HEPATOLOGY
Volume 44, Issue 6, December 2006
Liver Biology and Pathobiology
Pathobiology
Monocrotaline promotes transplanted cell engraftment and advances liver repopulation in rats via liver conditioning (p 1411-1420)
Brigid Joseph, Vinay Kumaran, Ekaterine Berishvili, Kuldeep K. Bhargava, Christopher J. Palestro, Sanjeev Gupta
Disruption of the hepatic endothelial barrier or Kupffer cell function facilitates transplanted cell engraftment in the liver. To determine whether these mechanisms could be activated simultaneously, we studied the effects of monocrotaline, a pyrollizidine alkaloid, with reported toxicity in liver sinusoidal endothelial cells and Kupffer cells. The effects of monocrotaline in Fischer 344 rats were examined by tissue morphology, serum hyaluronic acid levels, and liver tests (endothelial and hepatocyte injury) or incorporation of carbon and 99mTc-sulfur colloid (Kupffer cell damage). To study changes in cell engraftment and liver repopulation, Fischer 344 rat hepatocytes were transplanted into syngeneic dipeptidyl peptidase IV-deficient rats followed by histological assays. We observed extensive endothelial injury without Kupffer cell or hepatocyte damage in monocrotaline-treated rats. Monocrotaline enhanced transplanted cell engraftment without changes in transplanted cell numbers or induction of proliferation in native hepatocytes over 3 months. In monocrotaline-treated rats, transplanted cells integrated into the liver parenchyma and survived in vascular spaces. To determine whether native hepatocytes suffered inapparent damage after monocrotaline, we introduced further liver injury with carbon tetrachloride subsequent to cell transplantation. Monocrotaline sensitized the liver to carbon tetrachloride-induced necrosis, which advanced transplanted cell proliferation, leading to significant liver repopulation. During this process, we observed proliferation of bile duct cells and small epithelial cells, although transplanted hepatocytes did not appear to reconstitute bile ducts. The studies showed that perturbation of multiple liver cell compartments by monocrotaline promoted transplanted cell engraftment and proliferation. In conclusion, development of drugs with monocrotaline-like effects will help advance liver cell therapy.
Role of neutrophils in a mouse model of halothane-induced liver injury (p 1421-1431)
Qiang You, Linling Cheng, Timothy P. Reilly, Dale Wegmann, Cynthia Ju
Drug-induced liver injury (DILI) is a major safety concern in drug development. Its prediction and prevention have been hindered by limited knowledge of the underlying mechanisms, in part the result of a lack of animal models. We developed a mouse model of halothane-induced liver injury and characterized the mechanisms accounting for tissue damage. Female and male Balb/c, DBA/1, and C57BL/6J mice were injected intraperitoneally with halothane. Serum levels of alanine aminotransferase and histology were evaluated to determine liver injury. Balb/c mice were found to be the most susceptible strain, followed by DBA/1, with no significant hepatotoxicity observed in C57BL/6J mice. Female Balb/c and DBA/1 mice developed more severe liver damage compared with their male counterparts. Bioactivation of halothane occurred similarly in all three strains based on detection of liver proteins adducted by the reactive metabolite. Mechanistic investigations revealed that hepatic message levels of tumor necrosis factor- (TNF-), interleukin-1 (IL-1); IL-6, and IL-8 were significantly higher in halothane-treated Balb/c mice compared to DBA/1 and C57BL/6J mice. Moreover, a higher number of neutrophils were recruited into the liver of Balb/c mice upon halothane treatment compared with DBA/1, with no obvious neutrophil infiltration detected in C57BL/6J mice. Neutrophil depletion experiments demonstrated a crucial role for these cells in the development of halothane-induced liver injury. The halothane-initiated hepatotoxicity and innate immune response-mediated escalation of tissue damage are consistent with events that occur in many cases of DILI. In conclusion, our model provides a platform for elucidating strain-based and gender-based susceptibility factors in DILI development.
JunD is a profibrogenic transcription factor regulated by Jun N-terminal kinase-independent phosphorylation (p 1432-1440)
David E. Smart, Karen Green, Fiona Oakley, Jonathan B. Weitzman, Moshe Yaniv, Gary Reynolds, Jelena Mann, Harry Millward-Sadler, Derek A. Mann
JunD is implicated in the regulation of hepatic stellate cell (HSC) activation and liver fibrosis via its transcriptional regulation of the tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. In the present study we found in vivo evidence of a role for JunD in fibrogenesis. Expression of JunD was demonstrated in alpha-SMA-positive activated HSCs of fibrotic rodents and human livers. The junD-/- mice were protected from carbon tetrachloride-induced fibrosis. The livers of injured junD-/- mice displayed significantly reduced formation of fibrotic crosslinked collagen and a smaller number of alpha-SMA-positive HSCs compared with those of wild-type (wt) mice. Hepatic TIMP-1 mRNA expression in injured junD-/- mice was 78% lower and in culture activated junD-/- HSCs was 50%-80% lower than that in wt mice. In examining the signal transduction mechanisms that regulate JunD-dependent TIMP-1 expression, we found a role for phosphorylation of the Ser100 residue of JunD but ruled out JNK as a mediator of this event, suggesting ERK1/2 is utilized. In conclusion, a signaling pathway for the development of fibrosis involves the regulation of TIMP-1 expression by phosphorylated JunD.
STAT1 inhibits liver fibrosis in mice by inhibiting stellate cell proliferation and stimulating NK cell cytotoxicity (p 1441-1451)
Won-Il Jeong, Ogyi Park, Svetlana Radaeva, Bin Gao
Liver fibrosis, a common scarring response to chronic liver injury, is a precursor to cirrhosis and liver cancer. Here, we identified signal transducer and activator of transcription 1 (STAT1) as an important negative regulator in liver fibrosis. Our findings show that disruption of the STAT1 gene accelerated liver fibrosis and hepatic stellate cell (HSC) proliferation in an in vivo model of carbon tetrachloride (CCl4)-induced liver fibrosis. In vitro treatment with IFN- inhibited proliferation and activation of wild-type HSCs, but not STAT1-/- HSCs. Moreover, compared to wild-type cells, cellular proliferation stimulated by serum or platelet-derived growth factor (PDGF) was enhanced and accelerated in STAT1-/- HSCs, which was partially mediated via elevated PDGF receptor expression on such cells. Polyinosinic-polycytidylic acid (poly I:C) or IFN- treatment inhibited liver fibrosis in wild-type mice but not in STAT1-/- mice. Induction of NK cell killing of activated HSCs by poly I:C was attenuated in STAT1-/- mice compared to wild-type mice, which was likely due to reduced NKG2D and TRAIL expression on STAT1-/- NK cells. Finally, activation of TGF-/Smad3 signaling pathway was accelerated, whereas induction of Smad7 was diminished in the liver of STAT1-/- mice after CCl4 administration compared to wild-type mice. In conclusion, activation of STAT1 attenuates liver fibrosis through inhibition of HSC proliferation, attenuation of TGF- signaling, and stimulation of NK cell killing of activated HSCs. STAT1 could be a new therapeutic target for treating liver fibrosis.
Reg2 inactivation increases sensitivity to Fas hepatotoxicity and delays liver regeneration post-hepatectomy in mice (p 1452-1464)
Hanh-Tu Lieu, Marie-Thérèse Simon, Thao Nguyen-Khoa, Messeret Kebede, Alexandre Cortes, Luis Tebar, Andrew J. H. Smith, Rosemary Bayne, Stephen P. Hunt, Christian Bréchot, Laurence Christa
Reg2/RegIII is the murine homologue of the human secreted HIP/PAP C-type lectin. HIP/PAP transgenic mice were protected against acetaminophen-induced acute liver failure and were stimulated to regenerate post-hepatectomy. To assess the role of Reg2, we used Reg2-/- mice in a model of fulminant hepatitis induced by Fas and in the post-hepatectomy regeneration. Within 4 hours of J0-2 treatment (0.5 g/g), only 50% of the Reg2-/- mice were alive but with an increased sensitivity to Fas-induced oxidative stress and a decreased level of Bcl-xL. In contrast, HIP/PAP transgenic mice were resistant to Fas, with HIP/PAP serving as a sulfhydryl buffer to slow down decreases in glutathione and Bcl-xL. In Reg2-/- mice, liver regeneration was markedly impaired, with 29% mortality and delay of the S-phase and the activation of ERK1/2 and AKT. Activation of STAT3 began on time at 3 hours but persisted strongly up to 72 hours despite significant accumulation of SOCS3. Thus, Reg2 deficiency induced exaggerated IL-6/STAT-3 activation and mito-inhibition. Because the Reg2 gene was activated between 6 and 24 hours after hepatectomy in wild-type mice, Reg2 could mediate the TNF-/IL-6 priming signaling by exerting a negative feed-back on STAT3/IL-6 activation to allow the hepatocytes to progress through the cell cycle. In conclusion, Reg2 deficiency enhanced liver sensitivity to Fas-induced oxidative stress and delayed liver regeneration with persistent TNF-/IL6/STAT3 signaling. In contrast, overexpression of human HIP/PAP promoted liver resistance to Fas and accelerated liver regeneration with early activation/deactivation of STAT3. Reg2/HIP/PAP is therefore a critical mitogenic and antiapoptotic factor for the liver.
Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma (p 1465-1477)
Boris Blechacz, Patrick L. Splinter, Suzanne Greiner, Rae Myers, Kah-Whye Peng, Mark J. Federspiel, Stephen J. Russell, Nicholas F. LaRusso
The oncolytic measles virus Edmonston strain (MV-Edm), a nonpathogenic virus targeting cells expressing abundant CD46, selectively destroys neoplastic tissue. Clinical development of MV-Edm would benefit from noninvasive monitoring strategies to determine the speed and extent of the spread of the virus in treated patients and the location of virus-infected cells. We evaluated recombinant MV-Edm expressing carcinoembryonic antigen (CEA) or the human sodium iodide symporter (hNIS) for oncolytic potential in hepatocellular carcinoma (HCC) and efficiency in tracking viruses in vivo by noninvasive monitoring. CD46 expression in human HCC and primary hepatocytes was assessed by flow cytometry and immunohistochemistry. Infectivity, syncytium formation, and cytotoxicity of recombinant MV-Edm in HCC cell lines were evaluated by fluorescence microscopy, crystal violet staining, and the MTS assay. Transgene expression in HCC cell lines after infection with recombinant MV-Edm in vitro and in vivo was assessed by CEA concentration, 125I-uptake, and 123I-imaging studies. Toxicology studies were performed in IfnarKO?CD46 transgenic mice. The CD46 receptor was highly expressed in HCC compared to nonmalignant hepatic tissue. Recombinant MV-Edm efficiently infected HCC cell lines, resulting in extensive syncytium formation followed by cell death. Transduction of HCC cell lines and subcutaneous HCC xenografts with recombinant MV-Edm resulted in high-level expression of transgenes in vitro and in vivo. MV-Edm was nontoxic in susceptible mice. Intratumoral and intravenous therapy with recombinant MV-Edm resulted in inhibition of tumor growth and prolongation of survival with complete tumor regression in up to one third of animals. In conclusion, engineered MV-Edm may be a potent and novel cancer gene therapy system for HCC. MV-Edm expressing CEA or hNIS elicited oncolytic effects in human HCC cell lines in vitro and in vivo, enabling the spread of the virus to be monitored in a noninvasive manner.
Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver (p 1478-1486)
Jeonghoon Heo, Valentina M. Factor, Tania Uren, Yasushi Takahama, Ju-Seog Lee, Marian Major, Stephen M. Feinstone, Snorri S. Thorgeirsson
We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to beating myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl4-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy.
Oxidative-stress and IL-6 mediate the fibrogenic effects of rodent Kupffer cells on stellate cells (p 1487-1501)
Natalia Nieto
The impact of Kupffer cells (KCs) on the hepatic stellate cell (HSC) fibrogenic response was examined in an in vitro coculture model of primary KCs and HSCs. Coculture with KCs induced a more activated phenotype and greater proliferation compared to HSC cultured alone. Similar results were obtained on Matrigel which maintains HSCs quiescent. The effect of KCs on HSC collagen I involved transcriptional regulation, as determined by nuclear in vitro transcription run-on assays, promoter studies, and Northern blot analysis, while stability of the COL1A1 and COL1A2 mRNA were similar. The minimal COL1A1 and COL1A2 promoter regions responsible for the KC effects were localized to the -515 and -378 base pair (bp) regions, respectively. Intracellular and extracellular collagen I protein, H2O2, and IL-6 increased in a time-dependent fashion, especially for HSCs in coculture. Catalase prevented these effects as well as the transactivation of both collagen promoters. The rate of collagen I protein synthesis and intracellular collagen I degradation remained similar but the t1/2 of the secreted collagen I was lower for HSC in coculture. MMP13, a protease that degrades extracellular collagen I, decreased in the cocultures, while TIMP1, a MMP13 inhibitor, increased; and these effects were prevented by catalase, anti-IL-6, and siRNA-IL-6. Cocultured HSC showed elevated phosphorylation of p38 which when inhibited by catalase, anti-IL-6, and siRNA-IL-6 it blocked TIMP1 upregulation and collagen I accumulation. In conclusion, these results unveil a novel dual mechanism mediated by H2O2 and IL-6 by which KCs may modulate the fibrogenic response in HSCs.
Liver Failure and Liver Disease
Liver Failure
Portopulmonary hypertension: Results from a 10-year screening algorithm (p 1502-1510)
Michael J. Krowka, Karen L. Swanson, Robert P. Frantz, Michael D. McGoon, Russell H. Wiesner
Portopulmonary hypertension (POPH) is the elevation of pulmonary artery pressure due to increased resistance to pulmonary blood flow in the setting of portal hypertension. Increased mortality has occurred with attempted liver transplantation in such patients and thus, screening for POPH is advised. We examined the relationship between screening echocardiography and right heart catheterization determinations of pressure, flow, volume, and resistance. A prospective, echocardiography-catheterization algorithm was followed from 1996 to 2005. Consecutive transplantation candidates underwent Doppler echocardiography to determine right ventricular systolic pressure (RVSP). Of 1,235 patients, 101 with RVSP >50 mm Hg underwent catheterization to measure mean pulmonary artery pressure (MPAP), flow via cardiac output (CO), central volume via pulmonary artery occlusion pressure (PAOP), and resistance via calculated pulmonary vascular resistance (PVR). Bland-Altman analysis suggested marked discordance between echocardiography-derived RVSP and catheterization results. All-cause pulmonary hypertension (MPAP >25 mm Hg) was documented in 90/101 (90%) patients. Using current pressure and resistance diagnostic guidelines (MPAP >25 mm Hg, PVR 240 dynes/s/cm-5), POPH was documented in 66/101 (65%) patients. Elevated MPAP was due to increased CO and/or PAOP in 35/101 (35%) patients with normal resistance (PVR <240 dynes/s/cm-5). The transpulmonary gradient (MPAP-PAOP) further characterized POPH in the presence of increased volume. Model for end stage liver disease (MELD) scores correlated poorly with MPAP and PVR. In conclusion, right heart catheterization is necessary to confirm POPH and frequently identifies other reasons for pulmonary hypertension (e.g., high flow and increased central volume) in liver transplantation candidates. Severity of POPH correlates poorly with MELD scores.
Accuracy of liver stiffness measurement for the diagnosis of cirrhosis in patients with chronic liver diseases (p 1511-1517)
Nathalie Ganne-Carrié, Marianne Ziol, Victor de Ledinghen, Catherine Douvin, Patrick Marcellin, Laurent Castera, Daniel Dhumeaux, Jean-Claude Trinchet, Michel Beaugrand
A proper diagnosis of cirrhosis is essential for the management of patients with chronic liver diseases. We assessed the accuracy of liver stiffness measurement by Fibroscan for the diagnosis of cirrhosis in 1,257 patients with chronic liver diseases of various causes enrolled in a prospective multicenter study as well as clarified causes of discrepancies between liver histology and Fibroscan. One hundred thirty-two patients had unsuitable biopsy specimens, and 118 had unreliable liver stiffness measurements. Because 232 patients overlapped with a previous study, analysis was performed in the 775 new patients then derived in the whole population (1,007; 165 cirrhosis). Diagnostic accuracy was assessed by receiver operator curve (ROC) analysis. Liver samples were re-analyzed in case of discrepancies. The area under the ROC (AUROC) was 0.95 (95% CI, 0.93-0.96) for the diagnosis of cirrhosis in either 775 or 1,007 patients. The cutoff value with optimal diagnosis accuracy was 14.6 kPa in 1,007 patients (positive and negative predictive values, 74% and 96%) with discrepancies among the etiological groups. Eighty patients were misclassified: (1) among 45 patients without cirrhosis with liver stiffness 14.6 kPa or greater, 27 (60%) had extensive fibrosis and 10 (22%) significant perisinusoidal fibrosis; and (2) among 35 patients with cirrhosis and liver stiffness less than 14.6 kPa, 10 (29%) had a macronodular pattern and 25 (71%) either none or mild activity. In conclusion, Fibroscan is a reliable method for the diagnosis of cirrhosis in patients with chronic liver diseases, better at excluding than at predicting cirrhosis using a threshold of 14.6 kPa. False-negatives are mainly attributable to inactive or macronodular cirrhosis.
Prediction of recurrence of hepatocellular carcinoma after curative ablation using three tumor markers (p 1518-1527)
Ryosuke Tateishi, Shuichiro Shiina, Haruhiko Yoshida, Takuma Teratani, Shuntaro Obi, Noriyo Yamashiki, Hideo Yoshida, Masatoshi Akamatsu, Takao Kawabe, Masao Omata
Three tumor markers for hepatocellular carcinoma (HCC) are available in daily practice in Japan: alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin (DCP), and lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3). To elucidate the predictability of these tumor markers on HCC recurrence after curative ablation, we enrolled 416 consecutive patients with naïve HCC who had been treated by percutaneous ablation at our department from July 1997 to December 2002. Tumor marker levels were determined immediately before and 2 months after the treatment. Complete ablation was defined on CT findings as nonenhancement in the entire lesion with a safety margin. Tumor recurrence was also defined as newly developed lesions on CT that showed hyperattenuation in the arterial phase with washout in the late phase. We assessed the predictability of recurrence via tumor markers in multivariate analysis, using proportional hazard regression after adjusting for other significant factors in univariate analysis. Until the end of follow-up, tumor recurrence was identified in 277 patients. Univariate analysis revealed the following factors to be significant for recurrence: platelet count; size and number of tumors; AFP, AFP-L3, and DCP preablation; and AFP and AFP-L3 postablation. Multivariate analysis indicated that AFP >100 ng/mL and AFP-L3 >15%, both pre- and postablation, were significant predictors. The positivity of AFP and AFP-L3 preablation that turned negative postablation was not significant. In conclusion, tumor markers pre- and post-ablation were significant predictors for HCC recurrence and can complement imaging modalities in the evaluation of treatment efficacy.
Role of the JAK2 mutation in the diagnosis of chronic myeloproliferative disorders in splanchnic vein thrombosis (p 1528-1534)
Massimo Primignani, Giovanni Barosi, Gaetano Bergamaschi, Umberto Gianelli, Federica Fabris, Raffaella Reati, Alessandra Dell'Era, Paolo Bucciarelli, Pier Mannuccio Mannucci
The diagnosis of an underlying chronic myeloproliferative disorder (CMPD) is often problematic in patients with primary extrahepatic portal vein obstruction (EHPVO) or Budd-Chiari syndrome (BCS); indeed, conventional clinical and hematological parameters usually yield insufficient information. To assess the diagnostic contribution of the gain-of-function mutation V617F of the JAK2 gene, 93 patients with EHPVO or BCS were investigated. JAK2 V617F was identified in 35.6% of 73 patients with EHPVO and in 40% of 20 patients with BCS. Taking the JAK2 mutation as a test with the highest positive predictive value for the diagnosis of CMPD, conventional clinical-hematological parameters had a sensitivity for CMPD lower than 48%. Bone marrow (BM) histology provided a diagnosis of CMPD in 41/74 (55.4%) patients, with a sensitivity of 93.5%. Clonality of hematopoiesis as assessed by granulocyte X-chromosome inactivation was present in 65.1% of 43 informative female patients, with a sensitivity of 86.6%. By resolving the sensitivity bias of the JAK2 mutation with the results of BM histology and clonality assay, CMPD was diagnosed in 53% of patients with EHPVO or BCS. In conclusion, CMPD is the major cause of primary EHPVO or BCS. JAK2 V617F is a very reliable and noninvasive molecular marker for CMPD and should be used as a first test for diagnosis.
Hyponatremia in cirrhosis: Results of a patient population survey (p 1535-1542)
Paolo Angeli, Florence Wong, Hugh Watson, Pere Ginès, CAPPS Investigators
Low serum sodium concentration is an independent predictor of mortality in patients with cirrhosis, but its prevalence and clinical significance is unclear. To evaluate prospectively the prevalence of low serum sodium concentration and the association between serum sodium levels and severity of ascites and complications of cirrhosis, prospective data were collected on 997 consecutive patients from 28 centers in Europe, North and South America, and Asia for a period of 28 days. The prevalence of low serum sodium concentration as defined by a serum sodium concentration 135 mmol/L, 130 mmol/L, 125 mmol/L, and 120 mmol/L was 49.4%, 21.6%, 5.7%, and 1.2%, respectively. The prevalence of low serum sodium levels (<135 mmol/L) was high in both inpatients and outpatients (57% and 40%, respectively). The existence of serum sodium <135 mmol/L was associated with severe ascites, as indicated by high prevalence of refractory ascites, large fluid accumulation rate, frequent use of large-volume paracentesis, and impaired renal function, compared with normal serum sodium levels. Moreover, low serum sodium levels were also associated with greater frequency of hepatic encephalopathy, spontaneous bacterial peritonitis, and hepatorenal syndrome, but not gastrointestinal bleeding. Patients with serum sodium <130 mmol/L had the greatest frequency of these complications, but the frequency was also increased in patients with mild reduction in serum sodium levels (131-135 mmol/L). In conclusion, low serum sodium levels in cirrhosis are associated with severe ascites and high frequency of hepatic encephalopathy, spontaneous bacterial peritonitis, and hepatorenal syndrome.
