HEPATOLOGY
Volume 45, Issue 3, March 2007
Original Articles
Lactulose improves cognitive functions and health-related quality of life in patients with cirrhosis who have minimal hepatic encephalopathy (p 549-559)
Srinivasa Prasad, Radha K. Dhiman, Ajay Duseja, Yogesh K. Chawla, Arpita Sharma, Ritesh Agarwal
Minimal hepatic encephalopathy (MHE) has a negative effect on patients' daily functioning. Thus far, no study has investigated the effect of treatment-related improvement in cognitive functions on health-related quality of life (HRQOL). We measured psychometric performance by number and figure connection tests parts A and B, picture completion, and block design tests and HRQOL by the Sickness Impact Profile (SIP) of 90 patients with cirrhosis on inclusion into the study and 3 months later. A Z score less than -2 on the neuropsychological (NP) tests was considered abnormal. Sixty-one (67.7%) patients had MHE. They were randomly assigned in a 1:1 ratio to receive treatment (lactulose) for 3 months (n = 31) or no treatment (n = 30) in a nonblinded design. The mean number of abnormal NP tests decreased significantly in patients in the treated group (baseline, 2.74 [95% CI 2.40-3.08]; after 3 months, .75 [95% CI .36-1.16]) compared with patients in the untreated group (baseline, 2.47 [95% CI 2.19-2.74]; after 3 months, 2.55 [95% CI 2.16-2.94]); multivariate analysis of variance (MANOVA) for time and treatment, P = 0.001. The mean total SIP score improved among patients in the treated group (baseline, 10.39 [95% CI 9.36-11.43]; after 3 months, 3.77 [95% CI 2.52-5.02]) compared with patients in the untreated group (baseline, 10.36 [95% CI 8.98-11.73]; after 3 months, 10.39 [95% CI 8.36-12.42]); MANOVA for time and treatment, P = 0.002. Improvement in HRQOL was related to the improvement in psychometry. Conclusion: Treatment with lactulose improves both cognitive function and HRQOL in patients with cirrhosis who have MHE.
Isoleucine infusion during simulated upper gastrointestinal bleeding improves liver and muscle protein synthesis in cirrhotic patients (p 560-568)
Steven W. M. Olde Damink, Rajiv Jalan, Nicolaas E. P. Deutz, Cornelis H. C. Dejong, Doris N. Redhead, Paula Hynd, Peter C. Hayes, Peter B. Soeters
Upper gastrointestinal (GI) bleeding in cirrhotic patients has a high incidence of mortality and morbidity. Postbleeding catabolism has been hypothesized to be partly due to the low biological value of hemoglobin, which lacks the essential amino acid isoleucine. The aims were to study the metabolic consequences of a simulated upper GI bleed in patients with cirrhosis of the liver and the effects of intravenous infusion of isoleucine. Portal drained viscera, liver, muscle, and kidney protein kinetics were quantified using a multicatheterization technique during routine portography. Sixteen overnight-fasted, metabolically stable patients who received an intragastric infusion of an amino acid solution mimicking hemoglobin every 4 hours were randomized to saline or isoleucine infusion and received a mixture of stable isotopes (L-[ring-2H5]phenylalanine, L-[ring-2H4]tyrosine, and L-[ring-2H2]tyrosine) to determine organ protein kinetics. This simulated bleed resulted in hypoisoleucinemia that was attenuated by isoleucine infusion. Isoleucine infusion during the bleed resulted in a positive net balance of phenylalanine across liver and muscle, whereas renal and portal drained viscera protein kinetics were unaffected. In the control group, no significant effect was shown. Conclusion: The present study investigated hepatic and portal drained viscera protein metabolism selectively in humans. The data show that hepatic and muscle protein synthesis is stimulated by improving the amino acid composition of the upper GI bleed by simultaneous intravenous isoleucine administration.
Final results of a double-blind, placebo-controlled trial of the antifibrotic efficacy of interferon-1b in chronic hepatitis C patients with advanced fibrosis or cirrhosis (p 569-578)
Paul J. Pockros, Lennox Jeffers, Nezam Afdhal, Zachary D. Goodman, David Nelson, Robert G. Gish, K. Rajender Reddy, Robert Reindollar, Maribel Rodriguez-Torres, Sarah Sullivan, Lawrence M. Blatt, Sima Faris-Young
Interferon-1b (IFN-1b) is a pleiotropic cytokine that displays antifibrotic, antiviral, and antiproliferative activity. A total of 502 patients with compensated liver disease and an Ishak fibrosis score of 4-6 were randomized in a double-blind, placebo-controlled study, and 488 of these patients received subcutaneous injections of IFN-1b 100 g (group 1, n = 169), IFN-1b 200 g (group 2, n = 157), or placebo (group 3, n = 162) 3 times a week for 48 weeks. Most patients (83.6%) had cirrhosis at baseline (Ishak score = 5 or 6). Posttreatment liver biopsies were assessed in a blinded fashion for a reduction of 1 or more Ishak points (primary endpoint). Four hundred twenty patients with pretreatment and posttreatment liver biopsies were evaluable and showed no improvement in Ishak score between the 3 treatment groups (12.1%, 12.4%, and 16% of patients in groups 1, 2, and 3, respectively; P > 0.05). Analysis of IFN--inducible biomarkers revealed that interferon-inducible T cell-alpha chemoattractant (ITAC), an IFN--inducible CXCR3 chemokine was an independent predictor of stable or improving Ishak score. IFN-1b was well tolerated. There were similar numbers of deaths in all 3 arms (5, 5, and 4, respectively), and most were related to complications of cirrhosis. Conclusion: IFN-1b therapy was not able to reverse fibrosis in patients with advanced liver disease for 1 year. Subgroups of patients with elevated ITAC levels and perhaps less advanced disease may be considered for future studies with IFN-
Sustained virological response to interferon- is associated with improved outcome in HCV-related cirrhosis: A retrospective study (p 579-587)
Savino Bruno, Tommaso Stroffolini, Massimo Colombo, Simona Bollani, Luisa Benvegnù, Giuseppe Mazzella, Antonio Ascione, Teresa Santantonio, Felice Piccinino, Pietro Andreone, Alessandra Mangia, Giovanni B. Gaeta, Marcello Persico, Stefano Fagiuoli, Piero L. Almasio, on behalf of the Italian Association of the Study of the Liver Disease (AISF)
The effect of achieving a sustained virological response (SVR) following interferon- (IFN) treatment on the clinical outcomes of patients with HCV-related cirrhosis is unknown. In an attempt to assess the risk of liver-related complications, HCC and liver-related mortality in patients with cirrhosis according to the response to IFN treatment, a retrospective database was developed including all consecutive patients with HCV-related, histologically proven cirrhosis treated with IFN monotherapy between January 1992 and December 1997. SVR was an undetectable serum HCV-RNA by PCR 24 weeks after IFN discontinuation. HCC was assessed by ultrasound every 6 months. Independent predictors of all outcomes were assessed by Cox regression analysis. Of 920 patients, 124 (13.5%) were classified as achieving a SVR. During a mean follow-up of 96.1 months (range: 6-167) the incidence rates per 100 person-years of liver-related complications, HCC and liver-related death were 0, 0.66, and 0.19 among SVR and 1.88, 2.10, and 1.44 among non-SVR (P < 0.001 by log-rank test). Multivariate analyses found that non-SVR was associated with a higher risk of liver-related complications (hazard ratio, HR, not applicable), HCC (HR 2.59; 95% CI 1.13-5.97) and liver-related mortality (HR 6.97; 95% CI 1.71-28.42) as compared to SVR. Conclusion: Thus, in patients with HCV-related, histologically proven cirrhosis, achievement of a SVR after IFN therapy was associated with a reduction of liver-related mortality lowering both the risk of complications and HCC development. Irrespective of SVR achievement, all patients should continue surveillance because the risk of occurrence of HCC was not entirely avoided.
Dysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection (p 588-601)
Amalia Penna, Massimo Pilli, Alessandro Zerbini, Alessandra Orlandini, Sergio Mezzadri, Luca Sacchelli, Gabriele Missale, Carlo Ferrari
The functional impairment of HCV-specific T cell responses is believed to be an important determinant of HCV persistence, but the functional T cell defects of patients with chronic hepatitis C (CH-C) are only partially defined. CD8 responses to HLA-A2-restricted epitopes of HCV and other unrelated viruses were studied in 23 HLA-A2-positive patients both ex vivo and after in vitro culture. Degranulation capacity, intracellular perforin, and granzyme-A content and cytokine production (IFN-, TNF-) by HCV- and non-HCV-specific CD8 cells were tested both ex vivo and in vitro, whereas cytolytic activity was studied after 10 days' expansion in vitro. Memory maturation and role of exhaustion were assessed ex vivo by HCV-specific CD8 staining for CD127 and PD-1, and in vitro after peripheral blood mononuclear cells (PBMC) culture in the presence of anti-PD-L1 monoclonal antibodies. IFN- production and cytolytic activity were expressed less efficiently by HCV-specific than by non-HCV specific CD8 cells derived from the same CH-C patients. The amount of stored granzyme-A within single cells was always lower in HCV-specific CD8 cells, which were less efficient also in the release of lytic granules and in the production of TNF-. The CD8 dysfunction was associated with high PD-1 expression by most HCV-specific CD8 cells, and PD-1/PD-L1 blockade by anti-PD-L1 antibodies in vitro was able to improve the HCV-specific CD8 function. Conclusion: Our study characterizes CD8 defects that may be important in maintaining HCV persistence; identification of strategies to correct these defects may help to define novel approaches to treat HCV infection.
Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity (p 602-613)
Christine S. Rollier, Glaucia Paranhos-Baccala, Ernst J. Verschoor, Babs E. Verstrepen, Joost A. R. Drexhage, Zahra Fagrouch, Jean-Luc Berland, Florence Komurian-Pradel, Blandine Duverger, Nourredine Himoudi, Caroline Staib, Marcus Meyr, Mike Whelan, Joseph A. Whelan, Victoria A. Adams, Esther Larrea, José I. Riezu, Juan José Lasarte, Birke Bartosch, Francois-L. Cosset, Willy J. M. Spaan, Helmut M. Diepolder, Gerd R. Pape, Gerd Sutter, Genevieve Inchauspe, Jonathan L. Heeney
Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P = 0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 (1) and PD-1 levels in liver correlated with clearance or chronicity. Conclusion: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.
Immunosuppression reactivates viral replication long after resolution of woodchuck hepatitis virus infection (p 614-622)
Stephan Menne, Paul J. Cote, Scott D. Butler, Ilia A. Toshkov, John L. Gerin, Bud C. Tennant
Resolution of hepatitis B virus (HBV) infection is characterized by coordinated humoral and cellular immune responses. Immunity is durable over decades, protecting the host from reinfection and potential activation of residual HBV. Woodchucks infected at birth with woodchuck hepatitis virus (WHV) cleared viremia and developed antibodies to surface antigen (anti-WHs). Woodchucks became seronegative for anti-WHs 3-6 years later, but in some, WHV DNA was detected in serum, liver, and/or peripheral blood mononuclear cells (PBMCs). Those with WHV DNA had increased in vitro cellular immune responses to viral antigens, CD4 and CD8 markers, and Th1-type cytokines, suggesting active WHV-specific T lymphocytes. Immunosuppression for 12 weeks using cyclosporine A in such woodchucks resulted in transient reactivation of WHV replication. Serum of 1 woodchuck that became positive for WHV DNA during immunosuppression was inoculated into WHV-susceptible woodchucks, and a productive infection was demonstrated. The results indicate that after infection durable cellular immunity to WHV is essential for the long-term control of viral replication and is probably maintained by continuous priming from residual virus. Conclusion: These experimental observations demonstrate the potential of immunosuppression to reactivate HBV after resolution of infection.
Impact of aboriginal ethnicity on HCV core-induced IL-10 synthesis: Interaction with IL-10 gene polymorphisms (p 623-630)
Koko Bate Aborsangaya, Iga Dembinski, Suresh Khatkar, Martin Prince Alphonse, Peter Nickerson, Julia D. Rempel
The host immune response is a critical determinant in viral infection outcome. Epidemiological studies indicate that North American indigenous peoples are more resistant to chronic HCV infection than other populations. Due to the prominence of IL-10 in chronic HCV infection, we investigated the genetic tendency to produce IL-10 in Caucasian (CA) and First Nation (FN) populations. Peripheral blood mononuclear cells (PBMCs) from CA subjects had a greater tendency to produce IL-10 defined by allelic polymorphisms, as well as genotypes and haplotypes, at the -1082, -819, and -592 positions of the IL-10 promoter. More importantly, we directly evaluated the influence of ethnicity on the ability of HCV core protein to induce IL-10 synthesis and found significantly higher IL-10 production by PBMCs isolated from healthy CA subjects compared with FN subjects. Further examination of the underlying relationship between core-induced IL-10 with the high, intermediate, and low phenotypes at the -1082, -819, and -592 position revealed that spontaneous and core-induced IL-10 synthesis tended to interact negatively with defined polymorphisms. This was particularly evident for the FN cohort, in which the relationship was strengthened by a stronger interaction of core with the low-IL-10-producing phenotypes. As with previous studies, concanavalin A induced IL-10 synthesis from the CA cohort positively associated with defined genetic phenotypes. Conclusion: Cells from FN subjects had a reduced capacity to produce IL-10 in response to HCV core protein, suggesting that reduced susceptibility of FN immunity to virally induced IL-10 synthesis might contribute to epidemiological observations of enhanced HCV clearance.
Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes (p 631-638)
Marta Casado, Belén Mollá, Rosa Roy, Amalia Fernández-Martínez, Carme Cucarella, Rafael Mayoral, Lisardo Boscá, Paloma Martín-Sanz
Cyclooxygenase-2 (COX-2) is upregulated in many cancers, and the prostanoids synthesized increase proliferation, improve angiogenesis, and inhibit apoptosis in several tissues. To explore the function of COX-2 in liver, transgenic (Tg) mice were generated containing a fusion gene (LIVhCOX-2) consisting of human COX-2 cDNA under the control of the human ApoE promoter. Six lines were developed; all of them expressed the LIVhCOX-2 transgene selectively in hepatocytes. The Tg mice exhibited a normal phenotype, and the increased levels of PGE2 found were due to the constitutively expressed COX-2. Histological analysis of different tissues and macroscopic examination of the liver showed no differences between wild-type (Wt) and Tg animals. However, Tg animals were resistant to Fas-mediated liver injury, as demonstrated by low levels of plasmatic aminotransferases, a lesser caspase-3 activation, and Bax levels and an increase in Bcl-2, Mcl-1, and xIAP proteins, when compared with the Wt animals. Moreover, the resistance to Fas-mediated apoptosis is suppressed in the presence of COX-2-selective inhibitors, which prevented prostaglandin accumulation in the liver of Tg mice. Conclusion: These results demonstrate that expression of COX-2-dependent prostaglandins exerted a protection against liver apoptosis.
Cardiotrophin-1 is an essential factor in the natural defense of the liver against apoptosis (p 639-648)
Juan M. Marquès, Idoia Belza, Bettina Holtmann, Diane Pennica, Jesus Prieto, Matilde Bustos
We previously reported that exogenous cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, exerts hepatoprotective effects. Because CT-1 is expressed in the normal liver, we hypothesized that this cytokine may constitute an endogenous defense of the liver against proapoptotic stimuli. Here, we found that CT-1-/- mice died faster than wild-type animals after challenge with a lethal dose of the Fas agonist Jo-2. At sublethal doses of Jo-2, all wild-type mice survived whereas CT-1-/- animals developed extensive hepatocyte apoptosis with 50% mortality at 24 hours. Pretreatment with CT-1 improved survival and reduced injury in both CT-1-/- and wild-type animals. Upon Fas ligation the activation of STAT-3, a molecule that defends the liver against apoptosis, was lower in CT-1-/- mice than in wild-type animals despite similar IL-6 up-regulation in the 2 groups. Analysis of liver transcriptome in CT-1-/- and wild-type mice showed that 9 genes reported to be associated with cell survival/death functions were differentially expressed in the 2 groups. Four of these genes [IGFBP1, peroxiredoxin3, TNFR1, and calpastatin (endogenous inhibitor of calpain)] could be validated by real-time PCR. All of them were down-regulated in CT-1-/- mice and were modulated by CT-1 administration. Treatment of CT-1-/- animals with the calpain inhibitor MDL28170 afforded significant protection against Fas-induced liver injury. Conclusion: CT-1-/- mice are highly sensitive to Fas-mediated apoptosis due in part to deficient STAT-3 activation and inadequate control of calpain activity during the apoptotic process. Our data show that CT-1 is a natural defense of the liver against apoptosis. This cytokine may have therapeutic potential.
TRAIL/bortezomib cotreatment is potentially hepatotoxic but induces cancer-specific apoptosis within a therapeutic window (p 649-658)
Ronald Koschny, Tom M. Ganten, Jaromir Sykora, Tobias L. Haas, Martin R. Sprick, Armin Kolb, Wolfgang Stremmel, Henning Walczak
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents a novel promising anticancer biotherapeutic. However, TRAIL-resistant tumor cells require combinatorial regimens to sensitize tumor but not normal cells for TRAIL-induced apoptosis. Here, we investigated the mechanism of the synergistic antitumor effect of bortezomib in combination with TRAIL in hepatoma, colon, and pancreatic cancer cells in comparison to the toxicity in primary human hepatocytes (PHH). TRAIL cotreatment at high but clinically relevant concentrations of bortezomib caused toxicity in PHH which potentially limits the clinical applicability of bortezomib/TRAIL cotreatment. However, at low concentrations of bortezomib TRAIL-resistant hepatoma, colon and pancreatic cancer cell lines but not PHH were efficiently sensitized for TRAIL-induced apoptosis. RNA interference and TRAIL receptor blockage experiments revealed that in bortezomib-treated hepatoma cells TRAIL-R1/TRAIL-R2 up-regulation, enhanced TRAIL DISC formation and cFLIPL down-regulation in addition to accumulation of Bak cooperatively sensitized for TRAIL. Bim, although accumulated upon bortezomib treatment, did not play a causal role for TRAIL sensitization in Hep3b cells. Combined treatment with bortezomib and TRAIL massively reduced the clonogenic capacity of hepatoma cells in vitro. Surviving clones could be resensitized for repeated TRAIL treatment. Conclusion: Bortezomib/TRAIL cotreatment bears the risk of severe hepatotoxicity at high but clinically relevant concentrations of bortezomib. However, within a wide therapeutic window bortezomib sensitized different cancer cells but not PHH for TRAIL-induced apoptosis.
A sensitive bead assay for antimitochondrial antibodies: Chipping away at AMA-negative primary biliary cirrhosis (p 659-665)
Sabine Oertelt, Roman Rieger, Carlo Selmi, Pietro Invernizzi, Aftab A. Ansari, Ross L. Coppel, Mauro Podda, Patrick S.C. Leung, M. Eric Gershwin
The antimitochondrial response in primary biliary cirrhosis (PBC) is the most highly directed and specific self-reacting antibody in human immunopathology. Originally, antimitochondrial antibodies (AMAs) were detected by indirect immunofluorescence (IIF) and found in approximately 90% of well-documented patients with PBC. The introduction of recombinant autoantigens and the use of immunoblotting have increased the sensitivity and specificity of AMAs, and they are now considered positive in approximately 95% of patients with PBC. Clearly, accurate autoantibody detection represents one of the fundamental requirements for reliable diagnostics in autoimmunity. To address the 5% of AMA-negative patients with PBC, we have generated and validated a bead assay for the detection of AMA. We enrolled 120 patients with PBC, including a non-random group of 30 rigorously proven AMA-negative patients, 50 healthy subjects, and 74 controls with autoimmune diseases (18 with primary sclerosing cholangitis, 16 with autoimmune hepatitis, and 40 with systemic lupus erythematosus). Individual bead assays were done with the three mitochondrial autoantigens, PDC-E2, BCOADC-E2, and OGDC-E2. As expected, 90 of 90 previously known AMA-positive patients remained positive with this assay but, interestingly, 20% of the rigorously defined AMA-negative patient group had antibodies to one or more of the mitochondrial autoantigens. Furthermore, 100% of these newly detected AMA-positive patients were anti-nuclear antibody (ANA) positive. Conclusion: The development of this assay reflects the potential for automated detection with rapid and reliable assaying and further highlights the diminished number of truly AMA-negative PBC patients.
Sertraline as a first-line treatment for cholestatic pruritus (p 666-674)
Marlyn J. Mayo, Iorna Handem, Sandra Saldana, Heidi Jacobe, Yonas Getachew, A. John Rush
Pruritus is frequently the most debilitating symptom of cholestatic liver diseases. Moreover, existing therapies are often ineffective. Recent small, retrospective case series reports suggest that serotonin reuptake inhibitors can improve pruritus. This study was undertaken to establish the dose of sertraline and to evaluate its efficacy for cholestatic pruritus. Twenty one subjects with chronic pruritus due to liver disease (including primary biliary cirrhosis, primary sclerosing cholangitis, chronic hepatitis C, and postnecrotic cirrhosis) initially underwent an open-label, dose escalation to determine the dose with optimal efficacy and tolerability. After a washout period, 12 of the subjects entered a randomized, double-blind, placebo-controlled trial. Participants quantified their pruritus using a 0-10 visual analog scale, and pruritus was assessed for distribution, timing, degree of disability, and physical evidence of scratching. The optimum sertraline dose (75-100 mg/day) was well tolerated. In the controlled portion of the study, itch scores improved in patients taking sertraline, but worsened in patients taking placebo (P = 0.009). Changes in itch distribution, duration, direction, and physical evidence of scratching paralleled changes in the visual analog pruritus score. Conclusion: Sertraline seems to be an effective, well-tolerated treatment for pruritus due to chronic liver disease. These results suggest that serotonergic pathways are important in the perception of itch.
Hepatic abnormalities in patients with chronic granulomatous disease (p 675-683)
Nadeem Hussain, Jordan J. Feld, David E. Kleiner, Jay H. Hoofnagle, Reyes Garcia-Eulate, Sushil Ahlawat, Deloris E. Koziel, Victoria Anderson, Dianne Hilligoss, Peter Choyke, John I. Gallin, T. Jake Liang, Harry L. Malech, Steven M. Holland, Theo Heller
Chronic granulomatous disease (CGD) is a rare congenital disorder characterized by repeated bacterial and fungal infections. Aside from a high incidence of liver abscess, little is known about hepatic involvement in CGD. The aim of this study was to describe the spectrum of liver abnormalities seen in CGD. The charts of 194 patients with CGD followed at the NIH were reviewed, with a focus on liver abnormalities. Liver enzyme elevations occurred on at least one occasion in 73% of patients during a mean of 8.9 years of follow-up. ALT elevations were generally transient. Although transient alkaline phosphatase (ALP) elevations were also common, persistent ALP elevations lasting up to 17.6 years were seen in 25% of patients. Liver abscess occurred in 35% of patients. Drug-induced hepatotoxicity was documented in 15% of patients but likely occurred more frequently. Hepatomegaly was found in 34% and splenomegaly in 56% of patients. Liver histology showed granulomata in 75% and lobular hepatitis in 90% of specimens. Venopathy of the portal vein was common (80%) and associated with splenomegaly. Venopathy of the central vein was also common (63%) and was associated with the number of abscess episodes. Nodular regenerative hyperplasia (NRH) was seen in 9 patients, including 6 of 12 autopsy specimens. Conclusion: Liver enzyme abnormalities occur frequently in patients with CGD. In addition to liver abscesses and granulomata, drug hepatotoxicity is likely underappreciated. Vascular lesions such as venopathy and - to a lesser extent - NRH are common. The cause and clinical consequences of venopathy await prospective evaluation.
SgIGSF is a novel biliary-epithelial cell adhesion molecule mediating duct/ductule development (p 684-694)
Akihiko Ito, Yuji Nishikawa, Kazuhiro Ohnuma, Ikuyo Ohnuma, Yu-ichiro Koma, Ayuko Sato, Katsuhiko Enomoto, Tohru Tsujimura, Hiroshi Yokozaki
Spermatogenic immunoglobulin superfamily (SgIGSF) is an intercellular adhesion molecule of the nectin-like family. While screening its tissue distribution, we found that it was expressed in fetal liver but not adult liver. In the present study, we examined which cells in developing and regenerating liver express SgIGSF via immunohistochemistry and Western blot analysis. In developing mouse liver, SgIGSF expression was transiently upregulated at perinatal ages and was restricted to the lateral membrane of biliary epithelial cells (BECs). In regenerating rat livers from the 2-acetylaminofluorene/partial hepatectomy model, SgIGSF was detected exclusively in oval cells that aligned in ductal and trabecular patterns by the second week posthepatectomy. In human livers, fetal and newborn bile ducts and cirrhotic bile ductules were clearly positive for SgIGSF, whereas disease-free adult bile ducts were negative. To investigate the role of SgIGSF in bile duct/ductule formation, we used an in vitro model in which rat hepatocyte aggregates embedded in collagen gels containing insulin and epidermal growth factor extend epithelial sheets and processes in the first week and form ductules within a month. The process and ductular cells were continuously positive for SgIGSF and cytokeratin 19, a BEC marker. When the aggregate culture was started in the presence of a function-blocking anti-SgIGSF antibody, the number of epithelial processes per aggregate was reduced by 80%. Conclusion: We propose that SgIGSF is a novel and functional BEC adhesion molecule that is expressed for a limited time during active bile duct/ductule formation.
The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells (p 695-704)
Verena Keitel, Roland Reinehr, Petros Gatsios, Claudia Rupprecht, Boris Görg, Oliver Selbach, Dieter Häussinger, Ralf Kubitz
Sinusoidal endothelial cells (SEC) constitute a permeable barrier between hepatocytes and blood. SEC are exposed to high concentrations of bile salts from the enterohepatic circulation. Whether SEC are responsive to bile salts is unknown. TGR5, a G-protein-coupled bile acid receptor, which triggers cAMP formation, has been discovered recently in macrophages. In this study, rat TGR5 was cloned and antibodies directed against the C-terminus of rat TGR5 were developed, which detected TGR5 as a glycoprotein in transfected HepG2-cells. Apart from Kupffer cells, TGR5 was detected in SEC of rat liver. SEC expressed TGR5 over the entire acinus, whereas endothelial cells of the portal or central veins were not immunoreactive toward TGR5 antibodies. In isolated SEC, TGR5 mRNA and protein were detected by reverse transcription (RT) PCR, immunofluorescence microscopy, and Western blot analysis. Bile salts increased cAMP in isolated SEC and induced mRNA expression of endothelial NO synthase (eNOS), a known cAMP-dependent gene. In addition, bile acids activated eNOS by phosphorylation of eNOS at amino acid position 1177. In line with eNOS activation, bile acids induced NO production in liver slices. This is the first report on the expression of TGR5 in SEC. Conclusion: The data suggest that SEC are directly responsive toward specific bile salts. Regulation of eNOS in SEC by TGR5 connects bile salts with hepatic hemodynamics. This is of particular importance in cholestatic livers when bile salt concentrations are increased.
