antibiotic prophylaxis. (HEPATOLOGY 2004;40:652-659.)
Gas exchange mechanism of orthodeoxia in hepatopulmonary syndrome (p 660-666)
Federico P. Gómez, Graciela Martínez-Pallí, Joan A. Barberà, Josep Roca, Miquel Navasa, Robert Rodríguez-Roisin
The mechanism of orthodeoxia (OD), or decreased partial pressure of arterial oxygen (PaO 2) from supine to upright, a characteristic feature of hepatopulmonary syndrome (HPS), has never been comprehensively elucidated. We therefore investigated the intrapulmonary (shunt and ventilation-perfusion [ A/] mismatching) and extrapulmonary factors governing PaO 2in 20 patients with mild to severe HPS (14 males, 6 females; 50 ± 3 years old SE) at upright and supine, in random order. We set out a cutoff value for OD, namely a PaO 2decrease 5% or 4 mm Hg (area under the receiver operating characteristic curve, 0.96 each). Compared to supine, 5 patients showed OD (PaO 2change, -11% ± 2%, -7 ± 1 mm Hg, P< .05) with further A/worsening (shunt + low A/mode increased from 19% ± 7% to 21% ± 7% of cardiac output [ T], P< .05), as opposed to 15 patients who did not (+2% ± 2%, +1± 1 mm Hg) with A/improvement (from 20% ± 4% to 16% ± 4% of T,P< .01). Cardiac output was significantly lower in OD patients in both positions. Changes in extrapulmonary factors at upright, such as increased minute ventilation and decreased T, were of similar magnitude in both subsets of patients. In conclusion , our data suggest that gas exchange response to OD in HPS points to a more altered pulmonary vascular tone inducing heterogeneous blood flow redistribution to lung zones with prominent intrapulmonary vascular dilatations. (HEPATOLOGY 2004;40:660-666.)
Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling (p 667-676)
Ju-Seog Lee, In-Sun Chu, Jeonghoon Heo, Diego F. Calvisi, Zongtang Sun, Tania Roskams, Anne Durnez, Anthony J. Demetris, Snorri S. Thorgeirsson
We analyzed global gene expression patterns of 91 human hepatocellular carcinomas (HCCs) to define the molecular characteristics of the tumors and to test the prognostic value of the expression profiles. Unsupervised classification methods revealed two distinctive subclasses of HCC that are highly associated with patient survival. This association was validated via 5 independent supervised learning methods. We also identified the genes most strongly associated with survival by using the Cox proportional hazards survival analysis. This approach identified a limited number of genes that accurately predicted the length of survival and provides new molecular insight into the pathogenesis of HCC. Tumors from the low survival subclass have strong cell proliferation and antiapoptosis gene expression signatures. In addition, the low survival subclass displayed higher expression of genes involved in ubiquitination and histone modification, suggesting an etiological involvement of these processes in accelerating the progression of HCC. In conclusion , the biological differences identified in the HCC subclasses should provide an attractive source for the development of therapeutic targets ( e.g. , HIF1a) for selective treatment of HCC patient. (HEPATOLOGY 2004;40:667-676.)
Reduced expression of cell cycle regulator p18 INK4C in human hepatocellular carcinoma (p 677-686)
Asahiro Morishita, Tsutomu Masaki, Hitoshi Yoshiji, Seiji Nakai, Tomohiro Ogi, Yoshiaki Miyauchi, Shuhei Yoshida, Toshiharu Funaki, Naohito Uchida, Yuko Kita, Fumi Funakoshi, Hisashi Usuki, Setsuo Okada, Kunihiko Izuishi, Seishiro Watanabe, Kazutaka Kurokohchi, Shigeki Kuriyama
Cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors (CdkIs) are frequently altered in human cancer. p18 INK4C , a member of the INK4 family of CdkIs, is a potential tumor-suppressor gene product. However, the expression of p18 INK4C in hepatocellular carcinoma (HCC) remains unknown. The aim of this study was to examine the expression of p18 INK4C in various liver diseases including HCC and to assess its clinical significance in HCC. To that end, we examined the expression of p18 INK4C by immunohistochemistry in various liver diseases, including 51 HCCs, and also studied the relationship between p18 INK4C expression, the phosphorylation of retinoblastoma protein (pRb), and the activity level of Cdk4 and Cdk6. Immunohistochemical analysis revealed the frequent loss of p18 INK4C expression in HCC, especially in poorly differentiated HCC. The loss of p18 INK4C expression was shown to be associated with a poor prognosis compared with that associated with p18 INK4C - positivity. Further, the kinase activity of Cdk4 was found to be higher in p18 INK4C -negative HCCs than in p18 INK4C - positive HCCs. However, the level of Cdk6 activity was similar in the 2 groups of HCCs. In p18 INK4C - positive HCCs, p18 INK4C dominantly interacted with Cdk4 rather than with Cdk6. pRb phosphorylated at serine(Ser) 780 was detected more frequently in p18 INK4C - negative than in p18 INK4C - positive HCCs. In conclusion , the loss of p18 INK4C expression may play a role in the differentiation and development of HCC through the up-regulation of Cdk4 activity. (HEPATOLOGY 2004;40:677-686.)
A novel IgM class autoantibody to a hepatocyte-related 190 kDa molecule in patients with type 1 autoimmune hepatitis (p 687-692)
Katsumi Yamauchi, Naoko Yamaguchi, Takaji Furukawa, Kazuko Takatsu, Toshimi Nakanishi, Mina Sasaki, Etsuko Isono, Katsutoshi Tokushige, Tatsuji Komatsu, Keiko Shiratori
It has been reported that autoantibodies to hepatocytes are frequently found in patients with autoimmune hepatitis (AIH). To elucidate the nature of these hepatocyte-specific autoantibodies, we attempted to generate a hepatocyte-specific monoclonal antibody (MoAb) from Epstein-Barr virus-transformed peripheral blood mononuclear cells obtained from a patient with AIH. We established a single clone, 2E3, that continued to produce an immunoglobulin M (IgM) antibody ( -type). This MoAb had the following properties: it reacted mainly with hepatocyte-derived cell lines, rather than with other cell lines, and it reacted with liver tissue but not with other tissues. By immunoblot analysis, we found that this MoAb recognized a 190 kDa molecule on hepatocytes. The MoAb was able to kill hepatocyte-derived cell lines in the presence of fresh human serum. This cytotoxic effect was completely abrogated by heat inactivation of human serum prior to its addition to cell lines. In addition, an IgM autoantibody that recognized a 190 kDa molecule was also found in patients with AIH but not in those with chronic hepatitis C; its titer correlated significantly with serum alanine aminotransferase (ALT) levels in patients with AIH. In conclusion , we generated a human MoAb that recognizes a 190 kDa molecule on hepatocytes. Because of its ability to mediate complement-dependent cytotoxicity and the presence of similar IgM autoantibody in patients with AIH, we hypothesize this autoantibody may play a role in the immunopathogenesis of AIH. (HEPATOLOGY 2004;40:687-692.)
Effect of high-dose ursodeoxycholic acid on its biliary enrichment in primary sclerosing cholangitis (p 693-698)
Daniel Rost, Gerda Rudolph, Petra Kloeters-Plachky, Adolf Stiehl
Ursodeoxycholic acid (UDCA) has beneficial effects in cholestatic liver diseases. In primary sclerosing cholangitis (PSC), there is evidence that high doses (±20 mg/kg) of UDCA may be more effective than average doses. Biliary enrichment of UDCA at such high doses may represent the decisive factor for its beneficial effect. Up to now it is not clear how high-dose UDCA correlates with its biliary enrichment and whether bacterial degradation of large amounts of UDCA may lead to an increased bacterial formation of more toxic hydrophobic bile acids. We determined the biliary bile acid composition in 56 patients with PSC including 30 patients with repeat bile samples treated with various doses of UDCA. At a UDCA dose of 10-13 mg/kg/d (n = 18) biliary UDCA represented 43.1% + 0.3% (mean + SD) of total bile acids; at a UDCA dose of 14-17 mg/kg (n = 14), its biliary content increased to 46.9% + 0.3%, at 18-21 mg/kg (n = 34) to 55.9% + 0.2%, at 22-25 mg/kg (n = 12) to 58.6% + 2.3%, and at 26-32 mg/kg (n = 8) to 57.7% + 0.4%. During UDCA treatment, the biliary content of all other bile acids was unchanged or decreased. In conclusion , biliary enrichment of UDCA increases with increasing dose and reaches a plateau at 22-25 mg/kg. There was no increase of toxic hydrophobic bile acids. If biliary enrichment of UDCA represents the decisive factor for its clinical effect, it seems likely that UDCA doses of up to 22-25 mg/kg may be more effective than lower doses. (HEPATOLOGY 2004;40:693-698.)
Viral Hepatitis
Hepatitis C recurrence is more severe after living donor compared to cadaveric liver transplantation (p 699-707)
Montserrat Garcia-Retortillo, Xavier Forns, Josep M. Llovet, Miquel Navasa, Anna Feliu, Anna Massaguer, Miquel Bruguera, Josep Fuster, Juan Carlos Garcia-Valdecasas, Antoni Rimola
Preliminary reports suggested that hepatitis C virus (HCV) infection has a more aggressive course following living donor liver transplantation (LDLT) compared to cadaveric liver transplantation (CLT). The aim of this prospective study was to establish if HCV disease recurrence differs between LDLT and CLT. A cohort of 116 consecutive HCV-infected patients undergoing 117 LTs in a single center from March 2000 to August 2003 were followed-up, including systematic liver biopsies. Severe recurrence (SR) was defined as biopsy-proven cirrhosis and/or the occurrence of clinical decompensation. After a median follow-up of 22 months (2.6-44 months), 26 (22%) patients developed SR (decompensation in 12), involving 17 (18%) of 95 patients undergoing CLT and 9 (41%) of 22 undergoing LDLT. The 2-year probability of presenting SR was significantly higher in LDLT compared to CLT (45% vs. 22%, P= .019). By univariate analysis LDLT ( P= .019) and an ALT higher than 80 IU/L 3 months after LT ( P= .022) were predictors of SR. In 93 patients from whom a liver biopsy was available 3 months after LT, a lobular necroinflammatory score >1 ( P< .01), LDLT ( P< .01), and biliary complications ( P= .046) were associated with SR. However, the only variables independently associated with SR were LDLT (odds ratio [OR], = 2.8; 95% CI,1.19-6.6; P= .024) and a lobular necroinflammatory score >1 (OR, 3.1; 95% CI, 1.2-8; P= .013). In conclusion , HCV recurrence is more severe in LDLT compared to CLT. Although our results were based on a single-center experience, they should be considered in the decision-making process of transplant programs, since severe HCV recurrence may ultimately compromise graft and patient survival. (HEPATOLOGY 2004; 40:699-707.)