Prevention of hepatocellular carcinoma recurrence with alpha-interferon after liver resection in HCV cirrhosis (p 1543-1554)
Vincenzo Mazzaferro, Raffaele Romito, Marcello Schiavo, Luigi Mariani, Tiziana Camerini, Sherrie Bhoori, Lorenzo Capussotti, Fulvio Calise, Riccardo Pellicci, Giulio Belli, Alessandro Tagger, Massimo Colombo, Ferruccio Bonino, Pietro Majno, Josep M. Llovet, HCC Italian Task Force
Tumor recurrence after resection of hepatocellular carcinoma (HCC) can occur early (<2 years) or late (>2 years) as metastases or de novo tumors. Interferon (IFN) has the potential for chemoprevention against hepatitis C virus (HCV)-related cirrhosis. A predetermined group of 150 HCV RNA-positive patients undergoing resection of early- to intermediate-stage HCC was stratified into 80 HCV-pure (hepatitis B anticore antibody [anti-HBc]-negative) and 70 mixed HCV+hepatitis B virus (HBV) (anti-HBc-positive) groups, then randomized to IFN- (3 million units 3 times every week for 48 weeks [n = 76]) versus control (n = 74). The primary end point was recurrence-free survival (RFS); secondary end points were disease-specific and overall survival. Intention-to-treat and subgroup analysis on adherent patients were conducted. Treatment effects on early/late recurrences were assessed using multiple Cox regression analysis. No patient experienced life-threatening adverse events. There were 28 adherent patients (37%). After 45 months of median follow-up, overall survival was 58.5%, and no significant difference in RFS was detectable between the two study arms (24.3% vs. 5.8%; P = .49). HCC recurred in 100 patients (48 IFN-treated, 52 controls), with a 50% reduction in late recurrence rate in the treatment arm. HCC multiplicity and vascular invasion were significantly related to recurrence (P = .01 and .0003). After viral status stratification, while no treatment effect was apparent in the mixed HCV+HBV population and on early recurrences (72 events), there was a significant benefit on late recurrences (28 events) in HCV-pure patients adherent to treatment (HR: 0.3; 95% CI: 0.09-0.9; P = .04). In conclusion, IFN does not affect overall prevention of HCC recurrence after resection, but it may reduce late recurrence in HCV-pure patients receiving effective treatment.
Aquaporin-1 and aquaporin-2 urinary excretion in cirrhosis: Relationship with ascites and hepatorenal syndrome (p 1555-1563)
Christina Esteva-Font, Maria E. Baccaro, Patricia Fernández-Llama, Laia Sans, Monica Guevara, Elisabet Ars, Wladimiro Jiménez, Vicente Arroyo, Jose A. Ballarín, Pere Ginès
Several experimental models of cirrhosis have shown dysregulation of renal aquaporins in different phases of liver disease. We investigated the urinary excretion of both aquaporin-1 and aquaporin-2 in patients with cirrhosis at different stages of the disease. Twenty-four-hour urine was collected from 11 healthy volunteers, 13 patients with compensated cirrhosis (without ascites), and 20 patients with decompensated cirrhosis (11 with ascites without renal failure and 9 with hepatorenal syndrome). Aquaporin-1 and aquaporin-2 excretion was analyzed by immunoblotting. Urinary aquaporin-2 excretion was reduced in patients with cirrhosis compared to healthy subjects. A progressive decrease in urinary aquaporin-2 excretion was observed as the severity of cirrhosis increased, from compensated cirrhosis to cirrhosis with ascites and hepatorenal syndrome. Patients with hyponatremia had lower urinary aquaporin-2 excretion than patients without hyponatremia. Vasopressin plasma level did not correlate with aquaporin-2 excretion. There were no differences between healthy subjects and patients with cirrhosis with or without ascites in urinary excretion of aquaporin-1, but urinary aquaporin-1 excretion of those with hepatorenal syndrome was extremely low. In conclusion, patients with cirrhosis appear to exhibit a decreased abundance of renal aquaporin-2 and therefore lower water permeability in the collecting tubules. This may represent an adaptive renal response to sodium retention, with expansion of extracellular fluid volume and dilutional hyponatremia observed in those who have cirrhosis with ascites. Finally, aquaporin-1 does not appear to play a role in the progressive dysregulation of extracellular fluid volume in cirrhosis.
Liver Disease
Smoking and increased severity of hepatic fibrosis in primary biliary cirrhosis: A cross validated retrospective assessment (p 1564-1571)
Claudia O. Zein, Kristi Beatty, Anthony B. Post, Laurie Logan, Sara Debanne, Arthur J. McCullough
An epidemiological association between cigarette smoking and primary biliary cirrhosis (PBC) has been demonstrated. Our aim was to determine the relationship between smoking and severity of liver fibrosis at presentation in patients with PBC. All patients with PBC seen at the three major teaching hospitals of Case Western Reserve University between October 1998 and December 2005 were identified. Data obtained at the time of the first evaluation leading to the PBC diagnosis on 97 patients were collected. The cumulative number of cigarette packs smoked per year (pack-years) was calculated. Advanced histological disease was defined as Ludwig stages 3 or 4. Analyses were performed to determine associations between advanced histological disease, smoking and other variables related to liver fibrosis. Smoking history was more common (P = .0008) in patients with advanced histological disease at presentation compared to those with early disease. Among smokers, mean lifetime tobacco consumption was higher (P = .04) in cases with advanced histological disease at presentation (30 pack-years) compared to cases with early disease (17 pack-years). Logistic regression demonstrated a significant association between a lifetime tobacco consumption of 10 pack-years and advanced histological disease at presentation (OR = 13.3). The association remained significant after adjusting for age, gender, and alcohol intake. The validity of these results was corroborated by cross-validation in an independent confirmatory set of 172 patients with PBC. In conclusion, smoking may accelerate the progression of PBC. This could be induced by exposure to chemicals in cigarette smoke.
Dynamic FDG-PET is useful for detection of cholangiocarcinoma in patients with PSC listed for liver transplantation (p 1572-1580)
Hanne Prytz, Susanne Keiding, Einar Björnsson, Ulrika Broomé, Sven Almer, Maria Castedal, Ole Lajord Munk, The Swedish Internal Medicine Liver Club
Five to 15% of patients with primary sclerosing cholangitis (PSC) develop cholangiocarcinoma (CC) with a median survival of 5 to 7 months, an outcome not significantly improved by liver transplantation. However, if CC is found incidentally during the procedure or in the explanted liver, 5-year survival rates of 35% are reported. A noninvasive method to detect CC small enough to allow for intended curative surgery is needed. Unfortunately, computed tomography (CT) and ultrasonography (US) have poor sensitivity for detection of CC in PSC; however, positron emission tomography (PET) using 2-[18F]fluoro-2-deoxy-D-glucose (FDG) differentiates well between CC and nonmalignant tissue. We examined whether PET findings are valid using a blinded study design comparing pretransplantation FDG-PET results with histology of explanted livers. Dynamic FDG-PET was performed in 24 consecutive patients with PSC within 2 weeks after listing for liver transplantation and with no evidence of malignancy on CT, magnetic resonance imaging, or ultrasonography. The PET Center staff was blinded to clinical findings, and surgeons and pathologists were blinded to the PET results. Three patients had CC that was correctly identified by PET. PET was negative in 1 patient with high-grade hilar duct dysplasia. In 20 patients without malignancies, PET was false positive in 1 patient with epitheloid granulomas in the liver. In conclusion, dynamic FDG-PET appears superior to conventional imaging techniques for both detection and exclusion of CC in advanced PSC. FDG-PET may be useful for screening for CC in the pretransplant evaluation of patients with PSC.
Outcome of acute idiosyncratic drug-induced liver injury: Long-term follow-up in a hepatotoxicity registry (p 1581-1588)
Raúl J. Andrade, M. Isabel Lucena, Neil Kaplowitz, Beatriz García-Muoz, Yolanda Borraz, Ketevan Pachkoria, Miren García-Cortés, M. Carmen Fernández, Gloria Pelaez, Luis Rodrigo, José A. Durán, Joan Costa, Ramón Planas, Anabel Barriocanal, Carlos Guarner, Manuel Romero-Gomez, Teresa Muoz-Yagüe, Javier Salmerón, Ramón Hidalgo
A chronic adverse reaction may occur in some instances of drug-induced liver injury (DILI), even despite drug cessation. In our study, we obtained records from a Spanish registry and evaluated cases of DILI with biochemical evidence of long-term damage. Chronic outcome was defined as a persistent biochemical abnormality of hepatocellular pattern of damage more than 3 months after drug withdrawal or more than 6 months after cholestatic/mixed damage. Data on 28 patients with a chronic clinical evolution (mean follow-up 20 months) between November 1995 and October 2005 were retrieved (18 female; overall mean age 55 yr) and accounted for 5.7% of total idiosyncratic DILI cases (n = 493) submitted to the registry. The main drug classes were cardiovascular and central nervous system (28.5% and 25%, respectively), which, in contrast, represented only 9.8% and 13%, respectively, of all DILI cases. The most frequent causative drugs were amoxicillin-clavulanate (4 of 69 cases), bentazepam (3 of 7 cases), atorvastatin (2 of 7 cases), and captopril (2 of 5 cases). Patients with cholestatic/mixed injury (18 of 194 cases [9%]) were more prone to chronicity than patients with hepatocellular injury (10 of 240 cases; P < .031). In the case of chronic hepatocellular injury, 3 patients progressed to cirrhosis and 2 to chronic hepatitis. In the cholestatic/mixed group, liver biopsy indicated cirrhosis in 1 patient and ductal lesions in 3 patients. In conclusion, cholestatic/mixed type of damage is more prone to become chronic while, in the hepatocellular pattern, the severity is greater. Cardiovascular and central nervous system drugs are the main groups leading to chronic liver damage.
Viral Hepatitis
Fulminant hepatitis A virus infection in the United States: Incidence, prognosis, and outcomes (p 1589-1597)
Ryan M. Taylor, Timothy Davern, Santiago Munoz, Stephen-Huy Han, Brendan McGuire, Anne M. Larson, Linda Hynan, William M. Lee, Robert J. Fontana, U.S. Acute Liver Failure Study Group
Acute liver failure (ALF) due to hepatitis A virus (HAV) infection is an uncommon but potentially lethal illness. The aim of this study was to identify readily available laboratory and clinical features associated with a poor prognosis among ALF patients with HAV infection. The presenting features of 29 adults with anti-HAV IgM positive ALF enrolled in the ALFSG_between 1998 and 2005 were reviewed. The HAV patients listed for transplantation by UNOS were also reviewed. Acute HAV accounted for 3.1% of patients enrolled in the ALFSG. At 3 weeks follow-up, 16 had spontaneously recovered (55%), 9 underwent transplantation (31%), and 4 had died (14%). A prognostic model incorporating 4 presenting features (serum ALT <2,600 IU/L, creatinine >2.0 mg/dL, intubation, pressors) had an AUROC for transplant/death of 0.899 which was significantly better than the King's College criteria (0.623, P = .018) and MELD scores (0.707, P = .0503). Between 1988 and 2005, the frequency of patients requiring liver transplantation for HAV in the UNOS database significantly decreased from 0.7 % to 0.1% (P < .001). In addition, the proportion of HAV cases enrolled in the ALFSG significantly decreased from 5% to 0.8% (P = .007). In conclusion, the frequency of HAV patients enrolling in the ALFSG and being listed for liver transplantation in the United States has declined in parallel. A prognostic index consisting of 4 clinical and laboratory features predicted the likelihood of transplant/death significantly better than other published models suggesting that disease specific prognostic models may be of value in non-acetaminophen ALF.
