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Revue de Presse du Mois Mois d'Août 2002

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HEPATOLOGY

Table of Contents for August 2002 · Volume 36 · Number 2

Liver Biology and Pathobiology

Hepatocytes undergo phenotypic transformation to biliary epithelium in organoid cultures
George K. Michalopoulos, William C. Bowen, Karen Mulè, Juan Carlos Lopez-Talavera, Wendy Mars
Organoid cultures of hepatocytes in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) display characteristic histologic organization. Biliary epithelium covers the surface of the tissue exposed to the culture medium. Hepatocytes, stellate cells and endothelial cells compose the underlying structures. In order to investigate the origin of the biliary epithelial cells in the organoid cultures, we utilized the retrorsine/DPPIV system of hepatocyte transplantation to create hybrid livers in which clones of DPPIV hepatocytes colonize variable portions of the lobules. We demonstrate that, as others have shown, biliary epithelium in this in vivo system remains that of the recipient (DPPIV negative) rat. Hepatocytes are the only cells positive for the DPPIV marker enzyme in the hybrid livers. Organoid cultures were prepared from the hybrid livers. Overall, 46.82% of the hepatocytes placed into culture were positive for DPPIV at time zero (after isolation). At 21 days in culture, 47.54% of the biliary epithelium on the surface of the organoid cultures was positive for DPPIV. Since the only DPPIV cells inoculated in the cultures were hepatocytes, this finding demonstrates that, in the conditions of the organoid cultures, hepatocytes do undergo phenotypic transition to biliary epithelial cells. (HEPATOLOGY 2002;36:278-283.)

Regulation of Ca2+ signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms
Keiji Hirata, Jean-François Dufour, Kazunori Shibao, Roy Knickelbein, Allison F. O'Neill, Hans-Peter Bode, Doris Cassio, Marie V. St-Pierre, Nicholas F. LaRusso, M. Fatima Leite, Michael H. Nathanson
Cytosolic Ca2+ (Cai2+) regulates secretion of bicarbonate and other ions in the cholangiocyte. In other cell types, this second messenger acts through Ca2+ waves, Ca2+ oscillations, and other subcellular Ca2+ signaling patterns, but little is known about the subcellular organization of Ca2+ signaling in cholangiocytes. Therefore, we examined Ca2+ signaling and the subcellular distribution of Ca2+ release channels in cholangiocytes and in a model cholangiocyte cell line. The expression and subcellular distribution of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) isoforms and the ryanodine receptor (RyR) were determined in cholangiocytes from normal rat liver and in the normal rat cholangiocyte (NRC) polarized bile duct cell line. Subcellular Ca2+ signaling in cholangiocytes was examined by confocal microscopy. All 3 InsP3R isoforms were expressed in cholangiocytes, whereas RyR was not expressed. The type III InsP3R was the most heavily expressed isoform at the protein level and was concentrated apically, whereas the type I and type II isoforms were expressed more uniformly. The type III InsP3R was expressed even more heavily in NRC cells but was concentrated apically in these cells as well. Adenosine triphosphate (ATP), which increases Ca2+ via InsP3 in cholangiocytes, induced Ca2+ oscillations in both cholangiocytes and NRC cells. Acetylcholine (ACh) induced apical-to-basal Ca2+ waves. In conclusion, Ca2+ signaling in cholangiocytes occurs as polarized Ca2+ waves that begin in the region of the type III InsP3R. Differential subcellular localization of InsP3R isoforms may be an important molecular mechanism for the formation of Ca2+ waves and oscillations in cholangiocytes. Because Cai2+ is in part responsible for regulating ductular secretion, these findings also may have implications for the molecular basis of cholestatic disorders. (HEPATOLOGY 2002;36:284-296.)

Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat
Domenico Alvaro, Paolo Onori, Veronica Drudi Metalli, Gianluca Svegliati-Baroni, Franco Folli, Antonio Franchitto, Gianfranco Alpini, Maria Grazia Mancino, Adolfo Francesco Attili, Eugenio Gaudio
The aim of this study was to explore the intracellular signaling pathways involved in the stimulatory effects of estrogens on cholangiocyte proliferation. We investigated the tyrosine kinase­receptor pathway by evaluating the protein expression of total and phosphorylated mitogen-activated protein kinase (MAPK) isoform p44/p42 (e.g., extracellular signal-regulated kinase [ERK]1/2), the steroid-receptor coactivator Src and Shc (Src-homology/collagen protein). The study was performed in 3-week-old bile duct­ligated (BDL) rats, BDL rats treated with the antiestrogens, tamoxifen or Ici 182,780, and normal control rats. Proliferation was also evaluated in normal purified cholangiocytes treated with 17 estradiol in the presence or absence of tamoxifen, Ici 182,780, ERK, or Src inhibitors. After bile duct ligation, cholangiocyte proliferation was associated with a marked immunohistochemical nuclear positivity for phosphorylated (p)-ERK1/2, which was inhibited by in vivo treatment with tamoxifen or Ici 182,780. Protein expression of total and p-ERK1/2, and Shc in cholangiocytes isolated from BDL rats was markedly increased compared with controls and was inhibited by in vivo treatment with antiestrogens. In vitro, 17 estradiol­induced proliferation of isolated normal cholangiocyte was associated with increased (P < .01) protein expression of p-ERK1/2, Src, and Shc. Specific inhibitors of ER (Ici 182,780), ERK (U0125), and Src (PP2) inhibited in vitro 17 estradiol­induced cholangiocyte proliferation. In conclusion, this study showed that estrogens induced cholangiocyte proliferation by activating the Src/Shc/ERK pathway. This might suggest that pharmacologic modulation of ER, ERK, and/or Src could be proposed for the treatment of human pathology characterized by dysregulation of cholangiocyte proliferation. (HEPATOLOGY 2002;36:297-304.)

Ursodeoxycholic acid (UDCA) prevents DCA effects on male mouse liver via up-regulation of CXP and preservation of BSEP activities
Moreno Paolini, Laura Pozzetti, Marco Montagnani, Giuseppa Potenza, Laura Sabatini, Alessandra Antelli, Giorgio Cantelli-Forti, Aldo Roda
To investigate whether ursodeoxycholic acid (UDCA) can prevent metabolic impairment induced by deoxycholic acid (DCA), we evaluated the effects of these bile acids on murine CYP enzymes and the relationship with canalicular bile salt export pump (Bsep) expression. In Swiss Albino CD1 mice, UDCA and DCA were injected intraperitoneally either singly, concurrently, or sequentially (UDCA 1 hour before DCA) at equimolar 24.4 mg/kg body weight (BW) doses. CYP content, NADPH-CYP-c-reductase, and individual mixed function oxidases (MFO) were measured 24 hours later. Modulations were observed mainly in males: whereas DCA decreased MFO activities to various isoenzymes with respect to controls (up to 43%, CYP1A2-linked activity), UDCA boosted them (up to 6-fold, testosterone 16-hydroxylase); concurrent administration of UDCA and DCA provided a preventive effect, enhancing MFO activity with respect to single administration of DCA by up to 4.4-fold in the CYP3A1/2 and CYP2B1/2 (6-hydroxylase) and by 2.1-fold in the CYP2E1 (p-nitrophenol hydroxylase). In males (but not females), sequential administration (UDCA then DCA) produced a rather similar protective pattern, but the extent of recovery was generally smaller. Western immunoblotting results for the most affected isoenzymes (CYP3A1/2 and CYP2E1) and Bsep confirmed that UDCA can both prevent and reduce the CYP-dependent MFO inactivation and Bsep down-regulation caused by DCA. These findings may shed further light on the mechanisms responsible for UDCA's protective role in the treatment of cholestatic liver disease. (HEPATOLOGY 2002;36:305-314.)