Revelation of simple and complex liver acini after portal transplantation of pancreatic islets or thyroid follicles in rats (p 705-715)
Frank Dombrowski, Matthias Evert
The microarchitecture of the liver is still not completely understood although various concepts of structural liver organization have been proposed. Among them, Rappaport's liver acinus stands out as one of the most accepted models. The correctness of this model, however, has also been doubted, and its applicability is hampered by the fact that the outlines of the liver acinus are disguised and nobody was ever able to give visual evidence by unmasking a simple liver acinus from the surrounding liver tissue. After intraportal transplantation of pancreatic islets or thyroid follicles into diabetic or thyroidectomized rats, respectively, the transplants engraft in small portal tracts and morphologically alter the downstream liver tissue due to excessive hormone secretion. Using a combined approach of perfusion fixation, stereomicroscopy, and light microscopy, we demonstrate in this study that these foci of altered liver tissue represent simple and complex liver acini, exactly as described by Rappaport. We present stereomicroscopical and histological examples of all important cut levels of altered simple and complex liver acini, including their topographical relation to the supplying and draining vessels and to the central vein liver lobule. Moreover, by computer-aided reconstruction of serial semi-thin sections, we were able to present the first 3-dimensional images of simple and complex liver acini. Conclusion: Our results prove the correctness of Rappaport's acinus model and confirm the simple liver acinus as the principal microcirculatory unit of the liver.
Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver (p 716-724)
Hongchao Zhou, Leslie E. Rogler, Lewis Teperman, Glyn Morgan, Charles E. Rogler
Hepatocyte function and regeneration are severely compromised in severe liver disease, and a common sequela is cirrhosis. Structural changes caused by cirrhosis create a cellular environment conducive to the formation of ductular reactions (DRs). Ductular reactions are primarily composed of oval cells also known as intermediate hepatobiliary cells. We have conducted single, double, and triple staining to study lineages of oval cells present in DRs. Staining with NCAM, CK19, and HepPar1 has revealed a distinctly bipolar structure to DRs that are embedded in cirrhotic tissue. Spatial analysis of cells that are singly HepPar1-positive, or CK19-positive, has revealed hepatocytic and biliary poles, respectively, in the DRs. Also, the location of singly NCAM-positive cells in DRs suggests that they may be bipotent liver stem/progenitor cells. The locations of other intermediate hepatobiliary cells, which have combinations of markers, suggest that CK19+/NCAM+ cells are transitional cells in the biliary lineage and that rare cells that are negative for all three markers are transitional cells in the hepatocytic lineage. A working cell lineage model for DRs is presented.
Diagnostic value of HSP70, glypican 3, and glutamine synthetase in hepatocellular nodules in cirrhosis (p 725-734)
Luca Di Tommaso, Giada Franchi, Young Nyun Park, Barbara Fiamengo, Annarita Destro, Emanuela Morenghi, Marco Montorsi, Guido Torzilli, Maurizio Tommasini, Luigi Terracciano, Luigi Tornillo, Raffaella Vecchione, Massimo Roncalli
Hepatocellular nodules in cirrhosis include regenerative (large regenerative, LRN) and dysplastic (low and high grade, LGDN and HGDN) nodules, early and grade 1 HCC (eHCC-G1), and overt HCC. The differential diagnosis may be particularly difficult when lesions such as HGDN and eHCC-G1 are involved. We investigated the diagnostic yield of a panel of 3 putative markers of hepatocellular malignancy such as HSP70, glypican 3 (GPC3), and glutamine synthetase (GS). We selected 52 surgically removed nonmalignant nodules (15 LRNs, 15 LGDNs, 22 HGDNs) and 53 HCCs (10 early, 22 grade 1, and 21 grade 2-3) and immunostained them for HSP70, GPC3, and GS. The sensitivity and specificity of the individual markers for the detection of eHCC-G1 were 59% and 86% for GS, 69% and 91% for GPC3, and 78% and 95% for HSP70. We identified 2 main phenotypes: (1) all negative, seen in 100% LRN and LGDN, 73% HGDN and 3% eHCC-G1; (2) all positive, a feature detected in less than half the eHCC-G1. Using a 3-marker panel, when at least 2 of them, regardless which, were positive, the sensitivity and specificity for the detection of eHCC-G1 were respectively 72% and 100%; the most sensitive combination was HSP70+/GPC3+ (59%) when a 2-marker panel was used. Conclusion: The adopted panel of 3 markers is very helpful in distinguishing eHCC-G1 from dysplastic nodules arising in cirrhosis.
Decreased hepatocyte membrane potential differences and GABAa-3 expression in human hepatocellular carcinoma (p 735-745)
Gerald Y. Minuk, Manna Zhang, Yuewen Gong, Leonard Minuk, Hans Dienes, Norman Pettigrew, Michael Kew, Jeremy Lipschitz, Dongfeng Sun
To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and -aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (-19.8 ± 1.3 versus -25.9 ± 3.2 mV, respectively [P < 0.05]), and GABAA-3 expression was down-regulated (GABAA-3 mRNA and protein expression in HCC; 5,693 ± 1,385 and 0.29 ± 0.11 versus 11,046 ± 4,979 copies/100 mg RNA and 0.62 ± 0.16 optical density in adjacent tumor tissues, respectively [P = 0.002 and P < 0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down-regulation, human malignant hepatocytes deficient in GABAA-3 receptor expression (Huh-7 cells) were transfected with GABAA-3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n = 16-17 group). Tumors developed after a mean (± SD) of 51 ± 6 days (range: 41-60 days) in 7/16 (44%) mice injected with vector-transfected cells and 70 ± 12 days (range: 59-86 days) in 4/17 (24%) mice injected with GABAA-3 cDNA-transfected cells (P < 0.005). Conclusion: The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA-3 receptor expression is down-regulated in human HCC, and (3) restoration of GABAA-3 receptor expression results in attenuated in vivo tumor growth in nude mice.
Combined GM-CSF and IL-12 gene therapy synergistically suppresses the growth of orthotopic liver tumors (p 746-754)
Chun-Jung Chang, Yi-Hsiang Chen, Kai-Wen Huang, Hao-Wei Cheng, Suit-Fong Chan, Kuo-Feng Tai, Lih-Hwa Hwang
Immunotherapy is often effective only for small tumor burdens and, in many cases, is restricted to subcutaneous tumors. Here, we investigated the antitumor effects of combination therapy with GM-CSF and IL-12 on orthotopic liver tumors with intermediate or large tumor volumes, or on chemically-induced multifocal liver tumors in animals. Adenoviruses encoding GM-CSF or IL-12 were injected intratumorally to animals bearing transplanted tumors, or injected via intrahepatic artery in animals with primary multifocal liver tumors induced by diethylnitrosamine. Our results demonstrated that IL-12, but not GM-CSF, monotherapy displayed significant therapeutic effects, whereas combination therapy with both cytokines displayed synergistic antitumor effects not only on transplanted tumor models with intermediate or large tumor loads, but also on carcinogen-induced multifocal liver tumors. Effector cell analyses, revealed by in vivo cell subset depletion, flow cytometry analysis, and immunohistochemical staining of tumor infiltrates, indicated that NK cells were the prominent antitumor effectors for the IL-12-mediated antitumor activity, whereas CD8+ T cells, NKT cells, and macrophages were more important than NK cells in the combination therapy-mediated antitumor effects. Both IL-12 monotherapy and combination therapy could induce various types of effectors and high levels of IFN-; however, the latter induced much higher levels than the former, which may explain why combination therapy is superior to IL-12 monotherapy. Conclusion: Combination therapy with GM-CSF and IL-12 represents a promising immunotherapy strategy for treating orthotopic, widespread liver tumors.
Prevention of hepatocyte allograft rejection in rats by transferring adenoviral early region 3 genes into donor cells (p 755-766)
Elena V. Mashalova, Chandan Guha, Namita Roy-Chowdhury, Laibin Liu, Ira J. Fox, Jayanta Roy-Chowdhury, Marshall S. Horwitz
Hepatocyte transplantation is being evaluated as an alternative to liver transplantation for metabolic support during liver failure and for definitive treatment of inherited liver diseases. However, as with liver transplantation, transplantation of allogeneic hepatocytes requires prolonged immunosuppression with its associated untoward effects. Therefore, we explored strategies for the genetic modification of donor hepatocytes that could eliminate allograft rejection, obviating the need for immunosuppression. Products of early region 3 (AdE3) of the adenoviral genome are known to protect infected cells from immune recognition and destruction. In the present study we showed that immortalized rat hepatocytes that had been stably transduced with AdE3 before transplantation into fully MHC-mismatched rats are protected from allograft rejection. Quantitative real-time PCR analysis showed that a similar number of engrafted AdE3-transfected hepatocytes had survived in syngeneic and allogeneic recipients. AdE3 expression did not reduce expression of MHC class I on the surfaces of donor hepatocytes. Consistent with this, the in vivo cytotoxic cell-mediated alloresponse was attenuated but not abolished in recipients of AdE3-transfected allogeneic hepatocytes. In contrast, graft survival correlated with a marked reduction in cell-surface localization of Fas receptor in the transplanted cells and inhibition of Fas-mediated apoptosis, which are related to the antiapoptotic functions of the AdE3 proteins. CONCLUSION:AdE3 gene products prevent hepatocyte allograft rejection mainly by protecting the cells from the effector limb of the host immune response and could be used as a tool to facilitate allogeneic hepatocyte transplantation.
Novel aspects of PPAR-mediated regulation of lipid and xenobiotic metabolism revealed through a nutrigenomic study (p 767-777)
Pascal G. P. Martin, Hervé Guillou, Frédéric Lasserre, Sébastien Déjean, Annaig Lan, Jean-Marc Pascussi, Magali SanCristobal, Philippe Legrand, Philippe Besse, Thierry Pineau
Peroxisome proliferator-activated receptor- (PPAR) is a major transcriptional regulator of lipid metabolism. It is activated by diverse chemicals such as fatty acids (FAs) and regulates the expression of numerous genes in organs displaying high FA catabolic rates, including the liver. The role of this nuclear receptor as a sensor of whole dietary fat intake has been inferred, mostly from high-fat diet studies. To delineate its function under low fat intake conditions (4.8% w/w), we studied the effects of five regimens with contrasted FA compositions on liver lipids and hepatic gene expression in wild-type and PPAR-deficient mice. Diets containing polyunsaturated FAs reduced hepatic fat stores in wild-type mice. Only sunflower, linseed, and fish oil diets lowered hepatic lipid stores in PPAR-/- mice, a model of progressive hepatic triglyceride accumulation. These beneficial effects were associated, in particular, with dietary regulation of 9-desaturase in both genotypes, and with a newly identified PPAR-dependent regulation of lipin. Furthermore, hepatic levels of 18-carbon essential FAs (C18:26 and C18:33) were elevated in PPAR-/- mice, possibly due to the observed reduction in expression of the 6-desaturase and of enoyl-coenzyme A isomerases. Effects of diet and genotype were also observed on the xenobiotic metabolism-related genes Cyp3a11 and CAR. Conclusion: Together, our results suggest that dietary FAs represent - even under low fat intake conditions - a beneficial strategy to reduce hepatic steatosis. Under such conditions, we established the role of PPAR as a dietary FA sensor and highlighted its importance in regulating hepatic FA content and composition.
Impairment of hepatic Stat-3 activation and reduction of PPAR activity in fructose-fed rats (p 778-788)
Núria Roglans, Laia Vilà, Mireia Farré, Marta Alegret, Rosa María Sánchez, Manuel Vázquez-Carrera, Juan Carlos Laguna
Fructose makes up a significant proportion of energy intake in westernized diets; its increased consumption has paralleled the growing prevalence of obesity and metabolic syndrome over the past two decades. In the current study, we demonstrate that fructose administration (10% wt/vol) in the drinking water of rats reduces the trans-activating and trans-repressing activity of the hepatic peroxisome proliferator-activated receptor (PPAR). As a consequence, fructose decreases hepatic fatty oxidation and increases pro-inflammatory transcription factor nuclear factor B (NF-B) activity. These changes were not observed in glucose-administered rats (10% wt/vol), although both carbohydrates produced similar changes in plasma adiponectin and in the hepatic expression of transcription factors and enzymes involved in fatty acid synthesis. Fructose-fed, but not glucose-fed, rats were hyperleptinemic and exhibited increased tyrosine phosphorylation of the signal transducer and activator of transcription-3 (STAT-3) transcription factor, although they did not present a similar increase in the serine phosphorylation of nuclear STAT3. Thus, an impairment in the hepatic transduction of the leptin signal could be responsible for the observed alterations in PPAR activity in fructose-fed rats. Because PPAR activity is lower in human than in rodent liver, fructose ingestion in humans should cause even worse effects, which would partly explain the link between increased consumption of fructose and widening epidemics of obesity and metabolic syndrome. Conclusion: Hypertriglyceridemia and hepatic steatosis induced by fructose ingestion result from a reduction in the hepatic catabolism of fatty acids driven by a state of leptin resistance.
