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Table of Contents for November 2004 - Volume 40 - Issue 5
Rapid Communication
Frequent inactivation of the tumor suppressor Kruppel-like factor 6 ( KLF6 ) in hepatocellular carcinoma (p 1047-1052)
Sigal Kremer-Tal, Helen L. Reeves, Goutham Narla, Swan N. Thung, Myron Schwartz, Analisa Difeo, Amanda Katz, Jordi Bruix, Paulette Bioulac-Sage, John A. Martignetti, Scott L. Friedman
Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, reflecting incomplete characterization of underlying mechanisms and lack of early detection. Krüppel-like factor 6 ( KLF6 ) is a ubiquitously expressed zinc finger transcription factor that is deregulated in multiple cancers through loss of heterozygosity (LOH) and/or inactivating somatic mutation. We analyzed the potential role of the KLF6 tumor suppressor gene in 41 patients who had HCC associated with hepatitis C virus (16 patients), hepatitis B virus (12 patients, one of whom was coinfected with hepatitis C virus), and other etiologies (14 patients) by determining the presence of LOH and mutations. Overall, LOH and/or mutations were present in 20 (49%) of 41 tumors. LOH of the KLF6 gene locus was present in 39% of primary HCCs, and the mutational frequency was 15%. LOH and/or mutations were distributed across all etiologies of HCC evaluated, including patients who did not have cirrhosis. Functionally, wild-type KLF6 decreased cellular proliferation of HepG2 cells, while patient-derived mutants did not. In conclusion , we propose that KLF6 is deregulated by loss and/or mutation in HCC, and its inactivation may contribute to pathogenesis in a significant number of these tumors. (HEPATOLOGY 2004;40:1047-1052.) 
Viral Hepatitis
Persistence of isolated antibodies to woodchuck hepatitis virus core antigen is indicative of occult infection (p 1053-1061)
Carla S. Coffin, Tram N.Q. Pham, Patricia M. Mulrooney, Norma D. Churchill, Tomasz I. Michalak
Antibodies against virus nucleocapsid (anticore) normally accompany hepadnaviral hepatitis but they may also occur in the absence of symptoms and other serological indicators of the infection. This situation can be encountered following a clinically and serologically unapparent exposure to hepatitis B virus (HBV) or after recovery from hepatitis B. In this study, woodchucks inoculated with woodchuck hepatitis virus (WHV) were investigated to determine the relationship between anticore detection and the molecular status of virus replication in a primary WHV surface antigen (WHsAg)-negative infection or long-after resolution of WHV hepatitis. Serial, parallel samples of sera, peripheral blood mononuclear cells (PBMC) and liver tissue, collected for more than 5 years after inoculation with virus, were examined for WHV DNA by highly sensitive polymerase chain reaction (PCR)/nucleic acid hybridization assays. Sera were also tested for WHV DNA after DNase treatment and for WHV DNA and WHsAg after concentration in sucrose. Liver and PBMC were examined for WHV covalently closed circular DNA and viral RNA transcripts by PCR-based techniques to assess virus replication status. The study showed that anticore antibodies existing in the absence of other serological markers are a reliable indicator of occult WHV infection. This state can be accompanied by traces of circulating particles behaving as intact virions and by intermittent minimal-to-mild liver inflammation. In conclusion , the long-term presence of anticore antibodies alone is a consequence of sustained restimulation of the immune system by virus nucleocapsid produced during low-level hepadnaviral assembly. (HEPATOLOGY 2004.)
An immunomodulatory role for CD4 +CD25 +regulatory T lymphocytes in hepatitis C virus infection (p 1062-1071)
Roniel Cabrera, Zhengkun Tu, Yiling Xu, Roberto J. Firpi, Hugo R. Rosen, Chen Liu, David R. Nelson
The CD4 +CD25 +regulatory T lymphocytes have been implicated in suppressing T cell immune responses. Our aim was to characterize the frequency, phenotype, function, and specificity of CD4 +CD25 +T cells in hepatitis C virus (HCV) infection. Peripheral CD4 +CD25 +cells from recovered (n = 15), chronic infected (n = 30), and normal control (n = 15) subjects were analyzed ex vivo for quantitation, phenotype, and effect on HCV-specific interferon gamma production and proliferation. CD4 +CD25 +specificity was determined by intracellular cytokine staining for interleukin 10 (IL-10). A higher proportion of CD4 +CD25 +were found in chronic infection (mean, 3.02%) when compared with recovered (1.64%, P= .001) and normal controls (2.27%, P= .02). CD4 +CD25 +cells display CD45RO high , CD45RA low , CD28 high , CD62L high , and CD95 high phenotype. HCV-specific interferon gamma activity was enhanced in peripheral blood mononuclear cells depleted of CD4 +CD25 +and suppressed in peripheral blood mononuclear cells enriched with CD4 +CD25 +. Depletion of CD4 +CD25 +cells also enhanced HCV-specific CD4 +and CD8 +T cell proliferation. Cytokine analysis suggested CD4 +CD25 +cells secrete transforming growth factor beta (TGF- 1) and IL-10. The inhibitory role for TGF- 1was confirmed by anti-TGF- 1. Transwell studies showed CD4 +CD25 +mediated suppression to be dose dependent and requiring cell contact. CD4 +CD25 +cells showed HCV-specificity through IL-10 production, with a frequency ranging from 1.9% to 5.3%. A positive correlation was detected between CD4 +CD25 +T cell frequency and HCV RNA titer, whereas an inverse relation was found with liver inflammatory activity. In conclusion, CD4 +CD25 +T lymphocytes constitute a highly differentiated population and appear to play a role in viral persistence by suppressing HCV-specific T cell responses in a cell-cell contact manner. (HEPATOLOGY 2004;40:1062-1071.)
Occult hepatitis B virus infection in a North American adult hemodialysis patient population (p 1072-1077)
Gerald Y. Minuk, Dong Feng Sun, Rebecca Greenberg, Manna Zhang, Kimberly Hawkins, Julia Uhanova, Adam Gutkin, Kevin Bernstein, Antonio Giulivi, Carla Osiowy
Hepatitis B virus (HBV) infections continue to occur in adult hemodialysis units. A possible contributing factor is the presence of occult HBV (serum hepatitis B surface antigen [HBsAg] negative but HBV DNA positive). Two hundred forty-one adult hemodialysis patients were screened for occult HBV. HBV DNA testing was performed by real-time polymerase chain reaction (PCR) with 2 independent primer sets (core promoter and surface). Two (0.8%) of the 241 patients were HBsAg positive. Of the remaining 239 HBsAg-negative patients, 9 (3.8%) were HBV DNA positive. Viral loads in these individuals were low (10 2-10 4viral copies/mL). Seven of the 9 (78%) were nt 587 mutation (sG145R mutant) positive. Demographic, biochemical, and HBV serological testing did not help to identify those with occult HBV. In conclusion , the prevalence of occult HBV in adult hemodialysis patients in this North American urban center is approximately 4 to 5 times higher than standard HBsAg testing would suggest. The majority of these infections are associated with low viral loads and a high prevalence of the sG145R mutant. Finally, the demographic, biochemical, and/or serological features of HBV DNA-positive subjects do not distinguish these individuals from the remainder of the dialysis patient population. (HEPATOLOGY 2004; 40:1072-1077.)
Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury (p 1078-1087)
Heike Bantel, Andreas Lügering, Jan Heidemann, Xandra Volkmann, Christopher Poremba, Christian P. Strassburg, Michael Peter Manns, Klaus Schulze-Osthoff
Chronic hepatitis C virus (HCV) infection is characterized by inflammatory liver damage and is associated with a high risk of development of cirrhosis and hepatocellular carcinoma. Although histological examination of liver biopsies is currently the gold standard for the detection of early liver damage, there is a strong need for better noninvasive methods. We recently demonstrated that the proapoptotic activation of caspases is considerably enhanced in histological sections from HCV-infected liver tissue, suggesting an important role of apoptosis in liver damage. Here, we investigated whether caspase activation is detectable also in sera from patients with chronic HCV infection. Using a novel enzyme-linked immunosorbent assay that selectively recognizes a proteolytic neoepitope of the caspase substrate cytokeratin-18, we demonstrate that caspase activity is markedly increased in the sera of HCV patients. Interestingly, while 27% of patients with chronic HCV infection showed normal aminotransferase levels despite inflammatory and fibrotic liver damage, more than 50% of those patients exhibited already elevated serum caspase activity. Moreover, 30% of patients with normal aminotransferase but elevated caspase activity revealed higher stages of fibrosis. In conclusion , compared with conventional surrogate markers such as aminotransferases, detection of caspase activity in serum might be a more sensitive method of detecting early liver injury. Thus, measurement of caspase activity might provide a novel diagnostic tool, especially for patients with normal aminotransferases but otherwise undiagnosed histologically active hepatitis and progressive fibrosis. (HEPATOLOGY 2004;40:1078-1087.)
Liver Biology and Pathobiology
ACAT2 deficiency limits cholesterol absorption in the cholesterol-fed mouse: Impact on hepatic cholesterol homeostasis (p 1088-1097)
Joyce J. Repa, Kimberly K. Buhman, Robert V. Farese Jr., John M. Dietschy, Stephen D. Turley
Acyl CoA:cholesterol acyltransferase (ACAT) 2 is the major cholesterol-esterifying enzyme in mouse enterocytes and hepatocytes. Male ACAT2 +/+ and ACAT2 -/ - mice were fed chow containing added cholesterol (0%-0.500% w/w) for 24 days. Over this range, fractional cholesterol absorption in the ACAT2 +/+ mice fell from 41.4% ± 6.6% to 21.0% ± 5.2%, and in their ACAT2 -/- counterparts it fell from 35.1% ± 4.5% to 7.9% ± 0.8%. The mass of dietary cholesterol absorbed (mg/d per 100 g body weight) increased from 1.2 ± 0.2 to 14.7 ± 4.4 in the ACAT2 +/+ mice and from 1.0 ± 0.2 to 5.5 ± 0.6 in those without ACAT2. In the ACAT2 +/+ mice, hepatic cholesterol concentrations increased as a function of intake despite compensatory changes in cholesterol and bile acid synthesis and in the expression of adenosine triphosphate-binding cassette transporter G5 (ABCG5) and ABC transporter G8 (ABCG8). In contrast, in ACAT2 -/- mice in which the amount of cholesterol absorbed at the highest intake was only 37% of that in the ACAT2 +/+ mice, suppression of synthesis was a sufficient adaptive response; there was no change in bile acid synthesis, ABCG5/G8 expression, or hepatic cholesterol concentration. The expression of adenosine triphosphate-binding cassette transporter A1 (ABCA1) in the jejunum was markedly elevated in the ACAT2 -/- mice, irrespective of dietary cholesterol level. In conclusion , although ACAT2 deficiency limits cholesterol absorption, the extent to which it impacts hepatic cholesterol homeostasis depends on cholesterol intake. Loss of ACAT2 activity may result in unesterified cholesterol being absorbed via an ABCA1-mediated basolateral efflux pathway. (HEPATOLOGY 2004;40:1088-1097.)
Intact signaling by transforming growth factor is not required for termination of liver regeneration in mice (p 1098-1105)
Shoshiro Oe, Eric R. Lemmer, Elizabeth A. Conner, Valentina M. Factor, Per Levéen, Jonas Larsson, Stefan Karlsson, Snorri S. Thorgeirsson
Transforming growth factor (TGF- ) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF- signaling in liver regeneration in vivo , the TGF- type II receptor gene ( Tgfbr2 ) was selectively deleted in hepatocytes by crossing floxed Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver-specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF- 1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7-fold higher in R2LivKO mice compared with controls. Accelerated S-phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion , TGF- regulates G 1to S phase transition of hepatocytes, but intact signaling by TGF- is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF- pathway is abolished, and may be a principal factor in the termination of liver regeneration. (HEPATOLOGY 2004.)
Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats (p 1106-1115)
Christopher J. Parsons, Blair U. Bradford, Clark Q. Pan, Ellen Cheung, Michael Schauer, Andreas Knorr, Barbara Krebs, Sabine Kraft, Stefan Zahn, Bodo Brocks, Nikki Feirt, Baisong Mei, Myung-Sam Cho, Roopa Ramamoorthi, Greg Roldan, Paul Ng, Peggy Lum, Claudia Hirth-Dietrich, Adrian Tomkinson, David A. Brenner
Liver fibrosis is characterized by increased synthesis, and decreased degradation, of extracellular matrix (ECM) within the injured tissue. Decreased ECM degradation results, in part, from increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), which blocks matrix metalloproteinase (MMP) activity. TIMP-1 is also involved in promoting survival of activated hepatic stellate cells (HSCs), a major source of ECM. This study examined the effects of blocking TIMP-1 activity in a clinically relevant model of established liver fibrosis. Rats were treated with carbon tetrachloride (CCl 4), or olive oil control, for 6 weeks; 24 days into the treatment, the rats were administered a neutralizing anti-TIMP-1 antibody derived from a fully human combinatorial antibody library (HuCAL), PBS, or an isotype control antibody. Livers from CCl 4-treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle -actin ( -SMA). Compared to controls, rats administered anti-TIMP-1 showed a reduction in collagen accumulation by histological examination and hydroxyproline content. Administration of anti-TIMP-1 resulted in a marked decrease in -SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP-2. In conclusion , administration of a TIMP-1 antibody attenuated CCl 4-induced liver fibrosis and decreased HSC activation and MMP-2 activity. (HEPATOLOGY 2004.)
