Volume 39, Issue 3 (March 2004)
Perspectives in Clinical Hepatology
Recombinant activated factor VII (rFVIIa) as a hemostatic agent in liver disease: A break from convention in need of controlled trials (p 592-598)
Stephen H. Caldwell, Charissa Chang, B. Gail Macik
The management of coagulopathy in patients with acute and chronic liver disease has undergone little change in many years despite advances in our understanding of the pathogenesis of this problem. In general, deficiency of clotting factors as a result of poor hepatic synthetic function accounts for most of the coagulopathy. However, other processes such as disseminated intravascular coagulation (DIC), hyperfibrinolysis, dysfibrinogenemia, hemolysis, and a decrease in number or function of platelets may be present and thus add to the complexity of the problem. Coexisting portal hypertension and the associated risks of volume expansion, renal failure, and endothelial dysfunction add even more difficulty to the management of these patients. The clinician's despair is only exacerbated by uncertainty regarding the significance of laboratory indices of coagulation and the lack of agreement between health care providers regarding how to use these indices. Simple, conventional interventions such as vitamin K or plasma administration often produce only limited amelioration, and the latter carries the potential disadvantage of volume overexpansion as well as the risk of infection and transfusion reactions. Into this complex and uncertain clinical situation has arrived the antihemophilic agent recombinant activated factor VII (rFVIIa). Its development has led to a fundamental re-evaluation of the classic understanding of the normal clotting cascade. Moreover, use of this product in liver disease patients is increasing despite the lack of definitive studies or literature to guide therapy. Herein we review the mechanism of action of this agent, report the clinical applications in patients with liver disease, address the limitations and risks associated with the drug, and discuss the issue of its cost-effectiveness. (HEPATOLOGY 2004;39:592-598) 
Liver Biology & Pathobiology
Dietary n-3 polyunsaturated fatty acids decrease hepatic triglycerides in Fischer 344 rats (p 608-616)
James R. Levy, John N. Clore, Wayne Stevens
Dietary fatty acid composition modifies hepatic lipid metabolism. To determine the effects of fatty acids on hepatic triglyceride storage, rats were fed diets enriched in carbohydrates (control), fish oil, or lard. After 4 weeks, the animals were fasted overnight. In the morning, the animals were either sacrificed or fed 8 g of their respective diets before sacrifice. Animals ingested more food calories with diets containing fish oil than with other diets. However, fish oil-fed animals weighed less and had less body fat. In fish oil-fed animals, liver triglyceride was lower by 27% ( P< .05) and 73% ( P< .01) than in control- and lard-fed animals, respectively. Fish oil altered the postprandial gene expression of hepatic regulators of fatty acid degradation and synthesis. Fish oil feeding blunted the normal postprandial decline in fatty acid degradation genes (PPAR , CPT1, and ACO) and blunted the normal postprandial rise in triglyceride synthesis genes (SREBP1-c, FAS, SCD-1). Therefore, the direct postprandial effect of fish oil ingestion decreases the propensity for hepatic triglyceride storage. In conclusion, n-3 polyunsaturated fatty acids decrease total body weight, total body fat, and hepatic steatosis. (HEPATOLOGY 2004;39:608-616.)
Gene expression pattern in hepatic stem/progenitor cells during rat fetal development using complementary DNA microarrays (p 617-627)
Petko M. Petkov, Jiri Zavadil, David Goetz, Tearina Chu, Robert Carver, Charles E. Rogler, Erwin P. Bottinger, David A. Shafritz, Mariana D. Dabeva
To identify new and differentially expressed genes in rat fetal liver epithelial stem/progenitor cells during their proliferation, lineage commitment, and differentiation, we used a high throughput method - mouse complementary DNA (cDNA) microarrays - for analysis of gene expression. The gene expression pattern of rat hepatic cells was studied during their differentiation in vivo : from embryonic day (ED) 13 until adulthood. The differentially regulated genes were grouped into two clusters: a cluster of up-regulated genes comprised of 281 clones and a cluster of down-regulated genes comprised of 230 members. The expression of the latter increased abruptly between ED 16 and ED 17. Many of the overexpressed genes from the first cluster fall into distinct, differentially expressed functional groups: genes related to development, morphogenesis, and differentiation; calcium- and phospholipid-binding proteins and signal transducers; and cell adhesion, migration, and matrix proteins. Several other functional groups of genes that are initially down-regulated, then increase during development, also emerged: genes related to inflammation, blood coagulation, detoxification, serum proteins, amino acids, lipids, and carbohydrate metabolism. Twenty-eight genes overexpressed in fetal liver that were not detected in adult liver are suggested as potential markers for identification of liver progenitor cells. In conclusion, our data show that the gene expression program of fetal hepatoblasts differs profoundly from that of adult hepatocytes and that it is regulated in a specific manner with a major switch at ED 16 to 17, marking a dramatic change in the gene expression program during the transition of fetal liver progenitor cells from an undifferentiated to a differentiated state.
Immortalized p19 ARF null hepatocytes restore liver injury and generate hepatic progenitors after transplantation (p 628-634)
Mario Mikula, Eva Fuchs, Heidemarie Huber, Hartmut Beug, Rolf Schulte-Hermann, Wolfgang Mikulits
Primary hepatocytes are blocked in mitotic activity and well-defined culture conditions only allow the limited expansion of these cells. Various genetic modifications have therefore been employed to establish immortalized hepatic cell lines, but, unfortunately, proper hepatocyte cultures conducting a faithful hepatic gene expression program and lacking malignancy are hardly available. Here we report the immortalization of primary hepatocytes isolated from p19 ARF null mice using the rationale that loss of p19 ARF lowers growth-suppressive functions of p53 and bypasses cellular senescence without losing genetic stability. The established hepatocytes, called MIM, express liver-specific markers, show a nontumorigenic phenotype, and competence to undergo Fas-mediated apoptosis. Intrasplenic transplantation of GFP-expressing parental MIM cells into Fas-injured livers of SCID mice revealed liver-reconstituting activity. In the regenerated liver, MIM cells localized in small-sized clusters and showed presence in structures comparable to canals of Hering, the site of oval cells. Transplantation of MIM-Bcl-X Lcells, which are protected against apoptosis, and successive Fas-induced liver damage, enhanced donor-derived liver repopulation by showing differentiation into cholangiocytes and cells expressing markers characteristic of both fetal hepatocytes and oval cells. In conclusion, these data indicate that long-term cultivated p19 ARF null hepatocytes are capable of generating hepatic progenitor cells during liver restoration, and thus represent a highly valuable tool to study the differentiation repertoire of hepatocytes. (HEPATOLOGY 2004;39:628-634.)
Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice (p 635-644)
Koji Nakamura, Hidenori Nonaka, Hiroki Saito, Minoru Tanaka, Atsushi Miyajima
Oncostatin M (OSM) is a member of the IL-6 family of cytokines. Mice deficient in the OSM receptor (OSMR -/- ) showed impaired liver regeneration with persistent parenchymal necrosis after carbon tetrachloride (CCl 4) exposure. The recovery of liver mass from partial hepatectomy was also significantly delayed in OSMR -/- mice. In contrast to wildtype mice, CCl 4administration only marginally induced expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 genes in OSMR -/- mice, correlating with the increased gelatinase activity of matrix metalloproteinase (MMP)-9 and matrix degradation in injured livers. The activation of STAT3 and expression of immediate early genes and cyclins were decreased in OSMR -/- liver, indicating that OSM signaling is required for hepatocyte proliferation and tissue remodeling during liver regeneration. We also found that CCl 4administration in IL-6 -/- mice failed to induce OSM expression and that OSM administration in IL-6 -/- mice after CCl 4injection induced the expression of cyclin D1 and proliferating cell nuclear antigen, suggesting that OSM is a key mediator of IL-6 in liver regeneration. Consistent with these results, administration of OSM ameliorated liver injury in wildtype mice by preventing hepatocyte apoptosis as well as tissue destruction. In conclusion, OSM and its signaling pathway may provide a useful therapeutic target for liver regeneration. (HEPATOLOGY 2004;39:635-644.)
Hepatocyte growth factor induces Mcl-1 in primary human hepatocytes and inhibits CD95-mediated apoptosis via Akt (p 645-654)
Henning Schulze-Bergkamen, Dirk Brenner, Andreas Krueger, Dorothee Suess, Stefanie C. Fas, Christian R. Frey, Andreas Dax, Dorothea Zink, Peter Büchler, Martina Müller, Peter H. Krammer
CD95 (APO-1/Fas)-mediated apoptosis of hepatocytes plays a central role in the pathophysiology of various human liver diseases. Hepatocyte growth factor (HGF) was shown to exert antiapoptotic functions in rodent hepatocytes. We previously showed that primary human hepatocytes (PHH) are a valuable tool for the investigation of apoptotic processes in liver cells. In this study, we analyzed the influence of HGF on CD95-mediated apoptosis of PHH and its molecular determinants. HGF significantly inhibited CD95-mediated apoptosis of PHH as well as cleavage of caspase-8 and poly (ADP-ribose)polymerase. HGF transcriptionally induced the expression of the anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1). In contrary, HGF did not alter the expression levels of Bcl-2 or Bcl-x L. HGF activated survival pathways such as the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK and the signal transducer and activator of transcription 3 (STAT3) pathway. Notably, HGF triggered serine 727 - but not tyrosine 705 - phosphorylation of STAT3. Pretreatment of PHH with the PI3K inhibitor LY294002 as well as adenoviral transduction of dominant negative Akt1 prevented HGF-mediated Mcl-1 induction and reversed the antiapoptotic effects of HGF. In conclusion, HGF confers survival of PHH by activation of the PI3K/Akt pathway. PI3K/Akt activation by HGF results in the induction of antiapoptotic proteins such as Mcl-1. Thus, application of HGF may be a therapeutic approach to prevent CD95-mediated hepatocellular damage in human liver diseases. (HEPATOLOGY 2004;39:645-654.)
Subliminal Fas stimulation increases the hepatotoxicity of acetaminophen and bromobenzene in mice (p 655-666)
Marina Tinel, Alain Berson, Nathalie Vadrot, Véronique Descatoire, Alain Grodet, Gérard Feldmann, Jean Paul Thénot, Dominique Pessayre
The hepatotoxicity of several drugs is increased by mild viral infections. During such infections, death receptor ligands are expressed at low levels, and most parenchymal cells survive. We tested the hypothesis that subliminal death receptor stimulation may aggravate the hepatotoxicity of drugs, which are transformed by cytochrome P-450 cytochrome P-450 into glutathione-depleting reactive metabolites. Twenty-four-hour-fasted mice were pretreated with a subtoxic dose of the agonistic Jo2 anti-Fas antibody (1 g per mouse) 3 hours before acetaminophen (500 mg/kg) or 1 hour before bromobenzene (400 mg/kg) administration. Administration of Jo2 alone increased hepatic inducible nitric oxide synthase nitric oxide synthase but did not modify serum alanine aminotransferase (ALT), hepatic adenosine triphosphate (ATP), glutathione (GSH), cytochrome P-450, cytosolic cytochrome c, caspase-3 activity or hepatic morphology. However, pretreating mice with Jo2 further decreased both hepatic GSH and ATP by 40% 4 hours after acetaminophen administration, and further increased serum ALT and the area of centrilobular necrosis at 24 hours. In mice pretreated with the Jo2 antibody before bromobenzene administration, hepatic GSH 4 hours after bromobenzene administration was 51% lower than in mice treated with bromobenzene alone, and serum ALT activity at 24 hours was 47-fold higher. In conclusion, administration of a subtoxic dose of an agonistic anti-Fas antibody before acetaminophen or bromobenzene increases metabolite-mediated GSH depletion and hepatotoxicity. Subliminal death receptor stimulation may be one mechanism whereby mild viral infections can increase drug-induced toxicity. (H EPATOLOGY 2004;39:655-666.)
Flow cytometric isolation of endodermal progenitors from mouse salivary gland differentiate into hepatic and pancreatic lineages (p 667-675)
Yuichiro Hisatomi, Kenji Okumura, Kimitoshi Nakamura, Shirou Matsumoto, Ayumi Satoh, Koji Nagano, Tetsuro Yamamoto, Fumio Endo
Experimental injury is useful to induce tissue stem cells, which may exist in small numbers under normal conditions. The salivary glands originate from the endoderm and consist of acinar and ductal epithelial cells, which have exocrine function. After salivary gland duct ligation, acinar cells disappear as a result of apoptosis, and duct epithelium subsequently proliferates. In this study, we analyzed the tissue stem cells induced by salivary gland duct ligation in mice using immunohistochemistry and flow cytometry. We sorted the Sca-1 +/c-Kit +fraction from adult mice salivary glands by way of fluorescence-activated cell sorting. The sorted cells were apparently homogeneous and were designated mouse salivary gland-derived progenitors (mSGPs). mSGP cells differentiated into a hepatic lineage when cultured in matrigel. In spherical culture in the presence of glucagon-like peptide-1 (GLP-1), these cells differentiated into a pancreatic endocrine lineage. When spheroidal bodies of mSGP, 20 to 30 m in diameter, were transplanted into liver via the portal vein, the cells integrated into hepatic cords and expressed albumin and 1-antitrypsin, suggesting that they had differentiated into hepatic-type cells. Moreover, ductlike structures formed by mSGP cells also appeared, epithelial cells of which were positive for cytokeratin 19. In conclusion, fluorescence-activated cell sorting (FACS) based on histologic evidence is efficient in isolating adult tissue stem cells of the salivary gland. Tissue stem cells of endodermal origin ( e.g. , hepatic oval cells, pancreatic epithelial progenitor cells, and salivary gland progenitor cells) have similarities in their molecular markers and tissue location. Our findings suggest the existence of common tissue stem cells in endoderm-derived organs. (H EPATOLOGY 2004;39:667-675.)
P-selectin suppresses hepatic inflammation and fibrosis in mice by regulating interferon and the IL-13 decoy receptor (p 676-687)
Thomas A. Wynn, Matthias Hesse, Netanya G. Sandler, Mallika Kaviratne, Karl F. Hoffmann, Monica G. Chiaramonte, Rachael Reiman, Allen W. Cheever, Joseph P. Sypek, Margaret M. Mentink-Kane
The selectin family of cell adhesion molecules is widely thought to promote inflammatory reactions by facilitating leukocyte recruitment. However, it was unexpectedly found that mice with targeted deletion of the P-selectin gene (PsKO mice) developed unpolarized type 1/type 2 cytokine responses and severely aggravated liver pathology following infection with the type 2-promoting pathogen Schistosoma mansoni . In fact, liver fibrosis, which is dependent on interleukin 13 (IL-13), increased by a factor of more than 6, despite simultaneous induction of the antifibrotic cytokine interferon gamma (IFN- ). Inflammation, as measured by granuloma size, also increased significantly in the absence of P-selectin. When infected PsKO mice were treated with neutralizing anti-IFN- monoclonal antibodies, however, granuloma size was restored to wild-type levels; this finding revealed the potent proinflammatory role of IFN- when expressed concomitantly with IL-13. Untreated PsKO mice also exhibited a significant (sixfold) reduction in decoy IL-13 receptor (IL-13 receptor alpha-2) expression when compared with infected wild-type animals. It is noteworthy, however, that when decoy receptor activity was restored in PsKO mice by treatment with soluble IL-13 receptor alpha-2-Fc, the exacerbated fibrotic response was completely inhibited. Thus, reduced expression of the decoy IL-13 receptor mediated by the elevated type 1 cytokine response probably accounts for the enhanced activity of IL-13 in PsKO mice and for the resultant increase in collagen deposition. In conclusion, the current study has revealed the critical role of P-selectin in the progression of chronic liver disease caused by schistosome parasites. By suppressing IFN- and up-regulating the decoy IL-13 receptor, P-selectin dramatically inhibits the pathologic tissue remodeling that results from chronic type 2 cytokine-mediated inflammation. (HEPATOLOGY 2004;39:676-687.)