Jun kinase modulates tumor necrosis factor­dependent apoptosis in liver cells
Christian Liedtke, Jörg Plümpe, Stefan Kubicka, Cynthia A. Bradham, Michael P. Manns, David A. Brenner, Christian Trautwein
Tumor necrosis factor (TNF) triggers distinct pathways in liver cells through TNF receptor 1 (TNF-R1) via adapter molecules, including the intracellular cascades leading to apoptosis, nuclear factor-B (NF-B), and Jun kinase (JNK) activation. TNF-dependent activation of NF-B induces the transcription of antiapoptotic genes that renders liver cells resistant against TNF-induced apoptosis. In contrast, the role of JNK during TNF-induced apoptosis is less clear, so we studied its role during this process. Hepatoma cells treated with TNF and cycloheximide undergo apoptosis, which is proceeded by a strong activation of JNK. Adenoviral vectors (adv) were tested to block TNF-dependent JNK activation selectively. An adv expressing dominant-negative (dn) TRAF2 inhibited only JNK and not ERK or NF-B activation. However, the effect of inhibiting JNK activation with a dn TAK1 virus was also specific but was stronger than that via dn TRAF2. In further experiments, the inhibitory effect of dn TAK1 on JNK was used to define its role during TNF-dependent apoptosis. Inhibition of JNK by adv dn TAK1 resulted in an earlier and stronger induction of apoptosis. Interestingly, TAM67, a dn form of c-Jun, did not mediate the JNK-dependent effect on TNF-dependent apoptosis, indicating that other molecular targets are essential to confer this mechanism. However, the modified apoptosis pattern could be inhibited by adv expressing Bcl-2 or dn FADD. In conclusion, we define TAK1 as a kinase specifically involved in TNF-induced JNK activation in hepatoma cells and show that JNK transduces antiapoptotic signals, which modulate the strength and time course of FADD-dependent cell death involving mitochondrial permeability transfer. (HEPATOLOGY 2002;36:315-325.)

Selective mitochondrial glutathione depletion by ethanol enhances acetaminophen toxicity in rat liver
Ping Zhao, Thomas F. Kalhorn, John T. Slattery
Chronic alcohol consumption may potentiate acetaminophen (APAP) hepatotoxicity through enhanced formation of N-acetyl-p-benzoquinone imine (NAPQI) via induction of cytochrome P450 2E1 (CYP2E1). However, CYP2E1 induction appears to be insufficient to explain the claimed magnitude of the interaction. We assessed the role of selective depletion of liver mitochondrial glutathione (GSH) by chronic ethanol. Rats were fed the Lieber-DeCarli diet for 10 days or 6 weeks. APAP toxicity in liver slices (% glutathione-S-transferase released to the medium, GST release) and NAPQI toxicity in isolated liver mitochondria (succinate dehydrogenase inactivation, SDH) from these rats were compared with pair-fed controls. Ethanol induced CYP2E1 in both the 10-day and 6-week groups by ~2-fold. APAP toxicity in liver slices was higher in the 6-week ethanol group than the 10-day ethanol group. Partial inhibition of NAPQI formation by CYP2E1 inhibitor diethyldithiocarbamate to that of pair-fed controls abolished APAP toxicity in the 10-day ethanol group only. Ethanol selectively depleted liver mitochondrial GSH only in the 6-week group (by 52%) without altering cytosolic GSH. Significantly greater GSH loss and APAP covalent binding were observed in liver slice mitochondria of the 6-week ethanol group. Isolated mitochondria of the 6-week ethanol group were ~50% more susceptible to NAPQI (25-165 µmol/L) induced SDH inactivation. This increased susceptibility was reproduced in pair-fed control mitochondria pretreated with diethylmaleate. In conclusion, 10-day ethanol feeding enhances APAP toxicity through CYP2E1 induction, whereas 6-week ethanol feeding potentiates APAP hepatotoxicity by inducing CYP2E1 and selectively depleting mitochondrial GSH. (HEPATOLOGY 2002;36:326-335.)

p53 May positively regulate hepatocyte proliferation in rats
Yukiko Inoue, Tomoaki Tomiya, Mikio Yanase, Masahiro Arai, Hitoshi Ikeda, Kazuaki Tejima, Itsuro Ogata, Satoshi Kimura, Masao Omata, Kenji Fujiwara
p53, known as a tumor suppressor gene, is a transcription factor that regulates various cellular functions. Recently, several growth factor gene promoters, including that of transforming growth factor (TGF-), were shown to be direct targets of p53-mediated transcription. Hepatic p53 mRNA is up-regulated during liver regeneration in rats. The aim of this study is to examine the role of p53 in hepatocyte proliferation. p53 protein levels were examined in rat hepatocytes cultured in the medium containing hepatocyte growth factor (HGF). p53 levels began to increase after 6 hours of incubation, reached a maximum at 18 hours, and decreased thereafter. DNA synthesis increased at 12 hours and peaked at 30 hours. When hepatocytes were incubated with p53 antisense oligonucleotide in addition to HGF, increases of p53 and TGF- levels were suppressed, and DNA synthesis was reduced. The increases of TGF- levels and DNA synthesis were also suppressed by a chemical inhibitor of p53, pifithrin-. In rats after two-thirds partial hepatectomy, hepatic p53 increased and reached maximal levels around 16 hours when hepatic HGF levels have been shown to reach a maximum followed by an increase in hepatic TGF- levels or hepatocyte proliferation. In contrast, sham-operated rats showed minor elevations of hepatic p53 levels. In conclusion, p53 production is stimulated by HGF and may contribute to the proliferation of rat hepatocytes. Considering previous findings indicating the importance of endogenous TGF- for the proliferation of hepatocytes stimulated by HGF, TGF- might play a role in HGF-p53 mediated hepatocyte proliferation. (HEPATOLOGY 2002;36:336-344.)

