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Table of Contents for December 2004 - Volume 40 - Issue 6
Rapid Communication
Treatment with pegylated interferon and ribavarin in HCV infection with genotype 2 or 3 for 14 weeks: A pilot study (p 1260-1265)
Olav Dalgard, Kristian Bjøro, Kjell Block Hellum, Bjørn Myrvang, Ståle Ritland, Kjell Skaug, Nils Raknerud, Helge Bell
The aim of this study was to determine the efficacy of 14 weeks of treatment in patients infected with hepatitis C virus (HCV) genotype 2 or 3 who achieve early virological response (EVR). In a noncontrolled multicenter trial, 122 treatment-naive patients received 1.5 g/kg pegylated interferon alfa-2b subcutaneously once weekly and 800 to 1,400 mg/d ribavirin based on body weight. Treatment was stopped at week 14 in patients with EVR, defined as undetectable HCV RNA at weeks 4 and 8. Patients without EVR were assigned to 24 weeks of treatment. The primary end point was sustained virological response (SVR), defined as undetectable HCV RNA 24 weeks after end of treatment. Among the 122 patients, 95 (78%) had EVR and received 14 weeks of treatment. The remaining 27 (22%) were treated for 24 weeks. SVR was obtained in 85 (90%) of 95 patients in the 14-week treatment group and 15 of (56%) 27 in the 24-week treatment group. Altogether, SVR was obtained in 100 of 122 patients (82%; 95% CI, 75%-89%). SVR after 14 weeks of treatment was achieved more frequently among genotype 3a patients with low viral load compared with high viral load (98% vs. 79%; P = .019). Logistic regression analysis showed that absence of bridging fibrosis/cirrhosis was the only independent predictor of SVR. In conclusion, patients with genotype 2 or 3 and EVR obtained a high SVR after 14 weeks of treatment. The results need to be confirmed in a randomized, controlled study before this treatment approach can be recommended, particularly for patients with genotype 3 and high viral load or severe fibrosis. (HEPATOLOGY 2004;40: 1260-1265.)
Liver Biology and Pathobiology
Transcription factor HNF-6/OC-1 inhibits the stimulation of the HNF-3/Foxa1 gene by TGF- in mouse liver (p 1266-1274)
Nicolas Plumb-Rudewiez, Frédéric Clotman, Hélène Strick-Marchand, Christophe E. Pierreux, Mary C. Weiss, Guy G. Rousseau, Frédéric P. Lemaigre
A network of liver-enriched transcription factors controls differentiation and morphogenesis of the liver. These factors interact via direct, feedback, and autoregulatory loops. Previous work has suggested that hepatocyte nuclear factor (HNF)-6/OC-1 and HNF-3/FoxA1 participate coordinately in this hepatic network. We investigated how HNF-6 controls the expression of Foxa1. We observed that Foxa1 expression was upregulated in the liver of Hnf6-/- mouse embryos and in bipotential mouse embryonic liver (BMEL) cell lines derived from embryonic Hnf6-/- liver, suggesting that HNF-6 inhibits the expression of Foxa1. Because no evidence for a direct repression of Foxa1 by HNF-6 was found, we postulated the existence of an indirect mechanism. We found that the expression of a mediator and targets of the transforming growth factor beta (TGF-) signaling was increased both in Hnf6-/- liver and in Hnf6-/- BMEL cell lines. Using these cell lines, we demonstrated that TGF- signaling was increased in the absence of HNF-6, and that this resulted from upregulation of TGF- receptor II expression. We also found that TGF- can stimulate the expression of Foxa1 in Hnf6+/+ cells and that inhibition of TGF- signaling in Hnf6-/- cells down-regulates the expression of Foxa1. In conclusion, we propose that Foxa1 upregulation in the absence of HNF-6 results from increased TGF- signaling via increased expression of the TGF- receptor II. We further conclude that HNF-6 inhibits Foxa1 by inhibiting the activity of the TGF- signaling pathway. This identifies a new mechanism of interaction between liver-enriched transcription factors whereby one factor indirectly controls another by modulating the activity of a signaling pathway. (HEPATOLOGY 2004;40:1266-1274.)
In vitro hepatic differentiation of human mesenchymal stem cells (p 1275-1284)
Kuan-Der Lee, Tom Kwang-Chun Kuo, Jacqueline Whang-Peng, Yu-Fen Chung, Ching-Tai Lin, Shiu-Huey Chou, Jim-Ray Chen, Yi-Peng Chen, Oscar Kuang-Sheng Lee
This study examined whether mesenchymal stem cells (MSCs), which are stem cells originated from embryonic mesoderm, are able to differentiate into functional hepatocyte-like cells in vitro. MSCs were isolated from human bone marrow and umbilical cord blood, and the surface phenotype and the mesodermal multilineage differentiation potentials of these cells were characterized and tested. To effectively induce hepatic differentiation, we designed a novel 2-step protocol with the use of hepatocyte growth factor and oncostatin M. After 4 weeks of induction, cuboidal morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including albumin production, glycogen storage, urea secretion, uptake of low-density lipoprotein, and phenobarbital-inducible cytochrome P450 activity. In conclusion, human MSCs from different sources are able to differentiate into functional hepatocyte-like cells and, hence, may serve as a cell source for tissue engineering and cell therapy of hepatic tissues. Furthermore, the broad differentiation potential of MSCs indicates that a revision of the definition may be required. (HEPATOLOGY 2004;40:1275-1284.)
IRS-2 mediates the antiapoptotic effect of insulin in neonatal hepatocytes (p 1285-1294)
Angela M. Valverde, Isabel Fabregat, Deborah J. Burks, Morris F. White, Manuel Benito
To assess the role of insulin action and inaction in the liver, immortalized hepatocyte cell lines have been generated from insulin receptor substrate (IRS)-2-/- and wild-type mice. Using this model, we have recently demonstrated that the lack of IRS-2 in neonatal hepatocytes resulted in insulin resistance. In the current study, we show that immortalized neonatal hepatocytes undergo apoptosis on serum withdrawal, with caspase-3 activation and DNA laddering occurring earlier in the absence of IRS-2. Insulin rescued wild-type hepatocytes from serum withdrawal-induced caspase-3 activation and DNA fragmentation in a dose-dependent manner, but it failed to rescue hepatocytes lacking IRS-2. In IRS-2-/- cells, insulin failed to phosphorylate Bad. Furthermore, in these cells, insulin was unable to translocate Foxo1 from the nucleus to the cytosol. Adenoviral infection of wild-type cells with constitutively active Foxo1 (ADA) induced caspase-8 and caspase-3 activities, proapoptotic gene expression, DNA laddering and apoptosis. Dominant negative Foxo1 regulated the whole pathway in an opposite manner. Prolonged insulin treatment (24 hours) increased expression of antiapoptotic genes (Bcl-xL), downregulated proapoptotic genes (Bim and nuclear Foxo1), and decreased caspase-3 activity in wild-type hepatocytes but not in IRS-2-/- cells. Infection of IRS-2-/- hepatocytes with adenovirus encoding IRS-2 reconstituted phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/Foxo1 signaling, restored pro- and antiapoptotic gene expression, and decreased caspase-3 activity in response to insulin, thereby blocking apoptosis. In conclusion, IRS-2 signaling is specifically required through PIP3 generation to mediate the survival effects of insulin. Epidermal growth factor, via PIP3/Akt/Foxo1 phosphorylation, was able to rescue IRS-2-/- hepatocytes from serum withdrawal-induced apoptosis, modulating pro- and anti-apoptotic gene expression and downregulating caspase-3 activity.
PGE1-induced NO reduces apoptosis by D-galactosamine through attenuation of NF-B and NOS-2 expression in rat hepatocytes (p 1295-1303)
Emilio Siendones, Dalia Fouad, María José M. Díaz-Guerra, Manuel de la Mata, Lisardo Boscá, Jordi Muntané
Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. We have previously shown that PGE1 preadministration protects against NO-dependent cell death induced by D-galactosamine (D-GalN) through a rapid increase of nuclear factor B (NF-B) activity, inducible NO synthase (NOS-2) expression, and NO production. The present study investigates whether PGE1-induced NO was able to abolish NF-B activation, NOS-2 expression, and apoptosis elicited by D-GalN. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. PGE1 (1 mol/L) was administered 2 hours before D-GalN (5 mmol/L) in primary culture rat hepatocytes. PGE1 reduced inhibitor B degradation, NF-B activation, NOS-2 expression, and apoptosis induced by D-GalN. The administration of an inhibitor of NOS-2 abolished the inhibitory effect of PGE1 on NF-B activation and NOS-2 expression in D-GalN-treated hepatocytes. Transfection studies using different plasmids corresponding to the NOS-2 promoter region showed that D-GalN and PGE1 regulate NOS-2 expression through NF-B during the initial stage of hepatocyte treatment. PGE1 was able to reduce the promoter activity induced by D-GalN. In addition, a NO donor reduced NOS-2 promoter activity in transfected hepatocytes. In conclusion, administration of PGE1 to hepatocytes produces low levels of NO, which inhibits its own formation during D-GalN-induced cell death through the attenuation of NF-B-dependent NOS-2 expression. Therefore, a dual role for NO in PGE1-treated D-GalN-induced toxicity in hepatocytes is characterized by a rapid NO release that attenuates the late and proapoptotic NOS-2 expression.
Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice (p 1304-1311)
Isao Sakaida, Shuji Terai, Naoki Yamamoto, Koji Aoyama, Tsuyoshi Ishikawa, Hiroshi Nishina, Kiwamu Okita
We investigated the effect of bone marrow cell (BMC) transplantation on established liver fibrosis. BMCs of green fluorescent protein (GFP) mice were transplanted into 4-week carbon tetrachloride (CCl4)-treated C57BL6 mice through the tail vein, and the mice were treated for 4 more weeks with CCl4 (total, 8 weeks). Sirius red and GFP staining clearly indicated migrated BMCs existing along with fibers, with strong expression of matrix metalloproteinase (MMP)-9 shown by anti-MMP-9 antibodies and in situ hybridization. Double fluorescent immunohistochemistry showed the expression of MMP-9 on the GFP-positive cell surface. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area coinciding with the location of MMP-9-positive BMCs. Four weeks after BMC transplantation, mice had significantly reduced liver fibrosis, as assessed by hydroxyproline content of the livers, compared to that of mice treated with CCl4 alone. Subpopulation of Liv8-negative BMCs was responsible for this fibrolytic effect. In conclusion, mice with BMC transplants with continuous CCl4 injection had reduced liver fibrosis and a significantly improved survival rate after BMC transplantation compared with mice treated with CCl4 alone. This finding introduces a new concept for the therapy of liver fibrosis.
Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice (p 1312-1321)
Ming-Chin Yu, Cheng-Hsu Chen, Xiaoyan Liang, Lianfu Wang, Chandrashekhar R. Gandhi, John J. Fung, Lina Lu, Shiguang Qian
In the injured liver, hepatic stellate cells (HSCs) secrete many different cytokines, recruit lymphocytes, and thus participate actively in the pathogenesis of liver disease. Little is known of the role of HSCs in immune responses. In this study, HSCs isolated from C57BL/10 (H2b) mice were found to express scant key surface molecules in the quiescent stage. Activated HSCs express major histocompatibility complex class I, costimulatory molecules, and produce a variety of cytokines. Stimulation by interferon (IFN-) or activated T cells enhanced expression of these molecules. Interestingly, addition of the activated (but not quiescent) HSCs suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti-CD3-mediated T-cell receptor ligation in a dose-dependent manner. High cytokine production by the T cells suggests that the inhibition was probably not a result of suppression of their activation. T-cell division was also found to be normal in a CFSE dilution assay. The HSC-induced T-cell hyporesponsiveness was associated with enhanced T-cell apoptosis. Activation of HSCs was associated with markedly enhanced expression of B7-H1. Blockade of B7-H1/PD-1 ligation significantly reduced HSC immunomodulatory activity, suggesting an important role of B7-H1. In conclusion, the bidirectional interactions between HSCs and immune cells may contribute to hepatic immune tolerance. (HEPATOLOGY 2004;40:1312-1321.)