Ischemic preconditioning affects interleukin release in fatty livers of rats undergoing ischemia/reperfusion (p 688-698)
Anna Serafín, Joan Roselló-Catafau, Neus Prats, Emilio Gelpí, Joan Rodés, Carmen Peralta
The present study evaluates the effect of ischemic preconditioning on interleukin-1 (IL-1) and interleukin-10 (IL-10) generation following hepatic ischemia/reperfusion (I/R) in normal and steatotic livers as well as the role of nitric oxide (NO) in this process. Increased IL-1 and IL-10 levels were observed in normal livers after I/R. Steatotic livers showed higher IL-1 levels than normal livers, and IL-10 at control levels. The injurious role of IL-1 and the benefits of IL-10 on hepatic I/R injury was shown with the use of IL-1 receptor antagonist (IL-1ra), anti-IL-10 polyclonal antibody against IL-10 (anti-IL-10) and exogenous IL-10. The effective dose of these treatments was different in both types of livers. Preconditioning prevented IL-1 release and increased IL-10 generation after I/R in normal and steatotic livers. IL-1 or anti-IL-10 pretreatments reversed the benefits of preconditioning. IL-1 action inhibition in a preconditioned group that was pretreated with anti-IL-10 did not modify the benefits of preconditioning. In addition, anti-IL-10 pretreatment in the preconditioned group resulted in IL-1 levels comparable to those observed after I/R. NO inhibition eliminated the benefits of preconditioning on IL-10 release, IL-1 levels, and hepatic injury. In conclusion, preconditioning, through IL-10 overproduction, inhibits IL-1 release and the ensuing hepatic I/R injury in normal and steatotic livers. IL-10 generation induced by preconditioning could be mediated by NO. (HEPATOLOGY 2004;39:688-698.)
Interleukin 18 causes hepatic ischemia/reperfusion injury by suppressing anti-inflammatory cytokine expression in mice (p 699-710)
Dan Takeuchi, Hiroyuki Yoshidome, Atsushi Kato, Hiroshi Ito, Fumio Kimura, Hiroaki Shimizu, Masayuki Ohtsuka, Yasuhiro Morita, Masaru Miyazaki
Hepatic ischemia/reperfusion injury is a clinically important problem. While the mechanisms of the initial event and subsequent neutrophil-dependent injury are somewhat understood, little is known about the regulation of endogenous hepatoprotective effects on this injury. Interleukin 12 (IL-12) plays a role in the induction of this injury, but involvement of interleukin 18 (IL-18) has not been clarified. Using a murine model of partial hepatic ischemia and subsequent reperfusion, the aim of the current study was to determine whether IL-18 is up-regulated during hepatic ischemia/reperfusion and to determine the role of endogenous IL-18 in the development and regulation of inflammatory hepatic ischemia/reperfusion injury. Hepatic IL-18 expression was up-regulated from 1 to 8 hours after reperfusion. Hepatic ischemia/reperfusion induced nuclear factor- B (NF- B) and activator protein 1 (AP-1) activation, as defined by electrophoretic mobility shift assay, and caused significant increases in liver neutrophil recruitment, apoptosis, hepatocellular injury, and liver edema as defined by liver myeloperoxidase content, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling (TUNEL) staining, serum aminotransferase levels, and liver wet-to-dry weight ratios. In mice treated with neutralizing antibody to IL-18, ischemia/reperfusion-induced increases in CXC chemokine expression, activation of NF- B and AP-1, and apoptosis were greatly reduced. Furthermore, under blockade of IL-18, anti-inflammatory cytokines such as IL-4 and IL-10 were greatly up-regulated. Signal transducer and activator of transcription 6 (STAT6) was significantly activated under blockade of IL-18. These conditions also caused significant reduction in liver neutrophil sequestration and liver injury. In conclusion, the data suggest that IL-18 is required for facilitating neutrophil-dependent hepatic ischemia/reperfusion injury through suppressing anti-inflammatory cytokine expression. (HEPATOLOGY 2004;39:699-710.)
Ursodeoxycholic acid protects against secondary biliary cirrhosis in rats by preventing mitochondrial oxidative stress (p 711-720)
Gaetano Serviddio, Javier Pereda, Federico V. Pallardó, Julian Carretero, Consuelo Borras, Juan Cutrin, Gianluigi Vendemiale, Giuseppe Poli, José Viña, Juan Sastre
Ursodeoxycholic acid (UDCA) improves clinical and biochemical indices in primary biliary cirrhosis and prolongs survival free of liver transplantation. Recently, it was suggested that the cytoprotective mechanisms of UDCA may be mediated by protection against oxidative stress, which is involved in the development of cirrhosis induced by chronic cholestasis. The aims of the current study were 1) to identify the mechanisms involved in glutathione depletion, oxidative stress, and mitochondrial impairment during biliary cirrhosis induced by chronic cholestasis in rats; and 2) to determine the mechanisms associated with the protective effects of UDCA against secondary biliary cirrhosis. The findings of the current study indicate that UDCA partially prevents hepatic and mitochondrial glutathione depletion and oxidation resulting from chronic cholestasis. Impairment of biliary excretion was accompanied by decreased steady-state hepatic levels of -glutamyl cysteine synthetase and -cystathionase messenger RNAs. UDCA treatment led to up-regulation of -glutamyl cysteine synthetase in animals with secondary biliary cirrhosis and prevented the marked increases in mitochondrial peroxide production and hydroxynonenal-protein adduct production that are observed during chronic cholestasis. A population of damaged and primarily apoptotic hepatocytes characterized by dramatic decreases in mitochondrial cardiolipin levels and membrane potential as well as phosphatidylserine exposure evolves in secondary biliary cirrhosis. UDCA treatment prevents the growth of this population along with the decreases in mitochondrial cardiolipin levels and membrane potential that are induced by chronic cholestasis. In conclusion, UDCA treatment enhances the antioxidant defense mediated by glutathione; in doing so, this treatment prevents cardiolipin depletion and cell injury in animals with secondary biliary cirrhosis. (HEPATOLOGY 2004;39:711-720)
TNF -induced ras activation due to ethanol promotes hepatocyte proliferation independently of liver injury in the mouse (p 721-731)
Fuyumi Isayama, Matthias Froh, Ming Yin, Lars O. Conzelmann, Richard J. Milton, Stephen E. McKim, Michael D. Wheeler
Tumor necrosis factor (TNF ) has been shown to be both proapoptotic and mitogenic for hepatocytes and necessary for alcohol-induced liver injury. Ras, a known proto-oncogene, is very important in the regulation of cellular responses to TNF . Therefore, the purpose of this study was to investigate the role of Ras in alcohol-induced pathogenesis. Male C57Bl/6 mice were fed ethanol or high-fat control diet via intragastric cannulation for 4 weeks. Ras activity was increased significantly after 4 weeks of ethanol and correlated with an increase in pathologic features. However, in mice deficient in the receptor-type 1 for TNF (TNFR1 -/- ), ethanol-induced liver injury and the increase in Ras activity were significantly blunted compared with wild-type mice. Furthermore, it was demonstrated that H-, K-, and R-Ras isoforms were increased after ethanol exposure in wild-type mice. In TNFR1 -/- mice, R-Ras activity remained elevated by ethanol, whereas H-Ras and K-Ras activity was blunted significantly under these conditions. Interestingly, hepatocellular proliferation, which was elevated approximately fivefold after 4 weeks of chronic ethanol in wild-type mice, was also blunted in TNFR1 -/- mice given ethanol. Inhibition of Ras with adenovirus containing a dominant-negative Ras had no effect on ethanol-induced liver injury, but significantly blunted ethanol-induced hepatocyte proliferation by more than 50%. Overexpression of mitochondrial superoxide dismutase using recombinant adenovirus blunted lipid peroxidation and attenuated hepatic injury resulting from ethanol, but had no effect on Ras activation and hepatocyte proliferation caused by ethanol. In conclusion, these data support the hypotheses that hepatocellular oxidative stress leads to cell death and that TNF -induced Ras activation is important in hepatic proliferation in response to ethanol-induced liver injury. (HEPATOLOGY 2004;39:721-731.)
Oxysterols induce cyclooxygenase-2 expression in cholangiocytes: Implications for biliary tract carcinogenesis (p 732-738)
Jung-Hwan Yoon, Ali E. Canbay, Nathan W. Werneburg, Sum P. Lee, Gregory J. Gores
Cyclooxygenase-2 (COX-2), which is expressed by cholangiocytes in biliary tract disorders, has recently been implicated in biliary tract carcinogenesis. The mechanisms responsible for this COX-2 expression remain unclear. In human diseases, bile contains oxygenated derivatives of cholesterol (oxysterols) which possess diverse biological properties. Therefore, we determined if oxysterols modulate COX-2 expression. The effect of an oxysterol (22(R)-hydroxycholesterol, 22-HC) on COX-2 expression in KMBC cells, a human cholangiocarcinoma cell line, was examined. 22-HC enhanced COX-2 protein expression. This oxysterol activated p42/44 and p38 MAPK, but not JNK 1/2. A p42/44 MAPK inhibitor did not block COX-2 induction, while p38 MAPK inhibitor effectively attenuated COX-2 induction. Although COX-2 mRNA levels were increased by 22-HC, this increase was not transcriptionally regulated, as 22-OH did not increase activity in a COX-2 promoter gene assay. In contrast, COX-2 mRNA stability was augmented by 22-HC treatment, and this effect was reversed by a p38 MAPK inhibitor. In conclusion, the results demonstrate that the oxysterol 22-HC stabilizes COX-2 mRNA via a p38 MAPK-dependent mechanism. This enhanced COX-2 protein expression by oxysterols may participate in the genesis and progression of cholangiocarcinoma. (HEPATOLOGY 2004;39:732-738.)
Liver Failure & Liver Disease
Minimal hepatic encephalopathy impairs fitness to drive (p 739-745)
Christian Wein, Horst Koch, Birthe Popp, Gerd Oehler, Peter Schauder
It has been suggested that the ability to drive a car is impaired in patients with cirrhosis of the liver and minimal hepatic encephalopathy (MHE). However, the only study using an on-road driving test did not reveal such an impairment. In a prospective controlled study, we evaluated patients with cirrhosis of the liver for MHE and the ability to drive a car. MHE was diagnosed using three psychometric tests: Number Connection Test Part A, Digit Symbol Test, and a Complex Choice Reaction Test. In a standardized on-road driving test (22 miles, 90 minutes), designed for patients with brain impairment, a professional driving instructor blind to the subjects' diagnosis and test results assessed the driving performance. Four global driving categories (car handling, adaptation to traffic situation, cautiousness, maneuvering), 17 specific driving actions ( e.g. , changing lanes, overtaking, etc.), and a total score of driving performance were rated using a 6-point scale. Of 274 consecutive patients with liver cirrhosis, 48 fulfilled the medical and driving inclusion criteria, 14 of them with and 34 without MHE. Forty-nine subjects in a stable phase of chronic gastroenterological diseases and with normal liver findings served as controls. The total driving score of patients with MHE was significantly reduced in comparison to either cirrhotic patients without MHE or to controls ( P< .05). Significant differences in ratings were found in the following driving categories: car handling, adaptation, and cautiousness. Significant differences were also found in specific driving actions. The instructor had to intervene in the driving of 5 of the 14 MHE patients to avoid an accident, significantly more than in cirrhotic patients without MHE and in controls. There was no significant difference in any driving category or specific driving action in cirrhotic patients without MHE compared to controls. In conclusion, fitness to drive a car can be impaired in patients with MHE. Therefore, patients with liver cirrhosis should be tested for MHE and informed in the case of abnormal test results. Therapy known to improve psychometric test results should be initiated. (HEPATOLOGY 2004;39:739 -745.)
Antibiotic prophylaxis after endoscopic therapy prevents rebleeding in acute variceal hemorrhage: A randomized trial (p 746-753)
Ming-Chih Hou, Han-Chieh Lin, Tsu-Te Liu, Benjamin Ing-Tieu Kuo, Fa-Yauh Lee, Full-Young Chang, Shou-Dong Lee
Bacterial infection may adversely affect the hemostasis of patients with gastroesophageal variceal bleeding (GEVB). Antibiotic prophylaxis can prevent bacterial infection in such patients, but its role in preventing rebleeding is unclear. Over a 25-month period, patients with acute GEVB but without evidence of bacterial infection were randomized to receive prophylactic antibiotics (ofloxacin 200 mg i.v. q12h for 2 days followed by oral ofloxacin 200 mg q12h for 5 days) or receive antibiotics only when infection became evident (on-demand group). Endoscopic therapy for the GEVB was performed immediately after infection work-up and randomization. Fifty-nine patients in the prophylactic group and 61 patients in the on-demand group were analyzed. Clinical and endoscopic characteristics of the gastroesophageal varices, time to endoscopic treatment, and period of follow-up were not different between the two groups. Antibiotic prophylaxis decreased infections (2/59 vs. 16/61; P< .002). The actuarial probability of rebleeding was higher in patients without prophylactic antibiotics ( P= .0029). The difference of rebleeding was mostly due to early rebleeding within 7 days (4/12 vs. 21/27, P= .0221). The relative hazard of rebleeding within 7 days was 5.078 (95% CI: 1.854-13.908, P< .0001). The multivariate Cox regression indicated bacterial infection (relative hazard: 3.85, 95% CI: 1.85-13.90) and association with hepatocellular carcinoma (relative hazard: 2.46, 95% CI: 1.30-4.63) as independent factors predictive of rebleeding. Blood transfusion for rebleeding was also reduced in the prophylactic group (1.40 ± 0.89 vs. 2.81 ± 2.29 units, P< .05). There was no difference in survival between the two groups. In conclusion, antibiotic prophylaxis can prevent infection and rebleeding as well as decrease the amount of blood transfused for patients with acute GEVB following endoscopic treatment. (HEPATOLOGY 2004;39:746-753.)
Body fat distribution, relative weight, and liver enzyme levels: A population-based study (p 754-763)
Saverio Stranges, Joan M. Dorn, Paola Muti, Jo L. Freudenheim, Eduardo Farinaro, Marcia Russell, Thomas H. Nochajski, Maurizio Trevisan
Regional body fat distribution may represent an independent risk factor for several conditions, especially metabolic and cardiovascular diseases; recent findings have shown that abdominal fat accumulation can be an independent predictor of hepatic steatosis. Very few studies, mostly using selected clinical samples, have focused on the relationship between indices of abdominal visceral fat accumulation and the most commonly used biochemical liver tests, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyltransferase (GGT). The aim of the present study was to evaluate the relation between central fat accumulation, as assessed by abdominal height, relative weight, as determined by body mass index (BMI), and liver function tests (ALT, AST, and GGT) in a random sample of 2,704 residents of Erie and Niagara Counties in New York State, 35-80 years of age and free from known hepatic disease. Multiple linear regression models were used, with liver enzymes as dependent variables with abdominal height and BMI as independent variables, and the inclusion of several covariates (age, race, education, smoking status, pack-years of smoking, drinking status, and total ounces of ethanol in the past 30 days). Abdominal height was consistently a better correlate of ALT and GGT levels than BMI in both sexes. In addition, abdominal height was the most powerful independent predictor of ALT in both sexes as well as of GGT among women. In conclusion, these findings support a role for central adiposity independent from BMI in predicting increased levels of hepatic enzymes, likely as a result of unrecognized fatty liver. (HEPATOLOGY 2004;39:754-763.)
MELD score as a predictor of pretransplant and posttransplant survival in OPTN/UNOS status 1 patients (p 764-769)
Walter K. Kremers, Marrije van IJperen, W. Ray Kim, Richard B. Freeman, Ann M. Harper, Patrick S. Kamath, Russell H. Wiesner
The Model for End-Stage Liver Disease (MELD) score is predictive of survival and is used to prioritize patients with chronic liver disease patients for orthotopic liver transplantation (OLT). The aims of this study are (1) to assess the ability of MELD score at listing to predict pretransplant and posttransplant survival for nonchronic liver disease patients listed with the Organ Procurement and Transplantation Network/ United Network for Organ Sharing (OPTN/UNOS) as Status 1; and (2) to compare survival associated with 4 diagnostic groups within the Status 1 designation. The study population consisted of adult patients listed for OLT at Status 1 in the UNOS national database between November 1, 1999 and March 14, 2002 (N = 720). Events within 30 days of listing were analyzed using Kaplan-Meier and Cox regression methodology. Patients meeting criteria for fulminant hepatic failure without acetaminophen toxicity (FHF-NA, n = 312) had the poorest survival probability while awaiting OLT; this was negatively correlated with MELD score ( P= .0001). These patients experienced the greatest survival benefit associated with OLT, with an estimated improvement of survival from about 58% to 91% ( P< .0001). Patients listed for primary nonfunction within 7 days of OLT (n = 268) did not show mortality to be related to MELD score ( P= .41) and did not show a significant association between survival and OLT ( P= .68). In conclusion, liver allocation within the Status 1 designation may need to be further stratified by diagnosis, and MELD score may be useful for prioritizing FHF-NA candidates. (HEPATOLOGY 2004;39:764-769.)