Hepatitis C virus NS5A-regulated gene expression and signaling revealed via microarray and comparative promoter analyses (p 708-718)
Sophie Girard, Erik Vossman, David E. Misek, Philippe Podevin, Samir Hanash, Christian Bréchot, Laura Beretta
Most individuals exposed to hepatitis C virus (HCV) become chronically infected and are predisposed to liver disease. The mechanisms underlying viral persistence and disease progression are unknown. A role for the HCV NS5A protein in viral replication and interferon resistance has been demonstrated. To identify mechanisms affected by NS5A, we analyzed the gene expression of Huh7 cells expressing NS5A and control cells using oligonucleotide microarrays. A set of 103 genes (43 up-regulated, 60 down-regulated) whose expression was modified by at least twofold was selected. These included genes involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, and genes regulating immune responses. The finding of modulated expression of genes related to the TGF- superfamily and liver fibrosis was observed. Interestingly, both the tumor necrosis factor and lymphotoxin beta receptors were down-regulated by NS5A. Similar data were obtained following expression of four NS5A mutants obtained from patients who were not responsive or were sensitive to interferon therapy. Through computational analysis, we determined that 39 of the 43 genes up-regulated by NS5A contained one or more nuclear factor B (NF- B) binding sites within their promoter region. Using the Gibbs sampling method, we also detected enrichment of NF- B consensus binding sites in the upstream regions of the 43 coexpressed genes. Activation of NF- B by NS5A was subsequently demonstrated in luciferase reporter assays. Adenovirus-mediated expression of I Breverted NS5A mediated up-regulation of gene expression. In conclusion , this study suggests a role of NS5A and NF- B in HCV pathogenesis and related liver disease. (HEPATOLOGY 2004;40:708-718.)
A dose-finding study of once-daily oral telbivudine in HBeAg-positive patients with chronic hepatitis B virus infection (p 719-726)
Ching-Lung Lai, Seng Gee Lim, Nathaniel A. Brown, Xiao-Jian Zhou, Deborah M. Lloyd, Yin-Mei Lee, Man-Fung Yuen, George C. Chao, Maureen W. Myers
Current therapy for chronic hepatitis B is suboptimal as a result of limited durable response rates, cumulative viral resistance, and/or poor tolerability. Telbivudine has potent antiviral activity against hepatitis B virus (HBV) in vitro and in the woodchuck model and has a promising preclinical safety profile. In this first clinical study of telbivudine, safety, antiviral activity, and pharmacokinetics were assessed in 43 adults with hepatitis B e antigen-positive chronic hepatitis B. This placebo-controlled dose-escalation trial investigated 6 telbivudine daily dosing levels (25, 50, 100, 200, 400, and 800 mg/d); treatment was given for 4 weeks, with 12 weeks' follow-up. Serum HBV DNA levels were monitored via quantitative polymerase chain reaction. The results indicate that telbivudine was well tolerated at all dosing levels, with no dose-related or treatment-related clinical or laboratory adverse events. telbivudine plasma pharmacokinetics were dose-proportional within the studied dose range. Marked dose-related antiviral activity was evident, with a maximum at telbivudine doses of 400 mg/d or more. In the 800mg/d cohort, the mean HBV DNA reduction was 3.75 log 10 copies/mL at week 4, comprising a 99.98% reduction in serum viral load. Correspondingly, posttreatment return of viral load was slowest in the high-dose groups. Viral dynamic analyses suggested a high degree of efficiency of inhibition of HBV replication by telbivudine and helped refine selection of the optimal dose. In conclusion , these results support expanded clinical studies of this new agent for the treatment of hepatitis B. (HEPATOLOGY 2004;40:719-726.)
Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients (p 727-737)
Danny Ka-Ho Wong, Man-Fung Yuen, HeJun Yuan, Simon Siu-Man Sum, Chee-Kin Hui, Jeff Hall, Ching-Lung Lai
This study examined a signal amplification assay, the Invader assay, for the quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver biopsies and sera. DNA was extracted from liver biopsy and serum samples were collected from 16 hepatitis B e antigen (HBeAg)-positive and 36 antibody-to-HBeAg-positive (anti-HBe-positive) chronic hepatitis B patients. The amount of total HBV DNA and cccDNA was measured using the Invader assay. Anti-HBe-positive patients had lower median total intrahepatic HBV DNA ( P< .001) and intrahepatic cccDNA levels ( P= .001) than HBeAg-positive patients. Intrahepatic cccDNA correlated positively with the total intrahepatic HBV DNA ( r= 0.950, P< .001). However, the proportion of intrahepatic HBV DNA in the form of cccDNA was inversely related to the amount of total intrahepatic HBV DNA ( r= -0.822, P< .001). A small amount of cccDNA was detected in 39 of 52 (75%) serum samples. Anti-HBe-positive patients had lower median serum cccDNA levels than HBeAg-positive patients ( P= .002). Serum HBV DNA correlated positively with intrahepatic total HBV DNA ( r= 0.778, P< .001) and intrahepatic cccDNA ( r= 0.481, P= .002). In conclusion , the Invader assay is a reliable assay for the quantitation of cccDNA. Serum and intrahepatic total HBV DNA and cccDNA levels become lower as the disease progresses from HBeAg-positive to anti-HBe-positive phase, with cccDNA becoming the predominant form of intrahepatic HBV DNA. (HEPATOLOGY 2004;40:727-737.)
Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B (p 738-746)
Renate G. van der Molen, Dave Sprengers, Rekha S. Binda, Esther C. de Jong, Hubert G. M. Niesters, Johannes G. Kusters, Jaap Kwekkeboom, Harry L. A. Janssen
Dendritic cells (DC) play an important role in the induction of T-cell responses. We hypothesize that the hampered antiviral T-cell response in chronic hepatitis B patients is a result of impaired dendritic cell function. In this study, we compared the number, phenotype and functionality of two important blood precursor DC, myeloid DC (mDC) and plasmacytoid DC (pDC), of chronic hepatitis B patients with healthy volunteers. No differences in percentages of mDC and pDC in peripheral blood mononuclear cells were observed between chronic hepatitis B patients and healthy controls. The allostimulatory capacity of isolated and in vitro matured mDC, but not of pDC, was significantly decreased in patients compared to controls. Accordingly, a decreased percentage of mDC expressing CD80 and CD86 was observed after maturation, compared to controls. In addition, mDC of patients showed a reduced capacity to produce tumor necrosis factor after a stimulus with synthetic double-stranded RNA and interferon . Purified pDC from patients produced less interferon , an important antiviral cytokine, in response to stimulation with Staphylococcus aureus Cowan strain I than pDC isolated from controls. In conclusion , mDC and pDC are functionally impaired in patients with chronic hepatitis B. This might be an important way by which hepatitis B virus evades an adequate immune response, leading to viral persistence and disease chronicity. (HEPATOLOGY 2004;40:738-746.)
A case-control study for differences among hepatitis B virus infections of genotypes A (subtypes Aa and Ae) and D (p 747-755)
Yasuhito Tanaka, Izumi Hasegawa, Takanobu Kato, Etsuro Orito, Noboru Hirashima, Subrat K. Acharya, Robert G. Gish, Anna Kramvis, Michael C. Kew, Namiko Yoshihara, Santosh Man Shrestha, Mobin Khan, Yuzo Miyakawa, Masashi Mizokami
There are two subtypes of hepatitis B virus genotype A (HBV/A) and they are provisionally designated Aa ( astanding for Africa/Asia) and Ae ( efor Europe). In a case-control study, 78 HBV/Aa, 78HBV/Ae, and 78HBV/D carriers from several countries were compared. The prevalence of HBe antigen (HBeAg) in serum was significantly lower in carriers of HBV/Aa than in carriers of HBV/Ae (31% vs. 49%; P= .033), with a difference more obvious in the carriers aged 30 years or younger (34% vs. 67%; P= .029). HBV DNA levels in the carriers of HBV/Aa (median, 3.46 log copies/mL; 95% CI, 2.93-3.95) were significantly lower than those of carriers of HBV/Ae (6.09 log copies/mL; 95% CI, 4.24-7.64) or of carriers of HBV/D (5.48 log copies/mL; 95% CI, 4.06-7.02), regardless of the HBeAg status ( P< .001). The most specific and frequent substitutions in 54 HBV/Aa isolates were double substitutions for T1809 (100%) and T1812 (96%) immediately upstream of the precore initiation codon, which would interfere with the translation of HBeAg in HBV/Aa infections. They were not detected in 57 HBV/Ae or 61 HBV/D isolates examined. The double mutation in the core promoter (T1762/A1764) was more frequent in both HBV/Aa (50%) and HBV/Ae (44%) than in HBV/D isolates (25%; P< .01), whereas the precore mutation (A1896) occurred in HBV/D isolates only (48%; P< .0001). In conclusion, the clearance of HBeAg from serum may occur by different mechanisms in HBV/Aa, HBV/Ae, and HBV/D infections, which may influence clinical manifestations in the Western countries where both genotypes A and D are prevalent. (HEPATOLOGY 2004;40:747-755.)
Hepatology Elsewhere
Reexamining the role of the humoral immune response in control of hepatitis C virus infection (p 756-758)
Lynn B. Dustin
Immunoglobulin GM and KM allotypes - genetic markers of and chains, respectively - are associated with immune responsiveness to several infectious pathogens and with survival in certain viral epidemics. We hypothesized that GM and KM allotypes affect the outcome of hepatitis C virus (HCV) infection. To test this hypothesis, we serologically allotyped 100 persons with well-documented clearance of HCV infection and 198 matched persistently infected persons. None of the GM or KM phenotypes by itself was associated with the clearance or persistence of HCV infection. Particular combinations of these phenotypes, however, were significantly associated with the outcome of HCV infection. Subjects with GM 1,17 5,13 and KM 1,3 phenotypes were over three times (odds ratio [OR], 3.57; 95% confidence interval [CI], 1.44-8.87) as likely to clear the infection as the subjects who lacked these phenotypes. This GM phenotype had a similar association with clearance in the absence of KM 3 (OR, 2.75; 95% CI, 1.21-6.23). The presence of GM 1,3,17 23 5,13 phenotype (in the absence of KM 3) was associated with persistence (OR, 0.21; 95% CI, 0.06-0.77), while its absence (in the presence of KM 1,3) was associated with the clearance of infection (OR, 2.03; 95% CI, 1.16-3.54). These results show epistatic interactions of genes on chromosomes 14 (GM) and 2 (KM) in influencing the outcome of an HCV infection. Further investigations involving candidate genes (GM, KM, HLA, and Fc receptors) and cellular and humoral immune responses to HCV epitopes are needed to understand the mechanisms underlying these associations.