Disruption of hepatic adipogenesis is associated with impaired liver regeneration in mice (p 1322-1332)
Eyal Shteyer, Yunjun Liao, Louis J. Muglia, Paul W. Hruz, David A. Rudnick
The liver responds to injury with regulated tissue regeneration. During early regeneration, the liver accumulates fat. Neither the mechanisms responsible for nor the functional significance of this transient steatosis have been determined. In this study, we examined patterns of gene expression associated with hepatic fat accumulation in regenerating liver and tested the hypothesis that disruption of hepatic fat accumulation would be associated with impaired hepatic regeneration. First, microarray-based gene expression analysis revealed that several genes typically induced during adipocyte differentiation were specifically upregulated in the regenerating liver prior to peak hepatocellular fat accumulation. These observations suggest that hepatic fat accumulation is specifically regulated during liver regeneration. Next, 2 methods were employed to disrupt hepatocellular fat accumulation in the regenerating liver. Because exogenous leptin supplementation reverses hepatic steatosis in leptin-deficient mice, the effects of leptin supplementation on liver regeneration in wild-type mice were examined. The data showed that leptin supplementation resulted in suppression of hepatocellular fat accumulation and impairment of hepatocellular proliferation during liver regeneration. Second, because glucocorticoids regulate cellular fat accumulation during adipocyte differentiation, the effects of hepatocyte-specific disruption of the glucocorticoid receptor were similarly evaluated. The results showed that hepatic fat accumulation and hepatocellular proliferation were also suppressed in mice with liver specific disruption of glucocorticoid receptor. In conclusion, suppression of hepatocellular fat accumulation is associated with impaired hepatocellular proliferation following partial hepatectomy, indicating that hepatocellular fat accumulation is specifically regulated during and may be essential for normal liver regeneration. (HEPATOLOGY 2004;40:1322-1332.)
Biliverdin therapy protects rat livers from ischemia and reperfusion injury (p 1333-1341)
Constantino Fondevila, Xiu-Da Shen, Seiichiro Tsuchiyashi, Kenichiro Yamashita, Eva Csizmadia, Charles Lassman, Ronald W. Busuttil, Jerzy W. Kupiec-Weglinski, Fritz H. Bach
Heme oxygenase (HO-1) provides a cellular defense mechanism during oxidative stress and catalyzes the rate-limiting step in heme metabolism that produces biliverdin (BV). The role of BV and its potential use in preventing ischemia/reperfusion injury (IRI) had never been studied. This study was designed to explore putative cytoprotective functions of BV during hepatic IRI in rat liver models of ex vivo perfusion and orthotopic liver transplantation (OLT) after prolonged periods of cold ischemia. In an ex vivo hepatic IRI model, adjunctive BV improved portal venous blood flow, increased bile production, and decreased hepatocellular damage. These findings were correlated with amelioration of histological features of IRI, as assessed by Suzuki's criteria. Following cold ischemia and syngeneic OLT, BV therapy extended animal survival from 50% in untreated controls to 90% to 100%. This effect correlated with improved liver function and preserved hepatic architecture. Additionally, BV adjuvant after OLT decreased endothelial expression of cellular adhesion molecules (P-selectin and intracellular adhesion molecule 1), and decreased the extent of infiltration by neutrophils and inflammatory macrophages. BV also inhibited expression of inducible nitric oxide synthase and proinflammatory cytokines (interleukin 1, tumor necrosis factor , and interleukin 6) in OLTs. Finally, BV therapy promoted an increased expression of antiapoptotic molecules independently of HO-1 expression, consistent with BV being an important mediator through which HO-1 prevents cell death. In conclusion, this study documents and dissects potent cytoprotective effects of BV in well-established rat models of hepatic IRI. Our results provide the rationale for a novel therapeutic approach using BV to maximize the function and thus the availability of donor organs. (HEPATOLOGY 2004;40:1333-1341.)
Role of hepatic fibrin in idiosyncrasy-like liver injury from lipopolysaccharide-ranitidine coexposure in rats (p 1342-1351)
James P. Luyendyk, Jane F. Maddox, Christopher D. Green, Patricia E. Ganey, Robert A. Roth
Coadministration of nonhepatotoxic doses of the histamine 2-receptor antagonist ranitidine (RAN) and bacterial lipopolysaccharide (LPS) results in hepatocellular injury in rats, the onset of which occurs in 3 to 6 hours. This reaction resembles RAN idiosyncratic hepatotoxicity in humans. Early fibrin deposition occurs in livers of rats cotreated with LPS/RAN. Accordingly, we tested the hypothesis that the hemostatic system contributes to liver injury in LPS/RAN-treated rats. Rats were given either LPS (44.4 ? 106 EU/kg) or its vehicle, then RAN (30 mg/kg) or its vehicle 2 hours later. They were killed 2, 3, 6, 12, or 24 hours after RAN treatment, and liver injury was estimated from serum alanine aminotransferase activity. A modest elevation in serum hyaluronic acid, which was most pronounced in LPS/RAN-cotreated rats, suggested altered sinusoidal endothelial cell function. A decrease in plasma fibrinogen and increases in thrombin-antithrombin dimers and in serum concentration of plasminogen activator inhibitor-1 occurred before the onset of liver injury. Hepatic fibrin deposition was observed in livers from LPS/RAN-cotreated rats 3 and 6 hours after RAN. Liver injury was abolished by the anticoagulant heparin and was significantly attenuated by the fibrinolytic agent streptokinase. Hypoxia, one potential consequence of sinusoidal fibrin deposition, was observed in livers of LPS/RAN-treated rats. In conclusion, the results suggest that the hemostatic system is activated after LPS/RAN cotreatment and that fibrin deposition in liver is important for the genesis of hepatic parenchymal cell injury in this model. (HEPATOLOGY 2004;40:1342-1351.)
Liver Failure and Liver Disease
Initial response to percutaneous ablation predicts survival in patients with hepatocellular carcinoma (p 1352-1360)
Margarita Sala, Josep M. Llovet, Ramon Vilana, Lluís Bianchi, Manel Solé, Carmen Ayuso, Concepció Brú, Jordi Bruix, for the Barcelona Cly'nic Liver Cancer (BCLC) Group
Outcome predictors in patients with hepatocellular carcinoma (HCC) who are treated with percutaneous ablation are ill defined, and it is unknown if successful therapy is associated with improved survival. In our study, 282 cirrhotic patients with early nonsurgical HCC were treated with percutaneous ablation during a 15-year period. Single tumors were seen in 244 patients, and 2 to 3 nodules were seen in 38 patients. Initial complete response was achieved in 192 patients and was independently related to the size of the main tumor (P = .015) and tumor stage (P = .0001) (2 cm, 96%; 2.1-3 cm, 78%; >3 cm, 56%; 2-3 nodules, 46%). At the end of follow-up, 80 patients presented sustained complete response. The 1-, 3-, and 5-year survival rates were 87%, 51%, and 27%, respectively. The independent predictors of survival were Child-Turcotte-Pugh class (P = .0001) and initial complete response (P = .006). Child-Turcotte-Pugh class A patients with initial complete response achieved 42% survival at 5 years; this figure increased to 63% in patients with tumors 2 cm or smaller. In conclusion, our results demonstrate that initial complete response to percutaneous ablation is associated with an improved survival in both Child-Turcotte-Pugh class A and B patients with nonsurgical HCC. Accordingly, initial complete tumor necrosis should be considered a relevant therapeutic target irrespective of tumor size and liver function. (HEPATOLOGY 2004;40:1352-1360.)
Randomized trial of leuprorelin and flutamide in male patients with hepatocellular carcinoma treated with tamoxifen (p 1361-1369)
Groupe d'Etude et de Traitement du Carcinome Hépatocellulaire (GRETCH)
The growth of hepatocellular carcinoma (HCC) is thought to be dependent on androgens, as androgen receptors are present in most of these tumors. The aim of this multicenter trial was to assess the effect of antiandrogens in patients who have advanced HCC. Male patients with advanced HCC were randomized into 2 groups treated with (1) leuprorelin (3.75 mg/mo subcutaneously), flutamide (750 mg/d orally), and tamoxifen (30 mg/d orally) or (2) tamoxifen alone (30 mg/d orally) administered until death. Survival was the main end point (log-rank test). The required sample size was 375 patients (alpha, 5%; beta, 10%; 1-year survival, 45% in treated group and 30% in controls). Between February 1994 and January 1998, 376 male patients (mean age, 66 years; treated group, n = 192; control group, n = 184) were included. No baseline imbalance was found between the groups. At the reference date (January 1, 2003), 183 deaths (95.3%) were observed in the treated group and 177 deaths (96.2%) were observed in controls. Thirteen patients were lost to follow-up. Median survival time was estimated to be 135.5 days (95% CI, 112-189) and 176 days (95% CI, 141-227) in treated and control groups, respectively (P = .21). Crude and adjusted relative risks of death in the treated group were estimated at 1.14 (95% CI, 0.93-1.40) and 1.08 (95% CI, 0.87-1.33; P = .48) respectively. Premature interruption of treatment was more frequent in the treated group (n = 45) than in controls (n = 22; P = .0045), mainly because of digestive side effects. In conclusion, no benefit in survival was found with antiandrogenic treatment in male patients with advanced HCC. (HEPATOLOGY 2004;40:1361-1369.)
Low-grade steatosis and major changes in portal flow as new prognostic factors in steroid-treated alcoholic hepatitis (p 1370-1378)
Christophe Duvoux, Catherine Radier, Françoise Roudot-Thoraval, François Maille, Marie-Christine Anglade, Jeanne Tran Van Nhieu, Isabelle Rosa, Sylvie Hospitel, Issam Abd-Alsamad, Véronique Sitruk, Olivier Seror, Marianne Ziol, Hughes Blondon, Daniel Dhumeaux, Jean-Philippe Richardet
The aim of this study was to assess the prevalence and prognostic value of major alterations of portal flow in patients with steroid-treated alcoholic hepatitis. Fifty patients with severe, histologically proven alcoholic hepatitis were enrolled. Clinical data, liver test results, and hepatic Doppler ultrasound findings were collected at inclusion and at month 2. Patients were followed for 1 year or until death. Major changes in portal flow were defined as reversed or alternating flow in the portal trunk and/or in intrahepatic portal branches. Changes in portal flow were observed in 24 (48.0%) of 50 and 17 (39.5%) of 43 patients at inclusion and month 2, respectively. Univariate analysis showed that age older than 50 years, steatosis less than 20% on initial liver biopsy, presence of major changes in portal flow, Child-Turcotte-Pugh score higher than 12, factor V level higher than 45%, and hepatofugal splenic blood flow were associated with a lower 1-year survival. Cox regression analysis showed that steatosis < 20% (relative hazard [RH] = 9.3, P = .0009) and major changes in portal flow (RH = 3.1, P = .04), were independently associated with poor survival. In conclusion, major changes in portal flow are frequent in patients with severe alcoholic hepatitis. Altered portal flow and steatosis < 20% are new prognostic factors in steroid-treated alcoholic hepatitis and must be taken into account in patient management. (HEPATOLOGY 2004;40:1370-1378).
Metronidazole and ursodeoxycholic acid for primary sclerosing cholangitis: A randomized placebo-controlled trial (p 1379-1386)
Martti Färkkilä, Anna-Liisa Karvonen, Heimo Nurmi, Hannu Nuutinen, Matti Taavitsainen, Pekka Pikkarainen, Päivi Kärkkäinen
No effective medical therapy is currently available for primary sclerosing cholangitis (PSC). Ursodeoxycholic acid (UDCA) improves liver enzymes, but its effect on liver histology is controversial. Metronidazole (MTZ) prevents PSC-like liver damage in animal models and reduces intestinal permeability. We recruited 80 patients with PSC into a randomized placebo-controlled study to evaluate the effect of UDCA and MTZ (UDCA/MTZ) compared with UDCA/placebo on the progression of PSC. Patients (41 UDCA/placebo and 39 UDCA/MTZ) were followed every third month. Assessment of liver function test, histological stage and grade, and cholangiography (via ERCP) at baseline showed no differences between the groups. After 36 months, serum aminotransferases -glutamyltransferase, and alkaline phosphatase (ALP) decreased markedly in both groups, serum ALP more significantly in the UDCA/MTZ group (-337 ± 54 U/L, P < .05) compared with the UDCA/placebo group. The New Mayo Risk Score decreased markedly only in the UDCA/MTZ group (-0.50 ± 0.13, P < .01). The number of patients with improvement of stage (P < .05) and grade (P < .05) was higher in the combination group. ERCP findings showed no progression or improvement in 77% and 68% of patients on UDCA/MTZ and UDCA/placebo, respectively. In conclusion, combining MTZ with UDCA in PSC improved serum ALP levels and New Mayo Risk Score, but no statistically significant effect on disease progression as assessed via liver histology or ERCP was seen. Long-term studies using a higher dose of UDCA combined with MTZ in larger patient populations are indicated. (HEPATOLOGY 2004;40:1379-1386.)
