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Volume 39, Issue 5 (may 2004)
Special Article
Screening in liver disease: Report of an AASLD clinical workshop (p 1204-1212)
Paul C. Adams, Michael J. Arthur, Thomas D. Boyer, Laurie D. DeLeve, Adrian M. Di Bisceglie, Mark Hall, Theodore R. Levin, Dawn Provenzale, Leonard Seeff
This report summarizes an AASLD Clinical Workshop that was presented at Digestive Diseases Week 2003 on screening in liver diseases. As newer diagnostic tests become available, many liver diseases and complications of liver disease can be detected at an early asymptomatic stage. In many cases, early detection can lead to earlier treatment and an improved outcome. However, screening for liver diseases in asymptomatic persons has the potential for adverse consequences, including discrimination and stigmatization. The cost of screening programs is significant, and access to screening tests varies in different countries. Future screening programs require careful planning and implementation to balance the benefits, risks, and cost-effectiveness. This review outlines the concepts of screening and their application to a broad range of liver diseases. (HEPATOLOGY 2004;39:1204-1212.) 
Viral Hepatitis
Short-term interferon-alfa therapy for acute hepatitis C: A randomized controlled trial (p 1213-1219)
Hideyuki Nomura, Suketo Sou, Hironori Tanimoto, Takashi Nagahama, Yoichi Kimura, Jun Hayashi, Hiromi Ishibashi, Seizaburo Kashiwagi
Acute hepatitis C often progresses to chronic infection. We undertook a randomized controlled trial to determine whether short-term therapy with interferon (IFN) during acute hepatitis C is effective in preventing the development of chronic hepatitis. Thirty patients with acute hepatitis C were randomized into 1 of 2 treatment groups. IFN therapy was initiated 8 weeks after the onset of acute hepatitis in the early-intervention group and after 1 year of observation in the late-intervention group. Short-term therapy consisted of natural IFN-alfa (6 million units) administered on consecutive days for a period of 4 weeks. Any signs of recrudescence of disease were immediately followed by interval IFN therapy (3 times weekly for 20 weeks). In the early-intervention group, short-term therapy was associated with a sustained virological response in 13 of 15 patients (87%). Follow-up treatment was associated with a sustained virological response in both of the remaining 2 patients (100%). The sustained virological response rate was significantly higher in the early-intervention group (87%, 13 of 15 patients after short-term therapy alone, and 100%, 15 of 15 patients after short-term with or without follow-up therapy) than in the late-intervention group (40%, 6 of 15 patients after short-term therapy alone, and 53%, 8 of 15 patients after short-term therapy with or without follow-up therapy, P= .021 and P= .006, respectively). In conclusion, short-term (4 weeks) IFN treatment of patients with acute hepatitis C may be associated with satisfactory results, if initiated at an early stage of the disease. (HEPATOLOGY 2004;39:1213-1219.)
Expression of the CXCR3 ligand I-TAC by hepatocytes in chronic hepatitis C and its correlation with hepatic inflammation (p 1220-1229)
Karla J. Helbig, Andrew Ruszkiewicz, Ljiljana Semendric, Hugh A.J. Harley, Shaun R. McColl, Michael R. Beard
The factors that regulate lymphocyte traffic in chronic hepatitis C (CHC) are not completely defined. Interferon (IFN)-inducible T cell chemoattractant (I-TAC) is a relatively new member of the CXCR3 chemokine ligand family that selectively recruits activated T cells to sites of inflammation. To determine if I-TAC plays a role in CHC, we investigated I-TAC expression in hepatitis C virus (HCV)-infected liver biopsy material. I-TAC messenger RNA (mRNA) levels were significantly increased in HCV-infected liver compared with normal liver, which correlated with both portal and lobular inflammation. I-TAC expression was localized to hepatocytes throughout the liver lobule, with those in close proximity to active areas of inflammation expressing the highest concentration of I-TAC. In vitro , I-TAC mRNA and protein expression was inducible in Huh-7 cells following either IFN- or - stimulation and synergistically with tumor necrosis factor (TNF)- . Furthermore, transfection of Huh-7 cells with either poly(I:C) or HCV RNA representing the HCV subgenomic replicon induced I-TAC mRNA expression. HCV replication was also found to modulate I-TAC expression, with stimulation of Huh-7 cells harboring either the HCV subgenomic or genomic replicon showing significantly increased synergistic effects compared with those previously seen in Huh-7 cells alone with IFN- and TNF- . In conclusion, these results suggest I-TAC, one of the most potent chemoattractants for activated T cells, is produced by hepatocytes in the HCV-infected liver and plays an important role in T cell recruitment and ultimately the pathogenesis of CHC. (HEPATOLOGY 2004;39:1220-1229.)
Steatosis and liver cell apoptosis in chronic hepatitis C: A mechanism for increased liver injury (p 1230-1238)
Meagan J. Walsh, Daina M. Vanags, Andrew D. Clouston, Michelle M. Richardson, David M. Purdie, Julie R. Jonsson, Elizabeth E. Powell
Steatosis is increasingly recognized as a cofactor influencing the progression of fibrosis in chronic hepatitis C; however, the mechanisms by which it contributes to liver injury remain uncertain. We studied 125 patients with chronic hepatitis C to assess the effect of steatosis on liver cell apoptosis and the expression of Bcl-2, Bcl-x L, Bax, and tumor necrosis factor alpha (TNF- ) and the relationship between liver cell apoptosis and disease severity. A significant increase in liver cell apoptosis was seen in liver sections with increasing grade of steatosis ( r= 0.42; P< .0001). Hepatic steatosis and previous heavy alcohol consumption were the only two variables independently associated with the apoptotic index. Increasing steatosis was associated with decreased Bcl-2 mRNA levels and an increase in the proapoptotic Bax/Bcl-2 ratio ( r= -0.32, P= .007; and r= 0.27, P= .02, respectively). In the absence of steatosis, increased liver cell apoptosis was not associated with stellate cell activation or fibrosis ( r= 0.26, P= .11; r= 0.06, P= .71, respectively). In contrast, in the presence of steatosis, increasing apoptosis was associated with activation of stellate cells and increased stage of fibrosis ( r= 0.35, P= .047; r= 0.33, P= .03, respectively), supporting the premise that the steatotic liver is more vulnerable to liver injury. In patients with hepatitis C virus genotype 3, there was a significant correlation between TNF- mRNA levels and active caspase-3 ( r= 0.54, P= .007). In conclusion, these observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C. Further investigation will be required to determine the molecular pathways responsible for the proapoptotic effect of steatosis and whether this increase in apoptosis contributes directly to fibrogenesis. (HEPATOLOGY 2004.39:1230-1238.)
Improved prediction of fibrosis in chronic hepatitis C using measures of insulin resistance in a probability index (p 1239-1247)
Archana Sud, Jason M. Hui, Geoffrey C. Farrell, Priyanka Bandara, James G. Kench, Carolyn Fung, Rita Lin, Dev Samarasinghe, Christopher Liddle, Geoffrey W. McCaughan, Jacob George
We sought to develop a clinically useful index comprising standard and physiologically relevant variables to predict the probability of significant hepatic fibrosis in subjects with chronic hepatitis C virus (HCV) infection. Fibrosis was graded as mild (stages F0 or F1) or significant (stages F2-F4). Thirty-five clinical and laboratory parameters were analyzed initially in 176 patients with detectable HCV RNA to derive a fibrosis probability index (FPI) to predict significant fibrosis. This index then was validated in a second group of 126 subjects. Among 18 variables associated with severe fibrosis on univariate analysis, multiple logistic regression analysis identified age, aspartate aminotransferase (AST), total cholesterol level, insulin resistance (by homeostasis model), and past alcohol intake as independent predictors of significant fibrosis. The area under the receiver operating characteristic (ROC) curves was 0.84 for the initial cohort and 0.77 for the validation cohort. In the initial cohort, the sensitivity of the FPI based on these five predictors was 96%, and the negative predictive value was 93% at a score of 0.2. At scores 0.8, the FPI was 94% specific and had a positive predictive value of 87%. In conclusion, an FPI using routinely assessed markers and incorporating a measure of insulin resistance can reliably predict the probability of significant hepatic fibrosis in most patients with chronic HCV infection. Such an index should prove useful to guide decision making regarding the need for liver biopsy, and potentially for avoiding or deferring biopsy in a large proportion of patients with mild liver disease. (HEPATOLOGY 2004;39:1239-1247.)
Low membrane protein sulfhydrils but not G6PD deficiency predict ribavirin-induced hemolysis in hepatitis C (p 1248-1255)
Ignazio Grattagliano, Stefan Russmann, Vincenzo O. Palmieri, Peter Jüni, Florian Bihl, Piero Portincasa, Giuseppe Palasciano, Bernhard H. Lauterburg
Hemolysis is a frequent adverse effect of ribavirin (RBV). It has been suggested that oxidative stress plays a role, but mechanisms and predictive risk factors for severe forms remain unknown. Markers of redox status were determined in erythrocytes of 34 patients with hepatitis C - four of them with glucose-6-phosphate-dehydrogenase (G6PD) deficiency - before and during treatment with RBV and interferon (IFN) and were compared with 10 healthy control subjects. In addition, erythrocytes were incubated with RBV, and the effects of dipyridamole (DPD), diethylmaleate (DEM), and glutathione ester (GSHE) were studied in vitro . Of the 30 patients without G6PD deficiency who were treated with RBV and IFN- , five developed major hemolysis ( hemoglobin > 6 g/dL) and 25 developed minor hemolysis ( hemoglobin < 2.5 g/dL). Patients with major hemolysis had lower median pretreatment values of membrane protein sulfhydrils than patients with minor hemolysis (28.4 vs. 36.7 nmol/mg, P< .001). Erythrocytes of G6PD-deficient patients were not more susceptible to RBV-induced hemolysis. In in vitro incubations of erythrocytes, DEM enhanced the RBV-induced decrease of glutathione, protein sulfhydrils, and osmotic resistance. Supplementation of GSHE and DPD prevented the RBV-induced decrease in osmotic resistance, adenosyl triphosphate (ATP), and 2,3-diphosphoglycerate (DPG), the loss of glutathione and protein sulfhydrils, and the formation of thiobarbituric acid reactive substances (TBARs). In conclusion, the data indicate that low membrane protein sulfhydrils prior to therapy but not G6PD deficiency are predictive of RBV-induced major hemolysis. In vitro , GSHE and DPD reduce the RBV-associated oxidative stress in erythrocytes and prevent the increase in osmotic fragility, suggesting that these compounds might decrease the risk of hemolysis in patients. (HEPATOLOGY 2004;39:1248-1255.)
Recently primed CD8 +T cells entering the liver induce hepatocytes to interact with naïve CD8 +T cells in the mouse (p 1256-1266)
Nektarios Dikopoulos, Ursula Wegenka, Andrea Kröger, Hansjörg Hauser, Reinhold Schirmbeck, Jörg Reimann
Large number of T cells traffic through the liver. In order to examine the effects of such traffic on the phenotype of hepatocytes, we vaccinated mice using DNA vaccines encoding antigens with MHC class I-binding epitopes. Small numbers of activated CD8 +T blasts (10 5-10 6/liver) changed the surface phenotype and cytokine expression profile of hepatocytes (HCs). HCs upregulate surface expression of major histocompatibility complex (MHC) class I molecules and CD1d but not MHC class II molecules Qa-1, CD80, CD86, CD54, or CD95; in addition, they expressed/secreted interleukin (IL)-10 and IL-4 but not IL-1, IL-6, IL-13, interferon (IFN)- , tumor necrosis factor (TNF), IL-4, or IL-27 ( i.e. , they acquire the HC* phenotype). HCs* (but not HCs) induced specific activation, proliferation, and IFN- , TNF, and IL-13 release of cocultured naïve CD8 +T cells. In contrast to the specific activation of naïve CD8 +T cells by dendritic cells (DCs), specific CD8 +T cell activation by HC* was not down-modulated by IFN- . Only recently activated CD8 +T blasts (but not recently activated CD4 +T blasts or activated cells of the innate immune system, including natural killer T [NKT] cells) induced the HC* phenotype that is prominent from day 10 to day 20 postvaccination ( i.e. , time points at which peak numbers of recently primed CD8 +T blasts are found in the liver). In conclusion, recently activated CD8 +T blasts that enter the liver postimmunization in small numbers can transiently modulate the phenotype of HC, allowing them to activate naïve CD8 +T cells with unrelated specificities. (HEPATOLOGY 2004;39:1256-1266.)
Liver Biology and Pathobiology
Activation of hepatic Nrf2 in vivo by acetaminophen in CD-1 mice (p 1267-1276)
Christopher E. P. Goldring, Neil R. Kitteringham, Robert Elsby, Laura E. Randle, Yuri N. Clement, Dominic P. Williams, Michael McMahon, John D. Hayes, Ken Itoh, Masayuki Yamamoto, B. Kevin Park
The transcription factor NF-E2-related factor 2 (Nrf2) plays an essential role in the mammalian response to chemical and oxidative stress through induction of hepatic phase II detoxification enzymes and regulation of glutathione (GSH). Enhanced liver damage in Nrf2-deficient mice treated with acetaminophen suggests a critical role for Nrf2; however, direct evidence for Nrf2 activation following acetaminophen exposure was previously lacking. We show that acetaminophen can initiate nuclear translocation of Nrf2 in vivo, with maximum levels reached after 1 hour, in a dose dependent manner, at doses below those causing overt liver damage. Furthermore, Nrf2 was shown to be functionally active, as assessed by the induction of epoxide hydrolase, heme oxygenase-1, and glutamate cysteine ligase gene expression. Increased nuclear Nrf2 was found to be associated with depletion of hepatic GSH. Activation of Nrf2 is considered to involve dissociation from a cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), through a redox-sensitive mechanism involving either GSH depletion or direct chemical interaction through Michael addition. To investigate acetaminophen-induced Nrf2 activation we compared the actions of 2 other GSH depleters, diethyl maleate (DEM) and buthionine sulphoximine (BSO), only 1 of which (DEM) can function as a Michael acceptor. For each compound, greater than 60% depletion of GSH was achieved; however, in the case of BSO, this depletion did not cause nuclear translocation of Nrf2. In conclusion, GSH depletion alone is insufficient for Nrf2 activation: a more direct interaction is required, possibly involving chemical modification of Nrf2 or Keap1, which is facilitated by the prior loss of GSH. (HEPATOLOGY 2004;39:1267-1276.)