MELD score as a predictor of liver failure and death in patients with acetaminophen-induced liver injury (p 789-796)
Lars E. Schmidt, Fin Stolze Larsen
The Model for End-Stage Liver Disease (MELD) scoring system has been established as a reliable measure of short-term mortality risk in patients with end-stage chronic liver disease. The aim of this study was to evaluate the prognostic value of the MELD scoring as a predictor of fulminant hepatic failure (FHF) and death in patients with acetaminophen poisoning. Prospectively, serial measurements of the 3 MELD components - INR, bilirubin, and creatinine - were performed in 460 patients with acetaminophen-induced liver injury. Starting on the first day after the day of overdose, MELD score was significantly higher in patients who eventually developed hepatic encephalopathy (HE) than in those who did not. HE developed in 63 of 142 patients with a MELD score above 18 at 48-72 hours after the overdose (positive predictive value 44%) compared with 2 of 182 patients with a MELD score of 18 or below (negative predictive value 99%). Among 124 patients with FHF, a threshold MELD score of 33 on the day after the onset of HE had sensitivity of 60%, specificity of 69%, positive predictive value of 65%, and negative predictive value of 63%. However, the discriminative power of MELD score was not superior to that of INR alone or of the King's College Hospital criteria. Conclusion: MELD score may be useful as a predictor of FHF in patients admitted with acetaminophen toxicity. However, as a predictor of death from FHF, MELD score did not provide more information than the King's College Hospital criteria or INR alone.
Copyright © 2007 by the American Association for the Study of Liver Diseases. All rights reserved.
GASTROENTEROLOGY
Volume 132, Issue 2, February 2007
Clinical - Alimentary Tract
Peptic Ulcer and Bleeding Events Associated With Rofecoxib in a 3-Year Colorectal Adenoma Chemoprevention Trial, 14 November 2006
Lanas A, Baron JA, Sandler RS, Horgan K, Bolognese J, Oxenius B, Quan H, Watson D, Cook TJ, Schoen R, Burke C, Loftus S, Niv Y, Ridell R, Morton D, Bresalier R
Background & Aims: Our aim was to establish the incidence of symptomatic upper gastrointestinal ulcers, ulcer perforation, ulcer obstruction, or bleeding episodes (PUBs) associated with the use of selective cyclooxygenase-2 inhibitors at standard clinical doses compared with placebo. We report here on the PUB outcomes associated with the use of rofecoxib 25 mg in a 3-year, multicenter, double-blind, placebo-controlled trial designed to determine the effect of rofecoxib on the risk of recurrent neoplastic polyps of the colon. Methods: A total of 2587 patients with a history of colorectal adenomas underwent randomization to 25 mg/day of rofecoxib or to placebo. Investigator-reported PUBs were adjudicated by an external blinded committee. Kaplan–Meier and Cox proportional hazards techniques were used to estimate incidence and relative risks of PUBs in an intention-to-treat analysis. Results: Patients assigned to rofecoxib had a higher incidence of confirmed PUBs than those randomized to placebo (.88 vs .18 events per 100 patient-years; relative risk, 4.9; 95% confidence interval, 1.98–14.54). The incidence of confirmed complicated PUBs (ulcer perforation, obstruction, or bleeds) was low, but was numerically higher in the rofecoxib than in the placebo group (.23 vs .06 events per 100 patient-years; relative risk, 3.8; 95% confidence interval, .72–37.46; P = .14). Rofecoxib increased the incidence of confirmed PUBs vs placebo in both low-dose aspirin users and nonusers. Conclusions: Among patients with a history of colorectal adenomas, the long-term use of 25 mg/day of rofecoxib was associated with an increased risk of clinically relevant upper gastrointestinal events when compared with placebo.
Risk of Upper Gastrointestinal Complications Among Users of Traditional NSAIDs and COXIBs in the General Population, 6 December 2006
García Rodríguez LA, Barreales Tolosa L
Background & Aims: Traditional nonaspirin, nonsteroidal anti-inflammatory drugs (tNSAIDs) have been associated with a 3- to 5-fold increased risk in upper gastrointestinal complications (UGIC). Whether use of selective inhibitors of cyclooxygenase-2 (COXIBs) will translate into a clinically relevant reduced toxicity has not been widely investigated in the general population. Methods: We conducted a nested case control study using The Health Improvement Network Database identifying 1561 cases of UGIC between January 2000 and 2005. A random sample of 10,000 controls was frequency matched to the cases by age, sex, and calendar year. Results: The adjusted relative risk (RR) of UGIC associated with current use was 3.7 (95% CI: 3.1–4.3) for tNSAIDs and 2.6 (95% CI: 1.9–3.6) for COXIBs. Daily dose was a predictor of increased risk for both tNSAIDs and COXIBs. Users of tNSAIDs with a prolonged plasma half-life or slow release formulations had an augmented risk of UGIC. Overall, the estimate of RR associated with COXIBs was 0.8 (95% CI: 0.6–1.1) compared with current use of tNSAIDs, and, among nonusers of aspirin, the corresponding estimate of RR associated with COXIBs was 0.6 (95% CI: 0.4–0.9). Conclusions: COXIBs present a better upper gastrointestinal safety than tNSAIDs, although the risk of UGIC for an individual drug is determined by its daily dose and plasma drug exposure in addition to its selectivity for cyclooxygenase-2. Also, concomitant use of aspirin is a strong effect modifier of COXIBs that negates the superior gastrointestinal safety over tNSAIDs in the absence of aspirin use.
Low Colectomy Rates in Ulcerative Colitis in an Unselected European Cohort Followed for 10 Years, 20 November 2006
Hoie O, Wolters FL, Riis L, Bernklev T, Aamodt G, Clofent J, Tsianos E, Beltrami M, Odes S, Munkholm P, Vatn M, Stockbrügger RW, Moum B, European Collaborative Study Group of Inflammatory Bowel Disease
Background & Aims: The colectomy rate in ulcerative colitis (UC) is related to morbidity and to treatment decisions made during disease course. The aims of this study were to determine the colectomy risk in UC in the first decade after diagnosis and to identify factors that may influence the choice of surgical treatment. Methods: In 1991-1993, 781 UC patients from 9 centers located in 7 countries in northern and southern Europe and in Israel were included in a prospective inception cohort study. After 10 years of follow-up, 617 patients had complete medical records, 73 had died, and 91 had been lost to follow-up. Results: There were no significant differences in age, sex, or disease extent at diagnosis between patients followed for 10 years and those lost to follow-up. The 10-year cumulative risk of colectomy was 8.7%: 10.4% in the northern and 3.9% in the southern European centers (P < .001). Colectomy was more likely in extensive colitis than in proctitis, with an adjusted hazard ratio (HR) of 4.1 (95% CI: 2.0–8.4). Compared with the southern centers, the adjusted HR was 2.7 (95% CI: 1.3–5.6) for The Netherlands and Norway together and 8.2 (95% CI: 3.6–18.6) for Denmark. Age at diagnosis, sex, and smoking status at diagnosis had no statistically significant influence on colectomy rates. Conclusions: The colectomy rate was found to be lower than that in previous publications, but there was a difference between northern and southern Europe. Colectomy was associated with extensive colitis, but the geographic variations could not be explained.
A Meta-Analysis of the Placebo Rates of Remission and Response in Clinical Trials of Active Ulcerative Colitis, 30 December 2006
Su C, Lewis JD, Goldberg B, Brensinger C, Lichtenstein GR
Background & Aims: Knowledge of the placebo outcomes and understanding specific study features that influence these outcomes is important for designing future clinical trials evaluating therapy of ulcerative colitis (UC). The aims of this study were to estimate the placebo rates of remission and response in placebo-controlled, randomized clinical trials for active UC and to identify factors influencing these rates. Methods: We performed a systematic review and meta-analysis of placebo-controlled, randomized clinical trials evaluating therapies for active UC identified from MEDLINE from 1966 through 2005. Results: Forty studies met the inclusion criteria. The pooled estimates of the placebo rates of remission and response were 13% (95% confidence interval, 9%–18%; range, 0%–40%; median, 12%) and 28% (95% confidence interval, 23%–33%; range, 0%–67%; median, 30%), respectively, both with significant heterogeneity. Studies that used more stringent definitions of outcomes had lower placebo rates of remission and response. Study duration, number of study visits, disease duration, baseline composite and rectal bleeding scores of the disease activity index, and inclusion of endoscopic mucosal healing as the remission definition all were associated with the placebo remission rate. Conclusions: Rates of remission in the placebo arm of UC clinical trials ranges from 0% to 40%. The placebo remission rates are influenced by the trial length, number of study visits, use of stricter remission definitions, and design features that enroll patients with more active disease. These factors should be considered when designing future placebo-controlled clinical trials in patients with active UC.
Homozygous PMS2 Deletion Causes a Severe Colorectal Cancer and Multiple Adenoma Phenotype Without Extraintestinal Cancer, 6 December 2006
Will O, Carvajal-Carmona LG, Gorman P, Howarth KM, Jones AM, Polanco-Echeverry GM, Chinaleong JA, Günther T, Silver A, Clark SK, Tomlinson I
Background & Aims: We report a patient of Indian descent with parental consanguinity, who developed 10 carcinomas and 35 adenomatous polyps at age 23 and duodenal adenocarcinoma at age 25. He also had dysmorphic features, mental retardation, and café-au-lait spots but no brain tumor. We aimed to establish his molecular diagnosis. Methods: Germ-line screening for APC and MYH/MUTYH mutations was normal as was immunohistochemistry for MLH1 and MSH2 proteins. Investigation by array-comparative genomic hybridization revealed deletion of a small region on chromosome 7. Using polymerase chain reaction, this region was refined to a 400-kilobase deletion, which included exons 9–15 of the PMS2 gene, and all coding regions of oncomodulin, TRIAD3, and FSCN1. Results: The deletion was confirmed as homozygous, and both parents were carriers. Immunohistochemistry showed absent PMS2 expression in all tumors and normal tissue. Most tumors showed microsatellite instability, more marked at dinucleotide than mononucleotide repeats. The tumors harbored no somatic mutations in APC, BRAF, AXIN2, or ?-catenin, but KRAS2 and TGFBR2 mutations were found. Conclusions: Our patient represents a novel phenotype for homozygous PMS2 mutation and perhaps the most severe colorectal cancer phenotype—in terms of numbers of malignancies at an early age—described to date. PMS2 mutations—and perhaps other homozygous mismatch repair mutations—should be considered in any patient presenting with multiple gastrointestinal tumors, since our patient could not be distinguished clinically from cases with attenuated familial adenomatous polyposis or MUTYH-associated polyposis.
Clinical - Liver, Pancreas, and Biliary Tract
Quantification of Liver Glucose Metabolism by Positron Emission Tomography: Validation Study in Pigs, 30 December 2006
Iozzo P, Jarvisalo MJ, Kiss J, Borra R, Naum GA, Viljanen A, Viljanen T, Gastaldelli A, Buzzigoli E, Guiducci L, Barsotti E, Savunen T, Knuuti J, Haaparanta–Solin M, Ferrannini E, Nuutila P
Background & Aims: The liver is inaccessible to organ balance measurements in humans. To validate [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography (PET) in the quantification of hepatic glucose uptake (HGU), we determined [18F]FDG modeling parameters, lumped constant (LC), and input functions (single arterial versus dual). Methods: Anesthetized pigs were studied during fasting (n = 6), physiologic (n = 4), and supraphysiologic (n = 4) hyperinsulinemia. PET was performed with C15O (blood pool) and [18F]FDG (glucose uptake). 6,6-Deuterated glucose ([2H]G) was coinjected with [18F]FDG and blood collected from the carotid artery and portal and hepatic veins to compute LC as ratio between tracers fractional extraction. HGU was estimated from PET images and ex vivo from high-performance liquid chromatography measurements of liver [18F]FDG versus [18F]FDG-6-phosphate and [18F]-glycogen. Endogenous glucose production was measured with [2H]G and hepatic blood flow by flowmeters. Results: HGU was increased in hyperinsulinemia versus fasting (P < .05). Fractional extraction of [18F]FDG and [2H]G was similar (not significant), intercorrelated (r = 0.98, P < .0001), and equally higher during hyperinsulinemia than fasting (P ≤ .05), with an LC of 0.98 ± 0.10 and 1.18 ± 0.26, respectively. [18F]FDG-PET modeling provided HGU values that did not differ from, and were correlated with, those from ex vivo measurements (r = 0.61, P ≤ .02); proportional estimates of liver perfusion and endogenous glucose production were also obtained. Single and dual input functions produced strongly intercorrelated results (r > 0.95, P < .0001), with a modest underestimation of HGU by the former. Conclusions: [18F]FDG-PET–derived parameters provide accurate quantification of HGU and estimates of liver perfusion and glucose production. In the liver, LC of [18F]FDG is nearly unitary. Using a single arterial input introduces only a small error in estimation of HGU.
Identification of a Hepatitis B Virus S Gene Mutant in Lamivudine-Treated Patients Experiencing HBsAg Seroclearance, 6 December 2006
Hsu CW, Yeh CT, Chang ML, Liaw YF
Background & Aims: Seroclearance of hepatitis B virus (HBV) surface antigen (HBsAg) is a rare event in chronic hepatitis B patients receiving lamivudine therapy. It is generally believed to be a benevolent sign, implicating clearance of viremia. The aim of this study is to examine the authenticity of this dogma. Methods: In a 5-year period, 11 patients treated with lamivudine experienced seroclearance of HBsAg. The clinical data were examined. The HBV S gene sequences derived from the patient’s serum samples before and after seroclearance of HBsAg were analyzed. Results: Serum HBV-DNA could be detected by nested polymerase chain reaction (PCR) in all 11 patients, by 1-step PCR in 8, and by Cobas Amplicor HBV-DNA test (>200 copies/mL) in 5. A mutation hot spot, P120A in the S gene, was identified in 6 of the 11 patients. Site-directed mutagenesis experiments indicated that the Ausria-II RIA test failed to detect this mutant. Decreased sensitivity of detection was also observed when other monoclonal antibodies were applied. Conclusions: Seroclearance of HBsAg during lamivudine therapy may not indicate viral clearance. Specifically, it may be caused by a point mutation in the S gene, which results in detection failure. In such patients, further verification and follow-up using a sensitive HBV-DNA test are advised.