Ursodeoxycholic acid for treatment of nonalcoholic steatohepatitis: Results of a randomized trial (p 770-778)
Keith D. Lindor, Kris V. Kowdley, E. Jenny Heathcote, M. Edwyn Harrison, Roberta Jorgensen, Paul Angulo, James F. Lymp, Lawrence Burgart, Patrick Colin
No effective medical therapy is available for all patients with nonalcoholic steatohepatitis (NASH). Ursodeoxycholic acid (UDCA) has been suggested to be of benefit based on open label clinical studies. We randomized 166 patients with liver biopsy-proven NASH to receive between 13 and 15 mg/kg/d of UDCA or placebo for 2 years. End points included changes in liver test results and liver histology at 2 years of therapy. The treatment groups were comparable at entry with regard to age, gender, risk factors for NASH, serum liver biochemistries, and baseline liver histology. A total of 126 patients completed 2 years of therapy. Pre- and posttreatment liver biopsies were available in 107 patients for review at the end of the study. UDCA was well tolerated and body weight was stable during the study duration. Serum liver biochemistries were stable or improved in both the UDCA and placebo-treated groups. Changes in the degree of steatosis, necroinflammation, or fibrosis that occurred with therapy were not significantly different between the UDCA and placebo groups. In conclusion, 2 years of therapy with UDCA at a dose of 13 to 15 mg/kg/d, although safe and well tolerated, is not better than placebo for patients with NASH. (HEPATOLOGY 2004;39:770-778.)
BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis (p 779-791)
Christiane Pauli-Magnus, Reinhold Kerb, Karin Fattinger, Thomas Lang, Birgit Anwald, Gerd A. Kullak-Ublick, Ulrich Beuers, Peter J. Meier
Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are characterized by a cholestatic pattern of liver damage, also observed in hereditary or acquired dysfunction of the canalicular membrane transporters bile salt export pump (BSEP, ABCB11 ) and multidrug resistance protein type 3 (MDR3, ABCB4 ). Controversy exists whether a genetically determined dysfunction of BSEP and MDR3 plays a pathogenic role in PBC and PSC. Therefore, 149 healthy Caucasian control individuals (control group) were compared to 76 PBC and 46 PSC patients with respect to genetic variations in BSEP and MDR3 . Sequencing spanned 10,000 bp including promoter and coding regions as well as 50-350 bp of flanking intronic regions. In all, 46 and 45 variants were identified in BSEP and MDR3 , respectively. No differences between the groups were detected either in the total number of variants ( BSEP : control group: 37, PBC: 37, PSC: 31; and MDR3 : control group: 35; PBC: 32, PSC: 30), or in the allele frequency of the common variable sites. Furthermore, there were no significant differences in haplotype distribution and linkage disequilibrium. In conclusion, this study provides an analysis of BSEP and MDR3 variant segregation and haplotype structure in a Caucasian population. Although an impact of rare variants on BSEP and MDR3 function cannot be ruled out, our data do not support a strong role of BSEP and MDR3 genetic variations in the pathogenesis of PBC and PSC. (HEPATOLOGY 2004;39:779-791.)
Impact of cytomorphological detection of circulating tumor cells in patients with liver cancer (p 792-797)
Giovanna Vona, Laurence Estepa, Christophe Béroud, Diane Damotte, Frédérique Capron, Bertrand Nalpas, Alexandra Mineur, Dominique Franco, Bernard Lacour, Stanislas Pol, Christian Bréchot, Patrizia Paterlini-Bréchot
The clinical impact of circulating tumor cell (CTC) detection is controversial, mainly due to drawbacks of molecular approaches applied to this field. We sought to determine if the specific identification and counting of circulating tumor cells by cytomorphologic analysis has clinical usefulness. Peripheral blood (6 mL), treated using isolation by size of epithelial tumor cells, was obtained from 44 patients with primary liver cancer (PLC) and without metastases, 30 patients with chronic active hepatitis, 39 with liver cirrhosis, and 38 healthy individuals, and followed up for a mean period of 1 year. We searched for -catenin mutations in 60 single microdissected CTCs. One patient with liver cancer developed extrahepatic metastases during follow-up. CTCs and microemboli were found in 23 of the 44 patients with liver cancer and in none of the patients with chronic active hepatitis, patients with cirrhosis, or healthy subjects. Their presence was significantly associated with tumor diffusion ( P= .0001) and portal tumor thrombosis ( P= .006). Both the presence ( P= .01) and number ( P= .02) of CTCs and microemboli were significantly associated with a shorter survival. -Catenin mutations were found in 3 of 60 CTCs, arguing against their impact on the initial step of tumor cell invasion. In conclusion, the highly sensitive and specific detection of CTCs and microemboli may have clinical implications for cancer staging and outcome prediction. We also show the feasibility of molecular studies of individual circulating tumor cells, aimed at identifying gene mutations involved in tumor invasion. (HEPATOLOGY 2004;39:792-797.)
Is fibrolamellar carcinoma different from hepatocellular carcinoma? A US population-based study (p 798-803)
Hashem B. El-Serag, Jessica A. Davila
There have been no population-based studies of the epidemiology and prognosis of patients with fibrolamellar carcinoma (FLC). We conducted a retrospective cohort study using information collected by population-based registries of the Surveillance, Epidemiology, and End Results (SEER) program. The demographic features, stage at diagnosis, and type of therapy, as well as age-adjusted incidence rates and observed and relative survival rates were compared between persons with FLC and those with hepatocellular carcinoma (HCC) diagnosed between 1986 and 1999. A multivariate Cox proportional hazards model was constructed to examine the effect of histology (FLC vs. HCC) on the risk of mortality. There were 68 microscopically confirmed cases of FLC and 7,896 cases of HCC. FLC constituted 0.85% of all cases of primary liver cancer and 13.4% of all cases below the age of 40. Compared to HCC, patients with FLC were more likely to be younger (mean age 39 vs. 65), female (51.5% vs. 26.3%), and white (85.3% vs. 56.9%). A greater proportion of case with FLC had localized disease (41.2% vs. 30.9%), or received potentially curative therapy (resection, transplantation), compared to cases with HCC. The age-adjusted incidence rate for FLC was 0.02 per 100,000; No significant differences in age-adjusted incidence rates were observed by gender or race. The 1- and 5-year observed and relative survival rates were significantly longer in patients with FLC than HCC. The 5-year relative survival rate was 31.8% (95% CI, 20.5%-43.1%) for FLC, compared with 6.8% (95% CI, 6.3 %-7.4 %) for HCC. Adjusting for differences in age, gender, race, stage of disease, receipt of resection or transplantation, and time of diagnosis, FLC was independently associated with a 46% reduction in risk of mortality within 5years compared with HCC. In conclusion, in a population-based study, we observed remarkable differences in the epidemiology and prognosis of FLC compared to HCC. (HEPATOLOGY 2004;39:798-803.)
Viral Hepatitis
Long-term follow-up of alpha-interferon treatment of patients with chronic hepatitis B (p 804-810)
Monika van Zonneveld, Pieter Honkoop, Bettina E. Hansen, Hubertus G.M. Niesters, Sarwa Darwish Murad, Robert A. de Man, Solko W. Schalm, Harry L.A. Janssen
Data on the long-term effects of interferon alfa (IFN) treatment on disease progression and mortality in patients with chronic hepatitis B (CHB) are limited. To evaluate factors that influence clinical outcome and survival, we performed a follow-up study on 165 hepatitis B e antigen (HBeAg) positive CHB patients treated with IFN between 1978 and 2002. The median IFN dose was 30 megaunits (MU)/week (range, 2-70 MU/week), and the median duration of therapy was 16 weeks (range, 1-92 weeks). Response to treatment was defined as HBeAg loss within 12 months after the end of IFN therapy. Median follow-up was 8.8 years (range, 0.3-24 years). Fifty-four patients (33%) responded to IFN treatment. Relapse (HBeAg reactivation) occurred in 7 of the 54 (13%) responders. Fifty-two percent of the responders lost hepatitis B surface antigen (HBsAg) as compared with 9% of the nonresponders ( P< .001). Liver histology showed a decreased necroinflammatory activity and less progression of fibrosis in responders. Twenty-six patients died during follow-up. Hepatocellular carcinoma (HCC) was found in 8 patients, 6 of whom were nonresponders. Of the two responders who developed HCC, one patient had relapsed after discontinuation of therapy. Multivariate analysis showed significantly improved survival (relative risk (RR) of death 0.28, 95% CI 0.10-0.78) and reduced risk of developing HCC (RR 0.084, 95% CI 0.09-0.75) in responders. In conclusion, response to IFN therapy results in a prolonged clinical remission with an increased rate of HBsAg seroconversion and improved liver histology. Our results indicate that after correction for baseline factors, response to IFN therapy increases survival and reduces the risk of developing HCC. (HEPATOLOGY 2004;39:804-810.)
Changing epidemiology of hepatitis B in a U.S. community (p 811-816)
W. Ray Kim, Joanne T. Benson, Terry M. Therneau, Heidi A. Torgerson, Barbara P. Yawn, L. Joseph Melton III
Despite a reduction in newly acquired hepatitis B virus (HBV) infections since the mid-1980s, HBV remains an important cause of liver disease in the U.S. We report the prevalence of chronic HBV infection in a U.S. community and describe demographic and clinical characteristics. The Rochester Epidemiology Project records healthcare encounters of residents of Olmsted County, Minnesota. For all cases with a potential diagnosis of hepatitis B in this database, complete medical records were reviewed to identify subjects who met the inclusion criteria, i.e. , a clinician diagnosis of chronic HBV infection and a laboratory record of positive hepatitis B surface antigen (HBsAg). There were 191 residents with chronic HBV infection in the community, consisting of 53% Asian, 29% African, 13% Caucasian, and 5% other or unknown race. The overall age- and sex-adjusted prevalence of HBV in this community was 0.15% in 2000. The race-specific prevalence was highest among Asians (2.1%), followed by African Americans (1.9%). The prevalence among Caucasians was 0.02%. Overall, 86% were born outside the U.S., 98% of whom were non-Caucasian. A total of 131 residents were tested for HBV replicative status, of whom 27% had viral replication. Of those tested for aminotransferases (n = 184), 28% had an abnormal value at least once. In a multivariable regression analysis, replicative status was the most influential (odds ratio [OR] = 5.98, P< .01) factor associated with abnormal aminotransferase values, followed by male gender (OR = 3.69) and age greater than 40 years (OR = 2.32 per decade). In conclusion, in this Midwestern community, chronic HBV infection was predominantly seen in immigrants from endemic parts of the world. (HEPATOLOGY 2004;39;811-816.)
Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes (p 817-825)
Delphine Ducoulombier, Anne-Marie Roque-Afonso, Gaëtana Di Liberto, François Penin, Rachid Kara, Yolande Richard, Elisabeth Dussaix, Cyrille Féray
Differences in the composition of the hepatitis C virus (HCV) quasispecies between plasma and blood mononuclear cells (BMC) strongly suggest that BMCs support viral replication. We examined the frequency of such compartmentalization, the cell types involved, the constraints exerted on the different variants, and the role of immunoglobulin-complexed variants. We screened the hypervariable region (HVR1) of HCV isolates from 14 HBsAg - and HIV-seronegative patients with chronic HCV infection. HCV RNA was amplified and cloned from plasma, the immunoglobulin G (IgG)-bound fraction, and total and sorted BMCs (CD19+, CD8+, CD4+, and CD14+ cells). Compartmentalization was estimated using a matrix correlation test. The ratio of nonsynonymous/synonymous substitutions (d N/d Sratio) was calculated for each compartment. HCV RNA was detected in 3/3 BMC, 11/11 CD19+, 10/11 CD14+, 4/11 CD8+ and 0/11 CD4+ cell samples. HVR1 sequences were significantly different between plasma and at least one cellular compartment in all nine cases analyzed, and between B cells (CD19+) and monocytes (CD14+) in all five available cases. IgG-bound variants were distinct from cellular variants. D N/d Sratios were similar (n = 3) or lower (n = 6) in cellular compartments compared with plasma and the IgG-bound fraction. In conclusion, HCV compartmentalization is a common phenomenon. B cells and monocytes harbor HCV variants showing a low rate of nonsynonymous mutations, a feature that might contribute to the persistence of HCV infection. (HEPATOLOGY 2004;39:817-825.)
Risks of a range of alcohol intake on hepatitis C-related fibrosis (p 826-834)
Alexander Monto, Keyur Patel, Alan Bostrom, Stephen Pianko, Paul Pockros, John G. McHutchison, Teresa L. Wright
Heavy alcohol use contributes to liver disease in the setting of chronic hepatitis C virus (HCV) infection. Whether this is true for light or moderate alcohol use has not been demonstrated. Light alcohol use has survival benefits at a population level and is practiced by most patients with chronic HCV infection. In this study, 800 patients with HCV undergoing liver biopsy at three sites had detailed alcohol histories recorded and the relationship between alcohol and hepatic fibrosis was assessed. On univariate analysis, heavy alcohol use (>50 g/day) was associated with an increase in mean fibrosis ( P= .01). Such an association could not be demonstrated for light and moderate alcohol use. For each category of alcohol intake (none, light, moderate, and heavy), a spectrum of fibrosis was observed. On multivariate analysis, age, serum alanine aminotransferase (ALT), and histological inflammation were the independent predictors of fibrosis ( P= <.0001, .0003, <.0001, respectively). In conclusion, heavy alcohol use exerts a greater effect on fibrosis than light or moderate use. There is a range of fibrosis at each level of alcohol use. Age, serum ALT, and inflammation are independently associated with fibrosis in multivariate analysis, highlighting the fact that variables other than alcohol intake predominate in the production of hepatic fibrosis. (HEPATOLOGY 2004;39:826-834.)
Unexpected host range of hepatitis C virus replicons (p 835-838)
Ralf Bartenschlager
The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known if extrahepatic tissues are also infected by the virus nor if nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and other species. We have tested this hypothesis and found that subgenomic HCV RNA can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.
Interfering with capsid formation: A practicable antiviral strategy against hepatitis B virus? (p 838-840)
Michael Kann
Chronic hepatitis B virus (HBV) infection is a major cause of liver disease. Only interferon- and the nucleosidic inhibitors of the viral polymerase, 3TC and adefovir, are approved for therapy. However, these therapies are limited by the side effects of interferon and the substantial resistance of the virus to nucleosidic inhibitors. Potent new antiviral compounds suitable for monotherapy or combination therapy are highly desired. We describe nonnucleosidic inhibitors of HBV nucleocapsid maturation that possess in vitro and in vivo antiviral activity. These inhibitors have potential for future therapeutic regimens to combat chronic HBV infection.
Save Article Copyright © 2004 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for March 2004 • Volume 126 • Number 3
Rapid Communications
Outcome of patients with obscure gastrointestinal bleeding after capsule endoscopy: Report of 100 consecutive cases
Marco Pennazio, Renato Santucci, Emanuele Rondonotti, Carla Abbiati, Gizela Beccari, Francesco P. Rossini, Roberto De Franchis
Background & Aims :Capsule endoscopy (CE) is a promising diagnostic tool for the study of patients with obscure gastrointestinal bleeding. However, the diagnostic yield of this technique has not been adequately studied. We evaluated sensitivity and specificity of CE and the outcome after CE in patients with obscure gastrointestinal bleeding. Methods :One hundred consecutive patients (all with recent negative upper and lower endoscopy; 26 with ongoing overt bleeding [group A], 31 with previous overt bleeding [group B], and 43 with guaiac-positive stools and iron-deficiency anemia [group C]) underwent CE. Results :The yield of positive findings on CE was 92.3% in group A, 12.9% in group B, and 44.2% in group C ( P< 0.0001, A vs. B, A vs. C). Angiodysplasia (29%) and Crohn’s disease (6%) were the most common diagnoses. Sensitivity, specificity, and positive and negative predictive values of CE were 88.9%, 95%, 97%, and 82.6%, respectively. CE results led to treatments resolving the bleeding in 86.9% of patients undergoing the procedure while actively bleeding. Capsule retention because of unsuspected stenosis occurred in 5 patients and required surgery, which resolved the clinical problem, in 4 patients. Conclusions :CE is an effective diagnostic tool for patients with obscure GI bleeding. The best candidates for the procedure are those with ongoing obscure-overt bleeding or with obscure-occult bleeding. If done early in the course of the workup, CE could shorten considerably the time to diagnosis, lead to definitive treatment in a relevant proportion of patients, and spare a number of alternative investigations with low diagnostic yield.