Prevalence of hepatic steatosis in an urban population in the United States: Impact of ethnicity (p 1387-1395)
Jeffrey D. Browning, Lidia S. Szczepaniak, Robert Dobbins, Pamela Nuremberg, Jay D. Horton, Jonathan C. Cohen, Scott M. Grundy, Helen H. Hobbs
Despite the increasing prevalence of nonalcoholic fatty liver disease (NAFLD), its pathogenesis and clinical significance remain poorly defined. In this study, we examined and compared the distribution of hepatic triglyceride content (HTGC) in 2,287 subjects from a multiethnic, population-based sample (32.1% white, 48.3% black, and 17.5% Hispanic) using proton magnetic resonance spectroscopy. HTGC varied over a wide range (0.0%-41.7%; median, 3.6%) in the population. Almost one third of the population had hepatic steatosis, and most subjects with hepatic steatosis had normal levels of serum alanine aminotransferase (79%). The frequency of hepatic steatosis varied significantly with ethnicity (45% in Hispanics; 33% in whites; 24% in blacks) and sex (42% in white men; 24% in white women). The higher prevalence of hepatic steatosis in Hispanics was due to the higher prevalence of obesity and insulin resistance in this ethnic group. However, the lower frequency of hepatic steatosis in blacks was not explained by ethnic differences in body mass index, insulin resistance, ethanol ingestion, or medication use. The prevalence of hepatic steatosis was greater in men than women among whites, but not in blacks or Hispanics. The ethnic differences in the frequency of hepatic steatosis in this study mirror those observed previously for NAFLD-related cirrhosis (Hispanics > whites > blacks). In conclusion, the significant ethnic and sex differences in the prevalence of hepatic steatosis documented in this study may have a profound impact on susceptibility to steatosis-related liver disease. (HEPATOLOGY 2004;40:1387-1395.)
Validation of a new prognostic staging system for hepatocellular carcinoma: The JIS score compared with the CLIP score (p 1396-1405)
Masatoshi Kudo, Hobyung Chung, Seiji Haji, Yukio Osaki, Hiroko Oka, Toshihito Seki, Hiroshi Kasugai, Yo Sasaki, Takashi Matsunaga
The Japan Integrated Staging score (JIS score), which combines the Child-Turcotte-Pugh classification and tumor-node-metastasis staging, has been proposed as a better prognostic staging system for hepatocellular carcinoma (HCC) than the Cancer of the Liver Italian Program (CLIP) scoring system. In this study, validation was performed among a larger patient population. A total of 4,525 consecutive patients with HCC who had been diagnosed at five institutions were included. Stratification ability, prognostic predictive power, and reproducibility were analyzed and compared with results from the CLIP scoring system. Only 45% (1,951 of 4,525) of all patients were categorized as early stage HCC according to JIS score (0 or 1), whereas 63% (2,878 of 4,525) of the patients were categorized as having a CLIP score of 0 or 1. Significant differences in survival curves were not observed among CLIP scores 3 to 6. In contrast, survival curves showed significant differences among all the JIS scores. The same JIS scoring subgroups showed a similar prognosis, and good internal reproducibility was observed in each of the institutions. Multivariate analysis of the prognosis in all 4,525 patients proved the JIS score to be the best prognostic factor. Furthermore, the Akaike information criteria proved that the JIS scoring system was statistically a better model for predicting outcome than the CLIP scoring system. In conclusion, the stratification ability and prognostic predictive power of the JIS score were much better than that of the CLIP score and were simple to obtain and remember. (HEPATOLOGY 2004; 40:1396-1405.)
Glutathione S-transferase and liver function in intrahepatic cholestasis of pregnancy and pruritus gravidarum (p 1406-1414)
Anthony T. Dann, Anna P. Kenyon, Paul T. Seed, Lucilla Poston, Andrew H. Shennan, Rachel M. Tribe
Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease associated with poor maternal and fetal outcome. The diagnosis is based on pruritus with abnormal liver function in the absence of other pathological conditions. However, pruritus in pregnancy is common, and it may be the only presenting feature in ICP. No reliable test currently exists that can discriminate between those women destined to develop ICP and those with the benign condition of pruritus gravidarum (PG). The purpose of this prospective study was to investigate longitudinally the serum concentration of glutathione S-transferase alpha (GSTA, a specific marker of hepatocellular integrity) and to compare this with the temporal profile of conventional liver function markers in women with ICP (n = 63), PG (n = 43), and normal pregnant controls (n = 26). Blood was sampled on at least 3 separate occasions between 16 weeks of gestation and 4 weeks postpartum. Serum concentrations of GSTA increased with gestation in ICP, being significantly higher from 24 (±2) weeks compared with controls (400% difference; 95% CI, 240%-734%; P < .001). GSTA was also higher in ICP versus PG (433% difference; 95% CI, 228%-790%; P < .001) throughout the gestational period studied. Significant differences in the ICP compared with control and PG groups were also found for total bile acids, alanine aminotransferase, aspartate aminotransferase, -glutamyl transpeptidase and alkaline phosphatase. In conclusion, the measurement of GSTA provides a test of liver dysfunction that distinguishes women with ICP from those with PG. Additionally, on the basis of this study, reference ranges for biochemical markers of liver function require reevaluation in pregnancy. (HEPATOLOGY 2004;40:1406-1414.)
Viral Hepatitis
Waning immunity to plasma-derived hepatitis B vaccine and the need for boosters 15 years after neonatal vaccination (p 1415-1420)
Chun-Yi Lu, Bor-Luen Chiang, Wei-Kuang Chi, Mei-Hwei Chang, Yen-Hsuan Ni, Hsu-Mei Hsu, Shiing-Jer Twu, Ih-Jen Su, Li-Min Huang, Chin-Yun Lee
Neonatal immunization with hepatitis B (HB) vaccine is highly effective; however, more needs to be learned about the duration of protection and indications for boosters. We measured antibody to HB core antigen (anti-HBc), HB surface antigen (HBsAg), and pre- and postbooster titers of HBsAg antibody (anti-HBs) 15 years after primary neonatal immunization with plasma-derived HB vaccines in 2 cohorts of 15-year-old children. Group A consisted of 78 children who were born to HB e antigen-positive HBsAg carrier mothers and had developed protective levels of anti-HBs antibodies (10 mIU/mL) following HB immunization. Group B consisted of 113 apparently healthy children whose anti-HBs titers after vaccination were unknown. Anti-HBs was undetectable (antibody titer <10 mIU/mL) in 29.9% in group A and 62.4% in group B (P < .001). Anti-HBc was detected in 33.3 % in group A and 4.4 % in group B (P < .001). After a single booster dose of HB vaccine, 2.7% in group A and 3.3% in group B remained anti-HBs-negative. A blunted serological response was noted in approximately 20% in both groups. One HBsAg carrier was detected in group A (1.3%) and 4 in group B (3.5%). Fifteen years after neonatal immunization with plasma-derived HB vaccine, a large proportion of children exhibited waning immunity. This poses the risk of breakthrough infection. A single booster augmented the serological response to the vaccine in most but not all subjects. In conclusion, our findings suggest that one or more booster immunizations are needed in seronegative subjects by at least 15 years following neonatal immunization with plasma-derived HB vaccine. (HEPATOLOGY 2004;40:1415-1420.)
Comparison of adefovir and tenofovir in the treatment of lamivudine-resistant hepatitis B virus infection (p 1421-1425)
Florian van Bömmel, Thomas Wünsche, Stefan Mauss, Petra Reinke, Alexandra Bergk, Dirk Schürmann, Bertram Wiedenmann, Thomas Berg
defovir dipivoxil was recently approved for the treatment of wild-type and lamivudine-resistant hepatitis B virus (HBV) infection. Tenofovir disoproxil fumarate, a congender of adefovir that is used in the treatment of HIV infected patients, has recently been shown to also be effective in patients with lamivudine-resistant HBV infection. We therefore compared the two substances in a study of 53 patients defined by high HBV DNA (>6 log10 copies/mL) levels and genotypic evidence of lamivudine resistance. Thirty-five patients received tenofovir for 72 to 130 weeks, and 18 received adefovir for 60 to 80 weeks. Changes in HBV DNA levels were followed for the complete period of 48 weeks. Early viral kinetics were compared on matched subgroups of 5 patients each. Individually, all tenofovir-treated patients showed a strong and early suppression of HBV DNA within a few weeks whether they were coinfected with HIV or were without comorbidity. In contrast, considerable individual variations in HBV DNA decline were observed in the adefovir group. Thus at week 48, only 44% of these patients had HBV DNA levels below 105 copies/mL in contrast to 100% of the tenofovir-treated patients (P = .001). No severe side effects were noticed in either group. No evidence of phenotypic viral resistance could be demonstrated in the tenofovir-treated patients in the long term (up to 130 weeks). In conclusion, tenofovir may become an effective alternative for the treatment of patients with lamivudine-resistant HBV infection. (HEPATOLOGY 2004;40:1421-1425.)
Progression of liver fibrosis in women infected with hepatitis C: Long-term benefit of estrogen exposure (p 1426-1433)
Vincent Di Martino, Pascal Lebray, Robert P. Myers, Emmanuelle Pannier, Valérie Paradis, Frédéric Charlotte, Joseph Moussalli, Dominique Thabut, Catherine Buffet, Thierry Poynard
Female sex is a protective factor for the progression of fibrosis in patients with chronic hepatitis C virus (HCV) infection. Experimental data suggest that estrogens may have an antifibrotic effect. The objective of this study was to evaluate the influence of past pregnancies, oral contraceptives, menopause, and hormone replacement therapy (HRT) on liver fibrosis progression in HCV-infected women. Four hundred seventy-two HCV-infected women received a survey regarding prior pregnancies, menopause, and the use of oral contraceptives and HRT. The impact of these variables on liver fibrosis and its progression were evaluated using multivariate analyses considering all putative confounding factors. Two hundred one women completed the survey (43% response rate), 157 of whom had an estimated date of HCV infection (96 postmenopausal women, 96 women with previous pregnancies, and 105 women with past use of oral contraceptives). Through multivariate analyses, the estimated rate of fibrosis progression was higher in postmenopausal (P < .05) and nulliparous (P = .02) women and was associated with greater histological activity (P < .001). Prior use of oral contraceptives had no significant influence. Among postmenopausal women, the estimated rate of fibrosis progression (±SE) was lower in women who received HRT compared with untreated patients (0.099 ± 0.016 vs. 0.133 ± 0.006 METAVIR units/yr; P = .02) and was similar to that of premenopausal women (0.093 ± 0.012 METAVIR units/yr; P value not significant). In conclusion, menopause appears to be associated with accelerated liver fibrosis progression in HCV-infected women, an effect that may be prevented by HRT. Pregnancies may have a beneficial impact on the long-term progression of liver fibrosis. (HEPATOLOGY 2004;40:1426-1433.)
Chronic hepatitis C virus infection: Does it really impact health-related quality of life? A study in rural Egypt (p 1434-1441)
Michaël Schwarzinger, Sahar Dewedar, Claire Rekacewicz, Khaled Mahmoud Abd Elaziz, Arnaud Fontanet, Fabrice Carrat, Mostafa Kamal Mohamed
Previous Western studies showed a consistent and marked reduction in health-related quality of life (HRQOL) in patients chronically infected with hepatitis C virus (HCV). However, these studies were conducted on patients whose knowledge of their serological status may have affected their HRQOL. This HRQOL survey conducted in the Egyptian rural population provides a unique opportunity to clarify this issue among a population whose serological status is unknown. HRQOL was assessed by an Arabic translation of the Short-Form 12, and a visual analog scale of the relative severity of one's health status. HCV chronic infection was defined by positive tests for anti-HCV antibody and HCV-RNA. HRQOL was compared according to HCV chronic infection status in linear mixed models adjusted for potential confounding factors, such as age, sex, education, and health care-related risk factors, and adjusted for interviewer as a random effect. One hundred forty-six Egyptians chronically infected with HCV had similar Short-Form 12 and visual analog scale scores, compared with 1,140 uninfected controls from the same rural community. In individuals chronically infected with HCV, serum aminotransferase levels did not correlate with HRQOL. In conclusion, this study did not find a significant reduction of HRQOL in patients chronically infected with HCV compared with uninfected, contemporaneous controls. This may be explained in part by a lower morbidity amongst patients chronically infected with HCV in rural Egypt and a higher morbidity amongst uninfected controls as compared with those of Western studies, as well as a lack of awareness of hepatitis C serological status. (HEPATOLOGY 2004;40:1434-1441.)