Pivotal role of superoxide anion and beneficial effect of antioxidant molecules in murine steatohepatitis (p 1277-1285)
Alexis Laurent, Carole Nicco, Jeanne Tran Van Nhieu, Didier Borderie, Christiane Chéreau, Filomena Conti, Patrick Jaffray, Olivier Soubrane, Yvon Calmus, Bernard Weill, Frédéric Batteux
Nonalcoholic fatty liver disease, frequently associated with obesity, can lead to nonalcoholic steatohepatitis (NASH) and cirrhosis. The pathophysiology of NASH is poorly understood, and no effective treatment is available. In view of a potential deleterious role for reactive oxygen species (ROS), we investigated the origin of ROS overproduction in NASH. Mitochondrial production of ROS and its alterations in the presence of antioxidant molecules were studied in livers from ob/ob mice that bear a mutation of the leptin gene and develop experimental NASH. N-acetyl-cysteine and the superoxide dismutase (SOD) mimics ambroxol, manganese [III] tetrakis (5,10,15,20 benzoic acid) (MnTBAP), and copper [II] diisopropyl salicylate (CuDIPS) were used to target different checkpoints of the oxidative cascade to determine the pathways involved in ROS production. Liver mitochondria from ob /ob mice generated more O 2°-than those of lean littermates ( P< .01). Ex vivo , all three SOD mimics decreased O 2°-generation ( P< .001) and totally inhibited lipid peroxidation ( P< .001) versus untreated ob /ob mice. Those modifications were associated with in vivo improvements: MnTBAP and CuDIPS reduced weight ( P< .02) and limited the extension of histological liver steatosis by 30% and 52%, respectively, versus untreated ob /ob mice. In conclusion, these data demonstrate deleterious effects of superoxide anions in NASH and point at the potential interest of nonpeptidyl mimics of SOD in the treatment of NASH in humans. (HEPATOLOGY 2004;39:1277-1285.)
Administration of the potent PPAR agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice (p 1286-1296)
Emilia Ip, Geoff Farrell, Pauline Hall, Graham Robertson, Isabelle Leclercq
Administration of a methionine and choline deficient (MCD) diet to rodents causes progressive fibrosing steatohepatitis pathologically similar to human metabolic steatohepatitis. We have previously shown that the peroxisome proliferator-activated receptor- (PPAR ) agonist, Wy-14,643, prevented the development of MCD diet-induced steatohepatitis. We have now tested whether Wy-14,643 ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice were fed the MCD diet for 51 days to induce severe steatohepatitis. They were then treated with Wy-14,643 together with the MCD diet for 5 or 12 days; positive controls continued on the MCD diet for 5 or 12 days. After 5 days of Wy-14,643 treatment, alanine aminotransferase (ALT) levels were significantly decreased, steatohepatitis less severe, and hepatic lipoperoxides significantly reduced. After 12 days, hepatic triglycerides were normalized and there was near resolution of histological changes. MCD dietary feeding was associated with increased expression of vascular cell adhesion molecule (VCAM)-1, and increased numbers of activated macrophages in the liver. Treatment with Wy-14,643 reduced VCAM-1 expression and macrophage numbers. MCD diet-fed mice developed hepatic fibrosis with increased hepatic collagen 1(I), tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and matrix metalloproteinase (MMP)-13 mRNA levels. After treatment with Wy-14,643, expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells and near resolution of liver fibrosis. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by treatment with Wy-14,643. It is likely that activation of PPAR reverses fibrosis indirectly by reducing stimuli, such as lipid peroxides, and activation of cells responsible for promoting hepatic fibrosis. (HEPATOLOGY 2004;39:1286-1296.)
Murine alanine aminotransferase: cDNA cloning, functional expression, and differential gene regulation in mouse fatty liver (p 1297-1302)
Sanjay B. Jadaho, Rong-Ze Yang, Qiang Lin, Hong Hu, Frank A. Anania, Alan R. Shuldiner, Da-Wei Gong
Alanine aminotransferase (ALT) is a widely used index of liver integrity or hepatocellular damage in clinics as well as a key enzyme in intermediatary metabolism. In this study, we have cloned the complementary DNAs of murine homologues of human alanine aminotransferase 1 and 2 (ALT1 and ALT2). The deduced peptides of murine ALT1 (mALT1) and ALT2 (mALT2) share 87% and 93% identity, respectively, with their human counterparts at the amino acid level. Murine ALT genes localize to separate chromosomes, with mALT1 gene ( gpt1 ) on chromosome 15 and mALT2 gene ( gpt2 ) on chromosome 8. The murine gpt1 and gpt2 differ in messenger RNA expression: gpt1 is mainly expressed in liver, bowel, and white adipose tissue and gpt2 is highly expressed in muscle, liver, and white adipose tissue. Expression of recombinant mALT1 and mALT2 proteins in Escherichia coli (E. coli ) produced functional enzymes that catalyze alanine transamination. The potential diagnostic value of ALT isoenzymes in liver disease was evaluated in an obese animal model. In fatty livers of obese mice, ALT2 gene expression is induced 2-fold, but ALT1 remains the same. Furthermore, in fatty liver, total hepatic ALT activity is elevated significantly by 30% whereas aspartate aminotransferase (AST) activity remains unchanged. In conclusion, these results indicate that ALT2 may be responsible for the increased ALT activity in hepatic steatosis and provide evidence that an ALT isoenzyme-specific assay may have more diagnostic value than the total ALT activity assay currently in clinical use. (HEPATOLOGY 2004;39:1297-1302.)
Hepatic transmethylation reactions in micropigs with alcoholic liver disease (p 1303-1310)
Jesus A. Villanueva, Charles H. Halsted
Alcoholic liver disease is associated with abnormal hepatic methionine metabolism, including increased levels of homocysteine and S-adenosylhomocysteine (SAH) and reduced levels of S-adenosylmethionine (SAM) and glutathione (GSH). The concept that abnormal methionine metabolism is involved in the pathogenesis of alcoholic liver disease was strengthened by our previous findings in a micropig model where combining dietary folate deficiency with chronic ethanol feeding produced maximal changes in these metabolites together with early onset of microscopic steatohepatitis and an eightfold increase in plasma aspartate aminotransferase. The goal of the present study was to determine potential mechanisms for abnormal levels of these methionine metabolites by analyzing the transcripts and activities of transmethylation enzymes in the livers of the same micropigs. Ethanol feeding or folate deficiency, separately or in combination, decreased transcript levels of methylenetetrahydrofolate reductase (MTHFR), methionine adenosyltransferase (MAT1A), glycine-N-methyltransferase (GNMT) and S-adenosylhomocysteine hydrolase (SAHH). Ethanol feeding alone reduced the activities of methionine synthase (MS) and MATIII and increased the activity of GNMT. Each diet, separately or in combination, decreased the activities of MTHFR and SAHH. In conclusion, the observed abnormal levels of methionine metabolites in this animal model of accelerated alcoholic liver injury can be ascribed to specific effects of ethanol with or without folate deficiency on the expressions and activities of hepatic enzymes that regulate transmethylation reactions. These novel effects on transmethylation reactions may be implicated in the pathogenesis of alcoholic liver disease. (HEPATOLOGY 2004;39:1303-1310.)
Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytes in vitro and in vivo to TNF-mediated apoptosis (p 1311-1320)
Hannes Hentze, Markus Latta, Gerald Künstle, Saravanakumar Dhakshinamoorthy, Poh Yong Ng, Alan G. Porter, Albrecht Wendel
Topoisomerases are nuclear enzymes that maintain and modulate DNA structure. Inhibitors of topoisomerases like camptothecin (CPT), etoposide, and others are widely used antitumor drugs that interfere with transcription, induce DNA strand breaks, and trigger apoptosis preferentially in dividing cells. Because transcription inhibitors (actinomycin D, galactosamine, -amanitin) sensitize primary hepatocytes to the cytotoxic action of tumor necrosis factor (TNF), we reasoned whether topoisomerase inhibitors would act similarly. CPT alone was not toxic to primary cultured murine hepatocytes. When incubated with CPT, murine hepatocytes displayed an inhibition of protein synthesis and were thereby rendered sensitive to apoptosis induction by TNF. Apoptosis was characterized by morphology (condensed/fragmented nuclei, membrane blebbing), caspase-3-like protease activity, fragmentation of nuclear DNA, and late cytolysis. Hepatocytes derived from TNF receptor-1 knockout mice were resistant to CPT/TNF-induced apoptosis. CPT treatment completely abrogated the TNF-induced NF-kappa B activation, and mRNA expression of the antiapoptotic factors TNF-receptor associated factor 2, FLICE-inhibitory protein, and X-linked inhibitor of apoptosis protein was also inhibited by CPT. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-chloromethylketone (zDEVD-fmk), as well as depletion of intracellular ATP by fructose prevented CPT/TNF-induced apoptosis. In vivo , CPT treatment sensitized mice to TNF-induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies. (HEPATOLOGY 2004;39:1311-1320.)
Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice (p 1321-1331)
Makoto Ochi, Hideki Ohdan, Hiroshi Mitsuta, Takashi Onoe, Daisuke Tokita, Hidetaka Hara, Kohei Ishiyama, Wendy Zhou, Yuka Tanaka, Toshimasa Asahara
Although it is known that activation of natural killer (NK) cells causes liver injury, the mechanisms underlying NK cell-induced killing of self-hepatocytes are not clear. We demonstrated that liver NK cells have cytotoxicity against normal syngeneic hepatocytes in mice. Polyinosinic-polycytidylic acid (poly I:C) treatment enhanced hepatocyte toxicity of liver NK cells but not that of spleen NK cells. Unlike NK cells in other tissues, approximately 30%-40% of liver NK cells constitutively express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). An in vitro NK cell cytotoxic assay revealed that hepatocyte toxicity of liver NK cells from both naïve and poly I:C-treated mice was inhibited partially by an anti-TRAIL monoclonal antibody (mAb) alone and completely by the combination with anti-Fas ligand (FasL) mAb and a perforin inhibitor, concanamycin A, indicating contribution of TRAIL to NK cell-mediated hepatocyte toxicity. The majority of TRAIL +NK cells lacked expression of Ly-49 inhibitory receptors recognizing self-major histocompatibility complex class I, indicating a propensity to targeting self-hepatocytes. Poly I:C treatment significantly upregulated the expression of Ly-49 receptors on TRAIL -NK cells. This might be a compensatory mechanism to protect self-class I-expressing cells from activated NK cell-mediated killing. However, such compensatory alteration was not seen at all in the TRAIL +NK cell fraction. Thus, liver TRAIL +NK cells have less capacity for self-recognition, and this might be involved in NK cell-dependent self-hepatocyte toxicity. In conclusion, our findings are consistent with a model in which TRAIL-expressing NK cells play a critical role in self-hepatocyte killing through poor recognition of MHC. (HEPATOLOGY 2004;39:1321-1331.)
Interleukin 22 (IL-22) plays a protective role in T cell-mediated murine hepatitis: IL-22 is a survival factor for hepatocytes via STAT3 activation (p 1332-1342)
Svetlana Radaeva, Rui Sun, Hong-na Pan, Feng Hong, Bin Gao
The central role of T cell activation in hepatocellular injury has been well documented. In this article, we provide evidence suggesting that T cells may also play a protective role in liver disease by releasing interleukin-22 (IL-22), a recently identified T cell-derived cytokine whose biological significance is unclear. IL-22 messenger RNA and protein expression are significantly elevated in T cell-mediated hepatitis induced by concanavalin A (ConA) but are less extensively elevated in the carbon tetrachloride-induced liver injury model. Activated CD3 +T cells are likely responsible for the production of IL-22 in the liver after injection of ConA. The IL-22 receptor is normally expressed at high levels by hepatocytes and further induced after ConA injection. IL-22 blockade with a neutralizing antibody reduces signal transducer and activator of transcription factor 3 (STAT3) activation and worsens liver injury in T cell-mediated hepatitis, whereas injection of recombinant IL-22 attenuates such injury. In vitro treatment with recombinant IL-22 or overexpression of IL-22 promotes cell growth and survival in human hepatocellular carcinoma HepG2 cells. Stable overexpression of IL-22 in HepG2 cells constitutively activates STAT3 and induces expression of a variety of antiapoptotic ( e.g. , Bcl-2, Bcl-xL, Mcl-1) and mitogenic ( e.g. , c-myc, cyclin D1, Rb2, CDK4) proteins. Blocking STAT3 activation abolishes the antiapoptotic and mitogenic actions of IL-22 in hepatic cells. In conclusion, the T cell-derived cytokine IL-22 is a survival factor for hepatocytes; this suggests that T cell activation may also prevent and repair liver injury by releasing hepatoprotective cytokine IL-22 in addition to its previously documented central role in hepatocellular injury. (HEPATOLOGY 2004;39:1332-1342.)
Cytokines increase CRE binding but decrease CRE-mediated reporter activity in rat hepatocytes by increasing c-Jun (p 1343-1352)
Baochun Zhang, Shubing Liu, Michele D. Perpetua, William H. Walker, Brian G. Harbrecht
The cyclic AMP response element (CRE) has been implicated in the regulation of the expression of many genes and cellular processes important in hepatocyte function. CRE sites exist in the promoter regions of several genes expressed during inflammation. Numerous studies on the role of CRE in hepatocyte gene expression have been performed in resting hepatocytes, but the role of CRE during inflammation is unknown. To evaluate the regulation of CRE-mediated transcription during sepsis, cultured hepatocytes were exposed to proinflammatory cytokines and lipopolysaccharide (LPS) was injected into rats. Nuclear proteins were collected and CRE binding activity measured by electromobility shift assay (EMSA) using a consensus CRE oligonucleotide. CRE binding activity was increased in vitro by cytokines and in vivo by LPS administration but CRE-dependent reporter activity was decreased by cytokine stimulation. A c-jun N-terminal kinase (JNK) inhibitor reversed the cytokine-induced increase in CRE binding and increased CRE-dependent reporter activity. Supershift assays indicated that cyclic AMP response element binding protein (CREB) and c-Jun proteins were included in the CRE binding complex. CREB induced and c-Jun suppressed reporter activity using a CRE-dependent construct transfected into cultured primary hepatocytes. In conclusion, these data demonstrate that proinflammatory cytokines regulate CRE binding and activity in cultured hepatocytes and suggest that sepsis-induced changes in CRE binding may participate in the cellular response to inflammation. (HEPATOLOGY 2004;39:1343-1352.)
2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: Confocal and electron microscopic studies (p 1353-1361)
Sándor Paku, Peter Nagy, László Kopper, Snorri S. Thorgeirsson
The 2-acetylaminofluorene (AAF)/partial hepatectomy (PH) model is one of the most extensively studied experimental systems for oval cell proliferation and differentiation. We have previously described the oval cells as forming ductular structures surrounded by basement membrane, representing extensions of the canals of Hering. Herein we analyze the differentiation of oval cells into hepatocytes after varying degrees of liver damage induced by AAF. At a low dose of AAF, most oval cells synchronously differentiate into small hepatocytes by 6 days after the PH, resulting in complete restoration of the liver structure in 10 days. Higher doses of AAF delay the differentiation process and the new hepatocytes form foci, in contrast to what is observed at the low dose. Qualitatively, the differentiation process seems to be identical at the cellular level under both conditions. The transition from the expanding oval cell population into hepatocytes was correlated with the upregulation of hepatocyte nuclear factor 4 and the disappearance of the basement membrane. Also, the differentiation of oval cells into hepatocytes coincided with the loss of alpha-fetoprotein and OV-6 staining, and the replacement of the biliary cell-specific 6 integrin and connexin 43 with the hepatocyte-specific 1 integrin and connexin 32. In addition, bile canaliculi form between the new hepatocytes. In conclusion, these results indicate the rate of oval cell differentiation into hepatocytes is context dependent and suggest that, under favorable conditions, oval cells can complete this process much faster than previously appreciated. (HEPATOLOGY 2004;39:1353-1361.)
Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver (p 1362-1370)
Toshitaka Hoppo, Hideaki Fujii, Tetsuro Hirose, Kentaro Yasuchika, Hisaya Azuma, Shinji Baba, Masato Naito, Takafumi Machimoto, Iwao Ikai
Previously, we reported a system to enrich mouse fetal hepatic progenitor cells (HPCs) by forming cell aggregates. In this study, we sorted two cell populations, CD49f +Thy1 -CD45 -cells (CD49f-postive cells) and CD49f ±Thy1 +CD45 -cells (Thy1-positive cells), from the cell aggregates using a flow cytometer. CD49f-positive cells stained positive for endodermal specific markers such as -fetoprotein (AFP), albumin (ALB), and cytokeratin 19 (CK19), and are thus thought to be HPCs. However, Thy1-positive cells were a morphologically heterogeneous population; reverse-transcription polymerase chain reaction (RT-PCR) and immunocytochemical analyses revealed the expression of mesenchymal cell markers such as -smooth muscle actin, desmin, and vimentin, but not of AFP, ALB, or CK19. Therefore, Thy1-positive cells were thought to be of a mesenchymal lineage. When these two cell populations were co-cultured, the CD49f-positive colonies matured morphologically and stored a significant amount of glycogen. Furthermore, real-time RT-PCR demonstrated an increased expression of tyrosine amino transferase and tryptophan oxygenase mRNA, and transmission electron microscopy confirmed that co-cultured cells produced mature hepatocytes. However, when CD49f-positive cells were cultured alone or when the two populations were cultured separately, the CD49f-positive cells did not mature. These results indicate that CD49f-positive cells are primitive hepatic endodermal cells with the capacity to differentiate into hepatocytes, and that Thy1-positive cells promote the maturation of CD49f-positive cells by direct cell-to-cell contact. In conclusion, we were able to isolate CD49f-positive primitive hepatic endodermal cells and Thy1-positive mesenchymal cells and to demonstrate the requirement of cell-to-cell contact between these cell types for the maturation of the hepatic precursors. (HEPATOLOGY 2004;39:1362-1370.)
An oncolytic adenoviral vector of Smac increases antitumor activity of TRAIL against HCC in human cells and in mice (p 1371-1381)
Zifei Pei, Liang Chu, Weiguo Zou, Zilai Zhang, Songbo Qiu, Rong Qi, Jinfa Gu, Cheng Qian, Xinyuan Liu
Hepatocellular carcinoma (HCC) displays a high resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death. To increase sensitivity of HCC cells to TRAIL, we have constructed an oncolytic adenoviral vector (ZD55) and used this vector to deliver second mitochondria-derived activator of caspases (Smac) and TRAIL genes (ZD55-Smac and ZD55-TRAIL, respectively) into HCC cells. Our data showed that human HCC cells express high levels of inhibitor of apoptosis proteins (IAPs). Transfected HCC cells expressing exogenous X-linked IAPs (XIAPs) displayed more resistance to TRAIL. The expression of Smac led to rapid and potent activation of apoptosis in HCC cells after infection with ZD55-Smac. The activation of caspases and induction of apoptosis could be enhanced further through coinfection with ZD55-TRAIL. The combined treatment of ZD55-Smac and ZD55-TRAIL resulted in significant reduction of XIAP expression levels. In addition, our in vivo data in mice showed only a partial response in the established tumor treated either by ZD55-Smac or ZD55-TRAIL alone. By contrast, complete tumor regression was observed by combination of ZD55-Smac and ZD55-TRAIL in all treated animals. This strong antitumoral activity achieved by this combination was due to a dramatic induction of tumor cell apoptosis in the treated tumors. In conclusion, our data indicate that Smac antagonizes the IAPs in HCC tumor cells and enhances tumor cell death induced by TRAIL in the oncolytic adenoviral vector. The combination of Smac and TRAIL delivered by way of the oncolytic adenoviral vector would provide a useful strategy for therapy of HCC and might also be applied to other IAPs abundant in cancers. (HEPATOLOGY 2004;39:1371-1381.)
Down-regulation of the organic cation transporter 1 of rat liver in obstructive cholestasis (p 1382-1389)
Gerald U. Denk, Carol J. Soroka, Albert Mennone, Hermann Koepsell, Ulrich Beuers, James L. Boyer
The liver plays a major role in biotransformation and elimination of various therapeutic agents and xenobiotics, many of which are organic cations and substrates of the organic cation transporter 1 (Oct1, Slc22a1). Oct1 is expressed at the basolateral membranes of hepatocytes and proximal renal tubules. Although Oct1 is the major uptake mechanism in hepatocytes for many pharmaceutical compounds, little is known about the effects of liver injury on this process. Our aim was to investigate the effects of obstructive cholestasis on Oct1 expression and function in liver and kidney. The effects of bile duct ligation (BDL) on Oct1 protein, messenger RNA (mRNA) expression, and tissue localization were determined in rat liver and kidney with Western analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunofluorescence. To assess Oct1 function, the model substrate tetraethylammonium ([ 14 C]TEA) was administered intravenously to BDL and control rats and distribution of radioactivity was determined. Oct1 protein significantly decreased in cholestatic livers to 42.1 ± 17.7% ( P< .001), 15.5 ± 4.7% ( P< .05), and 8.6 ± 2.7% ( P< .05) of controls after 3, 7, and 14 days, respectively, but not in kidneys. Hepatic Oct1 mRNA decreased to 77.2 ± 12.7%, 40.7 ± 8.1% ( P< .05), and 50.3 ± 7.5% ( P< .05) 3, 7, and 14 days after BDL, respectively. Tissue immunofluorescence corroborated these data. Hepatic accumulation of [ 14 C]TEA in 14-day BDL rats was reduced to 29.6 ± 10.9% of controls ( P< .0005). In conclusion, obstructive cholestasis down-regulates Oct1 and impairs Oct1-mediated uptake in rat liver, suggesting that hepatic uptake of small cationic drugs may be impaired in cholestatic liver injury. (HEPATOLOGY 2004;39:1382-1389.)
Liver Failure and Chronic Liver Disease
A double-blind randomized controlled trial of infliximab associated with prednisolone in acute alcoholic hepatitis (p 1390-1397)
Sylvie Naveau, Sylvie Chollet-Martin, Sébastien Dharancy, Philippe Mathurin, Pauline Jouet, Marie-Astrid Piquet, Thierry Davion, Frédéric Oberti, Philippe Broët, Dominique Emilie
Tumor necrosis factor- (TNF- ) may contribute to the progression of acute alcoholic hepatitis (AAH). The aim of this study was to evaluate the efficacy of an association of infliximab and prednisolone at reducing the 2-month mortality rate among patients with severe AAH. Patients with severe AAH (Maddrey score 32) were randomly assigned to group A receiving intravenous infusions of infliximab (10 mg/kg) in weeks 0, 2, and 4; or group B receiving a placebo at the same times. All patients received prednisolone (40 mg/day) for 28 days. Blood neutrophil functional capacities were monitored over 28 days. After randomization of 36 patients, seven patients from group A and three from group B died within 2 months. The probability of being dead at 2 months was higher (not significant [NS]) in group A (39% ± 11%) than in group B (18% ± 9%). The study was stopped by the follow-up committee and the sponsor (Assistance Publique-Hôpitaux de Paris). The frequency of severe infections within 2 months was higher in group A than in group B ( P< .002). This difference was potentially related to a significantly lower ex vivo stimulation capacity of neutrophils. There were no differences between the two groups in terms of Maddrey scores at any time point. In conclusion, three infusions of 10 mg/kg of infliximab in association with prednisolone may be harmful in patients with severe AAH because of the high prevalence of severe infections. (HEPATOLOGY 2004;39:1390-1397.)
Asialoglycoprotein receptor facilitates hemolysis in patients with alcoholic liver cirrhosis (p 1398-1407)
Philip Hilgard, Thomas Schreiter, Richard J. Stockert, Guido Gerken, Ulrich Treichel
Hemolysis in patients with advanced alcoholic liver disease is a common clinical problem and indicates an unfavorable prognosis. In many cases, the etiology of the hemolysis remains unknown. We observed three patients with alcoholic liver disease, suffering from severe hemolytic anemia, requiring multiple blood transfusions. Steroid therapy was ineffective and two of the patients died. All patients had a soluble variant of the human asialoglycoprotein receptor (s-ASGP-R) in their serum, as well as high titers of autoantibodies against this receptor (anti-ASGP-R). Consecutively, examination of 60 patients with alcoholic liver disease revealed a high incidence for s-ASGP-R (36%) and anti-ASGP-R (27%) in patients with alcoholic liver cirrhosis (ALC) compared to patients with cirrhosis due to viral hepatitis. The potential etiology of hemolysis was studied in vitro on erythrocytes from patients with ALC and from healthy donors. Isolated ASGP-R but not anti-ASGP-R bound to the surface of erythrocytes preferentially of blood group A1 and caused dose-dependent agglutination and hemolysis, while this phenomenon was much lower using erythrocytes of the blood group B and almost absent with blood group O-erythrocytes. Furthermore, agglutination and hemolysis only occurred in erythrocytes from ALC-patients or after the pre-treatment of cells with neuraminidase. ASGP-R induced agglutination and hemolysis was blocked by the competitive ASGP-R inhibitor asialofetuin. In conclusion, our results indicate a new, non-immunological mechanism for hemolysis in patients with alcoholic liver disease, mediated through agglutination by a soluble variant of the human asialoglycoprotein receptor and mechanical shear stress. (HEPATOLOGY 2004;39:1398-1407.)
In vitro comparison of the molecular adsorbent recirculation system (MARS) and single-pass albumin dialysis (SPAD) (p 1408-1414)
Igor M. Sauer, Max Goetz, Ingo Steffen, Gesa Walter, Daniel C. Kehr, Ruth Schwartlander, Yoon J. Hwang, Andreas Pascher, Joerg C. Gerlach, Peter Neuhaus
The detoxification capacities of single-pass albumin dialysis (SPAD), the molecular adsorbents recirculation system, (MARS) and continuous veno-venous hemodiafiltration (CVVHDF) were compared in vitro . In each experiment 4,100 mL of toxin-loaded human plasma was processed for 6.5 hours. MARS treatment (n = 6) was undertaken in combination with CVVHDF. For SPAD (n = 6) and CVVHDF (n = 6) a high-flux hollow fiber hemodiafilter (identical to the MARS filter) was used. Levels of ammonia, urea, creatinine, bilirubin, and bile acids were determined. Concentrations before and after application of detoxification procedures were expressed as differences and were compared using the Kruskal-Wallis test. Post hoc comparisons for pairs of groups were adjusted according to Bonferroni-Holm. Time, group, and interaction effects were tested using the nonparametric ANOVA model for repeated measurements. SPAD and CVVHDF induced a significantly greater reduction of ammonia levels than MARS. No significant differences were found among SPAD, MARS, and CVVHDF with respect to other water-soluble substances. SPAD induced a significantly greater reduction in bilirubin levels than MARS. Reductions in bile acid levels were similar for SPAD and MARS. When operating MARS in continuous veno-venous hemodialysis mode, as recommended by the manufacturer, no significant differences in the removal of bilirubin, bile acids, urea, and creatinine were found. However, MARS in continuous veno-venous hemodialysis mode was significantly less efficient in removing ammonia than MARS in CVVHDF mode. In conclusion, the detoxification capacity of SPAD is similar to or even greater than that of MARS. (HEPATOLOGY 2004;39:1408-1414.)
Caspase induction by IgA antimitochondrial antibody: IgA-mediated biliary injury in primary biliary cirrhosis (p 1415-1422)
Shuji Matsumura, Judy Van de Water, Patrick Leung, Joseph A. Odin, Kazuhide Yamamoto, Gregory J. Gores, Keith Mostov, Aftab A. Ansari, Ross L. Coppel, Yasushi Shiratori, M. Eric Gershwin
Anti-mitochondrial antibodies (AMAs) have long been recognized as a serological hallmark of primary biliary cirrhosis (PBC). Although high titers of immunoglobulin (Ig)A AMAs are found in bile, saliva, and urine of patients, a pathogenic role for this antibody has remained elusive. Functional studies of this IgA in general have been impeded by low quantities of antibody and the inability to recover antigen-specific IgA in dimeric form. Using a newly defined synthetic group A. Streptococcus derived peptide, we purified large quantities of dimeric and monomeric IgA from patient sera. The purified IgA was incubated with Madine-Darby canine kidney (MDCK) cells transfected with the human polymeric Ig receptor (pIgR) and the cells studied by flow cytometric analysis for binding of carboxyfluorescein conjugated VAD-fmk peptide to activated caspase enzymes. A total of 87% of PBC patients that were anti-PDC-E2 positive had serum IgA that increased caspase activation in MDCK-pIgR+ cells compared to serum-derived IgA from controls with a maximum reaction 48 hours after addition of IgA. The titer of anti-PDC-E2 IgA among the PBC patients strongly correlated with caspase activation (cc = 0.88). Pre-absorption of the IgA using recombinant 2-oxo-acid dehydrogenase complex significantly diminished this activation. IgG from the same PBC patients did not induce caspase activation. These data suggest that during transcytosis through pIgR-positive cells, exposure to PDC-E2-specific dimeric IgA results in the initiation of caspase activation. In conclusion, we propose that due to an even greater concentration of dimeric IgA in biliary and mucosal secretions, constant transcytosis would render the exposed cells more susceptible to apoptosis resulting in subsequent bile duct damage. (H EPATOLOGY 2004;39:1415-1422.)