Cost-effectiveness of hematologic growth factors for anemia occurring during hepatitis C combination therapy (p 1598-1606)
Richard A. Del Rio, Anthony B. Post, Mendel E. Singer
In hepatitis C virus (HCV)-infected patients who develop anemia during combination therapy, erythropoietic growth factors maintain higher drug treatment levels compared to ribavirin dose reduction, which may lead to an increase in treatment response rates. This study estimated the cost-effectiveness of growth factor therapy in maintaining anemic HCV-infected patients on target drug levels during combination therapy. A decision analysis using a Markov model was developed with 7 health states: Sustained viral response, chronic HCV, compensated cirrhosis, decompensated cirrhosis, hepatocellular carcinoma, liver transplantation, and death. Data sources included population-based studies of growth factor therapy, previously published estimates of costs and natural history of hepatitis C, and recent prospective studies. Our reference case was a 45-year-old Caucasian man with HCV infection (genotype 1, 2, or 3) who developed anemia while undergoing combination therapy with ribavirin and pegylated interferon. We compared growth factor injections (darbepoetin alpha or epoetin alpha) during combination therapy with standard ribavirin dose reduction. Compared to a ribavirin dose reduction strategy, the cost of darbepoetin per additional quality-adjusted life-year was $34,793 for genotype 1 and $33,832 for genotypes 2 or 3 versus $60,600 and $64,311 for epoetin. For all genotypes, the results were sensitive to changes in the cure rates of HCV therapy, the utility of chronic HCV, the costs of growth factors, and the age at which therapy is begun. In conclusion, use of erythropoietic growth factors, specifically darbepoetin, for patients with anemia occurring during HCV combination therapy appears to be cost-effective for genotypes 1, 2, or 3.
Intrahepatic virus-specific IL-10-producing CD8 T cells prevent liver damage during chronic hepatitis C virus infection (p 1607-1616)
Michal Abel, Damien Sène, Stanislas Pol, Marc Bourlière, Thierry Poynard, Frédéric Charlotte, Patrice Cacoub, Sophie Caillat-Zucman
CD8 T cell killing of hepatitis C virus (HCV)-infected hepatocytes is thought to contribute to liver damage during chronic HCV infection, whereas the participation of HCV-nonspecific immune cells is unclear. To visualize the spatial relationship of HCV-specific CD8 T cells with parenchymal target cells, and to examine their local functional activity in relation to hepatocellular necrosis and fibrosis, we used HLA tetramers and confocal microscopy in biopsies from 23 HLA-A2 or HLA-B7 patients with chronic HCV infection. Intrahepatic tetramer+ (HCV-specific) CD8 T cells protected from hepatic necroinflammatory disease activity, independently of age, gender, viral load, and viral genotype. Indeed, tetramer+ cells were scattered in the liver within regions of weak fibrosis (low laminin expression) and low hepatocellular apoptosis (TUNEL method), and expressed IL-10 but not IFN. By contrast, tetramer-negative CD8 T cells were associated with active necroinflammatory liver disease, colocalized with strong laminin expression and hepatocellular apoptosis, and expressed more frequently IFN than IL-10. Overall, liver regions harboring HCV-specific CD8 T cells tended to be healthier than areas containing only inflammatory cells of undefined specificity. In conclusion, HCV-specific IL-10-producing CD8 T cells, although not cytotoxic and unable to control viral replication, can attenuate hepatocellular necrosis, liver fibrosis, and inflammation mediated by bystander T cells, and may thus represent antigen-induced regulatory CD8 T cells. Therapeutic modulation of the intrahepatic balance between specific and bystander CD8 T cells might be beneficial in patients with chronic hepatitis C.
IP-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with HCV genotype 1 infection (p 1617-1625)
Martin Lagging, Ana I. Romero, Johan Westin, Gunnar Norkrans, Amar P. Dhillon, Jean-Michel Pawlotsky, Stefan Zeuzem, Michael von Wagner, Francesco Negro, Solko W. Schalm, Bart L. Haagmans, Carlo Ferrari, Gabriele Missale, Avidan U. Neumann, Elke Verheij-Hart, Kristoffer Hellstrand, DITTO-HCV Study Group
Plasma from 173 patients with HCV genotype 1 infection was analyzed for IP-10 levels prior to treatment with pegylated interferon--2a and ribavirin. Significantly lower IP-10 levels were observed in patients achieving a rapid viral response (RVR) (P < .0001), even in those with body mass index (BMI) 25 kg/m2 (P = .004) and with baseline viral load 2 million IU/mL (P = .001). Similarly, significantly lower IP-10 levels were observed in patients obtaining a sustained viral response (SVR) (P = .0002), including those having higher BMI (P < .05), higher viral load (P = .0005), and both higher BMI and viral load (P < .03). In multivariate logistic regression analyses, a low IP-10 value was independently predictive of both RVR and SVR. A baseline cutoff IP-10 value of 600 pg/mL yielded a negative predictive value (NPV) of 79% (19/24) for all genotype 1-infected patients, which was comparable with that observed using a reduction in HCV-RNA by at least 2 logs after 12 weeks of therapy (NPV 86%; 19/22); by combining the two, 30 of 38 patients (NPV 79%) potentially could have been spared unnecessary therapy. In patients having both higher BMI and viral load, cut-off levels of 150 and 600 pg/mL yielded a positive predictive value (PPV) of 71% and NPV of 100%, respectively. In conclusion, pretreatment IP-10 levels predict RVR and SVR in patients infected with HCV genotype 1, even in those with higher BMI and viral load. A substantial proportion of the latter patients may achieve SVR in spite of unfavorable baseline characteristics if their pretreatment IP-10 level is low. Thus, pretreatment IP-10 analysis may prove helpful in decision-making regarding pharmaceutical intervention.
Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system (p 1626-1634)
Muriel Lavie, Cécile Voisset, Ngoc Vu-Dac, Virginie Zurawski, Gilles Duverlie, Czeslaw Wychowski, Jean Dubuisson
Serum amyloid A (SAA) is an acute phase protein produced by the liver. SAA concentration increases markedly in the serum following inflammation and infection. Large increases in SAA concentration during the acute phase response suggest that SAA has a beneficial role in host defense. This study sought to determine the effect of SAA on hepatitis C virus (HCV) infectivity using retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the recently developed cell culture system for HCV (HCVcc). SAA inhibited HCVpp and HCVcc infection in a dose-dependent manner by affecting an early step of the virus life cycle. Further characterization with HCVpp indicated that SAA blocks virus entry by interacting with the viral particle. In addition, the antiviral activity of SAA was strongly reduced when high-density lipoproteins (HDL) were coincubated with SAA. However, HDL had only a slight effect on the antiviral activity of SAA when HCVpp was first preincubated with SAA. Furthermore, analyses of SAA in sera of chronic HCV patients revealed the presence of variable levels of SAA with abnormally elevated concentrations in some cases. However, no obvious clinical correlation was found between SAA levels and HCV viral loads. In conclusion, our data demonstrate an antiviral activity for SAA and suggest a tight relationship between SAA and HDL in modulating HCV infectivity.
The protein kinase IKK can inhibit HCV expression independently of IFN and its own expression is downregulated in HCV-infected livers (p 1635-1647)
Myriam Vilasco, Esther Larrea, Damien Vitour, Stephanie Dabo, Adrien Breiman, Béatrice Regnault, Jose-Ignacio Riezu, Pierre Eid, Jesus Prieto, Eliane F. Meurs
During a viral infection, binding of viral double-stranded RNAs (dsRNAs) to the cytosolic RNA helicase RIG-1 leads to recruitment of the mitochondria-associated Cardif protein, involved in activation of the IRF3-phosphorylating IKK/TBK1 kinases, interferon (IFN) induction, and development of the innate immune response. The hepatitis C virus (HCV) NS3/4A protease cleaves Cardif and abrogates both IKK/TBK1 activation and IFN induction. By using an HCV replicon model, we previously showed that ectopic overexpression of IKK can inhibit HCV expression. Here, analysis of the IKK transcriptome profile in these HCV replicon cells showed induction of several genes associated with the antiviral action of IFN. Interestingly, IKK still inhibits HCV expression in the presence of neutralizing antibodies to IFN receptors or in the presence of a dominant negative STAT1 mutant. This suggests that good IKK expression levels are important for rapid activation of the cellular antiviral response in HCV-infected cells, in addition to provoking IFN induction. To determine the physiological importance of IKK in HCV infection, we then analyzed its expression levels in liver biopsy specimens from HCV-infected patients. This analysis also included genes of the IFN induction pathway (RIG-I, MDA5, LGP2, Cardif, TBK1), and three IKK-induced genes (IFN-, CCL3, and ISG15). The results show significant inhibition of expression of IKK and of the RNA helicases RIG-I/MDA5/LGP2 in the HCV-infected patients, whereas expression of TBK1 and Cardif was not significantly altered. In conclusion, given the antiviral potential of IKK and of the RNA helicases, these in vivo data strongly support an important role for these genes in the control of HCV infection.
Fibrosis in genotype 3 chronic hepatitis C and nonalcoholic fatty liver disease: Role of insulin resistance and hepatic steatosis (p 1648-1655)
Elisabetta Bugianesi, Gulio Marchesini, Elena Gentilcore, Ian Homer Y. Cua, Ester Vanni, Mario Rizzetto, Jacob George
Hepatic steatosis has been associated with fibrosis, but it is unknown whether the latter is independent of the etiology of fat infiltration. We analyzed the relationship between clinical characteristics, insulin resistance (HOMA-R) and histological parameters in 132 patients with viral steatosis caused by genotype 3 chronic hepatitis C (CHC-3) and 132 patients with metabolic steatosis caused by nonalcoholic fatty liver disease (NAFLD), matched by age, BMI, and degree of liver fat accumulation. Tests of liver function were comparable in the two study populations. The prevalence of features of insulin resistance was higher in NAFLD, as was HOMA-R (P = .008). Logistic regression analysis confirmed that steatosis was associated with a high viral load and low serum cholesterol in CHC-3, and with high aminotransferase, glucose, ferritin and hypertriglyceridemia in NAFLD. At univariate analysis, advanced fibrosis was associated with steatosis in NAFLD, but not in CHC-3. Other parameters related to fibrosis severity were HOMA-R and a low platelet count in CHC-3, and high aminotransferases, HOMA-R, ferritin and low HDL-cholesterol in NAFLD. On multivariate analysis, only low platelet count (OR = 0.78; 95% CI, 0.67-0.92) and HOMA-R (OR = 2.98; 1.13-7.89) were independent predictors of advanced fibrosis in CHC-3. In NAFLD, severe fibrosis was predicted by fat grading (OR = 3.03; 1.41-6.53), ferritin (OR = 1.13; 1.03-1.25) and HOMA-R (OR = 1.16; 1.02-1.31). In conclusion, insulin resistance is an independent predictor of advanced fibrosis in both NAFLD and CHC-3, but the extent of steatosis contributes to advanced disease only in NAFLD. Virus-induced hepatic steatosis as seen in CHC-3 does not contribute significantly to liver fibrosis.