Viral kinetics during antiviral therapy in patients with chronic hepatitis C and persistently normal ALT levels (p 1442-1449)
Bernd Kronenberger, Eva Herrmann, Florence Micol, Michael von Wagner, Stefan Zeuzem
The aim of the present study was to compare viral kinetics between patients with chronic hepatitis C and persistently normal alanine aminotransferase (ALT) levels and those with elevated ALT levels. Kinetic parameters were derived from nonlinear, least square fitting of serum hepatitis C virus RNA quantifications collected from patients with chronic hepatitis C and persistently normal (n = 20) and elevated (n = 19) ALT levels before and during treatment with 180 g pegylated interferon -2a once weekly plus daily ribavirin. Patients with chronic hepatitis C and persistently normal ALT levels showed a trend to lower pretreatment infected cell loss () (P = .13) but no differences in efficacy of blocking virus production () and infected cell loss during treatment (m) compared with patients with elevated ALT levels. Differences were significant for (P = .02) and (P = .04) when applying updated healthy levels for ALT (0.75 times and 0.63 times upper limit of normal for male and female patients, respectively). A significant reduction of the kinetic parameters , , and m was observed in patients with elevated -glutamyltranspeptidase (GGT) levels compared with patients with normal GGT levels (P = .02, P = .005, and P = .02, respectively). In conclusion, viral kinetics are similar in patients with chronic hepatitis C and persistently normal ALT levels and those with elevated ALT levels. However, in patients with elevated GGT levels, a major association with reduced efficacy of blocking virus production and lower infected cell loss was observed. These data show that virological response in patients with chronic hepatitis C is less associated with baseline ALT than with GGT levels. (HEPATOLOGY 2004;40:1442-1449.)
Epoetin alfa improves quality of life in anemic HCV-infected patients receiving combination therapy (p 1450-1458)
Paul J. Pockros, Mitchell L. Shiffman, Eugene R. Schiff, Mark S. Sulkowski, Zobair Younossi, Douglas T. Dieterich, Teresa L. Wright, Samir H. Mody, K. Linda Tang, Betty L. Goon, Peter J. Bowers, Gerhard Leitz, Nezam H. Afdhal, PROACTIVE Study Group
Anemia and decreased health-related quality of life (HRQL) are common in patients receiving combination therapy of interferon alfa (IFN) and ribavirin (RBV) for chronic hepatitis C virus (HCV) infection. In a randomized, prospective study evaluating the effectiveness of epoetin alfa in maintaining RBV dose, alleviating anemia, and improving HRQL in anemic (Hb 12 g/dL) HCV-infected patients receiving combination therapy, patients receiving epoetin alfa had significant improvements in HRQL compared with placebo. In this study, 185 patients were randomized to 40,000 units of epoetin alfa subcutaneously weekly or placebo for an 8-week double-blind phase (DBP), followed by an 8-week open-label phase during which all patients received epoetin alfa. To further assess the impact of epoetin alfa on HRQL, post hoc analyses were conducted in the same patient population to compare the HRQL of these patients at randomization with norms of other populations, and to determine the critical relationship between hemoglobin (Hb) levels and HRQL. Mean HRQL scores of anemic HCV-infected patients receiving combination therapy at randomization were significantly lower than those of both the general population and patients who had other chronic conditions. Patients receiving epoetin alfa who had the greatest Hb increases from randomization to the end of the DBP also had the largest improvements in HRQL. Hb improvement was an independent predictor of HRQL improvement in these patients. In conclusion, epoetin alfa provided clinically significant HRQL improvement in HCV-infected patients receiving IFN/RBV therapy. (HEPATOLOGY 2004;40:1450-1458.)
Copyright © 2004 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for December 2004 - Volume 127 - Number 6
Rapid Communications
Is there endoscopic capacity to provide colorectal cancer screening to the unscreened population in the United States?
Laura C. Seeff, Diane L. Manninen, Fred B. Dong, Sajal K. Chattopadhyay, Marion R. Nadel, Florence K.L. Tangka, Noelle-Angelique M. Molinari
Background & Aims: Screening rates for colorectal cancer remain low compared with screening rates for other cancers. The size of the unscreened population and the capacity to provide widespread screening are unknown. We estimated the number of average-risk persons aged 50 years or older not screened for colorectal cancer, the number of procedures required for this population, and the endoscopic capacity to satisfy this unmet need. Methods: Using data from the US Census Bureau and the Centers for Disease Control and Prevention’s National Health Interview Survey, we designed a forecasting model to estimate the number of persons in the United States currently not screened for colorectal cancer and the number of examinations needed to screen these persons. Test need was compared with available capacity, based on results from the national Survey of Endoscopic Capacity, assuming different proportions of available capacity were used for colorectal cancer screening. Results: Approximately 41.8 million average-risk people aged 50 years or older have not been screened for colorectal cancer according to national guidelines. Sufficient capacity exists to screen the unscreened population within 1 year using fecal occult blood testing followed by diagnostic colonoscopy for positive tests. Depending on the proportion of available capacity used for colorectal cancer screening, it could take up to 10 years to screen the unscreened population using flexible sigmoidoscopy or colonoscopy. Conclusions: The capacity exists for widespread screening with fecal occult blood testing. The capacity for screening with flexible sigmoidoscopy or colonoscopy depends on the proportion of available capacity used for colorectal cancer screening.
How many endoscopies are performed for colorectal cancer screening? Results from CDC’s survey of endoscopic capacity
Laura C. Seeff, Thomas B. Richards, Jean A. Shapiro, Marion R. Nadel, Diane L. Manninen, Leslie S. Given, Fred B. Dong, Linda D. Winges, Matthew T. McKenna
Background & Aims: Estimates of the current number of endoscopic colorectal cancer screening and follow-up examinations being performed are limited. A national study was therefore conducted among US physician practices. Methods: Approximately 1800 medical practices were surveyed from a list of all practices known to have purchased or leased lower endoscopic equipment between 1996 and 2000. Questions were asked regarding the current number of lower endoscopic procedures performed and the potential maximum number that could be performed. Results: In 2002, a total of 8207 practices reported performing flexible sigmoidoscopy or colonoscopy in the United States. Gastroenterologists performed 43.7% (95% confidence interval [CI], 37.250.2) of all sigmoidoscopies and 82.5% (95% CI, 80.384.7) of all colonoscopies. Primary care physicians performed 24.9% (95% CI, 20.329.5) of all sigmoidoscopies and 2.0% (95% CI, 1.42.6) of all colonoscopies. All physicians combined performed approximately 2.8 million (95% CI, 2.43.1) flexible sigmoidoscopies and 14.2 million (95% CI, 12.116.4) colonoscopies but reported that they could increase to approximately 9.5 million flexible sigmoidoscopies (95% CI, 8.410.5) and 22.4 million colonoscopies (95% CI, 20.124.8) in 1 year. Conclusions: Approximately 2.8 million flexible sigmoidoscopies and 14.2 million colonoscopies were estimated to have been performed in 2002. Physicians reported that they could perform an additional 6.7 million flexible sigmoidoscopies and 8.2 million colonoscopies in 1 year. These additional procedures could be used for the unscreened population and should be considered in the estimate of the national capacity to provide colorectal cancer screening to all eligible persons in the United States.
The mismatch repair complex hMutS? recognizes 5-fluorouracil-modified DNA: Implications for chemosensitivity and resistance
Akihiro Tajima, Martin T. Hess, Betty L. Cabrera, Richard D. Kolodner, John M. Carethers
Background & Aims:: Recent evidence suggests that patients with advanced microsatellite unstable (MSI) colorectal cancers lack a survival benefit with 5-fluorouracil (5-FU)-based chemotherapy. Additionally, tumor cells with MSI (caused by defective DNA mismatch repair) are more resistant to 5-FU in culture compared with microsatellite stable cells, despite similar amounts of 5-FU incorporation into the cell’s DNA. We examined whether the component of the DNA mismatch repair (MMR) system that normally recognizes single base pair mismatches could specifically recognize 5-FU incorporated into DNA as a potential mechanism for chemosensitivity. Methods: We synthesized oligonucleotides with and without incorporated 5-FU and created oligonucleotides with a single base pair mismatch (as a positive control) to perform electromobility gel shift assays (EMSA) with a purified, baculovirus-synthesized hMutS? MMR complex. We also utilized surface plasmon resonance to measure relative binding differences between the oligonucleotides and hMutS? in real time. Results: Using EMSA, we demonstrate that hMutS? recognizes and binds 5-FU-modified DNA. The reaction is specific as added ATP dissociates the hMutS? complex from the 5-FU-modified strand. Using surface plasmon resonance, we demonstrate greater binding between hMutS? and 5-FU-modified DNA compared with complementary DNA or DNA containing a C/T mismatch. Conclusions: The MMR complex hMutS? specifically recognizes and binds to 5-FU-modified DNA. Because MMR components are required for the induction of apoptosis by many DNA-damaging agents, the chemosensitivity of 5-FU for patients with advanced colorectal cancer may be in part due to recognition of 5-FU incorporated into tumor DNA by the MMR proteins.
Clinical-alimentary Tract
Contributions of gastric volumes and gastric emptying to meal size and postmeal symptoms in functional dyspepsia
Silvia Delgado-Aros, Michael Camilleri, Filippo Cremonini, Irene Ferber, Debra Stephens, Duane D. Burton
Background & aims: The aim was to assess relative contributions of gastric volumes (GV) and gastric emptying (GE) to meal size and postprandial symptoms in patients with functional dyspepsia. Methods: Patients with chronic upper gastrointestinal symptoms were prospectively evaluated. GV during fasting and after 300 mL Ensure was measured with 99mTc-single-photon emission computed tomography imaging and solid GE (99mTc-egg) by scintigraphy. Maximum tolerated volume (MTV) and symptoms were measured after Ensure challenge. Results: Of 57 adult patients evaluated, 39 (23 women, 16 men) met Rome II criteria for functional dyspepsia and had no other diagnosis to account for dyspepsia. The most frequent symptoms were abdominal pain (90%), pain predominantly after meals (76%), nausea (85%), and early fullness after meals (79%). Relative to established laboratory normal values, MTV was abnormal in 82%, aggregate symptom score >209 in 72%, GE (at 1 hour) accelerated in 41%, GE (at 4 hours) delayed in 41%, and postmeal GV reduced in 52%. Lower body mass was associated with lower MTV and higher postchallenge symptoms. Lower fasting (not postprandial) GV and faster GE were independent predictors of lower MTV, explaining 18% of the variance after adjusting for body weight (32% of variance). GE was an independent predictor of postchallenge symptoms (10% of variance) after adjusting for volume ingested (10%), age (20%), and weight (10%). Conclusions: In adults with functional dyspepsia seen in a tertiary referral practice, decreased meal size and postmeal symptoms are associated with low fasting GV and faster GE. These data provide physiologic targets for ameliorating symptoms of functional dyspepsia.