Regenerative activity and liver function following partial hepatectomy in the rat using 31P-MR spectroscopy
Ian R. Corbin, Richard Buist, Vyacheslav Volotovskyy, Jim Peeling, Manna Zhang, Gerald Y. Minuk
The aim of the present study was to determine whether alterations in hepatic energy expenditure following partial hepatectomy (PHx), as documented by in vivo hepatic 31P-MRS, correlate with standard parameters of hepatic regeneration and/or liver function. In addition, we sought to determine whether changes in hepatic energy levels are proportional to the extent of hepatic resection. Adult male Sprague-Dawley rats (4-7 per group) underwent a 40%, 70%, or 90% PHx or sham surgeries. Magnetic resonance spectroscopy (MRS) examinations were performed on each animal 24 or 48 hours thereafter. After MRS examinations, [3H]thymidine incorporation into hepatic DNA, proliferating cell nuclear antigen (PCNA) protein expression, and serum bilirubin determinations were performed on each rat. Twenty-four hours following surgery, rats that had undergone 70% PHx had unchanged adenosine triphosphate (ATP) levels but significantly lower ATP/inorganic phosphate (Pi) ratios (P < .05), whereas, at 48 hours post-PHx, both ATP and ATP/Pi levels were lower than in sham- and nonoperated controls (P < .05). Hepatic regeneration and liver dysfunction mirrored these changes; correlations existed between ATP/Pi ratios and [3H]thymidine incorporation (r = ­0.61, P < .005), PCNA protein expression (r = ­0.62, P < .005), and serum bilirubin (r = ­0.49, P < .05). For rats that had undergone graded resections, depleted energy levels 48 hours post-PHx were proportional to the extent of resection, degree of enhanced regenerative activity, and liver dysfunction. In conclusion, 31P-MRS-generated ATP/Pi index is a noninvasive, robust determination that correlates with standard parameters of hepatic regeneration and function. (HEPATOLOGY 2002;36:345-353.)

Normal hepatocytes correct serum bilirubin after repopulation of gunn rat liver subjected to irradiation/partial resection
Chandan Guha, Bhupesh Parashar, Niloy J. Deb, Madhur Garg, Giridhar R. Gorla, Anupam Singh, Namita Roy-Chowdhury, Bhadrasain Vikram, Jayanta Roy-Chowdhury
The treatment of inherited metabolic liver diseases by hepatocyte transplantation (HT) would be greatly facilitated if the transplanted normal hepatocytes could be induced to proliferate preferentially over the host liver cells. We hypothesized that preparative hepatic irradiation (HIR) should inhibit host hepatocyte proliferation in response to partial hepatectomy (PH). Normal nonirradiated hepatocytes transplanted in this setting should have a selective growth advantage over the host liver cells and should progressively repopulate the liver. To test this hypothesis, we transplanted 5 million hepatocytes from normal Wistar-Roman High Avoidance (RHA) rats into the livers of congeneic bilirubin-uridine 5'-diphosphoglucuronate glucuronosyltransferase (UGT1A1)-deficient jaundiced Gunn rats by intrasplenic injection after one of the following treatments: (1) 68% PH, (2) HIR (50 Gy), or (3) HIR + PH. In rats receiving either PH or HIR alone before HT, serum bilirubin concentrations declined by 25% to 30% in 28 weeks. In contrast, serum bilirubin levels were normalized completely in rats receiving HIR + PH before HT. Massive repopulation of the Gunn rat liver by the UGT1A1-positive Wistar-RHA hepatocytes was shown by UGT1A1 enzyme assay, immunoblot analysis, and immunohistochemical staining of the recipient liver. High-performance liquid chromatography analysis of the bile collected from Gunn rats 5 months after PH, HIR, and HT showed normalization of the pigment profile, with bilirubin diglucuronide and monoglucuronide as the predominant pigments. In conclusion, a preparative regimen of HIR + PH results in massive repopulation of the liver with functionally normal transplanted hepatocytes, resulting in complete correction of a metabolic deficiency. Noninvasive strategies to replace PH for providing proliferative stimuli to the transplanted cells should make this regimen valuable in augmenting the effects of HT for the treatment of liver diseases. (HEPATOLOGY 2002;36:354-362.)

