Volume 44, Issue 4, Pages 775-1056 (October 2006)
Liver Failure and Liver Disease
Liver Failure
Hemodynamic response-guided therapy for prevention of variceal rebleeding: An uncontrolled pilot study (p 806-812)
Antonio González, Salvador Augustin, Mercedes Pérez, Joan Dot, Esteban Saperas, Alejandro Tomasello, Antoni Segarra, Josep Ramón Armengol, Joan Ramón Malagelada, Rafael Esteban, Jaime Guardia, Joan Genescà
The clinical usefulness of assessing hemodynamic response to drug therapy in the prophylaxis of variceal rebleeding is unknown. An open-labeled, uncontrolled pilot trial was performed to evaluate the feasibility and efficacy of using the hemodynamic response to pharmacological treatment to guide therapy in this setting. Fifty patients with acute variceal bleeding underwent a hepatic venous pressure gradient (HVPG) measurement 5 days after the episode. Nadolol and nitrates were initiated, and a second HVPG was measured 15 days later. Responder patients (20% decrease in HVPG from baseline) were maintained on drugs, partial responders (10% and <20%) had banding ligation added to the drugs, and nonresponders (<10%) received a transjugular intrahepatic portal-systemic shunt (TIPS). Mean follow-up was 22 months. Eight patients (16%) did not receive the second HVPG, 6 of them because of early variceal rebleeding. Of the other 42 patients, 24 were classified as responders (57%); 10 as partial responders (24%), who had banding added; and 8 as nonresponders (19%), who received a TIPS. Patients with cirrhosis of viral etiology compared to alcoholic cirrhosis tended to present more early rebleedings, less response to drugs and needed more TIPS. Variceal rebleeding occurred in 22% of all patients but only in 12% of patients whose hemodynamic response was assessed. The 3 therapeutic groups were not different. In conclusion, using hemodynamic response to pharmacological treatment to guide therapy in secondary prophylaxis to prevent variceal bleeding is feasible and effectively protects patients from rebleeding. In this context, viral cirrhosis seems to present a worse outcome than alcoholic cirrhosis.
Contribution of hepatic adenosine A1 receptors to renal dysfunction associated with acute liver injury in rats (p 813-822)
Zhi Ming, Yi-Jun Fan, Xi Yang, W. Wayne Lautt
Acute liver injury is associated with renal insufficiency, whose mechanism may be related to activation of the hepatorenal reflex. We previously showed that intrahepatic adenosine is involved in activation of the hepatorenal reflex to restrict urine production in both healthy rats and in rats with cirrhosis. The aim of the present study was to test the hypothesis that activation of intrahepatic adenosine receptors is involved in the pathogenesis of the renal insufficiency seen in acute liver injury. Acute liver injury was induced by intraperitoneal injection of thioacetamide (TAA, 500 mg/kg) in rats. The animals were instrumented 24 hours later to monitor systemic, hepatic, and renal circulation and urine production. Severe liver injury developed following TAA insult, which was associated with renal insufficiency, as demonstrated by decreased (25%) renal arterial blood flow, a lower (30%) glomerular filtration rate, and decreased urine production. Further, the increase in urine production following volume expansion challenge was inhibited. Intraportal, but not intravenous, administration of a nonselective adenosine receptor antagonist, 8-phenyltheophylline, improved urine production. To specify receptor subtype, the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an adenosine A1 receptor antagonist) and 3,7-dimethyl-1-propargylxanthine (DMPX, an adenosine A2 receptor antagonist) were compared. Intraportal but not intravenous administration of DPCPX greatly improved impaired renal function induced by acute liver injury, and this beneficial effect was blunted in rats with liver denervation. In contrast, neither intraportal nor intravenous administration of DMPX showed significant improvement in renal function. In conclusion, an activated hepatorenal reflex, triggered by intrahepatic adenosine A1 receptors, contributed to the pathogenesis of the water and sodium retention associated with acute liver injury.
Derivation and validation of a new global method for assessing nutritional status in patients with cirrhosis (p 823-835)
Marsha Y. Morgan, Angela M. Madden, Clare T. Soulsby, Richard W. Morris
Accurate assessments of nutritional status are difficult to obtain in patients with cirrhosis. The aim of this study was to devise and validate a global nutritional assessment scheme for use in this patient population. Measures of body mass index (BMI) and mid-arm muscle circumference (MAMC) were combined with details of dietary intake in a semistructured, algorithmic construct to provide a nutritional assessment scheme for use in patients with cirrhosis; evaluated individuals were classified as adequately nourished, moderately malnourished (or suspected to be), or severely malnourished. There was good interobserver agreement in the nutritional categorization of 50 patients with cirrhosis (34 men, 16 women) using this scheme ( = 0.79) and significant associations with the contributing objective variables - namely, BMI (Spearman's correlation r = -0.78; P < .001) and relative MAMC (r = -0.69; P < .001) - confirming its internal validity. There was a significant association between nutritional categorization in 20 patients with cirrhosis (10 men, 10 women) and estimates of total body protein obtained using a four-component model (r = -0.45; P = .046), confirming the external validity of the scheme. Finally, a significant association was found between poor nutritional status in 116 patients with cirrhosis (65 men, 51 women), followed for 14 to 52 months, and shorter subsequent survival (P = .0005), confirming the scheme's predictive validity. In conclusion, a global assessment scheme has been devised that provides a simple, reproducible, valid, and predictive method of assessing nutritional status in patients with cirrhosis.
Significance of circulating endothelial progenitor cells in hepatocellular carcinoma (p 836-843)
Joanna W. Y. Ho, Roberta W. C. Pang, Cecilia Lau, Chris K. Sun, Wan Ching Yu, Sheung Tat Fan, Ronnie T. P. Poon
This study evaluated the significance of circulating bone marrow-derived endothelial progenitor cells (EPCs) in patients with hepatocellular carcinoma (HCC), a solid tumor with rich neovasculature. Eighty patients with HCC were recruited for the study, and 16 patients with liver cirrhosis and 14 healthy subjects were also included for comparison. Blood samples were taken before treatment. Total mononuclear cells were isolated from peripheral blood, preplated to eliminate mature circulating endothelial cells, and colony-forming units (CFUs) formed by circulating EPCs were counted. To validate the CFU scores, FACS quantification of EPCs using CD133, VEGFR2, and CD34 as markers was performed in 30 cases. Our study showed significantly higher mean CFU scores in patients with HCC compared to patients with cirrhosis and healthy controls (P = .001 and .009, respectively). Furthermore, the CFU scores of patients with HCC positively correlated with levels of serum -fetoprotein (r = .303, P = .017), plasma VEGF (r = .242, P = .035), and plasma interleukin-8 (IL-8) (r = .258, P = .025). Patients with unresectable HCC had higher CFU scores than patients with resectable tumors (P = .027). Furthermore, for those who underwent curative surgery, higher preoperative CFU scores were observed in patients with recurrence within 1 year compared with those who were disease-free after 1 year (P = .013). In conclusion, higher circulating levels of EPCs are seen in patients with advanced unresectable HCC as compared to patients with resectable HCC or those with liver cirrhosis. Our evidence supports the potential use of circulating level of EPCs as a prognostic marker in patients with HCC.
Effects of clonidine on diuretic response in ascitic patients with cirrhosis and activation of sympathetic nervous system (p 844-849)
Anne Lenaerts, Thierry Codden, Jean-Claude Meunier, Jean-Pol Henry, Gaston Ligny
The effects of the addition of clonidine to diuretics on the mobilization of ascites in the short term (diuretic response and requirement of diuretics) and the long term (readmissions for tense ascites and requirement of diuretics) were examined in patients with cirrhosis and with increased sympathetic nervous system (SNS) activity. We also studied neurohormonal, hemodynamic effects and side effects of clonidine and diuretics. Patients were randomized to receive placebo (group1, n = 32) or clonidine (0.075 mg) twice daily (group 2, n = 32) for 3 months. After 8 days and for 10 days duration, spironolactone (200 mg/day) was added in both groups. After this period, the dosages of diuretics were individually increased until diuretic response. Responding patients were discharged and followed at the outpatient clinic. During the first hospitalization, the time needed for diuretic response was shorter in group 2 than in group 1. The mean requirement for diuretics was significantly higher in group 1 than in group 2, and the diuretic complications (hyperkalemia and renal impairment) were significantly lower in group 2. Clonidine induced a permanent decrease in SNS activity and delayed decrease in renin/aldosterone levels. During the follow-up, the time to the first readmission for tense ascites was shorter in group 1 than in group 2. Readmissions related to tense ascites or diuretic complications were significantly lower in group 2. The mean requirement for diuretics was significantly higher in group 1 than in group 2. In conclusion, the additional administration of clonidine to diuretics induced an earlier diuretic response associated with fewer diuretic requirements and complications.
Liver Disease
Determinants of the clinical expression of amoxicillin-clavulanate hepatotoxicity: A prospective series from Spain (p 850-856)
M. Isabel Lucena, Raúl J. Andrade, M. Carmen Fernández, Ketevan Pachkoria, Gloria Pelaez, José A. Durán, Macarena Villar, Luis Rodrigo, Manuel Romero-Gomez, Ramón Planas, Anabel Barriocanal, Joan Costa, Carlos Guarner, Sonia Blanco, José M. Navarro, Fernando Pons, Agustin Castiella, Susana Avila, on behalf of the Spanish Group for the Study of Drug-Induced Liver Disease (Grupo de Estudio para las Hepatopatías Asociadas a Medicamentos (GEHAM)
Amoxicillin-clavulanate (AC) hepatotoxicity has been reported to exhibit a higher predominance of cholestatic types of damage, especially in males. However, the determinants of its clinical expression are unknown. This study prospectively evaluated the profile of AC hepatotoxicity. Data on all cases of hepatotoxicity reported to the Spanish Registry attributed to AC and assessed as definite or probable on the Council for International Organizations of Medical Sciences (CIOMS) scale were collated and compared to published case series. Hepatotoxicity related to amoxicillin-clavulanate was identified in 69 patients (36 males; mean age 56 years) representing 14% of all cases of hepatotoxicity submitted to the Registry. There was an overall sex distribution and the predominant pattern of lesion was hepatocellular (36%) which occurred at a shorter duration of treatment (P < .03). Mean time lapse between therapy initiation and jaundice onset was 16 days. Late onset of symptoms following end of treatment occurred in half the cases. Multiple logistic regression analysis identified advancing age as the factor associated with the development of cholestatic/mixed type of injury (odds ratio for an age interval for 1 year: 1.045 [95% CI = 1.013-1.078; P = .005). An unfavorable outcome was seen in 7% of patients. In conclusion, age is the most important determinant in the biochemical expression of AC hepatotoxicity; younger age is associated with cytolytic damage and shorter treatment duration, whereas cholestatic/mixed type of damage is related to older age and prolonged AC therapy.
Alpha1-Antitrypsin mutations in NAFLD: High prevalence and association with altered iron metabolism but not with liver damage (p 857-864)
Luca Valenti, Paola Dongiovanni, Alberto Piperno, Anna Ludovica Fracanzani, Marco Maggioni, Raffaela Rametta, Paola Loria, Maria Antonietta Casiraghi, Elda Suigo, Roberto Ceriani, Erica Remondini, Paola Trombini, Silvia Fargion
Hyperferritinemia, a common feature of nonalcoholic fatty liver disease (NAFLD), has been associated with steatohepatitis and fibrosis. Heterozygosity for 1-antitrypsin (AAT) mutations is a cofactor of liver damage, and AAT influences inflammation and iron metabolism. This study evaluated the prevalence of the common AAT PiS/PiZ mutants in 353 patients with NAFLD, 195 of whom had hyperferritinemia, versus 114 matched controls and their influence on iron metabolism and the severity of liver damage in the 212 patients submitted to biopsy. PiS and PiZ alleles were searched for by restriction analysis. Thirty-eight patients (10.8%) carried non-MM genotypes versus 4/114 (3.5%) controls (P = .02). Patients carrying AAT mutations had higher ferritin (573 [454-966] vs. 348 [201-648]; P = .001) with similar transferrin saturation. The difference was more evident in males (P < .0001) and significant in patients not carrying HFE genotypes associated with iron overload (P = .015). The prevalence of non-MM genotypes was higher in patients with hyperferritinemia than in those without (28/195, 14% vs. 10/158, 6%, P = .016), and AAT mutations were associated with higher prevalence of sinusoidal siderosis (17/27, 63% vs. 70/180, 39%; P = .02), and sinusoidal/total iron score (46.3 ± 38% vs. 25.1 ± 35%, P = .01). Although ferritin was independently associated with fibrosis (P = .047), AAT mutations favoring sinusoidal iron deposition did not affect liver damage. In conclusion, AAT mutations are associated with hyperferritinemia and sinusoidal iron accumulation, but not with more severe liver damage in NAFLD.
Long-term follow-up of patients with NAFLD and elevated liver enzymes (p 865-873)
Mattias Ekstedt, Lennart E. Franzén, Ulrik L. Mathiesen, Lars Thorelius, Marika Holmqvist, Göran Bodemar, Stergios Kechagias
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of elevated liver enzymes in patients of developed countries. We determined the long-term clinical and histological courses of such patients. In a cohort study, 129 consecutively enrolled patients diagnosed with biopsy-proven NAFLD were reevaluated. Survival and causes of death were compared with a matched reference population. Living NAFLD patients were offered repeat liver biopsy and clinical and biochemical investigation. Mean follow-up (SD) was 13.7 (1.3) years. Mortality was not increased in patients with steatosis. Survival of patients with nonalcoholic steatohepatitis (NASH) was reduced (P = .01). These subjects more often died from cardiovascular (P = .04) and liver-related (P = .04) causes. Seven patients (5.4%) developed end-stage liver disease, including 3 patients with hepatocellular carcinoma. The absence of periportal fibrosis at baseline had a negative predictive value of 100% in predicting liver-related complications. At follow-up, 69 of 88 patients had diabetes or impaired glucose tolerance. Progression of liver fibrosis occurred in 41%. These subjects more often had a weight gain exceeding 5 kg (P = .02), they were more insulin resistant (P = .04), and they exhibited more pronounced hepatic fatty infiltration (P = .03) at follow-up. In conclusion, NAFLD with elevated liver enzymes is associated with a clinically significant risk of developing end-stage liver disease. Survival is lower in patients with NASH. Most NAFLD patients will develop diabetes or impaired glucose tolerance in the long term. Progression of liver fibrosis is associated with more pronounced insulin resistance and significant weight gain.