The zonal distribution of megamitochondria with crystalline inclusions in nonalcoholic steatohepatitis (p 1423-1429)
Tri H. Le, Stephen H. Caldwell, Jan A. Redick, Bonnie L. Sheppard, Christine A. Davis, Kristen O. Arseneau, Julia C. Iezzoni, Elizabeth E. Hespenheide, Abdullah Al-Osaimi, Theresa C. Peterson
Megamitochondria with crystalline inclusions (MMC) have been previously described in nonalcoholic fatty liver; however, their distribution within hepatic zones is unknown. We sought to determine this distribution from the core liver biopsy specimens of 31 patients: 8 males and 23 females, age range 21 to 72 years. Twenty-nine showed evidence of nonalcoholic steatohepatitis (NASH) on biopsy with steatosis, inflammation, varying degree of fibrosis, ballooned hepatocytes, and Mallory hyaline, and two patients had cryptogenic cirrhosis thought to represent burned out NASH. Identified by transmission electron microscopy, the abundance of MMC was compared between low-stage (fibrosis stages 1 and 2) and high-stage (fibrosis stages 3 and 4) groups and between zones with or without difference in fibrosis stage. Regardless of stage, the MMC were distributed equally in all zones and were abundant similarly in low- and high-stage groups. This abundance did not correlate with the degree of oxidative stress (4-hydroxynonenal staining) or with the abundance of ballooned hepatocytes. Consistent with age as a risk factor for more severe disease, the median age for the low-stage group was significantly lower than that of the high-stage group ( P= .003). In conclusion, in NASH, the MMC seem to be distributed randomly among zones and without variation in abundance, regardless of the fibrosis stage. The exact function of these structures remains to be defined. In this study, their presence did not seem to correlate with the light microscopic injury pattern represented by ballooned hepatocytes or degree of oxidative stress defined by immunostaining for 4-hydroxynonenal. (H EPATOLOGY 2004;39:1423-1429.)
Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity (p 1430-1440)
Guruprasad P. Aithal, Lesley Ramsay, Ann K. Daly, Nhareet Sonchit, Julian B. S. Leathart, Graeme Alexander, J. Gerald Kenna, John Caldwell, Christopher P. Day
Diclofenac is a nonsteroidal anti-inflammatory drug that causes rare but serious hepatotoxicity, the mechanism of which is unclear. The purpose of the present study was to explore the potential role played by the immune processes. Antibodies to diclofenac metabolite-modified liver protein adducts were detected in the sera of seven out of seven patients with diclofenac-induced hepatotoxicity, 12 of 20 subjects on diclofenac without hepatotoxicity, and none of four healthy controls. The antibodies recognized adducts expressed in livers from rats treated with multiple doses of diclofenac, but not in those given single doses. In addition, several potential diclofenac adducts were identified in the liver of a patient with diclofenac-induced hepatic failure, but not from a normal human donor liver, by immunoblotting with an adduct-selective rabbit antiserum. To determine whether or not polymorphisms in genes encoding cytokine-related proteins influence susceptibility to hepatotoxicity, genotyping for the polymorphisms -627 in the interleukin (IL)-10 gene, -590 in the IL-4 gene, and codon 551 in the IL-4 receptor (IL-4R) were performed on DNA from 24 patients on diclofenac with hepatotoxicity, 48 subjects on diclofenac without hepatotoxicity, and healthy controls. The frequencies of the variant alleles for IL-10 and IL-4 were higher in patients (OR [odds ratio]: 2.8 for IL-10; 2.6 for IL-4; 5.3 for IL-10 + IL-4) compared with healthy controls and subjects on diclofenac without hepatotoxicity (OR: 2.8 for IL-10; 1.2 for IL-4; 5.0 for IL-10 + IL-4). In conclusion, the observed polymorphisms, resulting in low IL-10 and high IL-4 gene transcription, could favor a T helper (Th)-2 mediated antibody response to neoantigenic stimulation associated with disease susceptibility. H EPATOLOGY 2004;39:1430-1440.)
Synbiotic modulation of gut flora: Effect on minimal hepatic encephalopathy in patients with cirrhosis (p 1441-1449)
Qing Liu, Zhong Ping Duan, Da Kang Ha, Stig Bengmark, Jelica Kurtovic, Stephen M. Riordan
Minimal hepatic encephalopathy (MHE) is an important disorder that may seriously impair daily functioning and quality of life in patients with cirrhosis. Treatment with lactulose is of benefit. The possible role of synbiotics (probiotics and fermentable fiber) has not been assessed. We screened 97 consecutive cirrhotic patients without overt hepatic encephalopathy for MHE using the number connection test and measurement of brainstem auditory evoked potentials. MHE, defined by abnormality on at least one test modality, was present in 58 (60%) patients. Fifty-five of these patients with MHE were randomized to receive a synbiotic preparation (n = 20), fermentable fiber alone (n = 20), or placebo (n = 15) for 30 days. Cirrhotic patients with MHE were found to have substantial derangements in the gut microecology, with significant fecal overgrowth of potentially pathogenic Escherichia coli and Staphylococcal species. Synbiotic treatment significantly increased the fecal content of non-urease-producing Lactobacillus species at the expense of these other bacterial species. Such modulation of the gut flora was associated with a significant reduction in blood ammonia levels and reversal of MHE in 50% of patients. Synbiotic treatment was also associated with a significant reduction in endotoxemia. The Child-Turcotte-Pugh functional class improved in nearly 50% of cases. Treatment with fermentable fiber alone was also of benefit in a substantial proportion of patients. In conclusion, treatment with synbiotics or fermentable fiber is an alternative to lactulose for the management of MHE in patients with cirrhosis. (H EPATOLOGY 2004;39:1441-1449.)
Hepatology Elsewhere
Treatment of the pruritus of cholestasis: From unbearable lightness to substance (p 1450-1453)
Nora V. Bergasa
Background & Aims To assess the efficacy and safety of naltrexone for the short and long term treatment of pruritus of cholestasis.
Methods Twenty patients with pruritus and cholestasis were included. A baseline pruritus score was obtained over 1 week. Patients were then randomized to receive 50 mg/day of naltrexone or placebo for 2 weeks. Subsequently, a 1-week washout period ensued and patients were crossed over to the other therapy for 2 additional weeks. Pruritus was assessed daily with a visual analogue scale (VAS) from 0 to 10. Patients whose pruritus decreased >50% of basal with naltrexone received naltrexone 50 mg/day for 2 additional months. Results Mean basal VAS was similar in both groups. VAS showed greater and more significant changes with naltrexone than with placebo ( P< .0003). In nine out of 20 patients (45%) receiving naltrexone, pruritus decreased >50% compared to basal value, including five whose pruritus disappeared completely. No significant changes were observed in serum biochemistry. Most of the adverse events that occurred during the first 48 h of naltrexone therapy were consistent with opioid withdrawal-like phenomena and spontaneously disappeared 2 days after starting treatment. Conclusions
Naltrexone can be considered as an alternative option to treat pruritus of cholestasis. In the current study, side effects were transient and did not require specific medication. Controlled trials have suggested that opiate antagonist therapy may be effective for the treatment of the symptoms of cholestasis. The oral opiate antagonist naltrexone in particular has started to enter into routine clinical use for amelioration of cholestatic itch. Attention regarding the side effects of opiate antagonist therapy has, to date, largely focused on an opiate withdrawal-type reaction (which can be controlled effectively by titrated therapy introduction regimens). Here we describe three cases of a further clinically important side effect: loss of control of pain resulting from other pathologies, which in each case necessitated the withdrawal of hitherto clinically effective opiate antagonist therapy. Of the 14 patients treated by our unit with opiate antagonist agents for the control of cholestatic symptoms, 13 (93%) showed resolution of or significant improvement in symptoms. Of the 13 patients showing a clinical response, seven (54%) subsequently had to discontinue therapy because of side effects (including the three patients with uncontrolled pain). It is our experience that in the routine clinical setting, opiate antagonists are highly effective for the treatment of cholestatic symptoms. In practice, however, their usefulness is limited by their side effect profile.
Save Article Copyright © 2004 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for may 2004 • Volume 126 • Number 5
Rapid Communication
Inducible cre-mediated control of gene expression in the murine gastrointestinal tract: Effect of loss of -catenin
Heather Ireland, Richard Kemp, Carol Houghton, Louise Howard, Alan R. Clarke, Owen J. Sansom, Douglas J. Winton
Background & Aims : A system for introducing specific gene mutations into the epithelia of the adult murine gastrointestinal tract by the transcriptional regulation of Cre recombinase is presented and applied to delete -catenin, a central mediator of Wnt signaling, within the small intestine (SI). Methods : In a transgenic line (Ahcre), cre expression is inducible from a cytochrome P450 promoter element that is transcriptionally up-regulated in response to lipophilic xenobiotics such as -napthoflavone. Results : Recombination at a lacZ reporter locus showed extensive expression of -galactosidase in liver, intestine, pancreas, gallbladder, esophagus, and stomach in response to -napthoflavone treatment. Expression patterns were stable in renewing epithelia for at least 6 months, implying that long-lived stem cells undergo recombination. Analysis of the intestinal epithelium showed dose responsiveness in the extent of recombination and that villus and crypt populations could be targeted differentially by varying the route of administration of -napthoflavone. The use of this system to delete -catenin in the SI caused crypt ablation, increased apoptosis, depleted numbers of goblet cells, and detachment of villus absorptive cells from the villus core as intact sheets. Conclusions : The Ahcre model provides a simple route for introducing specific gene mutations into many of the epithelia of the gastrointestinal tract of the mouse. It has been used here to show that -catenin is required for the maintenance of intestinal cell proliferation and is implicated in goblet cell differentiation and enterocyte-matrix attachment.
Clinicalalimentary Tract
Wide variation in adenoma detection rates at screening flexible sigmoidoscopy
Wendy Atkin, Pauline Rogers, Christopher Cardwell, Claire Cook, Jack Cuzick, Jane Wardle, Rob Edwards
Background & Aims : Wide between-center variation in adenoma detection rates (ADRs) was observed in the U.K. Flexible Sigmoidoscopy Screening Trial (overall, 12.1%; range, 8.6%15.9%; P< 0.0001). The aim of this study was to determine whether the observed differences could be attributed to varying performance by endoscopists, to examine the effect of experience on performance, and to identify an attainable, standard ADR to which endoscopists could aspire. Methods : Thirteen medical endoscopists, one per trial center, each performed about 3000 examinations (200 per month) using the same equipment and protocol. Overall and monthly ADRs were compared using multivariable logistic regression. Results : Differences in ADRs were not explained by patient characteristics, incidence of colorectal cancer in the local population, or the endoscopists’ medical specialty or previous experience. Average ADRs increased significantly with screening experience (up to 400 examinations). Endoscopists were classified as higher, intermediate, or lower adenoma detectors, and performance levels were maintained over time. Higher detectors had ADRs of 15% overall (men, 20%; women, 10%) and also detected more adenomas per case (higher/lower detectors, 21.7/10.4 adenomas per 100 examinations). Conclusions : The differences in ADRs were due to variation in performance of the endoscopists. Long-term follow-up will determine whether this variation is clinically important. We suggest that the standards in higher detecting centers should be achievable by all endoscopists screening unscreened populations aged older than 55 years. Endoscopists should aim to stay above the lower 95% confidence interval band for 200 examinations (10% overall; 5% in women, 15% in men).
A meta-analysis of the placebo rates of remission and response in clinical trials of active crohn’s disease
Chinyu Su, Gary R. Lichtenstein, Karen Krok, Colleen M. Brensinger, James D. Lewis
Background & Aims: Placebo-controlled, randomized clinical trials (PC-RCTs) are commonly used to assess therapies for Crohn’s disease (CD). Knowledge of the placebo rates of remission and response and understanding of design factors that influence these rates is important for designing future clinical trials evaluating pharmacotherapy of CD. The aims of this study were to estimate rates of remission and response in patients with active CD receiving placebo and to identify factors influencing these rates. Methods: We performed a systematic review and meta-analysis of PC-RCTs evaluating therapies for active CD identified from MEDLINE from 1966 to 2001. Results: The pooled estimates of the placebo rates of remission and response were 18% (95% confidence interval, 14%24%; range, 0%50%) and 19% (95% confidence interval, 13%28%; range, 0%46%), respectively, both with significant heterogeneity among studies ( P< 0.01 for remission, P< 0.03 for response). In multivariate models, study duration, number of study visits, and entry Crohn’s Disease Activity Index score were important predictors of the placebo remission rate, with study duration the most important. However, no single factor could account for all of the heterogeneity. Factors that influence the placebo response rates were similar to those affecting the placebo remission rates. The absolute benefit of active treatment beyond placebo was generally larger when outcome was measured by response than remission. Conclusions: Placebo remission and response rates in PC-RCTs for active CD are variable. Study duration, number of study visits, and disease severity at entry have a large influence on placebo remission rates.
Fecal DNA testing compared with conventional colorectal cancer screening methods: A decision analysis
Kenneth Song, A. Mark Fendrick, Uri Ladabaum
Background & Aims : Fecal DNA testing is an emerging tool to detect colorectal cancer (CRC). Our aims were to estimate the clinical and economic consequences of fecal DNA testing vs. conventional CRC screening. Methods : Using a Markov model, we estimated CRC incidence, CRC mortality, and discounted cost/life-year gained for screening by fecal DNA testing (F-DNA), fecal occult blood testing (FOBT) and/or sigmoidoscopy, or colonoscopy (COLO) in persons at average CRC risk from age 50 to 80 years. Results : Compared with no screening, F-DNA at a screening interval of 5 years decreased CRC incidence by 35% and CRC mortality by 54% and gained 4560 life-years per 100,000 persons at $47,700/life-year gained in the base case. However, F-DNA gained fewer life-years and was more costly than conventional screening. The average number of colonoscopies per person was 3.8 with COLO and 0.8 with F-DNA. In most 1-way sensitivity analyses and Monte Carlo simulation iterations, F-DNA remained reasonably cost-effective compared with no screening, but COLO and FOBT dominated F-DNA. Assuming fecal DNA testing sensitivities of 65% for CRC and 40% for large polyp, and 95% specificity, a screening interval of 2 years and a test cost of $195 would be required to make F-DNA comparable with COLO. Conclusions : Fecal DNA testing every 5 years appears effective and cost-effective compared with no screening, but inferior to other strategies such as FOBT and COLO. Fecal DNA testing could decrease the national CRC burden if it could improve adherence with screening, particularly where the capacity to perform screening colonoscopy is limited.
Fecal lactoferrin for diagnosis of symptomatic patients with ileal pouch-anal anastomosis
Mansour A. Parsi, Bo Shen, Jean-paul Achkar, Feza F. Remzi, John R. Goldblum, James Boone, Dahai Lin, Jason T. Connor, Victor W. Fazio, Bret A. Lashner
Background & aims : Increased stool frequency, urgency, and abdominal pain in patients with ileal pouch-anal anastomosis (IPAA) may be due to inflammatory conditions, including pouchitis, cuffitis, or Crohn’s disease or noninflammatory conditions such as irritable pouch syndrome. Distinction among these entities requires pouch endoscopy and biopsy. Noninvasive means of diagnosis are preferable. Methods : Sixty consecutive subjects with IPAA for inflammatory bowel disease had measurements of fecal lactoferrin and 1-antitrypsin and underwent pouch endoscopy with biopsy, with calculation of the pouchitis disease activity index in a prospective cross-sectional study. Results : Symptomatic patients with an inflammatory condition had significantly higher fecal lactoferrin concentrations (median, 176.0 µg/mL, interquartile range [IQR] 79.0450.8) compared with those with a noninflammatory condition (median, 4.8 µg/mL; IQR, 1.211.0) or those who were asymptomatic (median, 7.8 µg/mL; IQR, 1.412.9), P< 0.001. At a cutoff level of 7 µg/mL, fecal lactoferrin could distinguish patients with irritable pouch syndrome from those with pouchitis, cuffitis, or Crohn’s disease with a sensitivity of 100% and specificity of 85%. Fecal 1-antitrypsin was not able to distinguish symptomatic patients with and without an inflammatory condition. Conclusions : Fecal lactoferrin can serve as a sensitive and noninvasive initial screening test in an algorithm for evaluation of symptomatic patients with IPAA. If fecal lactoferrin levels are low (<7 µg/mL), IPS can be diagnosed. If fecal lactoferrin levels are high, pouch endoscopy with biopsy is warranted to distinguish among different causes of inflammation. Longitudinal studies are needed to define better the role of this test in the management of patients with IPAA.