Loss of activin receptor type 2 protein expression in microsatellite unstable colon cancers
Barbara Jung, Ryan T. Doctolero, Akihiro Tajima, Alexie K. Nguyen, Temitope Keku, Robert S. Sandler, John M. Carethers
Background & Aims :Colorectal tumors manifesting high-frequency microsatellite instability (MSI-H) develop genetically as a consequence of mutations in genes harboring repetitive DNA sequences. The activin type 2 receptor ( ACVR2 ), possessing 2 polyadenine coding sequences, was identified as a mutational target, but it is not clear if expression is abrogated. Here, we analyzed MSI-H colorectal cancers for ACVR2 mutation and expression to assess if biallelic inactivation occurs. Methods :All 54 MSI-H colon cancers and 20 random microsatellite stable (MSS) tumors from a population-based cohort of 503 patients were analyzed for mutations in 2 A 8tracts (exon 3 and 10) of ACVR2 and the A 10 tract of transforming growth factor receptor 2 ( TGFBR2 ). Additionally, we sequenced exon 10 of ACVR2 in select cancers. ACVR2 expression was determined by immunohistochemistry using an antibody targeting an epitope beyond the predicted truncated protein. Results :Forty-five of 54 MSI-H cancers (83%) showed mutation (A 8to A 7) in the polyadenine tract of exon 10 compared with no MSS tumors. Of tumors with mutant ACVR2 , 62% lacked protein expression but all MSS and MSI-H tumors with wild-type ACVR2 expressed protein. We found no evidence of loss of heterozygosity at the ACVR2 locus in MSS tumors. Comparatively, 69% of MSI-H cancers had frameshift mutation in TGFBR2 .Conclusions :ACVR2 mutations are highly frequent in MSI-H colon cancers and in most cases cause loss of ACVR2 expression, indicating biallelic inactivation of the gene. Loss of activin signaling through mutation of ACVR2 , similar to observations with TGFBR2 , may be important in the genesis of MSI-H colorectal cancer.
Clinical-alimentary Tract
Heartburn severity underestimates erosive esophagitis severity in elderly patients with gastroesophageal reflux disease
David A. Johnson, M. Brian Fennerty
Background & Aims :Gastroesophageal reflux disease is common in adults of all ages, but its complications are more frequent in elderly patients. Although heartburn is the most common symptom of reflux disease, it is unclear whether the severity of heartburn reliably indicates the severity of erosive esophagitis. We therefore assessed the relationship between age, severe heartburn symptoms, and severe erosive esophagitis. Methods :This post hoc analysis of baseline data on the severity of both heartburn and erosive esophagitis pooled data from 5 prospective, randomized, controlled clinical trials that assessed the effect of proton pump inhibitors on healing of erosive esophagitis and symptom resolution. The clinical trials were conducted in 683 private and academic offices and hospital-based gastroenterology practices and involved 11,945 patients aged 18 years and older with gastroesophageal reflux disease and erosive esophagitis. Results :A progressive increase in the prevalence of severe erosive esophagitis was observed with each decade of age, ranging from 12% in patients aged <21 years to 37% in patients aged >70 years. Among patients with severe esophagitis, severe heartburn was less frequent in the older age groups: ranging from 82% of patients aged <21 years to 34% of those aged >70 years. Each of these associations was statistically significant ( P< 0.001). Conclusions :Although the prevalence of severe erosive esophagitis increases with age, the severity of heartburn is an unreliable indicator of the severity of erosive disease. More aggressive investigation and treatment may be necessary for elderly patients, regardless of the reported severity of heartburn.
Seasonal variation in flares of inflammatory bowel disease
James D. Lewis, Faten N. Aberra, Gary R. Lichtenstein, Warren B. Bilker, Colleen Brensinger, Brian L. Strom
Background & Aims :Previous research has yielded conflicting data as to whether the natural history of inflammatory bowel disease follows a seasonal pattern. The purpose of this study was to determine whether relapse of inflammatory bowel disease follows a seasonal pattern either across a cohort of patients or within individual patients. Methods :We used 1988 to 1997 data from the General Practice Research Database to conduct a retrospective cohort study of 1587 patients with Crohn’s disease (mean age at start of follow-up, 41 ± 17 years) and 2773 patients with ulcerative colitis (mean age at start of follow-up, 48 ± 16 years). Flares of disease were identified by receipt of a new prescription for either corticosteroids or 5-ASA medications following an interval of at least 4 months without prescriptions for either class of medication. Logistic regression was used to adjust the association of season of the year and flare of disease for potential confounding variables. Results :There was no association between season of the year and flare of Crohn’s disease ( P= 0.66). Season of the year was only weakly associated with flares of ulcerative colitis ( P= 0.02). Compared with winter, spring had very slightly higher rates of flares (OR = 1.13, 95% CI: 1.051.23). We did not observe seasonal patterns within individual patients experiencing multiple flares ( P> 0.05 for Crohn’s disease and ulcerative colitis). Conclusions :Although we observed a slight increase in exacerbations of ulcerative colitis in the spring, in general, these data do not support an association between season of the year and flares.
Induction and regulation of Smad7 in the gastric mucosa of patients with Helicobacter pylori infection
Giovanni Monteleone, Giovanna Del Vecchio Blanco, Giampiero Palmieri, Piero Vavassori, Ivan Monteleone, Alfredo Colantoni, Serena Battista, Luigi Giusto Spagnoli, Marco Romano, Melissa Borrelli, Thomas T. MacDonald, Francesco Pallone
Background & Aims :Helicobacter pylori (Hp ) infection causes a chronic gastric inflammation, which can lead to peptic ulceration and cancer. The inflammatory response is multifactorial and is characterized by exaggerated Th1 cytokine production. How the Th1 response is induced and maintained in the stomach of Hp -infected patients remains unclear. Transforming growth factor (TGF)- 1 negatively regulates Th1 cell development, and TGF- 1-deficient mice spontaneously develop gastritis. Here, we examined TGF- 1 signaling in Hp -associated gastritis. Methods :Gastric biopsy specimens taken from patients with or without Hp infection were analyzed for the content of activated TGF- 1 by ELISA and Smad3 and 7 expression by Western blotting. Induction of Smad7 by interferon (IFN)- was examined in normal gastric mucosal biopsy specimens, whereas the effect of Smad7 inhibition on the ongoing Th1 response was analyzed in Hp -colonized biopsy specimens. Results :Activated TGF- 1 was abundant in the mucosa of controls and Hp -infected patients, with no significant difference between the 2 groups. Despite this, in whole biopsy specimens and isolated mucosal cells from Hp -infected patients, there was defective TGF- 1-associated Smad3 phosphorylation, which was associated with high expression of the inhibitor Smad7. Blocking Smad7 with antisense oligonucleotides restored TGF- 1 signaling in biopsy specimens from Hp -infected patients and concomitantly reduced interferon- and T-bet. Smad7 was inducible in normal gastric biopsy specimens by interferon- through a STAT1-dependent mechanism, and neutralization of interferon- in biopsy specimens from Hp -infected patients reduced Smad7 expression. Conclusions :These data suggest that, in Hp -infected gastric mucosa, interferon- induces the expression of Smad7, which then prevents endogenous TGF- 1 from down-regulating the ongoing tissue-damaging Th1 response.
The development and maintenance of human visceral pain hypersensitivity is dependent on the N-methyl- D-aspartate receptor
Robert Paul Willert, Clifford J. Woolf, Anthony Robert Hobson, Claire Delaney, David G. Thompson, Qasim Aziz
Background & Aims :Visceral hypersensitivity is a common feature of functional gastrointestinal disorders. One speculated mechanism is an activity-dependent increase in spinal cord neuronal excitability (central sensitization), which is dependent on activation of the N-methyl- D-aspartate (NMDA) receptor. Our aims were to determine whether the development and maintenance of human visceral hypersensitivity is NMDA receptor mediated. Methods :Healthy subjects were studied using a randomized, double-blind, placebo-controlled, crossover design. Pain thresholds to electrical stimulation were determined both in the proximal esophagus and in the foot (control) before and after a 30-minute distal esophageal infusion of 0.15 mol/L HCl acid. Ketamine (NMDA receptor antagonist) or saline (vehicle) was given intravenously either prior to or following acid infusion, and pain thresholds were measured for the following 120 minutes. Protocol 1: In 6 subjects, the effect of ketamine in the esophagus was assessed without acid infusion. Protocol 2: In 14 subjects, ketamine was given prior to esophageal acid. Protocol 3: In 12 subjects, ketamine was given after esophageal acid. Results :Protocol 1: In the absence of esophageal acid, ketamine had no effect on either esophageal or foot pain thresholds (area-under-the-curve, [AUC] P= 0.36 esophagus, P= 0.34 foot, ANOVA) within 30 minutes of cessation of the infusion. Protocol 2: Acid-induced esophageal hypersensitivity was prevented by ketamine (AUC, P< 0.0001, ANOVA) without affecting foot pain thresholds (AUC, P= 0.06, ANOVA). Protocol 3: Ketamine delivered after acid reversed the induction of esophageal hypersensitivity induced by acid (AUC, P< 0.0001, ANOVA). Conclusions :The induction and maintenance of acid-induced esophageal hypersensitivity is prevented and reversed by ketamine. This finding strongly indicates that central sensitization is a mechanism of visceral hypersensitivity.
Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome
Giovanni Barbara, Vincenzo Stanghellini, Roberto De Giorgio, Cesare Cremon, Graeme S. Cottrell, Donatella Santini, Gianandrea Pasquinelli, Antonio M. Morselli-Labate, Eileen F. Grady, Nigel W. Bunnett, Stephen M. Collins, Roberto Corinaldesi
Background & Aims :The mechanisms underlying abdominal pain perception in irritable bowel syndrome (IBS) are poorly understood. Intestinal mast cell infiltration may perturb nerve function leading to symptom perception. We assessed colonic mast cell infiltration, mediator release, and spatial interactions with mucosal innervation and their correlation with abdominal pain in IBS patients. Methods :IBS patients were diagnosed according to Rome II criteria and abdominal pain quantified according to a validated questionnaire. Colonic mucosal mast cells were identified immunohistochemically and quantified with a computer-assisted counting method. Mast cell tryptase and histamine release were analyzed immunoenzymatically. Intestinal nerve to mast cell distance was assessed with electron microscopy. Results :Thirty-four out of 44 IBS patients (77%) showed an increased area of mucosa occupied by mast cells as compared with controls (9.2% ± 2.5% vs. 3.3 ± 0.8%, respectively; P< 0.001). There was a 150% increase in the number of degranulating mast cells (4.76 ± 3.18/field vs. 2.42 ± 2.26/field, respectively; P= 0.026). Mucosal content of tryptase was increased in IBS and mast cells spontaneously released more tryptase (3.22 ± 3.48 pmol/min/mg vs. 0.87 ± 0.65 pmol/min/mg, respectively; P= 0.015) and histamine (339.7 ± 59.0 ng/g vs. 169.3 ± 130.6 ng/g, respectively; P= 0.015). Mast cells located within 5 µm of nerve fibers were 7.14 ± 3.87/field vs. 2.27 ± 1.63/field in IBS vs. controls ( P< 0.001). Only mast cells in close proximity to nerves were significantly correlated with severity and frequency of abdominal pain/discomfort ( P< 0.001 and P= 0.003, respectively). Conclusions :Colonic mast cell infiltration and mediator release in proximity to mucosal innervation may contribute to abdominal pain perception in IBS patients.
Clinical-liver, Pancreas, and Biliary Tract
Antiviral action of ribavirin in chronic hepatitis C
Jean-Michel Pawlotsky, Harel Dahari, Avidan U. Neumann, Christophe Hezode, Georgios Germanidis, Isabelle Lonjon, Laurent Castera, Daniel Dhumeaux
Background & Aims :In the patients with chronic hepatitis C, the addition of ribavirin to interferon (IFN)- significantly increases the virologic responses. Our aim was to assess the antiviral action of ribavirin on hepatitis C virus (HCV) as a function of ribavirin pharmacokinetics and to evaluate the influence of this antiviral effect on IFN- efficacy. Methods :Forty-five patients with chronic hepatitis C (genotype 1b) received various schedules of IFN- and/or ribavirin administration. Frequent blood sampling was performed for HCV RNA kinetics and ribavirin pharmacokinetics assessment. Results :Ribavirin monotherapy induced a significant, moderate, early, and transient viral load decrease in approximately half of the patients. The occurrence of this effect was associated with longer ribavirin clearance half-lives and higher serum ribavirin concentrations. Ribavirin antiviral effect partly reduced the rebound preceding the second IFN- injection in patients receiving standard IFN- 3 times per week plus ribavirin. The magnitude of the rebound was inversely related to ribavirin concentrations. These patients subsequently experienced a slow, but significant, second slope of viral decrease and cleared HCV RNA. The addition of ribavirin to daily IFN- monotherapy did not have any impact on the second phase of viral decline. Conclusions :Ribavirin exerts a significant, moderate, and transient antiviral effect in a significant proportion of patients with chronic hepatitis C. The antiviral effect of ribavirin correlates with ribavirin pharmacokinetics and is partly responsible for the improved efficacy of the combination of standard IFN- and ribavirin compared with IFN- monotherapy by increasing the incidence of the initial response.
Computed tomography and magnetic resonance imaging in the assessment of acute pancreatitis
Marianna Arvanitakis, Myriam Delhaye, Viviane De Maertelaere, Monia Bali, Catherine Winant, Emmanuel Coppens, Jacques Jeanmart, Marc Zalcman, Daniel Van Gansbeke, Jacques Devière, Celso Matos
Background & Aims :This study aimed to compare the accuracy of magnetic resonance imaging (MRI) with computed tomography (CT) in assessing acute pancreatitis (AP) and to explore the correlation between MRI findings and clinical outcome. Methods :Patients with AP were investigated by contrast-enhanced CT and MRI on admission and 7 and 30 days thereafter. MRI was performed with intravenous secretin and contrast medium. Balthazar’s grading system was used to measure CT and MRI severity indices (CTSI and MRSI, respectively). Results :Thirty-nine patients (median age, 47 years; range, 1586) were studied. AP was of biliary etiology in 19 patients (49%). On admission, AP was assessed clinically as severe in 7 patients (18%). A strong correlation was demonstrated between CTSI and MRSI on admission and 7 days later. MRSI on admission correlated with the following: the Ranson score, C-reactive protein levels 48 hours after admission, duration of hospitalization, and clinical outcome regarding morbidity, including local and systemic complications. Considering the Ranson score as the gold standard, MRI detected severe AP with 83% (5896, 95% CI) sensitivity, 91% (6898) specificity vs. 78% (5293) and 86% (6396) for CT. Magnetic resonance cholangiopancreatography after IV secretin injection showed pancreatic duct leakage in 3 patients (8%). Conclusions :MRI is a reliable method of staging AP severity, has predictive value for the prognosis of the disease, and has fewer contraindications than CT. It can also detect pancreatic duct disruption, which may occur early in the course of AP.
Cholic acid supplementation enhances cholesterol absorption in humans
Laura A. Woollett, Donna D. Buckley, Lihang Yao, Peter J.H. Jones, Norman A. Granholm, Elizabeth A. Tolley, Patrick Tso, James E. Heubi
Background & Aims :Qualitative and quantitative changes in intralumenal bile acid composition may alter cholesterol absorption and synthesis and low-density lipoprotein (LDL) receptor expression. The role of cholic acid (CA) in cholesterol absorption in humans remains unclear and, thus, was examined in the current study. Methods :In a crossover design outpatient study, 12 adults aged 2436 years took 15 mg/kg/day (CA) or no bile acid supplement (control) while being fed a controlled diet (AHA heart-healthy diet). A liquid meal of defined composition was given on day 14 of the diet, and lumenal samples were collected. Thereafter, cholesterol absorption and cholesterol fractional synthetic rate (FSR) were assessed by stable isotopic methods from days 16 to 20. Results :With CA treatment, bile was enriched significantly with CA ( P< 0.0004) to 60.2% ± 2.4% (mean ± SEM) compared with 43.3% ± 2.4% for controls. CA plus diet treatment significantly increased ( P= 0.013) cholesterol absorption (72.6% ± 2.9%) compared with diet treatment alone (60.4% ± 2.9%). Percentage micellar cholesterol was increased by CA plus diet treatment vs. diet alone after meal ingestion ( P= 0.004). Plasma total and high-density lipoprotein (HDL) and LDL cholesterol was unchanged with CA treatment. Conclusions :Thus, enrichment in lumenal bile with CA results in an increase in cholesterol absorption, an effect potentially mediated by enhanced cholesterol solubilization in micelles.