Correlation of paired liver biopsies in morbidly obese patients with suspected nonalcoholic fatty liver disease (p 874-880)
Raphael B. Merriman, Linda D. Ferrell, Marco G. Patti, Shiobhan R. Weston, Mark S. Pabst, Bradley E. Aouizerat, Nathan M. Bass
In the absence of surrogate markers, the evaluation of suspected nonalcoholic fatty liver disease (NAFLD) is highly dependent on histological examination. The extent of sampling variability affecting the reliability of a single liver biopsy in patients with suspected NAFLD is poorly characterized. This prospective study aimed to correlate precise histological findings in paired biopsies - right and left lobe - in the diagnosis of NAFLD in morbidly obese subjects undergoing bariatric surgery employing both Brunt and Matteoni classifications and the NAFLD Activity Score (NAS). We also aimed to determine whether the composite histopathological findings of the two biopsies would improve diagnostic accuracy. Consecutive subjects had an intraoperative biopsy from both right and left lobes, evaluated and scored in a blinded manner. Intraobserver agreement was also assessed. Kappa coefficients of agreement were calculated. Forty-one subjects had acceptable biopsies. Agreement for steatosis was excellent and moderate for fibrosis. Concordance was only fair for most features of necroinflammation. Intraobserver agreement was only moderate for lobular inflammation. Excellent agreement was seen for the diagnosis of NASH using Brunt criteria and good agreement when using Matteoni and NAS scoring systems. Composite biopsy data particularly improved identification of hepatocyte ballooning. The diagnostic accuracy also improved substantially when composite features were compared with single-sided biopsy features, especially for the Matteoni and NAS scoring systems. In conclusion, significant sampling variability occurs in NAFLD, particularly for features of necroinflammation. This should be factored into the design of clinical trials and studies of the natural history of the disease.
Tensin2 variant 3 is associated with aggressive tumor behavior in human hepatocellular carcinoma (p 881-890)
Judy Wai Ping Yam, Frankie Chi Fat Ko, Chung-Yiu Chan, Tai-On Yau, Edmund Kwok Kwan Tung, Thomas Ho-Yin Leung, Dong-Yan Jin, Irene Oi-Lin Ng
Tensins are a new family of proteins that act as an important link among extracellular matrix, actin cytoskeleton, and signal transduction and have been implicated in human cancers. Tensin2 was initially identified in a search for new tensin family members that share extensive sequence homology with tensin1. Tensin2 was highly expressed in liver tissues. A recent study reported that one of the splicing variants of tensin2, variant 3, promotes cell migration. In the present study, we aimed to elucidate the role of variant 3 in hepatocarcinogenesis by assessing the expression of variant 3 mRNA in hepatocellular carcinoma (HCC) tissue and ectopically expressing variant 3 in HCC cell lines. Analysis of variant 3 expression in human HCC tissue revealed it was overexpressed in 46% (23/50) of tumor tissues as compared with the corresponding nontumorous livers. High expression of variant 3 was significantly associated with venous invasion (P = .037), tumor microsatellite formation (P = .022), and tumor nonencapsulation (P = .049). Our ectopic expression study showed that variant 3 significantly promoted the cell growth and motility of HCC cells. The clonal transfectants of variant 3 were more closely packed and resulted in a higher saturation density than in the control vector transfectants. Variant 3 expression also enhanced the proliferation rate in culture and in vivo tumorigenicity in nude mice. In conclusion, we reveal a novel role for variant 3 in the progression of HCC and suggest the feasibility of elevated variant 3 expression as a tumor progression marker for HCC.
Uracil-tegafur as an adjuvant for hepatocellular carcinoma: A randomized trial (p 891-895)
Kiyoshi Hasegawa, Tadatoshi Takayama, Masayoshi Ijichi, Yutaka Matsuyama, Hiroshi Imamura, Keiji Sano, Yasuhiko Sugawara, Norihiro Kokudo, Masatoshi Makuuchi
Frequent recurrence of hepatocellular carcinoma (HCC) after surgery remains a major clinical problem. This randomized controlled trial evaluated whether postoperative adjuvant therapy with oral uracil-tegafur (UFT) prevents recurrence of HCC. A total of 160 patients who underwent curative hepatic resection for HCC were randomly assigned to receive either 300 mg/day of UFT for 1 year after surgery (n = 79, UFT group) or surgery alone (n = 80, control group). The primary endpoint was recurrence-free survival, and the secondary endpoint was overall survival. Other study variables included liver function and type of recurrence. During a median follow-up of 4.8 years (range: 0.5-7.9), recurrence-free survival curves in the groups were similar (P = .87). Overall survival was slightly but not significantly worse in the UFT group than in the control group (P = .08). The rates of recurrence-free and overall survival at 5 years were 29% and 58%, respectively, in the UFT group, as compared with 29% and 73%, respectively, in the control group. The hazard ratio for recurrence in the UFT group, relative to the control, was 1.01 (95% confidence interval: 0.84-1.22, P = .87). The proportion of patients with advanced recurrence (i.e., multiple, extrahepatic, or associated with vascular invasion) was significantly higher in the UFT group (74%, 43 of 58 patients with recurrence) than in the control group (53%, 30 of 57) (P = .02). In conclusion, our results offer no evidence to support potential benefits of adjuvant chemotherapy with UFT after surgery in patients with HCC and suggest that such treatment may even worsen overall survival.
Viral Hepatitis
Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes (p 896-906)
Sean E. Doyle, Heidi Schreckhise, Kien Khuu-Duong, Katherine Henderson, Robert Rosler, Harold Storey, Lena Yao, Hong Liu, Fariba Barahmand-pour, Pallavur Sivakumar, Chung Chan, Carl Birks, Don Foster, Christopher H. Clegg, Perdita Wietzke-Braun, Sabine Mihm, Kevin M. Klucher
Interleukin-28A (IL-28A), IL-28B and IL-29 are a family of class II cytokines that stimulate antiviral responses through a heterodimeric receptor that is distinct from the type I interferon (IFN) receptor. To better understand how this newly described family of cytokines regulates the antiviral state, we compared various cellular responses elicited by IL-29 and IFN-. Here we show that these cytokines stimulate similar patterns of signal transducer and activator of transcription 1 (STAT-1), -2, -3, and -5 phosphorylation and nearly identical patterns of gene expression when analyzed in two distinct cell types by microarray analysis. Interestingly, the IL-29 receptor is preferentially expressed on primary hepatocytes within normal liver and pegylated forms of IL-29 and IFN- induced equivalent 25 oligoadenylate synthetase (OAS) and MX1 gene expression in this cell type. Pegylated IL-29 also produced a significant reduction in human hepatitis B and hepatitis C viral load in vitro and reduced the cytopathic effect caused by the fully replicating flavivirus, West Nile virus. In conclusion, IL-29 and IFN- stimulate identical antiviral responses despite their utilization of different receptors. This fact, combined with significant receptor expression in hepatitis virus-infected livers, suggests that IL-29 may have therapeutic value against chronic viral hepatitis in human patients.
Favorable effect of adefovir on the number and functionality of myeloid dendritic cells of patients with chronic HBV (p 907-914)
Renate G. van der Molen, Dave Sprengers, Paula J. Biesta, Johannes G. Kusters, Harry L. A. Janssen
In patients with chronic hepatitis B virus (HBV), 2 predominant precursor dendritic cell (DC) subtypes, the myeloid dendritic cell (mDC) and the plasmacytoid dendritic cell (pDC), were recently found to be functionally impaired. HBV DNA was found to be present in the DC subtypes, but no viral replication could be detected. The question remains whether simply the presence of the virus and viral proteins causes this dysfunction of DCs. To address this issue, the effect of viral load reduction resulting from treatment with the nucleotide analogue adefovir dipivoxil on the number and functionality of circulating DCs was studied during 6 months of treatment. Treatment resulted in a mean 5 log10 decrease in the viral load and normalization of alanine aminotransferase within 3 months. The number of mDCs, but not of pDCs, increased significantly over 6 months of treatment to a level comparable to that of uninfected healthy controls. The allostimulatory capacity of isolated and in vitro matured mDCs increased significantly after 3 months of treatment. Accordingly, mDCs exhibited an increased capacity to produce tumor necrosis factor alpha and interleukin-12 after 3-6 months of treatment. There was no change in interferon alpha production by pDCs during treatment. In conclusion, adefovir treatment results in an improvement in the number and functionality of mDCs, but not of pDCs. Our findings provide clues for the reasons why current antiviral therapy does not lead to consistently sustained viral eradication.
Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens (p 915-924)
Masaya Sugiyama, Yasuhito Tanaka, Takanobu Kato, Etsuro Orito, Kiyoaki Ito, Subrat K. Acharya, Robert G. Gish, Anna Kramvis, Takashi Shimada, Namiki Izumi, Masahiko Kaito, Yuzo Miyakawa, Masashi Mizokami
Various genotypes of the hepatitis B virus (HBV) induce liver disease of distinct severity, but the underlying virological differences are not well defined. Huh7 cells were transfected with plasmids carrying 1.24-fold the HBV genome of different genotypes/subgenotypes (2 strains each for Aa/A1, Ae/A2, Ba/B2 and D; 3 each for Bj/B1 and C). HBV DNA levels in cell lysates, determined by Southern hybridization, were the highest for C followed by Bj/Ba and D/Ae (P < .01), and the lowest for Aa (P < .01), whereas in culture media, they were the highest for Bj, distantly followed by Ba/C/D and further by Ae/Aa (P < .01). The intracellular expression of core protein was more than 3-fold lower for Ae/Aa than the others. Hepatitis B e antigen (HBeAg) was excreted in a trend similar to that of HBV DNA with smaller differences. Secretion of hepatitis B surface antigen (HBsAg) was most abundant for Ae followed by Aa, Ba, Bj/C and remotely by D, which was consistent with mRNA levels. Cellular stress determined by the reporter assay for Grp78 promoter was higher for C and Ba than the other genotypes/subgenotypes (P < .01). Severe combined immunodeficiency mice transgenic for urokinase-type plasminogen activator (uPA/SCID), with the liver replaced for human hepatocytes, were inoculated with virions passed in mouse and recovered from culture supernatants. HBV DNA levels in their sera were higher for C than Ae by 2 logs during 4-7 weeks after inoculation. In conclusion, virological differences among HBV genotypes were demonstrated both in vitro and in vivo. These differences may influence HBV infections with distinct genotypes in clinical and epidemiological settings.
Modeling hepatic fibrosis in African American and Caucasian American patients with chronic hepatitis C virus infection (p 925-935)
Robert J. Fontana, David E. Kleiner, Richard Bilonick, Norah Terrault, Nezam Afdhal, Steven H. Belle, Lennox J. Jeffers, Darmendra Ramcharran, Marc G. Ghany, Jay H. Hoofnagle, Virahep-C Study Group
Assessment of histological stage is an integral part of disease management in patients infected with the hepatitis C virus (HCV). The aim of this study was to develop a model incorporating objective clinical and laboratory parameters to estimate the probability of severe fibrosis (i.e., Ishak fibrosis 3) in previously untreated African American (AA) and Caucasian American (CA) patients with HCV genotype 1. The Ishak fibrosis scores of 205 CA and 194 AA patients enrolled in the Viral Resistance to Antiviral Therapy of Chronic Hepatitis C study (Virahep-C) were modeled using simple and multiple logistic regression. The model was then validated in an independent cohort of 461 previously untreated patients with HCV. The distribution of fibrosis scores was similar in the AA and CA patients as was the proportion of patients with severe fibrosis (35% vs. 39%, P = .47). After accounting for the number of portal areas in the biopsy, patient age, serum aspartate aminotransferase, alkaline phosphatase, and platelet count were independently associated with severe fibrosis in the overall cohort, and the relationship with fibrosis was similar in both the AA and CA subgroups. The area under the receiver operating characteristic curve (AUROC) of the Virahep-C model (0.837) was significantly better than in other published models (P = .0003). The AUROC of the Virahep-C model was 0.851 in the validation population. In conclusion, a model consisting of widely available clinical and laboratory features predicted severe hepatic fibrosis equally well in AA and CA patients with HCV genotype 1 and was superior to other published models. The excellent performance of the Virahep-C model in an external validation cohort suggests the findings are replicable and potentially generalizable.
HCV core expression in hepatocytes protects against autoimmune liver injury and promotes liver regeneration in mice (p 936-944)
Hiroki Kawamura, Sugantha Govindarajan, Fred Aswad, Keigo Machida, Michael M.C. Lai, Vicky M.-H. Sung, Gunther Dennert
Hepatitis C virus (HCV) infection causes acute and chronic liver disease often leading to liver cirrhosis and hepatocellular carcinoma. Numerous studies have shown that despite induction of virus specific immunity, a curative response is often not attained; this has led to the hypothesis that HCV genes modulate immunity, thereby enabling chronic infections. This study examined the effects on immune-mediated liver injury in transgenic mice expressing core protein throughout the body and bone marrow chimeras expressing core protein in either the lymphoid compartment or liver parenchyma. Presence of core protein in the liver parenchyma but not in lymphoid cells protects from autoimmune hepatitis induced by mitogen concanavalin A (ConA). Consistent with this observation, core transgenic hepatocytes are relatively resistant to death induced by anti-Fas antibody and tumor necrosis factor (TNF). This protective effect is associated with preferential activation of signal transducer and activation of transcription factor 3 (STAT3) versus STAT1 in livers of ConA-injected animals. In agreement with this effect of core protein on the Janus kinase (JAK)-STAT signaling pathway, transgenic mice accelerate liver regeneration after partial hepatectomy but are not protected from hepatocyte death. In conclusion, HCV core inhibits STAT1 and stimulates STAT3 activation, which protects infected hepatocytes from attack by the cell-mediated immune system and promotes their proliferation.