Save Article Copyright © 2004 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for September 2004 • Volume 127 • Number 3
Rapid Communication
Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo
Ralf Kiesslich, Juergen Burg, Michael Vieth, Janina Gnaendiger, Meike Enders, Peter DeLaney, Adrian Polglase, Wendy McLaren, Daniela Janell, Steven Thomas, Bernhard Nafe, Peter R. Galle, Markus F. Neurath
Background & Aims: Aconfocal laser endoscopy system has recently been developed that may allow subsurface imaging of living cells in colonic tissue in vivo. The aim of the present study was to assess its potential for prediction of histology during screening colonoscopy for colorectal cancer. Methods: Twenty-seven patients underwent colonoscopy with the confocal endoscope using acriflavine hydrochloride or fluorescein sodium with blue laser illumination. Furthermore, 42 patients underwent colonoscopy with this system using fluorescein sodium. Standardized locations and circumscript lesions were examined by confocal imaging before taking biopsy specimens. Confocal images were graded according to cellular and vascular changes and correlated with conventional histology in a prospective and blinded fashion. Results: Acriflavine hydrochloride and fluorescein sodium both yielded high-quality images. Whereas acriflavine hydrochloride strongly labeled the superficial epithelial cells, fluorescein sodium offered deeper imaging into the lamina propria. Fluorescein sodium was thus used for the prospective component of the study in which 13,020 confocal images from 390 different locations were compared with histologic data from 1038 biopsy specimens. Subsurface analysis during confocal laser endoscopy allowed detailed analysis of cellular structures. The presence of neoplastic changes could be predicted with high accuracy (sensitivity, 97.4%; specificity, 99.4%; accuracy, 99.2%). Conclusions: Confocal laser endoscopy is a novel diagnostic tool to analyze living cells during colonoscopy, thereby enabling virtual histology of neoplastic changes with high accuracy. These newly discovered diagnostic possibilities may be of crucial importance in clinical practice and lead to an optimized rapid diagnosis of neoplastic changes during ongoing colonoscopy.
Clinical-alimentary Tract
The incidence of colorectal cancer following a negative screening sigmoidoscopy: Implications for screening interval
V. Paul Doria-Rose, Theodore R. Levin, Joe V. Selby, Polly A. Newcomb, Kathryn E. Richert-Boe, Noel S. Weiss
Background & Aims: Current guidelines recommend a 5-year interval for colorectal cancer (CRC) screening by sigmoidoscopy. However, the optimal screening interval is uncertain. We estimated the annual incidence of distal and proximal CRC in the first 5 years following a negative sigmoidoscopy examination to gauge the potential benefit of rescreening in <5 years. Methods: A cohort of 72,483 participants in the Colon Cancer Prevention program of Kaiser Permanente of Northern California (KP) was defined using computerized databases. Men and women aged 50 years and older who had a negative screening flexible sigmoidoscopy examination between 1994 and 1996 and were considered not to be at high risk for developing CRC were included. Subjects were censored at the time of diagnosis (for cases), death, termination of KP membership, or subsequent colon examination. Results: Thirty cases of distal and 80 cases of proximal CRC occurred. Age-adjusted incidence rates of distal CRC ranged from a low of 2.8 per 100,000 person-years in the first year of follow-up to a high of 13.0 per 100,000 in the fourth year (rate difference, 10.2; 95% confidence interval, 1.119.3). However, for the entire follow-up period, incidence of distal CRC remained much lower than age-adjusted rates of 70.6 in the general population (Surveillance, Epidemiology, and End Results registry). The incidence of proximal CRC was also decreased modestly over population rates of disease. Conclusions: Screening by sigmoidoscopy more frequently than every 5 years would likely lead, at best, to only modest improvements as compared with a 5-year screening interval.
Postoperative maintenance of Crohn’s disease remission with 6-mercaptopurine, mesalamine, or placebo: A 2-year trial
Stephen B. Hanauer, Burton I. Korelitz, Paul Rutgeerts, Mark A. Peppercorn, Ronald A. Thisted, Russell D. Cohen, Daniel H. Present
Background & Aims: No therapy has been shown to reliably prevent the evolution of postoperative recurrence of Crohn’s disease. The aim of the current trial was to compare 6-mercaptopurine (6-MP) and mesalamine with placebo for the prevention of clinical, endoscopic, and radiographic recurrence of Crohn’s disease after resection and ileocolic anastomosis. Methods: Five centers randomized 131 patients to receive 6-MP (50 mg), mesalamine (3 g), or placebo daily in a double-blind, double-dummy trial. Patients had clinical assessments at 7 weeks and then every 3 months; colonoscopy at 6, 12, and 24 months; and small bowel series at 12 and 24 months. End points were clinical, endoscopic, and radiographic recurrence rates at 24 months. Results: Clinical recurrence rates (intent to treat) by life-table analysis at 24 months were 50% (95% confidence interval [CI], 34%68%), 58% (95% CI, 41%75%), and 77% (95% CI, 61%91%) in patients receiving 6-MP, mesalamine, and placebo, respectively. Endoscopic recurrence rates were 43% (95% CI, 28%63%), 63% (95% CI, 47%79%), and 64% (95% CI, 46%81%), and radiographic recurrence rates were 33% (95% CI, 19%54%), 46% (95% CI, 29%66%), and 49% (95% CI, 30%72%), respectively. 6-MP was more effective than placebo ( P< 0.05) at preventing clinical and endoscopic recurrence over 2 years. Patient withdrawals resulted in 69% of the study population evaluable for the clinical recurrence end point. Conclusions: 6-MP, 50 mg daily, was more effective than placebo at preventing postoperative recurrence of Crohn’s disease and should be considered as a maintenance therapy after ileocolic resection.
Azathioprine and mesalamine for prevention of relapse after conservative surgery for Crohn’s disease
Sandro Ardizzone, Giovanni Maconi, Gianluca M. Sampietro, Antonio Russo, Elisa Radice, Elisabetta Colombo, Venerina Imbesi, Mirko Molteni, Pier Giorgio Danelli, Angelo M. Taschieri, Gabriele Bianchi Porro
Background & Aims: Because the reoperation rate for Crohn’s disease is high after resective surgery, use of conservative surgery has increased. Mesalamine was investigated for the prevention of postoperative relapse, with disappointing results. The role of azathioprine in the postoperative setting is unknown. We aimed to compare the efficacy and safety of azathioprine and mesalamine in the prevention of clinical and surgical relapse in patients who have undergone conservative surgery for Crohn’s disease. Methods: In a prospective, open-label, randomized study, 142 patients received azathioprine (2 mg · kg ?1 · day ?1 ) or mesalamine (3 g/day) for 24 months. Clinical relapse was defined as the presence of symptoms with a Crohn’s Disease Activity Index score >200 and surgical relapse as the presence of symptoms refractory to medical treatment or complications requiring surgery. Results: After 24 months, the risk of clinical relapse was comparable in the azathioprine and mesalamine groups, both on intention-to-treat (odds ratio [OR], 2.04; 95% confidence interval [CI], 0.894.67) and per-protocol analyses (OR, 1.79; 95% CI, 0.803.97). No difference was observed with respect to surgical relapse at 24 months between the 2 groups. In a subgroup analysis, azathioprine was more effective than mesalamine in preventing clinical relapse in patients with previous intestinal resections (OR, 4.83; 95% CI, 1.4715.8). More patients receiving azathioprine withdrew from treatment due to adverse events than those receiving mesalamine (22% vs. 8%; P= 0.04). Conclusions: While no difference was observed in the efficacy of azathioprine and mesalamine in preventing clinical and surgical relapses after conservative surgery, azathioprine is more effective in those patients who have undergone previous intestinal resection.
Clinical-alimentary Tract
A circulating ligand for galectin-3 is a haptoglobin-related glycoprotein elevated in individuals with colon cancer
Robert S. Bresalier, James C. Byrd, David Tessler, Joseph Lebel, John Koomen, David Hawke, Elizabeth Half, Kai-Feng Liu, Nachman Mazurek, The Great Lakes-New England Clinical and Epidemiology Center of the Early Detection Research Network
Background & Aims: Galectin-3 is a ?-galactoside-binding protein implicated in tumor progression and metastasis of colorectal cancers. To determine whether circulating galectin-3 ligands are related to the presence of colon cancer, we sought to identify and quantify ligands in serum that bind to galectin-3. Methods: Sera from patients with colon cancer, adenomas, and normal individuals were desialylated, reduced, and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and blots probed with biotinylated galectin-3. Results: In colon cancer sera, the major galectin-3 ligand was a 40-kilodalton band distinct from mucin, carcinoembryonic antigen, and Mac-2 binding protein. Serum 40-kilodalton ligand was 10- to 30-fold higher in patients with colon cancer than in healthy subjects. Ligand was purified by gel filtration, affinity precipitation on galectin-3/agarose, and SDS-PAGE. When tryptic peptides were analyzed by matrix-assisted laser-desorption ionization mass spectrometry and protein database searching, the 40-kilodalton ligand was identified as haptoglobin ? subunit. In confirmation of this finding, depletion of haptoglobin by immunoprecipitation also eliminated the 40-kilodalton ligand. Colon cancer sera had only a modest increase in total haptoglobin as compared with healthy subjects, suggesting that the structure rather than the amount of haptoglobin is altered in patients with colon cancer. Immunohistochemical staining confirmed the absence of haptoglobin in normal colon and the ectopic expression of haptoglobin in colon cancers and adenomatous polyps. Conclusions: A major circulating ligand for galectin-3, which is elevated in the sera of patients with colon cancer, is a cancer-associated glycoform of haptoglobin.
Clinical-liver, Pancreas, and Biliary Tract
The impact of competing risks on the observed rate of chronic hepatitis C progression
W. Ray Kim, John J. Poterucha, Joanne T. Benson, Terry M. Therneau
Background & aims In previous studies about the natural history of chronic hepatitis C (CHC), age at the time of infection correlated with the rate at which hepatic fibrosis progresses. The presence of a competing risk, namely higher mortality from natural causes, may contribute to this observation. A simulation experiment was conducted to measure the magnitude of the effect of competing risks on the observed rate of fibrosis progression of CHC. Methods A computer-based probabilistic model was created in which fibrosis of CHC progressed from stage 0 to 4 (cirrhosis) in 20-year-old and 50-year-old male and female cohorts. The rate of fibrosis progression was randomly assigned to each simulated individual from a distribution common to all age- and sex-specific cohorts. The cohorts also experienced mortality from natural causes according to the 2000 census data. Results The observed median time to reach cirrhosis for the 50-year-old cohorts was 20.4 ± 0.2 years compared with 29.7 ± 0.2 for the 20-year-old cohorts ( P< 0.01). The median time to reach cirrhosis in men was 24.2 ± 0.6 years compared with 25.9 ± 0.6 in women ( P= 0.01). Overall, the observed rate of progression was slowest among young women. Similarly, accelerating mortality from natural causes, simulating the impact of comorbid conditions that shorten survival, reduced the observed time to reach cirrhosis. Conclusions Even if the underlying rate of fibrosis progression in CHC was held constant, the time to reach cirrhosis will be observed to be substantially shorter in subjects with a higher competing mortality.