Alpha-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca 2+ - and PKC-dependent stimulation of cAMP (p 1116-1127)
Gene D. LeSage, Domenico Alvaro, Shannon Glaser, Heather Francis, Luca Marucci, Tania Roskams, Jo Lynne Phinizy, Marco Marzioni, Antonio Benedetti, Silvia Taffetani, Barbara Barbaro, Giammarco Fava, Yoshiyuki Ueno, Gianfranco Alpini
Acetylcholine potentiates secretin-stimulated ductal secretion by Ca 2+ -calcineurin-mediated modulation of adenylyl cyclase. D2 dopaminergic receptor agonists inhibit secretin-stimulated ductal secretion via activation of protein kinase C (PKC)- . No information exists regarding the effect of adrenergic receptor agonists on ductal secretion in a model of cholestasis induced by bile duct ligation (BDL). We evaluated the expression of -1A/1C, -1 and -1 adrenergic receptors in liver sections and cholangiocytes from normal and BDL rats. We evaluated the effects of the -1 and -1 adrenergic receptor agonists (phenylephrine and dobutamine, respectively) on bile and bicarbonate secretion and cholangiocyte IP 3and Ca 2+ levels in normal and BDL rats. We measured the effect of phenylephrine on lumen expansion in intrahepatic bile duct units (IBDUs) and cyclic adenosine monophosphate (cAMP) levels in cholangiocytes from BDL rats in the absence or presence of BAPTA/AM and Gö6976 (a PKC- inhibitor). We evaluated if the effects of phenylephrine on ductal secretion were associated with translocation of PKC isoforms leading to increased protein kinase A activity. -1 and -1 adrenergic receptors were present mostly in the basolateral domain of cholangiocytes and, following BDL, their expression increased. Phenylephrine, but not dobutamine, increased secretin-stimulated choleresis in BDL rats. Phenylephrine did not alter basal but increased secretin-stimulated IBDU lumen expansion and cAMP levels, which were blocked by BAPTA/AM and Gö6976. Phenylephrine increased IP 3and Ca 2+ levels and activated PKC- and PKC- -II. In conclusion , coordinated regulation of ductal secretion by secretin (through cAMP) and adrenergic receptor agonist activation (through Ca 2+ /PKC) induces maximal ductal bicarbonate secretion in liver diseases.
Cooperative effect of biliverdin and carbon monoxide on survival of mice in immune-mediated liver injury (p 1128-1135)
Gabriele Sass, Stefan Seyfried, Miguel Parreira Soares, Kenichiro Yamashita, Elzbieta Kaczmarek, Winfried L. Neuhuber, Gisa Tiegs
nduction of the heme-degrading enzyme heme oxygenase-1 (HO-1) has been shown to be beneficial in terms of improvement of liver allograft survival and prevention of CD95-mediated apoptosis in the liver. In the present study, we investigated the effects of HO-1, and its products carbon monoxide (CO), biliverdin (BV), and iron/ferritin, in a mouse model of inflammatory liver damage inducible by lipopolysaccharide (LPS) in mice sensitized with the hepatocyte-specific transcription inhibitor D-galactosamine (GalN). Our results show that HO-1 induction by cobalt-protoporphyrin-IX (CoPP) reduced cytokine expression, protected mice from liver injury, and prolonged survival. While in contrast to ferritin overexpression, single administration of the CO donor methylene chloride (MC) or of BV also protected mice from liver damage, only coadministration of both HO products prolonged survival and reduced the expression of cytokines, e.g. , tumor necrosis factor (TNF) and interferon (IFN- ). In conclusion , HO-1-induced prolongation of survival, but not the protection from liver damage, seems to be dependent on down-regulation of cytokine synthesis. (HEPATOLOGY 2004;40:1128-1135.)
Opioid receptor blockade reduces Fas-induced hepatitis in mice (p 1136-1143)
Martial Jaume, Sébastien Jacquet, Pierre Cavaillès, Gaëtane Macé, Lionel Stephan, Catherine Blanpied, Cécile Demur, Pierre Brousset, Gilles Dietrich
Fas (CD95)-induced hepatocyte apoptosis and cytotoxic activity of neutrophils infiltrating the injured liver are two major events leading to hepatitis. Because it has been reported that opioids, via a direct interaction, sensitize splenocytes to Fas-mediated apoptosis by upregulating Fas messenger RNA (mRNA) and modulated neutrophil activity, we assumed that opioids may participate in the pathophysiology of hepatitis. Using the hepatitis model induced by agonistic anti-Fas antibody in mice, we showed that opioid receptor blockade reduced liver damage and consequently increased the survival rate of animals when the antagonist naltrexone was injected simultaneously or prior to antibody administration. Treatment of mice with morphine enhanced mortality. Naloxone methiodide - a selective peripheral opioid antagonist - had a protective effect, but the absence of opioid receptors in the liver, together with lack of morphine effect in Fas-induced apoptosis of primary cultured hepatocytes, ruled out a direct effect of opioids on hepatocytes. In addition, the neutralization of opioid activity by naltrexone did not modify Fas mRNA expression in the liver as assessed with real-time quantitative polymerase chain reaction. Injured livers were infiltrated by neutrophils, but granulocyte-depleted mice were not protected against the enhancing apoptotic effect of morphine. In conclusion , opioid receptor blockade improves the resistance of mice to Fas-induced hepatitis via a peripheral mechanism that does not involve a down-modulation of Fas mRNA in hepatocytes nor a decrease in proinflammatory activity of neutrophils. (HEPATOLOGY 2004.)
Impact of cirrhosis on the development of experimental hepatic metastases by B16F1 melanoma cells in C57BL/6 mice (p 1144-1150)
Ke Qi, Hongming Qiu, Dongfeng Sun, Gerald Y. Minuk, Michael Lizardo, John Rutherford, F. William Orr
Metastases rarely occur in human livers with cirrhosis in clinical studies. We postulated that this phenomenon would also occur in experimental cirrhosis. Cirrhosis was established in C57BL/6 mice by carbon tetrachloride (CCl 4) gastrogavage. B16F1 melanoma cells were injected into the mesenteric vein to induce hepatic metastases. Contrary to our postulate, there was greater than 4-fold increase in metastasis in animals with cirrhosis compared to controls. Intravital videomicroscopy showed that the hepatic sinusoids were narrower and more tumor cells were retained in the terminal portal vein (TPV) in cirrhotic livers. Immunohistochemistry demonstrated that the expression of vascular adhesion molecules was significantly increased in cirrhosis. Using confocal microscopy and the fluorescent nitric oxide (NO) probe 4,5-diaminofluorescein diacetate, a significantly lower level of NO release was detected in livers with cirrhosis both in basal conditions and after tumor cell arrest. Eight hours after mesenteric vein tumor cell injection, the percentage of apoptotic tumor cells in the sinusoids was 17% ± 2% in livers with cirrhosis and 30% ± 5% in normal livers. More mitotic and Ki-67 labeled tumor cells were seen in livers with cirrhosis. In conclusion , the changes in architecture and adhesion molecule expression in livers with cirrhosis may cause more tumor cells to arrest in the TPV. Lower levels of NO production may reduce apoptosis of B16F1 cells in livers with cirrhosis. As a result, these changes may promote the growth of metastasis in this cirrhotic model. (HEPATOLOGY 2004.)
A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations (p 1151-1159)
Scott T. Magness, Ramón Bataller, Liu Yang, David A. Brenner
Activation of hepatic stellate cells (HSCs) and other resident mesenchymal cells into myofibroblasts expressing alpha smooth muscle actin ( SMA) and collagen I is a key event in liver fibrogenesis. However, the temporal expression profiles of SMA and collagen I genes in these cells is unknown. To address this question, we studied SMA and collagen 1(I) transcriptional patterns in primary cultures of HSCs, and additionally, in an in vivo model of secondary biliary fibrosis using transgenic mice that express the Discomsoma sp. red fluorescent protein (RFP) and the enhanced green fluorescent protein (EGFP) reporter genes under direction of the mouse SMA and collagen 1(I) promoter/enhancers, respectively. The SMA-RFP mice were crossed with collagen-EGFP mice to generate double transgenic mice. Reporter gene expression in cultured HSCs demonstrated that both transgenes were induced at day 3 with continued expression through day 14. Interestingly, SMA and collagen 1(I) transgenes were not coexpressed in all cells. Flow cytometry analysis showed three different patterns of gene expression: SMA-RFP positive cells, collagen-EGFP positive cells, and cells expressing both transgenes. SMA-only and SMA/collagen expressing cells showed higher expression levels of synaptophysin, reelin, MMP13, TIMP1, and ICAM-1 compared to collagen-only expressing cells, as assessed by real-time PCR. Following bile duct ligation, SMA and collagen 1(I) transgenes were differentially expressed by peribiliary, parenchymal and vascular fibrogenic cells. Peribiliary cells preferentially expressed collagen 1(I), while parenchymal myofibroblasts expressed both SMA and collagen 1(I). In conclusion , these data demonstrate heterogeneity of gene expression in myofibroblastic cells during active fibrogenesis. These reporter mice provide a useful tool to further characterize fibrogenic cell types and to evaluate antifibrotic drugs. (HEPATOLOGY 2004.)
Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor B (p 1160-1169)
Motoaki Imose, Masahito Nagaki, Kiminori Kimura, Shinji Takai, Motohiro Imao, Takafumi Naiki, Yosuke Osawa, Takahiko Asano, Hideki Hayashi, Hisataka Moriwaki
Leflunomide is a novel immunosuppressive and anti-inflammatory agent for the treatment of autoimmune disease. The aim of this study was to investigate whether leflunomide protects from liver injury induced by concanavalin A (Con A), a T-cell-dependent model of liver damage. BALB/c mice were injected with 25 mg/kg Con A in the presence or absence of 30 mg/kg leflunomide. Liver injury was assessed biochemically and histologically. Levels of circulating cytokines and expressions of cytokine messenger RNA (mRNA) in the liver and the spleen were determined. Treatment with leflunomide markedly reduced serum transaminase activities and the numbers of dead liver cells. Leflunomide significantly inhibited increases in plasma tumor necrosis factor alpha (TNF- ) and interleukin 2 concentrations, and also reduced TNF- mRNA expression in the liver after administration of Con A. These findings were supported by the results in which leflunomide administration decreased the number of T lymphocytes infiltrating the liver as well as inhibiting their production of TNF- . Activation of nuclear factor B (NF- B), which regulates TNF- production, was inhibited in the liver of mice treated with leflunomide, resulting in a reduction of TNF- production from lymphocytes infiltrating the liver. In conclusion , leflunomide is capable of regulating T-cell-mediated liver injury in vivo and that this event may depend on the decrease of TNF- production in the liver through inhibition of NF- B activation caused by leflunomide. (HEPATOLOGY 2004.)
Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes (p 1170-1179)
Kazuyoshi Kon, Jae-Sung Kim, Hartmut Jaeschke, John J. Lemasters
Acetaminophen overdose causes massive hepatic failure via mechanisms involving glutathione depletion, oxidative stress, and mitochondrial dysfunction. The ultimate target of acetaminophen causing cell death remains uncertain, and the role of apoptosis in acetaminophen-induced cell killing is still controversial. Our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen-induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. After administration of 10 mmol/L acetaminophen, necrotic killing increased to more than 49% and 74%, respectively, after 6 and 16 hours. MPT inhibitors, cyclosporin A (CsA), and NIM811 temporarily decreased necrotic killing after 6 hours to 26%, but cytoprotection was lost after 16 hours. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization approximately 4.5 hours after acetaminophen administration. CsA delayed these changes, indicative of the MPT, to approximately 11 hours after acetaminophen administration. Apoptosis indicated by nuclear changes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and caspase-3 activation also increased after acetaminophen administration. Fructose (20 mmol/L, an adenosine triphosphate-generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis from 37% to 59% after 16 hours. In the presence of fructose plus glycine, CsA decreased apoptosis and delayed but did not prevent the MPT. In conclusion , after acetaminophen a CsA-sensitive MPT occurred after 3 to 6 hours followed by a CsA-insensitive MPT 9 to 16 hours after acetaminophen. The MPT then induces ATP depletion-dependent necrosis or caspase-dependent apoptosis as determined, in part, by ATP availability from glycolysis. (HEPATOLOGY 2004;40:1170-1179.)
Pivotal role of nuclear factor B signaling in anti-CD40-induced liver injury in mice (p 1180-1189)
Kiminori Kimura, Masahito Nagaki, Shinji Takai, Shinichi Satake, Hisataka Moriwaki
Nuclear factor B (NF- B) has a central role in coordinating the expression of a wide variety of genes that control immune responses and is also recognized as an antiapoptotic transcription factor. Here, we focused on the role of the NF- B signaling pathway in the interaction between inflammatory cells and hepatocytes in liver inflammation. We found that pretreatment of mice with adenoviruses expressing a mutant form of the inhibitor B superrepressor (Ad5I B), a NF- B inhibitor, reduced the migration of inflammatory cells and cytokine and chemokine expression in the liver 12 hours after a single intravenous injection of an anti-CD40 antibody ( CD40) compared with mice infected with control adenoviruses (Ad5LacZ). We also confirmed reductions in cytokine production by macrophages, T cells, and natural killer (NK) cells in the liver of Ad5I B-treated mice by FACS analysis. However, CD40 treatment in Ad5I B-infected mice induced elevation of serum alanine aminotransferase at 24 hours, and the liver injury was associated with massive hepatocyte apoptosis. Furthermore, interferon gamma (IFN- ) production by NK cells and T cells was increased and stimulated tumor necrosis factor alpha (TNF- ) production by macrophages in the Ad5I B-infected liver. Moreover, the liver injury was completely suppressed by the administration of anti-IFN- and anti-TNF- . These results suggest that inhibition of NF- B activity suppressed CD40-induced liver inflammation at an early phase, resulting in a reduction in cytokine and chemokine production, whereas it sensitized hepatocytes to TNF- -induced apoptosis and exacerbated liver injury at the late phase. In conclusion , NF- B exerts pivotal activities at inflammatory sites, and caution should be exercised in NF- B-targeted therapy of liver disease. (HEPATOLOGY 2004;40:1180-1189.)
Concanavalin a injection activates intrahepatic innate immune cells to provoke an antitumor effect in murine liver (p 1190-1196)
Takuya Miyagi, Tetsuo Takehara, Tomohide Tatsumi, Takahiro Suzuki, Masahisa Jinushi, Yoshiyuki Kanazawa, Naoki Hiramatsu, Tatsuya Kanto, Shingo Tsuji, Masatsugu Hori, Norio Hayashi
Concanavalin A (ConA), directly injected into mice, induces T cell-mediated liver injury. However, it remains unclear whether ConA injection can activate innate immune cells, including natural killer (NK) cells and natural killer T (NKT) cells, both of which exist abundantly in the liver. Here we report that ConA injection stimulated interferon (IFN)- production from liver NKT cells as early as 2 hours after injection and augmented YAC-1 cytotoxicity of liver NK cells. ConA-induced NK cell activation required other types of immune cells and critically depended on IFN- . Because a nonhepatotoxic low dose of ConA was capable of fully activating both NKT cells and NK cells, we next addressed the possibility of ConA injection displaying an antitumor effect in the liver without liver injury. A nonhepatotoxic low-dose ConA injection augmented the cytotoxicity of liver NK cells against Colon-26 colon cancer cells and suppressed hepatic metastasis of Colon-26 cells in a NK cell- and IFN- -dependent manner. In conclusion , a nonhepatotoxic low dose of ConA might serve as an immunomodulator that can preferentially activate the innate immune cells to induce an antitumor effect against metastatic liver tumor. (HEPATOLOGY 2004;40:1190-1196.)