Clinical-liver, Pancreas, and Biliary Tract
Patients with elevated liver enzymes are not at higher risk for statin hepatotoxicity
Naga Chalasani, Hisham Aljadhey, Joe Kesterson, Michael D. Murray, Stephen D. Hall
Background & Aims : Studies that evaluate the risk of hepatotoxicity from statins in hyperlipidemic subjects with elevated baseline serum transaminases are lacking. We conducted a study to test the hypothesis that patients with elevated baseline liver enzymes have higher risk of statin hepatotoxicity. Methods : Our study consisted of the following 3 cohorts of patients seen between January 1, 1998 and June 31, 2002: Cohort 1: 342 hyperlipidemic patients with elevated baseline enzymes (AST >40 IU/L or ALT >35 IU/L) who were prescribed a statin; cohort 2: 1437 hyperlipidemic patients with normal transaminases who were prescribed a statin; and cohort 3: 2245 patients with elevated liver enzymes but who were not prescribed a statin. The effect of statins on liver biochemistries was assessed over a 6-month period after statins were prescribed. Elevations in liver biochemistries during follow-up were categorized into mild-moderate or severe based on predefined criteria. Results : The incidence of mild-moderate elevations and severe elevations in liver biochemistries in cohort 1 were 4.7% and 0.6%, respectively. Compared with cohort 1, individuals in cohort 2 had lower incidence of mild-moderate elevations (1.9%, P= 0.002) but not severe elevations (0.2%, P= 0.2). However, between cohorts 1 and 3, there were no differences in the incidence of mild-moderate elevations (4.7% vs. 6.4%, respectively, P= 0.2) or severe elevations (0.6% vs. 0.4%, respectively, P= 0.6). Statin discontinuation during the follow-up was similar between cohorts 1 and 2 (11.1% vs. 10.7%, respectively, P= 0.8). Conclusions : These data suggest that individuals with elevated baseline liver enzymes do not have higher risk for hepatotoxicity from statins.
The effect of alcohol consumption on the prevalence of iron overload, iron deficiency, and iron deficiency anemia
George N. Ioannou, Jason A. Dominitz, Noel S. Weiss, Patrick J. Heagerty, Kris V. Kowdley
Background & Aims: Our aim was to investigate the relationship between alcohol consumption and iron overload, iron deficiency, or iron deficiency anemia in the U.S. population. Methods: Adult participants of the Third National Health and Nutrition Examination Survey who did not consume alcohol (n = 8839) were compared with participants who consumed 1 (n = 4976), >1 to 2 (n = 1153), or >2 (n = 915) alcoholic drinks/day during the preceding 12 months. We examined the following markers of iron overload: elevated serum transferrin-iron saturation (TS) level (>45%, >50%, and >60%), elevated serum ferritin level (>300, >400, >500, and >600 ng/mL), and combinations of both elevated serum TS and ferritin levels. Iron deficiency was defined as the presence of at least 2 of the following: serum ferritin level <12 ng/mL, serum TS level <15%, and erythrocyte protoporphyrin level >1.24 µmol/L. Iron deficiency anemia was defined as the presence of both iron deficiency and anemia. Results: Compared with nondrinkers, the prevalence of all markers of iron overload was significantly elevated among those who consumed >2 alcoholic drinks/day after adjusting for potential confounders. Consumption of any amount of alcohol was associated with a 40% reduction in the risk of iron deficiency anemia. Conclusions: Consumption of up to 2 alcoholic drinks/day seems to be associated with reduced risk of iron deficiency and iron deficiency anemia without a concomitant increase in the risk of iron overload. Consumption of >2 alcoholic drinks/day is associated with a significant elevation in the risk of iron overload.
Epoetin alfa maintains ribavirin dose in HCV-infected patients: A prospective, double-blind, randomized controlled study
Nezam H. Afdhal, Douglas T. Dieterich, Paul J. Pockros, Eugene R. Schiff, Mitchell L. Shiffman, Mark S. Sulkowski, Teresa Wright, Zobair Younossi, Betty L. Goon, K. Linda Tang, Peter J. Bowers
Background & Aims : Combination therapy with interferon (IFN- ) and ribavirin (RBV) or pegylated IFN- (PEG-IFN- )/RBV for chronic hepatitis C virus (HCV) infection often causes anemia, prompting RBV dose reduction/discontinuation. This study assessed whether epoetin alfa could maintain RBV dose, improve quality of life (QOL), and increase hemoglobin (Hb) in anemic HCV-infected patients. Methods : HCV-infected patients (n = 185) on combination therapy who developed anemia (Hb 12 g/dL) were randomized into a U. S. multicenter, placebo-controlled, clinical trial of epoetin alfa, 40,000 U subcutaneously, once weekly vs. matching placebo. The study design used an 8-week, double-blind phase (DBP) followed by an 8-week, open-label phase (OLP), in which placebo patients were crossed over to epoetin alfa. Results : At the end of the DBP, RBV doses were maintained in 88% of patients receiving epoetin alfa vs. 60% of patients receiving placebo ( P< 0.001). Mean QOL scores at the end of the DBP improved significantly on all domains of the Linear Analog Scale Assessment (LASA) and on 7 of the 8 domains of the Short Form-36, version 2 (SF-36v2). Mean Hb increased by 2.2 ± 1.3 g/dL (epoetin alfa) and by 0.1 ± 1.0 g/dL (placebo) in the DBP ( P< 0.001). Similar results were demonstrated in patients who switched from placebo to epoetin alfa in the OLP. Epoetin alfa was well tolerated; the most common adverse effects were headache and nausea. Conclusions : Epoetin alfa maintained RBV dose and improved QOL and Hb in anemic HCV-infected patients receiving combination therapy.
Association of antioxidant enzyme gene polymorphisms and glutathione status with severe acute pancreatitis
Sakhawat H. Rahman, Khadija Ibrahim, Michael Larvin, Andrew Kingsnorth, Michael J. McMahon
Background & aims : Genetic variations in antioxidant metabolism may explain varying biological responses to acute pancreatitis (AP). We studied the contribution of oxidative stress to the pathogenesis of severe pancreatitis by examining the prevalence of functional gene polymorphisms of antioxidant enzymes and evidence of heightened oxidative stress. Methods : DNA from 320 patients with AP (90 severe) and 263 controls was genotyped for glutathione S-transferase (Mu-1 [M-1], theta-1 [T-1], and pi-1 [P-1: Ile-105Val]), manganese superoxide dismutase (Ala-9Val), and catalase (C-260T) polymorphisms. Erythrocyte reduced glutathione (GSH) concentration was determined 24 and 72 hours after the onset of pain in 46 patients (11 severe). Disease severity was assessed using Atlanta clinical criteria, Acute Physiology Scores (APS), and peak serum C-reactive protein levels. Results : The functional GSTT-1*A genotype was more prevalent in severe (96%) compared with mild attacks of AP (78%; odds ratio [OR], 5.9; 95% confidence interval [CI ], 217; P< 0.0001) and controls (76%; OR, 6.6; 95% CI, 2.318.7; P< 0.0001). Compared with null genotype, GSTT-1*A was associated with higher peak C-reactive protein levels (184 vs. 94 g/dL; P= 0.0005) and APS (24 hours, P= 0.04; 48 hours, P= 0.015). Reduced glutathione (GSH) at 24 hours was lower in mild (median, 382 nmol/g) and severe attacks (median, 407 nmol/g) compared with controls (median, 3685 nmol/g; P< 0.001). Levels increased at 72 hours in mild ( P= 0.012) but not severe attacks and inversely correlated with APS ( r= 0.49; P= 0.04). Conclusions : The functional GSTT-1*A genotype was associated with severe attacks of pancreatitis. Heightened oxidative stress characterized by glutathione depletion may be of importance in mediating the progression from mild to severe pancreatitis.
Telangiectatic focal nodular hyperplasia: A variant of hepatocellular adenoma
Valerie Paradis, Asmae Benzekri, Delphine Dargère, Ivan Bièche, Ingrid Laurendeau, Valerie Vilgrain, Jacques Belghiti, Michel Vidaud, Claude Degott, Pierre Bedossa
Background & Aims : “Telangiectatic focal nodular hyperplasia” designate atypical lesions considered as variants of focal nodular hyperplasia (FNH). However, because “telangiectatic FNH” share several morphologic patterns with hepatocellular adenomas, classification of such lesions deserve further clarification. Therefore, the aim of the present study was to reconsider the classification of telangiectatic FNH with the help of a molecular approach. Methods : Ten telangiectatic FNH, 6 typical FNH, and 6 hepatocellular adenomas were studied. DNA, RNA, and protein from each lesion were extracted. Clonality was assessed by the study of the X chromosome inactivation pattern (HUMARA assay). Angiopoietin ( ANGPT-1 and ANGPT-2 ) mRNA, genes the expression of which is typically modified in FNH, were quantified by a real-time RT-PCR procedure. Protein profiles were analyzed by SELDI-TOF PROTEINCHIP (Cyphergen Biosystem, Inc., Fremont, CA) technology. Results : Although all informative cases of FNH (5 of 6) and hepatocellular adenomas (6 of 6) were polyclonal and monoclonal, respectively, clonal analysis showed a nonrandom pattern of X chromosome inactivation consistent with a monoclonal lesion in 6 of 8 cases of telangiectatic FNH. The mean value of the ANGPT-1/ANGPT-2 mRNA ratio was 21.4 in FNH, 2.6 in adenomas, and 2.1 in telangiectatic FNH ( P0.001 in telangiectatic FNH vs. FNH). SELDI-TOF PROTEINCHIP profiling and hierarchical clustering analysis showed that all except 1 telangiectatic FNH clustered within the group of hepatocellular adenomas. Conclusions : These results show that telangiectatic FNH display a molecular pattern closer to that of hepatocellular adenomas than to FNH and suggest that these lesions should instead be referred to as “telangiectatic hepatocellular adenomas.”
Diagnosis of pancreatic cystic neoplasms: A report of the cooperative pancreatic cyst study
William R. Brugge, Kent Lewandrowski, Elizabeth Lee-Lewandrowski, Barbara A. Centeno, Tara Szydlo, Susan Regan, Carlos Fernandez del Castillo, Andrew L. Warshaw
Background & Aims: Cysts of the pancreas display a wide spectrum of histology, including inflammatory (pseudocysts), benign (serous), premalignant (mucinous), and malignant (mucinous) lesions. Endoscopic ultrasonography (EUS) may offer a diagnostic tool through the combination of imaging and guided, fine-needle aspiration (FNA). The purpose of this investigation was to determine the most accurate test for differentiating mucinous from nonmucinous cystic lesions. Methods: The results of EUS imaging, cyst fluid cytology, and cyst fluid tumor markers (CEA, CA 72-4, CA 125, CA 19-9, and CA 15-3) were prospectively collected and compared in a multicenter study using histology as the final diagnostic standard. Results: Three hundred forty-one (341) patients underwent EUS and FNA of a pancreatic cystic lesion; 112 of these patients underwent surgical resection, providing a histologic diagnosis of the cystic lesion (68 mucinous, 7 serous, 27 inflammatory, 5 endocrine, and 5 other). Receiver operator curve analysis of the tumor markers demonstrated that cyst fluid CEA (optimal cutoff of 192 ng/mL) demonstrated the greatest area under the curve (0.79) for differentiating mucinous vs. nonmucinous cystic lesions. The accuracy of CEA (88 of 111, 79%) was significantly greater than the accuracy of EUS morphology (57 of 112, 51%) or cytology (64 of 109, 59%) ( P< 0.05). There was no combination of tests that provided greater accuracy than CEA alone ( P< 0.0001). Conclusions: Of tested markers, cyst fluid CEA is the most accurate test available for the diagnosis of mucinous cystic lesions of the pancreas
Basic-alimentary Tract
Growth arrest, apoptosis, and telomere shortening of Barrett’s-associated adenocarcinoma cells by a telomerase inhibitor
Masood A. Shammas, Hemanta Koley, David G. Beer, Cheng Li, Raj K. Goyal, Nikhil C. Munshi
Background & Aims : Barrett’s esophageal adenocarcinoma (BEAC) is a complication of gastroesophageal reflux disease, with no effective chemotherapy and poor prognosis. BEAC cells, like many other types of cancers, may reactivate telomerase to achieve unlimited proliferative potential, making telomerase a unique therapeutic target. The purpose of this study was to evaluate effects of telomerase inhibition on BEAC. Methods : We examined the effect of a selective G-quadruplex intercalating telomerase inhibitor, 2,6-bis[3-{N-Piperidino}propionamido]anthracene-9,10-dione (PPA), on telomerase activity, telomere length, colony size distribution, and proliferative potential in 2 BEAC cell lines, BIC-1 and SEG-1. Results : Telomerase activity was >10-fold and >600-fold elevated in the adenocarcinoma cells as compared with normal gastric/intestinal cells and normal diploid fibroblasts, respectively. Telomeres were short, being less than 4 kilobase pair in both tumor cell lines. Exposure to PPA effectively inhibited telomerase activity and shortened telomeres. PPA also arrested cell proliferation and reduced colony number and size after a lag period of about 10 cell generations, consistent with the attrition of telomeres. The growth arrest was not due to senescence but was due to apoptosis. Expression analysis of the cells following PPA treatment did not show significant change in the expression of genes involved in cell-cycle proliferation and apoptosis. Exposure to PPA had no effect on proliferative potential of normal intestinal cells. Conclusions : We conclude that telomerase inhibition by PPA induces cell growth arrest in BEAC cells and demonstrate the potential of telomerase inhibitors in chemoprevention and treatment of Barrett’s-associated esophageal adenocarcinoma.