Involvement of 85-kd cytosolic phospholipase A2 and cyclooxygenase-2 in the proliferation of human cholangiocarcinoma cells (*Human Study*)
Tong Wu, Chang Han, John G. Lunz, III, George Michalopoulos, James H. Shelhamer, A. Jake Demetris
Cyclooxygenase 2 (COX-2) and cytosolic phospholipase A2 (cPLA2) are the crucial rate-limiting enzymes in prostaglandin (PG) metabolism that show increased expression in a number of human cancers, including cholangiocarcinomas; and treatment of cholangiocarcinoma cell lines with COX-2 inhibitors can decrease proliferation. Cholangiocarcinomas also produce and proliferate in response to nonneoplastic biliary epithelial cell mitogens, such as interleukin 6 (IL-6) and hepatocyte growth factor (HGF). This study was designed to determine whether there is any relationship between eicosanoid metabolism and growth stimulation by IL-6 and HGF, two important biliary epithelial cell and cholangiocarcinoma mitogens. Incubation of SG231, a well-characterized human cholangiocarcinoma cell line, with HGF, IL-6, PGE2, or PGF2 resulted in significantly increased cell growth. HGF and IL-6 also induced a rapid release of arachidonic acid (AA) from SG231 and increased the synthesis of PGE2 and PGF2. The cPLA2 inhibitor arachidonyl fluorophosphonate (MAFP) and the COX-2 inhibitor NS-398 significantly inhibited HGF- and IL-6-induced release of AA, PG synthesis, and proliferation in SG231 cells as well as two other human cholangiocarcinoma cell lines, HuCCT1 and CC-LP-1 cells. Thus, PGs alone can induce cholangiocarcinoma growth, and the HGF- and IL-6-induced proliferation is mediated, at least in part, by PGs. HGF and IL-6 also induced a rapid phosphorylation of cPLA2 (within 1 minute) but did not alter cPLA2 and COX-2 protein expression. The HGF- and IL-6-induced cPLA2 phosphorylation was blocked by the inhibitors of p38 and p42/44 MAP kinases, protein kinase C, calmodulin kinase, and tyrosine kinase, showing that HGF- and IL-6-induced AA release and PG production are mediated by phosphorylation of cPLA2. In conclusion, molecular pathways link classic biliary epithelial cell mitogens to PG metabolism constituents in cholangiocarcinoma growth, which may be exploited as potential therapeutic targets. (HEPATOLOGY 2002;36:363-373.)

Liver Failure and Liver Disease

Effects of noradrenalin and albumin in patients with type 1 hepatorenal syndrome: A pilot study (*Human Study*)
Christophe Duvoux, David Zanditenas, Christophe Hézode, Anthony Chauvat, Jean-Luc Monin, Françoise Roudot-Thoraval, Ariane Mallat, Daniel Dhumeaux
Treatment of hepatorenal syndromes (HRSs) is currently based on vasopressin analogs. The aim of this pilot study was to evaluate the efficacy and safety of noradrenalin (NA) in the treatment of type 1 HRS. Between 1998 and 2000, 12 consecutive patients with type 1 HRS (7 men, 5 women; mean age, 54 ± 11 years; mean Child-Pugh score, 11.3 ± 1.7) were treated with intravenous NA (0.5-3 mg/h), in combination with intravenous albumin and furosemide. NA was given for 10 ± 3 days, at a mean dose of 0.8 ± 0.3 mg/h. Reversal of HRS was observed in 10 of 12 patients (83%; 95% confidence interval, 52%-98%) after a median of 7 days (range, 5-10 days). Serum creatinine levels fell from 358 ± 161 to 145 ± 78 µmol/L (P < .001), creatinine clearance rose from 13 ± 9 to 40 ± 15 mL/min (P = .003), and urinary sodium output increased from 8 ± 14 to 52 ± 72 mEq/d (P = .002). Changes in renal function under NA treatment were associated with an increase in mean arterial pressure (MAP; 65 ± 7 to 73 ± 9 mm Hg, P = .01) and a marked reduction in active renin (565 ± 989 to 164 ± 196 ng/L, P = .001) and aldosterone plasma concentrations (1,945 ± 1,931 to 924 ± 730 ng/mL, P = .02). There was one episode of reversible myocardial hypokinesia (in a patient on 1.5 mg/h NA) that did not recur after a dose reduction. In conclusion, NA combined with albumin and furosemide appears effective and safe for the treatment of type 1 HRS. (HEPATOLOGY 2002;36:374-380.)