Hepatitis C virus E2 and CD81 interaction may be associated with altered trafficking of dendritic cells in chronic hepatitis C (p 945-954)
Jacob Nattermann, Henning Zimmermann, Agathe Iwan, Marie von Lilienfeld-Toal, Ludger Leifeld, Hans Dieter Nischalke, Bettina Langhans, Tilman Sauerbruch, Ulrich Spengler
Dendritic cells (DC) are crucially involved in the induction of immune responses; however, reports on DC functions in chronic hepatitis C are controversial. Function of DC includes proper cell trafficking between sites of infection and lympho-cellular compartments. Thus, we analyzed DC compartmentalization and changes in DC migration in hepatitis C virus (HCV)-infected patients. We found significantly lower numbers of circulating BDCA1+ and BDCA2+ DC in HCV(+) patients (n = 20) than in healthy controls (n = 12) (P < .05). Analyzing liver samples from HCV(+) patients (n = 15), HCV(-) controls (n = 15), and disease controls (n = 10), we demonstrated chronic hepatitis C to be associated with intrahepatic DC enrichment (P < .05). In vitro studies indicated that HCV E2-induced secretion of RANTES efficiently attracts CCR5(+) immature DC. Incubation of DC with sera derived from HCV(+) patients made DC unresponsive to CCL21, the chemokine recruiting DC to lymphoid tissues for T cell priming. Unlike attraction of CCR5+ DCs via RANTES, direct inhibition of DC migration in response to CCL21 was specific for patients with chronic hepatitis C and could be attributed to interaction of HCV E2 with CD81 on DC. In conclusion, migration of DC is markedly affected by interaction of HCV E2 with CD81. Failure of DC to recirculate to lymphoid tissue may be critically involved in impaired T cell priming during HCV infection.
Liver Biology and Pathobiology
Biology
STAT1 contributes to dsRNA inhibition of liver regeneration after partial hepatectomy in mice (p 955-966)
Rui Sun, Ogyi Park, Norio Horiguchi, Shailin Kulkarni, Won-Il Jeong, Hao-Yu Sun, Svetlana Radaeva, Bin Gao
Increasing evidence suggests that liver regeneration is suppressed in patients with chronic HCV infection; however, the underlying mechanisms remain unclear. Previously, we demonstrated that injection of the synthetic double-stranded RNA (dsRNA) poly I:C to mimic viral infection suppresses liver regeneration in the partial hepatectomy (PHx) model, whereby IFN- contributes to the inhibition. In this study, we examined the role of the IFN--activated downstream signal (STAT1) and genes (IRF-1, p21cip1, and SOCS1) in liver regeneration and hepatocyte proliferation. Results show that disruption of the STAT1 gene abolished poly I:C suppression of liver regeneration and the inhibitory effect of poly I:C on liver regeneration was diminished in IRF-1-/- and p21cip1-/-mice. Treatment with IFN- in vitro inhibited cell proliferation of wild-type mouse hepatocytes, but not STAT1-/- hepatocytes. The inhibitory effect of IFN- on cell proliferation was also diminished in IRF-1-/- and p21cip1-/- hepatocytes, but enhanced in SOCS1-/- hepatocytes. Hepatocyte proliferation was unaffected by treatment with poly I:C alone, but when hepatocytes were co-cultured with liver lymphocytes, proliferation was inhibited by IFN-/STAT1-dependent mechanisms. Moreover, in HCV-infected livers with cirrhosis, activation of STAT1 was detected and correlated positively with liver injury (elevated serum levels of AST) but negatively with hepatocyte proliferation (hepatocyte PCNA and Ki-67 positive immunostaining). In conclusion, STAT1 is involved in dsRNA suppression of liver regeneration; not only does STAT1 activation contribute to liver injury, it may also block liver repair through inhibition of hepatocyte proliferation in HCV-infected patients, playing an important role in the pathogenesis of disease.
Hepatic expression of glutamine synthetase in rats is controlled by STAT5 and TCF transcription factors (p 967-975)
Max Werth, Rolf Gebhardt, Frank Gaunitz
In mammalian liver, high glutamine synthetase (GS) expression is restricted to hepatocytes surrounding the terminal venules. The most important enhancer of the GS gene is located 2520 base pairs (bp) upstream from the transcriptional start point. The nature of the transcription factors that bind to the enhancers has remained enigmatic. In this study, we purified nuclear proteins binding to the element. Supershift assays and footprint experiments with purified protein identified activated STAT5 as a transcription factor binding to a site within the enhancer. In addition, a second binding site close to the STAT5 site was observed that also binds a protein present in nuclear extracts. Sequence analysis indicated that the second site may bind a member of the LEF/TCF transcription factor family. Reporter gene assays demonstrate that the STAT5 binding site mediates enhancement of expression whereas the LEF/TCF site functions as a silencer of growth hormone-mediated enhancement in normal hepatocytes. LEF/TCF-sites are known to function as silencers in the absence and as enhancers in the presence of activated -catenin. In conclusion, the GS 5 enhancer contains elements important for GS expression in cells carrying an activated form of -catenin as previously shown in experimentally induced hepatocellular carcinomas.
Pathobiology
Indomethacin increases liver damage in a murine model of liver injury from alpha-1-antitrypsin deficiency (p 976-982)
David A. Rudnick, Olga Shikapwashya, Keith Blomenkamp, Jeffrey H. Teckman
Homozygous (PIZZ) alpha-1-antitrypsin (1-AT) deficiency is associated with the development of liver damage in children as well as chronic liver injury and hepatocellular carcinoma in adults. The 1-AT mutant Z gene encodes a mutant protein that accumulates in the endoplasmic reticulum of hepatocytes rather than being secreted appropriately into serum. Liver injury is caused by the accumulation of 1-AT mutant Z protein in hepatocytes, which triggers downstream intracellular injury pathways. However, development of clinical liver disease among PIZZ homozygotes is highly variable, suggesting other genetic or environmental factors contribute to liver injury. In this study, we tested whether nonsteroidal anti-inflammatory drugs (NSAIDs) could be a comorbid factor in the development of liver injury in 1-AT deficiency using the PiZ mouse. This mouse model is transgenic for the mutant Z allele of the human 1-AT gene, in which 1-ATZ expression is regulated by the human promoter regulatory sequences. Our results showed that administration of indomethacin to PiZ mice resulted in increased hepatic injury, indicated by increased hepatocellular proliferation and increased activation of caspase 9. This indomethacin-induced injury was associated with activation of IL-6-STAT3 signaling, increased expression of 1-AT mRNA, and greater accumulation of mutant polymerized 1-ATZ protein in livers of indomethacin-treated PiZ mice compared to vehicle-treated PiZ animals. In conclusion, environmental factors, such as exogenous medication administration, can significantly potentiate the liver injury associated with 1-ATZ hepatic accumulation; NSAIDs may be especially injurious to patients with 1-AT deficiency, possibly by increasing the expression and accumulation of the hepatotoxic mutant protein.
Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats (p 983-991)
Mitsuteru Kitade, Hitoshi Yoshiji, Hideyuki Kojima, Yasuhide Ikenaka, Ryuichi Noguchi, Kosuke Kaji, Junichi Yoshii, Koji Yanase, Tadashi Namisaki, Kiyoshi Asada, Masaharu Yamazaki, Tatsuhiro Tsujimoto, Takemi Akahane, Masahito Uemura, Hiroshi Fukui
Nonalcoholic steatohepatitis (NASH) may cause fibrosis, cirrhosis, and hepatocellular carcinoma (HCC); however, the exact mechanism of disease progression is not fully understood. Angiogenesis has been shown to play an important role in the progression of chronic liver disease. The aim of this study was to elucidate the role of angiogenesis in the development of liver fibrosis and hepatocarcinogenesis in NASH. Zucker rats, which naturally develop leptin receptor mutations, and their lean littermate rats were fed a choline-deficient, amino acid-defined diet. Both Zucker and littermate rats showed marked steatohepatitis and elevation of oxidative stress markers (e.g., thiobarbital acid reactive substances and 8-hydroxydeoxyguanosine). In sharp contrast, liver fibrosis, glutathione-S-transferase placental form (GST-P)-positive preneoplastic lesions, and HCC developed in littermate rats but not in Zucker rats. Hepatic neovascularization and the expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, only increased in littermate rats, almost in parallel with fibrogenesis and carcinogenesis. The CD31-immunopositive neovessels were mainly localized either along the fibrotic septa or in the GST-P-positive lesions. Our in vitro study revealed that leptin exerted a proangiogenic activity in the presence of VEGF. In conclusion, these results suggest that leptin-mediated neovascularization coordinated with VEGF plays an important role in the development of liver fibrosis and hepatocarcinogenesis in NASH.
Activation of Wnt/-catenin pathway during hepatocyte growth factor-induced hepatomegaly in mice (p 992-1002)
Udayan Apte, Gang Zeng, Peggy Muller, Xinping Tan, Amanda Micsenyi, Benjamin Cieply, Chunsun Dai, Youhua Liu, Klaus H. Kaestner, Satdarshan P. S. Monga
Hepatocyte growth factor (HGF) and -catenin both play a crucial role in stimulating hepatocyte proliferation, but whether these 2 pathways cooperate in inducing hepatocyte proliferation is unclear. We have previously reported that -catenin forms a complex with c-Met (HGF receptor) that undergoes dissociation because of -catenin tyrosine phosphorylation on stimulation by HGF. It is also known that delivery of the human HGF gene cloned in a plasmid under a CMV promoter results in hepatomegaly in mice. In addition, recently characterized -catenin transgenic mice also showed hepatomegaly. The present study was based on the hypothesis that HGF-induced hepatomegaly is mediated, at least in part, by activation of the Wnt/-catenin pathway. Here we report that delivery of the human HGF gene delivery in mice led to hepatomegaly via -catenin activation in the liver in 1- and 4-week studies. The mechanisms of -catenin activation in the 1-week study included loss of c-Met--catenin association as well as canonical -catenin activation, leading to its nuclear translocation. In the 4-week study, -catenin activation was observed via canonical mechanisms, whereas the c-Met--catenin complex remained unchanged. In both studies there was an associated increase in the E-cadherin--catenin association at the membrane. In addition, we generated liver-specific -catenin knockout mice, which demonstrated significantly smaller livers. HGF gene delivery failed to induce hepatomegaly in these -catenin conditionally null mice. In conclusion, -catenin- and HGF-mediated signaling pathways cooperate in hepatocyte proliferation, which may be crucial in liver development, regeneration following partial hepatectomy, and pathogenesis of hepatocellular carcinoma.
Oncogene-specific gene expression signatures at preneoplastic stage in mice define distinct mechanisms of hepatocarcinogenesis (p 1003-1011)
Cédric Coulouarn, Luis E. Gomez-Quiroz, Ju-Seog Lee, Pal Kaposi-Novak, Elizabeth A. Conner, Tatyana A. Goldina, Galina E. Onishchenko, Valentina M. Factor, Snorri S. Thorgeirsson
We applied a genome-wide microarray analysis to three transgenic mouse models of liver cancer in which targeted overexpression of c-Myc, E2f1, and a combination of the two was driven by the albumin promoter. Although gene expression profiles in HCC derived in all three transgenic lines were highly similar, oncogene-specific gene expression signatures were identified at an early dysplastic stage of hepatocarcinogenesis. Overexpression of E2f1 was associated with a strong alteration in lipid metabolism, and Srebp1was identified as a candidate transcription factor responsible for lipogenic enzyme induction. The molecular signature of c-Myc overexpression included the induction of more than 60 genes involved in the translational machinery that correlated with an increase in liver mass. In contrast, the combined activity of c-Myc and E2f1 specifically enhanced the expression of genes involved in mitochondrial metabolism - particularly the components of the respiratory chain - and correlated with an increased ATP synthesis. Thus, the results suggest that E2f1, c-Myc, and their combination may promote liver tumor development by distinct mechanisms. In conclusion, determination of tissue-specific oncogene expression signatures might be useful to identify conserved expression modules in human cancers.
Transcriptomic and genomic analysis of human hepatocellular carcinomas and hepatoblastomas (p 1012-1024)
Jian-Hua Luo, Baoguo Ren, Sergei Keryanov, George C. Tseng, Uma N. M. Rao, Satdarshan P. Monga, Steven Strom, Anthony J. Demetris, Michael Nalesnik, Yan P. Yu, Sarangarajan Ranganathan, George K. Michalopoulos
This study analyzed gene expression patterns and global genomic alterations in hepatocellular carcinomas (HCC), hepatoblastomas (HPBL), tissue adjacent to HCC and normal liver tissue derived from normal livers and hepatic resections. We found that HCC and adjacent non-neoplastic cirrhotic tissue have considerable overlap in gene expression patterns compared to normal liver. Several genes including Glypican 3, spondin-2, PEG10, EDIL3 and Osteopontin are over-expressed in HCC vs. adjacent tissue whereas Ficolin 3 is the most consistently under-expressed gene. HCC can be subdivided into three clusters based on gene expression patterns. HCC and HPBL have clearly different patterns of gene expression, with genes IGF2, Fibronectin, DLK1, TGFb1, MALAT1 and MIG6 being over-expressed in HPBL versus HCC. In addition, specific areas of the genome appear unstable in HCC, with the same regions undergoing either deletion or increased gene dosage in all HCC. In conclusion, a set of specific genes and areas of genomic instability are found across the board in liver neoplasia.
Comparison of gene expression profiles between Opisthorchis viverrini and non-Opisthorchis viverrini associated human intrahepatic cholangiocarcinoma (p 1025-1038)
Natini Jinawath, Yaovalux Chamgramol, Yoichi Furukawa, Kazutaka Obama, Tatsuhiko Tsunoda, Banchob Sripa, Chawalit Pairojkul, Yusuke Nakamura
Intrahepatic cholangiocarcinoma (ICC) is the second most common primary cancer in the liver, and its incidence is highest in the northeastern part of Thailand. ICCs in this region are known to be associated with infection with liver flukes, particularly Opisthorchis viverrini (OV), as well as nitrosamines from food. To clarify molecular mechanisms of ICC associated with or without liver flukes, we analyzed gene expression profiles of OV-associated ICCs from 20 Thai patients and compared their profiles with those of 20 Japanese ICCs that were not associated with OV, by means of laser microbeam microdissection and a cDNA microarray containing 27,648 genes. We identified 77 commonly upregulated genes and 325 commonly downregulated genes in the two ICC groups. Unsupervised hierarchical cluster analysis separated the 40 ICCs into two major branches almost completely according to the fluke status. The putative signature of OV-associated ICC exhibited elevated expression of genes involved in xenobiotic metabolism (UGT2B11, UGT1A10, CHST4, SULT1C1), whereas that of non-OV-associated ICC represented enhanced expression of genes related to growth factor signaling (TGFBI, PGF, IGFBP1, IGFBP3). Additional random permutation tests identified a total of 49 genes whose expression levels were significantly different between the two groups. We also identified genes associated with macroscopic type of ICCs. In conclusion, these data may not only contribute to clarification of common and OV-specific mechanisms underlying ICC, but also may serve as a starting point for the identification of novel diagnostic markers or therapeutic targets for the disease.