Basic - Alimentary Tract
Extracellular Superoxide Production by Enterococcus faecalis Promotes Chromosomal Instability in Mammalian Cells, 6 December 2006
Wang X, Huycke MM
Background & Aims: We investigated whether Enterococcus faecalis, a Gram-positive intestinal commensal that produces extracellular superoxide, could promote chromosomal instability (CIN) in mammalian cells. Methods: We measured the ability of E faecalis to promote CIN using hybrid hamster cells (ALN) containing human chromosome 11. Results: E faecalis promoted CIN in ALN cells with average mutant fractions per 105 survivors (±SD) of 72.3 ± 6.7 at 1 ? 109 cfu mL?1 compared with 22.2° ± 4.5 for the no bacteria control. ?-Irradiation at 2 Gray similarly resulted in 74.7 ± 5.7 mutant clones per 105 survivors. Deletions in chromosome 11 consistent with CIN were verified in 80% of mutant clones. E faecalis-treated ALN cells were protected from CIN by superoxide dismutase, ?-tocopherol, and cyclooxygenase-2 (COX-2) inhibitors. In a dual-chamber tissue culture model designed to mimic stromal-epithelial cell interactions, macrophages pretreated with E faecalis grown on permeable supports increased mutant fractions 2.5-fold for ALN cells. COX-2 was up-regulated by superoxide from E faecalis and mutant fractions decreased when COX-2 was silenced using short interfering RNA. Escherichia coli, a Gram-negative commensal that produces negligible extracellular superoxide, only modestly promoted CIN in this model. Conclusions: These findings indicate that macrophage COX-2 is induced by superoxide from E faecalis and promotes CIN in mammalian cells through diffusible factors. This mechanism links the oxidative physiology of E faecalis to propagation of genomic instability through a bystander effect, and offers a novel theory for the role of commensal bacteria in the etiology of sporadic colorectal cancer.
Soluble Proteins Produced by Probiotic Bacteria Regulate Intestinal Epithelial Cell Survival and Growth, 23 November 2006
Yan F, Cao H, Cover TL, Whitehead R, Washington MK, Polk DB
Background & Aims: Increased inflammatory cytokine levels and intestinal epithelial cell apoptosis leading to disruption of epithelial integrity are major pathologic factors in inflammatory bowel diseases. The probiotic bacterium Lactobacillus rhamnosus GG (LGG) and factors recovered from LGG broth culture supernatant (LGG-s) prevent cytokine-induced apoptosis in human and mouse intestinal epithelial cells by regulating signaling pathways. Here, we purify and characterize 2 secreted LGG proteins that regulate intestinal epithelial cell antiapoptotic and proliferation responses. Methods: LGG proteins were purified from LGG-s, analyzed, and used to generate polyclonal antibodies for immunodepletion of respective proteins from LGG-conditioned cell culture media (CM). Mouse colon epithelial cells and cultured colon explants were treated with purified proteins in the absence or presence of tumor necrosis factor (TNF). Akt activation, proliferation, tissue injury, apoptosis, and caspase-3 activation were determined. Results: We purified 2 novel proteins, p75 (75 kilodaltons) and p40 (40 kilodaltons), from LGG-s. Each of these purified protein preparations activated Akt, inhibited cytokine-induced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. TNF-induced colon epithelial damage was significantly reduced by p75 and p40. Immunodepletion of p75 and p40 from LGG-CM reversed LGG-CM activation of Akt and its inhibitory effects on cytokine-induced apoptosis and loss of intestinal epithelial cells. Conclusions: p75 and p40 are the first probiotic bacterial proteins demonstrated to promote intestinal epithelial homeostasis through specific signaling pathways. These findings suggest that probiotic bacterial components may be useful for preventing cytokine-mediated gastrointestinal diseases.
NOD2 Variants and Antibody Response to Microbial Antigens in Crohn’s Disease Patients and Their Unaffected Relatives, 14 November 2006
Devlin SM, Yang H, Ippoliti A, Taylor KD, Landers CJ, Su X, Abreu MT, Papadakis KA, Vasiliauskas EA, Melmed GY, Fleshner PR, Mei L, Rotter JI, Targan SR
Background & Aims: The Cdcs1 locus of the C3Bir mouse confers severe colitis associated with a decrease in innate immune function and an increase in adaptive T-cell responses to commensal bacterial products. The aim of our study was to determine if defects in innate immunity are similarly associated with increased adaptive immune responses to microbial antigens in Crohn’s disease patients. Methods: Sera from 732 patients, 220 unaffected relatives, and 200 healthy controls were tested for antibodies to oligomannan, the Pseudomonas fluorescens–related protein, Escherichia coli outer membrane porin C, CBir1 flagellin, and DNA from the same subjects was tested for 3 Crohn’s disease–associated variants of the NOD2 gene, and 5 toll-like receptor (TLR) 2, 2 TLR4, and 2 TLR9 variants. The magnitude of responses to microbial antigens was examined according to variant status. Results: NOD2 variant carriage increased in frequency with increasing number of positive antibodies and increasing cumulative quantitative response as measured by quartile sum (P for trend, .0008 and .0003, respectively). Mean antibody and quartile sums were higher for patients carrying any NOD2 variant versus those carrying none (2.24 vs 1.92 and 10.60 vs 9.72; P = .0008 and P = 0.0003, respectively). The mean quartile sum was higher for unaffected relatives carrying any NOD2 variant versus those carrying none (10.67 vs 9.75, respectively; P = .02). No association was found between any TLR variant and the magnitude of response. Conclusions: Patients with Crohn’s disease and unaffected relatives carrying variants of the NOD2 gene have increased adaptive immune responses to microbial antigens.
Cell-Cell Contacts Prevent Anoikis in Primary Human Colonic Epithelial Cells, 20 November 2006
Hofmann C, Obermeier F, Artinger M, Hausmann M, Falk W, Schoelmerich J, Rogler G, Grossmann J
Background & Aims: Colonic epithelial cells (CECs) receive important survival signals from the extracellular matrix and undergo detachment-induced apoptosis (anoikis) as soon as they lose their cell-matrix anchorage. In contrast to the established role of cell-matrix contact, the role of cell-cell contacts as a physiologic survival factor for CECs is less clear. Methods: Intact CEC crypts gently centrifuged to form a cell aggregate in which cell-cell contacts were maintained. Induction of apoptosis was assessed by Western Blot analysis, colorimetric assays, DNA electrophoresis, 4?,6-diamidino-2-phenylindole staining, and flow cytometry. Activation of survival pathways was analyzed by Western blot. The role of mitogen-activated protein kinase/extracellular signal–regulated kinase (MEK)/extracellular signal–regulated kinase (Erk)1/2, epidermal growth factor receptor, phosphatidylinositol 3-kinase (PI3-K), and Src signaling was investigated using specific inhibitors. Results: Despite a complete loss of cell-matrix adhesion after CEC isolation, activation of caspases was blocked and anoikis was prevented when cell-cell contacts were preserved. CECs with preserved cell-cell contacts exhibited a rapid dephosphorylation of focal adhesion kinase. Aggregated CECs had stable levels of active ?-catenin and phosphorylated Akt, Erk1/2, and epidermal growth factor receptor, but CECs undergoing anoikis rapidly degraded ?-catenin and dephosphorylated Akt. Inhibition of Src- and PI3-K–dependent signaling reversed the antiapoptotic effect of cell-cell contact preservation, while inhibition of the MEK pathway had no effect. Conclusions: Integrity of cell-cell contacts compensates for the loss of cell-matrix contact-mediated survival signals in CECs and prevents apoptosis. Cell-cell contact-triggered CEC survival involves antiapoptotic signaling through ?-catenin-, Src-, and PI3-K/Akt- but not through MEK- and focal adhesion kinase–dependent pathways.
Adiponectin Deficiency Protects Mice From Chemically Induced Colonic Inflammation, 23 November 2006
Fayad R, Pini M, Sennello JA, Cabay RJ, Chan L, Xu A, Fantuzzi G
Background & Aims: Adiponectin (APN) is an adipokine that regulates insulin sensitivity and is anti-inflammatory in atherosclerosis. The goal of this study was to investigate the role of APN in intestinal inflammation. Methods: APN knockout (KO) mice and their wild-type (WT) littermates received dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce intestinal inflammation. Clinical and histologic scores and proliferation of epithelial cells were assessed. Cytokines and APN levels were measured. Expression of APN and heparin binding epidermal growth factor (HB-EGF) was analyzed by immunohistochemistry. Expression of APN and its receptors, HB-EGF, and basic fibroblast growth factor (bFGF) messenger RNA was assessed by reverse-transcription polymerase chain reaction. Association of serum APN with HB-EGF and bFGF was studied by coimmunoprecipitation. Results: APN KO mice are protected from chemically induced colitis; administration of APN restores inflammation. APN is expressed in the colon, luminal APN associates with colonic epithelial cells. In vitro, APN increases production of proinflammatory cytokines from colonic tissue. Expression of colonic APN overlaps with that of bFGF and HB-EGF, which play a protective role in colitis. Circulating APN binds to bFGF and HB-EGF, likely inhibiting their protective activity. Inhibition of EGF receptor signaling, which is required for biologic activity of HB-EGF, restores inflammation in APN KO mice. Conclusions: APN deficiency is associated with protection from chemically induced colitis. APN exerts proinflammatory activities in the colon by inducing production of proinflammatory cytokines and inhibiting bioactivity of protective growth factors. Thus, in colitis, APN exerts an opposite role compared with atherosclerosis.
The Vanilloid Receptor Initiates and Maintains Colonic Hypersensitivity Induced by Neonatal Colon Irritation in Rats, 14 November 2006
Winston J, Shenoy M, Medley D, Naniwadekar A, Pasricha PJ
Background & Aims: Robust chemical or mechanical irritation of the colon of neonatal rats leads to chronic visceral hypersensitivity. The clinical and physiologic relevance of such noxious stimulation in the context of human irritable bowel syndrome is questionable. The aims of this study were to determine whether mild chemical irritation of the colon of neonatal rats produced persistent changes in visceral sensitivity and to evaluate the role of transient receptor potential vanilloid 1 (TRPV1) in the initiation and maintenance of visceral hypersensitivity. Methods: Ten-day-old rat pups received an intracolonic infusion of 0.5% acetic acid in saline. TRPV1 inhibitors were administered 30 minutes before acetic acid sensitization. Sensitivity of the colon to balloon distention (CRD) in adults was measured by grading their abdominal withdrawal reflex and electromyographic responses. In adult rats, TRPV1 antagonist was injected intraperitoneally 30 minutes before CRD. Results: Neonatal acetic acid treatment resulted in higher sensitivity to CRD in adult rats compared with controls in the absence of histopathologic signs of inflammation. Treatment of colons of adult rats with acetic acid did not produce persistent sensitization. Antagonism of the TRPV1 before neonatal administration of acetic acid and after established visceral hypersensitivity attenuated sensitivity to CRD. TRPV1 expression was increased in dorsal root ganglia–containing colon afferent neurons. Conclusions: We have described a new model for persistent colonic sensory dysfunction following a transient noxious stimulus in the neonatal period and a potentially important role for TRPV1 in initiation and maintenance of persistent visceral hypersensitivity.
The Intestinal Wnt/TCF Signature, 23 August 2006
Van der Flier LG, Sabates–Bellver J, Oving I, Haegebarth A, De Palo M, Anti M, Van Gijn ME, Suijkerbuijk S, Van de Wetering M, Marra G, Clevers H
Background & Aims: In colorectal cancer, activating mutations in the Wnt pathway transform epithelial cells through the inappropriate expression of a TCF4 target gene program, which is physiologically expressed in intestinal crypts. Methods: We have now performed an exhaustive array-based analysis of this target gene program in colorectal cancer cell lines carrying an inducible block of the Wnt cascade. Independently, differential gene-expression profiles of human adenomas and adenocarcinomas vs normal colonic epithelium were obtained. Results Expression analyses of approximately 80 genes common between these data sets were performed in a murine adenoma model. The combined data sets describe a core target gene program, the intestinal Wnt/TCF signature gene set, which is responsible for the transformation of human intestinal epithelial cells. Conclusions The genes were invariably expressed in adenomas, yet could be subdivided into 3 modules, based on expression in distinct crypt compartments. A module of 17 genes was specifically expressed at the position of the crypt stem cell.
Activin Type 2 Receptor Restoration in MSI-H Colon Cancer Suppresses Growth and Enhances Migration With Activin, 20 November 2006
Jung BH, Beck SE, Cabral J, Chau E, Cabrera BL, Fiorino A, Smith EJ, Bocanegra M, Carethers JM
Background & Aims: Colon cancers with high-frequency microsatellite instability (MSI-H) develop frameshift mutations in tumor suppressors as part of their pathogenesis. ACVR2 is mutated at its exon 10 polyadenine tract in >80% of MSI-H colon cancers, coinciding with loss of protein. ACVR2 transmits the growth effects of activin via phosphorylation of SMAD proteins to affect gene transcription. The functional effect of activin in colon cancers has not been studied. We developed and characterized a cell model in which we studied how activin signaling affects growth. Methods: hMLH1 and ACVR2 mutant HCT116 cells were previously stably transferred with chromosome 2 (HCT116+chr2), restoring a single regulated copy of wild-type ACVR2 but not hMLH1. Both HCT116+chr2 and parental HCT116 cells (as well as HEC59 and ACVR2 and hMSH2 complemented HEC59+chr2 cells) were assessed for genetic complementation and biologic function. Results: HCT116+chr2 cells and HEC59+chr2 cells, but not ACVR2-mutant HCT116 or HEC59 cells, acquired wild-type ACVR2 as well as expression of ACVR2 wild-type messenger RNA. Complemented ACVR2 protein complexed with ACVR1 with activin treatment, generating nuclear phosphoSMAD2 and activin-specific gene transcription. ACVR2-restored cells showed decreased growth and reduced S phase but increased cellular migration following activin treatment. ACVR2 small interfering RNA reversed these effects in complemented cells. Conclusions: ACVR2-complemented MSI-H colon cancers restore activin-SMAD signaling, decrease growth, and slow their cell cycle following ligand stimulation but show increased cellular migration. Activin is growth suppressive and enhances migration similar to transforming growth factor ? in colon cancer, indicating that abrogation of the effects of activin contribute to the pathogenesis of MSI-H colon cancers.