Guanylin regulates chloride secretion in the human gallbladder via the bile fluid
Hasan Kulaksiz, Thorsten Schlenker, Daniel Rost, Adolf Stiehl, Martin Volkmann, Thomas Lehnert, Yalcin Cetin, Wolfgang Stremmel
Background & Aims :The biliary epithelium of bile ducts and gallbladder modifies the composition of primary hepatic bile by absorption and secretion of an electrolyte-rich fluid. The underlying transport mechanisms, however, are still incompletely understood. We investigated the expression, the cellular localization, and the functional role of guanylin, a bioactive intestinal peptide involved in the cystic fibrosis transmembrane conductance regulator (CFTR)-regulated electrolyte/water secretion, in the human gallbladder. Methods :Peptide-specific antibodies were raised to localize guanylin and its affiliated signaling proteins, i.e., the guanylin receptor, guanylate cyclase C (GC-C), cGMP-dependent protein kinase type II (cGKII), and CFTR in the human gallbladder and cholangiocarcinoma cells (Mz-Cha-1) by RT-PCR, Western blot, and immunocytochemistry. A sensitive ELISA was used to assess the range of guanylin concentration in human bile fluid. The functional role of guanylin was investigated in subconfluent Mz-Cha-1 cell monolayers by isotope efflux experiments. Results :Guanylin and its affiliated signaling proteins are highly expressed in the human gallbladder. Guanylin is localized to secretory epithelial cells of the gallbladder and is present in the bile, whereas GC-C, cGKII, and CFTR are confined exclusively to the apical membrane of the same epithelial cells. Functional studies in Mz-Cha-1 cells identify guanylin as a specific regulator of biliary Cl secretion that very likely is mediated by an intracellular increase of cGMP-concentration. Conclusions :Based on the present findings and on the functional role of guanylin in other epithelia, it is likely that gallbladder epithelial cells synthesize and release guanylin into the bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.
Bile acid synthesis is increased in chilean hispanics with gallstones and in gallstone high-risk Mapuche Indians
Cecilia Gälman, Juan Francisco Miquel, Rosa Maria Pérez, Curt Einarsson, Lars Ståhle, Guillermo Marshall, Flavio Nervi, Mats Rudling
Background & Aims :Gallstone disease is an important, costly health-care problem in Western societies. It is still unclear whether hepatic lipid regulatory enzymes play primary or secondary roles in gallstone formation. In this study, the aim was to investigate whether the synthesis of bile acids and cholesterol is increased in gallstone disease and to test whether such a metabolic change, if present, might occur before gallstone formation. Methods :A total of 125 Chilean Hispanic women (80 without gallstones and 45 with gallstones) matched for age and body mass index were investigated, along with 40 Chilean Mapuche Indian women (20 without gallstones and 20 with gallstones), a population group in which the prevalence for gallstone disease is very high. Fasting blood plasma samples were assayed for 7 -hydroxy-4-cholesten-3-one and lathosterol, 2 strong indicators for hepatic bile acid and body cholesterol synthesis, respectively. Results :Plasma 7 -hydroxy-4-cholesten-3-one levels, corrected for plasma cholesterol, were significantly increased by 50% in Hispanic women with gallstones as compared with gallstone-free Hispanics ( P< 0.006). As compared with Hispanic women without gallstones, plasma 7 -hydroxy-4-cholesten-3-one levels were increased by 100% ( P< 0.002) in Mapuche Indian women, independently of whether gallstones were present. Plasma lathosterol, corrected for plasma cholesterol, was significantly increased by 22% in Hispanic women with gallstones and in Mapuche Indian women compared with Hispanic women. Conclusions :The results indicate that the synthesis of bile acids and cholesterol is induced in gallstone disease and precedes gallstone development. These inductions presumably occur as a response to an increased intestinal loss of bile acids.
Simvastatin enhances hepatic nitric oxide production and decreases the hepatic vascular tone in patients with cirrhosis
Carmen Zafra, Juan G. Abraldes, Juan Turnes, Annalisa Berzigotti, Mercedes Fernández, Juan C. Garca-Pagán, Juan Rodés, Jaime Bosch
Background & Aims :In cirrhosis, an insufficient release of nitric oxide contributes to increased hepatic resistance and portal pressure and enhances the postprandial increase in portal pressure. We hypothesized that simvastatin, which enhances Akt-dependent endothelial nitric oxide synthase phosphorylation, may increase hepatic nitric oxide release and decrease hepatic resistance in patients with cirrhosis and portal hypertension. Methods :In protocol 1, 13 patients had measurements of the hepatic venous pressure gradient, hepatic blood flow, mean arterial pressure, cardiac output, and nitric oxide products before and 30 and 60 minutes after 40 mg of simvastatin. In protocol 2, 17 patients were randomized to receive placebo or simvastatin (40 mg) 12 hours and 1 hour before the study. After baseline measurements of the hepatic venous pressure gradient, hepatic blood flow, and nitric oxide products, a standard liquid meal was given, and measurements were repeated at 15, 30, and 45 minutes. Results :In protocol 1, acute simvastatin did not modify the hepatic venous pressure gradient but increased the hepatic blood flow (21% ± 13% at 30 minutes; P= 0.01) and decreased hepatic sinusoidal resistance by 14% ± 11% ( P= 0.04). Nitric oxide product levels significantly increased in hepatic venous blood (from 31.4 ± 12.3 nmol · mL 1 to 35.8 ± 10.7 nmol · mL 1 ;P= 0.04), but not in peripheral blood. Systemic hemodynamics were not modified. In protocol 2, simvastatin pretreatment significantly attenuated the postprandial increase in hepatic venous pressure gradient (mean peak increase, 10% ± 9% vs. 21% ± 6% in placebo; P= 0.01). Hepatic blood flow increased similarly in the 2 groups. Hepatic nitric oxide products increased in the simvastatin group but not in the placebo group. Conclusions :Simvastatin administration increases the hepatosplanchnic output of nitric oxide products and decreases hepatic resistance in patients with cirrhosis.
Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity
Frank Chen, M. Ananthanarayanan, Sukru Emre, Ezequiel Neimark, Laura N. Bull, A.S. Knisely, Sandra S. Strautnieks, Richard J. Thompson, Margret S. Magid, Ronald Gordon, N. Balasubramanian, Frederick J. Suchy, Benjamin L. Shneider
Background & Aims :The mechanisms by which mutations in the familial intrahepatic cholestasis-1 gene cause Byler’s disease (progressive familial intrahepatic cholestasis type 1) are unknown. Methods :Interactions among the apical sodium-dependent bile acid transporter, the farnesoid X receptor (FXR), and familial intrahepatic cholestasis-1 were studied in the ileum of children with progressive familial intrahepatic cholestasis type 1 and in Caco-2 cells. Results :Increased ileal apical sodium-dependent bile acid transporter messenger RNA (mRNA) expression was detected in 3 patients with progressive familial intrahepatic cholestasis type 1. Paradoxically, ileal lipid-binding protein mRNA expression was repressed, suggesting a central defect in bile acid response. Ileal FXR and short heterodimer partner mRNA levels were reduced in the same 3 patients. In Caco-2 cells, antisense-mediated knock-down of endogenous familial intrahepatic cholestasis-1 led to up-regulation of apical sodium-dependent bile acid transporter and down-regulation of FXR, ileal lipid-binding protein, and short heterodimer partner mRNA. In familial intrahepatic cholestasis-1-negative Caco-2 cells, the activity of the human apical sodium-dependent bile acid transporter promoter was enhanced, whereas the human FXR and bile salt excretory pump promoters’ activities were reduced. Overexpression of short heterodimer partner but not of the FXR abrogated the effect of familial intrahepatic cholestasis-1 antisense oligonucleotides. FXR cis -element binding and FXR protein were reduced primarily in nuclear but not cytoplasmic extracts from familial intrahepatic cholestasis-1-negative Caco-2 cells. Conclusions :Loss of familial intrahepatic cholestasis-1 leads to diminished nuclear translocation of the FXR, with the subsequent potential for pathologic alterations in intestinal and hepatic bile acid transporter expression. Marked hypercholanemia and cholestasis are predicted to develop, presumably because of both enhanced ileal uptake of bile salts via up-regulation of the apical sodium-dependent bile acid transporter and diminished canalicular secretion of bile salts secondary to down-regulation of the bile salt excretory pump.
Basic-alimentary Tract
Urea movement across mouse colonic plasma membranes is mediated by UT-A urea transporters
Gavin S. Stewart, Robert A. Fenton, Frank Thévenod, Craig P. Smith
Background & Aims :Urea is a major nitrogen source for commensal bacteria that inhabit the large intestine. UT-A urea transporters mediate urea movement across plasma membranes. The aim of this study was to determine whether UT-A proteins are expressed in the mouse colon and, if so, whether they have a functional role in transcellular urea transport. Methods :Mouse colonic UT-A transporters were investigated with Northern blot analysis, immunoblotting, immunolocalization, and refractive light flux experiments. Results :Northern blot analysis showed that 4 UT-A transcripts were present in mouse colon. Two peptide-targeted polyclonal antibodies showed the presence of UT-A immunoreactive proteins in mouse colon. Antiserum ML446 targeted to the N-terminus of mouse UT-A1 detected proteins of 34 and 48 kilodaltons. Antiserum ML194 targeted to the C-terminus of mouse UT-A1 detected proteins of 48, 75, and 100 kilodaltons. Immunolocalization studies using ML446 showed the presence of UT-A proteins in cells throughout the colonic crypts. ML194 specifically stained cells located in the proliferative and stem regions of the lower portion of colonic crypts. Differential centrifugation and immunoblotting of colonic epithelia showed that UT-A proteins were present in plasma membrane-enriched fractions. Refractive light flux experiments using colonic plasma membrane vesicles showed a significant urea flux, which was completely inhibited by the UT-A inhibitor phloretin. Conclusions :Functional UT-A transporters are expressed in the plasma membranes of mouse colon, indicating that these proteins may play a key role in host/bacterial interaction.
Regulated alkali secretion acts in tandem with unstirred layers to regulate mouse gastric surface pH
Heidi K. Baumgartner, Marshall H. Montrose
Background & Aims :The physical basis for the protective pH gradient at the gastric surface is unconfirmed. This study examined the role of mucus, the unstirred layer, and acid/alkali secretion in controlling gastric surface pH in vivo. Methods :Stomachs of anesthetized mice were exteriorized, and exposed gastric mucosa was imaged by confocal microscopy. Results :Accessibility of molecules at the gastric surface was determined by monitoring the decrease in probe fluorescence over time after dyes were removed from perfusate. On dye removal, Cl-NERF (400 molecular weight) fluorescence decreased more slowly at the gastric surface in the presence of mucus (rate constant [k] = 0.08 ± 0.02 per second) than in the absence of mucus (k = 0.15 ± 0.02 per second) or 90 µm distant from the surface (k = 0.22 ± 0.03 per second). In contrast, 70-kilodalton Cl-NERF/dextran washed from the gastric surface more slowly in the presence (k = 0.05 ± 0.01 per second) or absence (k = 0.09 ± 0.01 per second) of mucus compared with 90 µm from the tissue surface (k = 0.36 ± 0.08 per second). Two-photon uncaging of fluorescein near nonsuperfused gastric surface showed that diffusion was not slowed at the gastric surface compared with diffusion in free solution. Surface pH was measured by Cl-NERF ratio imaging. Increasing the superfusion rate decreased the thickness of the surface pH gradient without significantly changing surface pH values, suggesting a pH set point of approximately 4 for control of surface pH. Increasing perfusate pH buffers decreased surface pH toward perfusate values. Conclusions :Proton concentration at the gastric surface is the result of regulating acid/alkali secretion to a set point in combination with an unstirred layer and not by trapping of proton or small-molecular-weight buffers in the unstirred layer.
Expression of functional neurokinin-1 receptors in regenerative glands during gastric wound healing in rodents
Adrian Schmassmann, Bea Waser, Beatrice Flogerzi, Jean Claude Reubi
Background & Aims :Although functions of the neurokinin-1 receptor have been well explored in neurogenic inflammation and immunoinflammatory responses, little is known about neurokinin-1 receptors during gastric wound healing. The aim of this study was to assess whether neurokinin-1 receptors play a role in gastric wound healing. Methods :In vitro neurokinin-1 receptor autoradiography and immunohistochemistry were performed to identify, locate, and quantify neurokinin-1 receptors during wound healing in rodents with cryoulcers in the gastric corpus and antrum. Moreover, to assess the functionality of these receptors, the effect of the neurokinin-1 receptor antagonist NKP608 on gastric wound healing was quantified in vivo in wild-type and cyclooxygenase-2 / mice. Results :Regenerative glands of the mucosal ulcer margin of rat cryoulcers of the gastric corpus showed strong expression of neurokinin-1 receptors in binding studies between days 3 and 22, with little expression on days 2984. In addition, strong immunoreactivity for neurokinin-1 receptors was detected on the cell membrane of these regenerative glands. Expression of neurokinin-1 receptors in regenerative glands was confirmed in the rat antrum and the mouse gastric corpus. Moreover, in vivo functional tests during gastric ulcer healing showed that cell proliferation in the regenerative epithelia of the ulcer margin was significantly decreased by NKP608 compared with placebo; furthermore, gastric ulcer healing was significantly delayed by NKP608 both in wild-type and cyclooxygenase-2 / mice. Conclusions :This report shows the time-limited overexpression of neurokinin-1 receptors in the mucosal repair tissue of the corpus and antrum. Our in vitro and in vivo data suggest that neurokinin-1 receptors are involved in gastric wound healing.
Chemotherapy- and radiotherapy-induced intestinal damage is regulated by intestinal trefoil factor
P.L. Beck, J.F. Wong, Y. Li, S. Swaminathan, R.J. Xavier, K.L. Devaney, Daniel K. Podolsky
Background & Aims :Injury to the intestinal mucosa is frequently a dose-limiting complication of radiotherapy and chemotherapy. Approaches to limit the damage to the intestine during radiation and chemotherapy have been largely ineffective. Trefoil factors are produced throughout the gastrointestinal tract and regulate cell migration, restitution, and repair. Studies were undertaken to define the role of intestinal trefoil factor in modulating the intestinal response to chemotherapy and radiation. Methods :The effect of intestinal trefoil factor on migration and cell survival in intestinal epithelial monolayer exposed to methotrexate was studied in vitro. Chemotherapy and radiation damage was assessed in wild-type and intestinal trefoil factor-null mice in the presence or absence of supplemental intestinal trefoil factor administered in drinking water. Results :Radiation and chemotherapy induced a marked reduction in goblet cell number and intestinal trefoil factor messenger RNA, as well as intestinal trefoil factor promoter activity. Intestinal trefoil factor improved intestinal epithelial cell viability and wound repair after chemotherapy exposure in vitro. Intestinal trefoil factor-deficient mice (intestinal trefoil factor / ) were more susceptible to chemotherapy- and radiation-induced mucositis. Oral recombinant intestinal trefoil factor reduced the severity of both chemotherapy-induced and chemotherapy/radiotherapy-induced intestinal mucositis. Conclusions :These studies suggest that intestinal trefoil factor is involved in protection against and recovery from intestinal mucositis induced by radiation and chemotherapy.
IP-10-induced recruitment of CXCR3 +host T cells is required for small bowel allograft rejection
Zheng Zhang, Levent Kaptanoglu, Yueming Tang, David Ivancic, Sambasiva M. Rao, Andrew Luster, Terrence A. Barrett, Jonathan Fryer
Background & Aims :Chemokines mediate cell trafficking in inflammatory states such as allograft rejection. However, their role in small-bowel allograft rejection has not been defined. The aim of this study was to examine the roles of type 1 helper T-cell chemokines in small-bowel allograft rejection. Methods :Mucosal histology, chemokine messenger RNA (real-time polymerase chain reaction), and cell isolates were examined in small-bowel allografts and isografts. Interferon- -inducible protein-10/ CXC chemokine receptor (CXCR) 3 interactions were specifically evaluated by using allografts from interferon- -inducible protein-10 / donors and adoptive transfer of CXCR3 / T cells into recombination activating gene (RAG)-1 / recipients of small-bowel allografts. Results :Type 1 helper T-cell cytokine (interferon- ) and chemokine (interferon- -inducible protein-10, monokine induced by interferon- , macrophage-inflammatory protein-1 , and regulated on activation, normal T cells expressed and secreted) messenger RNA up-regulation was detected (real-time polymerase chain reaction) by postoperative day 3 in small-bowel allografts. Interferon- -inducible protein-10 +/+ small-bowel allograft rejection was associated with a dramatic (>7-fold) increase in CXCR3 +host T cells in the graft lamina propria. With interferon- -inducible protein-10 / small-bowel allografts, CXCR3 +host T-cell infiltration of the graft lamina propria was markedly decreased and rejection was significantly delayed. Whereas adoptive transfer of wild-type B6 (CXCR3 +/+ ) T cells into B6 (RAG-1 / ) recipients induced rapid rejection of CB6F1 small-bowel allografts, rejection was significantly delayed (29.2 ± 8.7 days vs. 16.5 ± 3.1 days; P< 0.01) in B6 (RAG-1 / ) mice reconstituted with T cells from B6 (CXCR3 / ) mice. Conclusions :Recruitment of CXCR3 +host T cells by donor derived interferon- -inducible protein-10 may precipitate small-bowel allograft rejection. These data highlight the importance of type 1 helper T cell-related chemokines in promoting cell-mediated rejection responses in small-bowel allografts and suggest that interferon- -inducible protein-10 is an attractive therapeutic target for humanized monoclonal antibody strategies.