Special Article
Coagulation disorders and hemostasis in liver disease: Pathophysiology and critical assessment of current management (p 1039-1046)
Stephen H. Caldwell, Maureane Hoffman, Ton Lisman, B. Gail Macik, Patrick G. Northup, K. Rajender Reddy, Armando Tripodi, Arun J. Sanyal, Coagulation in Liver Disease Group
Normal coagulation has classically been conceptualized as a Y-shaped pathway, with distinct intrinsic and extrinsic components initiated by factor XII or factor VIIa/tissue factor, respectively, and converging in a common pathway at the level of the FXa/FVa (prothrombinase) complex. Until recently, the lack of an established alternative concept of hemostasis has meant that most physicians view the cascade as a model of physiology. This view has been reinforced by the fact that screening coagulation tests (APTT, prothrombin time - INR) are often used as though they are generally predictive of clinical bleeding. The shortcomings of this older model of normal coagulation are nowhere more apparent than in its clinical application to the complex coagulation disorders of acute and chronic liver disease. In this condition, the clotting cascade is heavily influenced by numerous currents and counter-currents resulting in a mixture of pro- and anticoagulant forces that are themselves further subject to change with altered physiological stress such as super-imposed infection or renal failure. This report represents a summary of a recent multidisciplinary symposium held in Charlottesville, VA. We present an overview of the coagulation system in liver disease with emphasis on the limitations of the current clinical paradigm and the need for a critical re-evaluation of the current tenets governing clinical practice. With the realization that there is often limited or conflicting data, we have attempted to represent diverse opinion and experience from the perspectives of both hepatology and hematology beginning with a brief update on the physiology of normal coagulation.
Copyright © 2006 by the American Association for the Study of Liver Diseases. All rights reserved.
Volume 131, Issue 4, Pages 985-1366 (October 2006)
Rapid Decline of Viral RNA in Hepatitis C Patients Treated With VX-950: A Phase Ib, Placebo-Controlled, Randomized Study
Hendrik W. Reesink?, Stefan Zeuzem‡, Christine J. Weegink?, Nicole Forestier?, Andre van Vliet§, Jeroen van de Wetering de Rooij§, Lindsay McNair?, Susan Purdy?, Robert Kauffman?, John Alam?, Peter L.M. Jansen?
Received 10 April 2006; accepted 15 June 2006
Background & Aims: VX-950 specifically inhibits the NS3·4A protease of hepatitis C and has antiviral activity in vitro. This phase I, placebo-controlled, double-blind study evaluated the antiviral activity, pharmacokinetics, and safety of VX-950 in patients with chronic hepatitis C (CHC). Methods: Thirty-four patients with genotype 1 CHC were randomized to receive placebo or VX-950 at doses of 450 mg or 750 mg every 8 hours or 1250 mg every 12 hours for 14 days. Of the 34 participants, 27 (79%) had failed prior treatment. Patients were monitored for safety and tolerability of VX-950. Plasma VX-950 concentrations and HCV RNA levels were measured. Results: VX-950 was well tolerated and had substantial antiviral effects: viral loads dropped ≥2 log10 in all 28 patients treated with VX-950 and ≥3 log10 in 26 (93%) of the 28 patients. In the 750-mg-dose group, which had the highest trough plasma drug concentrations, the median reduction of HCV RNA was 4.4 log10 after 14 days. In the 450-mg and 1250-mg groups, the maximal effect was seen between days 3 and 7 of dosing, and median HCV RNA increased between days 7 and 14; median reductions at day 14 were 2.4 log10 and 2.2 log10, respectively. Median alanine aminotransferase levels decreased during dosing in all VX-950 groups. Conclusions: VX-950 was well tolerated and demonstrated substantial antiviral activity. Some patients had viral breakthrough during dosing, related to selection of variants with decreased sensitivity to VX-950. The results support further studies of VX-950 in patients with CHC.
Relationship of Abdominal Bloating to Distention in Irritable Bowel Syndrome and Effect of Bowel Habit
Lesley A. Houghton?, Richard Lea?, Anvrag Agrawal?, Brian Reilly‡, Peter J. Whorwell?
Received 20 September 2005; accepted 28 June 2006
Background & Aims: The relationship between the sensation of bloating, often ranked as the most bothersome symptom by patients with irritable bowel syndrome (IBS), and actual distention manifest as an increase in abdominal girth is controversial. Investigation of this problem has been hampered by the lack of a reliable ambulatory technique to measure abdominal girth. The aim of this study was to use the technique of abdominal inductance plethysmography to compare diurnal variation in girth in IBS patients and healthy volunteers, relating these changes to the sensation of bloating. Methods: Abdominal girth was recorded for 24 hours in 20 IBS-constipation (age, 18–73 y), 20 IBS-diarrhea (age, 25–62 y) and 10 IBS-alternating (age, 21–59 y) female patients meeting Rome II criteria and 20 healthy female controls (age, 18–67 y). All subjects pursued normal daily activities, recording their symptoms of bloating and pain together with bowel habit. Results: All patients with IBS, irrespective of bowel habit, reported significantly greater bloating than controls (P < .0001). Forty-eight percent of patients also showed distention beyond the 90% control range, with this being most prominent in IBS-constipation. Bloating correlated strongly only with distention in IBS-constipation (r ≥ 0.48; P ≤ .02). Neither bloating nor distention in IBS was related to body mass index, age, parity, or psychologic status. Conclusions: Abdominal distention is a clearly definable phenomenon in IBS that can reach 12 cm. However, it only occurs in half of patients reporting bloating, and the 2 only correlate in IBS-constipation. Bloating and tentiodisn may differ pathophysiologically and this appears to be reflected in the bowel habit subtype.
Risk Stratification for Colon Neoplasia: Screening Strategies Using Colonoscopy and Computerized Tomographic Colonography
Otto S. Lin?‡, Richard A. Kozarek?, Drew B. Schembre?, Kamran Ayub?, Michael Gluck?, Nico Cantone?, Maw–Soan Soon‡, Jason A. Dominitz§
Received 16 August 2005; accepted 21 June 2006
Background & Aims: We developed a risk index to identify low-risk patients who may be screened for colorectal cancer with computerized tomographic colonography (CTC) instead of colonoscopy. Methods: Asymptomatic persons aged 50 years or older who had undergone screening colonoscopy were randomized retrospectively to derivation (n = 1512) and validation (n = 1493) subgroups. We developed a risk index (based on age, sex, and family history) from the derivation group. The expected results of 3 screening strategies—universal colonoscopy, universal CTC, and a stratified strategy of colonoscopy for high-risk and CTC for low-risk patients—were then compared. Outcomes for the 3 strategies were extrapolated from the known colonic findings in each patient, using sensitivity/specificity values for CTC from the medical literature. Results were validated in the validation subgroup. Results: In the derivation subgroup, universal colonoscopy detected 94% of advanced neoplasia and universal CTC detected only 70% and resulted in the largest total number of procedures and number of patients undergoing both procedures. The stratified strategy detected 92% of advanced neoplasia, requiring colonoscopy in 68% and CTC in 36% of patients, with only 4% having to undergo both procedures. In the validation subgroup, universal colonoscopy detected 94% and universal CTC detected 71% of advanced neoplasia, whereas the stratified strategy detected 89%, requiring colonoscopy in 64% and CTC in 40%. Unlike universal CTC, the stratified strategy was independent of assumptions for CTC sensitivity, specificity, and threshold for colonoscopy. Conclusions: The stratified strategy based on our risk index may optimize the yield of colonoscopic resources and reduce the number of patients undergoing colonoscopy.
Increased Serum Levels of Complement C3a Anaphylatoxin Indicate the Presence of Colorectal Tumors
Jens K. Habermann?‡§1, Uwe J. Roblick‡§1, Brian T. Luke?, Darue A. Prieto¶, William J.J. Finlay#, Vladimir N. Podust??, John M. Roman¶, Elisabeth Oevermann‡, Thomas Schiedeck‡, Nils Homann‡‡, Michael Duchrow‡, Thomas P. Conrads¶, Timothy D. Veenstra¶, Stanley K. Burt?, Hans–Peter Bruch‡, Gert Auer§, Thomas Ried?
Received 3 February 2006; accepted 15 June 2006
Background & Aims: Late diagnosis of colorectal carcinoma results in a significant reduction of average survival times. Yet despite screening programs, about 70% of tumors are detected at advanced stages (International Union Against Cancer stages III/IV). We explored whether detection of malignant disease would be possible through identification of tumor-specific protein biomarkers in serum samples. Methods: A discovery set of sera from patients with colorectal malignancy (n = 58) and healthy control individuals (n = 32) were screened for potential differences using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Candidate proteins were identified and their expression levels were validated in independent sample sets using a specific immunoassay (enzyme-linked immunosorbent assay). Results: By using class comparison and custom-developed algorithms we identified several m/z values that were expressed differentially between the malignant samples and the healthy controls of the discovery set. Characterization of the most prominent m/z values revealed a member of the complement system, the stable form of C3a anaphylatoxin (ie, C3a-desArg). Based on a specific enzyme-linked immunosorbent assay, serum levels of complement C3a-desArg predicted the presence of colorectal malignancy in a blinded validation set (n = 59) with a sensitivity of 96.8% and a specificity of 96.2%. Increased serum levels were also detected in 86.1% of independently collected sera from patients with colorectal adenomas (n = 36), whereas only 5.6% were classified as normal. Conclusions: Complement C3a-desArg is present at significantly higher levels in serum from patients with colorectal adenomas (P < .0001) and carcinomas (P < .0001) than in healthy individuals. This suggests that quantification of C3a-desArg levels could ameliorate existing screening tests for colorectal cancer.
Relative Contribution of Genetic and Nongenetic Modifiers to Intestinal Obstruction in Cystic Fibrosis
Scott M. Blackman?‡, Rebecca Deering–Brose?§, Rita McWilliams??¶, Kathleen Naughton?, Barbara Coleman?, Teresa Lai?, Marilyn Algire?, Suzanne Beck#, Julie Hoover-Fong?, Ada Hamosh?, M. Daniele Fallin¶, Kristen West?, Dan E. Arking?, Aravinda Chakravarti?, David J. Cutler?, Garry R. Cutting?
Received 22 September 2005; accepted 28 June 2006
Background & Aims: Neonatal intestinal obstruction (meconium ileus [MI]) occurs in 15% of patients with cystic fibrosis (CF). Our aim was to determine the relative contribution of genetic and nongenetic modifiers to the development of this major complication of CF. Methods: A total of 65 monozygous twin pairs, 23 dizygous twin/triplet sets, and 349 sets of siblings with CF were analyzed for MI status, significant covariates, and genome-wide linkage. Results: Specific mutations in the CF transmembrane conductance regulator (CFTR), the gene responsible for CF, correlated with MI, indicating a role for CFTR genotype. Monozygous twins showed substantially greater concordance for MI than dizygous twins and siblings (P = 1 ? 10?5), showing that modifier genes independent of CFTR contribute substantially to this trait. Regression analysis revealed that MI was correlated with distal intestinal obstruction syndrome (P = 8 ? 10?4). Unlike MI, concordance analysis indicated that the risk for development of distal intestinal obstruction syndrome in CF patients is caused primarily by nongenetic factors. Regions of suggestive linkage (logarithm of the odds of linkage >2.0) for modifier genes that cause MI (chromosomes 4q35.1, 8p23.1, and 11q25) or protect from MI (chromosomes 20p11.22 and 21q22.3) were identified by genome-wide analyses. These analyses did not support the existence of a major modifier gene on chromosome 19 in a region previously linked to MI. Conclusions: The CFTR gene along with 2 or more modifier genes are the major determinants of intestinal obstruction in newborn CF patients, whereas intestinal obstruction in older CF patients is caused primarily by nongenetic factors.
Effect of Ribavirin in Genotype 1 Patients With Hepatitis C Responding to Pegylated Interferon Alfa-2a Plus Ribavirin
Jean–Pierre Bronowicki?, Denis Ouzan‡, Tarik Asselah§, Hervé Desmorat?, Jean–Pierre Zarski¶, Juliette Foucher#, Marc Bourlière??, Christophe Renou‡‡, Albert Tran§§, Pascal Melin¶¶, Christophe Hézode??, Michelle Chevalier##, Magali Bouvier–Alias???, Stéphane Chevaliez???, François Montestruc‡‡‡, Isabelle Lonjon–Domanec‡‡‡, Jean–Michel Pawlotsky???
Received 26 July 2005; accepted 15 June 2006
Background & Aims: Pegylated interferon alfa-ribavirin combination is the standard treatment for chronic hepatitis C, but the mechanisms by which ribavirin enhances the rate of sustained hepatitis C virus (HCV) eradication remain unknown. We aimed to investigate the role of ribavirin in HCV clearance during therapy and to evaluate the consequences of ribavirin discontinuation in patients infected with genotype 1 hepatitis C who cleared HCV RNA at week 24. Methods: A total of 516 patients were treated with pegylated interferon alfa-2a, 180 ?g/wk, plus ribavirin, 800 mg/day. Seventy percent were RNA negative at week 24. They were randomized to continue with the combination or receive pegylated interferon alone. Results: Responders at week 24 who stopped ribavirin had a significantly higher rate of breakthroughs during, and relapses after, therapy (sustained virologic response, 52.8% vs 68.2%; P = .004), but their side-effect profile and quality of life tended to improve. Multiple logistic regression analysis in the pegylated interferon alfa monotherapy group allowed identification of responders at week 24 who could stop ribavirin without losing their chance of a sustained virologic response, based on baseline viral load and age. Forty-eight weeks of ribavirin may not be needed when HCV RNA is undetectable at week 2. Conclusions: We made 3 conclusions from this study. First, ribavirin primarily acts by sustaining the virologic response to pegylated interferon alfa; second, ribavirin must be administered for the full treatment duration in most genotype 1–infected patients who respond; third, baseline parameters may help identify patients who could discontinue ribavirin or reduce the dose without losing their chance of success.