Liver Failure and Liver Disease
PTFE-covered stents improve TIPS patency in Budd-Chiari syndrome (p 1197-1202)
Manuel Hernández-Guerra, Juan Turnes, Pablo Rubinstein, Simon Olliff, Elwyn Elias, Jaime Bosch, Juan Carlos García-Pagán
Transjugular intrahepatic portosystemic shunt (TIPS) have been shown to be an efficient portal-systemic derivative treatment for Budd-Chiari syndrome (BCS) patients uncontrolled by medical therapy. However, the main drawback of TIPS for this condition is a very high rate of shunt dysfunction. Recently, polytetrafluoroethylene (PTFE)-covered stents have been shown to reduce the incidence of TIPS dysfunction in patients with cirrhosis. The aim of the study was to assess the incidence of TIPS dysfunction in 2 cohorts of BCS patients treated with bare or PTFE-covered stents. The study included 25 TIPS procedures (16 bare stents and 9 covered stents) with a median follow-up period of 20.4 months (range, 3.9-124.8). Fourteen of 16 patients (87%) receiving bare stents had TIPS dysfunction compared to 3 of the 9 patients (33%) receiving PTFE-covered stents ( P= .005). The actuarial rates of primary patency in the bare-stent group were 19% at 1 year compared with 67% in the PTFE-covered stent group ( P= .02; log-rank test). The number of additional interventional procedures to maintain TIPS patency was significantly greater in the bare-stent than in the PTFE-covered stent group (1.9 ± 1.2 vs. 0.6 ± 0.9; P= .007). The number of patients with clinical relapses was greater in the bare-stent group compared to the PTFE-covered stent group (13 vs. 5 episodes in 9 and 3 patients, respectively). In conclusion , PTFE-covered stents have a considerable advantage over bare stents for the TIPS treatment of BCS patients, with a lower dysfunction rate, a lower number of reinterventions, and fewer prosthesis requirements. PTFE-covered stents are preferable in patients with Budd-Chiari Syndrome. (HEPATOLOGY 2004;40:1197-1202.)
Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of 1-antitrypsin (p 1203-1210)
Sabina Janciauskiene, Sten Eriksson, Francesco Callea, Meera Mallya, Aiwu Zhou, Kuniaki Seyama, Satoru Hata, David A. Lomas
Several point mutations of 1-antitrypsin cause a perturbation in protein structure with consequent polymerization and intracellular accumulation. The retention of polymers of 1-antitrypsin within hepatocytes results in protein overload that in turn is associated with juvenile hepatitis, cirrhosis, and hepatocellular carcinoma. The detection of 1-antitrypsin polymers and understanding the molecular basis of polymer formation is of considerable clinical importance. We have used a monoclonal antibody (ATZ11) that specifically recognizes a conformation-dependent neoepitope on polymerized 1-antitrypsin to detect polymers within hepatocytes of individuals with 1-antitrypsin deficiency. Paraffin-embedded liver tissue specimens were obtained from individuals who were homozygous for the Z (Glu342Lys), Mmalton (52Phe del), and Siiyama (Ser53Phe) alleles of 1-antitrypsin that result in hepatic inclusions and profound plasma deficiency. Immunohistological staining with a polyclonal anti-human 1-antitrypsin antibody showed hepatic inclusions in all 3 cases, while ATZ11 reacted with hepatic inclusions formed by only Z 1-antitrypsin. Polymers of plasma M and Z 1-antitrypsin prepared under different conditions in vitro and polymers of recombinant mutants of 1-antitrypsin demonstrated that the monoclonal antibody detected a neoepitope on the polymerized protein. It did not detect polymers formed by a recombinant shutter domain mutant (that mirrors the effects of the Siiyama and Mmalton variants), polymers formed by cleaving 1-antitrypsin at the reactive loop, or C-sheet polymers formed by heating 1-antitrypsin in citrate. In conclusion , the ATZ11 monoclonal antibody detects Z 1-antitrypsin in hepatic inclusions by detecting a neoepitope that is specific to the polymeric conformer and that is localized close to residue 342. (HEPATOLOGY 2004;40:1203-1210.)
A novel mechanism of liver allograft rejection facilitated by antibodies to liver sinusoidal endothelial cells (p 1211-1221)
Suchitra Sumitran-Holgersson, Xupeng Ge, Azza Karrar, Bo Xu, Silvia Nava, Ulrika Broomé, Grzegorz Nowak, Bo-Göran Ericzon
Liver sinusoidal endothelial cells (LSECs) may be implicated in the induction of liver allograft rejections. We studied the clinical consequences of LSEC-reactive antibodies and their functional capacity in modulating T-cell responses during acute rejections. Pre- and posttransplant sera and T lymphocytes from 95 liver transplant patients were used in this study. LSECs were isolated from normal healthy liver. Binding of antibodies to LSECs was detected using flow cytometry. To study whether LSEC antibodies facilitated cell-mediated immunity, a mixed cell culture (MCC) assay was used. Cytokines in the supernatants of MCC were measured by enzyme-linked immunosorbent assay. Liver biopsy sections were stained to detect the deposition of immunoglobulins in LSECs during rejections. The 2-year patient survival was 86.3%. A significantly higher number of patients with rejections had LSEC antibodies (35/50; 70%) than those without rejections (8/45; 18%) ( P< .0001). Purified fractions of LSEC antibodies induced the expression of the costimulatory molecule CD86 on LSECs. A significantly higher number of patients with LSEC antibodies and rejections had an increased proliferation of T cells and markedly decreased levels of transforming growth factor (TGF- ) in the MCC than those without antibodies and rejections ( P< .0001, P< .0001, respectively). Deposition of antibodies in LSECs during rejection episodes was observed in the biopsies of patients with LSEC antibodies but not in those without LSEC antibodies. In conclusion , antibodies to LSECs may facilitate acute liver allograft rejections by down-regulating the immune modulating cytokine TGF- and thus up-regulating alloreactive T-cell proliferation. (HEPATOLOGY 2004;40:1211-1221.)
Concise Communication
Therapeutic efficacy of an angiotensin II receptor antagonist in patients with nonalcoholic steatohepatitis (p 1222-1225)
Shiro Yokohama, Masashi Yoneda, Masakazu Haneda, Satoshi Okamoto, Mituyoshi Okada, Kazunobu Aso, Takenao Hasegawa, Yoshihiko Tokusashi, Naoyuki Miyokawa, Kimihide Nakamura
The therapeutic efficacy of angiotensin II receptor antagonist, losartan, was studied in patients with nonalcoholic steatohepatitis (NASH). Seven patients with both NASH and hypertension were treated with losartan (50 mg/d) for 48 weeks. Treatment with losartan resulted in a significant decrease in blood markers of hepatic fibrosis, plasma TGF- 1 and serum ferritin concentration concurrently with an improvement in serum aminotransferase levels. Histological assessment showed improvement of hepatic necroinflammation in five patients, reduction of hepatic fibrosis in four patients, and disappearance of iron deposition in two patients. No side effect of treatment was noted at any time during the study. In conclusion , the present data raise the possibility that an angiotensin II receptor antagonist may be therapeutically efficacious for NASH. (HEPATOLOGY 2004.)
Copyright © 2004 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for November 2004 - Volume 127 - Number 5
Clinical-alimentary Tract
Endoscopic balloon dilation compared with sphincterotomy for extraction of bile duct stones
James A. DiSario, Martin L. Freeman, David J. Bjorkman, Padraic MacMathuna, Bret T. Petersen, Philip E. Jaffe, Thomas G. Morales, Lee J. Hixson, Stuart Sherman, Glen A. Lehman, M. Mazen Jamal, Firas H. Al-Kawas, Mukul Khandelwal, Joseph P. Moore, Gregory A. Derfus, Priya A. Jamidar, Francisco C. Ramirez, Michael E. Ryan, Karen L. Woods, David L. Carr-Locke, Stephen C. Alder
Background & Aims: Endoscopic retrograde cholangiopancreatography is commonly performed to remove bile duct stones. The aim of this study was to determine short-term outcomes of endoscopic balloon dilation of the sphincter of Oddi compared with sphincterotomy for stone extraction. Methods: A randomized, controlled multicenter study of 117 patients assigned to dilation and 120 to sphincterotomy was performed in a spectrum of clinical and academic practices. Results: Characteristics of the patients, procedures, and endoscopists were similar except that dilation patients were younger. Procedures were successful in 97.4% and 92.5% of the dilation and sphincterotomy patients, respectively. Overall morbidity occurred in 17.9% and 3.3% ( P< .001; difference, 14.6; 95% confidence interval, 722.3) and severe morbidity, including 2 deaths, in 6.8% and 0%( P< .004; difference, 6.8; 95% confidence interval, 2.311.4) for dilation and sphincterotomy, respectively. Complications for dilation and sphincterotomy, respectively, included: pancreatitis, 15.4% and .8% ( P< .001; difference, 14.6; 95% confidence interval, 7.821.3); cystic duct fistula, 1.7% and 0%; cholangitis, .9% and .8%; perforation, 0% and .8%; and cholecystitis, 0% and .8%. There were 2 deaths (1.7%) due to pancreatitis following dilation and none with sphincterotomy. The study was terminated at the first interim analysis. Dilation patients required significantly more invasive procedures, longer hospital stays, and longer time off from normal activities. Conclusions: In a broad spectrum of patients and practices, endoscopic balloon dilation compared with sphincterotomy for biliary stone extraction is associated with increased short-term morbidity rates and death due to pancreatitis. Balloon dilation of the sphincter of Oddi for stone extraction should be avoided in routine practice.
Clinical-alimentary Tract
Computed tomographic colonography without cathartic preparation for the detection of colorectal polyps
Riccardo Iannaccone, Andrea Laghi, Carlo Catalano, Filippo Mangiapane, Antonietta Lamazza, Alberto Schillaci, Giovanni Sinibaldi, Takamichi Murakami, Paolo Sammartino, Masatoshi Hori, Francesca Piacentini, Italo Nofroni, Vincenzo Stipa, Roberto Passariello
Background & Aims :We prospectively compared the performance of low-dose multidetector computed tomographic colonography (CTC) without cathartic preparation with that of colonoscopy for the detection of colorectal polyps. Methods :A total of 203 patients underwent low-dose CTC without cathartic preparation followed by colonoscopy. Before CTC, fecal tagging was achieved by adding diatrizoate meglumine and diatrizoate sodium to regular meals. No subtraction of tagged feces was performed. Colonoscopy was performed 37 days after CTC. Three readers interpreted the CTC examinations separately and independently using a primary 2-dimensional approach using multiplanar reconstructions and 3-dimensional images for further characterization. Colonoscopy with segmental unblinding was used as reference standard. The sensitivity of CTC was calculated both on a per-polyp and a per-patient basis. For the latter, specificity, positive predictive values, and negative predictive values were also calculated. Results :CTC had an average sensitivity of 95.5% (95% confidence interval [CI], 92.1%99%) for the identification of colorectal polyps ≥8 mm. With regard to per-patient analysis, CTC yielded an average sensitivity of 89.9% (95% CI, 86%93.7%), an average specificity of 92.2% (95% CI, 89.5%94.9%), an average positive predictive value of 88% (95% CI, 83.3%91.5%), and an average negative predictive value of 93.5% (95% CI, 90.9%96%). Interobserver agreement was high on a per-polyp basis (? statistic range, .61.74) and high to excellent on a per-patient basis (? statistic range, .79.91). Conclusions :Low-dose multidetector CTC without cathartic preparation compares favorably with colonoscopy for the detection of colorectal polyps.
An analysis of the potential impact of computed tomographic colonography (virtual colonoscopy) on colonoscopy demand
Chin Hur, G. Scott Gazelle, Michael E. Zalis, Daniel K. Podolsky
Background & Aims: There has been much speculation about the potential impact on the use of conventional colonoscopy if “virtual” computed tomographic colonography (CTC) became a widely accepted modality for colorectal cancer (CRC) screening. However, no formal analysis of the impact of CTC on colonoscopy demand has been reported. Methods: A mathematical model to predict colonoscopy demand based on several relevant input parameters was constructed. Current national colonoscopy practice, estimated using various published reports, was used as the foundation to project colonoscopy demand if CTC were implemented as the primary CRC screening modality. Results: In the base-case analysis, if CTC were used as the primary modality for CRC screening, 1.78 million colonoscopies could be eliminated from the total 6.47 million in 2003. Depending on the polyp size threshold used to define a CTC study as positive (6 or 10 mm), this loss would be partially offset by 1.21 million (6 mm) or .34 million (10 mm) follow-up colonoscopies for CTC examinations with positive findings, resulting in a net loss of .57 million (8.8% decrease) (6 mm) or 1.44 million (22.3% decrease) (10 mm). Extensive sensitivity analyses showed that the findings of this model were robust and insensitive to most parameters tested but were sensitive to a few parameters, including the percentage of CTC examinations with positive findings. Conclusions: Wide-scale implementation of CTC for CRC screening would likely lead to a decrease in use of conventional colonoscopy. The percentage of CTC studies with positive findings seemed to be a pivotal variable, which would be determined in large part by the polyp size ultimately established to define a positive finding.