Natural history of chronic HBV carriers in northern Italy: Morbidity and mortality after 30 years
Mauro Manno, Calogero Cammà, Filippo Schepis, Fabio Bassi, Roberta Gelmini, Francesco Giannini, Francesca Miselli, Antonella Grottola, Ilva Ferretti, Chiara Vecchi, Marisa de Palma, Erica Villa
Background & aims: Increased morbidity and mortality from liver disease have been reported in chronic hepatitis B surface antigen (HBsAg) carriers, but data on survival are equivocal. To assess the impact of hepatitis B virus (HBV) infection on survival and liver-related complications, we re-evaluated, after a mean follow-up of 30 years, a cohort of 296 blood donors excluded from donation 30 years ago when HBsAg screening became mandatory. Methods: Clinical and ultrasound examination and biochemical and virologic tests were performed. The cause of death was recorded and survival was compared with a control population of 157 HBV-negative blood donors selected at baseline. Results: Thirty-two (10.8%) cases and 14 controls (8.9%) ( P= 0.625) had died; 3 of 32 (9.3%) and 1 of 14 (7.1%) deaths were liver-related. Hepatocellular carcinoma (HCC) caused death in 2 of 296 and 1 of 157 subjects (0.6% in each group). Alcohol-induced cirrhosis occured in the remaining subject. By Cox regression analysis, survival was independently predicted by older age, abnormal ?-glutamyl transpeptidase (GGT) levels, and presence of medical comorbidities at baseline. Unequivocal liver disease was found in 4 carriers only. No disease decompensation occurred during follow-up. Fifty-nine (32.2%) carriers cleared HBsAg (yearly incidence, 1.0%). Full-length serum HBV DNA was present in 32.2% of persistently HBsAg-positive individuals (average titer always <10 5copies/mL). Conclusions: Over a 30-year period, chronic HBV carrier blood donors from Northern Italy did not develop clinically significant liver disease, hepatocellular cancer, or other liver-related morbidity or mortality at a higher rate than uninfected controls. The presence of medical comorbidities, older age at diagnosis, and abnormal GGT levels were independent predictors of death among chronic HBV carriers.
Analysis of hepatitis C virus quasispecies transmission and evolution in patients infected through blood transfusion
Tomasz Laskus, Jeffrey Wilkinson, Juan F. Gallegos-Orozco, Marek Radkowski, Debra M. Adair, Marek Nowicki, Eva Operskalski, Zelma Buskell, Leonard B. Seeff, Hugo Vargas, Jorge Rakela
Background & Aims: Studies on hepatitis C virus (HCV) quasispecies dynamics in the natural course of infection are rare owing to difficulties in obtaining samples from the early phase of infection. Methods: We studied 15 patients from the Transfusion-Transmitted Viruses Study who seroconverted to anti-HCV after receiving infected blood. Follow-up serum samples were collected every 23 weeks for 6 months, at 10 months, and at 1116 years. Viral quasispecies in the second envelope hypervariable region 1 (E2/HVR1) and 5? untranslated region (5?UTR) were analyzed with single-strand conformation polymorphism (SSCP) and heteroduplex mobility assay (HMA). Results: Seven patients cleared infection within 724 weeks (mean, 14.0 wk) and 3 patients eventually became anti-HCV negative. In 6 patients with resolving hepatitis the SSCP band pattern remained stable, whereas in one patient minor changes appeared before clearance. In contrast, in all 8 patients progressing to chronicity, major changes in the E2/HVR1 quasispecies developed at 822 weeks (mean, 13.1 wk). Shannon entropy and medium mobility shift values derived from HMA gels remained stable in patients with resolving hepatitis but changed in those who developed chronic infection. Only 2 patients showed minor changes in 5?UTR. A decrease in E2/HVR1 complexity at the time of transmission (bottleneck) was found in 5 patients altogether. Conclusions: Changes in E2/HVR1 quasispecies 822 weeks after infection, likely caused by mounting immune pressure, were predictive of ensuing chronic infection, whereas stability was associated with resolution. Our study also showed that composition of HCV quasispecies may be preserved during transmission from host to host.
Basic-alimentary Tract
Activation of PPAR ? and ? by conjugated linoleic acid mediates protection from experimental inflammatory bowel disease
Josep Bassaganya-Riera, Kathryn Reynolds, Susan Martino-Catt, Yongzhi Cui, Lothar Hennighausen, Frank Gonzalez, Jurg Rohrer, Alejandro Uribe Benninghoff, Raquel Hontecillas
Background & Aims: The molecular targets for the protective actions of conjugated linoleic acid (CLA) on experimental inflammatory bowel disease (IBD) are unknown. We used a loss-of-function approach to investigate whether CLA ameliorated colitis through a peroxisome proliferator-activated receptor ? (PPAR ?)-dependent mechanism. Methods: The expression of PPAR ?, ?, and their target genes in the colon of mice fed control or CLA-supplemented diets was assayed after a 7-day dextran sodium sulfate (DSS) challenge by quantitative real-time polymerase chain reaction (PCR). Additionally, nuclear factor-? B (NF-?B) p65 activation was quantified in the colon. To determine the involvement of PPAR ? in the mechanism of action of CLA directly, specific deletions of PPAR ? in the colon were performed in mice by using the Cre-lox recombination system. Colonic PPAR ? null mice and wild-type littermates were fed either a CLA-supplemented or a control diet for 42 days and challenged with 2.5% DSS. The therapeutic efficacy of CLA also was examined by using the CD4 +CD45RB hi transfer colitis model. Results: CLA induced PPAR ? and ?, transcriptionally modulated PPAR ? and ?-responsive gene clusters involved in lipid metabolism (uncoupling protein [UCP]1, UCP3, PPAR ? coactivator 1? [PGC-1?], and CD36) and epithelial cell maturation (Gob-4 and Keratin 20). Additionally, CLA repressed tumor necrosis factor ? (TNF-?) expression and NF-?B activation while inducing the immunoregulatory cytokine transforming growth factor ? 1 (TGF-? 1). Clinically, CLA ameliorated DSS- and CD4 +-induced colitis. Loss of the PPAR ? gene in the colon abrogated the beneficial effects of CLA in DSS colitis. Conclusions: Our studies provide molecular evidence in vivo, suggesting that CLA ameliorates colitis through a PPAR ?-dependent mechanism.
Interleukin 10-deficient mice develop osteopenia, decreased bone formation, and mechanical fragility of long bones
Rivka Dresner-Pollak, Nir Gelb, Daniel Rachmilewitz, Fanny Karmeli, Miron Weinreb
Background & Aims: Bone loss is a common complication of human inflammatory bowel disease (IBD), but its mechanisms are not understood completely. We investigated bone metabolism in interleukin-10-deficient ( IL-10?/? ) mice, an animal model with IBD features. Methods: IL-10?/? male mice (8- and 12-weeks-old) and their age-matched wild-type counterparts (C57BL/6J) were studied. Bone mass of the femur was determined by ashing. Tibial cancellous and cortical bone mass and formation was measured by static and dynamic histomorphometry. Biomechanical strength of the femur was tested. Primary bone marrow stromal cell cultures were used to assess osteoblast generation. Serum levels of 25-OH vitamin D 3, insulin-like growth factor-1 (IGF-1), parathyroid hormone, osteocalcin, and deoxy-pyridinoline cross-links were measured. The presence of colitis was determined histologically, and by IL-12 and interferon-? (IFN-?) secretion from cultured colonic explants. Results: Eight- and 12-week-old IL-10?/? mice developed osteopenia of both cancellous and cortical bone, evidenced by lower femoral ash weight, cancellous bone area and surface, trabecular number, and decreased cortical bone area and width. Osteopenia was associated with mechanical fragility, manifested by decreased stiffness and mechanical load at fracture, and was caused by suppressed bone formation, indicated by decreased cancellous double-labeled surface, mineralizing surface, serum osteocalcin level, and mineralized nodule number in bone marrow stromal cell cultures. IL-10?/? mice with colitis had significantly less bone mass compared with IL-10?/? mice without colitis. Conclusions: IL-10?/? mice develop the hallmarks of osteoporosis, that is, reduced bone mass, increased mechanical fragility, and suppressed bone formation. The presence of colitis is an important contributor to osteoporosis in IL-10?/? mice.
ClC-2 chloride secretion mediates prostaglandin-induced recovery of barrier function in ischemia-injured porcine ileum
Adam J. Moeser, Melissa M. Haskell, Donnie E. Shifflett, Dianne Little, Bruce D. Schultz, Anthony T. Blikslager Background & Aims Ischemia results in the breakdown of the intestinal barrier, predisposing patients to sepsis and multiple organ failure. Prostaglandins play a critical role in mediating recovery of barrier function in ischemia-injured intestine through a mechanism involving stimulation of Cl ?secretion. In the present study, we investigated the contributory role of individual Cl ?channels in the recovery of barrier function in ischemia-injured porcine ileum. Methods Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers. Short-circuit current (Isc) and transepithelial resistance (TER) were measured in response to prostaglandin E 2(PGE 2) and pharmacologic inhibitors of epithelial Cl ?channels. Immunoassays were used to assess the expression and localization of ion channels. Results Application of PGE 2to ischemia-injured ileal mucosa stimulated increases in Isc, an indicator of Cl ?secretion, that was followed by marked increases in TER, an indicator of barrier function recovery. In vitro studies revealed that although PGE 2induced Cl ?secretion via at least 3 distinct secretory pathways, recovery of barrier function was initiated by Cl ?secretion via ClC-2 Cl ?channels co-expressed with occludin and localized to tight junctions within restituting epithelium. Intravenous administration of furosemide to pigs subjected to 1 hour of ileal ischemia impaired recovery of barrier function, as evidenced by decreased TER and increased mucosal-to-serosal 3H-mannitol flux after a 2-hour reperfusion/recovery period, confirming an important role for Cl ?secretory pathways in vivo. Conclusions ClC-2mediated intestinal Cl ?secretion restores TER in ischemia-injured intestine. These data may provide the basis for targeted pharmacologic therapy for diseases associated with impaired barrier function.
Transmembrane tumor necrosis factor is a potent inducer of colitis even in the absence of its secreted form
Nadia Corazza, Thomas Brunner, Caroline Buri, Silvia Rihs, Martin A. Imboden, Inge Seibold, Christoph Mueller
Background & Aims: Tumor necrosis factor (TNF) is cleaved proteolytically from a 26-kilodalton transmembrane precursor protein into secreted 17-kilodalton monomers. Transmembrane (tm) and secreted trimeric TNF are biologically active and may mediate distinct activities. We assessed the consequences of a complete inhibition of TNF processing on the course of colitis in recombination activating gene (RAG)2 ?/? mice on transfer of CD4 CD45RB hi T cells. Methods: TNF ?/? mice, transgenic for a noncleavable mutant TNF gene, were used as donors of CD4 T cells, and, on a RAG2 ?/? background, also as recipients. Kinetics of disease development were compared in the absence of TNF, in the absence of secreted TNF, and in the presence of secreted and tmTNF. The analysis at the end of the observation period included the histopathologic assessment of the intestine and the localization of TNF and interferon ? (IFN?)-expressing cells. Results: The complete prevention of TNF secretion in tmTNF transgenic RAG2 ?/? mice neither prevented nor delayed disease induction by transferred transgenic for a noncleavable transmembrane mutant of mouse TNF (tmTNF tg) CD4 CD45RB hi T cells. tmTNF expression by transferred CD4 T cells, however, was not required for disease induction because severe colitis and weight loss also were observed in tmTNF RAG2 ?/? recipients of TNF ?/? CD4 CD45RB hi T cells. In the presence of tmTNF, the absence of secreted TNF did not affect frequency and distribution of TNF and interferon-? messenger RNA (mRNA)-expressing cells. Conclusions: These results indicate that specific inhibitors of TNF processing are not appropriate for modulating the pro-inflammatory and disease-inducing effects of TNF in chronic inflammatory disorders of the intestine.