Norfloxacin vs Ceftriaxone in the Prophylaxis of Infections in Patients With Advanced Cirrhosis and Hemorrhage
Javier Fernández?, Luis Ruiz del Arbol‡, Cristina Gómez§, Rosa Durandez?, Regina Serradilla‡, Carlos Guarner§, Ramón Planas?, Vicente Arroyo?, Miguel Navasa?
Received 2 February 2006; accepted 15 June 2006
Background & Aims: Oral norfloxacin is the standard of therapy in the prophylaxis of bacterial infections in cirrhotic patients with gastrointestinal hemorrhage. However, during the last years, the epidemiology of bacterial infections in cirrhosis has changed, with a higher incidence of infections caused by quinolone-resistant bacteria. This randomized controlled trial was aimed to compare oral norfloxacin vs intravenous ceftriaxone in the prophylaxis of bacterial infection in cirrhotic patients with gastrointestinal bleeding. Methods: One hundred eleven patients with advanced cirrhosis (at least 2 of the following: ascites, severe malnutrition, encephalopathy, or bilirubin >3 mg/dL) and gastrointestinal hemorrhage were randomly treated with oral norfloxacin (400 mg twice daily; n = 57) or intravenous ceftriaxone (1 g/day; n = 54) for 7 days. The end point of the trial was the prevention of bacterial infections within 10 days after inclusion. Results: Clinical data were comparable between groups. The probability of developing proved or possible infections, proved infections, and spontaneous bacteremia or spontaneous bacterial peritonitis was significantly higher in patients receiving norfloxacin (33% vs 11%, P = .003; 26% vs 11%, P = .03; and 12% vs 2%, P = .03, respectively). The type of antibiotic used (norfloxacin), transfusion requirements at inclusion, and failure to control bleeding were independent predictors of infection. Seven gram-negative bacilli were isolated in the norfloxacin group, and 6 were quinolone resistant. Nonenterococcal streptococci were only isolated in the norfloxacin group. No difference in hospital mortality was observed between groups. Conclusions: Intravenous ceftriaxone is more effective than oral norfloxacin in the prophylaxis of bacterial infections in patients with advanced cirrhosis and hemorrhage.
Utility of a New Model to Diagnose an Alcohol Basis for Steatohepatitis
Winston Dunn?, Paul Angulo?, Schuyler Sanderson‡, Laith H. Jamil?, Linda Stadheim?, Charles Rosen§, Michael Malinchoc?, Patrick S. Kamath?, Vijay H. Shah?
Received 23 February 2006; accepted 21 June 2006
Background & Aims: Distinguishing an alcohol basis from a nonalcoholic basis for the clinical and histologic spectrum of steatohepatitic liver disease is difficult because of unreliability of alcohol consumption history. Unfortunately, various biomarkers have had limited utility in distinguishing alcoholic liver disease (ALD) from nonalcoholic fatty liver disease (NAFLD). Thus, the aim of our study was to create and validate a model to diagnose ALD in patients with steatohepatitis. Methods: A cross-sectional cohort study was performed at the Mayo Clinic, Rochester, Minnesota, to create a model using multivariable logistic regression analysis. This model was validated in 3 independent data sets comprising patients of varying severity of steatohepatitis spanning over 10 years. Results: Logistic regression identified mean corpuscular volume, aspartate aminotransferase (AST)/alanine aminotransfersase (ALT) ratio, body mass index, and gender as the most important variables that separated patients with ALD from NAFLD. These variables were used to generate the ALD/NAFLD Index (ANI), with ANI of greater than zero incrementally favoring ALD and ANI of less than zero incrementally favoring a diagnosis of NAFLD, thus making ALD unlikely. ANI had a c-statistic of 0.989 in the derivation sample, and 0.974, 0.989, 0.767 in the 3 validation samples. ANI performance characteristics were significantly better than several conventional and recently proposed biomarkers used to differentiate ALD from NAFLD, including the histopathologic marker protein tyrosine phosphatase 1b, AST/ALT ratio, ?-glutamyl transferase, and carbohydrate-deficient transferrin. Conclusions: ANI, derived from easily available objective variables, accurately differentiates ALD from NAFLD in hospitalized, ambulatory, and pretransplantation patients and compares favorably with other traditional and proposed biomarkers.
Advanced Cytologic Techniques for the Detection of Malignant Pancreatobiliary Strictures
Laura E. Moreno Luna?, Benjamin Kipp‡, Kevin C. Halling‡, Thomas J. Sebo‡, Walter K. Kremers§, Lewis R. Roberts?, Emily G. Barr Fritcher‡, Michael J. Levy?, Gregory J. Gores?
Received 6 April 2006; accepted 5 July 2006
Background & Aims: Two advanced cytologic techniques for detecting aneuploidy—digital image analysis (DIA) and fluorescence in situ hybridization (FISH)—have recently been developed to help identify malignant pancreatobiliary strictures. The aim of this study was to assess the clinical utility of cytology, DIA, and FISH for the identification of malignant pancreatobiliary strictures. Methods: Brush cytologic specimens from 233 consecutive patients undergoing endoscopic retrograde cholangiopancreatography for pancreatobiliary strictures were examined by all 3 (cytology, DIA, and FISH) techniques. Strictures were stratified as proximal (n = 33) or distal (n = 114) based on whether they occurred above or below the cystic duct, respectively. Strictures in patients with primary sclerosing cholangitis (n = 86) were analyzed separately. Results: Despite the stratification, the performances of the tests were similar. Conventional cytology has a low sensitivity (4%–20%) but 100% specificity. Because of the high specificity for cytology, we assessed the performance of the other tests when conventional cytology was negative. In this clinical context, FISH had an increased sensitivity (35%–60%) when assessing for chromosomal gains (polysomy) while preserving the specificity of cytology. The sensitivity and specificity of DIA was intermediate as compared with routine cytology and FISH but was additive to FISH values demonstrating only trisomy of chromosome 7 or chromosome 3. Conclusions: These findings suggest that FISH and DIA increase the sensitivity for the diagnosis of malignant pancreatobiliary tract strictures over that obtained by conventional cytology while maintaining an acceptable specificity.
STAT3 Activation Regulates Growth, Inflammation, and Vascularization in a Mouse Model of Gastric Tumorigenesis
Louise M. Judd?, Karin Bredin?, Anastasia Kalantzis?, Brendan J. Jenkins‡, Matthias Ernst‡, Andrew S. Giraud?
Received 31 October 2005; accepted 28 June 2006
Background & Aims: The gp130757F/F mouse is a well-characterized and robust model of distal gastric tumorigenesis displaying many of the characteristics of human intestinal type gastric cancer. Key to the development of tumors in this model, and in many examples of human tumor development, is hyperactivation of the transcription factor STAT3. This study addressed the requirement for STAT3 activation in tumor initiation and characterized some of the genes downstream of STAT3 required for tumor development. Furthermore, the interaction among STAT3, the microbial environment, and tumorigenesis was evaluated. Methods: The role of STAT3 in gastric tumor development was assessed in detail in gp130757F/Y757F:STAT3+/? mice displaying reduced STAT3 activity. Tumor size was quantified morphologically, and the effects on endocrine cell populations, neovascularization, and inflammatory cell infiltration as well as the outcome of STAT3 activation on transcription of a number of genes relevant in growth and inflammation were quantified. Results: Loss of one STAT3 allele in gp130757F/F mice reduced the frequency and rate of tumor development because of inhibition of proliferation-induced glandular hyperplasia. There was also a concomitant reduction in the degree of inflammatory infiltration and cytokine and chemokine expression, angiogenesis, and expression of metalloproteinases and growth factors. Antimicrobial treatment of gp130757F/F mice slowed tumor growth coincident with reduced macrophage and neutrophil infiltration. Conclusions: Activation of STAT3 and the microbial environment are pivotal for gastric tumor initiation and development in the gp130757F/F mouse, thus supporting the notion that STAT3 activation may play a role in human gastric cancer development.
Carcinogenesis in Mouse Stomach by Simultaneous Activation of the Wnt Signaling and Prostaglandin E2 Pathway
Hiroko Oshima?‡, Akihiro Matsunaga‡, Takashi Fujimura§, Tetsuya Tsukamoto?, Makoto M. Taketo‡, Masanobu Oshima?‡
Received 9 May 2006; accepted 21 June 2006
Background & Aims: Accumulating evidence indicates that prostaglandin E2 (PGE2), a downstream product of cyclooxygenase 2 (COX-2), plays a key role in gastric tumorigenesis. The Wnt pathway is also suggested to play a causal role in gastric carcinogenesis. However, the molecular mechanism remains poorly understood of how the Wnt and PGE2 pathways contribute to gastric tumorigenesis. To investigate the role of Wnt and PGE2 in gastric cancer, we have generated transgenic mice that activate both pathways and examined their phenotypes. Methods: We constructed K19-Wnt1 transgenic mice expressing Wnt1 in the gastric mucosa using the keratin 19 promoter. We then crossed K19-Wnt1 mice with another transgenic line, K19-C2mE, to obtain K19-Wnt1/C2mE compound transgenic mice. The K19-C2mE mice express COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) in the stomach, showing an increased gastric PGE2 level. We examined the gastric phenotypes of both K19-Wnt1 and K19-Wnt1/C2mE mice. Results:K19-Wnt1 mice had a significant suppression of epithelial differentiation and developed small preneoplastic lesions consisting of undifferentiated epithelial cells with macrophage accumulation. Importantly, additional expression of COX-2 and mPGES-1 converted the preneoplastic lesions in the K19-Wnt1 mice into dysplastic gastric tumors by 20 weeks of age. Notably, we found mucous cell metaplasia in the glandular stomach of the K19-Wnt1/C2mE mice as early as 5 weeks of age, before the dysplastic tumor development. Conclusions: Wnt signaling keeps the gastric progenitor cells undifferentiated. Simultaneous activation of both Wnt and PGE2 pathways causes dysplastic gastric tumors through the metaplasia-carcinoma sequence.
APC and Oncogenic KRAS Are Synergistic in Enhancing Wnt Signaling in Intestinal Tumor Formation and Progression
Klaus–Peter Janssen?‡1, Paola Alberici§1, Hafida Fsihi?, Claudia Gaspar§, Cor Breukel?, Patrick Franken§, Christophe Rosty¶, Miguel Abal?, Fatima El Marjou?, Ron Smits§, Daniel Louvard?, Riccardo Fodde§, Sylvie Robine?
Received 17 February 2006; accepted 21 June 2006
Background & Aims: Synchronous activation of the Wnt signaling pathway, mostly because of loss of function of the APC tumor suppressor, and of the oncogenic KRAS-signaling pathway is very frequent in colorectal cancer and is associated with poor prognosis. Methods: We have generated a compound transgenic mouse model, KRASV12G/Apc+/1638N, to recapitulate the human disease and compared it with single transgenic littermates. Results: Compound mutant mice are characterized by a 10-fold increase in tumor multiplicity and by accelerated tumor progression, resulting in strongly enhanced morbidity and mortality. Tumors from compound mutant mice proliferate faster and show decreased levels of apoptosis. Several lines of evidence indicate that the observed increase in tumor multiplicity and malignant transformation is caused by the synergistic activation of Wnt signaling in cells with oncogenic KRAS and loss-of-function Apc mutations. Activated KRAS is known to induce tyrosine phosphorylation of ?-catenin, leading to its release from E-cadherin at the adherens junction. This results in an increased ?-catenin pool in the cytoplasma, its subsequent translocation to the nucleus, and the transcriptional activation of Wnt downstream target genes. Accordingly, intestinal tumors from KRASV12G/Apc+/1638N mice show a significant increase in cells with nuclear accumulation of ?-catenin when compared with Apc+/1638N animals. Moreover, Apc/KRAS-mutant embryonic stem cells show a significantly enhanced ?-catenin/T-cell factor–mediated transcriptional activation, accompanied by increased ?-catenin nuclear localization. Conclusions: This KRAS-induced increase in Wnt/?-catenin signaling may enhance the plasticity and self-renewal capacity of the tumor, thus resulting in the drastically augmented tumor multiplicity and malignant behavior in compound mutant animals.
Human Thioredoxin-1 Ameliorates Experimental Murine Colitis in Association With Suppressed Macrophage Inhibitory Factor Production
Hiroyuki Tamaki?, Hajime Nakamura‡, Akiyoshi Nishio?, Hiroshi Nakase?, Satoru Ueno?, Norimitsu Uza?, Masahiro Kido?, Satoko Inoue?, Sakae Mikami?, Masanori Asada?, Keiichi Kiriya?, Hiroshi Kitamura?, Shinya Ohashi?, Toshiro Fukui?, Kimio Kawasaki?, Minoru Matsuura?, Yasuyuki Ishii§, Kazuichi Okazaki?, Junji Yodoi¶, Tsutomu Chiba?
Received 10 November 2005; accepted 15 June 2006
Background & Aims: Thioredoxin-1 (TRX) is a small multifunctional protein with antioxidative and redox-regulating functions. In this study, we investigated the significance of TRX in patients with inflammatory bowel disease (IBD) and the ability and mechanism to ameliorate experimental colitis. Methods: Serum TRX and macrophage migration inhibitory factor (MIF) levels were measured in patients with IBD. The effects of TRX were evaluated in a dextran sulfate sodium (DSS)-induced colitis model by comparing TRX-overexpressing transgenic (TRX-TG) and control mice. We further evaluated the effect of recombinant human TRX (rhTRX) administration on DSS-induced colitis and colonic inflammation of interleukin (IL)-10 knockout (IL-10 KO) mice. Colonic inflammation was examined clinically and histologically. Proinflammatory cytokine levels were examined in colonic tissues, and MIF levels were measured in colonic tissues and sera in mice. The effect of TRX on MIF production was also analyzed in vitro. Results: Serum TRX and MIF levels were significantly higher in patients with IBD than normal controls, and TRX levels correlated with disease activity. TRX significantly ameliorated DSS-induced colitis and colonic inflammation of IL-10 KO mice. Increase of tumor necrosis factor-? and interferon-? in colonic tissues was significantly suppressed in TRX-TG mice compared with wild-type mice. MIF levels in colonic tissues and sera were significantly lower in TRX-TG mice than in wild-type mice, irrespective of DSS administration. Anti-TRX treatment exacerbated DSS-induced colitis. In vitro studies demonstrated that rhTRX suppressed MIF production in human monocyte cells. Conclusions: TRX might have a potential as a novel therapeutic agent for the treatment of IBD.