Risks of clinically significant upper gastrointestinal events with etodolac and naproxen: A historical cohort analysis
Rick A. Weideman, Kevin C. Kelly, Salahuddin Kazi, Anh Cung, Kevin W. Roberts, Herbert J. Smith, George A. Sarosi, Bertis B. Little, Byron Cryer
Background & Aims :Etodolac is a generic nonsteroidal anti-inflammatory drug (NSAID). Previous in vitro studies have shown that etodolac is a selective inhibitor of cyclooxygenase (COX)-2 with selectivity in between that of other COX-2 inhibitors such as celecoxib and rofecoxib. However, there are no outcomes data assessing clinically significant upper gastrointestinal (CSUGI) events with etodolac. Methods :A historical cohort study was performed at the Dallas Veterans Affairs Medical Center in which 16,286 veteran patients (5596 patient-years) received etodolac or naproxen during a 3-year period without concurrent use of other ulcerogenic drugs other than low-dose aspirin. The primary outcome was the CSUGI event rate of the etodolac and naproxen groups without concomitant low-dose aspirin. Results :The incidence of CSUGI events was .78% and .24% for naproxen and etodolac, respectively. In the NSAID-naive subset, the incidence of CSUGI events was .99% and .24% for naproxen and etodolac, respectively. Compared with naproxen, etodolac was associated with a reduction in upper gastrointestinal events, corresponding to an odds ratio of .39 (95% confidence interval, .20.76; P= .006). Concomitantly used low-dose aspirin increased event rates with naproxen 2-fold and etodolac 9-fold. Hence, there was no significant difference in gastrointestinal event rates between etodolac and naproxen when low-dose aspirin was taken concomitantly. Conclusions :Etodolac is a generic COX-2 selective inhibitor that reduces CSUGI events compared with the nonselective NSAID naproxen. However, concomitant use of low-dose aspirin negates the gastrointestinal safety advantages of etodolac.
The efficacy of proton pump inhibitors in nonulcer dyspepsia: A systematic review and economic analysis
Paul Moayyedi, Brendan C. Delaney, Nimish Vakil, David Forman, Nicholas J. Talley
Background & Aims :The evidence that proton pump inhibitor (PPI) therapy affects symptoms of nonulcer dyspepsia is conflicting. We conducted a systematic review to evaluate whether PPI therapy had any effect in nonulcer dyspepsia and constructed a health economic model to assess the cost-effectiveness of this approach. Methods :Electronic searches were performed using the Cochrane Controlled Trials Register, MEDLINE, EMBASE, CINAHL, and SIGLE until September 2002. Dyspepsia outcomes were dichotomized into cured/improved versus same/worse. Results were incorporated into a Markov model comparing health service costs and benefits of PPI with antacid therapy over 1 year. Results :Eight trials were identified that compared PPI therapy with placebo in 3293 patients. The relative risk of remaining dyspeptic with PPI therapy versus placebo was .86 (95% confidence interval, .78.95; P= .003, random-effects model) with a number needed to treat of 9 (95% confidence interval, 525). There was statistically significant heterogeneity between trials (heterogeneity ? 2= 30.05; df = 7; P< .001). The PPI strategy would cost an extra $278/month free from dyspepsia if the drug cost $90/month. If a generic price of $19.99 is used, then a PPI strategy costs an extra $57/month free from dyspepsia. A third-party payer would be 95% certain that PPI therapy would be cost-effective, provided they were willing to pay $94/month free from dyspepsia. Conclusions :PPI therapy may be a cost-effective therapy in nonulcer dyspepsia, provided generic prices are used.
Clinical-liver, Pancreas, and Biliary Tract
Moderate hypothermia in patients with acute liver failure and uncontrolled intracranial hypertension
Rajiv Jalan, Steven W.M. Olde Damink, Nicolaas E.P. Deutz, Peter C. Hayes, Alistair Lee
Background & Aims :About 20% of patients with acute liver failure (ALF) die from increased intracranial pressure (ICP) while awaiting transplantation. This study evaluates the clinical effects and pathophysiologic basis of hypothermia in patients with ALF and intracranial hypertension that is unresponsive to standard medical therapy. Methods :Fourteen patients with ALF who were awaiting orthotopic liver transplantation (OLT) and had increased ICP that was unresponsive to standard medical therapy were studied. Core temperature was reduced to 32°C33°C using cooling blankets. Results :Thirteen patients were successfully bridged to OLT with a median of 32 hours (range, 10118 hours) of cooling. They underwent OLT with no significant complications related to cooling either before or after OLT and had complete neurologic recovery. ICP before cooling was 36.5 ± 2.7 mm Hg and was reduced to 16.3 ± .7 mm Hg at 4 hours, which was sustained at 24 hours (16.8 ± 1.5 mm Hg) ( P< .0001). Mean arterial pressure and cerebral perfusion pressure increased significantly, and the requirement for inotropes was reduced significantly. Hypothermia produced sustained and significant reduction in arterial ammonia concentration and its brain metabolism, cerebral blood flow, brain cytokine production, and markers of oxidative stress. Conclusions :Moderate hypothermia is an effective and safe bridge to OLT in patients with ALF who have increased ICP that is resistant to standard medical therapy. Hypothermia reduces ICP by impacting on multiple pathophysiologic mechanisms that are believed to be important in its pathogenesis. A large multicenter trial of hypothermia in ALF is justified.
Short-term antiviral efficacy of BILN 2061, a hepatitis C virus serine protease inhibitor, in hepatitis C genotype 1 patients
Holger Hinrichsen, Yves Benhamou, Heiner Wedemeyer, Markus Reiser, Roel E. Sentjens, José L. Calleja, Xavier Forns, Andreas Erhardt, Jens Crönlein, Ricardo L. Chaves, Chan-Loi Yong, Gerhard Nehmiz, Gerhard G. Steinmann
Background & Aims: Novel, potent, and well-tolerated hepatitis C virus (HCV) drugs are needed. BILN 2061 is a potent and specific inhibitor of HCV serine protease in vitro. Preclinical toxicology data and studies in healthy volunteers supported the administration of BILN 2061 to patients with HCV infection. Methods: The antiviral efficacy, pharmacokinetics, and tolerability of 25, 200, and 500 mg BILN 2061 twice daily given as monotherapy for 2 days in 31 patients infected with chronic genotype 1 HCV infection and with minimal liver fibrosis (Ishak score of 02) were assessed in a placebo-controlled, double-blind pilot study. In 2 subsequent placebo-controlled studies of similar design, 200 mg BILN 2061 twice daily was administered for 2 days to 10 patients with advanced liver fibrosis (Ishak score of 3 or 4) and to 10 patients with compensated cirrhosis (Ishak score of 5 or 6). Results: Viral RNA reductions of 23 log 10 copies/mL were achieved in most of the patients. There was a trend toward a higher number of patients receiving 500 mg BILN 2061 achieving a viral RNA reduction ≥3 log 10 copies/mL as compared with patients receiving 25 mg BILN 2061. Advanced fibrosis or compensated cirrhosis did not affect the antiviral efficacy of BILN 2061. BILN 2061 was well tolerated in all studies. Conclusions: BILN 2061 is a well-tolerated and very active compound that reduced serum viral RNA concentrations after 2 days of treatment in patients infected with genotype 1 HCV independent of the degree of fibrosis. Nevertheless, further clinical trials are on hold pending resolution of animal toxicity issues.
Occult hepatitis B virus infection in chronic liver disease: Full-length genome and analysis of mutant surface promoter
Vaishali Chaudhuri, Ruchi Tayal, Baibaswata Nayak, Subrat Kumar Acharya, Subrat Kumar Panda
ackground & Aims: Genome sequence of hepatitis B virus (HBV) from occult chronic infection is scarce. Fifty-six (9.4%) of 591 patients seronegative for hepatitis B surface antigen (HBsAg) with chronic liver disease were positive for HBV DNA. The complete HBV genome from 9 of these patients (S1-S9) and 5 controls positive for HBsAg (SWT.1-SWT.5) were analyzed. Methods: Overlapping genome fragment amplification, cloning, and sequencing was performed on these cases. Functional analysis of surface promoter was conducted using fusion construct. Results: All patients with occult infection except one (S8) had a low viral titer. Eight patients had infection with genotype A (S1-S5, SWT.12, SWT.5) and 6 had infection with genotype D (S6-S9, SWT.34). S4 and S5.1 of genotype A had the characteristic nucleotide deletions in core and pre-S1 region seen in genotype D. The major observations in patients with occult HBV infection were as follows: frequent quasispecies variation, deletions in pre-S2/S region affecting the surface promoters (nt 3025-54) and pre-S protein (S3, S5, S6, S8), truncated precore (S6, S8, S7.1) and core (S9) owing to stop signal, alternate start codon for the Polymerase gene (S3, S9), and YMDD mutation (S1, S4, S9) in patients not on antiviral therapy. HBsAg and core proteins could be shown immunohistochemically in 3 of 5 liver biopsy specimens available. The mutant surface promoters (pre-S2 and S) on functional analysis showed alterations in HBsAg expression. Conclusions: These changes in the regulatory region with possible alterations in the ratio of large and small surface proteins along with other mutations in the genome may decrease the circulating HBsAg level synergistically, making the immunodetection in serum negative
Hepatitis C infection and the increasing incidence of hepatocellular carcinoma: A population-based study
Jessica A. Davila, Robert O. Morgan, Yasser Shaib, Katherine A. McGlynn, Hashem B. ElSerag
Background & Aims: A significantincrease in the incidence of hepatocellular carcinoma (HCC) has been reported in the United States. The risk factors underlying this increase remain unclear. Methods: By using Surveillance, Epidemiology, and End-Results program (SEER)-Medicarelinked data, we conducted a population-based study to examine temporal changes in risk factors for patients 65 years and older diagnosed with HCC between 1993 and 1999. Only patients with continuous Medicare enrollment for 2 years before and up to 2 years after HCC diagnosis were examined. Univariate and multiple logistic regression analyses were used to evaluate changes in risk factors over time (January 1993June 1996 and July 1996December 1999). Results: The age-adjusted incidence of HCC among persons 65 years of age and older significantly increased from 14.2 per 100,000 in 1993 to 18.1 per 100,000 in 1999. We identified 2584 patients with continuous Medicare enrollment 2 years before and up to 2 years after HCC diagnosis. The proportion of hepatitis C virus (HCV)-related HCC increased from 11% during January of 1993 to June of 1996 to 21% during July of 1996 to December of 1999, whereas hepatitis B virus (HBV)-related HCC increased from 6% to 11% ( P< .0001). In multiple logistic regression analyses that adjusted for age, sex, race, and geographic region, the risk for HCV-related HCC and HBV-related HCC increased by 226% and 67%, respectively. Idiopathic HCC decreased from 43% to 39%. This decrease did not fully account for the significant increases observed for HCV and HBV. No significant changes over time were observed for alcohol-induced liver disease, nonspecific cirrhosis, or nonspecific hepatitis. Conclusions: There has been a significant recent increase in HCV- and HBV-related HCC. Increasing rates of HCV-related HCC can explain a substantial proportion of the reported increase in HCC incidence during recent years.
No evidence of maternal cell colonization in reverted liver nodules of tyrosinemia type I patients
Anne Bergeron, Francine Lettre, Pierre Russo, Jean Morissette, Robert M. Tanguay
Background & Aims: Hereditary tyrosinemia type I (HTI) is a recessively inherited disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine catabolic pathway. The mosaic pattern of FAH expression observed in the livers of >85% of studied patients was shown to result from the correction of the mutation in one of the FAH alleles. Bilateral cell trafficking can occur between mother and fetus and such an event could be responsible for the chimerism observed in some diseases. It also has been reported that the liver repopulation observed in a HTI murine model by serial transplantation of bone marrowderived cells was caused by a fusion of these cells to host hepatocytes. These observations led us to test the possibility that the transfer of nucleated heterozygous maternal cells in the fetal circulation could be responsible for the mosaic liver expression of FAH in HTI patients. Methods: We used polymorphic markers of short cytosine-adenine DNA repeats to compare DNA from corrected liver sections of 4 HTI patients with DNA from their parents’ blood. Results: Genotyping showed that only one maternal allele is present in DNA isolated from FAH-expressing liver nodules of each proband for at least 1 marker. Conclusions: The corrected liver nodules in HTI patients are not of maternal origin and do not support cell trafficking and cell fusion as mechanisms of correction of the gene defect in hepatocytes of tyrosinemia patients.
Claudin-1 gene mutations in neonatal sclerosing cholangitis associated with ichthyosis: A tight junction disease
Smail Hadj-Rabia, Lekbir Baala, Pierre Vabres, Dominique Hamel-Teillac, Emmanuel Jacquemin, Monique Fabre, Stanislas Lyonnet, Yves de Prost, Arnold Munnich, Michelle Hadchouel, Asma Smahi
Background & Aims :Most human and animal cholestatic disorders are associated with changes in hepatocyte cytoskeleton and tight junctions (TJs). These changes are usually secondary and nonspecific phenomena, both in intra- and extrahepatic cholestasis. Recently, missense mutations in TJ protein 2 (ZO-2) have been identified in patients with familial hypercholanemia. In the liver, TJs separate bile flow from plasma and are composed of strands of claudins and occludin. We previously assigned a syndrome associating ichthyosis and neonatal sclerosing cholangitis (NISCH syndrome) to chromosome 3q27-q28. We considered claudin-1 to be a strong candidate gene based on its mapping to the minimum interval and on the expression pattern of the mouse ortholog. Methods :The 4 exons and intron-exon junctions of claudin-1 gene were amplified using standard polymerase chain reaction protocols and specific primers. Western blot analysis on cultured fibroblasts and immunohistochemistry on liver tissue section were performed using rabbit anticlaudin-1 antibodies. Results :We described in 4 patients, of 2 inbred kindred of Moroccan origin, a 2-bp deletion (200201 TT) in exon 1 of the claudin-1 gene arising in a premature stop codon and resulting in total absence of claudin-1 protein in the liver and skin. Conclusions :Lack of claudin-1 in NISCH syndrome may lead to increased paracellular permeability between epithelial cells. Bile duct injury may be related to the absence of claudin-1 expression in cholangiocytes. Our observation, in conjunction with ZO-2associated hypercholanemia, emphasizes the role played by TJ components in hereditary cholestasis.