Effect of acute physical and psychological stress on gut autonomic innervation in irritable bowel syndrome
Charles D.R. Murray, Joanna Flynn, Laura Ratcliffe, Meron R. Jacyna, Michael A. Kamm, Anton V. Emmanuel
Background & Aims: Stress is an important causative factor in irritable bowel syndrome (IBS). It remains unknown whether stress-related changes in gut function are mediated by altered autonomic efferent gut-specific innervation. We studied the effect of acute physical and psychological stress on autonomic innervation and visceral sensitivity in healthy volunteers and patients with IBS. Methods: Twenty-four patients (20 women) with constipation-predominant IBS and 12 healthy volunteers (8 women) underwent either physical (cold water hand immersion) or psychological (dichotomous listening) stress on separate occasions. Assessments included stress perception (visual analogue scale), gut-specific autonomic innervation (rectal mucosal blood flow [RMBF] by laser Doppler flowmetry), and viscerosomatic sensitivity (anal and rectal electrosensitivity). Results: Patients with IBS had a heightened baseline perception of stress (P < .01). RMBF decreased during physical stress (29.6% ± 2.8% and 28.7% ± 3.9%) and psychological stress (24.4% ± 2.1% and 23.5% ± 4.3%) in patients with IBS and controls, respectively (mean ± SEM). During physical stress, rectal perception (23.2% ± 6% vs .6% ± 3% [IBS vs control group, P < .05]) and rectal pain thresholds (27.0% ± 4% vs 1.3% ± 5%, P < .001) decreased in patients with IBS only. Psychological stress reduced thresholds for rectal perception (19.4% ± 6% vs 8% ± 6%, P < .01) and rectal pain (28.4% ± 4% vs 3.4% ± 3.8%, P < .001) in patients with IBS only. Acute stress elevated anal perception thresholds in patients with IBS but not controls (physical stress: 14.7% ± 14% vs ?9.3% ± 11%, P < .05; psychological stress: 24.7% ± 9% vs 11% ± 11%, P < .05). Conclusions: Acute stress alters gut-specific efferent autonomic innervation in both controls and patients with IBS, although normalization is delayed in IBS. By contrast, only patients with IBS show heightened visceral sensation, suggesting involvement of a different regulatory mechanism, either central or peripheral.
Clinical-liver, Pancreas, and Biliary Tract
Serum markers detect the presence of liver fibrosis: A cohort study
William M.C. Rosenberg, Michael Voelker, Robert Thiel, Michael Becka, Alastair Burt, Detlef Schuppan, Stefan Hubscher, Tania Roskams, Massimo Pinzani, Michael J.P. Arthur
Background & Aims: Histologic examination of a liver biopsy specimen is regarded as the reference standard for detecting liver fibrosis. Biopsy can be painful and hazardous, and assessment is subjective and prone to sampling error. We developed a panel of sensitive automated immunoassays to detect matrix constituents and mediators of matrix remodeling in serum to evaluate their performance in the detection of liver fibrosis. Methods: In an international multicenter cohort study, serum levels of 9 surrogate markers of liver fibrosis were compared with fibrosis stage in liver biopsy specimens obtained from 1021 subjects with chronic liver disease. Discriminant analysis of a test set of samples was used to identify an algorithm combining age, hyaluronic acid, amino-terminal propeptide of type III collagen, and tissue inhibitor of matrix metalloproteinase 1 that was subsequently evaluated using a validation set of biopsy specimens and serum samples. Results: The algorithm detected fibrosis (sensitivity, 90%) and accurately detected the absence of fibrosis (negative predictive value for significant fibrosis, 92%; area under the curve of a receiver operating characteristic plot, .804; standard error, .02; P < .0001; 95% confidence interval, .758.851). Performance was excellent for alcoholic liver disease and nonalcoholic fatty liver disease. The algorithm performed equally well in comparison with each of the pathologists. In contrast, pathologists’ agreement over histologic scores ranged from very good to moderate (? = .97.46). Conclusions: Assessment of liver fibrosis with multiple serum markers used in combination is sensitive, specific, and reproducible, suggesting they may be used in conjunction with liver biopsy to assess a range of chronic liver diseases.
Radiofrequency ablation improves prognosis compared with ethanol injection for hepatocellular carcinoma ≤4 cm
Shi-Ming Lin, Chun-Jung Lin, Chen-Chun Lin, Chao-Wei Hsu, Yi-Cheng Chen
Background & Aims: The aim of this study was to compare the clinical outcome of percutaneous radiofrequency (RF) ablation, conventional percutaneous ethanol injection (PEI), and higher-dose PEI in treating hepatocellular carcinoma (HCC) 4 cm or less. Methods: A total of 157 patients with 186 HCCs 4 cm or less were randomly assigned to 3 groups (52 patients in the conventional PEI group, 53 in the higher-dose PEI group, and 52 in the RF group). Clinical outcomes in terms of complete tumor necrosis, overall survival, local tumor progression, additional new tumors, and cancer-free survival were compared across 3 groups. Results: The rate of complete tumor necrosis was 88% in the conventional PEI group, 92% in the higher-dose PEI group, and 96% in the RF group. Significantly fewer sessions were required to achieve complete tumor necrosis in the RF group than in the other 2 groups (P < .01). The local tumor progression rate was lowest in the RF group (vs the conventional PEI group, P = .012; vs the higher-dose PEI group, P = .037). The overall survival rate was highest in the RF group (vs the conventional PEI group, P = .014; vs the higher-dose PEI group, P = .023). The cancer-free survival rate was highest in the RF group (vs the conventional PEI group, P = .019; vs the higher-dose PEI group, P = .024). Multivariate analysis determined that tumor size, tumor differentiation, and the method of treatment (RF vs both methods of PEI) were significant factors in relation to local tumor progression, overall survival, and cancer-free survival. Conclusions: The results show that RF ablation yielded better clinical outcomes than conventional and higher-dose PEI in treating HCC 4 cm or less.
Peginterferon alfa-2a (40 kilodaltons) and ribavirin in patients with chronic hepatitis C and normal aminotransferase levels
Stefan Zeuzem, Moisés Diago, Edward Gane, K. Rajender Reddy, Paul Pockros, Daniele Prati, Mitchell Shiffman, Patrizia Farci, Norman Gitlin, Christopher B. O’Brien, François Lamour, Pilar Lardelli
Background & Aims: Patients with chronic hepatitis C and persistently normal alanine aminotransferase (ALT) levels have been routinely excluded from large randomized treatment trials; consequently, the efficacy and safety of antiviral therapy in this population are unknown. Methods: Patients with at least 3 normal ALT values over an 18-month period were randomized (3:3:1) to treatment with peginterferon alfa-2a 180 ?g/wk plus ribavirin 800 mg/day for 24 weeks (212 patients), the same combination for 48 weeks (210 patients), or no treatment (69 patients) in a multinational study. All patients were monitored for 72 weeks. The primary measure of efficacy was sustained virologic response (SVR), defined as undetectable serum hepatitis C virus (HCV) RNA by qualitative polymerase chain reaction at the end of 24 weeks of untreated follow-up. Results: No patient cleared HCV RNA in the untreated control group. SVR rates of 30% and 52% were obtained in the 24- and 48-week treatment groups, respectively. In patients infected with HCV genotype 1, SVR rates of 13% and 40% were obtained with 24 and 48 weeks of treatment, respectively (P < .0001). In patients infected with genotypes 2 or 3, SVR rates were 72% and 78% with 24 and 48 weeks of treatment, respectively (P = .452). Treatment-related flares in ALT activity were not observed. Conclusions: The efficacy and safety of peginterferon alfa-2a and ribavirin combination therapy in patients with chronic hepatitis C and persistently normal ALT levels are similar to that in patients with elevated ALT levels. The indication for treatment of hepatitis C can be evaluated independently from baseline ALT activity.
Clinical relevance of hepatitis B virus genotype in children with chronic infection and hepatocellular carcinoma
Yen-Hsuan Ni, Mei-Hwei Chang, Kuan-Jan Wang, Hong-Yuan Hsu, Huey-Ling Chen, Jia-Horng Kao, Shiou-Hwei Yeh, Yung-Ming Jeng, Keh-Sung Tsai, Ding-Shinn Chen
Background & Aims: The aim of this study was to investigate the influence of hepatitis B virus (HBV) genotypes on the clinical outcome of chronic childhood HBV infection and hepatocellular carcinoma (HCC). Methods: A total of 460 HBV carrier children were followed-up for 15 years and 26 children with HBV-related HCC were recruited. HBV genotyping was examined at enrollment and the latest follow-up of these carrier children and at diagnosis in HCC children. Viral load was checked at enrollment for the carrier children. These carriers were grouped based on their initial hepatitis B e antigen (HBeAg) and antibody to hepatitis B e antigen (anti-HBe) status. The HBeAg positive (+) group was divided further into an HBeAg(+/+) group and HBeAg(+/?) group, depending on whether spontaneous HBeAg seroconversion occurred during the follow-up period. Results: Genotype B constituted 73%, 86%, and 76% in the HBeAg(+/+), HBeAg(+/?), and anti-HBe(+) groups, respectively. Genotype C was found in 27%, 8%, and 6% in the HBeAg(+/+), HBeAg(+/?), and anti-HBe(+) group, respectively. Genotype C carriers were more prevalent in the HBeAg(+/+) group than the other 2 groups (P = .01), and had a delayed HBeAg seroconversion compared with the genotype B carriers (P < .001). Changes of genotype during the follow-up period were rare (2.8%). In those with HCC, genotype B was also the major type (74%). There was no difference in the baseline viral load between genotypes B and C. Conclusions: Although HBV genotype B dominates in children with chronic HBV infection and HCC in Taiwan, genotype C delays HBeAg seroconversion in pediatric chronic HBV infection.
Basic-alimentary Tract
The ion channel ASIC1 contributes to visceral but not cutaneous mechanoreceptor function
Amanda J. Page, Stuart M. Brierley, Christopher M. Martin, Carlos Martinez-Salgado, John A. Wemmie, Timothy J. Brennan, Erin Symonds, Taher Omari, Gary R. Lewin, Michael J. Welsh, L. Ashley Blackshaw
Background & Aims: Visceral mechanoreceptors are critical for perceived sensations and autonomic reflex control of gastrointestinal function. However, the molecular mechanisms underlying visceral mechanosensation remain poorly defined. Degenerin/epithelial Na+ channel (DEG/ENaC) family ion channels are candidate mechanosensory molecules, and we hypothesized that they influence visceral mechanosensation. We examined the influence of the DEG/ENaC channel ASIC1 on gastrointestinal mechanosensory function, on gastric emptying, and on fecal output. We also compared its role in gastrointestinal and somatic sensory function. Methods: To assess the role of ASIC1 we studied wild-type and ASIC1?/? mice. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis determined expression of ASIC1 messenger RNA and protein in vagal and spinal sensory ganglia. Colonic, gastroesophageal, and cutaneous afferent fibers were characterized by functional subtype and their mechanical stimulus-response relationships were determined. Gastric emptying was determined by using a 13CO2 breath test. Behavioral tests assessed somatic mechanical and thermal sensitivity. Results: ASIC1 was expressed in sensory ganglia and was lost after disruption of the ASIC1 gene. Loss of ASIC1 increased mechanosensitivity in all colonic and gastroesophageal mechanoreceptor subtypes. In addition, ASIC1?/? mice showed almost double the gastric emptying time of wild-type mice. In contrast, loss of ASIC1 did not affect function in any of the 5 types of cutaneous mechanoreceptors, nor did it affect paw withdrawal responses or fecal output. Conclusions: ASIC1 influences visceral but not cutaneous mechanoreceptor function, suggesting that different mechanisms underlie mechanosensory function in gut and skin. The role of ASIC1 is highlighted by prolonging gastric emptying of a meal in ASIC1?/? animals.
Intestinal surgical resection disrupts electrical rhythmicity, neural responses, and interstitial cell networks
Hiroe Yanagida, Haruko Yanase, Kenton M. Sanders, Sean M. Ward
Background & Aims: Surgical manipulations of the gastrointestinal (GI) tract, including intestinal resection and anastomosis, lead to motility disorders including a decrease in phasic and segmental contractions. The aims of the present investigation were to determine mechanisms underlying the loss of motility in a murine model of intestinal resection and to follow-up the recovery of intestinal motility after surgical manipulation. Methods: Segments of ileum were removed from mice and the intestines were reconstructed. After surgery, the structure and activity of the ileal muscles, 05 cm oral and aboral to the site of resection, were examined at 5 and 24 hours with intracellular microelectrode recordings, isometric force measurements, Kit-like immunohistochemistry, and electron microscopy. Results: Five hours after surgery there was loss of electrical slow waves and phasic contractions in muscles near the site of resection. This defect decreased as a function of distance above and below the resection. Tissues in the affected region were poorly responsive to carbachol and transmural nerve stimulation. Kit-like immunohistochemistry revealed disruption in interstitial cell of Cajal (ICC) networks at the level of the myenteric and deep muscular plexuses. Lesions in ICCs decreased with distance from the site of resection. Slow waves and mechanical activity recovered at the site of anastomosis 24 hours after surgery and recovered more rapidly when tissues were incubated in the inducible nitric oxide synthase (iNOS) inhibitor, L-N6-(1-Iminoethyl) lysine hydrochloride (L-NIL). Conclusions: Loss of intestinal motility after surgery is associated with acute disruption of ICC networks, slow waves, and phasic contractions. This activity partially recovered within 24 hours after surgery.