The Vagus Nerve: A Tonic Inhibitory Influence Associated With Inflammatory Bowel Disease in a Murine Model
Jean Eric Ghia, Patricia Blennerhassett, Harry Kumar–Ondiveeran, Elena F. Verdu, Stephen M. Collins
Received 29 November 2005; accepted 21 June 2006
Background & Aims: The recently proposed Inflammatory Reflex describes an interaction between the vagus nerve and peripheral macrophages, resulting in attenuation of proinflammatory cytokine release in response to systemic exposure to bacterial endotoxin. The purpose of this study was to determine whether a similar vagus/macrophage axis modulates the inflammatory responses in the colon in mice. Methods: We assessed the Disease Activity Index (DAI), macroscopic and histologic damage, serum amyloid-P level, and myeloperoxidase activity in colitis induced by administration of dextran sodium sulfate (DSS) in healthy and vagotomized C57BL/6 and in mice deficient in macrophage-colony stimulating factor (M-CSF)–induced and in hapten-induced colitis. A pyloroplasty was performed in vagotomized mice. Results: DAI, macroscopic and histologic scores, myeloperoxidase activity, levels of serum amyloid-P, and colonic tissue levels of interleukin (IL)-1?, IL-6, and tumor necrosis factor-? were increased significantly in vagotomized mice 5 days post-DSS and 3 days after hapten-induced colitis compared with sham-operated mice that received DSS or the hapten. Pretreatment with nicotine significantly decreased each of these markers in vagotomized mice with DSS colitis, and all markers except DAI and IL-6 in sham-operated DSS-treated mice. Conversely, hexamethonium treatment significantly increased each of these markers in the sham-operated DSS-treated mice. Vagotomy had no effect on the colitis in M-CSF–deficient mice. Conclusions: The vagus nerve plays a counterinflammatory role in acute colitis via a macrophage-dependent mechanism, involving hexamethonium-sensitive nicotinic receptors. The identification of a counterinflammatory neural pathway would open new therapeutic avenues for treating acute exacerbations of inflammatory bowel disease.
Altered Gastrointestinal and Metabolic Function in the GPR39-Obestatin Receptor–Knockout Mouse
Dieder Moechars?1, Inge Depoortere‡1, Benoit Moreaux?, Betty de Smet‡, Ilse Goris?, Luc Hoskens?, Guy Daneels?, Stefan Kass?, Luc Ver Donck?, Theo Peeters‡, Bernard Coulie?
Received 27 January 2006; accepted 15 June 2006
Background & Aims: The G-protein–coupled receptor GPR39 is a member of a family that includes the receptors for ghrelin and motilin. Recently the peptide obestatin was identified as a natural ligand for GPR39. The objective of this study was to gain insight into the biological function of the GPR39 receptor. Methods: GPR39?/? mice were generated and analyzed. Results: Endogenous GPR39 expression was detected in the brain (septum-amygdala) and the gastrointestinal system (parietal cells, enterocytes, neurons, and pancreas). Gastric emptying of a solid meal (measured by the 14C octanoic breath test) in GPR39?/? mice was accelerated significantly with a gastric half-emptying time of 49.5 ± 2.2 minutes compared with 86.9 ± 8.4 minutes in GPR39+/+ mice. A more effective expulsion of distally located pellets (30%–75% of length) was observed in the colon of GPR39?/? mice. Four hours after pylorus ligation, the volume of gastric secretion was increased significantly (GPR39?/?: 638 ± 336 ?L; GPR39+/+: 225 ± 170 ?L), but gastric acid secretion was unchanged. The mature body weight and body fat composition of GPR39?/? mice was significantly higher compared with GPR39+/+ mice, but this was not related to hyperphagia because 24-hour food intake did not differ between both genotypes. In contrast, deficiency of the GPR39 receptor led to reduced hyperphagia after fasting. The cholesterol levels were increased significantly in the GPR39?/? mice. Conclusions: Our data partially confirm and extend the described in vivo effects of obestatin and suggest that this peptide plays a functional role in the regulation of gastrointestinal and metabolic function through interaction with the GPR39 receptor.
Nitric Oxide-Induced Down-Regulation of ?-Catenin in Colon Cancer Cells by a Proteasome-Independent Specific Pathway
Laurent Prévotat, Rodolphe Filomenko, Eric Solary, Jean-François Jeannin, Ali Bettaieb
Received 24 October 2005; accepted 21 June 2006
Background & Aims: We have previously reported that nitric oxide could induce the death of colon cancer cells. Because an inappropriate activation of ?-catenin has been associated with intestinal cell malignant transformation, we explored whether nitric oxide could affect ?-catenin expression and function. Methods: Human colon cancer cell lines were treated with the nitric oxide donor glyceryl trinitrate (GTN) before analyzing ?-catenin expression by immunofluorescence, immunoblotting, and immunoprecipitation methods and its transcriptional activity using a luciferase reporter gene driven by a T-cell factor-responsive promotor. Results: GTN induces ?-catenin degradation and down-regulates its transcriptional activity in colon cancer cells. This effect is preceded by GTN-induced tyrosine nitration of ?-catenin, together with its dephosphorylation on serine 33, 37, and 45 and threonine 41. GTN-induced ?-catenin degradation involves proteases that are sensitive to a broad-spectrum caspase inhibitor, z-VAD-fmk, and to serine protease inhibitors N-tosyl-L-phenylalaline chloromethyl ketone (TPCK) and [4-(2-aminoethyl)-benzenesulfonylfluoride] (AEBSF), whereas the ubiquitin/proteasome pathway is not involved. Interestingly, only TPCK and AEBSF restore ?-catenin transcriptional activity and preserve ?-catenin nuclear localization in GTN-treated colon cancer cells. Conclusions: Exposure of colon cancer cells to nitric oxide unraveled a so-far-unidentified mechanism of ?-catenin regulation. The protein is nitrated and dephosphorylated, and its transcriptional activity is reduced through degradation by a TPCK and AEBSF-sensitive protease.
IFN-Gamma-Induced TNFR2 Expression Is Required for TNF-Dependent Intestinal Epithelial Barrier Dysfunction
Fengjun Wang?‡1, Brad T. Schwarz‡1, W. Vallen Graham‡, Yingmin Wang‡, Liping Su‡, Daniel R. Clayburgh‡, Clara Abraham§, Jerrold R. Turner‡
Received 4 March 2006; accepted 21 June 2006
Background & Aims: Tumor necrosis factor (TNF) plays a critical role in intestinal disease. In intestinal epithelia, TNF causes tight junction disruption and epithelial barrier loss by up-regulating myosin light chain kinase (MLCK) activity and expression. The aim of this study was to determine the signaling pathways by which TNF causes intestinal epithelial barrier loss. Methods: Caco-2 cells that were either nontransfected or stably transfected with human TNF receptor 1 (TNFR1) or TNFR2 and mouse colonocytes were used for physiologic, morphologic, and biochemical analyses. Results: Colitis induced in vivo by adoptive transfer of CD4+CD45RBhi T cells was associated with increased epithelial MLCK expression and myosin II regulatory light chain (MLC) phosphorylation as well as morphologic tight junction disruption. In vitro studies showed that TNF caused similar increases in MLCK expression and MLC phosphorylation, as well as barrier dysfunction, in Caco-2 monolayers only after interferon (IFN)-? pretreatment. This reductionist model was therefore used to determine the molecular mechanism by which IFN-? and TNF synergize to cause intestinal epithelial barrier loss. IFN-? priming increased TNFR1 and TNFR2 expression, and blocking antibody studies showed that TNFR2, but not TNFR1, was required for TNF-induced barrier dysfunction. Transgenic TNFR2, but not TNFR1, expression allowed IFN-?-independent TNF responses. Conclusions: IFN-? primes intestinal epithelia to respond to TNF by inducing TNFR2 expression, which in turn mediates TNF-induced MLCK-dependent barrier dysfunction. The data further suggest that epithelial TNFR2 blockade may be a novel approach to restore barrier function in intestinal disease.
Glial-Derived Neurotrophic Factor Modulates Enteric Neuronal Survival and Proliferation Through Neuropeptide Y
Mallappa Anitha?1, Bindu Chandrasekharan?1, Joana R. Salgado‡, Eric Grouzmann‡, Simon Mwangi?, Shanthi V. Sitaraman?, Shanthi Srinivasan?
Received 4 November 2005; accepted 28 June 2006
Background & Aims: Glial-derived neurotrophic factor (GDNF) promotes the survival and proliferation of enteric neurons. Neuropeptide Y (NPY) is an important peptide regulating gastrointestinal motility. The role of NPY on the survival and proliferation of enteric neurons is not known. We examined the effects of GDNF on the expression and release of NPY from enteric neurons and the role of NPY in promoting enteric neuronal proliferation and survival. Methods: Studies were performed in primary enteric neuronal cultures and NPY knockout mice (NPY?/?). GDNF-induced expression of NPY was assessed by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and enzyme-linked immunosorbent assay. Using NPY-siRNA and NPY-Y1 receptor antagonist, we examined the role of NPY in mediating the survival and proliferation effects of GDNF. Gastrointestinal motility was assessed by measuring gastric emptying, intestinal transit, and isometric muscle recording from intestinal muscle strips. Results: GDNF induced a significant increase in NPY messenger RNA and protein expression in primary enteric neurons and the release of NPY into the culture medium. NPY (1 ?mol/L) significantly increased proliferation of neurons and reduced apoptosis. In the presence of NPY-siRNA and NPY-Y1 receptor antagonist or in enteric neurons cultured from NPY?/? mice, GDNF-mediated neuronal proliferation and survival was reduced. NPY increased the phosphorylation of Akt, a downstream target of the PI-3-kinase pathway. In NPY?/? mice, there were significantly fewer nNOS-containing enteric neurons compared with wild-type (WT) mice. NPY?/? mice had accelerated gastric emptying and delayed intestinal transit compared with WT mice. Conclusions: We demonstrate that NPY acts as an autocrine neurotrophic factor for enteric neurons.
The Motogenic Effects of Cyclic Mechanical Strain on Intestinal Epithelial Monolayer Wound Closure Are Matrix Dependent
Jianhu Zhang?‡, Cheri R. Owen?‡, Matthew A. Sanders?‡, Jerrold R. Turner§, Marc D. Basson?‡
Received 17 March 2006; accepted 28 June 2006
Background & Aims: Complex deformation during normal digestion due to peristalsis or villous motility may be trophic for the intestinal mucosa. Because tissue fibronectin is increased in inflammatory states that may accompany mucosal injury, we evaluated the effects of cyclic mechanical strain and fibronectin on intestinal epithelial monolayer wound closure in Caco-2 and IEC-6 intestinal epithelial cells. Methods: Wounds created in intestinal epithelial monolayers were subjected to cyclic deformation. Wound closure was assessed by morphometry using microscopic imaging. Cell signals were assessed by Western blot and confocal microscopy. Results: Mechanical strain stimulated wound closure on fibronectin but inhibited closure on collagen in Caco-2 and IEC-6 cells. The effect was independent of proliferation or cell spreading. Myosin light chain (MLC) and extracellular signal–regulated kinase (ERK) were phosphorylated in response to strain in confluent monolayers on both collagen and fibronectin. Blocking MLC or ERK phosphorylation inhibited the motogenic effect of strain on fibronectin. Although phosphorylated MLC was redistributed to the leading edge of migrating cells following 6 hours of strain on collagen and fibronectin, phosphorylated ERK was redistributed to the lamellipodial edge only on fibronectin. Conclusions: Strain promotes intestinal epithelial wound closure by a pathway requiring ERK and MLC kinase. Fibronectin-dependent ERK redistribution in response to strain in confluent migrating cells may explain the matrix dependence of the motogenic effect. Repetitive deformation stimulates intestinal epithelial proliferation on a collagen substrate, but not fibronectin. Deformation may exert matrix-dependent effects on intestinal epithelial cells, promoting epithelial restitution in fibronectin-rich tissue and proliferation in fibronectin-poor mucosa.
TUCAN (CARD8) Genetic Variants and Inflammatory Bowel Disease
Dermot P.B. McGovern?‡, Helen Butler?, Tariq Ahmad‡, Marta Paolucci?, David A. van Heel§, Kenichi Negoro?, Pirro Hysi?, Jiannis Ragoussis?, Simon P.L. Travis‡, Lon R. Cardon?, Derek P. Jewell‡
Received 6 October 2005; accepted 5 July 2006
Background & Aims: The identification of the association between Crohn’s disease (CD) and NOD2 (CARD15) confirmed both the heritability of CD and highlighted the role of the nuclear factor ?B (NF?B) pathway in disease pathogenesis. Other susceptibility loci exist. TUCAN (CARD8) is located beneath a CD peak of linkage on chromosome 19q. TUCAN is expressed in the gut and is a negative regulator of NF?B, making it an excellent candidate gene for gastrointestinal inflammation. Methods: Ten single nucleotide polymorphisms (SNP) across TUCAN were genotyped in 365 controls, 372 patients with CD, and 373 patients with ulcerative colitis. A diagnostic panel for CD was constructed using smoking status and TUCAN, NOD2, IBD5, NOD1, and TNFSF15 data. Results: We demonstrate significant association between a TUCAN SNP and CD (OR 1.35, P = .0083). The association was more pronounced with disease affecting sites other than the colon (odds ratio, 1.52) and NOD2-negative CD (odds ratio, 1.50). Combination of these data with smoking and NOD2, IBD5, NOD1, and TNFSF15 status demonstrated very strong associations with CD and high sensitivities (96.3%), specificities (99.4%), and likelihood ratios (12.8) for CD, although further work will be needed before this model can be translated into direct clinical utility. Conclusions: We have shown an association between a likely functional polymorphism in TUCAN and CD. The combination of these data in a genetic panel suggests that clinicians may soon be able to translate genetic advances into direct benefits for patients.