Basic-alimentary Tract
Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor
F. Gregory Buchanan, Woogki Chang, Hongmiao Sheng, Jinyi Shao, Jason D. Morrow, Raymond N. D u bois
Background & Aims :The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Methods :Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A 2[cPLA 2]). Results: The inducible expression of Ha-Ras V12 increased the production of prostaglandin (PG)F 2? and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras V12 also up-regulated the mRNA and protein levels of cPLA 2, COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA 2. Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI 2alone also induced the expression of VEGF. Conclusions: Inducible Ha-Ras V12 increases the production of PGI 2through the coordinate up-regulation of cPLA 2, COX-2, and prostacyclin synthase (PGIS). The production of PGI 2leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.
hPepT1 transports muramyl dipeptide, activating NF-?B and stimulating IL-8 secretion in human colonic Caco2/bbe cells
Stephan R. Vavricka, Mark W. Musch, Jonathan E. Chang, Yasushi Nakagawa, Kittiporn Phanvijhitsiri, Tonya S. Waypa, Didier Merlin, Olaf Schneewind, Eugene B. Chang
Background & Aims: Bacterial proteoglycan-derived muramyl dipeptide (MDP) activates the intracellular NOD2/CARD15 gene product. How intestinal epithelial cells take up MDP is poorly understood. We hypothesized that the intestinal apical di-/tripeptide transporter, hPepT1, transports MDP, thereby activating downstream pathways similar to nuclear factor ? B (NF-?B). Methods: Time- and concentration-dependent 3H-MDP uptakes were studied in Caco2/bbe (C2) cell monolayers where hPepT1 expression was either over- or underexpressed, using an inducible adenovirus system or silencing RNA (siRNA), respectively. NF-?B activation and interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) release were determined by enzyme-linked immunosorbent assay. NOD2/CARD15 expression was inhibited by siRNA. MDP in human duodenal, cecal, and stool samples was measured. Results: MDP, but not its isoforms, inhibited uptake of glycosylsarcosine in C2 cells, indicating stereoselective and competitive inhibition. Approximately 90% of the MDP was cytosolic, showing uptake rather than binding. The K mfor MDP uptake was 4.3 mmol/L. Cells overexpressing hPepT1 showed increased Gly-Sar and MDP uptake, whereas decreased uptake was observed after hPepT1 siRNA-inhibition. MDP treatment activated NF-?B, resulting in IL-8 release, an effect blocked by siRNA-inhibited expression of NOD2/CARD15. MDP content in cecal and stool samples (in normal subjects) was 2087 ?mol/L, but undetectable in duodenal fluid. Conclusions: In colonic epithelial cells, MDP is taken up by hPepT1 and activates NF-?B and chemokine production. Because hPepT1 expression in chronic colonic inflammation is increased, this may play an important role in promoting colonocyte participation in host defense and pathogen clearance through increased uptake of MDP.
H+/amino acid transporter 1 (PAT1) is the imino acid carrier: An intestinal nutrient/drug transporter in human and rat
Catriona M.H. Anderson, Danielle S. Grenade, Michael Boll, Martin Foltz, Katherine A. Wake, David J. Kennedy, Lars K. Munck, Seiji Miyauchi, Peter M. Taylor, Frederick Charles Campbell, Bjarne G. Munck, Hannelore Daniel, Vadivel Ganapathy, David T. Thwaites
Background & Aims: Hepatocyte growth factor activator (HGFA) is a serum proteinase that specifically converts an inactive single-chain form of hepatocyte growth factor (HGF) into an active 2-chain form. HGFA is produced in its precursor form and then activated in injured tissues. To address the precise role of HGFA and to investigate the mechanisms of HGF activation in injured tissues, we generated mice deficient in HGFA. Methods: HGFA-deficient mice were generated using targeted gene disruption. The regenerating process of intestinal mucosa damaged by oral administration of dextran sodium sulfate (DSS) or by rectal administration of acetic acid was examined in both HGFA-deficient and control mice. HGF processing activity was analyzed using Western blotting and an HGF activation assay. Results: Homozygous mutant mice were viable and fertile without obvious abnormalities. When mice were treated with 3% DSS in drinking water for 6 days followed by distilled water without DSS, 72% of HGFA-deficient mice died through day 12 while 75% of control mice survived injury. Similar results were also observed in the acetic acid-induced intestinal injury; the survival rate was 36.6% in HGFA-deficient mice and 84.2% in control mice. In HGFA-deficient mice, the injured mucosa was not sufficiently covered by regenerated epithelium and the activation of HGF was impaired in the injured colon. Conclusions: These results indicate that HGFA is required for repair of injured intestinal mucosa but is not essential for normal development during embryogenesis or after birth.
Regeneration of injured intestinal mucosa is impaired in hepatocyte growth factor activator-deficient mice
Hiroshi Itoh, Seiji Naganuma, Naoki Takeda, Shiro Miyata, Shunro Uchinokura, Tsuyoshi Fukushima, Shuichiro Uchiyama, Hiroyuki Tanaka, Koki Nagaike, Takeshi Shimomura, Keiji Miyazawa, Gen Yamada, Naomi Kitamura, Masashi Koono, Hiroaki Kataoka
Background & Aims: Hepatocyte growth factor activator (HGFA) is a serum proteinase that specifically converts an inactive single-chain form of hepatocyte growth factor (HGF) into an active 2-chain form. HGFA is produced in its precursor form and then activated in injured tissues. To address the precise role of HGFA and to investigate the mechanisms of HGF activation in injured tissues, we generated mice deficient in HGFA. Methods: HGFA-deficient mice were generated using targeted gene disruption. The regenerating process of intestinal mucosa damaged by oral administration of dextran sodium sulfate (DSS) or by rectal administration of acetic acid was examined in both HGFA-deficient and control mice. HGF processing activity was analyzed using Western blotting and an HGF activation assay. Results: Homozygous mutant mice were viable and fertile without obvious abnormalities. When mice were treated with 3% DSS in drinking water for 6 days followed by distilled water without DSS, 72% of HGFA-deficient mice died through day 12 while 75% of control mice survived injury. Similar results were also observed in the acetic acid-induced intestinal injury; the survival rate was 36.6% in HGFA-deficient mice and 84.2% in control mice. In HGFA-deficient mice, the injured mucosa was not sufficiently covered by regenerated epithelium and the activation of HGF was impaired in the injured colon. Conclusions: These results indicate that HGFA is required for repair of injured intestinal mucosa but is not essential for normal development during embryogenesis or after birth.
Evidence for a new human CYP1A1 regulation pathway involving PPAR-? and 2 PPRE sites
E. Sérée, P.-H. Villard, J.-M. Pascussi, T. Pineau, P. Maurel, Q.B. Nguyen, F. Fallone, P.-M. Martin, S. Champion, B. Lacarelle, J.-F. Savouret, Y. Barra
Background & Aims: Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferatoractivated receptors in cytochrome P450 1A1 gene induction. Methods: The role of peroxisome proliferatoractivated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. Results: We show that peroxisome proliferatoractivated receptor-? agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferatoractivated receptor-? agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O-deethylase activity (10-fold at 200 ?mol/L). Transfection of peroxisome proliferatoractivated receptor-? complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions ?931/?919 and ?531/?519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferatoractivated receptor-?/retinoid X receptor-? heterodimer. Conclusions: We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferatoractivated receptor-? and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferatoractivated receptor-? ligands, which are common environmental compounds, may be involved in carcinogenesis.
Profiling of microdissected gastric epithelial cells reveals a cell typespecific response to Helicobacter pylori infection
Anne Mueller, D. Scott Merrell, Jan Grimm, Stanley Falkow
Background & Aims: Helicobacter pylori colonizes the epithelial lining of the human stomach and is associated with disorders ranging from chronic gastritis to peptic ulcers and gastric cancer. We have explored the transcriptional response of the epithelium globally by applying a whole-genome approach to a murine model of infection. Methods: The 3 major epithelial lineages of the stomachthe parietal, mucus-producing, and chief cellswere harvested from cryosections of infected and uninfected murine stomachs by laser microdissection and subjected to gene expression profiling. The localization and quantity of selected transcripts were verified by in situ hybridization and quantitative real-time reverse-transcription polymerase chain reaction. Results: Each cell type is characterized by a transcriptional signature profile. The parietal cell profile is highly enriched for factors involved in mitochondrial energy generation, whereas the chief cell predominantly expresses digestive enzymes and glycosylation-associated proteins. In contrast, the mucus cell signature is distinguished by an abundance of cell-surface receptors, signaling molecules, and factors involved in antigen presentation. All of these indicate a role in sampling, sensing, and responding to environmental stimuli. In line with this biological function, we measured a strong transcriptional response to Helicobacter pylori infection only in this cell type. The genes that are differentially expressed upon infection are implicated in a proinflammatory and mucosal defense response as well as modulation of angiogenesis, iron availability, and tumor suppression. Conclusions: Laser microdissectionassisted transcriptional profiling is a useful tool to explore the biology of specific cell populations and is sensitive enough to measure the transcriptional response to bacterial infection in vivo.
The lymphotoxin-? receptor is critical for control of murine Citrobacter rodentiuminduced colitis
Thomas W. Spahn, Christian Maaser, Lars Eckmann, Jan Heidemann, Andreas Lügering, Rodney Newberry, Wolfram Domschke, Hermann Herbst, Torsten Kucharzik
Background & Aims :Lymphotoxin is a tumor necrosis factor-family cytokine. Blocking of lymphotoxin ? 1?2/lymphotoxin-? receptor interactions prevents experimental colitis in mice, and this suggests a potential treatment principle of human inflammatory bowel disease. Infection of mice with Citrobacter rodentium serves as an animal model for human infectious colitis induced by enteropathogenic Escherichia coli . We studied the role of lymphotoxin ? 1?2/lymphotoxin-? receptor signaling in Citrobacter rodentium induced colitis. Methods :Mice with disrupted lymphotoxin ? 1?2/lymphotoxin-? receptor interactions secondary to gene defects (lymphotoxin-? ?/? , lymphotoxin-? ?/? , and lymphotoxin-? receptor ?/? ) or treatment with the antagonist lymphotoxin-? receptor-immunoglobulin G fusion protein were infected with Citrobacter rodentium . Body weight, fecal excretion of Citrobacter rodentium , and disease-related mortality were monitored. Spleen and liver organ cultures of mice assessed systemic infection. Intestinal inflammation and lymphoid architecture were histologically recorded in the large intestine, mesenteric lymph nodes, and spleen of infected mice. Results :Inhibition of lymphotoxin ? 1?2/lymphotoxin-? receptor interactions was associated with increased severity of Citrobacter rodentium induced colitis, as indicated by increased disease-related mortality, more severe weight loss, intestinal bacterial abscesses, and a higher burden of Citrobacter rodentium in the spleen and liver of ?/? and lymphotoxin-? receptor-immunoglobulin G-treated mice. There was a reduction of CD11c +dendritic cells in the spleen of naive and infected ?/? and lymphotoxin-? receptor-immunoglobulin G-treated mice. In infected lymphotoxin-? receptor ?/? mice, anti Citrobacter rodentium immunoglobulin G2a levels were decreased, whereas immunoglobulin G1 levels were increased. Citrobacter rodentium induced interleukin-4 secretion was increased in lymphotoxin-? receptor ?/? mice. Conclusions :Lymphotoxin ? 1?2/lymphotoxin-? receptor interactions are critical for immunity against Citrobacter rodentium in mice. Impaired anti-enteropathogenic Escherichia coli immunity may be anticipated in anti-lymphotoxin-? receptor-directed therapy for human inflammatory bowel disease.
Probiotics inhibit nuclear factor-?B and induce heat shock proteins in colonic epithelial cells through proteasome inhibition
Elaine O. Petrof, Keishi Kojima, Mark J. Ropeleski, Mark W. Musch, Yun Tao, Claudio de Simone, Eugene B. Chang
Background & Aims: The extent and severity of mucosal injury in inflammatory bowel diseases are determined by the disequilibrium between 2 opposing processes: reparative and cytoprotective mechanisms vs. inflammation-induced injury. Probiotics may provide clinical benefit by ameliorating colitis; however, their mechanisms of action remain largely unknown. Our objective was to investigate microbial-epithelial interactions that could explain the beneficial therapeutic effects of probiotics. Methods: The effect of VSL#3-conditioned media on the nuclear factor-?B pathway in young adult mouse colonic epithelial cells was assessed by using monocyte chemoattractant protein-1 enzyme-linked immunosorbent assays; I?B?, I?B?, and p105 immunoblot analysis; and nuclear factor-?B luciferase reporter gene and proteasome assays. Effects on heat shock proteins were determined by electrophoretic mobility shift assay and immunoblot for heat shock proteins 25 and 72 in young adult mouse colonic cells. Cytoprotection against oxidant injury was determined by chromium 51 release and filamentous and globular actin assays. Results: VSL#3 produces soluble factors that inhibit the chymotrypsin-like activity of the proteasome in gut epithelial cells. Proteasome inhibition is an early event that begins almost immediately after exposure of the epithelial cells to the probiotic-conditioned media. In addition, these bacteria inhibit the proinflammatory nuclear factor-?B pathway through a mechanism different from the type III secretory mechanisms described for other nonpathogenic enteric flora. They also induce the expression of cytoprotective heat shock proteins in intestinal epithelial cells. Conclusions: The resulting inhibition of nuclear factor-?B and increased expression of heat shock proteins may account for the anti-inflammatory and cytoprotective effects reported for probiotics and may be a novel mechanism of microbial-epithelial interaction. These effects seem to be mediated through the common unifying mechanism of proteasome inhibition.