Basic-liver, Pancreas, and Biliary Tract
Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity
Zhang-Xu Liu, Sugantha Govindarajan, Neil Kaplowitz
Background & Aims: Inflammatory mediators released by nonparenchymal inflammatory cells in the liver have been implicated in the progression of acetaminophen (APAP) hepatotoxicity. Among hepatic nonparenchymal inflammatory cells, we examined the role of the abundant natural killer (NK) cells and NK cells with T-cell receptors (NKT cells) in APAP-induced liver injury. Methods: C57BL/6 mice were administered a toxic dose of APAP intraperitoneally to cause liver injury with or without depletion of NK and NKT cells by anti-NK1.1 monoclonal antibody (MAb). Serum alanine transaminase (ALT) levels, liver histology, hepatic leukocyte accumulation, and cytokine/chemokine expression were assessed. Results: Compared with APAP-treated control mice, depletion of both NK and NKT cells by anti-NK1.1 significantly protected mice from APAP-induced liver injury, as evidenced by decreased serum ALT level, improved survival of mice, decreased hepatic necrosis, inhibition of messenger RNA (mRNA) expression for interferon-? (IFN-?), Fas ligand (FasL), and chemokines including KC (Keratinocyte-derived chemokine); MIP-1? (macrophage inflammatory protein-1?); MCP-1 (monocyte chemoattractant protein-1); IP-10 (interferon-inducible protein); Mig (monokine induced by IFN-?) and decreased neutrophil accumulation in the liver. Hepatic NK and NKT cells were identified as the major source of IFN-? by intracellular cytokine staining. APAP induced much less liver injury in Fas-deficient (lpr) and FasL-deficient (gld) mice compared with that in wild-type mice. Conclusions: NK and NKT cells play a critical role in the progression of APAP-induced liver injury by secreting IFN-?, modulating chemokine production and accumulation of neutrophils, and up-regulating FasL expression in the liver, all of which may promote the inflammatory response of liver innate immune system, thus contributing to the severity and progression of liver injury downstream of the metabolism of APAP and depletion of reduced glutathione (GSH) in hepatocytes.
The role of notch signaling in the development of intrahepatic bile ducts
Yuzo Kodama, Makoto Hijikata, Ryoichiro Kageyama, Kunitada Shimotohno, Tsutomu Chiba
ackground & Aims: Mutations in Jagged1, a Notch ligand, cause Alagille syndrome (AGS), a disorder characterized by a paucity of intrahepatic bile ducts (IHBD). The mechanism underlying the contribution of the Notch signaling pathway to IHBD formation, however, remains unknown. Here we investigated the role of Notch signaling in IHBD development. Methods: The expression patterns of Jagged1, Notch2, and Hes1 during mouse liver development were analyzed by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), immunoblot, and immunohistochemistry. The hepatocyte maturation level and IHBD development were studied in Hes1 null mice in comparison with wild-type mice. The effect of Jagged1 on biliary differentiation was assessed by using an in vitro 2-cell coculture system with WB-F344 cells, a cell line derived from normal adult rat liver. Results: Jagged1 was expressed in the portal mesenchyme during the neonatal period. During the same period, Notch2 and Hes1 expression was observed in the biliary epithelial cells adjacent to the Jagged1-positive cells. During ductal plate remodeling, Notch2 and Hes1 were up-regulated exclusively in the biliary epithelial cells that form tubular structures. In contrast, the tubular formation of IHBD was completely absent in Hes1 null mice. Coculture with Balb3T3 cells stably overexpressing Jagged1 induced transactivation of the Hes1 promoter and increased expression of biliary lineage markers, such as cytokeratin-19 and ?-glutamyl transpeptidase, in WB-F344 cells. Conclusions: Our results suggest that Notch signaling has an important role in the differentiation of biliary epithelial cells and is essential for their tubular formation during IHBD development.
Translational regulation of XIAP expression and cell survival during hypoxia in human cholangiocarcinoma
Carla Marienfeld, Yoko Yamagiwa, Yoshiyuki Ueno, Valorie Chiasson, Linda Brooks, Fanyin Meng, Tushar Patel
Background & Aims: Tumor progression is promoted by the ability of tumor cells to resist adverse environmental conditions such as hypoxia. We have shown that translational dysregulation contributes to transformed cell growth in malignant cholangiocytes. Translational regulation of gene expression can contribute to an immediate and rapid response to environmental changes such as hypoxia. Thus, our aims were to assess translational mechanisms involved in cell survival during hypoxia and to identify specific translationally regulated proteins involved in the cellular response to hypoxia. Methods: Cell viability and apoptosis in response to hypoxia were assessed in human cholangiocarcinoma cells. Translational processes were deregulated by cycloheximide or rapamycin or by targeted deletion of eukaryotic initiation factor (eIF)-4E, a rate-limiting translational initiation factor using small interfering RNA (siRNA). A protein antibody microarray was used to screen for eIF-4E-dependent proteins expressed during hypoxia. Expression of the X-linked inhibitor of apoptosis (XIAP) was decreased using siRNA. Results: Malignant cholangiocytes are resistant to hypoxia-induced apoptosis. Furthermore, cell survival during hypoxia required protein translation. eIF-4E was over expressed in malignant cholangiocytes. Reduction in eIF-4E expression by siRNA decreased tumor cell resistance to hypoxia, increased caspase-3 activation and apoptosis, and decreased cell survival compared with controls. XIAP was identified as a translationally regulated protein expressed during hypoxia. Modulation of XIAP expression by siRNA decreases cell death during hypoxia in vitro and in vivo. Conclusions: Human cholangiocarcinoma cells are highly resistant to hypoxia. Translational regulation of survival proteins such as XIAP is a mechanism mediating cholangiocarcinoma survival during hypoxia.
The role of AMP-activated protein kinase in the action of ethanol in the liver
Min You, Michinaga Matsumoto, Christine M. Pacold, Won Kyoo Cho, David W. Crabb
Background & Aims: Our previous work has shown that ethanol induces the fatty acid synthesis pathway by activation of sterol regulatory element-binding protein 1 (SREBP-1). In the present study, we studied the mechanisms of this activation by identifying a new target of ethanol, AMP-activated protein kinase (AMPK). Methods: The effects of ethanol on AMPK, acetyl-CoA carboxylase (ACC), and SREBP-1 were assessed in rat hepatic cells and in the livers of ethanol-fed mice. Results: In rat hepatoma H4IIEC3 or McA-RH 7777 cell lines, ethanol-induced transcription of an SREBP-regulated promoter was suppressed by the presence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or metformin, 2 known AMPK activators. Consistent with this, over expression of a constitutively active form of AMPK blocked the effect of ethanol, whereas coexpression of a dominant-negative form of AMPK augmented the effect. Moreover, activation of AMPK by metformin or AICAR largely blocked the ability of ethanol to increase levels of mature SREBP-1 protein. These findings suggest that the effect of ethanol on SREBP-regulated promoter activation was partially mediated through AMPK inhibition. We further demonstrated that AMPK was inhibited by ethanol in hepatic cells. In parallel, ethanol increased the activity of ACC and suppressed the rate of palmitic acid oxidation. Finally, feeding mice a low-fat diet with ethanol resulted in significantly reduced hepatic AMPK activity, increased ACC activity, and enhanced malonyl CoA content. Conclusions: Taken together, our findings suggest that AMPK may play a key role in regulating the effects of ethanol on SREBP-1 activation, fatty acid metabolism, and development of alcoholic fatty liver.
Case Reports
BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level
Burkhard M. Helmke, Jan Mollenhauer, Christel Herold-Mende, Axel Benner, Marianne Thome, Nikolaus Gassler, Wolfgang Wahl, Stefan Lyer, Annemarie Poustka, Herwart F. Otto, Martin Deichmann
Background & Aims: Anorectal melanoma (AM) is a rare but highly malignant tumor, displaying histologic and immunohistochemical features very similar to cutaneous melanoma (CM). Because BRAF mutations were recently identified in the majority of CM and nevi, we investigated AM for BRAF mutations and mutations of NRAS, an additional component of the MAPK-signalling pathway. Methods: DNA from formalin-fixed and paraffin-embedded AM was PCR amplified and sequenced. Results: We detected BRAF mutations in 2 of 19 cases and NRAS mutations in none of the cases. Mutations in exon 15 of BRAF were present in only 1 tumor (1 of 19 cases). The A1800T base exchange represented a novel mutation and resulted in a K600N transition in an AM from a 96-year-old white man who presented with rectal bleeding and painful sitting of a few weeks’ duration. The second positive AM case, a 69-year-old white man who presented with painless rectal bleeding and clinical symptoms of an intestinal constipation showed a novel missense mutation (C1327T leading to R443W conversion) in BRAF exon 11. None of the AM cases displayed the oncogenic V599E mutation preponderating in CM. Conclusions: With regard to the frequency of V599E BRAF mutations, AM significantly differs from CM (P ≤ .0001), which suggests that BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level.
Special Reports and Reviews
Colorectal cancer vaccines: Principles, results, and perspectives
Simone Mocellin, Carlo Riccardo Rossi, Mario Lise, Donato Nitti
In the search for novel therapeutic approaches to treat patients with colorectal carcinoma, anticancer vaccination holds promise. A large body of preclinical and clinical evidence has demonstrated that the immune system can be polarized against malignant cells by means of several active specific immunotherapy strategies. Although no vaccination regimen can be currently recommended outside clinical trials, tumor response and immunologic findings observed in animal models and humans prompt researchers to explore further the antitumor potential of such biotherapy in an effort to reproduce in a larger set of patients the cascade of molecular events that characterizes the successful tumor immune rejection currently observed in a minority of vaccinated subjects. In this work, we summarize the principles and the main results of cancer vaccine strategies so far implemented for the treatment of patients with colorectal carcinoma. We also discuss the most recent preclinical tumor immunology insights that might change the way to design the next generation of cancer vaccines, hopefully improving the effectiveness of such a biotherapeutic approach.
Copyright © 2001-2004 by the American Gastroenterological Association. All rights reserved.
Estrogen receptors in cholangiocytes and the progression of primary biliary cirrhosis
Domenico Alvaroae*, Pietro Invernizzib, Paolo Onoric, Antonio Franchittod, Adriano De Santis a, Andrea Crosignanib, Roberta Sferrac, Stefano Ginanni-Corradinia, Maria Grazia Mancinoa, Marco Maggionib, Adolfo F. Attilia, Mauro Poddab, Eugenio Gaudiod
Estrogen receptors (ER) in cholangiocytes of primary biliary cirrhosis (PBC) patients and their relationship with cell proliferation and death were evaluated.
Methods: Liver biopsies from PBC patients with different histological stages were investigated by immunohistochemistry for ER-? and -?, cytokeratin-19, proliferating cellular nuclear antigen (PCNA), Fas and terminal deoxynucleotide transferase end labelling (TUNEL). Normal livers and livers from primary sclerosing cholangitis and alcoholic cirrhosis were investigated as controls.
Results: ER-? and -? were observed in cholangiocytes of PBC patients but not in normal liver. In PBC, positivity for ER-? was high (5065%) in all histological stages while, positivity for ER-? increased from 1% in stage I to 12% in stage III (positivity correlated and co-localized in the same cell with PCNA). In stage IV of PBC, cholangiocytes were negative for ER-? in association with a lower PCNA positivity and with maximal degree of ductopenia. ER-? positivity in cholangiocytes of PBC patients was markedly lower than primary sclerosing cholangitis and alcoholic cirrhosis. Conclusions :ER are expressed in PBC and other pathologies associated with cholangiocyte proliferation but not in normal subjects. The low expression of ER-? in PBC and their disappearance in the advanced histological stages suggests that an estrogenic deficiency could favour the evolution of this disease toward ductopenia.