Disruption of intestinal motility by a calcium channel-stimulating autoantibody in type 1 diabetes
Michael W. Jackson, Tom P. Gordon, Sally A. Waterman
Background & Aims :Autonomic neuropathy, including gastrointestinal dysfunction, is a common complication of type 1 diabetes; however, its cause is uncertain. This study aimed to test whether functional autoantibodies cause the gastrointestinal dysfunction. Methods :We used isolated mouse colon undergoing colonic migrating motor complex (MMC) activity to test for autoantibodies that disrupt colonic motility. Purified immunoglobulin G (IgG) from patients with type 1 diabetes or from controls was tested either in vitro or after passive transfer. Pharmacological studies of the interaction between the IgG and L-type calcium channel activator (Bay K8644) and inhibitors (nicardipine and verapamil) were performed. The effect of IgG on nerve-evoked contraction of the vas deferens longitudinal smooth muscle was also assessed. Results :MMC activity was disrupted by IgG (0.2 mg/mL) from 8 of 16 patients with type 1 diabetes but not by control IgG. Passive transfer of diabetic IgG to mice also disrupted MMCs, showing access to the antigen in vivo. The acute effect of the autoantibody was mimicked by the dihydropyridine agonist, Bay K8644 (210 nmol/L), and both Bay K8644 and the autoantibody competitively inhibited the effect on MMC contraction of the dihydropyridine antagonist, nicardipine. Diabetic IgG, but not control IgG, altered the nerve-evoked contractile activity of vas deferens smooth muscle effects mimicked by Bay K8644. Conclusions :A novel functional autoantibody that activates smooth muscle L-type calcium channels at the dihydropyridine binding site is produced specifically by patients with type 1 diabetes and may mediate gastrointestinal and autonomic dysfunction in these patients.
Hyperexpression of inducible costimulator and its contribution on lamina propria T cells in inflammatory bowel disease
Toshiro Sato, Takanori Kanai, Mamoru Watanabe, Atsushi Sakuraba, Susumu Okamoto, Takaaki Nakai, Akira Okazawa, Nagamu Inoue, Teruji Totsuka, Motomi Yamazaki, Richard A. Kroczek, Tsuneo Fukushima, Hiromasa Ishii, Toshifumi Hibi
Background & Aims :To investigate the role of inducible costimulator (ICOS), a new member of the CD28 family involved in regulation of T-cell activation and chronic intestinal inflammation, we assessed its expression and functional role in patients with inflammatory bowel disease (IBD). Methods :Expression of ICOS, CD28, and cytotoxic T-lymphocyte antigen (CTLA) 4 on intestinal lamina propria mononuclear cells (LPMC) from patients with ulcerative colitis (UC), Crohn’s disease (CD), and normal controls was determined using flow cytometry and immunohistochemistry. Expressions of the ICOS ligand, B7h, on lamina propria B cells, macrophages, and epithelial cells (EC) in the intestinal mucosa were also determined using flow cytometry. The functional costimulatory effect of ICOS on LPMC was assessed by the proliferative response and cytokine production. Results :CD4 +LPMC expressing ICOS was significantly increased in the inflamed mucosa of IBD patients but not in inflammatory or normal controls. B7h was also significantly up-regulated on B cells, macrophages, and EC in inflamed mucosa of IBD patients. Proliferative responses of anti-CD3/ICOS costimulation were significantly higher compared with those of anti-CD3 monoclonal antibody (mAb) alone. Anti-CD3/ICOS-stimulated-LPMC from UC secreted significantly increased amounts of interleukin (IL)-5 among the 3 groups. In contrast, anti-CD3/ICOS-stimulated-LPMC from CD secreted significantly increased amounts of interferon (IFN)- in the presence of IL-12. Conclusions :Highly expressed ICOS in activated CD4 +LPMC of IBD patients contributes to the dysregulated immune responses in IBD. Because ICOS hyperexpression was limited to inflammatory sites in IBD patients, ICOS would be a feasible therapeutic target for the treatment of IBD.
Basic-liver, Pancreas, and Biliary Tract
Hepatitis C virus infection and diabetes: Direct involvement of the virus in the development of insulin resistance
Yoshizumi Shintani, Hajime Fujie, Hideyuki Miyoshi, Takeya Tsutsumi, Kazuhisa Tsukamoto, Satoshi Kimura, Kyoji Moriya, Kazuhiko Koike
Background & Aims :Epidemiological studies have suggested a linkage between type 2 diabetes and chronic hepatitis C virus (HCV) infection. However, the presence of additional factors such as obesity, aging, or cirrhosis prevents the establishment of a definite relationship between these 2 conditions. Methods :A mouse model transgenic for the HCV core gene was used. Results :In the glucose tolerance test, plasma glucose levels were higher at all time points including in the fasting state in the core gene transgenic mice than in control mice, although the difference was not statistically significant. In contrast, the transgenic mice exhibited a marked insulin resistance as revealed by the insulin tolerance test, as well as significantly higher basal serum insulin levels. Feeding with a high-fat diet led to the development of overt diabetes in the transgenic mice but not in control mice. A high level of tumor necrosis factor- , which has been also observed in human chronic hepatitis C patients, was considered to be one of the bases of insulin resistance in the transgenic mice, which acts by disturbing tyrosine phosphorylation of insulin receptor substrate-1. Moreover, administration of an anti-tumor necrosis factor- antibody restored insulin sensitivity. Conclusions :The ability of insulin to lower the plasma glucose level in the HCV transgenic mice was impaired, as observed in chronic hepatitis C patients. These results provide a direct experimental evidence for the contribution of HCV in the development of insulin resistance in human HCV infection, which finally leads to the development of type 2 diabetes.
Placenta-derived CD95 ligand causes liver damage in hemolysis, elevated liver enzymes, and low platelet count syndrome
Susanne Strand, Dennis Strand, Rudolf Seufert, Amrit Mann, Johannes Lotz, Manfred Blessing, Michael Lahn, Andreas Wunsch, Dieter C. Broering, Uwe Hahn, Eva-Maria Grischke, Xavier Rogiers, Gerd Otto, Gregory J. Gores, Peter R. Galle
Background & Aims :The HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome is a life-threatening complication during pregnancy. The associated liver disease may be severe, and maternal hepatic complications may progress to the point that transplantation becomes necessary. CD95 (APO-1, Fas)-mediated apoptosis of liver cells is one of the major pathogenic mechanisms during liver disease. The interaction of CD95 with its ligand, CD95L(FasL), induces apoptosis and thus the source of the death-inducing ligand is critical for understanding the pathomechanism of liver damage involving the CD95-system. Methods :Sera from HELLP patients were analyzed and used in cell culture experiments to study CD95-mediated apoptosis. We established a mouse model for placenta-induced liver damage and used a new therapeutical agent, LY498919, to block CD95 apoptosis. Results :We describe apoptosis in the liver of HELLP patients and cytotoxic activity for primary human hepatocytes in HELLP serum. Blocking of CD95 signaling reduced the cytotoxic activity of HELLP serum. In addition, cytotoxic activity increased as HELLP syndrome developed. Furthermore, CD95L was found to be produced in the placenta and extracts of placenta were cytotoxic for human hepatocytes. Injection of mouse placenta extract in mice induces liver damage that could be prevented by blocking CD95L. Conclusions :Taken together, these data suggest that CD95L derived from the placenta acts systemically and is a primary cause of liver damage in HELLP syndrome. Our results also show that blocking of CD95L can reduce liver cell apoptosis, indicating that such a strategy may have therapeutic advantages.
Impaired clearance of virus-infected hepatocytes in transgenic mice expressing the hepatitis C virus polyprotein
Olivier Disson, Delphine Haouzi, Solange Desagher, Kim Loesch, Michael Hahne, Eric J. Kremer, Chantal Jacquet, Stanley M. Lemon, Urszula Hibner, Hervé Lerat
Background & Aims :Multiple molecular mechanisms are likely to contribute to the establishment of persistent infection by hepatitis C virus (HCV). The aim of this work was to study the evasion of cell-mediated antiviral immune responses in transgenic mice with livertargeted expression of the hepatitis C viral genome. These mice develop steatosis and hepatocellular carcinoma and constitute a murine model of chronic HCV infection. Methods :Mice of the FL-N/35 lineage were infected with replication-deficient adenoviral vectors encoding -galactosidase, and the persistence of infected cells was measured by histochemistry and enzymatic assays. Results :Hepatocytes from the HCV +transgenic mice are deficient in eliminating an adenoviral infection, despite an apparently normal T-cell response. The defect in adenoviral clearance was associated with resistance of transgenic hepatocytes to apoptosis induced by Fas/APO1/CD95 death receptor stimulation, a major pathway of cell killing by cytotoxic T lymphocytes. The attenuation of Fas-mediated apoptosis observed in the murine model was associated with a reduced abundance of Bid, a BH3-only member of the Bcl-2 family of apoptosis regulators. Conclusions :Our results suggest that viral evasion of cell-mediated immune responses leading to apoptotic death of hepatocytes may contribute to viral persistence. Such a mechanism might also contribute to the development of liver cancer in HCV.
Pioglitazone prevents alcohol-induced fatty liver in rats through up-regulation of c-Met
Kengo Tomita, Toshifumi Azuma, Naoto Kitamura, Jiro Nishida, Gen Tamiya, Akira Oka, Sayaka Inokuchi, Takeshi Nishimura, Makoto Suematsu, Hiromasa Ishii
Background & Aims :Treatment of steatosis is important in preventing development of fibrosis in alcoholic liver diseases. This study aimed to examine if pioglitazone, an antidiabetic reagent serving as a ligand of peroxisome proliferator-activated receptor gamma (PPAR ), could prevent alcoholic fatty liver. Methods :Rats fed with an ethanol-containing liquid diet were given the reagent at 10 mg/kg per day intragastrically for 6 weeks. Hepatic genes involved in actions of the reagent were mined by transcriptome analyses, and their changes were confirmed by real-time polymerase chain reaction and Western blotting analyses. The direct effects of pioglitazone on primary-cultured hepatocytes were also assessed in vitro. Results :Pioglitazone significantly attenuated steatosis and lipid peroxidation elicited by chronic ethanol exposure without altering insulin resistance. Mechanisms for improving effects of the reagent appeared to involve restoration of the ethanol-induced down-regulation of c-Met and up-regulation of stearoyl-CoA desaturase (SCD). Such effects of pioglitazone on the c-Met signaling pathway resulted from its tyrosine phosphorylation and resultant up-regulation of the apolipoprotein B (apoB)-mediated lipid mobilization from hepatocytes through very low-density lipoprotein (VLDL) as well as down-regulation of sterol regulatory element binding protein (SREBP) -1c and SCD levels and a decrease in triglyceride synthesis in the liver. Conclusions :Pioglitazone activates c-Met and VLDL-dependent lipid retrieval and suppresses triglyceride synthesis and thereby serves as a potentially useful stratagem to attenuate ethanol-induced hepatic steatosis.
Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice
Mercedes Fernandez, Francesco Vizzutti, Juan Carlos Garcia-Pagan, Juan Rodes, Jaime Bosch
Background & Aims :Portal hypertension is a frequent syndrome that develops in patients with chronic liver diseases, which are one of the most common causes of death in adults worldwide. The most serious clinical consequences of portal hypertension are related to the development of portal-systemic collateral vessels. Those include hepatic encephalopathy and massive bleeding from ruptured gastroesophageal varices. The high relevance of these collateral vessels prompted us to investigate the mechanism underlying its formation in a murine model of portal hypertension. Methods :To determine whether the development of portal-systemic collateral vessels in portal hypertension is a vascular endothelial growth factor (VEGF)-dependent angiogenic process, we assessed the effects of a monoclonal antibody against VEGF receptor-2 on the formation of these collateral vessels in mice with portal hypertension induced by partial portal vein ligation. We also studied the effects of a selective and specific inhibitor of VEGF receptor-2 autophosphorylation in partial portal vein-ligated rats. Results :A significant and marked inhibition in the formation of portal-systemic collateral vessels was observed in both partial portal vein-ligated mice and rats treated with anti-VEGF receptor-2 monoclonal antibodies or with the inhibitor of VEGF receptor-2 autophosphorylation, respectively, compared with animals receiving control solutions. Conclusions :Our present study shows that formation of collateral vessels is an angiogenesis-dependent process that can be markedly inhibited by blockade of the VEGF signaling pathway. These findings will make angiogenesis a focal point of research in portal hypertension and may lead to novel approaches for therapy of patients with chronic liver diseases.
Copyright © 2001-2004 by the American Gastroenterological Association. All rights reserved.
Table of Contents for Volume 40, Issue 3, March 2004
Biliary Tract and Cholestasis
Susceptibility to primary sclerosing cholangitis is associated with polymorphisms of intercellular adhesion molecule-1
Xuesong Yang, Susan N. Cullen, Jin H. Li, Roger W. Chapman and Derek P. Jewell
Background/Aims: Intercellular adhesion molecule-1 ( ICAM-1 , CD54) gene polymorphisms have been implicated in the susceptibility to a range of inflammatory diseases, including inflammatory bowel disease (IBD). Primary sclerosing cholangitis (PSC) is an immune-mediated chronic cholestatic liver disease associated with IBD. ICAM-1 is expressed on proliferating bile ducts and interlobular bile ducts in late stage PSC and serum levels of soluble intercellular adhesion molecules are increased in PSC. The aim of this study was to analyse ICAM-1 gene polymorphisms in PSC patients. Methods In this study, 104 patients with PSC and 213 healthy controls were recruited from Oxfordshire Caucasians. PCR with sequence-specific primers (PCR-SSP) was used to detect both ICAM-1 biallelic polymorphisms G241R and K469E. The results were controlled for the HLA haplotypes associated with PSC. Results The E/E frequency of K469E in PSC was 12% (12/104), significantly lower than that in controls (24%, 51/213; P=0.009; Pc=0.02; OR, 0.41). The occurrence of the haplotype G241-E469/G241-E469 in PSC was 4% (4/104), significantly lower than the control group (13%, 28/213; P=0.01; Pc=0.04; OR, 0.26). There was no difference between PSC and control groups in the frequencies of the genotype R241G or in allele frequencies of K469E. Conclusions The E469E homozygote status for ICAM-1 is associated with protection against PSC.
Keywords: Intercellular adhesion molecule-1 ;Primary sclerosing cholangitis ;PCR with sequence-specific primer
Primary biliary cirrhosis: an infectious disease caused by Chlamydia pneumoniae ?
Ahmad S. Abdulkarim et al.
Background/Aims The etiology and pathogenesis of primary biliary cirrhosis (PBC) remain elusive. Both an infectious etiology and molecular mimicry have been implicated. The aim is to study the prevalence of Chlamydial antigens and RNA in the liver tissue of patients with PBC. Methods We compared the prevalence of Chlamydial antigen and RNA in 25 explants with PBC who underwent orthotopic liver transplantation with 105 explanted livers from other chronic liver disease. We also studied 14 liver biopsies from patients with early stages of PBC. Donor livers were also studied. Results In all 39 patients with PBC, Chlamydia pneumoniae antigens were present but not Chlamydia trachomatis , and only 9/105 (8.5%) of patients in the other categories were positive ( P<0.01) for C. pneumoniae . Eight explanted PBC livers were tested for C. pneumoniae 16S RNA by in situ hybridization and were positive. Conclusions The presence of C. pneumoniae antigen and RNA in liver tissue of patients with PBC suggests that C. pneumoniae antigen may trigger an immune response based on molecular mimicry.
Keywords: Chlamydia pneumoniae ;Primary biliary cirrhosis ;Pathogenesis
Experimental cholestatic liver disease through bile-duct ligation in rats results in skeletal fragility and impaired osteoblastogenesis
Miron Weinreb, Rivka Dresner Pollak and Zvi Ackerman
Background/Aims Patients with cholestatic liver disease have `low-turnover` osteoporosis. Since we reported that bile-duct ligated (BDL) rats develop bone disease with low bone formation and mass, we examined whether their reduced bone mass results in skeletal fragility, and whether the reduction in osteoprogenitor cells could explain the depressed bone formation. Methods Four-week-old rats were pair-fed and subjected to BDL or sham surgery. After 4 weeks, ex vivo bone marrow stromal cell cultures were used to estimate the number of osteoprogenitors and tibial strength was measured by mechanical testing. The serum levels of albumin, bilirubin, alanine amino-transferase (ALT), alkaline phosphatase (ALP) and nitrite were measured. Results BDL rats had elevated levels of bilirubin, ALT, ALP and nitrite. Tibiae of BDL rats were weaker than those of sham rats, exhibiting lower maximal force ( 34%) and stiffness ( 37%). The number of mineralized bone-like nodules in cultures from BDL rats was 65% lower than that in cultures from sham-operated rats, attesting to a diminished number of osteoprogenitors. Conclusions Skeletal fragility diminished osteoprogenitor pool and elevated plasma levels of nitrite are three additional characteristics of the bone disease that develops in BDL rats, thus increasing the validity of this animal model as representing the human bone disease in patients with cholestatic liver disease.