Bioactivation of nitroglycerin and vasomotor response to nitric oxide are impaired in cirrhotic rat livers
Amy A. Dudenhoefer, Maurício R. Loureiro-Silva, Gregory W. Cadelina, Tarun Gupta, Roberto J. Groszmann
Nitroglycerin (NTG), a nitric oxide (NO) donor, has been shown to reduce portal pressure in cirrhotic patients. Using the in situ perfusion of normal and cirrhotic rat livers, we compared the vascular relaxation induced by either NTG or the spontaneous nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Normal and cirrhotic livers were perfused (40 mL/min, 37°C) with Krebs' solution in a recirculating system. After preconstriction with methoxamine (10­4 mol/L), a dose-response study was performed using 6 cumulative doses of NTG or SNAP (10­7 to 3 ¥ 10­5 mol/L). NOx (NO2­ + NO) production in the perfusate was measured by chemiluminescence. Cirrhotic livers exhibited lower vasorelaxant responses, compared with normal livers, to both NTG (P < .0001) and SNAP (P = .0020). In normal livers, NTG and SNAP induced similar vasorelaxant responses (P = .44). In cirrhotic livers, NTG induced less vasorelaxation than SNAP (P < .0001). In the presence of NTG (P = .0045), but not SNAP (P = .99), NOx production in experiments with cirrhotic livers was lower than in experiments with normal livers. In conclusion, in cirrhotic rat livers, the vasorelaxant response to NTG is impaired owing to both a decreased metabolism of this NO donor and an inability of the hepatic vasculature to respond to NO. (HEPATOLOGY 2002;36:381-385.)

Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes
Jin Cai, Masahiro Ito, Hideo Nagata, Karen A. Westerman, Daryl LaFleur, Jayanta Roy Chowdhury, Philippe Leboulch, Ira J. Fox
The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, 35 immortalized rat hepatocyte clones were developed by transduction with the gene encoding the simian virus 40 T antigen (SV40Tag). The SV40Tag sequence and a suicide gene, herpes simplex virus thymidine kinase (HSV-tk), were flanked by loxP sequences so that they could be excised by Cre/lox recombination. When transplanted into the spleens of portacaval-shunted rats, 3 of the 35 immortalized hepatocyte clones prevented the development of hyperammonemia-induced hepatic encephalopathy. The protection was reversed by treatment with ganciclovir, which kills HSV-tk­expressing cells. Transplantation of alginate-encapsulated, immortalized hepatocytes into the spleens of cirrhotic rats resulted in significant improvement in prothrombin time, serum albumin and bilirubin levels, hepatic encephalopathy score, and duration of survival. The metabolic support provided by the immortalized cells equaled that observed after transplantation of primary rat hepatocytes. In conclusion, immortalized hepatocytes can function as well as primary hepatocytes following transplantation and can be engineered to contain safeguards that could make them clinically useful. Further investigation is warranted regarding the mechanisms of loss of mass or function of the transplanted hepatocytes over time and how the relatively few engrafted hepatocytes can ameliorate liver decompensation in cirrhosis. (HEPATOLOGY 2002;36:386-394.)