Processing of protein glycation, oxidation and nitrosation adducts in the liver and the effect of cirrhosis
Naila Ahmeda, Paul J. Thornalleya*, Reinhard Lüthenb, Dieter Häussingerb, Katarina Sebekovac, Reinhard Schinzeld, Wolfram Voelkere, August Heidlande
Plasma proteins are modified non-enzymatically in vivo by glycation, oxidation and nitrosation processes. Hepatic extraction of albumin glycated in vitro was reported but it is not clear if plasma proteins glycated in vivo also undergo hepatic extraction. We investigated the hepatic extraction of glycated, oxidised and nitrosated proteins in vivo.
Methods : Protein glycation, oxidation and nitrosation marker residues and free adducts were determined in portal, hepatic and peripheral venous blood plasma of cirrhotic patients and hepatic and peripheral venous blood plasma (as a surrogate of portal venous blood) of control subjects by liquid chromatography-mass spectrometry.
Results : There was no evidence for extraction of glycated, oxidised or nitrosated proteins or related free adducts by the liver in control subjects. There was limited extraction of methylglyoxal-modified proteins in cirrhotic patients and twofold increases in the concentrations of fructosyl-lysine and advanced glycation endproduct residues of plasma protein, with respect to controls. Remarkably, glyoxal-derived hydroimidazolone free adduct was increased 1416-fold probably as a consequence of hepatic lipid peroxidation.
Conclusions : We found no evidence for hepatic extraction of glycated, oxidised and nitrosated proteins or related free adducts in subjects with normal liver function and limited extraction of methylglyoxal-modified protein in cirrhotic subjects.
Epidemiology of liver disease in cystic fibrosis: a longitudinal study
Thierry Lamireauac, Sylvie Monnereauc, Steven Martina, Jacques-Edouard Marcotteb, Maria Winnockd, Fernando Alvareza*
To describe the prevalence of liver disease in a cohort of 241 cystic fibrosis (CF) patients.
Methods : 241 CF patients were followed-up every 3 months with clinical and biological assessment, and every year with ultrasonography of the liver. The presence of liver disease was studied using a multivariate Cox's regression analysis including variables such as history of meconium ileus, pulmonary function, pancreatic insufficiency and CFTR gene mutations.
Results : The prevalence of liver disease was 18, 29, and 41% after 2, 5 and 12 years, respectively, and did not increase thereafter. In multivariate analysis, the probability of liver disease was independently associated with history of meconium ileus (P=0.001) and pancreatic insufficiency (P=0.004). CFTR mutations and severity of pulmonary disease were not associated with liver disease. Cirrhosis occurred in 19 (7.8%) patients at a median age of 10 years, and liver transplantation was required in five patients.
Conclusions :This study shows that CF related-liver disease occurs mainly in the first decade of life with a prevalence of 41% of patients at 12 years of age. A history of meconium ileus and pancreatic insufficiency are predictive of liver disease. Preventive treatment with ursodesoxycholic acid could be considered in patients with meconium ileus.
Curcumin inhibits NF-?B activation and reduces the severity of experimental steatohepatitis in mice
Isabelle A. Leclercqa*, Geoffrey C. Farrellc, Christine Sempouxb, Aileen dela Peñac, Yves Horsmansa
While oxidative stress is a feature of non-alcoholic steatohepatitis, the causal link between oxidative stress and inflammatory recruitment has yet to be demonstrated. We analysed the role of NF-?B redox-sensitive signalling pathway of inflammatory recruitment in experimental steatohepatitis.
Methods : Mice were fed the methionine and choline deficient (MCD) or the control diet, with or without curcumin, an NF-?B inhibitor, for up to 4 weeks. Histopathology, lipoperoxides, NF-?B/DNA binding and expression of NF-?B-regulated genes were assessed.
Results : MCD-fed mice developed steatohepatitis accompanied by dramatic accumulation of hepatic lipoperoxides, activation of NF-?B and induction of pro-inflammatory ICAM-1, COX-2, MCP-1 and CINC mRNA. Curcumin significantly reduced MCD-induced inflammation but had no effect on steatosis or on the level of hepatic lipid peroxides. Curcumin prevented the MCD-induced activation of NF-?B and decreased downstream induction of ICAM-1, COX-2 and MCP-1. However, it failed to reduce activation of AP-1, MAPK pathways or CINC expression. Conclusions :Curcumin alleviates the severity of hepatic inflammation in experimental steatohepatitis induced by the MCD diet, an effect likely to be mediated via inhibition of NF-kB activation and dependent pro-inflammatory genes. The NF-?B pathway is one among several possible signalling pathways by which inflammation is recruited in experimental steatohepatitis.
Evaluation of a panel of non-invasive serum markers to differentiate mild from moderate-to-advanced liver fibrosis in chronic hepatitis C patients
Keyur Patela, Stuart C. Gordonb, Ira Jacobsonc, Christophe Hézodede, Esther Ohf, Katie M. Smithf, Jean-Michel Pawlotskyd, John G. McHutchisona*
Background/Aims : In chronic hepatitis C (CHC) infection, a liver biopsy provides important information that guides treatment decisions, but is invasive, expensive and associated with possible complications. Extracellular matrix remodeling proteins may be useful non-invasive markers of fibrosis. The aim of this study was to evaluate the diagnostic accuracy of a panel of these markers in CHC patients, develop a predictive algorithm that differentiates no/mild (METAVIR F0F1) from moderate/severe (F2F4) fibrosis, and validate the model in external cohorts.
Methods : A combination of matrix markers were initially evaluated and optimized in 294 CHC patients from a single center, and validated in an external cohort of 402 patients.
Results : Hyaluronic acid, TIMP-1 and alpha2-macroglobulin were selected as having the best predictive accuracy for F2F4 fibrosis (combined AUROC=0.831). At an index cut-off >0.36 and prevalence for F2F4 of 52%, results in all 696 patients indicated positive and negative predictive values of 74.3 and 75.8% with an accuracy of 75%.
Conclusions :The three-marker panel may reliably differentiate CHC patients with moderate/severe fibrosis from those with no/mild fibrosis, although accurate delineation between stages was not possible. Prospective studies are required to determine the potential utility of the marker panel in guiding treatment decisions and following disease progression.
Leptin, insulin resistance, and liver fibrosis in human nonalcoholic fatty liver disease
Paul Anguloa*, Laura M. Albaa, Lydia M. Petrovicb, Leon A. Adamsa, Keith D. Lindora, Michael D. Jensenc
Background/Aims : Data from animal models of fibrosis and fatty liver suggest that leptin may mediate the profibrogenic responses in the liver, but the association of leptin and liver fibrosis in human nonalcoholic fatty liver disease (NAFLD) remains undefined. We aimed at determining the relation between leptin and liver fibrosis in human NAFLD.
Methods : Human plasma leptin and several indicators of insulin resistance were measured in 88 NAFLD patients and matched controls.
Results :Leptin levels were significantly greater in patients with more advanced fibrosis (P=0.005). By multivariate analysis, the significant association between leptin and fibrosis was abolished (adjusted P=0.3) when controlling for confounders including age, gender, BMI, diabetes and insulin resistance. Only age (adjusted P=0.006) and insulin sensitivity (adjusted P=0.04) correlated significantly with fibrosis stage. A second liver biopsy was performed in 39 out of the 88 patients at 27.9±16 months. Leptin levels were not significantly different between patients who had fibrosis progression (n=10) and those who did not (n=29).
Conclusions : human NAFLD, no relationship between leptin levels and fibrosis stage was demonstrated. The correlation of leptin and fibrosis severity seems to be an indicator of the factors that determine leptin production.
Development of a scaled up liver device incorporating cryo-preserved pig liver micro-organs
Amikam Gershonowitza, Etty Grad Itacha, Daniel Shouvalb*, Dov Mitrania, Yaron Ilanb, Eduardo Mitranic
Currently there is no effective therapy for most patients with fulminant or end stage liver disease.
Methods : Pig liver micro-organs (LMOs), which preserve liver micro-architecture and ensure a maximal 150200?m distance from a source of nutrients and gases have been prepared and a method to cryo-preserve them has been developed. A new scaled-up extra-corporeal liver device termed aLIVE-H in which LMOs are exposed to liver-like hemodynamic conditions has also been developed. The purpose of this work is to test the safety and function of cryo-preserved LMOs and how the hemodynamic properties of the scaled up aLIVE device affect their function. Results : Pig LMOs in aLIVE-H, transcribe albumin and Factor V at similar levels, irrespective of their position within the bioreactor, indicating that the hemodynamic features of the aLIVE-H device allow for homogeneous plasma distribution and proper function at different locations. Cryo-preserved LMOs transcribe albumin and Factor V at levels comparable to those transcribed by a normal pig liver. Connecting the aLIVE-H bioreactor to normal pigs did not affect key blood components and biochemical parameters.
Conclusions : An extra-corporeal liver device aLIVE-H which imitates the hemodynamic and functional properties of the normal liver and incorporates cryo-preserved LMOs has been developed and characterized. aLIVE-H was found to perform key synthetic liver functions.
Stat3 confers resistance against hypoxia/reoxygenation-induced oxidative injury in hepatocytes through upregulation of Mn-SOD
Keita Teruiab, Shin Enosawaa, Sanae Hagaa, Hui Qi Zhanga, Hiroaki Kurodab, Katsunori Kouchib, Tadashi Matsunagab, Hideo Yoshidab, John F. Engelhardtc, Kaikobad Iranid, Naomi Ohnumab, Michitaka Ozakiae*
Hypoxia/reoxygenation (H/R) causes oxidative stress to the cell and induces apoptotic cell death. Signal transducer and activator of transcription-3 (Stat3) is one of the most important molecules involved in the initiation of liver development and regeneration, and has recently been shown to protect cells against various pathogens. In order to investigate the hepatoprotective effects of Stat3, we examined whether it protects against H/R-induced injury in primary hepatocytes.
Methods : Primary cultured hepatocytes were prepared from SD rats. Adenoviruses and cytokines were added 2 days and 1h prior to the H/R insult, respectively. Hepatocytes and culture media were harvested for the assays before and after H/R insult.
Results : Interleukin-6 and cardiotropin-1, which may function mainly through Stat3 activation, protected cells from H/R-induced apoptosis. Adenoviral overexpression of the constitutively activated form of Stat3 (Stat3-C) reduced H/R-induced apoptosis as well as generation of reactive oxygen species (ROS) in hepatocytes. Interestingly, Stat3-C induced Mn-SOD, but not Cu/Zn-SOD, both at the protein and mRNA levels. Overexpression of Mn-SOD significantly reduced H/R-induced ROS and apoptosis by inhibiting redox-sensitive activation of caspase-3 activity.
Conclusions : Stat3 protects hepatocytes from H/R-induced cell injury at least partly by upregulating Mn-SOD and inactivating caspase-3.
Involvement of natural killer cells in PolyI:C-induced liver injury
Zhongjun Donga, Haiming Weia, Rui Sunab, Zhiqing Huc, Bin Gaod*, Zhigang Tianab*
The roles of T cells, natural killer T cells (NKT) and macrophages in autoimmune hepatitis have been well documented. However, the roles of natural killer (NK) cells in liver injury remain obscure. Here we examined the effect of Polyinosinic: polycytidylic acid (PolyI:C)-activated NK cells on liver injury.
Methods : Mice were intraperitoneally injected with PolyI:C at a dose of 20?g/g body wt. The percentage and absolute number of NK cells in the liver were analyzed with flow cytometry. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) assay and HE staining were used to evaluate the liver injury.
Results : Following PolyI:C injection, NK cells accumulation and activation occurred in the liver. Meanwhile, slight elevation of ALT/AST in the serum, mild inflammation and focal necrosis in the liver were also observed. Depletion of NK cells markedly attenuated PolyI:C-induced liver injury. Neutralization of endogenous Interleukin-12 produced by Kupffer cells abrogated the accumulation of NK cells in the liver and subsequent liver injury. The liver injury was also alleviated by neutralization of vascular cell adhesive molecule-1.
Conclusions : These findings suggest that PolyI:C preferentially recruits and activates hepatic NK cells, which may be responsible for the mild hepatitis.