A Simple Method for the Routine Detection of Somatic Quantitative Genetic Alterations in Colorectal Cancer, 6 December 2006
Killian A, Di Fiore F, Le Pessot F, Blanchard F, Lamy A, Raux G, Flaman J, Paillot B, Michel P, Sabourin J, Tuech J, Michot F, Kerckaert J, Sesboüé R, Frebourg T
Background & Aims: Several quantitative genetic alterations have been suggested to have in colorectal cancer (CRC) either a prognostic or a therapeutic predictive value. Routine detection of these alterations is limited by the absence of simple methods.
Methods: The somatic quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) is based on the simultaneous amplification under quantitative conditions of several dye-labeled targets both from tumor and nonmalignant tissues. For each patient, the resulting QMPSF fluorescent profiles are superimposed, and quantitative changes are simply detected by an increase or decrease of the corresponding fluorescent peaks. Two assays were developed and applied to 57 CRC: a “bar code” exploring several loci with known prognostic value and a “kinogram” studying the copy number change of kinase genes, against which inhibitors have been developed.
Results: The bar code revealed that the most frequent alterations were the gain of AURKA/20q13 (53%) and MYC/8q24 (39%) and heterozygous deletion of DCC/18q21.3 (39%) and TP53/17p13 (23%). The kinogram detected a gene copy number increase for AURKA, PTK2, MET, and EGFR in 53%, 37%, 33%, and 28% of the tumors, respectively. QMPSF results were validated by comparative genomic hybridization and multiplex real-time polymerase chain reaction on genomic DNA.
Conclusions: The somatic QMPSF is a simple method able to detect simultaneously on a routine basis several quantitative changes in tumors. Its flexibility will allow the integration of clinically relevant genes. This high throughput method should be a valuable complementary tool of fluorescent in situ hybrization and comparative genomic hybridization.
Basic-Liver, Pancreas, and Biliary Tract
Discordant Role of CD4 T-Cell Response Relative to Neutralizing Antibody and CD8 T-Cell Responses in Acute Hepatitis C, 6 December 2006
Kaplan DE, Sugimoto K, Newton K, Valiga ME, Ikeda F, Aytaman A, Nunes FA, Lucey MR, Vance BA, Vonderheide RH, Reddy KR, McKeating JA, Chang K
Background & Aims: Acute hepatitis C virus (HCV) infection becomes chronic in the majority of patients. Although HCV-specific CD4 T-cell response is associated with HCV clearance, less is known about virus-specific CD8 T-cell or neutralizing antibody (nAb) responses and the role of CD4 help in their induction during acute infection.
Methods: HCV-specific CD4, CD8, and HCV pseudoparticle (HCVpp) nAb responses were monitored in acutely HCV-infected patients to define their relative contributions to viral clearance.
Results: Our results show that the outcome of acute hepatitis C is associated with a functional hierarchy in HCV-specific CD4 T-cell response and the scope of virus-specific, total T-cell interferon-? response. HCV-specific CD8 T-cell response was readily detectable in acutely HCV-infected patients regardless of virologic outcome or virus-specific CD4 T-cell response. In contrast, HCVpp-specific nAbs were readily detected in patients with chronic evolution and impaired virus-specific CD4 T-cell response but not in patients who cleared infection with robust virus-specific CD4 T-cell response.
Conclusions: The outcome of acute hepatitis C is associated with efficient virus-specific CD4 T-cell response(s) without which HCV-specific CD8 T-cell and heterologous nAb responses may develop but fail to clear viremia. Furthermore, HCV-specific nAb responses may not be induced despite robust virus-specific CD4 T-cell response.
Hepatitis C Virus Continuously Escapes From Neutralizing Antibody and T-Cell Responses During Chronic Infection In Vivo, 6 December 2006
von Hahn T, Yoon JC, Alter H, Rice CM, Rehermann B, Balfe P, McKeating JA
Background & Aims: Broadly reactive neutralizing antibodies (nAbs) and multispecific T-cell responses are generated during chronic hepatitis C virus (HCV) infection and yet fail to clear the virus. This study investigated the development of autologous nAb and HCV-glycoprotein–specific T-cell responses and their effects on viral sequence evolution during chronic infection in order to understand the reasons for their lack of effectiveness.
Methods: Numerous E1E2 sequences were amplified and sequenced from serum samples collected over a 26-year period from patient H, a uniquely well-characterized, chronically infected individual. HCV pseudoparticles (HCVpp) expressing the patient-derived glycoproteins were generated and tested for their sensitivity to neutralization by autologous and heterologous serum antibodies.
Results: A strain-specific nAb response developed early in infection (8 weeks postinfection), whereas cross-reactive antibodies able to neutralize HCVpp-bearing heterologous glycoproteins developed late in infection (>33 wk postinfection). The humoral response continuously failed to neutralize viruses bearing autologous glycoprotein sequences that were present in the serum at a given time. The amplified glycoprotein sequences displayed high variability, particularly in regions corresponding to defined linear B-cell epitopes. Mutations in defined neutralizing epitopes were associated with a loss of recognition by monoclonal antibodies against these epitopes and with decreased neutralization of corresponding HCVpp. Viral escape from CD4 and CD8 T-cell responses also was shown for several novel epitopes throughout the glycoprotein region.
Conclusions: During chronic infection HCV is subjected to selection pressures from both humoral and cellular immunity, resulting in the continuous generation of escape variants.
Genetic Study of Variation in Normal Mouse Iron Homeostasis Reveals Ceruloplasmin as an HFE-Hemochromatosis Modifier Gene, 23 November 2006
Gouya L, Muzeau F, Robreau A, Letteron P, Couchi E, Lyoumi S, Deybach J, Puy H, Fleming R, Demant P, Beaumont C, Grandchamp B
Background & Aims: Genetic hemochromatosis is one of the most common genetic disorders, with progressive tissue iron overload leading to severe clinical complications. In Northern European populations, genetic hemochromatosis is usually caused by homozygosity for the C282Y mutation in the HFE protein. However, penetrance of this mutation is incomplete, suggesting that other genetic and environmental factors contribute to its differential biologic or clinical expression.
Methods: To identify genes modifying iron homeostasis, we screened the 27 recombinant congenic strains of the C3H/DiSnA-C57BL/10ScSnA/Dem series for tissue and serum iron indices and genotyped 18 microsatellite markers in (C3H/DiSnA ? HcB-2) F2 hybrid mice.
Results: We identified 1 locus encompassing the Ceruloplasmin (Cp) gene with a strong linkage with liver iron, serum iron, and transferrin levels but not with spleen iron. Sequencing of Cp showed an R435X nonsense mutation in exon 7 in C3H/DiSnA mice. To evaluate whether Cp might act as a modifier gene of genetic hemochromatosis, we intercrossed C3H Hfe?/? and C3HDiSnA CpR435X/R435X mice. As expected, we found that double-mutant mice deposited more iron in the liver than mice defective for either one or both genes. In contrast, Hfe?/? ? CpR435/R435X or CpR435X/R435X ? Hfe+/? showed 30% decrease in liver iron when compared with single mutant mice.
Conclusions: This study highlights the existence of complex interactions between Cp and HFE and represents the first example of a modifier gene with a protective effect, in which heterozygosity reduces the iron load in the context of HFE deficiency.
Hepatic Expression of Candidate Genes in Patients With Alcoholic Hepatitis: Correlation With Disease Severity, 30 December 2006
Colmenero J, Bataller R, Sancho–Bru P, Bellot P, Miquel R, Moreno M, Jares P, Bosch J, Arroyo V, Caballería J, Ginès P
Background & Aims: Alcoholic hepatitis (AH) is a form of acute-on-chronic liver failure for which current therapy is not fully effective. We investigated the hepatic expression of candidate genes in patients with AH to identify new targets for therapy. Methods: Hepatic expression of candidate genes (n = 46) was assessed by quantitative polymerase chain reaction in patients with AH (n = 23) and in normal livers (n = 6). Disease severity was assessed by the Maddrey’s discriminant function and the occurrence of clinical complications. Histologic analysis included the assessment of myofibroblasts (smooth muscle ?-actin), collagen deposition (Sirius red), and inflammatory infiltrate (CD43). Portal hypertension was assessed by hepatic venous pressure gradient. Predictive association between gene expression and disease severity was assessed by k-nearest neighbor analysis. Results: Patients with AH showed profound hepatocellular dysfunction advanced fibrosis, and severe portal hypertension. Livers with AH showed up-regulation of genes encoding extracellular matrix proteins (procollagen I), fibrogenesis mediators, inflammatory cytokines, and apoptosis regulators. Key components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were markedly up-regulated, whereas cytochrome p450 2E1 and angiotensinogen were down-regulated. The expression of tissue inhibitor of metalloproteinases-1, growth-related oncogene ?, and several components of NADPH oxidase (dual oxidases 1 and 2) correlated with histologic findings and parameters indicative of disease severity. Conclusions: Genes involved in hepatic fibrogenesis, inflammatory response, and oxidative stress are overexpressed in AH. Some candidate genes correlate with histologic findings and disease severity, suggesting that they may be potential targets for therapy.
Chronic Ethanol Consumption Impairs Cellular Immune Responses Against HCV NS5 Protein Due to Dendritic Cell Dysfunction, 14 November 2006
Aloman C, Gehring S, Wintermeyer P, Kuzushita N, Wands JR
Background & Aims: Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8+ cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. Methods: Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. Results: Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1? and IL-10 and decreased tumor necrosis factor ?, IL-12, interferon ?, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. Conclusions: Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.
Nitroflurbiprofen, a Nitric Oxide-Releasing Cyclooxygenase Inhibitor, Improves Cirrhotic Portal Hypertension in Rats, 30 December 2006
Laleman W, Van Landeghem L, Van der Elst I, Zeegers M, Fevery J, Nevens F
Background & Aims: We studied whether administration of nitroflurbiprofen (HCT-1026), a cyclooxygenase inhibitor with nitric oxide (NO)-donating properties, modulates the increased intrahepatic vascular tone in portal hypertensive cirrhotic rats. Methods: In vivo hemodynamic measurements (n = 8/condition) and evaluation of the increased intrahepatic resistance by in situ perfusion (n = 5/condition) were performed in rats with thioacetamide-induced cirrhosis that received either nitroflurbiprofen (45 mg/kg), flurbiprofen (30 mg/kg, equimolar concentration to nitroflurbiprofen), or vehicle by intraperitoneal injection 24 hours and 1 hour prior to the measurements. Additionally, we evaluated the effect of acute administration of both drugs (250 ?mol/L) on the intrahepatic vascular tone in the in situ perfused cirrhotic rat liver (endothelial dysfunction and hyperresponsiveness to methoxamine) and on hepatic stellate cell contraction in vitro. Typical systemic adverse effects of nonsteroidal anti-inflammatory drugs, such as gastrointestinal ulceration, renal insufficiency, and hepatotoxicity, were actively explored. Results: In vivo, nitroflurbiprofen and flurbiprofen equally decreased portal pressure (8 ± 0.8 and 8.4 ± 0.1 mm Hg, respectively, vs 11.8 ± 0.6 mm Hg) and reduced the total intrahepatic vascular resistance. Systemic hypotension was not aggravated in the different treatment groups (P = .291). In the perfused cirrhotic liver, both drugs improved endothelial dysfunction and hyperresponsiveness. This was associated with a decreased hepatic thromboxane A2-production and an increased intrahepatic nitrate/nitrite level. In vitro, nitroflurbiprofen, more than flurbiprofen, decreased hepatic stellate cells contraction. Flurbiprofen-treated rats showed severe gastrointestinal ulcerations (bleeding in 3/8 rats) and nefrotoxicity, which was not observed in nitroflurbiprofen-treated cirrhotic rats. Conclusions: Treatment with nitroflurbiprofen, an NO-releasing cyclooxygenase inhibitor, improves portal hypertension without major adverse effects in thioacetamide-induced cirrhotic rats by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperreactivity to vasoconstrictors.
Isolation of Mouse Pancreatic Ductal Progenitor Cells Expressing CD133 and c-Met by Flow Cytometric Cell Sorting, 23 November 2006
Oshima Y, Suzuki A, Kawashimo K, Ishikawa M, Ohkohchi N, Taniguchi H
Background & Aims: Islet transplantation has become available across the globe since a novel protocol was reported. However, because donors are in short supply, only a minority of patients benefit from this procedure. Pancreatic progenitor cells are a promising resource for regeneration of new islets, but whether progenitor cells reside in ductal epithelium is not clear. Methods: Mouse pancreas was examined by immunohistochemistry with cell surface markers specific for ductal cells. We developed an isolation method for ductal cells by flow cytometric cell sorting using a newly identified specific marker for ductal cells. By using an in vitro colony assay, we characterized their proliferative and multipotent capacity. Results: CD133 is expressed specifically in ductal epithelium. Flow cytometric analysis revealed that purified ductal cells are highly enriched in the CD133+CD34?CD45?Ter119? fraction. An analysis of clonal epithelial colonies formed by individual cells revealed that progenitor cells with multilineage differentiation capacity are present in neonatal ductal epithelium. Moreover, these progenitor cells express c-Met. In adult mice, progenitor cells that show a high proliferative capacity but appear committed to a ductal lineage are co-purified with CD133+CD34?CD45?Ter119? cells. Conclusions: We established a system for isolating and culturing mouse pancreatic ductal cells that relies on flow cytometric cell sorting. Clonal analysis revealed that a population of progenitor cells is present among CD133+ ductal cells. Isolation of these cells will facilitate future studies into the roles of pancreatic progenitor cells in regeneration and carcinogenesis.