Lactobacillus paracasei normalizes muscle hypercontractility in a murine model of postinfective gut dysfunction
Elena F. Verdú, Premysl Bercík, Gabriela E. Bergonzelli, Xian-Xi Huang, Patricia Blennerhasset, Florence Rochat, Muriel Fiaux, Robert Mansourian, Irène Corthésy-theulaz, Stephen M. Collins
Background & Aims: The effects of probiotics on gut dysfunction in postinfective irritable bowel syndrome are unknown. We tested whether probiotics influence persistent muscle hypercontractility in mice after recovery from infection with Trichinella spiralis and analyzed the underlying mechanisms. Methods: Mice were gavaged with Lactobacillus paracasei, Lactobacillus johnsonii, Bifidobacterium longum , or Bifidobacterium lactis in spent culture medium from days 10 to 21 after infection. Additional mice received heat-inactivated Lactobacillus paracasei, Lactobacillus paracasei -free spent culture medium, or heat-inactivated Lactobacillus paracasei -free spent culture medium. Lactobacilli enumeration, immunohistochemistry, and cytokine detection (enzyme-linked immunosorbent assay) were performed. Mice were also treated with Lactobacillus paracasei or Lactobacillus paracasei -free spent culture medium from days 18 to 28 after infection. Contractility was measured on days 21 and 28 after infection. Results: Lactobacillus paracasei , but not Lactobacillus johnsonii, Bifidobacterium lactis , or Bifidobacterium longum , attenuated muscle hypercontractility. This was associated with a reduction in the Trichinella spiralis -associated T-helper 2 response and a reduction in transforming growth factor-?1, cyclooxygenase-2, and prostaglandin E 2levels in muscle. Attenuation of muscle hypercontractility by Lactobacillus paracasei -free spent culture medium was abolished after heat treatment. Improvement of muscle hypercontractility at day 28 after infection was also observed after the administration of Lactobacillus paracasei or Lactobacillus paracasei -free spent culture medium from day 18 after infection. Conclusions: Probiotics show strain-dependent attenuation of muscle hypercontractility in an animal model of postinfective irritable bowel syndrome. This likely occurs via both a modulation of the immunologic response to infection and a direct effect of Lactobacillus paracasei or a heat-labile metabolite on postinfective muscle hypercontractility. Lactobacillus paracasei may be useful in the treatment of postinfective irritable bowel syndrome.
Ursodeoxycholate/Sulindac combination treatment effectively prevents intestinal adenomas in a mouse model of polyposis
Russell F. Jacoby, Carolyn E. Cole, E.T. Hawk, R.A. Lubet
Background & Aims: Preclinical studies in animal models, human epidemiological data, and clinical trials in patients with adenomatous polyposis have consistently indicated that sulindac and other nonsteroidal antiinflammatory drugs or cyclooxygenase inhibitors have the greatest potential efficacy among current candidates for colon tumor chemopreventive agents. However, at highly effective doses they all have some risk of toxicity, and their therapeutic profile might be improved by use at lower, more tolerable doses, in combination with a second agent acting via other mechanisms. Methods: Sulindac was tested in combination with ursodeoxycholic acid (ursodiol), a naturally occurring 7-B-epimer of the bile component chenodeoxycholic acid, for prevention of adenomas in the Min mouse model of adenomatous polyposis. Results: Ursodeoxycholic acid caused a dose-dependent decrease in the number of intestinal tumors. Unlike sulindac and other nonsteroidal anti-inflammatory drugs, which are quite beneficial in the distal intestine but are somewhat less effective in the proximal small intestine (especially the clinically important periampullary duodenum), ursodeoxycholate had equal efficacy throughout the entire intestine, both proximal and distal. Combined treatment with low-dose sulindac was less toxic, with normal weight gain and fewer gastrointestinal ulcerations than high-dose sulindac. Combined treatment with sulindac and ursodeoxycholate was more effective than either agent alone for the prevention of tumors throughout the entire intestine. Conclusions: These experiments provide the first evidence that ursodeoxycholic acid is effective for preventing adenomas in an animal model. Cyclooxygenase inhibition, when combined with this naturally occurring bile component, may become a promising approach for colon cancer prevention.
Helicobacter pylori and H 2O2increase AP endonuclease-1/redox factor-1 expression in human gastric epithelial cells
Song-Ze Ding, Ann M. O’Hara, Tim L. Denning, Bernadette Dirden-Kramer, Randy C. Mifflin, Victor E. Reyes, Kieran A. Ryan, Susan N. Elliott, Tadahide Izumi, Istvan Boldogh, Sankar Mitra, Peter B. Ernst, Sheila E. Crowe Background & Aims: Helicobacter pylori infection causes inflammation, accumulation of reactive oxygen species, and oxidative DNA damage in the gastric mucosa. Apurinic/apyrimidinic endonuclease-1 (APE-1)/redox factor-1 (Ref-1) repairs damaged DNA and reductively activates transcription factors, including activator protein-1. Considering that H. pylori generate reactive oxygen species and that reactive oxygen species modulate APE-1/Ref-1 in other cell types, we examined the effect of H. pylori , oxidative stress, and antioxidants on APE-1/Ref-1 expression in human gastric epithelial cells. Methods: Human gastric epithelial cell lines or cells isolated from mucosal biopsy samples were stimulated with H. pylori, Campylobacter jejuni , and/or H 2O2in the presence or absence of antioxidants. APE-1/Ref-1 expression was assayed by Western blot or reverse-transcription polymerase chain reaction, and its cellular distribution was determined by using indirect conventional and confocal immunofluorescence. New protein synthesis was detected by [S 35 ]methionine labeling. APE-1/Ref-1 function was assessed by using a luciferase-linked reporter construct containing 3 activator protein 1 binding sites. Results: APE-1/Ref-1 protein and messenger RNA were detected in resting gastric epithelial cells. APE-1/Ref-1 protein expression was increased after stimulation with H 2O2or live cag pathogenicity island-bearing H. pylori , but not cag pathogenicity island-negative H. pylori or C. jejuni .H. pylori - or reactive oxygen species-mediated increases in APE-1/Ref-1 expression involved de novo protein synthesis that was inhibited by antioxidants. H. pylori or H 2O2also induced nuclear accumulation of APE-1/Ref-1, and overexpression of APE-1/Ref-1 increased activator protein 1 binding activity. Conclusions: The data show that H. pylori or reactive oxygen species enhance APE-1/Ref-1 protein synthesis and nuclear accumulation in human gastric epithelial cells and implicate APE-1/Ref-1 in the modulation of the pathogenesis of H. pylori infection.
Differing roles of protein kinase C-? in disruption of tight junction barrier by enteropathogenic and enterohemorrhagic Escherichia coli
Farol L. Tomson, Athanasia Koutsouris, V.K. Viswanathan, Jerrold R. Turner, Suzana D. Savkovic, Gail Hecht
Background & Aims :Enteropathogenic Escherichia coli and enterohemorrhagic E. coli harbor highly homologous pathogenicity islands yet show key differences in their mechanisms of action. Both disrupt host intestinal epithelial tight junctions, but the effects of enteropathogenic E. coli are more profound than those of enterohemorrhagic E. coli . The basis for this is not understood. The atypical protein kinase C isoform, protein kinase C-?, associates with and regulates the tight junction complex. The aim of this study was to compare the role of protein kinase C-? in the disruption of tight junctions after infection with enteropathogenic E. coli and enterohemorrhagic E. coli .Methods :Model intestinal epithelial monolayers infected by enteropathogenic E. coli or enterohemorrhagic E. coli were used for these studies. Results :Neither bisindolylmaleimide nor Gö6976, which block several protein kinase C isoforms but not protein kinase C-?, protected against the decrease in transepithelial electrical resistance after enteropathogenic E. coli infection. Rottlerin at concentrations that block novel and atypical isoforms, including protein kinase C-?, significantly attenuated the decrease in transepithelial electrical resistance. The specific inhibitory peptide, myristoylated protein kinase C-? pseudosubstrate, also significantly decreased the enteropathogenic E. coli associated decrease in transepithelial electrical resistance and redistribution of tight junction proteins. In contrast to enteropathogenic E. coli , the level of protein kinase C-? enzyme activity stimulated by enterohemorrhagic E. coli was transient and minor, and protein kinase C-? inhibition had no effect on the decrease in transepithelial electrical resistance or the redistribution of occludin. Conclusions :The differential regulation of protein kinase C-? by enteropathogenic E. coli and enterohemorrhagic E. coli may in part explain the less profound effect of the latter on the barrier function of tight junctions.
Basic-liver, Pancreas, and Biliary Tract
Immune stimulation of hepatic fibrogenesis by CD8 cells and attenuation by transgenic interleukin-10 from hepatocytes
Rifaat Safadi, Masayuki Ohta, Carlos E. Alvarez, M. Isabel Fiel, Meena Bansal, Wajahat Z. Mehal, Scott L. Friedman
Backgrounds & Aims :Immunomodulatory cytokines, including interleukin-10 (IL-10), may mediate hepatic fibrosis. Methods :We generated transgenic (TG) mice with hepatocyte expression of rat IL-10 (rIL-10) to assess its impact on lymphocyte subsets and activation of hepatic stellate cells following liver injury from carbon tetrachloride (CCl 4) or thioacetamide (TAA). Results :Fibrosis was reduced in the TG animals in both models, which was not explained solely by differences in liver injury. By fluorescence-activated cell sorter (FACS), there were less CD4+ T cells in naive TG mice, and, following fibrosis induction, CD4+ T cells decreased only in wild-type (WT) mice, whereas increases in CD8+ T cells seen in WT animals were significantly attenuated in TG mice. Subtotal irradiation diminished fibrosis equally in both WT and TG groups, suggesting that rIL-10’s antifibrotic effect was lymphocyte mediated. To assess the role of lymphocytes on stellate cell activation, either whole splenic lymphocytes, CD4+, or CD8+ T-cell subsets from WT animals with CCl 4fibrosis were adoptively transferred to severe combined immunodeficiency (SCID) recipients, which led to stellate cell activation and fibrogenic stimulation as assessed by expression of transforming growth factor (TGF)-?1 and collagen I messenger RNA (mRNA) and by immunoblot of ?-smooth muscle actin. Moreover, serum aminotransferase levels and stellate cell activation mRNA were significantly higher among the CD8+ T-cell recipients. Conclusions :Transgenic expression of rIL-10 in liver leads to reduced fibrosis and alterations in liver lymphocyte subsets both in untreated liver and following fibrosis induction. In this model, fibrosis may be a CD8+ T-cell-mediated disease that is attenuated by rIL-10.