Basic-liver, Pancreas, and Biliary Tract
Ischemic preconditioning protects hepatocytes via reactive oxygen species derived from Kupffer cells in rats
Kazuaki Tejima, Masahiro Arai, Hitoshi Ikeda, Tomoaki Tomiya, Mikio Yanase, Yukiko Inoue, Kayo Nagashima, Takako Nishikawa, Naoko Watanabe, Masao Omata, Kenji Fujiwara
Background & Aims :Hepatic ischemic preconditioning decreases sinusoidal endothelial cell injury and Kupffer cell activation after cold ischemia/reperfusion, leading to improved survival of liver transplant recipients in rats. Ischemic preconditioning also protects livers against warm ischemia/reperfusion injury, in which hepatocyte injury is remarkable. We aimed to determine whether ischemic preconditioning directly protects hepatocytes and to elucidate its mechanisms. Methods :Rats were injected with gadolinium chloride to deplete Kupffer cells or with N-acetyl- L-cysteine, superoxide dismutase, or catalase to scavenge reactive oxygen species. Livers were then preconditioned by 10 minutes of ischemia and 10 minutes of reperfusion. Subsequently, livers were subjected to 40 minutes of warm ischemia and 60 minutes of reperfusion in vivo or in a liver perfusion system. In other rats, livers were preconditioned by H 2O2perfusion instead of ischemia. In the other experiments, livers were perfused with nitro blue tetrazolium to detect reactive oxygen species formation. Results :Ischemic preconditioning decreased injury in hepatocytes, but not in sinusoidal endothelial cells. Kupffer cell depletion itself did not change hepatocyte injury after ischemia/reperfusion, indicating no contribution of Kupffer cells to ischemia/reperfusion injury. However, Kupffer cell depletion reversed hepatoprotection by ischemic preconditioning. Reactive oxygen species formation occurred in Kupffer cells after ischemic preconditioning. Scavenging of reactive oxygen species reversed the effect of ischemic preconditioning, and H 2O2preconditioning mimicked ischemic preconditioning. Conclusions :Ischemic preconditioning directly protected hepatocytes after warm ischemia/reperfusion, which is not via suppression of changes in sinusoidal cells as in cold ischemia/reperfusion injury. This hepatocyte protection was mediated by reactive oxygen species produced by Kupffer cells.
The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis
Stefano Fiorucci, Elisabetta Antonelli, Giovanni Rizzo, Barbara Renga, Andrea Mencarelli, Luisa Riccardi, Stefano Orlandi, Roberto Pellicciari, Antonio Morelli
Background & Aims: The farnesoid X receptor (FXR) is an endogenous sensor for bile acids and inhibits bile acid synthesis by inducing small heterodimer partner (SHP) gene expression. The aim of this study was to investigate whether FXR is expressed by and modulates function of hepatic stellate cells (HSCs). Methods: The antifibrotic activity of FXR ligand was tested in 2 rodent models: the porcine serum and bile duct ligation (BDL). Results: Twelve-week administration of 110 mg/kg 6-ethyl chenodeoxycholic acid (6-ECDCA), a synthetic FXR ligand, to porcine serum-treated rats prevented liver fibrosis development and reduced liver expression of ?1(I) collagen, TGF-?1 and ?-SMA mRNA by ?90%. Therapeutic administration of 6-ECDCA, 3 mg/kg, to BDL rats reduced liver fibrosis and ?1(I) collagen, transforming growth factor (TGF)-?1, ?-SMA, and tissue metalloproteinase inhibitor (TIMP)-1 and 2 messenger RNA (mRNA) by 70%80%. No protection was observed in BDL rats treated with CDCA, 3 mg/kg, and ursodeoxycholic acid, 15 mg/kg. FXR expression was detected in HSCs. Exposure of HSCs to FXR ligands caused a 3-fold increase of SHP, reduced ?1(I)collagen and TGF-?1 by ?60%70% and abrogates ?1(I) collagen mRNA up-regulation induced by thrombin and TGF-?1. By retrovirus infection and small interference RNA, we generated SHP overexpressing and SHP-deficient HSC-T6. Using these cell lines, we demonstrated that SHP binds JunD and inhibits DNA binding of adaptor protein (AP)-1 induced by thrombin. Conclusions: By demonstrating that an FXR-SHP regulatory cascade promotes resolution of liver fibrosis, this study establish that FXR ligands might represent a novel therapeutic option to treat liver fibrosis.
Hepatitis C core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation
Angela Dolganiuc, Shilpa Oak, Karen Kodys, Douglas T. Golenbock, Robert W. Finberg, Evelyn Kurt-Jones, Gyongyi Szabo
Background & Aims: Recent evidence suggests that toll-like receptors (TLRs) recognize certain viruses. We reported that hepatitis C virus (HCV) core and nonstructural 3 (NS3) proteins activate inflammatory pathways in monocytes. The aim of this study was to investigate the role of TLRs in innate immune cell activation by core and NS3 proteins. Methods: Human monocytes, human embryonic kidney cells transfected with TLR2, and peritoneal macrophages from TLR2, MyD88 knockout, and wild-type mice were studied to determine intracellular signaling and proinflammatory cytokine induction by HCV proteins. Results: HCV core and NS3 proteins triggered inflammatory cell activation via the pattern recognition receptor TLR2 and failed to activate macrophages from TLR2 or MyD88-deficient mice. HCV core and NS3 induced interleukin (IL)-1 receptor-associated kinase (IRAK) activity, phosphorylation of p38, extracellular regulated (ERK), and c-jun N-terminal (JNK) kinases and induced AP-1 activation. Activation of nuclear factor-?B by core and NS3 was associated with increased I?B? phosphorylation. TLR2-mediated cell activation was dependent on the conformation of core and NS3 proteins and required sequences in the regions of aa 2122 in core and aa 14501643 in NS3. Although cellular uptake of core and NS3 proteins was independent of TLR2 expression, cell activation required TLR2. HCV core protein and TLR2 showed intracellular colocalization. The hyper-elevated TNF-? induction by TLR2 ligands in monocytes of HCV-infected patients was not due to increased TLR2 expression. Conclusions: HCV core and NS3 proteins trigger inflammatory pathways via TLR2 that may affect viral recognition and contribute to activation of the innate immune system.
Negative regulation of liver regeneration by innate immunity (natural killer cells/interferon-?)
Rui Sun, Bin Gao
Background & Aims: Hepatic lymphocytes are composed mainly of natural killer (NK) cells and NKT cells, which play key roles in innate immune responses against pathogens and tumors in the liver. This report analyzes the effects of activation of innate immunity by viral infection or the toll-like receptor 3 (TLR3) ligand on liver regeneration. Methods: The partial hepatectomy (PHx) method was used as a model of liver regeneration. Murine cytomegalovirus (MCMV) infection and the TLR3 ligand polyinosinic-polycytidylic acid [poly(I:C)] were used to activate innate immunity. Results: NK cells are activated after PHx, as evidenced by producing interferon (IFN)-?. Infection with MCMV or injection of poly(I:C) further activates NK cells to produce IFN-? and attenuates liver regeneration in the PHx model. Depletion of NK cells or disruption of either the IFN-? gene or the IFN-? receptor gene enhances liver regeneration and partially abolishes the negative effects of MCMV and polyI:C on liver regeneration, whereas NKT cells may only play a minor role in suppression of liver regeneration. Adoptive transfer of IFN-? +/+ NK cells, but not IFN-? ?/? NK cells, restores the ability of polyI:C to attenuate liver regeneration in NK-depleted mice. Finally, administration of polyI:C or IFN-? enhances expression of several antiproliferative proteins, including STAT1, IRF-1, and p21cip1/waf1 in the livers of partially hepatectomized mice. Conclusions: Our findings suggest that viral infection and the TLR3 ligand negatively regulate liver regeneration via activation of innate immunity (NK/IFN-?), which may play an important role in the pathogenesis of viral hepatitis.
Involvement of the Src family kinase yes in bile salt-induced apoptosis
Roland Reinehr, Stephan Becker, Matthias Wettstein, Dieter Häussinger
ackground & Aims: Hydrophobic bile acids induce CD95 (Fas, APO-1)-dependent hepatocyte apoptosis, which involves epidermal growth factor receptor (EGFR)-catalyzed CD95 tyrosine phosphorylation. The mechanisms underlying bile salt-induced EGFR activation remain unclear. Methods: Bile acid-induced EGFR activation was studied in 24-hour cultured rat hepatocytes and perfused rat liver. Results: The proapoptotic bile salts taurolithocholate-3-sulfate (TLCS), glycochenodesoxycholate (GCDC) and taurochenodeoxycholate (TCDC), but not taurocholate (TC), activate within 1 minute the Src kinase family member Yes, followed by an association of Yes with EGFR and subsequent EGFR activation. EGFR phosphorylation by TLCS involves tyrosines 845 and 1173 but not 1045. Yes/EGFR association and EGFR activation were sensitive to inhibition by SU6656 but not by PP-2. cAMP had no effect on TLCS and GCDC-induced Yes activation but induced Ser/Thr phosphorylation of Yes and prevented Yes/EGFR association and subsequent EGFR activation. Both SU6656 and cAMP had no effect on bile salt-induced c-Jun N-terminal kinase activation, but blocked bile salt-induced CD95 tyrosine phosphorylation, membrane trafficking of CD95, formation of the death-inducing signaling complex, and apoptosis. In 4-day cultured hepatocytes, knockdown of either Yes or EGFR strongly attenuated bile salt-induced CD95 activation and apoptosis. Conclusions: The data identify the Src kinase Yes as an upstream target of proapoptotic bile acids, which triggers EGFR activation, subsequent CD95 tyrosine phosphorylation, and apoptosis. The antiapoptotic effect of cAMP involves a protein kinase A-dependent inhibition of Yes/EGFR association, thereby preventing EGFR activation, which is required for CD95 activation.
Copyright © 2001-2004 by the American Gastroenterological Association. All rights reserved.
Isoflurane pretreatment lowers portal venous resistance by increasing hepatic heme oxygenase activity in the rat liver in vivo
Rene Schmidta, Alexander Hoetzela, Tilo Baechlea, Torsten Loopa, Matjaz Humara, Michael Bauerb, Heike L. Pahla, Klaus K. Geigera, Benedikt H.J. Pannena*
Background/Aims
The heme oxygenase (HO) system contributes to the maintenance of hepatic perfusion and integrity. It was the objective of this study to determine the influence of isoflurane (ISO) on hepatic HO-1 induction and its impact on hepatic hemodynamics.
Methods
Rats were pretreated with or without ISO for 5h. After hemodynamic measurements by pressure-, laser doppler-, and ultrasound based techniques, the liver was harvested. HO-1 was analyzed by an HO activity assay, Northern- and Western blotting.
Results
ISO pretreatment induced hepatic HO-1 mRNA and protein resulting in an increase of HO activity. No effect on hsp-27, hsp-70 and hsp-90 mRNA could be observed. ISO lowered portal resistance. HO inhibition by tin protoporphyrine IX increased portal resistance in ISO pretreated animals up to control levels. This was associated with an increase in portal pressure and a reduction of portal flow. Microvascular flux was also impaired after HO blockade and ISO. However, hepatic arterial and systemic hemodynamics remained unchanged, indicating a specific effect within the portal vascular bed.
Conclusions
ISO pretreatment induces hepatic HO-1 mRNA and protein followed by an increase in HO activity, thereby reducing portal resistance. These findings indicate a beneficial effect of ISO on hepatic hemodynamics in vivo.
Patterns of transgene expression and viral clearance from the transplanted liver following ex vivo adenovirus-mediated gene transfer
Gideon Zamir, Andrew E. Gelman, Kim M. Olthoff, Fotini Debonera, Xavier Aldeguer, Abraham Shaked*
Background/Aims
In the rat liver transplant model, the liver graft can be transduced ex vivo by adenovirus encoding CTLA-4Ig (AdCTLA-4Ig) to achieve high level of immunosuppression in the liver after transplantation. To characterize the pattern of transgene expression following ex vivo gene transfer to the liver and examine whether immunosuppression would promote adenovirus persistence, we followed the life span of vector DNA and transgene expression in the transplanted liver.
Methods
Rat liver grafts were perfused ex vivo with adenovirus carrying the reporter gene ?-galactosidase (AdlacZ). The period of transgene expression was assessed at predetermined intervals after transplantation into syngeneic, allogeneic or nude (athymic) recipients. Clearance of vector DNA was assessed by PCR analysis of liver DNA after transplantation.
Results
Graft transduction with AdCTLA-4Ig or systemic cyclosporine treatment effectively abrogated the alloimmune response but did not result in sustained lacZ expression. The course of viral DNA clearance from the liver was also unaffected by immunosuppression as was the implied nucleolytic cleavage of viral DNA.
Conclusions
In the transplant setting, local expression of CTLA-4Ig or systemic immunosuppression does not solve the problem of viral clearance from the liver. Non-adaptive immune mechanisms may have a significant role in the host response to adenovirus after liver transplantation.
Polyunsaturated fatty acid deficiency reverses effects of alcohol on mitochondrial energy metabolism
Marie-Astrid Piquetab*, Michel Rouletc, Véronique Nogueirab, Céline Filippib, Brigitte Sibilleb, Isabelle Hourmand-Olliviera, Marianne Piletc, Vincent Rouleaud, Xavier M. Leverveb
Received 21 February 2004; received in revised form 26 June 2004; accepted 2 July 2004 published online 19 August 2004.
Background/Aims
Polyunsaturated fatty acids (PUFA) deficiency is common in patients with alcoholic liver disease. The suitability of reversing such deficiency remains controversial. The aim was to investigate the role played by PUFA deficiency in the occurrence of alcohol-related mitochondrial dysfunction.
Methods
Wistar rats were fed either a control diet with or without alcohol (control and ethanol groups) or a PUFA deficient diet with or without alcohol (PUFA deficient and PUFA deficient+ethanol groups). After 6 weeks, liver mitochondria were isolated for energetic studies and fatty acid analysis.
Results
Mitochondria from ethanol fed rats showed a dramatic decrease in oxygen consumption rates and in cytochrome oxidase activity. PUFA deficiency showed an opposite picture. PUFA deficient+ethanol group roughly reach control values, regarding cytochrome oxidase activity and respiratory rates. The relationship between ATP synthesis and respiratory rate was shifted to the left in ethanol group and to the right in PUFA-deficient group. The plots of control and PUFA deficient+ethanol groups were overlapping. Phospholipid arachidonic over linoleic ratio closely correlated to cytochrome oxidase and oxygen uptake.
Conclusions
PUFA deficiency reverses alcohol-related mitochondrial dysfunction via an increase in phospholipid arachidonic over linoleic ratio, which raises cytochrome oxidase activity. Such deficiency may be an adaptive mechanism.
Pirfenidone inhibits the induction of iNOS stimulated by interleukin-1? at a step of NF-?B DNA binding in hepatocytes
Hideki Nakanishi1, Masaki Kaibori1, Shigeru Teshima1, Hideyuki Yoshida1, A-Hon Kwon1, Yasuo Kamiyama1, Mikio Nishizawa2, Seiji Ito2, Tadayoshi Okumura2*
Background/Aims
Pirfenidone has antiinflammatory effects in animals with endotoxemia. We reported that pirfenidone inhibits the enhancement of inflammatory cytokines and inducible nitric oxide synthase (iNOS) in liver of endotoxin-treated rats, leading to the prevention of hepatic injury. However, the mechanisms involved in suppression of these gene inductions are obscure. Studies were performed to investigate whether pirfenidone directly influences iNOS induction in hepatocytes.