Entecavir resistance is rare in nucleoside naïve patients with hepatitis B (p 1656-1665)
Richard J. Colonno, Ronald Rose, Carl J. Baldick, Steven Levine, Kevin Pokornowski, Cheng F. Yu, Ann Walsh, Jie Fang, Mayla Hsu, Charles Mazzucco, Betsy Eggers, Sharon Zhang, Mary Plym, Kenneth Klesczewski, Daniel J. Tenney
Comprehensive monitoring of genotypic and phenotypic antiviral resistance was performed on 673 entecavir (ETV)-treated nucleoside naïve hepatitis B virus (HBV) patients. ETV reduced HBV DNA levels to undetectable by PCR (<300 copies/mL, <57 IU/mL) in 91% of hepatitis B e antigen (HBeAg)-positive and -negative patients by Week 96. Thirteen percent (n = 88) of the comparator lamivudine (LVD)-treated patients experienced a virologic rebound (1 log increase from nadir by PCR) in the first year, with 74% of these having LVD resistance (LVDr) substitutions evident. In contrast, only 3% (n = 22) of ETV-treated patients exhibited virologic rebound by Week 96. Three ETV rebounds were attributable to LVDr virus present at baseline, with one having a S202G ETV resistance (ETVr) substitution emerge at Week 48. None of the other rebounding patients had emerging genotypic resistance or loss of ETV susceptibility. Genotyping all additional ETV patients with PCR-detectable HBV DNA at Weeks 48, 96, or end of dosing identified seven additional patients with LVDr substitutions, including one with simultaneous emergence of LVDr/ETVr. Generally, ETV patients with LVDr were detectable at baseline (8/10) and most subsequently achieved undetectable HBV DNA levels on ETV therapy (7/10). No other emerging substitutions identified decreased ETV susceptibility. In conclusion, ETVr emergence in ETV-treated nucleoside naïve patients over a 2-year period is rare, occurring in two patients with LVDr variants. These findings suggest that the rapid, sustained suppression of HBV replication, combined with a requirement for multiple substitutions, creates a high genetic barrier to ETVr in nucleoside naïve patients.
Characterization of HBV DNA+/HBsAg- blood donors in Poland identified by triplex NAT (p 1666-1674)
Ewa Brojer, Piotr Grabarczyk, Grzegorz Liszewski, Maria Mikulska, Jean-Pierre Allain, Magdalena Letowska, Polish Blood Transfusion Service Viral Study Group
Nucleic acid testing (NAT) for hepatitis B virus (HBV) has been performed in Poland since 2005 on samples seronegative for hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (anti-HCV), and anti-human immunodeficiency virus (anti-HIV). Tools included 24-donation pool testing (PT) using Cobas Amplicor or in individual donations (ID) by Procleix Ultrio. Seven of 761,666 (1:108,800) and 21/250,191 (1:11,900) HBV DNA-positive donations were identified and confirmed by alternative methods. HBV DNA load ranged between 11.6 and 4.6 ? 104 IU/mL in 11 samples and could not be quantified in 17 samples. HBV genotypes A (56%) and D (4%) were found. The analysis of combined results from index, follow-up, and look-back samples identified four groups: (1) Two cases tested HBsAg positive with alternative, more sensitive, assays; (2) Four cases were in the pre-seroconversion window period; (3) Eight cases had a fluctuating pattern of HBV DNA and anti-HBs detection (recovered infection); and (4) twelve cases carried anti-HBc without anti-HBs, which might correspond to either chronic or recovered occult HBV infection. One donor with no HBV markers in the follow-up was excluded, and another was in the window period preceding anti-HBs. HBV NAT identified more confirmed positive donors than HCV or HIV NAT, and 1:250,000 could not be detected by anti-HBc screening. Serological and molecular studies on follow-up and look-back samples are important to classify donors. In conclusion, further studies are needed to determine whether the considerably higher yield of HBV DNA detection obtained with individual donation screening improves blood safety compared with anti-HBc screening.
Impact of disease severity on outcome of antiviral therapy for chronic hepatitis C: Lessons from the HALT-C trial (p 1675-1684)
Gregory T. Everson, John C. Hoefs, Leonard B. Seeff, Herbert L. Bonkovsky, Deepa Naishadham, Mitchell L. Shiffman, Jeffrey A. Kahn, Anna S. F. Lok, Adrian M. Di Bisceglie, William M. Lee, Jules L. Dienstag, Marc G. Ghany, Chihiro Morishima, HALT-C Trial Group
In patients with chronic hepatitis C, advanced fibrosis and cirrhosis are associated with lower rates of sustained virologic response (SVR) to interferon (IFN)-based therapy. In this study, we assessed virologic response to retreatment with peginterferon alfa-2a and ribavirin (RBV), as a function of the baseline fibrosis score (Ishak staging) and platelet count, in 1,046 patients enrolled in the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial. All patients had failed prior treatment with IFN or peginterferon ± RBV and had Ishak fibrosis scores 3. Four groups of patients with increasingly severe liver disease were compared: (A) bridging fibrosis (Ishak 3 and 4) with platelet counts >125,000/mm3 (n = 559); (B) bridging fibrosis with platelet counts 125,000/mm3 (n = 96); (C) cirrhosis (Ishak 5 and 6) with platelet counts >125,000/mm3 (n = 198); and (D) cirrhosis with platelet counts 125,000/mm3 (n = 193). SVR rates were 23%, 17%, 10%, and 9% in groups A, B, C, and D, respectively (P < .0001 for trend). Reduction in SVR as a function of increasingly severe disease was independent of age, percent African American, HCV genotype, HCV level, and type of prior therapy. Dose reduction lowered SVR frequencies, but to a lesser extent than disease severity. By logistic regression, cirrhosis (P < .0001) was the major determinant that impaired virologic response, independent of dose reduction or platelet count. In conclusion, disease severity is a major independent determinant of rate of SVR in patients with advanced chronic hepatitis C. New strategies are needed to optimize antiviral therapy in these difficult-to-cure patients.
Copyright © 2006 by the American Association for the Study of Liver Diseases. All rights reserved.
GASTROENTEROLOGY
Volume 131, Issue 6, December 2006
Basic - Alimentary Tract
Genetic Variation in Myosin IXB Is Associated With Ulcerative Colitis, 12 September 2006
van Bodegraven AA, Curley CR, Hunt KA, Monsuur AJ, Linskens RK, Onnie CM, Crusius JBA, Annese V, Latiano A, Silverberg MS, Bitton A, Fisher SA, Steinhart AH, Forbes A, Sanderson J, Prescott NJ, Strachan DP, Playford RJ, Mathew CG, Wijmenga C, Daly MJ, Rioux JD, van Heel DA
Background & Aims: Common germline genetic variation in the 3? region of myosin IXB (MYO9B) has been associated recently with susceptibility to celiac disease, with a hypothesis that MYO9B variants might influence intestinal permeability. These findings suggested the current study investigating a possible further role for MYO9B variation in inflammatory bowel disease. Methods: Eight single-nucleotide polymorphisms (SNPs) were selected to tag common haplotypes from the 35-kb 3? region of MYO9B. These included the strongest celiac disease–associated variants reported in a Dutch cohort. These SNPs were studied in 3 independently collected and genotyped case-control cohorts of European descent (UK, Dutch, and Canadian/Italian), comprising in total 2717 inflammatory bowel disease patients (1197 with Crohn’s disease, 1520 with ulcerative colitis) and 4440 controls. Results: Common variation in MYO9B was associated with susceptibility to inflammatory bowel disease in all 3 cohorts examined (most associated SNP, rs1545620; meta-analysis P = 1.9 ? 10-6; odds ratio, 1.2), with the same alleles showing association as reported for celiac disease. Conclusions: MYO9B genetic variants predispose to inflammatory bowel disease. Interestingly, rs1545620 is a nonsynonymous variant leading to an amino acid change (Ala1011Ser) in the third calmodulin binding IQ domain of MYO9B. Unlike previous variants (in other genes) reported to predispose to inflammatory bowel disease, the association at MYO9B was considerably stronger with ulcerative colitis, although weaker association with Crohn’s disease also was observed. These data imply shared causal mechanisms underlying intestinal inflammatory diseases.
CD8+CD28? Regulatory T Lymphocytes Prevent Experimental Inflammatory Bowel Disease in Mice, 12 September 2006
MÉnager–Marcq I, PomiÉ C, Romagnoli P, van Meerwijk JPM
Background & Aims: Immune responses to innocuous intestinal antigens appear tightly controlled by regulatory T lymphocytes. While CD4+ T lymphocytes have recently attracted the most attention, CD8+ regulatory T-cell populations are also believed to play an important role in control of mucosal immunity. However, CD8+ regulatory T-cell function has mainly been studied in vitro and no direct in vivo evidence exists that they can control mucosal immune responses. We investigated the capacity of CD8+CD28? T cells to prevent experimental inflammatory bowel disease (IBD) in mice. Methods: CD8+CD28? regulatory T cells were isolated from unmanipulated mice and tested for their capacity to inhibit T-cell activation in allogeneic mixed lymphocyte cultures in vitro and to prevent IBD induced by injection of CD4+CD45RBhigh cells into syngeneic immunodeficient RAG-2 mutant mice. Results: CD8+CD28? T lymphocytes inhibited proliferation and interferon gamma production by CD4+ responder T cells in vitro. CD8+CD28? regulatory T cells freshly isolated from spleen or gut efficiently prevented IBD induced by transfer of colitogenic T cells into immunodeficient hosts. Regulatory CD8+CD28? T cells incapable of producing interleukin-10 did not prevent colitis. Moreover, IBD induced with colitogenic T cells incapable of responding to transforming growth factor ? could not be prevented with CD8+CD28? regulatory T cells. CD8+CD28+ T cells did not inhibit in vitro or in vivo immune responses. Conclusions: Our findings show that naturally occurring CD8+CD28? regulatory T lymphocytes can prevent experimental IBD in mice and suggest that these cells may play an important role in control of mucosal immunity.
Inhibition of Smad7 With a Specific Antisense Oligonucleotide Facilitates TGF-Beta1–Mediated Suppression of Colitis, 21 September 2006
Boirivant M, Pallone F, Di Giacinto C, Fina D, Monteleone I, Marinaro M, Caruso R, Colantoni A, Palmieri G, Sanchez M, Strober W, MacDonald TT, Monteleone G
Background & Aims: Defective transforming growth factor (TGF)-Beta1 signaling due to high levels of Smad7 is a feature of inflammatory bowel disease (IBD). In this study, we analyzed the effect of reducing Smad7 levels with antisense oligonucleotide on mouse models of colitis. Methods: Mucosal samples taken from colitic tissue of mice with colitis due to either haptenating reagents (trinitrobenzene sulfonic acid [TNBS] or oxazolone) or to transfer of T cells (SCID transfer colitis) were analyzed for Smad3 and/or Smad7 expression by Western blotting and, in some cases, content of TGF-Beta1 by enzyme-linked immunosorbent assay. The effect of oral Smad7 antisense oligonucleotide on mucosal inflammation was assessed. Results: TGF-Beta1 levels were increased in the inflamed tissues of mice with colitis induced by either TNBS or oxazolone. Nevertheless, TGF-Beta1 did not exert a regulatory effect, probably because TGF-Beta1 signaling was blocked, as indicated by the presence of reduced Smad3 phosphorylation and high levels of Smad7. Oral administration of Smad7 antisense oligonucleotide to colitic mice restored TGF-Beta1 signaling via Smad3 and ameliorated inflammation in hapten-induced colitis. In addition, Smad7 antisense oligonucleotide had a therapeutic effect on relapsing TNBS-induced colitis but not on cell-transfer colitis. Conclusions: These data suggest that colitis models associated with high endogenous TGF-Beta1 levels and defective TGF-Beta1 signaling due to high levels of Smad7 can be ameliorated by down-regulation of Smad7 and by oral administration of Smad7 antisense oligonucleotide. This may represent a new approach to the control of IBD, particularly during active phases when its Smad7 profile resembles that of hapten-induced colitis.