Trypsin mediates nociception via the proteinase-activated receptor 2: A potentially novel role in pancreatic pain
Willemijntje A. Hoogerwerf, Mohan Shenoy, John H. Winston, Shu-Yuan Xiao, Zhijun He, Pankaj J. Pasricha
Background & Aims: The pathogenesis of pain in pancreatitis remains poorly understood. We hypothesized that trypsin, a key inflammatory mediator in this condition, can also activate nociceptive neurons via the proteinase-activated receptor 2. Methods: Double immunohistochemical staining of T8 to T12 dorsal root ganglia sections was performed with antibodies against proteinase-activated receptor 2 and vanilloid receptor 1, a marker for primary nociceptive neurons. In vivo nociceptive activity was measured by FOS immunoreactivity in thoracic spinal dorsal horn segments after intrapancreatic administration of proteinase-activated receptor 2 agonists. Pain behavior was assessed by visceromotor reflex activity in response to noxious stimulation of the pancreas with proteinase-activated receptor 2 agonists. Results: Proteinase-activated receptor 2 was expressed by virtually all nociceptive neurons in thoracic dorsal root ganglia. Intraductal trypsin, in subinflammatory concentrations, activated spinal dorsal horn neurons in a dose-dependent manner, as measured by FOS expression. Both trypsin and a proteinase-activated receptor 2specific peptide agonist induced a behavioral pain response when infused into the pancreatic duct of awake rats. Preinfusion of the pancreatic duct with proteinase-activated receptor 2specific activating peptide desensitized the response to trypsin. Conclusions: Our findings suggest a novel proteinase-activated receptor 2mediated role for trypsin in the pathogenesis of pancreatic pain and one that is independent of its inflammatory effect.
Inducible histamine protects mice from P. acnes-primed and LPS-induced hepatitis through H2-receptor stimulation
Minori Yokoyama, Akira Yokoyama, Shuji Mori, Hideo K. Takahashi, Tadashi Yoshino, Takeshi Watanabe, Takehiko Watanabe, Hiroshi Ohtsu, Masahiro Nishibori
Background & Aims: Inducible histamine and histamine H2-receptors have been suggested to be involved in innate immune response. Methods: We examined a functional role of inducible histamine in the protection against hepatic injury and lethality in Propionibacterium acnes -primed and lipopolysaccharide-induced hepatitis, using histidine decarboxylase knockout and H2-receptor knockout mice. Results: Lipopolysaccharide challenge after Propionibacterium acnes priming increased histidine decarboxylase activity in the liver of wild-type mice, associated with a marked elevation of histamine turnover. Histidine decarboxylase-like immunoreactivity was observed in CD68-positive Kupffer cells/macrophages. Treatment of wild-type mice with famotidine or ranitidine but not d-chlorpheniramine augmented hepatic injury and inhibited the survival rate significantly. The same dose of Propionibacterium acnes and lipopolysaccharide induced severe hepatitis and high lethality in histidine decarboxylase knockout and H2-receptor knockout mice; the former were rescued by the subcutaneous injection of histamine. Immunohistochemical study supported the protective role of histamine against the apoptosis of hepatocytes. Histamine suppressed the expression of IL-18 and tumor necrosis factor ? in the liver, leading to the reduced plasma levels of cytokines including IL-18, TNF-?, IL-12, IFN-?, and IL-6. Conclusions: These findings as a whole indicated that endogenously produced histamine in Kupffer cells/macrophages plays a very important role in preventing excessive innate immune response in endotoxin-induced fulminant hepatitis through the stimulation of H2-receptors
Calcium-dependent regulation of secretion in biliary epithelial cells: The role of apamin-sensitive SK channels
Andrew P. Feranchak, R. Brian Doctor, Marlyn Troetsch, Kathryn Brookman, Sylene M. Johnson, J. Gregory Fitz
Background & Aims: Increases in intracellular Ca 2+ are thought to complement cAMP in stimulating Cl ?secretion in cholangiocytes, although the site(s) of action and channels involved are unknown. We have identified a Ca 2+ -activated K +channel (SK2) in biliary epithelium that is inhibited by apamin. The purpose of the present studies was to define the role of SK channels in Ca 2+ -dependent cholangiocyte secretion. Methods: Studies were performed in human Mz-Cha-1 cells and normal rat cholangiocytes (NRC). Currents were measured by whole-cell patch clamp technique and transepithelial secretion by Ussing chamber. Results: Ca 2+ -dependent stimuli, including purinergic receptor stimulation, ionomycin, and increases in cell volume, each activated K +-selective currents with a linear IV relation and time-dependent inactivation. Currents were Ca 2+ dependent and were inhibited by apamin and by Ba 2+ . In intact liver, immunoflourescence with an antibody to SK2 showed a prominent signal in cholangiocyte plasma membrane. To evaluate the functional significance, NRC monolayers were mounted in a Ussing chamber, and the short-circuit current ( Isc ) was measured. Exposure to ionomycin caused an increase in Isc 2-fold greater than that induced by cAMP. Both the basal and ionomycin-induced Isc were inhibited by basolateral Ba 2+ , and ?58% of the basolateral K +current was apamin sensitive. Conclusions: These studies demonstrate that cholangiocytes exhibit robust Ca 2+ -stimulated secretion significantly greater in magnitude than that stimulated by cAMP. SK2 plays an important role in mediating the increase in transepithelial secretion due to increases in intracellular Ca 2+ . SK2 channels, therefore, may represent a target for pharmacologic modulation of bile flow.
Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning
Rita Carini, Maria Grazia De Cesaris, Roberta Splendore, Gianluca Baldanzi, Maria Paola Nitti, Elisa Alchera, Nicoletta Filigheddu, Cinzia Domenicotti, Maria Adelaide Pronzato, Andrea Graziani, Emanuele Albano
Background & Aims: Ischemic preconditioning has been proved effective in reducing ischemia/reperfusion injury during liver surgery. However, the mechanisms involved are still poorly understood. Here, we have investigated the role of phosphatidylinositol 3-kinase (PI3K) in the signal pathway leading to hepatic preconditioning. Methods: PI3K activation was evaluated in isolated rat hepatocytes preconditioned by 10-minute hypoxia followed by 10-minute reoxygenation. Results: Hypoxic preconditioning stimulated phosphatidylinositol-3,4,5-triphosphate production and the phosphorylation of PKB/Akt, a downstream target of PI3K. Conversely, PI3K inhibition by wortmannin or LY294002 abolished hepatocyte tolerance against hypoxic damage induced by preconditioning. PI3K activation in preconditioned hepatocytes required the stimulation of adenosine A 2A receptors and was mimicked by adenosine A 2A receptors agonist CGS21680. In the cells treated with CGS21680, PI3K activation was prevented either by inhibiting adenylate cyclase and PKA with, respectively, 2,5-dideoxyadenosine and H89 or by blocking G?i-protein and Src tyrosine kinase with, respectively, pertussis toxin and PP2. H89 also abolished the phosphorylation of adenosine A 2A receptors. However, the direct PKA activation by forskolin failed to stimulate PI3K. This suggested that PKA-phosphorylated adenosine A 2A receptors may activate PI3K by coupling it with G?i-protein through Src. We also observed that, by impairing PI3K-mediated activation of phospholypase C? (PLC?), wortmannin and LY294002 blocked the downstream transduction of preconditioning signals via protein kinase C (PKC) ?/? isozymes. Conclusions: PI3K is activated following hepatocyte hypoxic preconditioning by the combined stimulation of adenosine A 2A receptors, PKA, G?i protein, and Src. By regulating PKC-?/?-dependent signals, PI3K can play a key role in the development of hepatic tolerance to hypoxia/reperfusion.
High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection
Georg M. Lauer, Eleanor Barnes, Michaela Lucas, Joerg Timm, Kei Ouchi, Arthur Y. Kim, Cheryl L. Day, Gregory K. Robbins, Deborah R. Casson, Markus Reiser, Geoffrey Dusheiko, Todd M. Allen, Raymond T. Chung, Bruce D. Walker, Paul Klenerman
Background & Aims: Cellular immune responses are thought to play a key role in the resolution of primary HCV infection. Although it has been consistently shown that CD4+ T-cell responses are maintained in those with spontaneous resolution but lost in those with persistent infection, the role of CD8+ T-cell responses remains controversial. Previous studies have largely focused on limited HLA alleles and predefined CD8+ T-cell epitopes, and, thus, comprehensive studies remain to be performed. Methods: To understand the composition of the immune response associated with spontaneous resolution, we comprehensively mapped CD8+ T-cell responses in 20 HLA-diverse persons with resolved HCV infection, using HCV peptides spanning the entire genome. We analyzed the magnitude, breadth, function, and phenotype using ELISpot, class-I tetramers, intracellular cytokine staining, and cytolytic assays. We studied in parallel HCV-specific responses and viral sequence variation in persistent infection. Results: Responses in individuals with resolved infection were strong and broad with robust proliferation in response to antigen. Responses in those persistently infected were rarely detected ex vivo and, when present, were narrowly directed and weak. However, they also proliferated in vitro. Dominant target epitopes differed among individuals in both cohorts, despite frequently shared HLA-alleles. Conclusions: These data indicate that persisting, strong CD8+ T-cell responses are observed in the majority of persons with resolved HCV infection and provide support for strategies to boost CD8+ T-cell responses for the prevention or treatment of HCV infection but also highlight the diversity of responses that may need to be elicited to provide protection.
PTF1?/p48 and cell proliferation
Annie Rodolosse, Elisabet Chalaux, Teresa Adell, Hélène Hagège, Anouchka Skoudy, Francisco X. Real
Background & Aims: The basic helix-loop-helix transcription factor pancreas-specific transcription factor 1? (PTF1?)/p48 is critical for committing cells to a pancreatic fate and for the maintenance of the differentiated state in acinar cells. The aim was to analyze the ability of p48 to modulate cell proliferation, its relationship with cell differentiation, and the mechanisms involved therein. Methods: Pancreatic and nonpancreatic cells were transfected with p48 cDNA, and the effects on cell proliferation were examined. The effects on cell cycle regulators were analyzed by Western blotting and RT-PCR; transient transfection assays were used to analyze promoter regulation. Results: p48 Inhibited proliferation of acinar and nonacinar cells by inducing a delay in G1-S progression through the up-regulation of p21 CIP1/WAF1 and p27 KIP1 and the down-regulation of cyclin D2. A 2-fold increase in p21 CIP1/WAF1 mRNA and in the activity of the p21 CIP1/WAF1 promoter was observed. The growth inhibition action of p48 was not associated with exocrine differentiation or with apoptosis. The antiproliferative effects were dependent on the COOH-terminal region of p48 and did not require the bHLH domain. Loss of p48 expression occurring during acinar-to-ductal transitions, characteristic of chronic pancreatitis, was associated with an increase of cell proliferation in ductal complexes. Conclusions: The results indicate that p48 couples cell proliferation and cell differentiation in the exocrine pancreas, thus contributing to tissue homeostasis. These effects may play a role in the increased risk for pancreatic cancer associated with chronic pancreatitis.
Copyright © 2001-2004 by the American Gastroenterological Association. All rights reserved.