Methods
Cultured hepatocytes were treated with interleukin-1? (IL-1?) in the presence and absence of pirfenidone, and iNOS induction and its signal including NF-?B were analyzed.
Results
Pirfenidone inhibited the induction of iNOS mRNA and protein, resulting in the decrease of nitric oxide production. Gel shift assay demonstrated that pirfenidone inhibited the activation of NF-?B. Consistent with this observation, transfection experiments revealed that pirfenidone decreased transcriptional activation of iNOS gene promoter. In contrast, pirfenidone had no effect on the degradation of I?B, and could not prevent nuclear translocation of p50/p65. Finally, pirfenidone inhibited the activation of Akt and up-regulation of IL-1 receptor stimulated by IL-1?.
Conclusions
Results indicate that pirfenidone inhibits the induction of iNOS gene expression at a step of NF-?B DNA binding, but not its nuclear translocation, partly through the inhibition of IL-1 receptor induction in hepatocytes.
Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury
Audrey Avrila, Virginie Picharda, Marie-Pierre Braletb, Nicolas Ferrya*
Background/Aims
Mature hepatocytes divide to restore liver mass after injury. However, when hepatocyte division is impaired by retrorsine poisoning, regeneration proceeds from another cell type: the small hepatocyte-like progenitor cells (SHPCs). Our aim was to test whether SHPCs could originate from mature hepatocytes.
Methods
Mature hepatocytes were genetically labeled using retroviral vectors harboring the ?-galactosidase gene. After labeling, retrorsine was administered to rats followed by a partial hepatectomy to trigger regeneration. A liver biopsy was performed one month after surgery and rats were sacrificed one month later.
Results
We observed the proliferation of small hepatocytes arranged in clusters in liver biopsies. These cells expressed Ki67 antigen and displayed a high mitotic index. At sacrifice, regeneration was completed and clusters had merged. A significant proportion of clusters also expressed ?-galactosidase demonstrating their origin from labeled mature hepatocytes. Finally, the overall proportion of ?-galactosidase positive cells was identical at the time of hepatectomy as well as in liver biopsy and at sacrifice.
Conclusions
The constant proportion of ?-galactosidase positive cells during the regeneration process demonstrates that mature hepatocytes are randomly recruited to proliferate and compensate parenchyma loss in this model. Furthermore, mature hepatocytes are the source of SHPC after retrorsine injury.
Genetic polymorphisms of ADH2, ADH3, CYP4502E1 Dra-I and Pst-I, and ALDH2 in Spanish men: lack of association with alcoholism and alcoholic liver disease
Francesc Vidalad*, Alfons Lorenzoa, Teresa Auguetad, Montserrat Olonab, Montserrat Brochcd, Cristina Gutiérrezcd, Carmen Aguilarc, Pere Estupiñàc, Mauro Santose, Cristóbal Richartad
Received 1 October 2002; received in revised form 1 May 2003; accepted 1 June 2003 published online 19 August 2004.
Background/Aims
The relationship between polymorphisms at the alcohol dehydrogenase 2 (ADH2), ADH3, CYP4502E1 and aldehyde dehydrogenase 2 (ALDH2) loci and the individual predisposition to alcoholism and alcoholic liver disease in Caucasians is controversial.
Methods
We determined the genotypes of ADH2, ADH3, CYP4502E1 (Pst-I and Dra-I) and ALDH2 in 519 male Spaniards: 264 alcoholic subjects (47 without liver disease, 118 with non-cirrhotic liver disease and 99 with cirrhosis) and 255 non-alcoholic subjects (64 healthy controls, 110 with non-cirrhotic non-alcoholic liver disease and 81 with cirrhosis unrelated to alcohol). Genotyping was performed using PCR-RFLP methods on white cell DNA.
Results
The distribution of the allelic variants (allele *1 and allele *2) in the whole subjects analyzed was: ADH2 93.1% and 6.9%; ADH3 55.7 and 44.3%; CYP4502E1 Dra-I 11.2 and 88.8%; CYP4502E1 Pst-I 96.2 and 3.8% and ALDH2 100 and 0%, respectively. No differences were observed in the allelic distributions of the alcoholic and non-alcoholic subjects for the loci examined. Allele distribution in alcoholics with no liver disease, with alcoholic steatosis or hepatitis, and with cirrhosis was also similar.
Conclusions
ADH2, ADH3, and CYP4502E1 Pst-I and Dra-I genetic variations are not related to alcoholism or susceptibility to alcoholic liver disease in our male population. ALDH2 locus is monomorphic.
Silent non-alcoholic fatty liver diseasea clinicalhistological study
Paolo Sorrentinoa, Giovanni Tarantinoa*, Paolo Concaa, Alessandro Perrellab, Maria Luigi Terraccianoc, Raffaella Vecchionec, Giovanna Gargiulod, Nicola Gennarellid, Roberto Lobellod
Received 11 February 2004; received in revised form 16 June 2004; accepted 12 July 2004 published online 19 August 2004.
Background/Aims
We studied the prevalence of non-alcoholic fatty liver disease and non-alcoholic-steatohepatitis in patients with metabolic-syndrome but normal liver enzymes. The histological findings of patients with normal liver enzymes and non-alcoholic-steatohepatitis were compared with those of a control-group with persistently abnormal liver enzymes.
Methods
Patients presenting with normal liver enzymes were enrolled in the study and underwent liver biopsy. Prevalence of non-alcoholic-steatohepatitis and risk factors for fibrosis and cirrhosis were evaluated. Data from a control-group with non-alcoholic-steatohepatitis and abnormal liver enzymes were used to compare the histological findings.
Results
Fifty-eight of the 80 patients enrolled had varying degrees of non-alcoholic steatohepatitis, of these 26 had fibrosis and 8 silent cirrhosis. The association of metabolic-syndrome, female-sex, a long-history of obesity and body mass index>45 were considered to be independent risk-factors for fibrosis. Comparing the histological findings of cases and controls we found a similar severity of steatosis and fibrosis, with a greater prevalence of ballooning degeneration and glycogenated-nuclei rather than lobular-inflammation.
Conclusions
In the subjects selected according to our criteria, liver enzyme levels could not be used as surrogate markers of non-alcoholic-steatohepatitis. Histological hallmarks of patients with metabolic-syndrome, normal liver enzymes and non-alcoholic-steatohepatitis consist to a lesser degree of lobular-inflammation and a more severe ballooning and glycogenated-nuclei.
The H1069Q mutation in ATP7B is associated with late and neurologic presentation in Wilson disease: results of a meta-analysis
Janneke M. Stapelbroeka, Casper W. Bollenb, Johannes K. Ploos van Amstelc, Karel J. van Erpecumd, Jan van Hattumd, Leonard H. van den Berge, Leo W.J. Klompf, Roderick H.J. Houwena*
Received 19 February 2004; received in revised form 1 July 2004; accepted 16 July 2004 published online 19 August 2004.
Background and Aims
Wilson disease is an hereditary disorder of copper metabolism, caused by mutations in the ATP7B gene, and leading to hepatic or neurologic disease. We examined whether H1069Q, the most common ATP7B mutation, is associated with a specific phenotype.
Methods
Genotyping results in 70 Dutch patients were related to clinical presentation. Subsequently a meta-analysis for genotypephenotype correlation was performed on all patients available from literature, combined with the current Dutch group, a total of 577 patients.
Results
The Dutch patients homozygous or heterozygous for the H1069Q mutation presented more frequently with neurologic disease (63% and 43% vs. 15%), and at a later age (20.9 and 15.9 vs. 12.6 years) than patients without the H1069Q mutation. In the meta-analysis the odds-ratio for neurologic presentation in homozygous or heterozygous H1069Q vs. non-H1069Q patients was 3.50 (95% CI 2.016.09) and 2.13 (95% CI 1.183.83), respectively. Age at presentation was 21.1, 19.2 and 16.5 years, respectively, corresponding to a weighted mean difference (WMD) of 4.41 (95% CI 1.567.26) for homozygous H1069Q vs. heterozygous patients and 6.68 (95% CI 4.339.38) for homozygous H1069Q vs. non-H1069Q patients.
Conclusions
Our results indicate that the H1069Q mutation is associated with a late and neurologic presentation.
Heme oxygenase-1 protects human hepatocytes in vitro against warm and cold hypoxia
Eda Tüzünera†, Liegang Liua, Masashi Shimadaa, Eser Yilmazb, Matthias Glanemanna, Utz Settmachera, Jan M. Langrehra, Sven Jonasa, Peter Neuhausa, Andreas K. Nusslera*
Background/Aims
Hepatic injury induced by ischemia/reperfusion following surgery, transplantation, or circulatory shock combined with resuscitation is a major clinical problem.
Methods
In this study, hypoxic and inflammatory conditions were mimicked by exposing human hepatocytes to N2 (at 4 and 37°C) or to cytokines/endotoxin to investigate the potential protective effects of heme oxygenase-1 (HO-1). Incubation of human hepatocytes with single cytokines (IFN-?, IL-1?, TNF-?) or LPS, as well as a combination of all four stimuli (CM, cytomix) caused a time-dependent HO-1 mRNA expression over 12h and a decline by 24h. In parallel, we observed a time-dependent membrane leakage for LDH and AST and a maximum HO-1 protein expression between 324h.
Results
Warm and cold hypoxia showed similar results in HO-1 mRNA and protein expression and the release of LDH and AST. CoPP, a potent HO-1 inducer, and bilirubin, a co-product of the HO-pathway, protected human hepatocytes from warm and cold hypoxia. HO-1 enzyme activity was highest during warm hypoxia, followed by cold hypoxia and CM which was confirmed by intracellular Fe2+ formation.
Conclusions
Taken together, we demonstrated, that HO-1 induction protected human hepatocytes against warm and cold hypoxia. Our results also suggest that HO-1 induction may have therapeutic potential against inflammatory insults.
Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins
Reto Gulera, Maria L. Ollerosa, Dominique Vesina, Roumen Parapanova, Christian Vesina, Salomé Kantengwaa, Laura Rubbia-Brandta, Noury Mensib, Anne Angelillo-Scherrerc, Eduardo Martinez-Soriaa, Fabienne Tacchini-Cottierd, Irene Garciaa*
Received 11 June 2004; received in revised form 19 July 2004; accepted 22 July 2004 published online 19 August 2004.
Background/Aims
Bacillus Calmette Guérin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which render mice highly sensitive to endotoxin-mediated hepatotoxicity. This work investigates the role of inducible nitric oxide synthase (iNOS) in liver damage induced by BCG and endotoxins in BCG-infected mice.
Methods
Liver injury and cytokine activation induced by BCG and by LPS upon BCG infection (BCG/LPS) were compared in wild-type and iNOS?/? mice.
Results
iNOS?/? mice infected with living BCG are protected from hepatic injury when compared to wild-type mice which express iNOS protein in macrophages forming hepatic granulomas. In addition, iNOS?/? mice show a decrease in BCG-induced IFN-? serum levels. LPS challenge in BCG-infected mice strongly activates iNOS in the liver and spleen of wild-type mice which show important liver damage associated with a dramatic increase in TNF and IL-6 and also Th1 type cytokines. In contrast, iNOS?/? mice are protected from liver injury after BCG/LPS challenge and their TNF, IL-6 and Th1 type cytokine serum levels raise moderately.
Conclusions
These results demonstrate that nitric oxide (NO) from iNOS is involved in hepatotoxicity induced by both mycobacterial infection and endotoxin effects upon BCG infection and that inhibition of NO from iNOS protects from liver injuries.
Interferon-?Con1 suppresses proliferation of liver cancer cell lines in vitro and in vivo
Toru Hisaka, Hirohisa Yano*, Sachiko Ogasawara, Seiya Momosaki, Naoyo Nishida, Yumi Takemoto, Sakiko Kojiro, Yuno Katafuchi, Masamichi Kojiro
Background/Aims
We investigated the effects of consensus interferon (IFN-?Con1), a nonnaturally occurring type I interferon with higher specific activity than other type I IFNs, on the growth of human liver cancer cells.
Methods
The effect of IFN-?Con1 on the proliferation of 13 liver cancer cell lines was investigated in vitro. Hepatocellular carcinoma (HCC) cells (KIM-1 and HAK-1B) were transplanted subcutaneously into the back of nude mice, then IFN-?Con1 was subcutaneously administered to the mice once a day for 2 weeks, and tumor volume and histology were examined.
Results
IFN-?Con1 expressed a dose-dependent growth inhibitory effect in all cell lines in vitro. KIM-1 tumor volume in mice that received 0.01?g (104IU)/mouse/day of IFN-?Con1 (similar to the clinical dose for chronic hepatitis C) was 62% of the control, 0.1?g/mouse/day resulted in 26%, and 1?g/mouse/day resulted in 10%. HAK-1B tumor volume under the same treatment was 61, 24 and 0% of the control, respectively. The number of apoptotic cells significantly increased and the number of blood vessels significantly decreased with the increase in IFN-?Con1 dose.
Conclusions
IFN-?Con1 suppressed HCC growth in nude mice. These data indicate the potential clinical application of IFN-?Con1 in the prevention and treatment of HCC.
Suppression of C/EBP ? expression in biliary cell differentiation from hepatoblasts during mouse liver development
Nobuyoshi Shiojiri*, Kentaro Takeshita, Harufumi Yamasaki, Takeyuki Iwata
Background/Aims
Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent mesenchyme, which leads to the suppression of mature hepatocyte marker expression. This study was undertaken to analyze C/EBP ? and ? expression, which may govern transcription of mature hepatocyte marker genes, during mouse liver development with special attention given to biliary differentiation.
Methods
Expression of C/EBP ? and ? was immunohistochemically examined. Expression of ?-fetoprotein, albumin and urea cycle enzymes, the genes of which have CCAAT motifs in their upstream regulatory sequences, was examined immunohistochemically or by using in situ hybridization.