Keywords: Bile-duct ligation ;Osteopenia ;Cirrhosis ;Bone marrow ;Osteoporosis ;Mechanical strength
Cell Biology, Metabolism and Transport
Hepatocyte growth factor-induced proliferation of hepatic stem-like cells depends on activation of NF- B
Peng Yao et al.
Background/Aims Hepatocyte growth factor (HGF) regulates proliferation of hepatic stem cells. Transcription factor nuclear factor kappa B (NF- B) has been demonstrated as a key mediator for cell growth regulation. We investigated the role of NF- B in HGF-mediated cellular proliferation responses in a rat liver-derived hepatic stem-like cell line WB-F344. Methods Cell proliferation was determined by incorporation of [ 3H]thymidine. Phosphorylation of ERK1/2, p38 MAPK, Akt and I Bby HGF stimulation was detected by Western blotting. NF- B activation was determined by electrophoretic mobility shift assay and NF- B-mediated SEAP reporter assay. NF- B activation was inhibited by treatment with an I Bdominant-negative vector or inhibitor BAY-11-7082. Results We found that stimulation of WB-F344 cells with HGF promoted cell proliferation and effectively protected WB-F344 cells from apoptosis induced by TNF- . We also observed activation of ERK1/2, p38 MAPK, Akt and NF- B signaling pathways by HGF in WB-F344 cells. HGF-induced cell proliferation was partly blocked by pre-treatment of the cells with inhibitors against MEK1 or p38 MAPK, and completely blocked using an inhibitor for NF- B activity. Furthermore, it was demonstrated that I B mutant that suppressed NF- B activity completely blocked HGF-induced cell proliferation. Conclusions NF- B activity is required for HGF-induced proliferation in hepatic stem-like cell line WB-F344, and this activity requires ERK1/2 and p38 MAPK pathways.
Keywords: Hepatocyte growth factor ;Nuclear factor B;Proliferation ;Hepatic stem cell
Activin A augments vascular endothelial growth factor activity in promoting branching tubulogenesis in hepatic sinusoidal endothelial cells
Daisuke Endo, Kimitaka Kogure, Yoshihisa Hasegawa, Masatoshi Maku-uchi and Itaru Kojima
Background/Aims The production of activin A is markedly up-regulated in hepatocytes after partial hepatectomy. This factor tonically inhibits growth of hepatocytes but little is known about its effect on sinusoidal endothelial cells (SEC). In the present study, we investigated whether or not activin A affects growth and differentiation of SEC. Methods Growth and survival of SEC were measured in monolayer culture. Capillary formation was studied using SEC cultured in a collagen gel. Results SEC could not survive in the absence of vascular endothelial growth factor (VEGF). Activin A had a small effect on prevention of cell death and also enhanced anti-apoptotic action of VEGF. In addition, activin A and VEGF acted synergistically to stimulate cell growth of SEC. In the collagen gel, VEGF induced capillary formation of SEC. Activin A had little effect on branching tubulogenesis by itself but markedly enhanced tubular formation induced by VEGF. Finally, VEGF induced the expression of activin A and activin A increased the expression of VEGF receptors in cultured SEC. Conclusions Activin A augments VEGF activity in promoting growth and tubulogenesis of SEC.
Keywords: Activin A ;Follistatin ;Hepatic sinusoid ;Tubulogenesis ;The activin-follistatin system ;Vascular endothelial growth factor
In vivo real-time microangiography of the liver in mice using synchrotron radiation
Shogo Kobayashi et al.
Background/Aims : The aim of this study was to visualize hepatic microvessels (less than 100 µm in diameter) in vivo, which could not be visualized by conventional X-ray angiography, by using synchrotron radiation (ultra-bright and monochromatic X-ray). Methods : Five female Balb/c nu/nu mice were used. To investigate the hepatic microvessels under nearly physiologic conditions, we performed in vivo aortography under anesthesia with 370mgI/ml nonionic iodine contrast medium using monochromatic 17-keV X-rays generated by a synchrotron. Images were captured with a pixel matrix size of 1024?1024 at a rate of 30pictures/s. The field of view was 7mm?7 mm and thus the pixel size was approximately 7 µm. Captured images were evaluated both qualitatively and quantitatively. Results : Small hepatic arterial and portal venous branches of the liver were visualized separately during one sequential aortogram. The minimum diameter of the vessels observed was approximately 20 µm, and the vessels which ran parallel to the hepatic artery were observed and it seemed to be intrahepatic peribiliary arterial plexus. Conclusions : Our new experimental model would be useful for visualization of changes in the hepatic microcirculation under nearly physiologic conditions. Keywords: Liver ;Microangiography ;Synchrotron radiation
Chronic Liver Diseases
Tetrathiomolybdate in the treatment of acute hepatitis in an animal model for Wilson disease
Dominik Klein et al.
Background/Aims Tetrathiomolybdate (TTM) is a potent copper-chelating agent that has been shown to be effective in Wilson disease patients with neurological symptoms. Here, we investigate the potential use of TTM in treating the acute hepatic copper toxicosis in Long-Evans Cinnamon (LEC) rats, an authentic model for Wilson disease. Methods After the onset of acute hepatitis, LEC rats were treated once with 10 mg TTM/kg. After 1 and 4 days, parameters of liver toxicity and the subcellular distribution and binding of copper and iron were studied. Results In 11 out of 12 rats TTM rapidly improved acute hepatitis. Hepatic copper decreased through removal from cytosolic metallothionein and lysosomal metallothionein polymers. The remaining lysosomal copper forms a metallothionein-copper-TTM complex. In an almost moribund rat, however, TTM caused severe hepatotoxicity with fatal outcome. Conclusions TTM is effective in treating acute hepatitis in LEC rats when applied before the animals become moribund. TTM appears to act by removing the presumable reactive copper associated to lysosomal metallothionein polymers. The remaining lysosomal copper seems to be inactivated by forming a complex with TTM. Moreover, TTM removes copper from cytosolic copper-containing metallothionein. As a consequence, metallothionein is degraded and the uptake of copper-metallothionein into the lysosomes and the formation of the metallothionein polymer associated copper is reduced. Keywords: Wilson disease ;Long-Evans Cinnamon rat ;Therapy ;Ammonium tetrathiomolybdate ;Copper toxicity ;Iron overload ;Hepatitis
Cirrhosis and its Complications
Inappropriately low angiotensin II generation: a factor determining reduced kidney function and survival in patients with decompensated cirrhosis
Giovanni Sansoé et al.
Abstract Background /Aims : Angiotensin II contributes to the post-glomerular arteriolar vasoconstriction which maintains the glomerular filtration rate (GFR) in renal hypoperfusion. To explore whether depressed angiotensin II generation, due to reduced angiotensinogen production or low angiotensin-converting enzyme (ACE) levels, could impair kidney function in advanced cirrhosis. Methods : We studied and prospectively followed up 21 diuretic-free ascitic cirrhotic patients, through these determinations: plasma levels of active renin (AR), renin activity (PRA), angiotensin II, ACE and aldosterone; renal clearances of sodium, inulin and para -aminohippurate; antipyrine clearance. Fifteen healthy subjects were also studied. Results : GFR distribution was bimodal, 10 patients had low GFR values (l-GFR group) and 11 had normal-GFR values (n-GFR group) (below and above 105 ml/min per 1.73 m 2body surface area). Antipyrine clearance and Child-Pugh score did not differ in the two patient groups. l-GFR group had higher AR and PRA values, lower ACE levels and a significantly higher AR/Angiotensin II ratio than n-GFR group (all P<0.01). All 21 patients showed increased values of the AR/PRA ratio, i.e. subnormal angiotensinogen levels ( P<0.03). The 18-month survival rates of l-GFR and n-GFR groups were 20 and 81% ( P<0.02). Conclusions : Low-GFR cirrhotic patients had a worse survival rate associated with more severe contraction of the effective arterial blood volume, higher AR/Angiotensin II ratio and lower ACE levels. Keywords: Liver cirrhosis ;Ascites ;Glomerular filtration rate ;Hepatorenal syndrome ;Renal perfusion Abbreviations: A, aldosterone ;Ang, II, angiotensin II ;ACE, angiotensin-converting enzyme ;Ap, antipyrine ;AR, active renin ;CAp, antipyrine clearance ;CIN, inulin clearance ;CK, renal potassium clearance ;CNa, renal sodium clearance ;CPAH, para-aminohippurate clearance ;FEK, fractional potassium excretion ;FENa, fractional sodium excretion ;FF, filtration fraction ;FlNa, filtered sodium load ;GFP, glomerular filtration pressure ;GFR, glomerular filtration rate ;IN, inulin ;MAP, mean arterial pressure ;PAH, para-aminohippurate ;P-Cr, creatinine plasma concentration ;P-K, potassium plasma concentration ;P-Na, sodium plasma concentration ;PRA, plasma renin activity ;RPF, renal plasma flow ;t1/2Ap, antipyrine plasma half-life ;U-K, urinary potassium concentration ;UKV, urinary potassium excretion ;U-Na, urinary sodium concentration ;UNaV, urinary sodium excretion
High-density lipoproteins reduce the effect of endotoxin on cytokine production and systemic hemodynamics in cirrhotic rats with ascites
María José Ramírez et al.
Abstract Background/Aims : Hypersensitivity to endotoxin is a recognized feature in cirrhosis. High-density lipoproteins (HDL) have a high capacity to inactivate endotoxin. The aim was to determine if HDL reduces the effect of endotoxin on cytokine production and systemic hemodynamics in experimental cirrhosis. Methods : The study was performed in control and rats with carbon-tetrachloride induced cirrhosis with ascites. Hemodynamic parameters were determined before and after several doses of endotoxin. The effects of 25 µg/kg of endotoxin on the serum concentration of TNF and mean arterial pressure (MAP) were determined after treatment with HDL (80 mg/kg) or saline. Results : Whereas endotoxin decreased MAP only at doses of 100 and 1000 µg/kg in control rats, in cirrhotic rats significant hypotension occurred at doses of 25, 50, 100 and 1000 µg/kg. Following the administration of endotoxin (25 µg/kg) the serum levels of TNF were 140 times higher in cirrhotic than in control rats (89,835±21,090 vs. 625±137pg/ml; P<0.001). Serum TNF was 80% lower in cirrhotic rats pretreated with HDL (18,890±5012pg/ml; P<0.001) than in those pretreated with saline. The administration of endotoxin (25 µg/kg) was associated with a significant lower decrease of MAP in cirrhotic rats pretreated with HDL than in those receiving saline (11.9±3.5 vs. 24.7±4.3%; P<0.05). Conclusions : HDL attenuates the increased effect of endotoxin on cytokine production and systemic hemodynamic in cirrhosis.
Keywords: Cirrhosis ;Spontaneous bacterial peritonitis ;Lipopolysaccharides ;Endotoxin ;Cytokines ;Lipoproteins ;High-density lipoprotein
Inflammation and Fibrosis
Connective tissue growth factor induces c-fos gene activation and cell proliferation through p44/42 MAP kinase in primary rat hepatic stellate cells
Runping Gao, DeAnna K. Ball, Bernard Perbal and David R. Brigstock
Background/Aims Connective tissue growth factor (CCN2) is expressed during activation of hepatic stellate cells (HSC) and promotes HSC proliferation, adhesion, and collagen production. The aim of the study was to investigate CCN2 signaling pathways in HSC. Methods Primary HSC were obtained by enzymatic perfusion of rat liver. DNA synthesis was evaluated by [ 3H]thymidine incorporation. Phosphorylation of Elk-1, extracellular signal-regulated kinase (ERK1/2) and focal adhesion kinase (FAK) was evaluated by Western blot. Transcriptional factor binding activity was determined by gel mobility shift assay while c-fos promoter and CCN2 promoter activity was evaluated using luciferase reporters. c-fos mRNA expression was evaluated by Northern blot. Results CCN2 stimulated DNA synthesis and phosphorylation of FAK , Elk-1 and ERK1/2, the latter of which was blocked by heparin. The serum response element binding activity and luciferase reporter activity of the c-fos promoter, together with expression of c-fos, were enhanced by CCN2. CCN2-induced c-fos gene activation, expression and cell proliferation were blocked by inhibiting ERK1/2 with PD98059. CCN2 promoter activity was enhanced by TGF- 1 or PDGF via a Smad7-dependent pathway. Conclusions CCN2-stimulated HSC DNA synthesis is associated with transient induction of c-fos gene activation and expression as well as activation of the ERK1/2 signal pathway.
Liver Cell Injury and Liver Failure
Effects of liver failure on branched-chain -keto acid dehydrogenase complex in rat liver and muscle: comparison between acute and chronic liver failure
Takashi Honda et al.
Background/Aims : Branched-chain -keto acid dehydrogenase (BCKDH) complex catalyses the committed step in the branched-chain amino acid (BCAA) catabolic pathway. In many cases of liver failure, the serum BCAAs/aromatic amino acids ratio (Fisher's ratio) decreases, and BCAAs have been administered to patients with liver failure to correct this ratio. We conducted an animal study to examine whether the effects on hepatic BCKDH complex differ between acute liver failure (ALF) and chronic liver failure (CLF). Methods : ALF and CLF was induced in rats by a single high-dose injection and 21 weeks of repeated low-dose injections of carbon tetrachloride, respectively. Plasma BCAA and branched-chain -keto acid (BCKA) levels, and activities and protein amounts of hepatic BCKDH complex and kinase were measured. Results : ALF was characterized by elevated plasma BCAA and BCKA levels and decreased hepatic BCKDH activity. CLF was characterized by decreased plasma BCAA and BCKA levels and increased hepatic BCKDH activity. This increase in BCKDH activity in CLF was associated with the decreased BCKDH kinase, which is responsible for the BCKDH inactivation. Conclusions : The results obtained in the present study suggest that BCAA catabolism is suppressed in ALF and increased in CLF.
Keywords: Branched-chain amino acid ;Branched-chain -keto acid ;Branched-chain -keto acid dehydrogenase ;Carbon tetrachloride ;Liver cirrhosis ;Muscle Abbreviations: AAA, aromatic amino acid ;ALP, alkaline phosphatase ;ALT, alanine aminotransferase ;AST, aspartate aminotransferase ;BCAA, branched-chain amino acid ;BCAT, branched-chain aminotransferase ;BCKA, branched-chain -keto acid ;BCKDH, branched-chain -keto acid dehydrogenase ;ECL, enhanced chemiluminescence ;PVDF, polyvinylidene difluoride
Induction of Mx-2 in rat liver by toxic injury
Danko S. Batusic, Thomas Armbrust, Bernhard Saile and Giuliano Ramadori
Background/Aims Mx proteins are supposed to be strictly regulated by viruses or interferon-alpha (IFN- ). We used a non-viral model of acute liver injury to study Mx expression. Methods We induced toxic liver injury by CCl 4, and studied the expression of IFN- , IFN- , and IFN-inducible antiviral genes (Mx-2; 2´-5´ oligoadenylate synthetase, 2-5 A; double-stranded RNA-activated protein kinase, PKR). Results Similar to 2-5 A and PKR, Mx-2 gene expression was biphasically induced after CCl 4administration with a maximum at 24 h, and a second peak at 72 h. On protein level, Mx-2 only was up-regulated. IFN- remained constant for the first 24 h while IFN- peaked at 6 h. Thereafter, IFN- increased to a maximum at 72 h while IFN- decreased to 77±4%. Small monocyte-like liver macrophages, but not large macrophages, expressed Mx-2 constitutively. In vitro, IFN- but not IFN- induced Mx-2 in different liver cell populations. IFN- , instead, reduced the susceptibility of liver macrophages to the actions of IFN- .Conclusions Our data suggest that Mx expression does not invariably result from the presence of a viral particle or IFN- synthesis but may represent an innate defensive armamentarium that may be up-regulated without antigen specificity upon liver injury.