CD36 Is Important for Chylomicron Formation and Secretion and May Mediate Cholesterol Uptake in the Proximal Intestine
Andromeda M. Nauli?, Fatiha Nassir‡, Shuqin Zheng?, Qing Yang?, Chun–Min Lo?, Sarah B. VonLehmden?, Dana Lee?, Ronald J. Jandacek?, Nada A. Abumrad‡, Patrick Tso?
Received 7 August 2005; accepted 5 July 2006
Background & Aims: Studies are aimed to determine the role of CD36 in intestinal lipid absorption. Methods: Knock-out (KO) and wild-type (WT) lymph fistula mice were used to study fatty acids (FA) and cholesterol uptake, and chylomicron formation and secretion. Uptake of FA and cholesterol was studied by using sucrose polybehenate and fecal dual isotope methods, respectively. Results: The CD36 KO exhibited significant accumulation of dietary cholesterol in the intestinal lumen at the end of 6-hour lipid infusion and significant reduction of dietary cholesterol transport into the lymph. Fecal dual isotope studies, however, did not show any significant difference in cholesterol uptake, suggesting that given sufficient time, the KO intestine could compensate for the reduced cholesterol uptake observed in the acute lymph fistula studies. Recovery of dietary FA in the intestinal lumen was comparable between WT and KO, consistent with the sucrose polybehenate study. However, the KO mice accumulated more, albeit not significantly, dietary triacylglycerols in the intestine, followed by a significant reduction in lymphatic transport. The ratio of intestinal dietary triacylglycerols to FA was not higher in WT than KO, arguing against impaired lipid esterification. It is rather a deficiency in the formation and secretion of chylomicrons, as supported by the significantly less apolipoprotein B-48 and the smaller, albeit not significantly, lipoprotein particles secreted into the lymph of the KO. Conclusions: CD36 may play an important role in chylomicron formation and secretion and may also facilitate cholesterol uptake in the proximal intestine.
Loss of Raf Kinase Inhibitor Protein Promotes Cell Proliferation and Migration of Human Hepatoma Cells
Han Chu Lee?1, Bo Tian?1, John M. Sedivy‡, Jack R. Wands?, Miran Kim?
Received 2 April 2006; accepted 15 June 2006
Background & Aims: The Raf kinase inhibitor protein (RKIP) has been identified as a suppressor of the mitogen-activated protein kinase (MAPK) pathway. Loss of RKIP function promotes tumor metastasis in prostate cancer and melanoma. The insulin-like growth factor I (IGF-I)–mediated MAPK cascade is often activated in hepatocellular carcinoma (HCC), but the role of RKIP in the molecular pathogenesis of these tumors is unknown. This study was performed to evaluate the role of RKIP in the development of HCC. Methods: The levels of RKIP expression in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry and Western blot analysis. The underlying mechanisms of RKIP were assessed with immunoblot analysis, Raf kinase activity assay, cell proliferation, and migration assays after either overexpression or knockdown of RKIP expression in HCC cell lines. Results: RKIP expression is down-regulated in human HCC compared with adjacent peritumoral tissues. Low RKIP levels were correlated with enhanced extracellular signal–regulated-kinase (ERK)/MAPK pathway activation. Reconstitution experiments antagonized IGF-I–mediated MAPK pathway activation, resulting in reduced nuclear accumulation of phospho-ERK. In contrast, knockdown of RKIP expression using small interfering RNA induced activation of the ERK/MAPK pathway. Ectopic expression of RKIP altered HCC cell proliferation and migration. Conclusions: Our findings indicate that down-regulation of RKIP expression is a major factor in activation of the IGF-I/ERK/MAPK pathway during human hepatocarcinogenesis.
Prickle-1 Negatively Regulates Wnt/?-Catenin Pathway by Promoting Dishevelled Ubiquitination/Degradation in Liver Cancer
David W. Chan, Chung–Yiu Chan, Judy W.P. Yam, Yick–Pang Ching, Irene O.L. Ng
Received 2 December 2005; accepted 5 July 2006
Background & Aims: Aberrant activation of Wnt signaling due to accumulation of ?-catenin has been linked to tumorigenesis. Mutations of ?-catenin, APC, and axins are important but not frequent enough to be accountable for the accumulation of ?-catenin in human hepatocellular carcinoma (HCC). In this study, we characterized the roles of Prickle-1, a Dishevelled (Dvl)-associated protein, in regulation of Wnt/?-catenin activity in HCC. Methods: The expression levels of human Prickle-1 and Dvl3 were examined in HCC cell lines and human HCC samples. The interaction and effects of Prickle-1 on Dvl3, the Wnt/?-catenin pathway, and cell growth were assessed in HCC cell lines. Results: We showed that Prickle-1 bound with Dvl3 and facilitated Dvl3 ubiquitination/degradation, and this was through its destruction box (D-box) motifs. Enforced expression of Prickle-1 significantly reduced the Wnt/?-catenin activity and tumorigenic properties of HCC cells. Clinicopathologic analysis showed that underexpression of Prickle-1 was significantly associated with overexpression of Dvl3, ?-catenin accumulation (P = .023), and larger tumor size (P = .030). Conclusions: Our results have elucidated a novel mechanistic relationship between Prickle-1 and Dvl3 in the Wnt/?-catenin pathway. The facilitation of Prickle-1 on Dvl3 degradation and the suppression of ?-catenin activity and cell growth suggest that Prickle-1 is a negative regulator of the Wnt/?-catenin signaling pathway and is a putative tumor suppressor in human HCCs.
Hyperlipidemic Mice Present Enhanced Catabolism and Higher Mitochondrial ATP-Sensitive K+ Channel Activity
Luciane C. Alberici?, Helena C.F. Oliveira‡, Patrícia R. Patrício‡, Alicia J. Kowaltowski§, Anibal E. Vercesi?
Received 24 February 2006; accepted 21 June 2006
Background & Aims: Changes in mitochondrial energy metabolism promoted by uncoupling proteins (UCPs) are often found in metabolic disorders. We have recently shown that hypertriglyceridemic (HTG) mice present higher mitochondrial resting respiration unrelated to UCPs. Here, we disclose the underlying mechanism and consequences, in tissue and whole body metabolism, of this mitochondrial response to hyperlipidemia. Methods: Oxidative metabolism and its response to mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoKATP) agonists and antagonists were measured in isolated mitochondria, livers, and mice. Results: Mitochondria isolated from the livers of HTG mice presented enhanced respiratory rates compared with those from wild-type mice. Changes in oxygen consumption were sensitive to adenosine triphosphate (ATP), diazoxide, and 5-hydroxydecanoate, indicating they are attributable to mitochondrial ATP-sensitive K+ channel (mitoKATP) activity. Indeed, mitochondria from HTG mice presented enhanced swelling in the presence of K+ ions, sensitive to mitoKATP agonists and antagonists. Furthermore, mitochondrial binding to fluorescent glibenclamide indicates that HTG mice expressed higher quantities of mitoKATP. The higher content and activity of liver mitoKATP resulted in a faster metabolic state, as evidenced by increased liver oxygen consumption and higher body co2 release and temperature in these mice. In agreement with higher metabolic rates, food ingestion was significantly larger in HTG mice, without enhanced weight gain. Conclusions: These results show that primary hyperlipidemia leads to an elevation in liver mitoKATP activity, which may represent a regulated adaptation to oxidize excess fatty acids in HTG mice. Furthermore, our data indicate that mitoKATP, in addition to UCPs, may be involved in the control of energy metabolism and body weight.
Acetaldehyde Inhibits PPAR? via H2O2-Mediated c-Abl Activation in Human Hepatic Stellate Cells
Elisabetta Ceni?‡, David W. Crabb§, Marco Foschi?, Tommaso Mello¶, Mirko Tarocchi?, Valentino Patussi?#, Luca Moraldi??, Renato Moretti??, Stefano Milani?‡, Calogero Surrenti?‡#, Andrea Galli?‡
Received 5 July 2005; accepted 5 July 2006
Background & Aims: Accumulating evidence indicates that acetaldehyde (AcCHO) is one of the main mediators of fibrogenesis in alcoholic liver disease. AcCHO stimulates synthesis of fibrillar collagens in hepatic stellate cells, but the molecular events directly involved in the activation of collagen genes are debatable. Methods: Peroxisome proliferator-activated receptor ? (PPAR?) is a nuclear receptor that is expressed in stellate cells, and its activation by specific ligands inhibits collagen synthesis. In this study, we evaluated the effects of AcCHO on PPAR? transcriptional activity and its correlation with the AcCHO-induced collagen synthesis in hepatic stellate cells. Results: AcCHO treatment inhibited ligand-dependent and -independent PPAR? transcriptional activity, and this effect was correlated with an increased phosphorylation of a mitogen-activated protein kinase site at serine 84 of the human PPAR?. Transfection of the PPAR?Ser84Ala mutant completely prevented the effect of AcCHO on PPAR? activity and in parallel abrogated the induction of collagen gene expression by AcCHO. The effect of AcCHO on PPAR? activity and phosphorylation was blocked by extracellular signal–regulated kinase (ERK) 1/2 and protein kinase C (PKC)? inhibitors as well as by catalase, suggesting that hydrogen peroxide is involved in the molecular cascade responsible for PPAR? phosphorylation via activation of the PKC?/ERK pathway. Furthermore, inhibition of c-Abl completely abrogated the effect of AcCHO on either PPAR? function or collagen synthesis; in addition, expression of the PPAR?Ser84Ala mutant prevented the profibrogenic signals mediated by c-Abl activation. Conclusions: Our results showed that the induction of collagen expression by AcCHO in stellate cells is dependent on PPAR? phosphorylation induced by a hydrogen peroxide–mediated activation of the profibrogenic c-Abl signaling pathway.
Selection of a Multiple Drug-Resistant Hepatitis B Virus Strain in a Liver-Transplanted Patient
Stéphanie Villet?, Christian Pichoud?, Jean-Pierre Villeneuve‡, Christian Trépo?§?, Fabien Zoulim?§?
Received 16 February 2006; accepted 2 June 2006
Background & Aims: Sequential anti-hepatitis B virus (HBV) therapy may lead to the selection of complex mutants. We analyzed the genetic and phenotypic evolution of the viral quasispecies of a patient who received successively lamivudine, add-on adefovir+lamivudine, followed by lamivudine+adefovir+hepatitis B immunoglobulins (HBIg) after orthotopic liver transplantation. Methods: For genotypic analysis, a 1310-bp region of the polymerase gene was amplified, cloned, and sequenced. Huh-7 cells were transfected to compare the replication fitness of HBV mutants and their susceptibility to drugs. Results: At baseline, all HBV genomes carried a wild-type (wt) RT gene but 22% harbored the sP120S and 55% the sC107stop mutations within the surface (S) gene associated with vaccine escape. Following viral breakthrough to lamivudine monotherapy, a complex mixture of lamivudine-resistant HBV strains prevailed. Interestingly, among these mutants emerged a population harboring only the rtL180M+A181V mutations, conferring lamivudine-resistance in vitro. After addition of adefovir to the ongoing treatment, viral load dropped, and the patient underwent an orthotopic liver transplantation and received HBIg. As viral load rose again, a single viral population was progressively selected, harboring the rtV173L+L180M+A181V+N236T and sP120S mutations. In vitro, this last mutant showed a level of replication reduced by only 30% compared to wt HBV and a strong resistance to both lamivudine (>1000-fold) and adefovir (>10-fold). It remained sensitive to tenofovir both in vitro and in vivo. Conclusions: We report the selection of a complex HBV mutant that escaped the antiviral pressure of lamivudine, adefovir, and HBIg, and provide insight on the process of selection via genotypic and phenotypic analysis.
© 2006 American Gastroenterological Association (AGA) Institute. Published by Elsevier Inc. All rights reserved.
Volume 45, Issue 4, Pages 463-632 (October 2006)
"Seuls les abstracts des articles Originaux sont mis sur cette page. Les autres articles sont en ligne en FREE sur le site du Journal"
Original Articles
Viral Hepatitis
PD-L1 is induced in hepatocytes by viral infection and by interferon-? and -? and mediates T cell apoptosis, 16 June 2006
Mühlbauer M, Fleck M, Schütz C, Weiss T, Froh M, Blank C, Schölmerich J, Hellerbrand C
Background/Aims
B7-H1 (PD-L1) is a B7-family member that binds to programmed death-1 (PD-1). Recently, deficiency of PD-L1 has been demonstrated to result in accelerated hepatocyte damage in experimental autoimmune hepatitis, and PD-L1 was suggested to play a critical role in regulating T cell homeostasis. Absence of PD-1 enhanced proliferation of T cells in adenovirus-infected livers and resulted in a rapid clearance of the virus. Here, we aimed to get more insight into hepatic PD-L1 expression, regulation and function.
Methods
PD-L1 expression was analyzed by quantitative PCR and FACS-analysis in primary human liver cells and hepatoma cells. Furthermore, coculture experiments with primary human T cells or Jurkat T cells were established.
Results
In addition to nonparenchymal liver cells, also hepatocytes constitutively expressed low levels of PD-L1. PD-L1 expression in hepatocytes was strongly enhanced by activated T cells and viral infection, and markedly augmented by further stimulation with type I or type II interferons. Moreover, PD-L1 expression on hepatocytes induced apoptosis in T cells.
Conclusions
Our results suggest a novel bidirectional interaction between hepatocytes and lymphocytes modulated by PD-L1 expression in hepatocytes, which may contribute to the unique immunological properties of the liver.
The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide, 23 June 2006
Perz JF, Armstrong GL, Farrington LA, Hutin YJF, Bell BP
pages 529-538
Background/Aims
End-stage liver disease accounts for one in forty deaths worldwide. Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) are well-recognized risk factors for cirrhosis and liver cancer, but estimates of their contributions to worldwide disease burden have been lacking.