Results
C/EBP ? started to be expressed in endodermal cells of 9.5-day liver primordium, and continued to be expressed in hepatoblasts and hepatocytes throughout development. Although biliary cell progenitors transiently expressed mature hepatocyte markers, their expression of C/EBP ? was weak or totally absent. The signals of C/EBP ? in hepatocytes were weak in fetal liver, but became stronger with postnatal development. Differentiated epithelial cells of intrahepatic biliary structures did not express C/EBP ?.
Conclusions
These data suggest that the suppression of C/EBP ? expression may be prerequisite to biliary cell differentiation in the hepatoblast population and one of its earliest signs.
Peripheral benzodiazepine receptor ligands induce apoptosis and cell cycle arrest in human hepatocellular carcinoma cells and enhance chemosensitivity to paclitaxel, docetaxel, doxorubicin and the Bcl-2 inhibitor HA14-1
Andreas P. Suttera, Kerstin Maasera, Patricia Grabowskia, Gesine Bradacsa, Kirsten Vormbrocka, Michael Höpfnera, Antje Krahna, Bernhard Heineb, Harald Steinb, Rajan Somasundarama, Detlef Schuppanc, Martin Zeitza, Hans Scherübla*
Background/Aims
Hepatocellular carcinoma (HCC) is one of the most common causes of cancer deaths worldwide. Thus, novel therapies are urgently needed. A promising approach is the use of peripheral benzodiazepine receptor (PBR) ligands which inhibit the proliferation of various tumors.
Methods
PBR expression both in human HCC cell lines and in tumor specimens of HCC patients was analyzed by RT-PCR and immunostaining. To evaluate PBR ligands for the treatment of HCC, we tested their effects on human HCC cells.
Results
PBR was localized to the mitochondria both of HCC cell lines and tumor tissues of HCC patients. In contrast, normal liver did not express PBR. PBR ligands inhibited the proliferation of HCC cell lines by inducing apoptosis and cell cycle arrest. Apoptosis was characterized by a breakdown of the mitochondrial membrane potential, caspase-3 activation and nuclear degradation. Furthermore, pro-apoptotic Bax was overexpressed while anti-apoptotic Bcl-2 and Bcl-XL were suppressed. Cell cycle was arrested both at the G1/S- and G2/M-checkpoints. Synergistic anti-neoplastic effects were obtained by a combination of PBR ligands with cytostatic drugs (paclitaxel, docetaxel, doxorubicin), or with an experimental Bcl-2 inhibitor.
Conclusions
This is the first report on the induction of apoptosis and cell cycle arrest by PBR ligands in HCC cells. Moreover, PBR ligands sensitized HCC cells to taxans and doxorubicin.
Enhanced epidermal growth factor receptor activation in human cholangiocarcinoma cells
Jung-Hwan Yoona, Geum-Youn Gwaka, Hyo-Suk Leea, Steven F. Bronkb, Nathan W. Werneburgb, Gregory J. Goresb*
Background/Aims
Epidermal growth factor receptor (EGFR) signaling has been implicated in the genesis and progression of cholangiocarcinoma. However, the characteristics of EGFR signaling in cholangiocarcinoma cells have not been characterized. Thus, we attempted to more fully characterize EGF/EGFR signaling in human cholangiocarcinoma cells.
Methods
EGFR phosphorylation and ubiquitination were evaluated using immunoblot techniques. EGFR internalization was analyzed by immunofluorescent staining of EGFR or by immunoblot analysis for biotinylated EGFR. Cell growth was assessed using the MTS assay.
Results
EGFR activation was sustained following EGF stimulation in cholangiocarcinoma cells as compared to hepatoma cells. This prolonged EGFR activation resulted in extended p42/44 MAPK activation in cholangiocarcinoma cells. Despite ubiquitination, EGFR activation-dependent internalization was defective in cholangiocarcinoma cells. Cell growth was increased in cholangiocarcinoma cells following EGF stimulation as compared to hepatoma cells, and this was significantly attenuated by EGFR kinase inhibitors. The EGFR kinase inhibitors also significantly decreased COX-2 expression in cholangiocarcinoma cells, while this was not evident in hepatoma cells.
Conclusions
The results demonstrate that cholangiocarcinoma cells exhibit sustained EGFR activation due to defective receptor internalization. As EGFR kinase inhibitors effectively attenuated cellular growth, these agents may be therapeutically efficacious in human cholangiocarcinoma.
Vitamin E down-modulates iNOS and NADPH oxidase in c-Myc/TGF-? transgenic mouse model of liver cancer
Diego F. Calvisi, Sara Ladu, Koji Hironaka, Valentina M. Factor, Snorri S. Thorgeirsson*
Background/Aims
Co-expression of c-Myc and TGF-? in the mouse liver accelerates hepatocarcinogenesis and enhances DNA damage due to chronic oxidative stress. Dietary supplementation with vitamin E (VE) inhibits hepatocarcinogenesis and reduces chromosomal alterations in the same mice. Here we investigated the sources of reactive oxygen species (ROS) production in c-Myc/TGF-? transgenic mice.
Methods
Inducible nitric oxide synthase (iNOS) and NADPH oxidase levels were determined in c-Myc, TGF-? and c-Myc/TGF-? mice by RT-PCR, western blot analysis and immunohistochemistry.
Results
iNOS and nitrotyrosines levels were higher in the three transgenic lines when compared with wild-type mice. Preneoplastic and neoplastic lesions from c-Myc, TGF-? and c-Myc/TGF-? transgenic mice displayed upregulation of NADPH oxidase subunits p47-, 67-phox, Rac1, HSP 70, and HO-1. Importantly, dietary supplementation with vitamin E abolished iNOS expression, lowered nitrotyrosines, p47-, p67-phox, and Rac1 levels, and suppressed HSP 70 and HO-1 proteins in c-Myc/TGF-? livers.
Conclusions
The results suggest that iNOS and NADPH oxidase are involved in ROS generation during c-Myc/TGF-? hepatocarcinogenesis and are inhibited by VE treatment. The data provide additional evidence for the potential use of VE in treatment of chronic liver diseases and HCC prevention.
Involvement of c-Myc in growth inhibition of Hep 3B human hepatoma cells by a vitamin K analog
Lisheng Ge, Ziqiu Wang, Meifang Wang, Siddhartha Kar, Brian I. Carr*
Background/Aims
A synthetic vitamin K analog, compound 5 (Cpd 5), is a potent inhibitor of cell growth. The aim was to investigate whether c-Myc was involved in Cpd 5-induced cell growth inhibition.
Methods
Human hepotoma cells (Hep 3B) were cultured and treated with Cpd 5, and c-Myc protein expression and phosphorylation were investigated using Western blot analysis.
Results
Cpd 5 was found to inhibit c-Myc protein expression and induce c-Myc phosphorylation in Hep 3B cells. The phosphorylation of c-Myc was induced by both Cpd 5-mediated persistent extracellular signal-regulated kinase (ERK) phosphorylation and Cpd 5 increased glycogen synthase kinase-3 (GSK-3) activity. When using GSK-3 inhibitor, SB216763, c-Myc phosphorylation was significantly decreased and c-Myc levels were restored in Cpd 5 treated cells, suggesting that Cpd 5-mediated increase of GSK-3 activity enhanced c-Myc degradation and resulted in reduction of c-Myc levels. The lower c-Myc levels were found to cause altered expression of two c-Myc target genes, growth arrest gene gadd45 and ornithine decarboxylase (ODC).
Conclusions
The results suggest that Cpd 5-mediated c-Myc phosphorylation resulted in enhanced c-Myc protein degradation and reduced c-Myc protein levels, which may contribute to cell growth inhibition by Cpd 5.
Fibrosis progression after liver transplantation in patients with recurrent hepatitis C
Ulf P. Neumanna*†, Thomas Bergb†, Marcus Bahraa, Daniel Seehofera, Jan M. Langrehra, Ruth Neuhausa, Cornelia Radkec, Peter Neuhausa
See Editorial, pages 862--863
Background/Aims
Aim of our study was to analyze fibrosis progression after liver transplantation (OLT) in hepatitis C virus (HCV)-infected patients based on protocol liver biopsies and to identify risk factors, which may play a role in the development of severe fibrosis stages.
Methods
One hundred and eighty-three liver graft recipients who had a histological follow-up evaluation of 1 year after OLT were analyzed. Overall 1039 protocol liver biopsies were performed after 1-, 3-, 5-, 7- and 10 years and staged according to the Scheuer score.
Results
The fibrosis progression rate was not linear. The fibrosis scores were 1.2 after one, 1.7 after three, 1.9 after five, 2.1 after 7 and 2.2 after 10 years. The 39 recipients with fibrosis stages 3 or 4 in the 1-year biopsy had a significantly reduced survival rate, while fibrosis stage 02 indicated excellent survival. Independent risk factors for progression of fibrosis at 1 year were HCV genotype 1 and 4 (P=0.01) and donor age>33 years (P=0.01), whereas risk factors for development of cirrhosis (30/183 recipients (16%)) were donor age (P=0.002) and multiple steroid pulses (P=0.05).
Conclusions
These data provide information on the course of recurrent hepatitis C and may be helpful to individualize the treatment of transplanted patients.
Regional and transient ischemia/reperfusion injury in the liver improves therapeutic efficacy of allogeneic intraportal hepatocyte transplantation in low-density lipoprotein receptor deficient Watanabe rabbits
Masoumeh Attarana†, Andrea Schneidera†, Christiane Grotea, Caroline Zwiensa, Peer Flemmingb, Klaus F. Gratzc, Andrea Jochheima, Matthias J. Bahra, Michael P. Mannsa, Michael Otta*
Background/Aims
Hepatocyte transplantation has the potential to become an alternative to organ transplantation for the treatment of hereditary liver disease. Currently used hepatocyte transplantation techniques are often not sufficient for phenotypic correction. In a pre-clinical model we investigated the effect of regional transient ischemia reperfusion injury and repeated infusions of allogeneic hepatocytes on LDL cholesterol levels in LDL receptor deficient hyperlipidemic Watanabe rabbits.
Methods
A catheter was surgically inserted into the inferior mesenteric vein. Blood supply to the right liver lobe was transiently interrupted. Nine infusions of 2.5?107 adult allogeneic hepatocytes from white New Zealand rabbits were applied over a period of 2 months.
Results
Compared to pretreatment levels LDL cholesterol decreased significantly in Watanabe rabbits with transient ischemia reperfusion injury and repeated hepatocyte transplantation (?42±3%). Repeated hepatocyte transplantation without transient ischemia reperfusion injury decreased LDL cholesterol levels only moderately (?11±4%). LDL receptor messenger RNA and proteins were detected in hepatocyte transplanted liver but not in the liver of sham treated animals.
Conclusions
Our data indicate that transient ischemia reperfusion injury of the recipient liver is safe and significantly improves the therapeutic efficacy of allogeneic hepatocyte transplantation in hyperlipidemic rabbits with congenital LDL receptor deficiency.
Hepatitis C virus infection affects the brainevidence from psychometric studies and magnetic resonance spectroscopy
Karin Weissenborna*, Jochen Krausea, Martin Bokemeyerab, Hartmut Heckerc, Andreas Schülerd, Jochen C. Ennena, Björn Ahla, Michael P. Mannsd, Klaus W. Bökerd
Received 26 January 2004; received in revised form 19 July 2004; accepted 22 July 2004 published online 19 August 2004.
Background/Aims
Up to 50% of patients infected with the hepatitis C virus (HCV) complain of chronic fatigue and difficulties in concentration and memory. The aim of the present study was to seek evidence for the presence of central nervous system involvement in HCV infected patients with only mild liver disease.
Methods
Thirty HCV infected patients with normal liver function, 15 of whom were identified as having mild and 15 moderate to severe fatigue using the fatigue impact scale, underwent neurological and neuropsychological examination, electroencephalography (EEG) and cerebral proton magnetic resonance imaging (MRI) and spectroscopy (MRS). Fifteen healthy volunteers, matched for age and educational attainment, served as controls.
Results
In comparison to the healthy controls the patients with HCV infection showed evidence of cognitive impairment, primarily attention and higher executive functions, higher levels of anxiety and depression and impairment of quality of life. In addition they showed a significant decrease of the N-acetyl-aspartate/creatine ratio in the cerebral cortex on 1H MRS while the EEG was slowed in 25%. In general the deficits were more marked in the patients with moderate rather than mild fatigue.
Conclusions
The data provide evidence of central nervous system involvement in patients with HCV infection.
Safety and efficacy of alamifovir in patients with chronic hepatitis B virus infection
Danny K.W. Soona, Stephen L. Lowea, Choo Hua Tenga, Kwee Poo Yeoa, James McGillb, Stephen D. Wisea*
Background/Aims
Alamifovir is a purine nucleotide analogue prodrug that shows potent activity against wild type and lamivudine resistant hepatitis B virus in preclinical studies. The aim of this study was to assess the safety and potential antiviral effects of alamifovir in humans.
Methods
A randomised, placebo controlled, dose escalation study of oral alamifovir was conducted in 66 chronic hepatitis B infected patients who were selected based on stable HBV DNA (>105copies/ml), with no significant liver pathology. They received either placebo or alamifovir at a total daily dose ranging from 2.5 to 20mg in single or divided doses for 28 days and were followed up for approximately 12 weeks after cessation of treatment.
Results
All doses showed significant antiviral activity, with mean plasma viral load reductions ranging from 1.5 to 2.6 log10 after 28 days of dosing. Once and twice daily regimen for the same daily dose (5mg BID vs 10mg QD, 10mg BID vs 20mg QD) showed no apparent difference in the rate and extent of viral decline, or viral reduction at day 28. Post-treatment viral suppression was dose dependent. There were no serious adverse events attributable to study drug, nor were significant dose related events identified.
Conclusions
Alamifovir has shown potent in vivo anti-HBV activity, with a favourable safety profile.
Copyright © 2001-2004 European Association for the Study of the Liver. All rights reserved.
Copyright © 2004 BMJ Publishing Group Ltd
The New England Journal of Medicine is owned, published, and copyrighted © 2004 Massachusetts Medical Society. All rights reserved.
Volume 364, Number 9441 02 October 2004
The Lancet, published, and copyrighted © 2004. All rights reserved.
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