Keywords: CCl 4;Mononuclear phagocytes ;Interferon- inducible antiviral genes ;Interferon
Toxicity of low dose azathioprine and 6-mercaptopurine in rat hepatocytes. Roles of xanthine oxidase and mitochondrial injury
Michael J. Tapner, Brett E. Jones, Wan M. Wu and Geoffrey C. Farrell
Background/Aims To study effects of pharmacologic concentrations of azathioprine and 6-mercaptopurine (6-MP) on rat hepatocytes. Methods Hepatocytes cultured on matrigel were incubated with azathioprine or 6-MP; effects of putative protective agents were studied. Viability (LDH leakage), reduced (GSH) and oxidized glutathione (GSSG), mitochondrial (mt) GSH, ATP and ultrastructural changes were determined. Results Azathioprine and 6-MP (0.5-5µmol/l) reduced viability 5-34% at day 1 and 42-92% by day 4. Allopurinol (20 µM) (xanthine oxidase inhibitor) and 2 mM Trolox (vitamin E analog) together provided near complete protection. During culture with azathioprine, GSSG increased before cell death and there was a disproportionate reduction of mtGSH and ATP, together with ultrastructural abnormalities in mitochondria. All changes were prevented by allopurinol and trolox. Discontinuation of 1µmol/l azathioprine restored ATP levels and arrested cell injury, while culture in glucose-enriched media augmented ATP levels and ameliorated cell death. Conclusions Clinically relevant concentrations of azathioprine and 6-MP are toxic to rat hepatocyte cultures by a mechanism that involves oxidative stress, mitochondrial injury and ATP depletion. This can lead to irreversible de-energization and cell death by oncosis (necrosis).
Keywords: Azathioprine ;6-Mercaptopurine ;Hepatotoxicity ;Mitochondrial injury ;Xanthine oxidase
Liver Growth and Cancer
Mechanism of liver regeneration after partial hepatectomy using mouse cDNA microarray
Shinji Togo et al.
Background/Aims : The liver has the capacity to regenerate after partial hepatectomy. In order to clarify the mechanism of liver regeneration, we observed the initial stage, especially the mechanism of gene expression during progress from G0 to S phase (0-24 h), and attempted to identify new genes controlling progress to the S phase. Methods : We applied large-scale gene expression analysis with complementary DNA microarrays in mouse hepatectomy models to clarify the mechanism of liver regeneration after partial hepatectomy. Results : As a result, 23 new immediate-early gene candidates such as interleukin-1 receptor associated kinase-1 and karyopherin -1, which are involved in transportation within the nucleus, were discovered. Candidates for new genes concerned with the progress to the S phase were discovered: inhibitor of DNA binding 2 (ID2) and inhibitor of DNA binding 3 (ID3), both new liver regeneration factors that promoted progress to the S phase, and GADD45 (growth arrest and DNA-damage-inducible protein) as a factor inhibiting that process. Conclusions : The above results not only suggest the importance of NF B in the initial stage of liver regeneration but also points to the orderly maintenance of the proliferation of the cells in liver regeneration.
Keywords: Liver regeneration ;cDNA microarray ;Partial hepatectomy
Rising incidence of intrahepatic cholangiocarcinoma in the United States: a true increase?
Yasser H. Shaib, Jessica A. Davila, Kathryn McGlynn and Hashem B. El-Serag
Background /Aims : The incidence of intrahepatic cholangiocarcinoma (ICC) has been reported to be increasing in the USA. The aim of this study is to examine whether this is a true increase or a reflection of improved detection or reclassification. Methods : Using data from the Surveillance Epidemiology and End Results (SEER) program, incidence rates for ICC between 1975 and 1999 were calculated. We also calculated the proportions of cases with each tumor stage, microscopically confirmed cases, and the survival rates. Results : A total of 2864 patients with ICC were identified. The incidence of ICC increased by 165% during the study period. Most of this increase occurred after 1985. There were no significant changes in the proportion of patients with unstaged cancer, localized cancer, microscopic confirmation, or with tumor size <5 cm during the period of the most significant increase. The 1-year survival rate increased significantly from 15.8% in 1975-1979 to 26.3% in 1995-1999, while 5-year survival rate remained essentially the same (2.6 vs. 3.5%). Conclusions : The incidence of ICC continues to rise in the USA. The stable proportions over time of patients with early stage disease, unstaged disease, tumor size <5 cm, and microscopic confirmation suggest a true increase of ICC.
Keywords: Intrahepatic cholangiocarcinoma ;SEER ;Incidence
Viral Hepatitis
Combination therapy with amantadine and interferon in naïve patients with chronic hepatitis C: meta-analysis of individual patient data from six clinical trials
Alessandra Mangia et al.
Background/Aims In chronic hepatitis C, clinical trials evaluating the efficacy of amantadine (AMA) and interferon (INF) compared to INF monotherapy, have produced conflicting results. We performed a meta-analysis of the individual patient's data from previous studies. Methods Nine hundred and seventy-two patients from six European centres were evaluated by means of individual patient meta-analysis, using mixed models with centres and the centre-treatment interaction fitted as random variables. Results At the end of therapy, virological responses were 38.5% (95% CI 34.1-42.8) after INF and AMA, and 29.5% (95% CI 25.5-33.6) after INF alone (P =0.003). Sustained response occurred in 111 (23.1%; 95% CI 19.3-20.2) and 85 patients (17.3%; 95% CI 14.0-20.7), respectively (P =0.03). Even accounting for the centre effect, therapy with AMA and INF was more effective than IFN alone (P =0.029). When genotypes and viraemia levels were combined, the response rate after combination therapy doubled that observed with IFN alone in all subgroups, except those with low viraemia and genotypes 2 or 3. Conclusions In chronic hepatitis C, therapy with AMA and INF is effective and may be an alternative to INF and ribavirin in patients who cannot tolerate ribavirin.
Keywords: Amantadine ;Interferon ;Chronic hepatitis C ;Meta-analysis ;Randomized clinical trials ;Therapy Abbreviations: HCV, hepatitis C virus ;HCV-RNA, hepatitis C virus ribonucleic acid ;IFN, -interferon ;RCT, randomized controlled trial ;ALT, alanine transaminase ;CI, confidence interval
The impact of steatosis on disease progression and early and sustained treatment response in chronic hepatitis C patients
Heather M. Patton et al.
Background /Aims : Questions remain regarding the etiology of steatosis in hepatitis C, and its impact on disease progression and treatment outcomes. Methods : We evaluated liver biopsies from 574 patients with chronic hepatitis C from a single center. Results : Severity of steatosis was associated with body mass index, HCV genotype 3 infection, age, and duration of infection ( P0.01). Serum HCV RNA levels were associated with severity of steatosis in HCV genotype 3 infection ( P0.03). In HCV genotype 1 infection, fibrosis was associated with severity of steatosis ( P<0.01), and patients who achieved SVR had lesser degrees of pre-treatment steatosis compared to nonresponders (4.6±1.6 vs. 10.1±1.1%, P=0.02). Genotype 1 infected patients with an early virologic response were more likely to have grade 0 steatosis compared to those without an early response (71 vs. 42%; P=0.003). Evaluation of paired biopsies demonstrated a marked decline in steatosis in genotype 3 patients who achieved SVR ( P<0.01). Conclusions : In conclusion, steatosis is an important cofactor in hepatitis C as it is associated with fibrosis and reduces the likelihood of achieving early and sustained virologic response in genotype 1 infected patients. Keywords: Hepatitis C virus ;Liver disease ;Steatosis ;Therapy ;Fibrosis
Complementary and alternative therapies in the treatment of chronic hepatitis C: a systematic review
Joanna Thompson Coon and Edzard Ernst
Background/Aims Hepatitis C is an escalating global health problem. The recommended treatment regimen is associated with considerable expense, adverse effects and poor efficacy in some patients. Complementary therapies are widely promoted for and used by patients with hepatitis C. The aim is to systematically assess the efficacy of complementary therapies in treating chronic hepatitis C. Methods Systematic searches were conducted in six databases, reference lists of all papers were checked for further relevant publications and information was requested from experts. No language restrictions were imposed. Results Twenty-seven eligible randomised clinical trials were located involving herbal products and supplements. No randomised clinical trials were identified for any other complementary therapy. In 14 of the trials, patients received interferon- in combination with the complementary therapy. Less than half the trials (11/27) were of good methodological quality. Compared with the control group, significant improvements in virological and/or biochemical response were seen in trials of vitamin E, thymic extract, zinc, traditional Chinese medicine, Glycyrrhiza glabra and oxymatrine. Conclusions We identified several promising complementary therapies, although extrapolation of the results is difficult due to methodological limitations. More research is warranted to establish the role of these and other therapies in the treatment of hepatitis C.
Keywords: Hepatitis C ;Complementary medicine ;Herbal medicine ;Systematic review
Coarse vs. fine needle aspiration biopsy for the assessment of diffuse liver disease from hepatitis C virus-related chronic hepatitis
Enrico Brunetti et al.
Background/Aims Liver biopsy is crucial in defining natural history and therapeutic choices in chronic hepatitis C and it is usually performed with coarse (>1 mm) needles (CN). As fine needles (FN) do not require anaesthesia, are used over a wider range of coagulation values and allow multiple passes, we compared the diagnostic yield of FN vs. CN biopsies. Methods Paired samples obtained with FN (0.8 mm) and CN (1.2 mm) on 149 consecutive outpatients from a tertiary care institution were evaluated prospectively. Histologic variables were quantitatively scored. Sensitivity, specificity, positive predictive value, negative predictive value and positive likelihood ratio were calculated as measures of diagnostic ability assuming CN as reference. Results FN biopsy was adequate in 83 cases, CN in 140 cases ( P<0.001). Considering the 83 paired adequate specimens, the best sensitivity of FN vs. CN was for portal inflammation (0.95%) and the worst for cirrhosis (0.33%). Overall discriminant ability of FN was unsatisfactory and histologic variables were systematically underscored. Tolerability was good for both procedures. Conclusions The advantages of FN biopsy are lost on its inferior diagnostic performance. Its use in diffuse liver diseases should be restricted to early non-fibrotic lesions.
Keywords: Liver histology ;Microhistology ;Chronic hepatitis ;Liver biopsy
Mutations in the basic core promoter region of hepatitis B virus. Relationship with precore variants and HBV genotypes in a Spanish population of HBV carriers
Rosendo Jardi et al.
Background/Aims : To determine the prevalence and significance of hepatitis B virus (HBV) basic core promoter (BCP) mutations and to establish their relationship with precore (preC) mutations, HBV genotypes and HBV-DNA levels. Methods : BCP and preC mutations and genotypes were determined by sequencing. Results : Genomic analysis was performed in 129 (71%) of 182 patients. BCP mutations were detected in 83% of 18 HBeAg-negative (e ) chronic hepatitis B (CHB) patients with fluctuating ALT levels, and in 76% of 58 e CHB with elevated ALT. The prevalence was lower and similar, 55% in 30 HBeAg-positive CHB (e+ CHB) with elevated ALT and in 23 e inactive carriers. Frequency of preC mutations was higher in e CHB (80%) than in e inactive carriers (65%). Among e CHB, patients with elevated ALT and preC mutations at nt 1896 showed highest HBV-DNA, regardless of BCP mutations. BCP mutations were similar in genotypes A and D, while preC mutations were most common in genotype D (82 vs. 40%). Simultaneous presence of the main BCP (1762, 1764) and preC (1896, 1899) mutations was associated with the degree of histological injury. Conclusions : Combined BCP and preC mutational and genotype analysis provides clinically relevant information in the study of HBV infection.
Keywords: Hepatitis B virus ;Mutations ;HBV-basic core promoter ;HBV-precore region ;HBV genotypes ;HBV-DNA ;Liver cirrhosis
Non-sequencing molecular approaches to identify preS2-defective hepatitis B virus variants proved to be associated with severe liver diseases
Giovanni Raimondo et al.
Background /Aims : PreS2-defective hepatitis B virus (HBV) variants may emerge during chronic HBV infection. These variants carry mutation(s) at the ATG-start-codon and/or in-frame deletion into the preS2 genomic region and are commonly detected by sequencing analyses. We evaluated the prevalence of these variants in a large series of chronic HBV infected patients through non-sequencing molecular approaches. Methods : We examined HBV isolates from 110 HBV carriers: 15 were inactive carriers (IC); 50 had chronic hepatitis (CH); 25 were cirrhotics; 19 had hepatocellular carcinoma (HCC). The entire preS2 genomic region was amplified by PCR technique. The amplicons were processed: (A) through electrophoresis on acrylamide gel to reveal deleted genomes; (B) through electrophoresis on agarose gel after digestion by Nla III enzyme that cuts the wild ATG-start-codon but not the mutated one. Results : We detected preS2 variants in 56/110 cases (51%). In particular, we found preS2-defective mutants in 2/15 IC, 25/50 CH, 13/26 cirrhotics, and 16/19 HCC. The presence of these variants was thus significantly associated with active infection and liver disease ( P<0.002). Moreover, among cases with liver disease preS2-mutants were more prevalent in HCC patients ( P<0.02). Conclusions : Our non-sequencing molecular methods are sensitive and specific, and simplify the identification of all preS2 HBV variant forms. Infection by these variants is significantly associated with active infection and HCC.
Keywords: HBV carriers ;Inactive HBV infection ;Hepatocelluar carcinoma ;HBV heterogeneity ;PreS2-defective HBV variants
Hepatitis B virus enhances transduction of human hepatocytes by SV40-based vectors
Uri Arad, Jonathan Axelrod, Orly Ben-nun-Shaul, Ariella Oppenheim and Eithan Galun
Background/Aims : Chronic HBV infection, a world-wide epidemic, can lead to chronic hepatitis and eventually to cirrhosis and hepatocellular carcinoma. The liver poses obstacles for many available gene-transfer vectors. SV40-based vectors can transduce human hepatic and hematopoietic cells. We studied the effect of HBV on the transduction - efficiency of human hepatic cells by SV40 - based vectors. Methods : A SV40-vector carrying the luciferase gene, and wild-type SV40, were used to assess transduction efficiency of human HBV-positive and HBV-negative hepatic cells. Transduction efficiency was measured as luciferase activity or by T-antigen staining. To evaluate whether differences in transduction efficiency are due to cell recognition and/or nuclear transport, MHC-I receptors were measured by FACS analysis and SV40-DNA was extracted from the nuclei of transduced cells and quantified. Results : Two HBV-positive cell-lines, HepG2.2.2.15 and FLC4-A10II, were transduced significantly more efficiently than their parental HBV-negative cell-lines. Transient transfection of HuH-7 cells with the HBV genome also increased transduction efficiency. The level of MHC-I, the cellular receptor for SV40, was comparable in all the cell-lines studied. However, soon after infection with SV40, the nuclei of HepG2.2.2.15 contained >6-fold more SV40-DNA than HepG2. Conclusions : HBV increases transduction by SV40-vectors. This is due to enhanced vector entry and/or transport into the nucleus. SV40-vectors appear to have a potential for gene therapy for the treatment of HBV infections.
Keywords: SV40 ;HBV ;Liver ;Gene therapy
Hepatitis C virus Core Antigen as a predictor of non-response in genotype 1 chronic hepatitis C patients treated with peginterferon -2b plus ribavirin
Background/Aims Chronic hepatitis C patients infected by genotype 1 are the least responsive to combination therapy and therefore monitoring response is important in identifying non-responders quickly, permitting therapy discontinuation and avoiding side effects and costs. We examined the usefulness of measuring total HCV Core Ag in early treatment with peginterferon -2b and ribavirin in genotype 1 patients in the prediction of response and compared the results with those from HCV RNA quantification. Methods Two hundred and sixty-eight serum samples from 46 genotype 1 patients receiving combination therapy were examined for HCV Core Ag and quantitative HCV RNA. Results At baseline, mean HCV RNA and HCV Core Ag concentrations were significantly lower in sustained virologic responders than in non-responders. The negative predictive value of HCV Core Ag testing in predicting non-response at week 12 is 100%, and for a 2log drop in HCV RNA, using two quantitative tests, it is 88%. Conclusions HCV Core Ag determination allows the identification of non-responders with only one test at week 12 and permits stopping therapy in these patients. HCV Core Antigen testing is cheaper and easier to perform than HCV RNA quantification.
Keywords: Pegylated interferon ;Ribavirin ;HCV Core Ag ;Hepatitis C virus RNA ;Hepatitis C ;Therapy ;Sustained virologic response
Copyright © 2001-2004 European Association for the Study of the Liver. All rights reserved.
Copyright © 2004 BMJ Publishing Group Ltd
Volume 350 March 11, 2004 Number 11
Hepatitis B Virus Infection Natural History and Clinical Consequences
Don Ganem, M.D., and Alfred M. Prince, M.D.
In the past 10 years, remarkable strides have been made in the understanding of the natural history and pathogenesis of hepatitis B virus (HBV) infection. In this article we will review these advances, with particular reference to the implications for antiviral therapy. History Clinical and epidemiologic studies began to differentiate among various types of acute hepatitis in the decades after World War II. The groundbreaking studies of Krugman and colleagues in 1967 firmly established the existence of at least two types of hepatitis, 1one of which (then called serum hepatitis, and now called hepatitis B) was parenterally transmitted. Links to . . .
The New England Journal of Medicine is owned, published, and copyrighted © 2004 Massachusetts Medical Society. All rights reserved.
The Lancet, published, and copyrighted © 2004. All rights reserved.
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