Therapeutic Treatment of Experimental Colitis With Regulatory Dendritic Cells Generated With Vasoactive Intestinal Peptide, 18 October 2006
Gonzalez–Rey E, Delgado M
Background & Aims: Crohn’s disease is a chronic debilitating disease characterized by severe T helper cell (Th)1-driven inflammation of the colon partially caused by a loss of immune tolerance against mucosal antigens. The use of regulatory dendritic cells (DCs) with the capacity to induce regulatory T cells has been proposed recently for the treatment of Crohn’s disease in a strategy to restore immune tolerance. Vasoactive intestinal peptide is an immunomodulatory neuropeptide that induces regulatory DCs. The aim of this study was to investigate the therapeutic effect of vasoactive intestinal peptide-induced regulatory DCs (DCVIP) in a murine model of colitis. Methods: We examined the therapeutic action of DCVIP in the colitis induced by intracolonic administration of trinitrobenzene sulfonic acid, evaluating diverse clinical signs of the disease including weight loss, diarrhea, colitis, and histopathology. We also investigated the mechanisms involved in the potential therapeutic effect of DCVIP, such as inflammatory cytokines and chemokines, Th1-type response, and the generation of regulatory T cells. Results: DCVIP injection significantly ameliorated the clinical and histopathologic severity of colitis, abrogating body weight loss, diarrhea, and inflammation, and increasing survival. The therapeutic effect was associated with down-regulation of both inflammatory and Th1-driven autoimmune response, by regulating a wide spectrum of inflammatory mediators directly through activated macrophages, and by generating interleukin-10–secreting regulatory T cells with suppressive capacity on autoreactive T cells. Conclusions: The possibility to generate/expand ex vivo regulatory DCVIP opens new therapeutic perspectives for the treatment of Crohn’s disease in human beings, and may minimize the dependence on nonspecific immunosuppressive drugs used currently for autoimmune disorders.
Saccharomyces boulardii Inhibits Inflammatory Bowel Disease by Trapping T Cells in Mesenteric Lymph Nodes, 10 November 2006
Dalmasso G, Cottrez F, Imbert V, Lagadec P, Peyron JF, Rampal P, Czerucka D, Groux H
Background & Aims:Saccharomyces boulardii is a nonpathogenic yeast used for treatment of diarrhea. We used a mice model of inflammatory bowel disease (IBD) to analyze the effects of S boulardii on inflammation. Methods: Lymphocyte-transferred SCID mice, displaying IBD, were fed daily with S boulardii. Weight loss and inflammatory status of the colon were monitored. Nuclear factor-?B activity was assessed in the colon. The CD4+ T-cell production of interferon (IFN) ? was evaluated by enzyme-linked immunosorbent assay, and a comprehensive reverse-transcription polymerase chain reaction (RT-PCR) analysis for both colon and mesenteric lymph nodes was performed. Finally, we analyzed cell migration mechanisms in vitro and in vivo. Results:S boulardii treatment inhibits IBD. S boulardii induces an accumulation of IFN-?-producing T-helper 1 cells within the mesenteric lymph nodes correlated with a diminution of CD4+ T-cell number and IFN-? production by CD4+ T cells within the colon. The influence of S boulardii treatment on cell accumulation in mesenteric lymph nodes was also observed in normal BALB/c mice and involves modifications of lymph node endothelial cell adhesiveness by a yeast secretion product. Conclusions:S boulardii has a unique action on inflammation by a specific alteration of the migratory behavior of T cells, which accumulate in mesenteric lymph nodes. Therefore, S boulardii treatment limits the infiltration of T-helper 1 cells in the inflammed colon and the amplification of inflammation induced by proinflammatory cytokines production. These results suggest that S boulardii administration may have a beneficial effect in the treatment of IBD.
Contribution of Sensory Neurons to Sex Difference in the Development of Stress-Induced Gastric Mucosal Injury in Mice, 12 September 2006
Shimozawa N, Okajima K, Harada N, Arai M, Ishida Y, Shimada S, Kurihara H, Nakagata N
Background & Aims: Sensory neurons play a critical role in reducing stress-induced gastric mucosal injury by releasing calcitonin gene-related peptide (CGRP) through an increase in gastric mucosal levels of prostacyclin (PGI2). Because estrogen enhances nerve growth factor–mediated CGRP production in sensory neurons, we hypothesized that stress-induced gastric mucosal injury occurs less in females than in males. Methods: Gastric ulcer index, gastric myeloperoxidase activity, and gastric tissue levels of CGRP and 6-keto-PGF1?, a stable metabolite of PGI2, were determined in male and female wild-type (CGRP+/+) mice and CGRP knockout (CGRP?/?) mice subjected to water-immersion restraint stress. Results: In CGRP+/+ mice, ulcer index and myeloperoxidase activities were lower and gastric tissue levels of CGRP and 6-keto-PGF1? were higher in female mice than in male mice, but there were no such sex differences in CGRP?/?mice. Sex differences in CGRP+/+ mice were eliminated by pretreatment with SB366791 (500 ?g/kg intraperitoneally), a vanilloid receptor antagonist, and by ovariectomy. Reversal of sex differences by ovariectomy was not observed in female CGRP+/+ mice with estradiol replacement (1 mg · kg?1· wk?1 for 3 weeks). Levels of CGRP messenger RNA in dorsal root ganglion neurons isolated from female CGRP+/+ mice were decreased by ovariectomy, and these decreases were reversed by estradiol replacement. Conclusions: Estrogen-mediated increases in CGRP levels in sensory neurons might contribute to reduce stress-induced gastric mucosal injury by attenuating inflammatory responses. This might at least partly explain the sex difference observed in the development of stress-induced gastric mucosal injury in mice.
Correlation Between the Single-Site CpG Methylation and Expression Silencing of the XAF1 Gene in Human Gastric and Colon Cancers, 4 October 2006
Zou B, Chim CS, Zeng H, Leung SY, Yang Y, Tu SP, Lin MCM, Wang J, He H, Jiang SH, Sun YW, Yu LF, Yuen ST, Kung HF, Wong BCY
Background & Aims: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) antagonizes the anti-caspase activity of XIAP. XAF1 messenger RNA is present in normal tissues but undetectable in various cancers and thus poses a potential tumor suppressor gene. The aim of this study was to examine the novel pattern of methylation of XAF1 in gastric and colon cancers and locate the important CpG sites for transcriptional regulation and tumor progression. Methods: XAF1 expression was detected by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis. Four different fragments around the transcription start site of XAF1 were cloned and examined putative promoter activities by luciferase reporter assay. Each CpG site in fragment F291 was mutated by site-directed mutagenesis technique, and the change of promoter activity of this fragment was detected by luciferase reporter assay. Methylation status of XAF1 was determined by methylation-specific PCR (MSP) and bisulfite DNA sequencing PCR analysis. Results: Down-regulation of XAF1 in association with hypermethylation was detected in 3 of 4 human gastric cancer cell lines and 6 of 8 colon cancer cell lines. Of the 4 promoter fragments, F291 showed the highest promoter activity, which could be down-regulated obviously by the mutation of particular CpG sites. Moreover, aberrant hypermethylation of these important CpG sites was strongly associated with the development of gastric and colon cancers. Conclusions: A cluster of methylated CpG sites instead of CpG islands located in the promoter area resulted in gene silencing of XAF1, and CpGs at ?2nd, ?1st, and +3rd positions are functionally more important in its transcriptional regulation.
Basic - Liver, Pancreas, and Biliary Tract
A Mouse Model of Hereditary Pancreatitis Generated by Transgenic Expression of R122H Trypsinogen, 4 October 2006
Archer H, Jura N, Keller J, Jacobson M, Bar–Sagi D
Background & Aims: Missense mutations in human cationic trypsinogen PRSS1 are frequently detected in patients with hereditary pancreatitis, a rare genetic disease of the pancreas characterized by autodigestive necrosis, chronic inflammation, and fibrosis. To examine the link between PRSS1 mutations and the initiation and progression of hereditary pancreatitis, we have sought to generate a transgenic mouse that carries a missense mutation in the PRSS1 that is most frequently observed in patients. Methods: A transgenic mouse was generated in which the expression of the mouse PRSS1 mutant R122H (R122H_mPRSS1) is targeted to pancreatic acinar cells by fusion to the elastase promoter. The expression of the mutant trypsinogen was assessed by immunohistochemical staining and real-time reverse transcription polymerase chain reaction analysis. The relationship between transgene expression and inflammation was analyzed by morphologic assessment of H&E-stained tissue sections, responsiveness to cerulein-induced pancreatitis, and immunohistochemical identification of cellular and biochemical components of the inflammatory response. Results: Pancreata from transgenic mice display early-onset acinar cell injury and inflammatory cell infiltration. With progressing age, the transgenic mice develop pancreatic fibrosis and display acinar cell dedifferentiation. Moreover, the expression of R122H_mPRSS1 transgene is associated with enhanced response to cerulein-induced pancreatitis. Finally, cell-specific activation of the inflammation-associated signaling pathways, c-jun-N-terminal kinase and extracellular signal–regulated kinase, was observed in response to expression of R122H_mPRSS1. Conclusions: These results underscore the importance of PRSS1 mutations as pathogenic mediators of hereditary pancreatitis and indicate that persistent pancreatic injury might be causally linked to chronic pancreatitis.
Primary Cilia Deletion in Pancreatic Epithelial Cells Results in Cyst Formation and Pancreatitis, 2 November 2006
Cano DA, Sekine S, Hebrok M
Background & Aims: Defects in cilia formation or function have been implicated in several human genetic diseases, including polycystic kidney disease (PKD), Bardet-Biedl syndrome, and primary ciliary dyskinesia. Pancreatic lesions are found in approximately 10% of PKD patients, suggesting a connection between cilia defects and pancreatic pathologies. Here, we investigate the role of cilia in pancreas formation and function by analyzing mice that lack cilia in pancreatic cells. Methods: Using Cre/lox technology, we conditionally inactivated Kif3a, the gene encoding for a subunit of the kinesin-2 complex that is essential for cilia formation, in pancreatic epithelia. Kif3a mice were studied by immunohistochemical and biochemical methods to assess the morphology and differentiation status of pancreatic cells. Results: Tissue-specific loss of Kif3a in pancreatic cells resulted in severe pancreatic abnormalities including acinar-to-ductal metaplasia, fibrosis, and lipomatosis. Ductal metaplasia appears to be due to expansion of ductal cells rather than transdifferentiation of acinar cells. Cyst formation, aberrant ductal morphology, and extensive fibrosis associated with severe adhesion to adjacent organs were commonly observed in aged Kif3a mutant mice. Deletion of Kif3a using different pancreas-specific Cre strains suggests that these pancreatic phenotypes might be caused by the absence of cilia in ductal cells. Activation of transforming growth factor ? and Mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways may play a role in these phenotypes. Conclusions: These results demonstrate that the absence of cilia in pancreatic cells produces pancreatic lesions that resemble those found in patients with chronic pancreatitis or cystic fibrosis.