Volume 41, September 2004
The effects of oxidative stress on the liver sieve
Victoria C. Cogger 1?,Michael Muller 1,Robin Fraser 1,2 ,Allan J. McLean 3,Jameel Khan 1and David G. Le Couteur 1
Received 28 September 2003;revised 22 April 2004;accepted 28 April 2004. published online 19 August 2004. Abstract
Background/Aims
Oxidative stress is implicated in the pathogenesis of age-related and disease-associated changes in the hepatic sinusoid. We studied the effects of oxidative stress on the morphology of the liver, focusing specifically on the hepatic sinusoidal endothelium (the ‘liver sieve’). Methods The effects of tert -butyl hydroperoxide on the intact liver and isolated sinusoidal endothelial cells were assessed by electron microscopy, immunohistochemistry and biochemical analysis. Results Immunohistochemistry revealed a dose-dependent increase in peri-sinusoidal 3-nitrotyrosine staining, particularly in the regions adjacent to the portal triads. Electron microscopy showed dose-dependent formation of large intracellular gaps in the sinusoidal endothelium with reduction in the diameter of the remaining endothelial fenestrations. Activated Kupffer cells extending processes through the fenestrations to contact hepatocytes were noted. Biochemical analysis of total liver tissue showed no significant changes in malondialdehyde content but a decrease in the ratio of GSH to GSSG. tert -Butyl hydroperoxide administered directly onto isolated liver sinusoidal endothelial cells was associated with similar gap formation, indicating a direct effect on the endothelial cells by tert -butyl hydroperoxide. Conclusions Oxidative stress selectively damages hepatic sinusoidal endothelial cells. This has implications for those processes associated with changes in the sinusoidal endothelium such as ageing, cirrhosis and exposure to hepatotoxins.
High-fat enteral nutrition reduces endotoxin, tumor necrosis factor-alpha and gut permeability in bile duct-ligated rats subjected to hemorrhagic shock
Misha D.P. Luyer 1,Wim A. Buurman 1?,M'hamed Hadfoune 1,Jan A. Jacobs 2,Cornelis H.C. Dejong 1and Jan Willem M. Greve 1Received 31 October 2003; revised 27 April 2004;accepted 28 April 2004. published online 19 August 2004. Abstract Background/Aims Cholestatic patients are prone to septic complications after major surgery due to an increased susceptibility to endotoxin and hypotension. High-fat enteral nutrition reduces endotoxin after hemorrhagic shock. However, it is unknown whether this nutritional intervention is protective in biliary obstruction. We investigated the effect of high-fat enteral nutrition on endotoxin, tumor necrosis factor-alpha (TNF-?) and intestinal permeability in cholestatic rats subjected to hemorrhagic shock. Methods Bile duct-ligated (BDL) rats were fasted or fed with low-fat or high-fat enteral nutrition before hemorrhagic shock. Blood and tissue samples were taken after 90 min. Results Plasma endotoxin decreased after hemorrhagic shock in BDL-rats fed with high-fat nutrition compared to fasted ( P<0.01) and low-fat treated rats ( P<0.05). Additionally, circulating TNF-? was reduced in BDL-rats pretreated with high-fat nutrition compared to fasted rats ( P<0.01). The increased intestinal permeability to macromolecules was reduced by high-fat enteral nutrition, whereas bacterial translocation did not significantly change. Simultaneously, tight junction distribution in ileum and colon was disrupted in non-treated BDL-rats but remained unchanged in high-fat pretreated BDL-rats. Conclusions High-fat enteral nutrition protects against endotoxin-mediated complications independently of intraluminal bile. These results provide a potential new strategy to prevent endotoxin-mediated complications in cholestatic patients undergoing major surgery.
Effect of intravenous albumin on systemic and hepatic hemodynamics and vasoactive neurohormonal systems in patients with cirrhosis and spontaneous bacterial peritonitis
Javier Fernández 1?,Miguel Navasa 1,Juan Carlos Garcia-Pagan 1,Juan G-Abraldes 1,Wladimiro Jiménez 2,Jaume Bosch 1and Vicente Arroyo 1
Received 17 December 2003;revised 29 April 2004;accepted 7 May 2004. published online 19 August 2004. Abstract
Background/Aims Albumin administration prevents renal failure and improves survival in spontaneous bacterial peritonitis. This study characterizes the mechanisms of action of albumin in this condition. Methods Systemic and splanchnic hemodynamics, plasma renin activity and plasma concentration of interleukin-6, serum concentration of nitric oxide and ascitic fluid levels of nitric oxide and interleukin-6 were assessed at diagnosis and resolution of infection in 12 patients with spontaneous bacterial peritonitis treated with ceftriaxone plus albumin. At infection resolution there was a significant improvement in circulatory function, as indicated by a significant increase in mean arterial pressure (+8%, P=0.02), a fall in heart rate (?10%, P=0.01), a suppression of plasma renin activity (?67%, P=0.002) and a decrease in creatinine levels. These changes were related to both an increase in cardiac work (stroke work index: +18%, P=0.005) and in peripheral vascular resistance (+14%, P=0.05). The improvement in cardiac function was due to an increase in filling. No significant changes were observed in portal pressure or hepatic blood flow. Conclusions These results indicate that the beneficial effects of albumin administration on systemic hemodynamics and renal function in spontaneous bacterial peritonitis are related to both an improvement in cardiac function and a decrease in the degree of arterial vasodilation.
A novel aminosterol reverses diabetes and fatty liver disease in obese mice
Nobuhiko Takahashi ,Yong Qi ,Hiral R. Patel and Rexford S. Ahima ?
Received 29 January 2004;revised 6 April 2004;accepted 4 May 2004. published online 19 August 2004. Abstract Background/Aims Non-alcoholic fatty liver disease (NAFLD) is common in obesity. However, weight reduction alone does not prevent the development or progression of NAFLD. Since NAFLD is associated with insulin resistance and diabetes, we hypothesized that improvement of these factors would reverse obesity-related NAFLD. Methods We examined the effects of an aminosterol, 1436, on glucose, lipids and liver metabolism in Lep ob/ob mice, a model of obesity, severe insulin resistance, diabetes, hyperlipidemia and hepatic steatosis. Results 1436 decreased body weight, specifically fat content, by inhibiting food intake and increasing energy expenditure. In contrast to weight loss from food restriction, this aminosterol specifically lowered circulating lipids, reversed hepatic steatosis and normalized alanine aminotransferase level. 1436 decreased glucose, increased adiponectin and enhanced insulin action in liver. These changes culminated in inhibition of hepatic triglyceride synthesis and increased fatty acid oxidation. Gene expression studies confirmed a reduction in lipogenic enzymes in liver, and elevation of enzymes involved in lipid catabolism. Conclusions These results demonstrate that 1436 is an effective treatment for insulin resistance and hepatic steatosis in Lep ob/ob mice, by decreasing hepatic lipid synthesis and stimulating lipolysis. In contrast, weight loss from food restriction has no substantial effect on insulin resistance, lipids and hepatic steatosis.
A gene-expression signature can quantify the degree of hepatic fibrosis in the rat
Tohru Utsunomiya a,Masahiro Okamoto a,Masaji Hashimoto b,Keiji Yoshinaga a,Takeshi Shiraishi a,Fumiaki Tanaka a,Koshi Mimori a,Hiroshi Inoue a,Goro Watanabe b,Graham F. Barnard c,Masaki Mori a*
Received 26 January 2004; received in revised form 5 March 2004; accepted 5 May 2004 published online 19 August 2004. Backgrounds/Aims A more accurate and objective quantification of hepatic fibrosis would provide clinically useful information for the monitoring of chronic liver disease progression and therapy recommendation. Methods Using a cDNA microarray of 14,814 clones, we analyzed the gene-expression profiles of fibrotic livers in a rat model. Results We identified 750 up- and 345 down-regulated genes by combining a signal-to-noise score and a random permutation test ( P<0.01). The functions of these genes provided insight into the underlying molecular mechanisms of both structural remodeling and functional deficits in cirrhosis. To quantify the extent of liver fibrosis, we have generated for the first time a ‘genetic fibrosis index’ based on gene-expression profiling of 95 genes by combining a Pearson correlation coefficient and a ‘leave-one-out’ cross-validation procedure. This technique based on a supervised learning analysis correctly quantified the various degrees of fibrosis in both 20 training samples ( R2=0.829, P<0.001) and 6 test samples ( R2=0.822, P<0.05). Conclusions Our method will assist researchers in identifying rational targets for intervention and might help clinicians to objectively monitor the severity of liver fibrosis.
Molecular mechanisms regulating the antifibrogenic protein heme-oxygenase-1 in human hepatic myofibroblasts
Liying Li a,Boris Julien a,Pascale Grenard a,Fatima Teixeira-Clerc a,Ariane Mallat ab,Sophie Lotersztajn a*Received 5 March 2004; received in revised form 15 May 2004; accepted 27 May 2004 published online 19 August 2004. Background/Aims Hepatic myofibroblasts are central in liver fibrogenesis associated with chronic liver diseases. We previously showed that heme-oxygenase-1 (HO-1) displays antifibrogenic properties in human hepatic myofibroblasts. Here, we further investigated the mechanisms regulating HO-1 expression.
Methods Expression of HO-1 was assayed in cultured human hepatic myofibroblasts by Northern and Western blot. Functional studies were also performed in cultured human hepatic myofibroblasts.
Results 15-Deoxy-? 12,14 -prostaglandin J2 (15-d-PGJ2) elicited inhibition of proliferation and of ?1(I) collagen mRNA expression. These effects were reproduced by the glutathione depletor diethyl maleate and blunted by the glutathione precursor N-acetyl cysteine, indicating the involvement of oxidative stress. Two consecutive events mediated inhibition of proliferation and of ?1(I) collagen mRNA expression by 15-d-PGJ2: (i) mild oxidative stress characterized by a transient GSH decrease and (ii) activation of p38 MAPK, resulting in increased HO-1 mRNA stability. Conclusions Our results provide new insights into the regulatory mechanisms governing HO-1 expression in human hepatic myofibroblasts and identify mild oxidative stress and p38 MAPK as two consecutive early signals promoting HO-1 induction that are crucial for its antifibrogenic properties, namely inhibition of growth and extracellular matrix gene expression.
Protein kinase A dependent signalling mediates anti-apoptotic effects of the atrial natriuretic peptide in ischemic livers
Stefanie Kulhanek-Heinze a,Alexander L. Gerbes b,Tobias Gerwig ab,Angelika M. Vollmar a,Alexandra K. Kiemer ab*
Received 16 September 2003; received in revised form 17 May 2004; accepted 27 May 2004 published online 19 August 2004.
Background/Aims Preconditioning of livers with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury (IRI) via the particulate guanylate cyclase. Recently, we have shown that ANP affects the p38 MAPK signalling cascade in the liver. Thus, aim of the present study was to elucidate the role of cGMP- and p38 MAPK-dependent signalling pathways in ANP-mediated anti-apoptotic effects. Methods Rat livers were perfused with KH-buffer with or without ANP, 8-Br-cGMP (±kinase inhibitors) and kept in UW solution (4°C, 24h). Caspase-3-like activity was measured by a fluorometric assay. Expression of cGMP-dependent protein kinases (PKG) in liver tissue was determined by RT-PCR, BAD phosphorylation by Western blot, and cAMP-dependent protein kinase (protein kinase A, PKA) activity by in vitro phosphorylation. Results Compared to control organs, ANP-preconditioning reduced post-ischemic caspase-3-like activity. Neither perfusion with a p38 MAPK inhibitor nor with a PKG inhibitor abolished the ANP-mediated anti-apoptotic action. The two PKG isoforms were demonstrated not to be expressed in the liver. In contrast, liver perfusion with a selective PKA inhibitor abrogated the anti-apoptotic effect of ANP. Phosphorylation of pro-apoptotic BAD by ANP-activated PKA might inhibit liver cell apoptosis. Conclusions ANP mediates its anti-apoptotic action during ischemic injury via a crosstalk with the PKA pathway.