Methods
The prevalence of serologic markers of HBV and HCV infections among patients diagnosed with cirrhosis or hepatocellular carcinoma (HCC) was obtained from representative samples of published reports. Attributable fractions of cirrhosis and HCC due to these infections were estimated for 11 WHO-based regions.
Results
Globally, 57% of cirrhosis was attributable to either HBV (30%) or HCV (27%) and 78% of HCC was attributable to HBV (53%) or HCV (25%). Regionally, these infections usually accounted for >50% of HCC and cirrhosis. Applied to 2002 worldwide mortality estimates, these fractions represent 929,000 deaths due to chronic HBV and HCV infections, including 446,000 cirrhosis deaths (HBV: n=235,000; HCV: n=211,000) and 483,000 liver cancer deaths (HBV: n=328,000; HCV: n=155,000).
Conclusions
HBV and HCV infections account for the majority of cirrhosis and primary liver cancer throughout most of the world, highlighting the need for programs to prevent new infections and provide medical management and treatment for those already infected.
Biochemical and histological effects of 26 weeks of glycyrrhizin treatment in chronic hepatitis C: A randomized phase II trial, 29 June 2006
Orlent H, Hansen BE, Willems M, Brouwer JT, Huber R, Kullak-Ublick GA, Gerken G, Zeuzem S, Nevens F, Tielemans WCM, Zondervan PE, Lagging M, Westin J, Schalm SW
pages 539-546
Background/Aims
Phase I/II studies of 4 weeks duration have confirmed the ALT lowering effect of glycyrrhizin in Western chronic hepatitis C patients. Our aim was to determine the dose frequency of glycyrrhizin required to maintain the ALT response beyond 4 weeks and evaluate its effect on liver histology and quality of life.
Methods
HCV-RNA-positive patients with elevated ALT and marked fibrosis or necro-inflammation who were not eligible for interferon therapy were treated for 4 weeks with six infusions weekly of glycyrrhizin. Patients with an ALT response at week 4 were randomized to continue treatment for 22 weeks in three dose frequency groups: 6?, 3? or once weekly.
Results
72/121 (60%) patients were randomized. At the end of treatment the ALT response was maintained in 60%, 24% and 9% of patients in the 6?, 3?, and once weekly groups, respectively (p<0.001). In ALT responders the necro-inflammation score improved non-significantly compared to ALT non-responders. Quality of life assessed by SF-36 increased in patients treated with the study drug, albeit unrelated to the occurrence of ALT response.
Conclusions
ALT responses induced by 4 weeks glycyrrhizin therapy can be maintained in a subset of chronic hepatitis C patients receiving at least three injections weekly. The observed ALT response did not translate in a significant histological improvement after 6 months treatment.
Cultivation of HepG2.2.15 on Cytodex-3: Higher yield of hepatitis B virus and less subviral particles compared to conventional culture methods, 23 June 2006
Lupberger J, Mund A, Kock J, Hildt E
pages 547-552
Background/Aims
Several novel systems are available to study human hepatitis B virus (HBV) replication in cell culture demanding for efficient cell culture based systems for HBV production. The aim was to enhance HBV production of the HBV stably producing cell line HepG2.2.15 by cultivation on spherical micro substrate.
Methods
HepG2.2.15 was cultivated on microcarrier substrate Cytodex-3. HBV specific transcripts, viral protein and genome secretion, cell proliferation and MAP kinase signaling were analyzed. Infectivity of HBV particles was analyzed using primary tupaia hepatocytes.
Results
Compared to stationary flask cultures, HepG2.2.15 on Cytodex-3 secreted 18-fold more HBV genomes, more HBeAg per culture volume and less HBV surface antigen per extracellular viral genome equivalent. This was reflected by a significantly higher infectivity of supernatant derived from carrier grown HepG.2.2.15 cells tested by infection of primary tupaia hepatocytes. The amount of phosphorylated ERK-2 was significantly elevated in cells cultivated on microcarrier.
Conclusions
The cultivation of HepG2.2.15 on Cytodex-3 increased production of infectious HBV particles and decreased secretion of subviral particles compared to the stationary cell cultivation. Microcarrier cultivation activates MAP kinase signaling that is crucial for HBV replication.
Quantification of hepatitis B virus covalently closed circular DNA in patients with hepatocellular carcinoma, 23 June 2006
Wong DKH, Yuen MF, Poon RTP, Yuen JCH, Fung J, Lai CL
Background/Aims
This study aimed to measure the intrahepatic total hepatitis B virus (HBV) DNA and covalently closed circular DNA (cccDNA) levels in tumor and non-tumor tissues in hepatocellular carcinoma (HCC) patients.
Methods
Intrahepatic total HBV DNA and cccDNA were measured in 25 HCC patients (21 hepatitis B surface antigen [HBsAg]-positive and 4 HBsAg-negative) by the Invader® assay.
Results
A low level of intrahepatic HBV DNA was detectable in all HBsAg-negative patients. For HBsAg-positive patients, the intrahepatic total HBV DNA levels in the tumor and non-tumor tissues were comparable (P=0.903). However, the tumor tissues had significantly higher levels of cccDNA (0.35 vs. 0.16copies/cell, P=0.030) and higher proportion of intrahepatic HBV DNA in the form of cccDNA (100% vs. 84%, P=0.004) than the non-tumor tissues. Seventeen out of 21 (81%) tumor tissues had intrahepatic HBV DNA solely in cccDNA form. Analysis of HBV mRNA expression indicated that HBV replication appeared to be lower in the tumor tissues than the non-tumor tissues.
Conclusions
Compared to the non-tumor tissues, the levels of HBV replication in the tumor tissues appeared to be lower, and cccDNA was the predominant form of HBV DNA in the tumor tissues.
Cirrhosis and its Complications
A randomized controlled trial comparing ligation and sclerotherapy as emergency endoscopic treatment added to somatostatin in acute variceal bleeding, 29 June 2006
Villanueva C, Piqueras M, Aracil C, Gómez C, López-Balaguer JM, Gonzalez B, Gallego A, Torras X, Soriano G, Sáinz S, Benito S, Balanzó J
Background/Aims
The currently recommended treatment for acute variceal bleeding is the association of vasoactive drugs and endoscopic therapy. However, which emergency endoscopic treatment combines better with drugs has not been clarified. This study compares the efficacy and safety of variceal ligation and sclerotherapy as emergency endoscopic treatment added to somatostatin.
Methods
Patients admitted with acute gastrointestinal bleeding and with suspected cirrhosis received somatostatin infusion (for 5 days). Endoscopy was performed within 6h and those with esophageal variceal bleeding were randomized to receive either sclerotherapy (N=89) or ligation (N=90).
Results
Therapeutic failure occurred in 21 patients treated with sclerotherapy (24%) and in nine treated with ligation (10%) (RR=2.4, 95% CI=1.1–4.9). Failure to control bleeding occurred in 15% vs 4%, respectively (P=0.02). Treatment group, shock and HVPG >16mmHg were independent predictors of failure. Side-effects occurred in 28% of patients receiving sclerotherapy vs 14% with ligation (RR=1.9, 95% CI=1.1–3.5), being serious in 13% vs 4% (P=0.04). Six-week survival probability without therapeutic failure was better with ligation (P=0.01).
Conclusions
The use of variceal ligation instead of sclerotherapy as emergency endoscopic therapy added to somatostatin for the treatment of acute variceal bleeding significantly improves the efficacy and safety.
Liver Failure, Growth and Cancer
Diagnostic benefit of biliary brush cytology in cholangiocarcinoma in primary sclerosing cholangitis, 21 June 2006
Boberg KM, Jebsen P, Clausen OP, Foss A, Aabakken L, Schrumpf E
pages 568-574
Background/Aims
Primary sclerosing cholangitis (PSC) is associated with a high risk of cholangiocarcinoma development. Efforts should be made to detect early neoplastic changes that can be radically treated by liver transplantation.
Methods
In this prospective case series we obtained brush cytology specimens from bile duct strictures in 61 consecutive PSC patients. The cytological classifications were compared with histopathological findings in bile ducts from explanted livers or clinical outcome.
Results
Among patients with cytological low-grade (n=9; 15%) or high-grade dysplasia/adenocarcinoma (n=13; 21%), 8 (36%) proved to have cholangiocarcinoma and 7 (32%) to have high-grade dysplasia (i.e. cholangiocarcinoma in situ) in bile ducts from explanted livers. The sensitivity, specificity, positive- and negative predictive values, and accuracy of brush cytology in diagnosis of biliary malignancy were 100%, 84%, 68%, 100%, and 88% for the combination of low-grade and high-grade dysplasia/adenocarcinoma and 73%, 95%, 85%, 91%, and 90% for high-grade dysplasia/adenocarcinoma only. All patients with high-grade biliary epithelial dysplasia in explanted bile ducts were tumour free at follow-up.
Conclusions
Brush cytology from bile duct strictures in PSC patients can detect cholangiocarcinoma in situ. Patients with cytological low-grade and high-grade dysplasia/adenocarcinoma are currently referred for liver transplantation in our hospital.
Cholestasis and Autoimmune Liver Disease
Autoimmune hepatitis (AIH) in the elderly: A systematic retrospective analysis of a large group of consecutive patients with definite AIH followed at a tertiary referral centre, 16 May 2006
Al-Chalabi T, Boccato S, Portmann BC, McFarlane IG, Heneghan MA
pages 575-583
Background/Aims
A few reports have suggested that AIH may be less severe in the elderly and may be underdiagnosed, but there is a paucity of data.
Methods
We have undertaken a systematic analysis of 164 consecutive patients (36 males, 128 females) with definite AIH (median score 23, range 18–28) attending our clinics, comparing those presenting at age >60 years (Group 1, n=43) with those presenting at <60 years (Group 2, n=121).
Results
Median (range) duration of follow-up was 9 years (1–28) in Group 1 and 14 years (1–33) in Group 2. Median ages (ranges) at presentation were: Group 1=65 (60–79) and Group 2=41 (6–59). Group 1 patients had a significantly increased incidence of ascites at presentation (p<0.001) and a lower incidence of relapse (42% vs. 70%, p=0.002), but there were no significant differences between the groups with respect to mode of onset (acute, insidious, asymptomatic), other clinical signs at presentation, biochemical parameters, types or titres of autoantibodies, incidence of histological cirrhosis, response to therapy or related side effects. There were also no significant differences in liver-related deaths or transplantation, or the frequencies of HLA DR3 or DR4 – although there was an increased frequency of the A1-B8-DR3/4 haplotype in Group 2 (40% vs. 22%, p=0.138).
Conclusions
These findings suggest that AIH often presents in older patients, who frequently have severe disease. Active management in these patients can lead to a normal life expectancy.
Utility of thiopurine methyltransferase genotyping and phenotyping, and measurement of azathioprine metabolites in the management of patients with autoimmune hepatitis, 23 June 2006
Heneghan MA, Allan ML, Bornstein JD, Muir AJ, Tendler DA
pages 584-591
Background/Aims
Azathioprine is a key drug in the management of autoimmune hepatitis (AIH), with effects mediated via conversion to 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP), the latter controlled by thiopurine methyltransferase (TPMT). Our aims were to evaluate the role of TPMT genotyping and phenotyping and to examine 6-TG and 6-MMP metabolite levels in patients with AIH.
Methods
TPMT genotyping and phenotyping was performed on 86 patients with AIH, and metabolites evaluated in assessable patients.
Results
Eighty-six patients with AIH received azathioprine; 22 developed toxicity and 4/22 were heterozygous for TPMT alleles. Cirrhosis was more common amongst patients who developed toxicity (12/22 (54.5%) versus 19/64 (29.6%), P=0.043). Patients who required persistent prednisone at equivalent azathioprine doses had a higher mean fibrosis stage (P=0.044). TPMT activity, but not metabolites, was lower in patients with stage III/IV fibrosis versus stage I/II fibrosis (30±1.92 versus 35.2±1.93, P=0.044). Azathioprine dose significantly correlated with measured 6-TG levels (r=0.409, P<0.0001) and 6-MMP levels (r=0.387, P<0.001).
Conclusions
Advanced fibrosis but not TPMT genotype or activity predicts azathioprine toxicity in AIH. Overlap in 6-TG and 6-MMP metabolite levels is noted whether or not steroid therapy is used to maintain remission.
Genetic and Metabolic Liver Disease
Diagnostic and predictive factors of significant liver fibrosis and minimal lesions in patients with persistent unexplained elevated transaminases. A prospective multicenter study, 16 June 2006
de Lédinghen V, Ratziu V, Causse X, Bail BL, Capron D, Renou C, Pilette C, Oules V, Gelsi E, Oberti F, Vallet-Pichard A, Provost NL, Cadranel JF, Association Française pour l’Etude du Foie (Groupe Epidémiologie et Evaluation), Association Nationale des Gastroentérologues des Hôpitaux généraux de France
pages 592-599
Background/Aims
In patients with unexplained elevated transaminases, prognosis of the liver disease and factors associated with increased risk of liver fibrosis and normal/subnormal liver are unknown. The aim of this prospective study was to identify diagnosis and clinical and biological factors associated with significant (bridging) fibrosis and minimal lesions of the liver in patients with persistent unexplained elevated ALT levels.
Methods
From July 2002 through October 2004, all consecutive asymptomatic patients with unexplained chronically elevated ALT levels were included. All patients had clinical, biological, ultrasonographic examination and a liver biopsy.
Results
272 patients (60.3% males, mean age 46.4 years, BMI 26.7) were included. Pathological findings were: minimal lesions (18.7%), steatosis (26.8%), NASH (32.7%), and miscellaneous (21.7%). Significant fibrosis was found in 27.4% of cases, including 9 cases of cirrhosis. By multivariate analysis, independent predictors of significant fibrosis were tobacco use (OR 2.5, 95% CI 1.34–4.74 p=0.04), BMI>25 (2.49, 1.31–4.73 p=0.005) and diabetes (4.41, 1.73–11.29 p=0.002). Independent factors associated with minimal lesions were female gender (OR 3.4 95% CI 1.73–6.75 p<0.0001) and BMI<25 (3.55, 1.8–6.98, p<0.0001).
Conclusions
In patients with unexplained chronically elevated transaminases, significant fibrosis is statistically associated with tobacco use, BMI>25 and diabetes, and minimal lesions are significantly associated with female gender and BMI<25.
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