The expression and function of costimulatory molecules B7H and B7-H1 on colonic epithelial cells
Atsushi Nakazawa, Iris Dotan, Jens Brimnes, Matthieu Allez, Ling Shao, Fumihiko Tsushima, Miyuki Azuma, Lloyd Mayer
Background & Aims: Previous studies have suggested that intestinal epithelial cells (IECs) may function as antigen-presenting cells for CD4 +and CD8 +T cells. However, these cells fail to express conventional costimulatory molecules (CD80, CD86), leading to the possibility that antigen presented by normal IECs could result in anergy. Other members of the B7 family have recently been identified. B7h interacts with inducible costimulator (ICOS) on T cells and provides a positive signal, whereas B7-H1 and B7-DC interact with PD-1 and transmit an inhibitory signal. Our aim was to determine whether IECs express novel B7 family members and whether these molecules play a role in IEC:T-cell interactions. Methods: B7h and B7-H1 expression was assessed in isolated IECs and IEC lines. The functional role of B7h and B7-H1 in the interaction between IECs and T cells was assessed in coculture experiments using purified anti-B7h or B7-H1 monoclonal antibodies (mAbs), B7h immunoglobulin (Ig), or B7-H1 fusion proteins. Results: B7h and B7-H1 messenger RNA was detected in IEC lines and IECs from healthy controls and patients with inflammatory bowel disease (IBD). IECs from patients with IBD but not healthy controls expressed B7h and B7-H1 protein on their surface. Proliferation of IEC-stimulated T cells was inhibited only by B7h immunoglobulin treatment, whereas interferon gamma secretion in these cocultures was inhibited by both anti-B7h mAb and B7h Ig. No difference was seen between IBD or normal IEC populations. Conclusions: These data suggest that the B7h-ICOS costimulatory pathway may be important in IEC:T-cell interactions.
DNA from probiotic bacteria modulates murine and human epithelial and immune function
Humberto Jijon, Jody Backer, Hugo Diaz, Helen Yeung, David Thiel, Conor McKaigney, Claudio De Simone, Karen Madsen
Background & Aims: The intestinal epithelium must discriminate between pathogenic and nonpathogenic bacteria and respond accordingly. The aim of this study was to examine whether bacterial DNA can serve as the molecular basis for bacterial recognition. Methods: HT-29 monolayers were treated with various bacterial DNA and interleukin (IL)-8 secretion measured by enzyme-linked immunosorbent assay, nuclear factor B activation by electrophoretic mobility shift assay and reporter assays, and I B levels by Western blotting. Cytokine secretion in response to bacterial DNA was measured in murine colonic segments and splenocytes. IL-10deficient mice were fed DNA from VSL probiotic compound daily for 2 weeks. Colons were removed and analyzed for cytokine production and inflammation. Results: HT-29 cells responded with IL-8 secretion to bacterial DNA in a differential manner. In the presence of proinflammatory stimuli, VSL3 DNA inhibited IL-8 secretion, reduced p38 mitogen-activated protein kinase activation, delayed nuclear factor B activation, stabilized levels of I B, and inhibited proteasome function. VSL3 DNA inhibited colonic interferon (IFN)- secretion in mouse colons and also attenuated a Bacteroides vulgatus induced IFN- release from murine splenocytes. In mice, VSL3 DNA attenuated a systemic release of tumor necrosis factor in response to Escherichia coli DNA injection. Treatment of IL-10deficient mice with oral VSL3 DNA resulted in a reduction in mucosal secretion of tumor necrosis factor and IFN- and an improvement in histologic disease. Conclusions: DNA from probiotic bacteria can limit epithelial proinflammatory responses in vivo and in vitro. Systemic and oral administration of VSL3 DNA ameliorates inflammatory responses.
Basic-liver, Pancreas, and Biliary Tract
Disruption of -catenin pathway or genomic instability define two distinct categories of liver cancer in transgenic mice
Diego F. Calvisi, Valentina M. Factor, Sara Ladu, Elizabeth A. Conner, Snorri S. Thorgeirsson
Background & Aims : Human liver cancer can be divided into 2 categories that are characterized by activation of -catenin and genomic instability. Here we investigate whether similar categories exist among 5 transgenic models of liver cancer, including c-myc, transforming growth factor- , E2F-1, c-myc/transforming growth factor- , and c-myc/E2F-1 mice. Methods : The random amplified polymorphic DNA method was used to assess the overall genomic instability, and chromosomal loci affected by genomic alterations were determined by microsatellite analysis. -Catenin mutations and deletions were analyzed by polymerase chain reaction and sequencing screening. Cellular localization of -catenin and expression of -fetoprotein, a prognostic marker of hepatocellular carcinoma, were investigated by immunohistochemistry. Results : Liver tumors from the transgenic mice could be divided into 2 broad categories characterized by extensive genomic instability (exemplified by the c-myc/transforming growth factor- mouse) and activation of -catenin (exemplified by the c-myc/E2F-1 mouse). The c-myc/transforming growth factor- tumors displayed extensive genomic instability with recurrent loss of heterozygosity at chromosomes 1, 2, 4, 6, 7, 9, 12, 14, and X and a low rate of -catenin activation. The genomic instability was evident from the early dysplastic stage and occurred concomitantly with increased expression of -fetoprotein. The c-myc/E2F-1 tumors were characterized by a high frequency of -catenin activation in the presence of a relatively stable genome and low -fetoprotein levels. Conclusions : We have identified 2 prototype experimental models, i.e., c-myc/transforming growth factor- and c-myc/E2F-1 mice, for the 2 categories of human hepatocellular carcinoma characterized by genomic instability and -catenin activation, respectively. These mouse models will assist in the elucidation of the molecular basis of human hepatocellular carcinoma.
Activated natural killer T cells induce liver injury by Fas and tumor necrosis factor- during alcohol consumption
Masahiro Minagawa, Qinggao Deng, Zhang-xu Liu, Hidekazu Tsukamoto, Gunther Dennert
Background & aims : Chronic alcohol abuse induces liver injury and increases the severity of viral hepatitis, but the precise mechanisms responsible are not well understood. In particular, little is known about the role of natural killer T cells in alcohol-induced liver injury. Natural killer T cells are mediators of important regulator and effector functions making use of Fas and tumor necrosis factor (TNF)- in apoptosis induction. This report analyzes the role of natural killer T cells, Fas, and TNF- in a model of chronic alcohol consumption. Methods : Mice fed alcohol by intragastric tube were assayed for serum alanine aminotransferase values, liver histology, and liver mononuclear cells before and after activation of natural killer T cells by ligand -galactosylceramide. Results : In alcohol-consuming animals, liver natural killer T cells increase, and further activation by -galactosylceramide causes lethal liver injury. This is explained by alcohol-induced hepatocyte sensitization to cell-mediated lysis, which develops concomitant to increased cytolytic activity of natural killer T cells. Natural killer T cell-mediated apoptosis proceeds by the Fas pathway, and Fas is essential for alcohol-associated liver injury. TNF- plays an additional role as a defect in TNF receptor-1 inhibits alcohol-associated liver injury. Alcohol-fed natural killer T cell-deficient J 281 / mice express a delay in alcohol-induced liver injury. Conclusions : Alcohol consumption induces an increase of natural killer T cells in the liver and a high sensitivity of hepatocytes to cell-mediated lysis. Stimulation of natural killer T cells during alcohol consumption induces serious liver injury by a mechanism that involves concomitant signals by Fas and tumor necrosis factor receptor-1 on alcohol-stressed hepatocytes.
Basic-liver Pancreas and Biliary Tract
Multigenic control of hepatic iron loading in a murine model of hemochromatosis
Mounia Bensaid, Séverine Fruchon, Christine Mazères, Seiamak Bahram, Marie-paule Roth, Hélène Coppin
Background & Aims : Hereditary hemochromatosis is a common disorder of iron homeostasis characterized by increased dietary iron absorption and progressive iron accumulation, mainly in the liver. Most patients are homozygous for the C282Y mutation in the HFE gene. However, not all individuals carrying the hemochromatosis-predisposing genotype in the general population become iron loaded. Genetic modifiers have been shown to influence disease penetrance, but their number and chromosomal locations remain unknown, and their identification is hampered by complex interactions with environmental factors. To circumvent these difficulties, we used 2 strains of mice made deficient for the Hfe gene that strongly differ in their propensity to develop hepatic iron loading. Methods : To localize the loci controlling hepatic iron loading in this murine model of hemochromatosis, we produced 1028 mice by an F2 intercross between the C57BL/6 and DBA/2 Hfe -deficient strains. We selected the 276 mice that contributed the most to the total linkage information for genotyping with 145 microsatellite markers. Results : We mapped 4 modifier loci on chromosomes 7, 8, 11, and 12, with logarithm of odds scores of 14.47, 12.96, 6.04, and 6.72, respectively, in regions containing several genes recently shown to exert important roles in the regulation of iron metabolism. Conclusions : Our data provide a clear demonstration of the polygenic pattern of hepatic iron loading inheritance in Hfe -deficient mice. Examination of candidate genes residing at the loci identified in this study and genetic analysis of the syntenic chromosomal regions in humans may provide important insight into the heterogeneous disease presentation observed among HFE C282Y homozygotes.
Liver biopsy discloses a new apolipoprotein A-I hereditary amyloidosis in several unrelated Italian families
Laura Obici, Giovanni Palladini, Sofia Giorgetti, Vittorio Bellotti, Gina Gregorini, Eloisa Arbustini, Laura Verga, Sabrina Marciano, Simona Donadei, Vittorio Perfetti, Laura Calabresi, Cesare Bergonzi, Francesco Scolari, Giampaolo Merlini
Background & Aims : Hereditary systemic amyloidoses are autosomal dominant, late-onset disorders caused by mutations in the genes for a group of plasma proteins including transthyretin, lysozyme, fibrinogen A chain, gelsolin, apolipoprotein A-I, and apolipoprotein A-II. We investigated both phenotypic and genotypic aspects of apolipoprotein A-I amyloidosis unexpectedly disclosed by liver biopsy in 13 unrelated individuals with asymptomatic, persistent elevation of alkaline phosphatase and -glutamyltransferase levels. Methods : Immunoelectron microscopy was used for in situ characterization of amyloid deposits on liver biopsy specimens. Mutation analysis was performed by sequencing of the apolipoprotein A-I gene in all patients. Wild-type/variant apolipoprotein A-I ratio in plasma high-density lipoproteins was assessed by a peptide mass fingerprinting approach after purification of total apolipoprotein A-I of 2 patients. Results : Family history was informative in 5 cases. Renal failure developed in 9 cases. Hypogonadism due to testicular involvement was observed. Amyloid fibrils specifically stained with anti-apolipoprotein A-I antibody. A novel (Leu75Pro) heterozygous mutation in the apolipoprotein A-I gene was present in affected individuals but not in controls. Variant apolipoprotein A-I was about 10% of the total protein in high-density lipoproteins. Conclusions : The high number of individuals with apparently sporadic disease might reflect widespread occurrence of this mutation in the population and a milder phenotype of this variant compared with other apolipoprotein A-I amyloidogenic mutants. These findings suggest that specific staining for amyloid should be performed on liver biopsy of individuals with asymptomatic chronic elevation of alkaline phosphatase and -glutamyltransferase levels.
Special Report and Review
Colorectal cancer chemopreventionan overview of the science
Ernest T. Hawk, Asad Umar, Jaye L. Viner
The development and dissemination of sophisticated detection technologies have recently exposed the high prevalence of preinvasive colorectal neoplasia in the adult U.S. population. Although cancer screening and surveillance provide opportunities for risk stratification, they achieve risk reduction only when coupled with effective interventions. This review surveys the lead compounds for colorectal cancer prevention and the measures by which they may be prioritized for clinical testing. Clinical trials remain the rate-limiting step in agent development, and novel trial designs are needed to hasten agent identification and testing for cancer prevention. Innovative research models include the nesting of prevention end points within cancer treatment trials and within trials testing promising preventive compounds intended for nononcologic indications.
Digestive and liver diseases statistics, 2004
Mark W. Russo, Jeffrey T. Wei, Michelle T. Thiny, Lisa M. Gangarosa, Alphonso Brown, Yehuda Ringel, Nicholas J. Shaheen, Robert S. Sandler
Background & Aims : Digestive and liver diseases are associated with substantial morbidity and mortality in the United States. Statistics about the incidence, prevalence, mortality, and resource utilization of digestive and liver diseases in the United States may be cumbersome to obtain because they are scattered in multiple sources. These data may be useful for policy makers, grant applicants, and authors. Methods : Data on the most common gastrointestinal and liver diseases were collected from large publicly available national databases. Information was collected on inpatient and outpatient gastrointestinal complaints and diagnoses, gastrointestinal cancers, and deaths from common liver diseases. Results : The leading gastrointestinal complaint prompting an outpatient visit is abdominal pain, with 12.2 million annual visits, followed by diarrhea, nausea, and vomiting. Abdominal pain is the leading outpatient gastrointestinal diagnosis, accounting for 5.2 million visits annually, followed by gastroesophageal reflux disease, with 4.5 million visits. Gallstone disease is the most common inpatient diagnosis, with 262,411 hospitalizations and a median inpatient charge of $11,584. Colorectal cancer is the most common gastrointestinal cause of death and is the most common gastrointestinal cancer, with an incidence of 54 per 100,000. Among gastrointestinal cancers, primary liver cancer had the highest increase in incidence from 1992 to 2000. Conclusions : Gastrointestinal and liver diseases are associated with significant outpatient and inpatient healthcare utilization. Following trends in utilization is important for determining allocation of resources for health care and research.
Copyright © 2001-2004 by the American Gastroenterological Association. All rights reserved.
Table of Contents for Volume 40, Issue 5, May 2004
Cirrhosis and its Complications
Risk factors and clinical presentation of portal vein thrombosis in patients with liver cirrhosis
Lucio Amitrano et al.
Background/Aims Portal vein thrombosis in patients with liver cirrhosis is usually associated to hepatocellular carcinoma. Clinical presentation of non-neoplastic portal vein thrombosis (PVT) in cirrhotic patients has not been specifically studied and risk factors of PVT in this group of patients are still poorly understood. Methods We studied all patients with PVT and liver cirrhosis admitted to our Unit from January 1998 to December 2002. They were paired (by gender, age and Child-Pugh score) to a group of cirrhotic patients without PVT and screened for acquired and inherited thrombophilic risk factors. These factors together with the site of thrombosis and the severity of the liver disease were correlated to the clinical presentation of PVT. Results Out of a total of 701 cirrhotic patients admitted to our hospital and routinely screened with Doppler ultrasound, 79 (11.2%) were found to have PVT. Of these, 34 (43%) were asymptomatic and 45 (57%) were symptomatic (31 presented with portal hypertensive bleed and 14 with abdominal pain, 10 of whom had intestinal infarction). Mesenteric vein involvement was never asymptomatic and lead to intestinal ischemia or infarction. Most patients were in class Child-Pugh B and C. Among thrombophilic risk factors studied only the mutation 20210 of the prothrombin gene resulted independently associated to PVT. Conclusions Portal vein thrombosis may be completely asymptomatic in patients with liver cirrhosis; however in more than half of cases presents with life-threatening complications such as gastrointestinal haemorrhage and intestinal infarction. Cirrhotic patients with PVT usually have an advanced liver disease and the presence of the mutation 20210 of the prothrombin gene increases more than fivefold the risk of PVT. Keywords: Liver cirrhosis ;Portal vein thrombosis
Increased brain serotonin turnover correlates with the degree of shunting and hyperammonemia in rats following variable portal vein stenosis
Violina Lozeva et al.