Usefulness of contrast-enhanced perfusional sonography in the assessment of hepatocellular carcinoma hypervascular at spiral computed tomography
Stefano Gaiani 1?,Natascia Celli 1,Fabio Piscaglia 1,Laura Cecilioni 1,Franco Losinno 2,Francesco Giangregorio 3,Mikaela Mancini 1,Patrizia Pini 1,Fabio Fornari 3and Luigi Bolondi 1
Received 22 November 2003;revised 16 April 2004;accepted 28 April 2004. published online 19 August 2004. Abstract Background/Aims Diagnosis of hepatocellular carcinoma (HCC) relies strongly on the detection of hypervascularity in the arterial phase and, in this setting, spiral computed tomography (CT) is the most widely used method. This prospective study aimed to investigate the usefulness of low mechanical index harmonic ultrasound (US), using a second generation contrast-enhanced technique, in the assessment of vascular pattern of HCC shown to be hypervascular at spiral CT. Methods A total of 79 cirrhotic patients with 103 nodules (mean±SD 28±13 mm) with arterial hypervascularity at spiral CT were studied. US examination was performed by perfusional sonography, using a new dedicated technology (CnTI-Esaote), operating at low mechanical index, after injection of a second generation contrast agent (SonoVue-Bracco), allowing detection of tumoral flow during arterial phase. Results Selective arterial enhancement on perfusional sonography was observed in 94 /103 nodules (91.3%), with a sensitivity of 66.6, 87.5, 91.7, and 97.3% in nodules ≤1 cm, >1≤2 cm, >2≤3 cm, and >3 cm respectively. Conclusions Perfusional sonography shows good diagnostic agreement with spiral CT in hypervascular HCC and may be proposed for the immediate vascular characterization of nodules detected at US and used as second imaging technique to confirm hypervascularity in cirrhotic nodules.
Transarterial chemo-lipiodolization can reactivate hepatitis B virus replication in patients with hepatocellular carcinoma
Jeong Won Jang ,Jong Young Choi *,Si Hyun Bae ,Chang Wook Kim ,Seung Kew Yoon ,Se Hyun Cho ,Jin Mo Yang ,Byung Min Ahn ,Chang Don Lee ,Young Sok Lee ,Kyu Won Chung ,Hee Sik Sun
Received 17 November 2003; received in revised form 6 April 2004; accepted 4 May 2004 published online 19 August 2004. Background/Aims Reactivation of hepatitis B virus (HBV) replication is a well-known complication in cancer patients receiving chemotherapy. The aims of this study were to determine the incidence of HBV reactivation in hepatocellular carcinoma (HCC) patients undergoing transarterial chemo-lipiodolization, and to clarify factors contributing to HBV reactivation. Methods From April 2001 to September 2002, 146 HBsAg positive patients newly diagnosed as HCC were enrolled in the study. Among these, 83 patients underwent transarterial chemo-lipiodolization using epirubicin and/or cisplatin, and 63 received other treatments. Results In total, HBV reactivation occurred in 30 (20.5%) patients (28 with chemo-lipiodolization and 2 with other treatments), and of the 30 patients, 19 (13.0%) (18 with chemo-lipiodolization and 1 with other treatments) developed hepatitis. Chemo-lipiodolization was significantly correlated with a higher incidence of hepatitis attributed to HBV reactivation than other treatments (21.7% vs. 1.6%, P<0.001), irrespective of HBeAg or HBV DNA. Among 83 patients undergoing chemo-lipiodolization, HBV reactivation occurred in 28 (33.7%) patients, and HBeAg seropositivity was the only independent predictor of HBV reactivation ( P=0.013). Three (10.7%) of them died of hepatic decompensation resulting from HBV reactivation. Conclusions Transarterial chemo-lipiodolization can reactivate HBV, and HBeAg-positive HCC patients receiving chemo-lipiodolization should be closely monitored for HBV reactivation.
Identification of genes up-regulated by histone deacetylase inhibition with cDNA microarray and exploration of epigenetic alterations on hepatoma cells
Tetsuhiro Chiba a,Osamu Yokosuka a*,Makoto Arai a,Motohisa Tada a,Kenichi Fukai a,Fumio Imazeki a,Masaki Kato b,Naohiko Seki b,Hiromitsu Saisho a
Received 13 December 2003; received in revised form 19 April 2004; accepted 27 May 2004 published online 19 August 2004. Background/Aims Epigenetics is the key factor in the regulation of gene expression. We conducted cDNA microarray analysis to screen for genes induced by histone deacetylase (HDAC) inhibition and examined epigenetic alterations. Methods Microarray analysis was performed in six hepatoma cell lines and primary hepatocytes treated with trichostatin A (TSA). mRNA expression of several genes was examined by reverse transcription-polymerase chain reaction in TSA-treated cells and hepatoma samples. Acetylated histones and methylation status in 5?CpG islands was assessed by chromatin immunoprecipitation (ChIP) assay and bisulfite genomic sequencing, respectively. Results Fifty-seven genes showed greater than 2-fold change after TSA treatment in multiple cell lines. Among them, four genes including p21 WAF1 exhibited substantial induction (greater than 5-fold changes). Decreased mRNA levels of these genes in hepatoma tissues were observed in more than half of patients. ChIP assay, in general, demonstrated a good correlation between mRNA expression and histone acetylation, but only a limited correlation with the methylated DNA in the promoter region. Conclusions We identified 57 up-regulated genes by TSA treatment in hepatoma cells and some of them appeared to be cancer-related genes in hepatomas. The alterations in acetylated histones are likely closely associated with gene expression.
Donor age influences 10-year liver graft histology independently of hepatitis C virus infection
Kinan Rifai a,Mylène Sebagh b,Vincent Karam a,Faouzi Saliba a,Daniel Azoulay a,René Adam a,Denis Castaing a,Henri Bismuth a,Michel Reynès b,Didier Samuel a,Cyrille Féray a*
Received 16 January 2004; received in revised form 24 March 2004; accepted 7 May 2004 published online 19 August 2004. Background/Aims Factors influencing the long-term histological outcome of liver graft are not known. We conducted a prospective study based on a 10-year liver biopsy in order to identify the main factors influencing long-term graft histology. Methods 270 of 423 patients who still had their first functional graft 10 years after liver transplantation accepted to undergo routine liver biopsy. All slides were blindly reviewed by two pathologists. Results Main histological findings were fibrosis in 143 patients (54%) and ductopenia in 76 patients (29%). Ductopenia was independently related to higher donor age (32±12 vs 28±13 years; P<0.02). Severity of fibrosis was influenced by hepatitis C virus (HCV) infection ( P<0.001), hepatitis B virus (HBV) recurrence ( P=0.001) and higher donor age ( P=0.03). Eighty biopsies (30%) showed minimal-change lesions which were associated with the absence of HCV infection (24/80 vs 99/185; P<0.001) or of HBV infection (1/80 vs 15/185; P=0.03) and lower donor age (25±11 vs 31±13 years; P<0.001). Conclusions Post-transplant infection by HCV or HBV are main factors influencing the histological course of liver graft. Donor age was also a strong factor in HCV infected patients as well as in HCV-negative patients. This variable should be taken into account, particularly for candidate recipients with long life expectancy.
Comparison of clinical outcome between patients continuing and discontinuing lamivudine therapy after biochemical breakthrough of YMDD mutants
Chien-Hung Chen 1,Chuan-Mo Lee 1?,Sheng-Nan Lu 1,Jing-Houng Wang 1,Hung-Da Tung 1,Chao-Hung Hung 1,Wei-Jen Chen 2and Chi-Sin Changchien 1
Received 19 January 2004;revised 12 April 2004;accepted 29 April 2004. published online 19 August 2004. Abstract Background/Aims The aim of this study was to compare the clinical outcome between patients continuing and discontinuing lamivudine therapy after the biochemical breakthrough of hepatitis B virus tyrosinemethionineaspartateaspartate (YMDD) mutant. Methods YMDD mutants were detected in 51 chronic hepatitis B patients who experienced a flare-up of alanine aminotransferase (ALT) during lamivudine treatment. Twenty-seven of them discontinued lamivudine therapy (group A), and 24 continued therapy (group B) after biochemical breakthrough. The follow-up period was 12 months in both the groups. Results There was no significant difference between groups A and B in the incidence and severity of ALT peaks and hepatic decompensation within the first 3 months after biochemical breakthrough. After the fourth month of biochemical breakthrough, however, group A experienced acute exacerbation more frequently [20/26 (77%) vs. 7/23 (30%); P=0.002] and higher ALT peaks than group B. The same result was found when the patients were divided into naïve and retreated or cirrhotic and non-cirrhotic groups. Hepatic decompensation at the onset of biochemical breakthrough was associated with higher mortality (OR=70, 95% CI=6.06807.75). Conclusions Patients who discontinued lamivudine therapy increased the frequency of flare-ups and higher ALT peaks than those who continued therapy after 4 months post-breakthrough.
Evaluation of amantadine in chronic hepatitis C: a meta-analysis
Pierre Deltenre ab,Jean Henrion b,Valérie Canva a,Sebastien Dharancy ac,Frédéric Texier a,Alexandre Louvet ac,Stephane De Maeght b,Jean-Claude Paris a,Philippe Mathurin ac*
Received 6 March 2004; received in revised form 13 May 2004; accepted 27 May 2004 published online 19 August 2004. Background/Aims The benefit of amantadine combination therapy, either with interferon (IFN) alone (double therapy) or with ribavirin and IFN (triple therapy) is unknown.
Methods We analyzed the effect of amantadine on the end-of-treatment virological response and the sustained response using meta-analysis of 31 randomized controlled trials. Results Overall analysis revealed a significant effect of amantadine. Triple therapy was the best regimen for improving the sustained response (mean difference: 8.4%, 95% CI: 2.413.8%, P=0.002). In subgroup analysis, amantadine did not have a significant effect upon naive patients or relapsers. In non-responders, combination therapy with amantadine was associated with a significant effect on the sustained response (mean difference: 8.3%, 95% CI: 1.914.6%, P=0.01). In sensitivity analysis, double therapy did not improve virological responses. Conversely, triple therapy tended to improve the end-of-treatment virological response and was associated with a significant effect upon the sustained response (mean difference: 12.7%, 95% CI: 3.821.6%, P=0.005). Conclusions Combination therapy with amantadine is of no effect upon naive patients or relapsers. In non-responders, triple therapy with amantadine improved the sustained response. New randomized controlled trials are required to confirm this meta-analysis.
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