The clinical importance of adrenal insufficiency in acute hepatic dysfunction (*Human Study*)
Rachael Harry, Georg Auzinger, Julia Wendon
Acute liver failure and septic shock share many clinical features, including hyperdynamic cardiovascular collapse. Adrenal insufficiency may result in a similar cardiovascular syndrome. In septic shock, adrenal insufficiency, defined using the short synacthen test (SST), is associated with hemodynamic instability and poor outcome. We examined the SST, a dynamic test of adrenal function, in 45 patients with acute hepatic dysfunction (AHD) and determined the association of these results with hemodynamic profile, severity of illness, and outcomes. Abnormal SSTs were common, occurring in 62% of patients. Those who required noradrenaline (NA) for blood pressure support had a significantly lower increment (median, 161 vs. 540 nmol/L; P < .001) following synacthen compared with patients who did not. Increment and peak were lower in patients who required ventilation for the management of encephalopathy (increment, 254 vs. 616 nmol/L, P < .01; peak, 533 vs. 1,002 nmol/L, P < .01). Increment was significantly lower in those who fulfilled liver transplant criteria compared with those who did not (121 vs. 356 nmol/L; P < .01). Patients who died or underwent liver transplantation had a lower increment (148 vs. 419 nmol/L) and peak (438 vs. 764 nmol/L) than those who survived (P < .01). There was an inverse correlation between increment and severity of illness (Sequential Organ Failure Assessment, r = ­0.63; P < .01). In conclusion, adrenal dysfunction assessed by the SST is common in AHD and may contribute to hemodynamic instability and mortality. It is more frequent in those with severe liver disease and correlates with severity of illness. (HEPATOLOGY 2002;36:395-402.)

Serum leptin in NASH correlates with hepatic steatosis but not fibrosis: A manifestation of lipotoxicity? (*Human Study*)
Shivakumar Chitturi, Geoffrey Farrell, Linda Frost, Adamandia Kriketos, Rita Lin, Christopher Liddle, Dev Samarasinghe, Jacob George
Nonalcoholic steatohepatitis (NASH) is a disorder characterized by hepatic steatosis, inflammation, and fibrosis. Leptin is an adipocyte-derived antiobesity hormone that in rodents prevents "lipotoxicity" by limiting triglyceride accumulation and also regulates matrix deposition (fibrosis) during wound healing. We therefore determined serum leptin levels in patients with NASH to determine whether relationships existed between leptin levels and severity of hepatic steatosis or fibrosis. We used a radioimmunoassay to determine serum [total] leptin concentrations in 27 men and 20 women with NASH and 47 controls matched for gender and body mass index (BMI; and partly for age). Serum leptin values were correlated with hepatic steatosis, fibrosis, and inflammation (each categorized semiquantitatively on liver histology), and with anthropometric indices, serum lipids, glucose, insulin, c-peptide, and alanine aminotransferase (ALT) levels. Compared with the controls, mean serum leptin levels were raised in both men and women with NASH (men 14 ± 11 ng/mL vs. 7.2 ± 4.1 ng/mL, P = .003; women 35 ± 16 ng/mL vs. 15 ± 8.2 ng/mL, P < .001). Leptin values correlated with serum c-peptide levels but not with BMI. In a multivariate analysis, serum leptin (P = .027), serum c-peptide (P = .001), and age (P = .027) were selected as independent predictors of the severity of hepatic steatosis. However, serum leptin was not an independent predictor of hepatic inflammation or fibrotic severity. In conclusion, hyperleptinemia occurs in NASH and is not explained simply by gender, obesity, or the presence of type 2 diabetes. Furthermore, leptin levels correlate directly with the severity of hepatic steatosis but not with inflammation or fibrosis. We propose that the relationship between leptin and steatosis reflects a pathogenic role of leptin in hepatic insulin resistance and/or a failure of the antisteatotic actions of leptin ("peripheral leptin resistance"). (HEPATOLOGY 2002;36:403-409.)

Racial differences in effectiveness of -fetoprotein for diagnosis of hepatocellular carcinoma in hepatitis C virus cirrhosis (*Human Study*)
Mindie H. Nguyen, Ruel T. Garcia, Peter W. Simpson, Teresa L. Wright, Emmet B. Keeffe
-Fetoprotein (AFP) is frequently used as a diagnostic marker for hepatocellular carcinoma (HCC). Most available data concerning AFP come from studies of patients with chronic hepatitis B or other chronic liver diseases of mixed etiologies. Limited data concerning the diagnostic value of AFP for hepatitis C virus (HCV)-related HCC have to date come only from Asian and European studies, and results are conflicting. There may be significant differences in AFP levels depending on racial backgrounds and etiologies of primary liver disease. We conducted a multicenter, retrospective, case-control study of 163 HCC patients with HCV infection and 149 control patients with HCV-related cirrhosis. The positive likelihood ratios for AFP at 0 to 20, 21 to 50, 51 to 100, and 101 to 200 ng/mL were 0.46, 1.31, 1.15, and 6.90, respectively. No controls had AFP greater than 200 ng/mL. The sensitivity of AFP for the diagnosis of HCC in African Americans with HCV infection was lower than that of patients of all other ethnic groups combined (57.1% vs. 81.6% for AFP > 10 ng/mL, P = .02, and 42.9% vs. 66.0% for AFP > 20 ng/mL, P = .05). The area under the receiver operating characteristics curve was 0.81 for non-African Americans but only 0.56 for African Americans. In conclusion, AFP greater than 200 ng/mL can be used to confirm HCC in patients with HCV-related cirrhosis and a hepatic mass. However, AFP is insensitive for the diagnosis of HCC in African Americans. (HEPATOLOGY 2002;36:410-417.)