Defective Hepatic Response to Interferon and Activation of Suppressor of Cytokine Signaling 3 in Chronic Hepatitis C, 6 December 2006
Huang Y, Feld JJ, Sapp RK, Nanda S, Lin JH, Blatt LM, Fried MW, Murthy K, Liang TJ
Background & Aims: Approximately half of hepatitis C virus (HCV)-infected patients do not respond to current interferon (IFN)-? combination therapy. To understand IFN-? resistance in vivo, we examined the dynamic responses to both type I and type II IFNs, human IFN (hIFN)-?, -?, and consensus IFN, in the chimpanzee model. Methods: Naive and HCV-infected chimpanzees were treated with 3 forms of hIFNs in vivo. Quantitative real-time polymerase chain reaction was performed to evaluate the expression of IFN-stimulated genes (ISGs) in both peripheral blood mononuclear cells and liver to compare the responses to hIFN between naive and infected chimpanzees. The hepatic expression of IFN signaling components and inhibitory regulators including suppressor of cytokine signaling 3 (SOCS3) were assessed. SOCS3 expression was also evaluated in the liver of HCV-infected patients undergoing IFN treatment. Results: The in vivo responses to all 3 hIFNs were much lower in the HCV-infected chimpanzees than those in the naive chimpanzees. This defect was particularly evident in the liver because induction of hepatic ISGs was barely detectable in the infected animals. Following IFN administration, the expression of SOCS3 was significantly up-regulated, possibly through induction of interleukin-6, in the liver of HCV-infected chimpanzees. HCV-infected humans also showed a differential pattern of hepatic SOCS3 expression in response to IFN that is associated with treatment response. Conclusions: Our data indicate a predominantly defective hepatic response to IFN in HCV-infected chimpanzees, which is probably mediated through the activation of SOCS3 and may explain the nonresponse of many HCV patients to IFN-based therapy.
© 2007 American Gastroenterological Association (AGA) Institute. Published by Elsevier Inc. All rights reserved.
JOURNAL OF HEPATOLOGY
Volume 46, Issue 3, March 2007
"Seuls les abstracts des articles Originaux sont mis sur cette page. Les autres articles sont en ligne en FREE sur le site du Journal"
Original Articles
Viral Hepatitis
Gender-dependent association of CTLA4 polymorphisms with resolution of hepatitis C virus infection, 2 November 2006
Schott E, Witt H, Hinrichsen H, Neumann K, Weich V, Bergk A, Halangk J, Müller T, Tinjala S, Puhl G, Neuhaus P, Wiedenmann B, Berg T
Background/Aims
A vigorous T-cell response is essential for the resolution of HCV infection. It is modified by co-stimulatory molecules that attenuate T-lymphocyte responses by binding to CTLA4. We investigated whether CTLA4 single nucleotide polymorphisms are associated with the resolution of infection or with the course of disease.
Methods
We enrolled 127 individuals with self-limited and 947 patients with chronic HCV infection, of whom 560 were treated with interferon-?-based therapies, and 200 healthy controls. We analyzed CTLA4 polymorphisms ?318C>T and +49A>G by melting curve analysis and reconstructed haplotypes.
Results
CTLA4 haplotypes were distributed differently between men but not women with self-limited and chronic infection (p=0.043) but were not predictive of the stage of fibrosis in chronic carriers. Haplotypes were distributed differently between male but not female end-of-treatment responders and non-responders (p=0.025). The influence of CTLA4 haplotypes was more pronounced in “hard-to-treat” situations, i.e., treatment with interferon-? monotherapy or infection with HCV genotypes 1/4. Logistic regression analysis confirmed gender-specific risk factors for a virological non-response.
Conclusions
CTLA4 polymorphisms are associated with the resolution of HCV infection. This study underlines the role of an efficient T-cell response in the clearance of HCV and sheds light on a gender-dependent difference of immune regulation.
Polymorphisms of interleukin-1? in Japanese patients with hepatitis B virus infection, 2 November 2006
Migita K, Maeda Y, Abiru S, Nakamura M, Komori A, Miyazoe S, Nakao K, Yatsuhashi H, Eguchi K, Ishibashi H
Background/Aims
Hepatitis B virus (HBV) induces liver cirrhosis (LC) and hepatocellular carcinoma (HCC) mainly by causing chronic necro-inflammatory hepatic disease. Our aim was to investigate the relationships between the polymorphisms of the interleukin-1B (IL-1B) promoter region and the interleukin-1 receptor antagonist gene (IL-1RN) and disease progression in an HBV-infected Japanese population.
Methods
Genomic DNA was extracted from the peripheral blood of 237 HBV carriers. Polymorphisms in IL-1B and IL-1RN were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR with confronting two-pair primers (PCR-CTPP) methods. These polymorphic sites include the promoter regions of IL-1B at positions ?511 and ?31, and IL-1RN variable tandem repeats.
Results
The IL-1B ?31 and ?511 loci were in complete linkage disequilibrium, and the frequency of the IL-1B ?31 T carrier (IL-1B ?31 T/T or T/C) was significantly higher in HBV carriers with LC compared to those without LC (LC; 86.1% vs non-LC; 72.1%, P=0.009). There was no difference in the genotype distribution of the IL-1RN polymorphism.
Conclusions
This is the first report describing the association between IL-1B polymorphism and HBV-related hepatic fibrosis, and our data suggest that IL-1B polymorphisms may be related to disease progression of HBV-related hepatitis in Japan.
Hepatitis E virus (HEV) infection in patients with cirrhosis is associated with rapid decompensation and death, 3 November 2006
Kumar Acharya S, Kumar Sharma P, Singh R, Kumar Mohanty S, Madan K, Kumar Jha J, Kumar Panda S
Background/Aims
India is hyper-endemic for hepatitis E virus (HEV). HEV infection in cirrhosis may cause high mortality. Prospective study evaluating HEV infection in cirrhotics is scarce.
Methods
Consecutive patients with cirrhosis and healthy controls were included. Cirrhotics were categorized to 3 groups, (Group I – rapid decompensation, Group II – chronically decompensated, Group III – cirrhotics without decompensation). Sera from cirrhotics and controls were tested for HEV-RNA (RT-PCR). HEV-RNA positivity among cirrhotics and controls was compared. Natural course and mortality rate between HEV infected and non-infected cirrhotics were assessed during a 12-month follow-up.
Results
107 cirrhotics and 200 controls were included. 30 (28%) cirrhotics and 9 (4.5%) controls had detectable HEV-RNA (p<0.001). HEV- RNA positivity among Group I (n=42), II (n=32) and III (n=33) cirrhotics was 21 (50%), 6 (19%) and 3 (10%), respectively (p=0.002). 70% (21/30) with HEV infection and 27% (21/77) without it had rapid decompensation (p=0.001). Mortality between HEV infected and non-infected cirrhotics at 4 weeks (43% vs. 22%, p=0.001) and 12 month (70% vs. 30%, p=0.001) was different. Multivariate analysis identified HEV infection, Child-Pugh’s score, renal failure, and sepsis as independent factors for mortality.
Conclusions
In India, cirrhotics were prone to HEV infection, which was associated with rapid decompensation and death.
Comparison of test performance profile for blood tests of liver fibrosis in chronic hepatitis C, 7 November 2006
Halfon P, Bacq Y, De Muret A, Penaranda G, Bourliere M, Ouzan D, Tran A, Botta D, Renou C, Bréchot MC, Degott C, Paradis V
ackground/Aims
We evaluated the test performance profile (TPP) of blood tests of liver fibrosis.
Methods
Three hundred and fifty-six patients with C chronic hepatitis were included in two centers. Metavir staging of liver specimens by two independent pathologists and the following tests were evaluated: Fibrotest (FT), APRI, FibroMeter (FM), and Hepascore (HS).
Results
Metavir stages were: F0: 4%, F1: 55%, F2: 26%, F3: 11%, and F4: 4%. The AUROCs were not significantly different, respectively, FT, FM, APRI, HS:?F2: 0.79, 0.78, 0.76, 0.76; ?F3: 0.81, 0.85, 0.81, 0.81; and F4: 0.86, 0.94, 0.92, 0.89. The TPP relies on the paired comparison of blood-test misclassification based on liver specimen, e.g. FT vs FM, respectively: F0+1: 18 vs 28% (p=0.0003), ?F2: 43 vs 31% (p=0.004). There was no center effect.
Conclusions
In those populations, the four blood tests had a similar performance for significant fibrosis (F?2), lying in the lower range of published results which is attributable to a low ?F2 prevalence, and for ?F3 and F4. However, FM and FT had performance profiles significantly different as a function of fibrosis stages or diagnostic target (fibrosis cut-off). This has to be considered during the interpretation process. Moreover, the performance should be reported with different diagnostic targets.
Predictive factors of early and sustained responses to peginterferon plus ribavirin combination therapy in Japanese patients infected with hepatitis C virus genotype 1b: Amino acid substitutions in the core region and low-density lipoprotein cholesterol levels, 7 November 2006
Akuta N, Suzuki F, Kawamura Y, Yatsuji H, Sezaki H, Suzuki Y, Hosaka T, Kobayashi M, Kobayashi M, Arase Y, Ikeda K, Kumada H
Background/Aims
We showed previously that amino acid (aa) substitutions in the HCV core region (HCV-CR) are predictors of non-virological response (NVR) to peginterferon (PEG-IFN) plus ribavirin (RBV) therapy. Here, we determined the predictive factors of sustained virological response (SVR) and early virologic response (EVR) to this treatment.
Methods
We evaluated the response to 48-week PEG-IFN-RBV therapy in 114 Japanese adults infected with HCV genotype 1b and determined the predictors of EVR and SVR.
Results
EVR was achieved by 70% and SVR by 45% of patients. 64% of patients who achieved EVR also showed SVR, while none of non-EVR achieved SVR. Multivariate analysis identified low-density lipoprotein cholesterol (LDL-C) (?86mg/dl), aa substitutions in HCV-CR (double-wild-type; arginine at aa 70/leucine at aa 91), gamma-glutamyl transpeptidase (GGT) (<109IU/l), RBV dose (?11.0mg/kg), and leukocyte count (?4500/mm3) as significant determinants of EVR, and aa substitutions in HCV-CR (double-wild-type), LDL-C (?86mg/dl), male gender, ICG R15 (<10%), GGT (<109IU/l), and RBV dose (?11.0mg/kg) as determinants of SVR. Prediction of response to therapy based on combination of these factors had high sensitivity, specificity, positive, and negative predictive values.
Conclusions
Our study identified aa substitutions in the core region and serum LDL-C as predictors of response to PEG-IFN-RBV therapy in Japanese patients infected with HCV genotype 1b.
The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus, 10 November 2006
Molina S, Castet V, Fournier-Wirth C, Pichard-Garcia L, Avner R, Harats D, Roitelman J, Barbaras R, Graber P, Ghersa P, Smolarsky M, Funaro A, Malavasi F, Larrey D, Coste J, Fabre JM, Sa-Cunha A, Maurel P
Background/Aims
The direct implication of low-density lipoprotein receptor (LDLR) in hepatitis C virus (HCV) infection of human hepatocyte has not been demonstrated. Normal primary human hepatocytes infected by serum HCV were used to document this point.
Methods
Expression and activity of LDLR were assessed by RT-PCR and LDL entry, in the absence or presence of squalestatin or 25-hydroxycholesterol that up- or down-regulates LDLR expression, respectively. Infection was performed in the absence or presence of LDL, HDL, recombinant soluble LDLR peptides encompassing full-length (r-shLDLR4-292) or truncated (r-shLDLR4-166) LDL-binding domain, monoclonal antibodies against r-shLDLR4-292, squalestatin or 25-hydroxycholesterol. Intracellular amounts of replicative and genomic HCV RNA strands used as end point of infection were assessed by RT-PCR.
Results
r-shLDLR4-292, antibodies against r-shLDLR4-292 and LDL inhibited viral RNA accumulation, irrespective of genotype, viral load or liver donor. Inhibition was greatest when r-shLDLR4-292 was present at the time of inoculation and gradually decreased as the delay between inoculation and r-shLDLR4-292 treatment increased. In hepatocytes pre-treated with squalestatin or 25-hydroxycholesterol before infection, viral RNA accumulation increased or decreased in parallel with LDLR mRNA expression and LDL entry.
Conclusions
LDLR is involved at an early stage in infection of normal human hepatocytes by serum-derived HCV virions.
Health-related quality of life in patients with chronic hepatitis C and advanced fibrosis, 23 November 2006
Bonkovsky HL, Snow KK, Malet PF, Back-Madruga C, Fontana RJ, Sterling RK, Kulig CC, Di Bisceglie AM, Morgan TR, Dienstag JL, Ghany MG, Gretch DR, HALT-C Trial Group
Background/Aims
Although the antiviral and histological benefits of peginterferon/ribavirin therapy are well established, the effects on health-related quality of life (HRQOL) and sexual health are less certain. This study assessed HRQOL and sexual health in patients with advanced fibrosis or cirrhosis in the HALT-C Trial.