Inactivation of Hepatic Microsomal Triglyceride Transfer Protein Protects Mice From Diet-Induced Gallstones, 14 August 2006
Amigo L, Castro J, Miquel JF, Zanlungo S, Young S, Nervi F
Background & Aims: Microsomal triglyceride transfer protein (MTTP) is critical for the production of very-low-density lipoproteins (VLDL). The current studies were undertaken to examine the in vivo role of MTTP in hepatic cholesterol and fatty acid metabolism, as well as in biliary lipid secretion. We also tested whether MTTP plays a role in diet-induced cholelithiasis in mice. Methods: We used mice in which Mttp had been inactivated in the liver (Mttp?/? mice). We measured several parameters of cholesterol metabolism, fatty acid synthesis, and biliary lipid levels in mice fed a normal or a lithogenic diet. We also assessed the incidence of diet-associated gallstones. Results: Hepatic Mttp inactivation markedly decreased plasma triglyceride and cholesterol levels and increased biliary cholesterol and bile acid output. Hepatic cholesterogenesis and fatty acid synthesis were significantly decreased in Mttp?/? mice compared with control mice. The incidence of gallstones decreased from 90% in control mice to 33% in Mttp?/? mice after 8 weeks of a lithogenic diet (P < .0001). The mechanism of the protective effect appears to be increased biliary phospholipid output in Mttp?/? mice, leading to significant unsaturation of gallbladder bile. Conclusions: These results indicate that modulation of Mttp expression in the liver affects hepatic lipid synthesis and storage as well as biliary lipid secretion. Our findings further indicate that inhibition of hepatic MTTP activity decreases the risk of experimental cholelithiasis by favoring phospholipid output into the bile.
Hepatitis C and Risk of Lymphoma: Results of the European Multicenter Case-Control Study EPILYMPH, 26 September 2006
Nieters A, Kallinowski B, Brennan P, Ott M, Maynadié M, Benavente Y, Foretova L, Cocco PL, Staines A, Vornanen M, Whitby D, Boffetta P, Becker N, De Sanjosé S
Background & Aims: Increasing evidence points toward a role of hepatitis C virus (HCV) infection in the etiology of malignant lymphomas. However, previous epidemiologic studies were limited in size to establish an association between HCV infection and specific lymphoma subtypes. We performed a large, multicenter, case-control study to address this question. Methods: The study comprised 5 European countries and included newly diagnosed cases of any lymphoid malignancy recruited between 1998 and 2004. Controls were matched to cases by 5-year age group, sex, and study center. In-person interviews were conducted to collect data on demographic, medical, and family history as well as environmental exposures. Serum samples of 1807 cases and 1788 controls (excluding human immunodeficiency virus-positive and organ-transplantation subjects) were screened for HCV infection using an enzyme immunoassay. Positive as well as randomly selected negative samples were subjected to HCV RNA detection and HCV genotyping. Results: HCV infection was detected in 53 (2.9%) lymphoma cases and in 41 (2.3%) control subjects (odds ratio [OR], 1.42; 95% confidence interval [CI]: 0.93–2.15). Restricted to individuals who tested positive for HCV-RNA (indicating persistent infection and active viral replication), the OR was 1.82 (95% CI: 1.13–2.91). In subtype-specific analyses, HCV prevalence was associated with diffuse large B-cell lymphoma (OR, 2.19; 95% CI: 1.23–3.91) but not with chronic lymphocytic leukemia or follicular, Hodgkin’s, or T-cell lymphoma. The sample size was not sufficient to derive any conclusions for rare lymphoma entities such as splenic marginal zone lymphoma. Conclusions: These results support a model that chronic HCV replication contributes to lymphomagenesis and establish a specific role of HCV infection in the pathogenesis of diffuse large B-cell lymphoma.
Interferons Alpha and h Inhibit Hepatitis C Virus Replication With Distinct Signal Transduction and Gene Regulation Kinetics, 4 October 2006
Marcello T, Grakoui A, Barba–Spaeth G, Machlin ES, Kotenko SV, Macdonald MR, Rice CM
Background & Aims: Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. Current therapy with pegylated interferon ? (IFN-?) in combination with ribavirin is associated with adverse effects and often fails to induce a sustained response. IFN-?s, recently discovered IFN gene family members, exhibit antiviral and cell stimulatory activities similar to IFN-?. We aimed to determine whether IFN-? exhibits antiviral activity toward HCV and to compare the signal transduction and effector gene pathways with those of IFN-?. Methods: Using the HCV replicon system and cell culture infectious reporter virus, we compared IFN-? and IFN-? effects on HCV RNA replication and protein expression, as measured by quantitative reverse-transcriptase polymerase chain reaction, luciferase expression, and Western blot. Receptor expression and signaling pathways were explored using flow cytometry and Western blot. IFN-?- and IFN-?-mediated gene expression changes were compared using microarray analyses. Results: IFN-? exhibited dose- and time-dependent HCV inhibition, independent of types I and II IFN receptors. The kinetics of IFN-?-mediated signal transducers and activators of transcription (STAT) activation and induction of potential effector genes were distinct from those of IFN-?. IFN-? induced steady increases in levels of known interferon stimulated genes (ISGs), whereas IFN-? ISGs peaked early and declined rapidly. IFN-? inhibited replication of HCV genotypes 1 and 2 and enhanced the antiviral efficacy of subsaturating levels of IFN-?. Conclusions: These results demonstrate distinct differences in IFN-?- and IFN-?-induced antiviral states. Understanding these differences may prove useful for developing new HCV treatment strategies.
Novel Model of Antigen-Specific Induction of Bile Duct Injury, 18 October 2006
Buxbaum J, Qian P, Khuu C, Shneider BL, Daikh DI, Gershwin ME, Allen PM, Peters MG
Background & Aims: Biliary-directed inflammation is an important cause of acute and chronic liver disease. We developed and characterized a transgenic mouse model of immune-mediated hepatobiliary injury. Methods: Ovalbumin (OVA)-BIL mice were developed using 3.0 kilobase of the rat apical sodium-dependent bile acid transporter promoter to drive aberrant expression of a membrane form of ovalbumin (OVA) on biliary epithelium. Liver inflammation resulted from adoptive transfer of OVA-specific T cells. Liver immune cells were characterized to determine the mechanism of the response by assessing activation, proliferation, and intracellular cytokine expression. Results: OVA-BIL transgenic mice were tolerant to OVA, without evidence of liver disease. Adoptive transfer of OVA-specific CD4+ and CD8+ T cells into naïve OVA-BIL mice led to biliary-centered necroinflammatory damage in a dose-dependent manner. This inflammation absolutely required CD8+ T cells and was augmented by CD4+ T cells. Adoptively transferred OVA CD8+ cells homed to and proliferated in the liver but not the spleen. These activated, adoptively transferred cytotoxic T lymphocytes produced elevated levels of tumor necrosis factor ? and interferon ?. Conclusions: T-cell recognition of antigen aberrantly expressed on bile duct epithelium induced an acute necroinflammatory response specific to the liver, with activation, proliferation, and cytokine production predominantly by the OVA-specific cytotoxic T cells. Thus, OVA BIL represents an antigen-specific animal model of inflammatory bile duct injury.
© 2006 American Gastroenterological Association (AGA) Institute. Published by Elsevier Inc. All rights reserved.
JOURNAL OF HEPATOLOGY
Volume 45, Issue 6, December 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
Hepatitis A virus infection suppresses hepatitis C virus replication and may lead to clearance of HCV, 21 September 2006
Deterding K, Tegtmeyer B, Cornberg M, Hadem J, Potthoff A, Böker KHW, Tillmann HL, Manns MP, Wedemeyer H
Background/Aims
The significance of hepatitis A virus (HAV) super-infection in patients with chronic hepatitis C had been a matter of debate. While some studies suggested an incidence of fulminant hepatitis A of up to 35%, this could not be confirmed by others.
Methods
We identified 17 anti-HCV-positive patients with acute hepatitis A from a cohort of 3170 anti-HCV-positive patients recruited at a single center over a period of 12 years.
Results
Importantly, none of the anti-HCV-positive patients had a fulminant course of hepatitis A. HCV-RNA was detected by PCR in 84% of the anti-HCV-positive/anti-HAV-IgM-negative patients but only in 65% of anti-HCV-positive patients with acute hepatitis A (p=0.03), indicating suppression of HCV replication during hepatitis A. Previous HAV infection had no effect on HCV replication. After recovery from hepatitis A, an increased HCV replication could be demonstrated for 6 out of 9 patients with serial quantitative HCV-RNA values available while 2 patients remained HCV-RNA negative after clearance of HAV throughout follow-up of at least 2 years.
Conclusions
HAV super-infection is associated with decreased HCV-RNA replication which may lead to recovery from HCV in some individuals. Fulminant hepatitis A is not frequent in patients with chronic hepatitis C recruited at a tertiary referral center.
Interleukin-10?1082 GG polymorphism influences the occurrence and the clinical characteristics of hepatitis C virus infection, 21 September 2006
Persico M, Capasso M, Persico E, Masarone M, Renzo Ad, Spano D, Bruno S, Iolascon A
Background/Aims
In this study, we determined the genotypic and allelic frequencies of the Interleukin (IL)-10?1082G/A IL-10?592A/C, and IL-10?819C/T polymorphisms, and their association with the risk to develop B cell Non Hodgkin Lymphoma (NHL) in hepatitis virus C (HCV) carriers.
Results
Genetic polymorphisms in the IL-10 gene promoter were studied in 250 consecutive patients with B-cell NHL with no clinical and/or laboratory findings of cryoglobulinemia, 142 NHL/HCV? and 108 NHL/HCV+ with chronic hepatitis (CH), 120 consecutive subjects with HCV-related CH, and 110 age, sex-matched healthy blood donors. The frequency of the IL-10?1082GG genotype vs remaining genotypes (IL-10?1082GA/AA) was higher in NHL/HCV+ patients than HCV-related CH patients (P=0.0002, OR=2.89, CI: 1.62–5.15) and in NHL/HCV+ than NHL/HCV? patients (P=0.0001, OR=2.99, CI: 1.72–5.19). Moreover, the IL-10?1082GG genotype was more prevalent in indolent NHL/HCV+ cases than aggressive NHL/HCV+ (P=0.0004, OR=4.97, CI: 2.10–11.79). Finally, we confirmed that IL-10?1082GG genotype is associated with higher IL-10 production compared to AA homozygous (P=0.037).
Conclusions
The high IL-10 production, due to IL-10?1082GG genotype, influences the clinical expression of the HCV infection by increasing susceptibility to develop NHL and might contribute to the indolent form of the disease.
Cirrhosis and its Complications
Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis, 21 September 2006
Neef M, Ledermann M, Saegesser H, Schneider V, Reichen J
Background/Aims
Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis.
Methods
Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-?1, transforming growth factor-?1 (TGF-?1) and ?2 was quantified by RT-PCR.