Suppression of transforming growth factor-? results in upregulation of transcription of regeneration factors after chronic liver injury
Toru Nakamuraab, Takato Uenoa*, Masaharu Sakamotoab, Ryuichiro Sakataab, Takuji Torimuraab, Osamu Hashimotoab, Hikaru Uenoc, Michio Sataab
To determine the effects of dominant-negative TGF-? receptor expression during liver regeneration in rats with dimethylnitrosamine (DMN)-induced liver injury.
Methods : Rats were first treated with DMN for 3 weeks, and then intravenously injected once with AdT?-TR, AdLacZ, or saline. Serial changes in hepatocyte proliferation and apoptosis were evaluated by immunohistochemistry using anti-Ki67 antibody, and TUNEL staining, respectively. The mRNA expression of regeneration factors (HGF, TGF-?, EGF, and IGF-I) and IL-6 were evaluated by real-time PCR and northern blotting. Results : Anti-TGF-? molecular intervention up-regulated hepatocyte proliferation and inhibited apoptosis. In the AdT?-TR-treated rats, EGF and IGF-I mRNA expression levels were significantly increased at day 1 and remained high for 3 days after gene transfer; TGF-? mRNA expression levels were significantly increased at 2 to 5 days after gene transfer; HGF mRNA expression levels were significantly up-regulated at day 2 only after gene transfer; while IL-6 mRNA expression level tended to increase at day 1, but decreased thereafter.
Conclusions : In rats with DMN-induced liver injury, anti-TGF-? molecular intervention therapy stimulates proliferation and reduces apoptosis of hepatocytes, and also up-regulates the transcription of various growth factors.
Progenitor cell expansion: an important source of hepatocyte regeneration in chronic hepatitis
Jennifer A. Eleazar, Lorenzo Memeo, Jeffrey S. Jhang, Mahesh M. Mansukhani, Steven Chin, Soo Mi Park, Jay H. Lefkowitch, Govind Bhagat*
Progenitor cell activation with subsequent maturation to hepatocytes and cells of the biliary lineage has been demonstrated in a variety of chronic liver diseases but the kinetics and magnitude of the progenitor cell response has not been adequately studied in detail in chronic hepatitis. We undertook this study to evaluate factors responsible for the progenitor cell/ductular response and further dissect the role of disease grade and stage as determinants of hepatocellular differentiation of bipotential progenitor cells in chronic hepatitis.
Methods :Cytokeratin 7 (and 19) stained biopsies from patients with chronic hepatitis C (n=47), hepatitis B (n=20), and autoimmune hepatitis (n=20) were studied. Ploidy analysis and proliferation indices were evaluated in a subset of cases. Results : Ductular reactions were present in the majority of cases (97%), appeared early in disease, and correlated with disease activity, while progenitor cell derived hepatocyes appeared later in disease and their extent correlated with disease stage. Proliferation indices of all cell types correlated with disease activity.
Conclusions : Progenitor cell derived hepatocytes accrue in chronic hepatitis, possibly related to native hepatocellular dysfunction. However, the fate of these hepatocytes is unclear.
Unique hypervascular nodules in alcoholic liver cirrhosis: identical to focal nodular hyperplasia-like nodules?
Osamu Nakashimaa, Mina Kurogia, Rin Yamaguchia, Hisamitsu Miyaakia, Masaru Fujimotoa, Hirohisa Yanoa, Tsutomu Kumabeb, Naohumi Hayabuchib, Junjiro Hisatomic, Michio Satac, Masamichi Kojiroa*
Currently, focal nodular hyperplasia (FNH)-like nodules in cirrhotic liver is spotlighted. Unique hypervascular nodules mimicking FNH-like nodule in alcoholic liver cirrhosis were clinicopathologically clarified.
Methods : Six resected and six biopsy cases of small hypervascular nodules found in alcoholic cirrhosis were studied clinicopathologically.
Results : All cases were male and consumed 90150g/day of ethanol for longer than 20 years, and hepatitis virus markers were negative. The nodules, 921mm in diameter, were detected by ultrasonography during follow-up of alcoholic cirrhosis, and showed hypervascularity on angiography. Six patients were diagnosed as hepatocellular carcinoma and six were as hyperplastic nodule by biopsy, and the former six cases received partial hepatectomy. All of the resected nodules were completely or incompletely encapsulated. Histologically, all resected and biopsy nodules showed moderate increase of cell-density with an irregular trabecular pattern, and scar-like fibrosis with anomalous blood vessels, and unpaired arteries. All nodules showed marked or mild iron deposits in hepatocytes and/or kupffer cells, and a diffuse capillarization of the sinusoids.
Conclusions : The nodules in the present series seem to fall in the same category as FNH-like nodules in cirrhotic liver, and should be taken account in screening programs including patients with alcoholic cirrhosis.
Immunoregulation of dendritic and T cells by alpha-fetoprotein in patients with hepatocellular carcinoma
Marcus Rittera†, Mona Y. Alia†, Christian F. Grimma†, Robert Wetha, Leonhard Mohra, Wulf O. Bocherb, Katja Endrulata, Heiner Wedemeyerc, Hubert E. Bluma, Michael Geisslera*
Novel immunotherapeutic and other strategies are being explored for the treatment of hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) may be a target antigen for immunotherapy. Little is known, however, about the immunobiology of AFP. Therefore, the impact of AFP on dendritic cells (DC), CD4+ and CD8+ T cells was studied in detail.
Methods : Immune cells from peripheral blood of 27 HCC patients were studied using FACS, ELISPOT, and proliferation assays.
Results : The in vitro generation, maturation, and T cell stimulatory capacity of DCs were not altered by AFP up to concentrations of 20?g/ml. Higher AFP concentrations (>20?g/ml) resulted in phenotypic changes on DCs without impairing their capacity to stimulate CD4+ T cells. Frequencies and function of DCs and AFP specific T cells were not reduced in HCC patients independent on serum AFP levels. Finally, T lymphocytic infiltrations in the liver were not dependent on AFP serum levels.
Conclusions : These studies clearly demonstrate that (i) DC-based immunotherapeutic approaches are a promising approach for HCC treatment and (ii) AFP-reactive T cell clones have not been deleted from the human T cell repertoire establishing AFP as a potential target for T cell based immunotherapy of HCC.
Targeting the epidermal growth factor receptor by gefitinib for treatment of hepatocellular carcinoma
Michael Höpfnera†, Andreas P. Suttera†, Alexander Huethera, Detlef Schuppanb, Martin Zeitza, Hans Scherübla*
Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. Due to very poor 5-year-survival new therapeutic approaches are mandatory. Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), potently suppresses the growth of various tumors, but its effect on HCC remains unexplored. We therefore studied the antineoplastic potency of gefitinib in human HCC cells.
Results : Gefitinib induced a time- and dose-dependent growth inhibition of the human HCC cell lines Huh-7 and HepG2. Gefitinib-treatment induced both mitochondria-dependent and -independent apoptosis. Changes in mitochondrial membrane potential and caspase-8 activation, followed by caspase-3 activation and nuclear degradation, were detected. Moreover, gefitinib induced cell cycle arrest at the G1/S checkpoint and decreased the phosphorylation of mitogen-activated protein kinase ERK1/2. Finally, gefitinib suppressed the expression of antiapoptotic Bcl-2 and Bcl-XL, further rendering HCC cells prone to apoptosis.
Conclusions : Our data demonstrate that the inhibition of EGFR-TK by gefitinib induced growth inhibition, apoptosis and cell cycle arrest in human HCC cells. Thus, EGFR-TK inhibition appears to be a promising novel approach for future treatment strategies of HCC.
Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation
Erwin Geukenab, Dorien Visserb, Folkert Kuipersc, Hans Blokzijld, Henri G.D. Leuveninkb, Koert P. de Jonga, Paul M.J.G. Peetersa, Peter L.M. Jansend, Maarten J.H. Slooffa, Annette S.H. Gouwe, Robert J. Portea*
Biliary strictures are a serious cause of morbidity after liver transplantation. We have studied the role of altered bile composition as a mechanism of bile duct injury after human liver transplantation.
Methods : In 28 liver transplant recipients, bile samples were collected daily posttransplantation for determination of bile composition. Hepatic expression of bile transporters was studied before and after transplantation. Histopathological criteria as well as biliary concentrations of alkaline phosphatase (ALP) and ?-glutamyltransferase (?-GT) were used to quantify bile duct injury.
Results : Early after transplantation, bile salt secretion increased more rapidly than phospholipid secretion, resulting in high biliary bile salt/phospholipid ratio (BA/PL). In parallel with this, mRNA levels of the bile salt transporters NTCP and BSEP increased significantly after transplantation, whereas phospholipid translocator MDR3 mRNA levels remained unchanged. Bile duct injury correlated significantly with bile salt secretion and was associated with a high biliary BA/PL ratio.
Conclusions : Bile salt secretion after human liver transplantation recovers more rapidly than phospholipid secretion. This results in cytotoxic bile formation and correlates with bile duct injury. These findings suggest that endogenous bile salts have a role in the pathogenesis of bile duct injury after liver transplantation.
Referral of chronic hepatitis B patients from primary to specialist care: making a simple guideline work
Maria Catharina Mosterta, Jan Hendrik Richardusbc, Robert A. de Mana*
To evaluate a guideline selecting patients at the primary care level for referral to a specialist, to identify bottlenecks and subsequently implement and evaluate improvements.
Methods : Retrospective patient files analysis and a prospective cohort study. The study was conducted in Municipal Public Health Service (PHS), University Medical Center. Patients diagnosed with chronic hepatitis B virus (HBV) infection were referred to the PHS. Improvement of bottlenecks were identified in the referral chain, based on the guideline. Number of patients receiving correct advice, number of patients reaching the hospital for specialist care, time between notification of the PHS and final arrival in the hospital.
Results : The guideline for the referral of chronic HBV patients appeared to be appropriate, although one-third of the selected patients was not seen by the specialist. After the intervention more HBV patients (76 versus 61%) received correct advice from the PHS, and the number of HBV patients seen by the specialist increased by 18%.
Conclusions : The referral guideline works, yet we could improve the efficiency of the guideline increasing the proportion of eligible patients reaching specialist care. In countries where mandatory reporting of HBV infections exists this guideline can be adapted to local health systems.
Predominance of type 1 (Th1) cytokine production in the liver of patients with HCV-associated mixed cryoglobulinemia vasculitis
David Saadounab, Olivier Boyerb, Hélène Trébeden-Nègreb, Nicolas Limala, Véronique Bon-Durandb, Marita Andreua, David Klatzmannb, Jean Charles Piettea, Patrice Cacoubab*
Patients with hepatitis C virus (HCV) mixed cryoglobulinemia (MC) vasculitis have a higher mortality rate and more frequent incidence of cirrhosis than their cryoglobulin-negative counterparts. To compare the cytokine profile of liver-infiltrating T cells in HCV-infected patients with or without MC vasculitis.
Methods : Hepatic biopsy specimens were obtained from HCV infected patients with and without MC vasculitis. Using intracellular staining and flow cytometry, we assessed the ability of freshly isolated liver T cells from these biopsies to produce IFN-?, TNF-?, IL-2, IL-4, and IL-10 in response to stimulation with PMA and ionomycin.
Results : HCV-MC vasculitis patients compared to HCV-MC negative controls have an enhanced hepatic T cells production of Th1-type cytokines [i.e. TNF-?(30.3±13% vs. 15.5±5%, P=0.01), IL-2 (20.2±9% vs. 10±4%, P=0.01) and IFN-? (22.2±11% vs. 9.4±4%, P=0.008)], whereas IL-10, a representative Th2-type cytokine, was significantly lower (7.2±4% vs. 17±7%, P=0.01).
Conclusions : T cell from the liver of HCV-MC vasculitis patients display a significantly augmented liver Th1 profile compared to MC-negative controls. This enhanced production of type-1 cytokines may account for a more severe course of liver disease.
Copyright © 2001-2004 European Association for the Study of the Liver. All rights reserved.
Copyright © 2004 BMJ Publishing Group Ltd
The New England Journal of Medicine is owned, published, and copyrighted © 2004 Massachusetts Medical Society. All rights reserved.
Volume 364, Number 9441 02 October 2004
The Lancet, published, and copyrighted © 2004. All rights reserved.
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