Background/Aims Hepatic encephalopathy (HE) is a serious neuropsychiatric complication of chronic liver disease. Brain monoamines have been implicated in the pathogenesis of HE. We examined the relationship between monoamine dysfunction and the degree of portal-systemic shunting (PSS) in rats with varying degrees of PSS. Methods Concentrations of catecholamines, serotonin, histamine, precursors and metabolites in frontal cortex of rats with varying degrees of PSS (9-99.8%) were measured by HPLC. Results The concentrations of the serotonin precursor, tryptophan, and its metabolite, 5-HIAA were increased up to 4-fold in brains of rats with various degrees of PSS and were significantly correlated with the degree of shunting and with arterial ammonia levels. Brain levels of histamine, its precursor, L-histidine, and metabolite, tele -methylhistamine were significantly increased only following total shunting. Concentrations of catecholamines and their metabolites were not significantly correlated with degree of PSS or hyperammonemia. Conclusions Given the established role of the serotonin system in the regulation of sleep, circadian rhythmicity and locomotion these findings suggest that selective alterations of this system could be implicated in the pathogenesis of HE. Therapeutic approaches aimed at the normalization of serotonin turnover could be beneficial in the prevention and treatment of early neuropsychiatric symptoms of HE.
Keywords: Histamine ;Catecholamines ;Portacaval anastomosis (PCA)
Deficient phospholipase C activity in blood polimorphonuclear neutrophils from patients with liver cirrhosis
Cirrhosis and its Complications
Cristina Garfia, Inmaculada García-Ruiz and José Antonio Solís-Herruzo
Background/Aims Circulating neutrophils from cirrhotic patients have a reduced capacity to generate superoxide anion (O 2), which might contribute to frequent bacterial infections in these patients. We studied the signal transduction pathways involved in the generation of O 2in neutrophils from 98 cirrhotic patients and 46 healthy controls. Methods We measured O 2production in neutrophils induced by fMLP, opsonized zymosan, TNF , NaF, AlF 4, A23187 and phorbol myristate acetate. Furthermore, we measured phospholipase C activity in neutrophils from healthy controls and end-stage cirrhotic patients. Results O2production was decreased in neutrophils from patients in response to fMLP, opsonized zymosan and TNF . Likewise, response of these cells to G-protein stimulation by fluorides was also decreased. These reduced responses correlated significantly with the degree of liver dysfunction. On the contrary, neutrophils from patients responded normally to A23187 and phorbol esters stimulation indicating that Ca 2+ - and PKC-dependent pathways are intact in these cells. Finally, phospholipase C activity was markedly reduced in neutrophils from end-stage liver cirrhosis. Conclusions These data confirm that O 2generation by neutrophils is decreased in patients with cirrhosis, particularly in those with more severe liver dysfunction, and suggest that this defect involves phosphatidylinositol specific phospholipase C activity.
Keywords: Cirrhosis ;Neutrophils ;Superoxide anion ;Phospholipase C ;Signal transduction ;fMLP ;Zymosan
Maintenance of hemodynamic response to treatment for portal hypertension and influence on complications of cirrhosis
Càndid Villanueva et al.
Background/Aims Following treatment with beta blockers in patients with cirrhosis and portal hypertension, reduction of hepatic venous pressure gradient (HVPG) to <12 mmHg or by >20% of baseline induces an extremely low risk of variceal bleeding. However, several factors such as compliance to therapy or alcohol abstinence may change the initial response after a long follow-up, and the effect of response on other complications of cirrhosis is less clear. The aim of this study was to assess the long-term maintenance of hemodynamic response and its influence on complications of cirrhosis. Methods One hundred and thirty two cirrhotic patients received nadolol and isosorbide mononitrate to prevent variceal rebleeding. HVPG was measured at baseline, after 1 to 3 months under treatment and again 12 to 18 months later. Results Sixty four patients were responders. After a longer follow-up, earlier response did not change in 81% of cases. Changes of response were mainly related to modifications in medication dose or in alcohol intake. As compared with poor-responders, responders had a lower probability of developing ascites ( P<0.001) and related conditions, a greater improvement of Child-Pugh score ( P=0.03), and a lower likelihood of developing encephalopathy ( P=0.001) and of requiring liver transplantation ( P=0.002). Eleven responders and 22 poor-responders died ( P=0.029). Conclusions Hemodynamic response to treatment of portal hypertension is usually sustained after a long-term follow-up. Response decreases the probability of developing complications of cirrhosis and the need for liver transplantation, and significantly improves survival.
Keywords: Cirrhosis ;Pharmacological Therapy ;Portal hypertension ;Hemodynamic measurements ;Complications of cirrhosis
Increased protein kinase A regulatory subunit content and cGMP binding in erythrocyte membranes in liver cirrhosis
Carmina Montoliu et al.
Background/Aims : Patients with liver disease show increased plasma cGMP and decreased intracellular cGMP in lymphocytes. The initial aim of this work was to assess whether decreased intracellular cGMP and increased plasma cGMP may be due to increased ATP-dependent release of cGMP from cells. The results obtained led to a new aim: to identify and quantify a protein responsible for the increased cGMP binding found in erythrocyte membranes from patients with liver disease. Methods : ATP-dependent cGMP transport was determined in inside-out vesicles from erythrocyte membranes. cGMP-binding proteins were isolated from the membranes and identified by MALDI-TOF peptide mass fingerprint. Protein kinase A was quantified by immunoblotting. Results : ATP-independent cGMP binding is increased in erythrocyte membranes from patients. There is a significant increase in the membrane content of a cGMP-binding protein with Mr 48,000, which was identified as the regulatory subunit of protein kinase A. Conclusions : The content of the regulatory subunit of protein kinase A is significantly increased (twice) in erythrocyte membranes from patients with liver cirrhosis. This protein binds cGMP strongly and may be responsible for the decrease in intracellular cGMP in liver disease.
Keywords: cGMP transport ;Nitric oxide ;Soluble guanylate cyclase ;cGMP homeostasis ;Hepatic encephalopathy
Inflammation and Fibrosis
Cellular retinol-binding protein-1 expression in normal and fibrotic/cirrhotic human liver: different patterns of expression in hepatic stellate cells and (myo)fibroblast subpopulations
Sébastien Lepreux et al.
Background/Aims Cellular retinol-binding protein-1 (CRBP-1) which is involved in vitamin A metabolism is highly expressed in liver cells, particularly in hepatic stellate cells (HSCs). In this work, the CRBP-1 expression was studied by immunohistochemistry in the different liver cell populations, including HSCs and portal fibroblasts, of normal liver and of fibrotic and cirrhotic liver. Methods Normal liver, fibrotic liver in different stages and cirrhotic liver sections were studied. Immunohistochemistry was performed using antibodies against CRBP-1, -smooth muscle actin (SMA), CD 68 and CD 34. Results In normal liver, quiescent HSCs expressed CRBP-1, while portal fibroblasts did not. In fibrotic or cirrhotic liver, activated HSCs co-expressed CRBP-1 and -SMA; a variable proportion of portal and septal (myo)fibroblasts, more important in cirrhosis, neo-expressed both CRBP-1 and -SMA. Biliary epithelial cells both in normal and pathological situations expressed CRBP-1. Neither Kupffer cells, nor endothelial cells showed CRBP-1 expression. Conclusions Our study demonstrates that CRBP-1 is a good marker to identify HSC in normal human liver. Furthermore, in fibrotic or cirrhotic liver, the different patterns of expression for CRBP-1 and -SMA allow the distinction of different subsets of fibroblastic cells involved in fibrogenesis and septa formation.
Keywords: Cellular retinol-binding protein-1 ;Hepatic stellate cell ;Portal fibroblast ;Myofibroblast ;-Smooth muscle actin ;Biliary epithelial cell ;Fibrosis ;Cirrhosis ;Liver
Polymorphisms of microsomal triglyceride transfer protein gene and manganese superoxide dismutase gene in non-alcoholic steatohepatitis
Chikako Namikawa et al.
Background/Aims The pathogenesis of non-alcoholic steatohepatitis (NASH) is poorly understood. The aim of this study was to examine genetic influences on NASH pathogenesis. Methods Blood samples from 63 patients with biopsy-proven NASH and 150 healthy controls were analyzed by the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Two functional polymorphisms were studied: the 493 G/T polymorphism in the promoter of microsomal triglyceride transfer protein (MTP) and the 1183 T/C polymorphism in the mitochondrial targeting sequence of manganese superoxide dismutase (MnSOD). Results NASH patients had a much higher incidence of the MTP gene G allele ( P=0.001) and of the G/G genotype ( P=0.002) compared to the controls. Fat occupied more area in liver lobules and the stage of NASH was advanced in patients with the G/G-genotype than in patients with G/T-genotype ( P=0.04). NASH patients also had a higher incidence of the MnSOD T/T genotype ( P=0.016). Conclusions The G allele in the MTP promoter leads to decreased MTP transcription, less export of triglyceride from hepatocytes, and greater intracellular triglyceride accumulation. The T allele in MnSOD mitochondrial targeting sequence leads to less transport of MnSOD to the mitochondria. Therefore, functional polymorphisms in MTP and MnSOD may be involved in determining susceptibility of NASH.
Keywords: Non-alcoholic steatohepatitis ;Oxidative stress ;4-Hydroxy-2-nnoneal ;Manganese superoxide dismutase ;Body mass index ;Radical oxygen species Abbreviations: A, Alanine ;BMI, body mass index ;C, cytosine ;G, guanine ;HCV, hepatitis C virus ;HNE, 4-hydroxy-2-nonenal ;MnSOD, manganese superoxide dismutase ;MTP, microsomal triglyceride transfer protein ;NASH, non-alcoholic steatohepatitis ;PCR, polymerase chain reaction ;RFLP, restriction fragment length polymorphism ;ROS, reactive oxygen species ;T, thymine ;V, Valine ;VLDL, very low density lipoprotein
Liver Growth and Cancer
Establishment of an orthotopic tumour model for hepatocellular carcinoma and non-invasive in vivo tumour imaging by high resolution ultrasound in mice
Volker Schmitz et al.
Background/Aims In this study we established an orthotopic tumour model for hepatocellular carcinoma and evaluated a non-invasive high resolution ultrasound technique for diagnosis and follow-up of intrahepatic HCC. Methods Orthotopic liver tumours were induced by intrahepatic tumour cell injection of 10 5Hepa129 hepatoma cells. Tumour establishment and growth were assessed by explorative laparotomy, ultrasound technique and hepatectomy one and two weeks after tumour cell implantation. Tumour establishment was confirmed histologically in liver sections. Results Our results show that the Hepa129 hepatoma cell line is suitable for orthotopic tumour establishment and that tumours can be diagnosed correctly by ultrasound imaging in all cases as confirmed by explorative laparotomy, hepatectomy and cross-sections. Tumour diameters obtained by explorative laparotomy correlated significantly with diameters assessed by ultrasound ( r=0.7; P<0.0001). Tumour burden was slightly overestimated (1.2-fold) by ultrasound one week after tumour induction and relative tumour extensions increased 1.7-fold and 1.8-fold within one week as determined by subsequent explorative laparotomy or ultrasound imaging, respectively. Conclusions These data demonstrate in a systematic study that ultrasound imaging can be used as a reliable tool to detect and to follow up orthotopic liver tumours in this tumour model in mice.
Keywords: Cancer ;Hepatocellular carcinoma ;High resolution ultrasound ;Tumour model ;Ultrasound imaging
Cortistatin production by HepG2 human hepatocellular carcinoma cell line and distribution of somatostatin receptors
George Notas et al.
Background/Aims Recently, trials of octreotide have shown a significant survival benefit in the treatment of advanced hepatocellular carcinoma but new data are controversial. We, therefore, examined the production of somatostatin and cortistatin, the expression and distribution of somatostatin receptors (sst) in HepG2 human hepatocellular carcinoma cells, and the possible antiproliferative effect of octreotide on these cells. Methods Radioimmunoassay and RT-PCR studies were performed for the detection of somatostatin and cortistatin. RT-PCR, radioligand binding and immunocytochemistry assays were employed for the detection of the ssts. Growth and viability of cells were measured by the tetrazolium salt assay. Results HepG2 cells were found to express sst 2, sst 3and sst 5receptors. Immunocytochemistry revealed a mainly intracellular distribution of all ssts with unique patterns for each of them. Membrane binding sites for somatostatin were mainly of the sst 3(39±8%) and sst 5(59±5%) types, while only minor sst 2binding could be detected (5±12%). Octreotide was found to inhibit the proliferation of HepG2 cells (IC 50 1.25?10 9M) via protein tyrosine phosphatases. HepG2 cells produced cortistatin while somatostatin expression was not detected. Conclusions In conclusion, HepG2 cells express cortistatin, which regulates somatostatin receptors. Cell proliferation was reduced by octreotide via a protein tyrosine phosphatase dependent mechanism.
Keywords: Cortistatin mRNA ;RT-PCR ;Immunohistochemistry ;Octreotide ;Radioligand binding ;Hepatocellular carcinoma
Overexpression of angiopoietin-1 and angiopoietin-2 in hepatocellular carcinoma
Takuji Torimura et al.
Background/Aims Hepatocellular carcinoma (HCC) is a highly vascular tumor. Angiopoietin-1 and Angiopoietin-2 have been shown to be involved in tumor angiogenesis. We investigated the expression of Angiopoietin-1 and Angiopoietin-2 in HCC. Methods The expression of Angiopoietin-1 and Angiopoietin-2 mRNAs in cultured hepatoma cells under hypoxic conditions and in HCC and noncancerous liver tissue was evaluated by real-time PCR. The expression of Angiopoietin-1, Angiopoietin-2, and their receptor Tie-2 in HCC was assessed by immunohistochemistry. The changes in Angiopoietin-1 and Angiopoietin-2 expression were evaluated in relation to tumor differentiation and changes in tumor vascularity. Results Hypoxic conditions did not up-regulate the expression of Angiopoietin-1 and Angiopoietin-2 mRNAs in hepatoma cells. Increased expression of Angiopoietin-1 and Angiopoietin-2 mRNAs was detected in HCC. Angiopoietin-1 and Angiopoietin-2 were detected in hepatoma cells, hepatic stellate cells, and smooth muscle cells, whereas Tie-2 was detected in endothelial cells, hepatic stellate cells and smooth muscle cells. Increased expression of Angiopoietin-2 and Angiopoietin-2 mRNA was associated with tumor dedifferentiation. The expression of Angiopoietin-1 and Angiopoietin-2 correlated with HCC vascularity. Conclusions Our findings indicate that the increased expression of Angiopoietin-1 and Angiopoietin-2 play a critical role in the process of vascular development in HCC.