Results
Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-?1 mRNA was downregulated in TAA, TGF-?2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats.
Conclusions
Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.
Impaired insulin action despite upregulation of proximal insulin signaling: Novel insights into skeletal muscle insulin resistance in liver cirrhosis, 28 September 2006
Jessen N, Buhl ES, Schmitz O, Lund S
Background/Aims
Disturbance in glucose metabolism is a common feature in liver diseases and this is associated with skeletal muscle insulin resistance. However, the underlying molecular mechanisms are unclear. To characterize skeletal muscle insulin resistance associated with liver disease, we examined muscles from animals after an acute, 5 weeks perturbation of the common bile duct. Clinical findings, elevated plasma levels of liver enzymes and histological examinations confirmed cirrhosis.
Methods/Results:
Cirrhotic animals were insulin resistant and this was associated with reduced glucose transport into muscles. Interestingly, activity in the proximal part of the insulin signaling cascade was not decreased, as evinced by increased activity of key enzymes in the signal to glucose transport. Expression of the glucose transporter, GLUT4, was normal. So together these results indicate that signaling downstream of PKB/Akt and/or the translocation of GLUT4 is impaired in skeletal muscle from cirrhotic animals.
Conclusions
In conclusion, in an animal model of liver cirrhosis whole body insulin resistance is associated with insulin resistance in skeletal muscles. Unlike other common forms of insulin resistance, muscles from cirrhotic animals have increased activity in the proximal insulin signaling cascade. This emphasizes the fact that skeletal muscle insulin resistance associated with liver cirrhosis is a unique entity.
Liver Failure, Growth and Cancer
Nucleotide change of codon 38 in the X gene of hepatitis B virus genotype C is associated with an increased risk of hepatocellular carcinoma, 21 September 2006
Muroyama R, Kato N, Yoshida H, Otsuka M, Moriyama M, Wang Y, Shao RX, Dharel N, Tanaka Y, Ohta M, Tateishi R, Shiina S, Tatsukawa M, Fukai K, Imazeki F, Yokosuka O, Shiratori Y, Omata M
Background/Aims
The hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, the HBV X gene, which encodes the pleiotropic transactivator HBx, has also been associated with the development of HCC. In this study, we investigated whether nucleotide changes in the X gene of genotype C are associated with the development of HCC.
Methods/Results
We sequenced the X gene in age- and sex-matched 39 HBV-infected patients with HCC and 36 HBV-infected patients without HCC. A novel nucleotide change that resulted in a proline to serine substitution at codon 38 in HBx (codon-38 change) was preferentially found in patients with HCC. Then, sera were collected from a new group of age- and sex-matched 52 patients with HCC and 51 patients without HCC. In this cohort also, the codon-38 change was associated with HCC. Multiple logistic regression analysis showed the prevalence of the codon-38 change was significantly associated with HCC in all patients (P=0.001, odds ratio: 4.89).
Conclusion
The codon-38 change in genotype C is an independent risk factor for the development of HCC and may serve as a useful molecular marker for predicting the clinical outcomes in patients infected with HBV.
Molecular and Cell Biology
Critical role of Toll-like receptors and the common TLR adaptor, MyD88, in induction of granulomas and liver injury, 31 July 2006
Velayudham A, Hritz I, Dolganiuc A, Mandrekar P, Kurt-Jones E, Szabo G
Background/Aims
Toll-like receptors (TLR) recognize pathogens and regulate innate immune activation. Here, we investigated the roles of TLR9 and the common TLR adaptor, MyD88, in liver injury.
Methods
C57BL6, TLR9?/?, IFN??/? or MyD88?/? mice were primed with Propionibacterium acnes, TLR9 (CpG) or TLR2 (lipoteichoic acid) ligands followed by LPS challenge. ALT, cytokines and liver histology were assessed.
Results
Selective priming through TLR9 but not TLR2 induced granulomas, elevated serum ALT, and sensitized C57BL6 mice to increased LPS-induced serum IL-6, IL-12 and IFN? levels. Further, TLR2 and TLR9 ligands synergized in induction of granulomas and sensitization to LPS-induced inflammation. IFN? induction by P. acnes, TLR2 and TLR9 ligands required MyD88. In MyD88?/? mice P. acnes failed to induce granulomas and both MyD88 and TLR9 deficiency prevented P. acnes-induced sensitization to LPS. Increased mRNA expression of genes of the TLR4 signaling complex (TLR4, CD14, MD-2, and MyD88) and the NADPH complexes (p47phox, p67phox, gp91phox, and p22phox) was induced by priming with P. acnes or TLR9 plus TLR2 suggesting mechanisms for LPS sensitization and liver injury.
Conclusions
TLR9±TLR2 activation via MyD88-dependent pathways plays a pivotal role in liver sensitization and granuloma formation.
Secretin receptors in the human liver: Expression in biliary tract and cholangiocarcinoma, but not in hepatocytes or hepatocellular carcinoma, 28 July 2006
Körner M, Hayes GM, Rehmann R, Zimmermann A, Scholz A, Wiedenmann B, Miller LJ, Reubi JC
Background/Aims
Gut hormone receptors are over-expressed in human cancer and allow receptor-targeted tumor imaging and therapy. A novel promising receptor for these purposes is the secretin receptor. The secretin receptor expression was investigated in the human liver because the liver is a physiological secretin target and because novel diagnostic and treatment modalities are needed for liver cancer.
Methods
Nineteen normal livers, 10 cirrhotic livers, 35 cholangiocarcinomas, and 45 hepatocellular carcinomas were investigated for secretin receptor expression by in vitro receptor autoradiography using 125I-[Tyr10] rat secretin and, in selected cases, for secretin receptor mRNA by RT-PCR.
Results
Secretin receptors were present in normal bile ducts and ductules, but not in hepatocytes. A significant receptor up-regulation was observed in ductular reaction in liver cirrhosis. Twenty-two (63%) cholangiocarcinomas were positive for secretin receptors, while hepatocellular carcinomas were negative. RT-PCR revealed wild-type receptor mRNA in the non-neoplastic liver, wild-type and spliced variant receptor mRNAs in cholangiocarcinomas found receptor positive in autoradiography experiments, and no receptor transcripts in autoradiographically negative cholangiocarcinomas.
Conclusions
The expression of secretin receptors in the biliary tract is the molecular basis of the secretin-induced bicarbonate-rich choleresis in man. The high receptor expression in cholangiocarcinomas may be used for in vivo secretin receptor-targeting of these tumors and for the differential diagnosis with hepatocellular carcinoma.
A critical role of CpG motifs in a murine peritonitis model by their binding to highly expressed toll-like receptor-9 on liver NKT cells, 21 September 2006
Tsujimoto H, Ono S, Matsumoto A, Kawabata T, Kinoshita M, Majima T, Hiraki S, Seki S, Moldawer LL, Mochizuki H
Backgrounds
Toll-like receptor (TLR)-9 plays a critical role in the recognition of the CpG motifs, which is frequently observed in bacterial DNA. To date, there have not been any reports regarding the role of bacterial DNA in the systemic circulation on the development of sepsis.
Methods
We examined the expression of TLR-9 in the liver and spleen in a murine peritonitis model (CLP mice). We also measured the cytokine response of mononuclear cells (MNCs) from normal and CLP mice to CpG oligodeoxynucleotides (ODN) in vitro and in vivo.
Results
TLR-9 expression on F4/80+ and NK1.1+CD3?+ cells in the liver of CLP mice was elevated compared to sham-operated mice. With regard to cytokine production, we found that CpG ODN markedly stimulated the production of inflammatory cytokines by murine macrophages and liver MNCs. The intravenous injection of CpG ODN in mice that underwent CLP 12h earlier led to their increased cytokine production and their increased mortality. In addition, the depletion of NK/NKT cells contributed to improve their survival rate.
Conclusions
Our results suggest that, in patients with overwhelming bacterial infection, bacterial DNA may induce liver toxicity that is mediated by liver NKT cells and macrophages that express high levels of TLR-9.
Cholestasis and Autoimmune Liver Disease
HLA Class II influences humoral autoimmunity in patients with type 2 autoimmune hepatitis, 28 September 2006
Djilali-Saiah I, Fakhfakh A, Louafi H, Caillat-Zucman S, Debray D, Alvarez F
Background/Aims
Type 2 autoimmune hepatitis (AIH) is characterized by the presence of anti-liver kidney microsome (anti-LKM-1) and/or anti-liver cytosol type 1 (anti-LC1) autoantibodies. However, the correlation between these autoantibodies and the genetic background has not been studied.
Methods
Frequencies of HLA class II alleles were compared between the 60 Caucasian children with type 2 AIH and 313 control subjects. The anti-LKM1 antibody reactivity directed against antigenic sites of CYP2D6 was analysed by ELISA.
Results
HLA-DQB1?0201 allele was found to be the primary genetic determinant of susceptibility to type 2 AIH by conferring the highest odd-ratio (OR=6.4). HLA-DRB1?03 allele was significantly increased (P<0.0001) among patients with both anti-LKM1 and anti-LC1 autoantibodies as well as in those with only anti-LC1+ compared to those with anti-LKM1+ alone. In contrast, HLA-DRB1?07 allele was significantly associated (P<0.0001) with anti-LKM1+ alone compared to groups with both anti-LKM and anti-LC1 or with LC1+ alone. Children with the DRB1?07 allele develop anti-LKM1 autoantibodies having a more restricted specificity (2 epitopes) than to those having HLA-DRB1?03 allele (5 epitopes).
Conclusions
The HLA-DR locus is involved in autoantibody expression, while the DQ locus appears to be a critical determinant for the development of type 2 AIH.
Genetic and Metabolic Liver Disease
Multiple voxel 1H MR spectroscopy of phosphorylase-b kinase deficient patients (GSD IXa) showing an accumulation of fat in the liver that resolves with aging, 21 August 2006
Sijens PE, Smit GP, Borgdorff MAJ, Kappert P, Oudkerk M
Background/Aims
Phosphorylase-b deficient patients suffer from glycogen storage disease (GSD IXa) leading to liver enlargement which usually resolves during puberty and adolescence. This pathology has not yet been documented by 1H MR spectroscopy (MRS) investigation.
Methods
MRS of eight GSD IXa patients was performed in this study to assess whether or not liver fat content is elevated in GSD IXa and decreases with aging. An improvement in our MRS method compared with previous liver fat MRS studies is that we measured a plane of liver voxels at once rather than a single MRS voxel, yielding a reliable determination of liver fat content.
Results
Fat contents of 3.4–10% were observed in young GSD IXa patients, as compared with 0.5–0.9% in controls, these dropped to control levels in patients past age 40 (r=?0.82; P<0.01).
Conclusions
Liver fat content is increased in glycogen storage disease (GSD IXa) and normalizes with ageing. Assessing liver fat levels in this population is a novel and interesting concept. This could potentially enhance the understanding of liver function in that 20% of the population who has increased liver fat.
Copyright © 2001-2006 European Association for the Study of the Liver. All rights reserved.
BRITISH MEDICAL JOURNAL
Copyright © 2006 BMJ Publishing Group Ltd
NEW ENGLAND JOURNAL
The New England Journal of Medicine is owned, published, and copyrighted © 2006 Massachusetts Medical Society. All rights reserved.
LANCET
The Lancet, published, and copyrighted © 2006. All rights reserved.