Methods
Subjects completed SF-36 and sexual health questionnaires prior to and after 24 weeks of peginterferon/ribavirin therapy (n=1144). Three hundred and seventy-three (33%) subjects were HCV RNA negative at week 20 and continued therapy through week 48; 258 were seen at week 72. One hundred and eighty achieved sustained virological responses (SVR) and 78 relapsed.
Results
At baseline, patients had poorer scores for all eight SF-36 domains compared to healthy controls. Patients with cirrhosis had lower HRQOL scores than those with bridging fibrosis, as did patients with higher depression scores. SVR patients had significant improvements in seven domains, whereas relapsers had significant worsening in one domain. Sexual scores improved in SVR patients and decreased in relapsers (p=0.03). In multivariate analyses, improvements in HRQOL and sexual scores were significantly associated with SVR but were less striking in patients with lower depression scores.
Conclusions
Achievement of SVR after peginterferon/ribavirin therapy improves HRQOL and sexual health in chronic hepatitis C patients with advanced fibrosis or cirrhosis.
Cirrhosis and its Complications
Reduced susceptibility to epinephrine-induced arrhythmias in cirrhotic rats: The roles of nitric oxide and endogenous opioid peptides, 2 November 2006
Tavakoli S, Hajrasouliha AR, Jabehdar-Maralani P, Ebrahimi F, Solhpour A, Sadeghipour H, Ghasemi M, Dehpour AR
Background/Aims
The clinical relevance of QT prolongation, the most widely recognized cardiac electrophysiological abnormality of cirrhosis, is still undefined. The aim of this study is to examine the susceptibility of chronic (4-week) bile duct-ligated rats to epinephrine-induced arrhythmias. The roles of nitric oxide and endogenous opioids were also evaluated.
Methods
Sham-operated and cirrhotic rats were treated with daily subcutaneous administrations of normal saline (1ml/kg/day), l-NAME (a non-selective nitric oxide synthase inhibitor, 3mg/kg/day), and naltrexone (20mg/kg/day) during the fourth week after operation. In order to evaluate the effects of acute nitric oxide synthesis inhibition, additional groups of animals were treated by acute intraperitoneal l-NAME injections (3mg/kg). Arrhythmias were induced by intravenous injections of 10?g/kg epinephrine.
Results
Despite QT prolongation (P<0.001), epinephrine induced fewer arrhythmias in cirrhotic rats compared to sham-operated animals (P<0.05). Chronic, but not acute, l-NAME administration corrected the QT prolongation in cirrhotic rats (P<0.001), and restored the susceptibility of cirrhotic rats to arrhythmias (P<0.05). Naltrexone injection without a significant effect on epinephrine-induced arrhythmias corrected QT interval in cirrhotic rats (P<0.001).
Conclusions
This study shows that despite QT prolongation, cirrhotic animals are resistant against epinephrine-induced arrhythmias. This resistance is mediated by chronic nitric oxide overproduction.
Impaired extracellular matrix degradation in aortic vessels of cirrhotic rats, 10 November 2006
Fernández-Varo G, Morales-Ruiz M, Ros J, Tugues S, Muñoz-Luque J, Casals G, Arroyo V, Rodés J, Jiménez W
Background/Aims
Thinning of the vascular wall occurs in conductance vessels of cirrhotic rats. Increased nitric oxide synthase (NOS) activity has been involved in the pathogenesis of this phenomenon. Therefore, we assessed the NO-regulated cell signaling pathways participating in vascular remodeling in cirrhosis.
Methods
Aortas were obtained from 15 control and 15 cirrhotic rats. Phosphorylated p38 MAPK and ERK1/2 were used to evaluate the activation of cell MAPK signaling pathways. Extracellular matrix (ECM) turnover was estimated by measuring matrix metalloproteinases (MMPs) activity and protein expression of collagen IV, MMP-2, MMP-9 and tissue inhibitor of MMPs (TIMP)-2. Thereafter, 12 control and 12 cirrhotic rats received N?-nitro-l-arginine-methyl-ester or vehicle daily for 11 weeks.
Results
Cirrhotic vessels showed a reduction in ERK1/2 phosphorylation, lower MMP activity, decreased MMPs expression and higher collagen IV and TIMP-2 abundance, compared to control rats. Chronic NOS inhibition normalized ERK1/2 phosphorylation and MMPs activity, increased MMPs abundance and decreased TIMP-2 expression in cirrhotic rats.
Conclusions
Vascular remodeling in cirrhotic rats is mediated by down-regulation of cell growth and impaired ERK1/2 activation and subsequent imbalance of ECM turnover. These results further stress the importance of vascular NO overactivity in the reduction of vascular wall thickness in cirrhosis.
Spatio-temporal decomposition of the electroencephalogram in patients with cirrhosis, 24 November 2006
Montagnese S, Jackson C, Morgan MY
Background/Aims
Slowing of the electroencephalogram (EEG) is a recognised feature of hepatic encephalopathy but its diagnostic sensitivity is indeterminate. Recent advances in EEG analysis should provide better quantifiable/more informative data. The aim of this study was to isolate and determine the scalp distribution of the posterior basic rhythm, in patients with cirrhosis, using a technique for spatio-temporal decomposition (SEDACA) of the EEG.
Methods
One hundred and ten patients with cirrhosis, classified, using clinical and psychometric criteria, as neuropsychiatrically unimpaired or as having minimal/overt hepatic encephalopathy were studied. Eyes-closed, awake EEGs were obtained and subjected to standard spectral analysis and spatio-temporal decomposition. Control data were obtained from 26 reference EEGs.
Results
The error in the estimate of the SEDACA-derived mean dominant frequency was lower than for the standard EEG derivation (P<0.00001). The SEDACA-derived spectral estimates correlated better with neuropsychiatric status and allowed differentiation of the patients with minimal hepatic encephalopathy from the reference population. The SEDACA-derived spatial information showed an anteriorization of the posterior basic rhythm, which became more prominent as the degree of neuropsychiatric impairment increased (P=0.00052).
Conclusions
Analysis of the EEG utilising SEDACA provides significantly more diagnostic information on the neuropsychiatric status of patients with cirrhosis than obtained conventionally.
Transplantation
Sustained virological response to antiviral therapy reduces mortality in HCV reinfection after liver transplantation, 24 November 2006
Picciotto FP, Tritto G, Lanza AG, Addario L, De Luca M, Di Costanzo G, Lampasi F, Tartaglione MT, Marsilia GM, Calise F, Cuomo O, Ascione A
Background/Aims
HCV infection recurs almost in all HCV-positive patients receiving liver transplantation and carries a poor prognosis. Aim of this study was to analyze efficacy and effect on survival of antiviral therapy in this clinical setting.
Methods
Pegylated-interferon ?-2b and ribavirin were administered at a dose of 1?g/kg of bwt weekly and 600–800mg/day. Planned duration of treatment was 24 or 48 weeks according to HCV genotype. Patients who failed to respond at week 24 were considered as non-responders.
Results
61 patients were enrolled. According to intention-to-treat analysis, 44 (72%) patients were considered as treatment failure (31 non-responders, 4 relapsers, 9 dropout). Sustained virological response was achieved in 17 cases (28%). Genotype 2, higher doses of antivirals and absence of histological cirrhosis were predictors of sustained virological response. In the follow up, patients with sustained virological response had a significantly lower mortality compared to patients with treatment failure (?2=6.9; P<0.01).
Conclusions
Response rate to antiviral therapy in HCV reinfection after liver transplantation is higher if a full dose of antiviral drugs is administered and if treatment starts before histological cirrhosis has developed. Sustained virological response improves patient survival.
Liver Failure, Growth and Cancer
Amanita phalloides poisoning: Reassessment of prognostic factors and indications for emergency liver transplantation, 23 November 2006
Escudié L, Francoz C, Vinel JP, Moucari R, Cournot M, Paradis V, Sauvanet A, Belghiti J, Valla D, Bernuau J, Durand F
Background/Aims
Amanita phalloides poisoning is an uncommon cause of acute liver failure with an especially rapid course. The aim of this study was to re-assess transplantation criteria in patients with mushroom poisoning.
Methods
Twenty-seven patients admitted for Amanita phalloides poisoning were studied. Previously reported transplantation criteria, including the recent Ganzert’s criteria, were tested retrospectively.
Results
The rate of fatal intoxication (death and/or transplantation) was 8/27 (30%). An interval between ingestion and diarrhea <8h was a very early predictor of a fatal outcome (accuracy of 78%). Later on, non-paracetamol and paracetamol King’s College criteria were superior to Clichy’s and Ganzert’s criteria (accuracy of 100% compared to 85% and 85%, respectively). Encephalopathy and renal insufficiency were not constant in the fatal intoxication group. Prothrombin index below 10% 4 days or more after ingestion had a 100% accuracy for predicting a fatal outcome.
Conclusions
Liver transplantation should be strongly considered in patients with an interval between ingestion and diarrhea <8h. Encephalopathy should not be an absolute prerequisite for deciding transplantation. From day 4 after ingestion, prothrombin index lower than 10% (≈INR of 6) alone is a reliable tool for deciding emergency transplantation.
Chemoembolization of hepatocellular carcinoma with drug eluting beads: Efficacy and doxorubicin pharmacokinetics, 30 November 2006
Varela M, Real MI, Burrel M, Forner A, Sala M, Brunet M, Ayuso C, Castells L, Montañá X, Llovet JM, Bruix J
Background/Aims
This study assesses the safety, pharmacokinetics and efficacy of transarterial chemoembolization using drug eluting beads (DEB), an embolizing device that slowly releases chemotherapy to decrease systemic toxicity.
Methods
Twenty-seven Child-Pugh A cirrhotics (76% male, 59% HCV) with untreated large/multifocal HCC received chemoembolization with doxorubicin loaded DEBs at doses adjusted for bilirubin and body surface (range: 47–150mg). Clinical and analytical data were recorded at 24 and 48h, 7, 14 and 30days after first and second TACE. Response rate was assessed by CT at 6months. Blood samples were obtained in 13 patients at 5, 20, 40, 60, 120min, 6, 24, 48 and 168h to determine doxorubicin Cmax and AUC.
Results
DEB-TACE was well tolerated with an acceptable safety profile. Two cases developed liver abscess, one leading to death. Response rate was 75% (66.6% on intention-to-treat). Doxorubicin Cmax and AUC were significantly lower in DEB-TACE patients (78.97±38.3ng/mL and 662.6±417.6ng/mLmin) than in conventional TACE (2341.5±3951.9ng/mL and 1812.2±1093.7ng/mLmin, p=0.00002 and p=0.001, respectively). After a median follow-up of 27.6months, 1- and 2-year survival is 92.5% and 88.9%, respectively.
Conclusions
Chemoembolization using DEBs is an effective procedure with a favorable pharmacokinetic profile.
Molecular and Cell Biology
Liver-homing of purified glucose oxidase: A novel in vivo model of physiological hepatic oxidative stress (H2O2), 17 November 2006
Rost D, Welker A, Welker J, Millonig G, Berger I, Autschbach F, Schuppan D, Mueller S
Background/Aims
Reactive oxygen species (ROS), such as H2O2, are implicated in normal and pathological liver function. However, due to the lack of suitable in vivo models of ROS generation the (patho) physiological relevance of H2O2 remains controversial.
Methods
We established a novel model of sustained hepatic H2O2 release using intravenous administration of purified Aspergillus niger glucose oxidase (GOX) in rats.
Results
GOX is rapidly cleared from the blood stream and almost exclusively localizes to Kupffer cells. GOX maintained its ability to generate H2O2 over 24h. While sublethal GOX doses induced hepatocellular necrosis, our morphological and functional studies show that lower levels of GOX which generate H2O2 comparable to release by inflammatory cells are non-toxic and do not induce histological inflammation. However, these non-toxic H2O2 levels increased oxidized glutathione and induced heme oxygenase 1 in the liver. In addition, several hepatocyte transporters were downregulated, while no decrease of bile formation, a sensitive marker of liver function, was observed.
Conclusions
Our in vivo model allows to study the effects of extracellular H2O2 in the liver as is released by inflammatory cells. Thus analysis of the role of this major ROS in the absence of confounding inflammatory cofactors will be possible.
Genetic and Metabolic Liver Disease
Glycogen storage disease type III-hepatocellular carcinoma a long-term complication?, 9 November 2006
Demo E, Frush D, Gottfried M, Koepke J, Boney A, Bali D, Chen YT, Kishnani PS
Background/Aims
Glycogen storage disease III (GSD III) is caused by a deficiency of glycogen-debranching enzyme which causes an incomplete glycogenolysis resulting in glycogen accumulation with abnormal structure (short outer chains resembling limit dextrin) in liver and muscle. Hepatic involvement is considered mild, self-limiting and improves with age. With increased survival, a few cases of liver cirrhosis and hepatocellular carcinoma (HCC) have been reported.
Methods
A systematic review of 45 cases of GSD III at our center (20 months to 67 years of age) was reviewed for HCC, 2 patients were identified. A literature review of HCC in GSD III was performed and findings compared to our patients.
Conclusions
GSD III patients are at risk for developing HCC. Cirrhosis was present in all cases and appears to be responsible for HCC transformation There are no reliable biomarkers to monitor for HCC in GSD III. Systematic evaluation of liver disease needs be continued in all patients, despite lack of symptoms. Development of guidelines to allow for systematic review and microarray studies are needed to better delineate the etiology of the hepatocellular carcinoma in patients with GSD III.
Copyright © 2007 European Association for the Study of the Liver. All rights reserved.
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Copyright © 2007 BMJ Publishing Group Ltd
NEW ENGLAND JOURNAL
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LANCET
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