Keywords: Angiogenesis ;Tumorigenesis ;Tumor differentiation ;Vascularity
Increased expression of H19 non-coding mRNA follows hepatocyte proliferation in the rat and mouse
Youhei Yamamoto, Yuji Nishikawa, Takuo Tokairin, Yasufumi Omori and Katsuhiko Enomoto
Background/Aims H19 is a paternally imprinted gene that is believed to function as non-coding mRNA. While H19 is only faintly expressed in the normal adult liver, it is abundantly expressed during the fetal period. We explored the possibility that H19 might participate in the regulation of hepatocyte proliferation. Methods Adult male rats and mice were subjected to a two-thirds partial hepatectomy, and after various time periods, hepatocytes were isolated and analyzed for H19 gene expression. The expression was also examined in cultured rat hepatocytes. Results The expression of H19 was dramatically increased after 2 days (rat) and 4 days (mouse), peaked at 3 days (rat) and 6 days (mouse), and then gradually declined. In both species, the increase in H19 gene expression was preceded by the induction of proliferating cell nuclear antigen and DNA synthesis. An allele-specific RT-PCR analysis in the mouse showed that the paternally imprinted status of the gene was maintained after a partial hepatectomy. H19 was strongly induced in spheroid cultures after transient hepatocyte proliferation, but not in conventional monolayer cultures, in which persistent proliferation occurred. Conclusions Our results demonstrated that H19 gene expression was dynamically regulated in adult hepatocytes in close association with their proliferation.
Keywords: H19 ;Non-coding RNA ;Genomic imprinting ;Liver regeneration ;Hepatocytes Abbreviations: PH, partial hepatectomy ;RT-PCR, reverse transcriptase-polymerase chain reaction ;PCNA, proliferating cell nuclear antigen ;FP, -fetoprotein ;IGF2, insulin-like growth factor 2 ;GAPDH, glyceraldehyde-3-phosphate dehydrogenase ;IGF2R, insulin-like growth factor 2 receptor ;BrdU, bromodeoxyuridine
Transplantation
Liver transplantation for primary sclerosing cholangitis; predictors and consequences of hepatobiliary malignancy
B. Brandsæter et al.
Background/Aims Hepatobiliary malignancies are frequently seen in primary sclerosing cholangitis (PSC) and they complicate the evaluation of patients and timing of liver transplantation. Methods Data from all Nordic PSC patients listed for liver transplantation during 1990-2001 were recorded prospectively. Predictors of hepatobiliary malignancy and patient survival rates have been analysed. Results Hepatobiliary malignancy was found in 52/255 (20%) patients accepted to the waiting list. Recent diagnosis of PSC, no ursodeoxycholic acid (UDCA) treatment, clinical suspicion and previous colorectal-cancer were predictors of malignancy. Among 89 patients with a strong suspicion of malignancy prior to acceptance, 35 (39%) had confirmed malignancy. A clinical suspicion had been raised in 35/52 (67%) patients with malignancy. Malignancy was found in 31/223 patients who received a liver allograft. The 1-, 3- and 5-year patient survival rates following transplantation for patients with PSC and cholangiocarcinoma were 65, 35 and 35%, respectively. Conclusions Hepatobiliary malignancy is suspected in 1/3 of the PSC patients and found in 1/5. Although cholangiocarcinoma is regarded as a contraindication to liver transplantation (LTX), PSC patients with cholangiocarcinoma had a 35% 5-year survival following transplantation.
Keywords: Primary sclerosing cholangitis ;Liver transplantation ;Cholangiocarcinoma ;Waiting list
Viral Hepatitis
Natural history of decompensated hepatitis C virus-related cirrhosis. A study of 200 patients
Ramon Planas et al.
Background/Aims Since few data are available concerning the clinical course of decompensated hepatitis C virus (HCV)-related cirrhosis, the aim of the present study was to define the natural long-term course after the first hepatic decompensation. Methods Cohort of 200 consecutive patients with HCV-related cirrhosis, and without known hepatocellular carcinoma (HCC), hospitalized for the first hepatic decompensation. Results Ascites was the most frequent first decompensation (48%), followed by portal hypertensive gastrointestinal bleeding (PHGB) (32.5%), severe bacterial infection (BI) (14.5%) and hepatic encephalopathy (HE) (5%). During follow-up (34±2 months) there were 519 readmissions, HCC developed in 33 (16.5%) patients, and death occurred in 85 patients (42.5%). The probability of survival after diagnosis of decompensated cirrhosis was 81.8 and 50.8% at 1 and 5 years, respectively. HE and/or ascites as the first hepatic decompensation, baseline Child-Pugh score, age, and presence of more than one decompensation during follow-up were independently correlated with survival. Conclusions Once decompensated HCV-related cirrhosis was established, patients showed not only a very high frequency of readmissions, but also developed decompensations different from the initial one. These results contribute to defining the natural course and prognosis of decompensated HCV-related cirrhosis.
Keywords: Ascites ;Hepatic encephalopathy ;Portal hypertensive gastrointestinal bleeding ;Hepatocellular carcinoma ;Bacterial infection
HVR-1 quasispecies modifications occur early and are correlated to initial but not sustained response in HCV-infected patients treated with pegylated- or standard-interferon and ribavirin
Isabella Abbate et al.
Background/Aims HVR-1 quasispecies composition and evolution were investigated in patients chronically infected with genotype 1b HCV, treated with PEG-IFN 2b or STD-IFN 2b plus RBV. Methods HVR-1 heterogeneity was assessed by calculating nucleotidic complexity, diversity, synonymous (S) and non-synonymous (NS) substitutions at baseline, after 4 weeks of therapy ( T1) and at follow-up ( T18). Evolution of viral quasispecies was analysed by constructing phylogenetic trees. Results No correlation of baseline viremia with heterogeneity was observed. Nucleotidic complexity was lower in patients showing early virological response, and tended to be inversely correlated to viral load decline at 4 weeks of treatment. In the majority of SR, profound changes of quasispecies composition occurred during 4 weeks of treatment, while in NR virtually no major changes of pre-therapy variants were observed. Relapse showed both patterns of quasispecies evolution. Virus quasispecies after follow-up was similar to that found at T1 in both Relapsers and NR patients. Conclusions Baseline parameters of HVR-1 heterogeneity seem to be involved in the early response to treatment, and early response is associated with profound variations in the HVR-1 quasispecies. Viral quasispecies surviving early therapeutic pressure are most likely able to give rise to either virus rebound or persistence at T18.
Keywords: HCV ;Interferon ;Quasispecies ;HVR-1
Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments
Sun Pyo Hong et al.
Background/Aims Mutations in hepatitis B virus (HBV) to lamivudine resistance that arise during prolonged treatment frequently cause amino acid substitutions in the YMDD motif of HBV DNA polymerase. Current methods of detecting such variants are time-consuming, labor intensive, and unsuitable for screening large numbers of samples. Here, we describe the development of a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) genotyping assay suitable for detecting HBV variants in a sensitive and specific manner. Methods The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif. Results The MALDI-TOF MS-based genotyping assay is sufficiently sensitive to detect as few as 100 copies of HBV genome per millilitre of serum, with superior specificity for determining mixtures of wild-type and variant viruses. When sera from 40 patients were analyzed, the MALDI-TOF MS-based assay correctly identified known viral variants and additional viral quasi-species not detected by previous methods, as well as their relative abundance. Conclusions The sensitivity, accuracy and amenability to high-throughput analysis makes the MALDI-TOF MS-based assay suitable for mass screening of HBV infected patients receiving lamivudine, and can help provide further understanding of disease progression and response to therapy. Keywords: Hepatitis B virus ;Lamivudine ;YMDD variant ;Genotyping ;Mass spectrometry
Association of serum interleukin-8 with virologic response to antiviral therapy in patients with chronic hepatitis C
Ulrike Mihm et al.
Background/Aims Upregulation of interleukin-8 by the hepatitis C virus non-structural-5A-protein leads to inhibition of the antiviral activity of interferon- in vitro. The clinical significance of interleukin-8 levels for virologic response to interferon- -based treatment in patients with chronic hepatitis C is unknown. Methods We investigated serum interleukin-8 in 59 healthy controls and 214 patients with chronic hepatitis C (genotype 1, n=152; genotype 2, 3, n=62) and different outcome to interferon- -based therapy. Results In patients with chronic hepatitis C higher interleukin-8 levels were observed compared with healthy controls ( P<0.0001). Hepatitis C genotype 1-infected patients with early and overall virologic response to interferon- -based therapy showed lower interleukin-8 levels than non-responders ( P=0.025 and P=0.035, respectively). In all patients, elevated interleukin-8 levels were associated with cirrhosis (genotype 1, P=0.0003; genotype 2, 3, P=0.009). Interleukin-8 levels in sustained virologic responders were still higher 24 weeks after the end-of-therapy compared with healthy controls ( P<0.0001). Conclusions In genotype 1 infected patients, low pretreatment serum interleukin-8 is associated with virologic response to interferon- -based therapy. Thus, the conclusion from in vitro studies that the upregulation of interleukin-8 by the hepatitis C virus contributes to the inhibition of the antiviral actions of interferon- may also be applicable in vivo.
Keywords: Hepatitis C virus RNA ;Virologic response ;Interferon resistance ;Non-structural 5A ;Interleukin-8
Copyright © 2001-2004 European Association for the Study of the Liver. All rights reserved.
1 May 2004 (Vol 328, No 7447)
Non-absorbable disaccharides for hepatic encephalopathy: systematic review of randomised trials
Bodil Als-Nielsen, Lise L Gluud, Christian Gluud
BMJ 2004;328:1046, doi:10.1136/bmj.38048.506134.EE
Objective To assess the effects of non-absorbable disaccharides (lactulose and lactitol) in patients with hepatic encephalopathy. Data sources Cochrane Hepato-Biliary Group controlled trials register, Cochrane Library, Medline, and Embase until March 2003; reference lists of relevant articles; authors and pharmaceutical companies. Review methods Randomised trials that compared non-absorbable disaccharides with placebo, no intervention, or antibiotics for hepatic encephalopathy were included. The primary outcome measures were no improvement of hepatic encephalopathy and all cause mortality. Results 22 trials were included. Compared with placebo or no intervention, non-absorbable disaccharides seemed to reduce the risk of no improvement in patients with hepatic encephalopathy (relative risk 0.62, 95% confidence interval 0.46 to 0.84, six trials). However, high quality trials found no significant effect (0.92, 0.42 to 2.04, two trials). Compared with placebo or no intervention, non-absorbable disaccharides had no significant effect on mortality (0.41, 0.02 to 8.68, four trials). Non-absorbable disaccharides were inferior to antibiotics in reducing the risk of no improvement (1.24, 1.02 to 1.50, 10 trials) and lowering blood ammonia concentration (weighted mean difference 2.35 µmol/l, 0.06 µmol/l to 13.45 µmol/l, 10 trials). There was no significant difference in mortality (0.90, 0.48 to 1.67, five trials). Conclusions There is insufficient evidence to support or refute the use of non-absorbable disaccharides for hepatic encephalopathy. Antibiotics were superior to non-absorbable disaccharides in improving hepatic encephalopathy, but it is unclear whether this difference is clinically important. Non-absorbable disaccharides should not serve as comparator in randomised trials on hepatic encephalopathy.
Hepatitis C carriers must be found and treated to avert crisis
Paul Stephenson
BMJ 2004;328:1031, doi:10.1136/bmj.328.7447.1031-a
Considerable extra resources to improve detection and treatment rates for hepatitis C are urgently needed to avoid a public health crisis that could overwhelm liver units, says new guidance from the Royal College of Physicians of Edinburgh. It calls for high priority to be given to finding cases among former injecting drug users, for the development of new, community based and specialist nurse led services, and for broader access to treatment. The guidance also says it is no longer essential to carry out a liver biopsy to determine the selection of patients. The hepatitis C virus is thought to have infected up to 600 000 people in the United Kingdom and up to 200 million worldwide. It is estimated that up to a fifth of carriers of the hepatitis C virus could develop cirrhosis and need liver transplantation. Consultant gastroenterologist Professor Peter Hayes from the Royal College of Physicians of Edinburgh said it was certain that "if we do not invest adequately now, we will not be able to afford the consequences of failing to tackle this epidemic." He said services had to be redesigned, that clinics had to improve their attendance rates (as non-attendance was around 50%), and that measures such as treatment in prisons needed to be examined. The guidance says a new, community focused model of care is needed, using outreach nurse led clinics in primary care, prisons, and drug treatment services. It also says healthcare workers need training and GPs need clear guidelines about suitability for referral. Charles Gore, chief executive of the national charity Hepatitis C Trust, said the guidance was very welcome and that public awareness was key to increasing detection rates. He said: "We are very aware that huge numbers are not yet diagnosed. One of the key things we want to do is identify people. You can't afford to ignore this. Managing end stage liver disease is so expensive and so labour intensive. "Managed clinical networks are a key point, and patient groups are saying this is what we need. You can really change your life expectancy by looking after yourself, and that doesn't cost much, except setting up support networks." The primary aim of treatment for hepatitis C is viral clearance. A sustained viral response is defined as the absence of hepatitis C virus RNA in serum 24 weeks after the end of treatment. Combination therapy with pegylated interferon alfa and ribavirin is recommended for all patients suitable for treatment. Patients with genotypes 1 and 4-6 should receive therapy for 48 weeks and genotypes 2 and 3 for 24 weeks. In genotype 1, quantitative polymerase chain reaction (a test of viral load) at 12 weeks will determine if patients continue therapy.
Copyright © 2004 BMJ Publishing Group Ltd
The New England Journal of Medicine is owned, published, and copyrighted © 2004 Massachusetts Medical Society. All rights reserved.
Volume 363, Number 9419 01 May 2004
Portopulmonary hypertension and hepatopulmonary syndrome
Marius M Hoeper, Michael J Krowka, Christian P Strassburg
Lancet 2004; 363: 1461-68
Departments of Respiratory Medicine (M M Hoeper MD) and Gastroenterology, Hepatology and Endocrinology (C Strassburg MD), Hannover Medical School, 30623 Hannover, Germany; and Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, MN, USA (M J Krowka MD) Correspondence to: Dr Marius M Hoeper (e-mail: hoeper.marius@mh-hannover.de )
The clinically and pathophysiologically distinct entities of portopulmonary hypertension and hepatopulmonary syndrome occur in a substantial proportion of patients who have advanced liver disease of different causes. These disorders are notoriously underdiagnosed, but they have a substantial impact on survival and require focused treatment. Abnormal intrapulmonary vascular dilatation, the hallmark of hepatopulmonary syndrome, can cause profound hypoxaemia that can be very difficult to treat. By contrast, portopulmonary hypertension results from excessive pulmonary vasoconstriction and vascular remodelling that eventually leads to right-heart failure. Insights into the pathogeneses of these syndromes have led to novel therapeutic approaches. However, in severely affected patients, effective treatment remains a difficult task. In selected patients, liver transplantation represents the only treatment option, but the decision to do isolated liver transplantation is particularly challenging in patients who have severe pulmonary disease involvement. Data from several centres have contributed to provide criteria that allow improved prediction of which patients may, or may not, benefit from liver transplantation alone.
The Lancet, published, and copyrighted © 2004. All rights reserved.
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