Expression, regulation, and function of V integrins in hepatocellular carcinoma: An in vivo and in vitro study (*Human Study*)
Mimoun Nejjari, Zakia Hafdi, Géraldine Gouysse, Michelangelo Fiorentino, Olivier Béatrix, Jérôme Dumortier, Céline Pourreyron, Chiara Barozzi, Antonia D'Errico, Walter F. Grigioni, Jean-Yves Scoazec
The expression of V integrins by neoplastic cells contributes to the promotion of local invasion and metastasis. The most characteristic extracellular ligands of V integrins are vitronectin and fibronectin. Hepatocytes are the main source of vitronectin, and the capacity to synthesize and secrete vitronectin is usually retained in hepatocellular carcinoma. The aim of this study was to explore the expression, regulation, and functional role of V integrins in hepatocellular carcinoma. We first analyzed the expression of V integrins and their ligands fibronectin and vitronectin in 80 cases of hepatocellular carcinoma. V integrin chain was detected in 44 cases and vitronectin in 50. Twenty-four of the 44 V-positive tumors contained large amounts of vitronectin. These cases presented more frequently with adverse histoprognostic factors, including infiltrative growth pattern (62.5%), lack of capsule (71%), presence of capsular invasion (57%), and satellite nodules (50%). We then used HepG2 and Hep3B cell lines as in vitro models to study V integrin regulation and function. HepG2 and Hep3B cells expressed V integrin chain and used V1 and V5 for adhesion and migration on vitronectin. Tumor necrosis factor (TNF) and transforming growth factor (TGF) significantly increased the expression levels of V integrins and stimulated the adhesion and migration of both HepG2 and Hep3B cell lines on vitronectin. The effects of growth factors on cell adhesion and migration were reproduced by incubation with conditioned medium from rat liver myofibroblasts. In conclusion, our results support the existence of an V integrin/vitronectin connection in hepatocellular carcinoma and suggest that this connection may be an adverse prognostic factor. (HEPATOLOGY 2002;36:418-426.)

Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma
Massimo Roncalli, Paolo Bianchi, Barbara Bruni, Luigi Laghi, Annarita Destro, Sonia Di Gioia, Leandro Gennari, Maurizio Tommasini, Alberto Malesci, Guido Coggi
One of the main regulatory pathways reported to be altered in hepatocellular carcinoma (HCC) is that of cell cycle control involving RB1 gene-related cell inhibitors. We investigated p14ARF, p15INK4B, p16INK4A, p18INK4C, and RB1 genes in a series of HCCs and associated cirrhosis with the goal of ascertaining their pattern of inactivation by gene methylation. Thirty-three HCCs, adjacent nonneoplastic cirrhotic tissues, and 6 HCC cell lines were studied. Cirrhoses (25 of 33, 76%), HCCs (31 of 33, 94%), and 3 of 6 (50%) cell lines showed 1 or more methylated genes. Cirrhoses (17 of 33, 51%) had more frequently than HCCs (11 of 33, 33%, P = .01) only 1 methylated gene. With the exception of p18INK4C the genes under study showed promoter methylation with frequency ranging from 82% (p16INK4A in HCC) to 33% and 39% (p15INK4B and p16INK4A in cirrhoses). In cases with only 1 methylated gene, p15INK4B in cirrhosis (8 of 17, 47%) and p16INK4A in HCC (10 of 11, 91%) were the more frequently altered. An optimal correlation was found between p15 and p16 gene methylation and complete protein loss in HCC detected by immunocytochemistry, whereas a partial loss of the same proteins was a feature of methylated cirrhoses. Inactivation by DNA methylation of several genes of the RB1 pathway is common to cirrhosis and HCC. An early pattern of methylatory events (1 methylated gene) is a feature of cirrhosis rather than HCC, whereas an advanced one (3 methylated genes) is characteristic of malignancy. Early methylation changes seem to involve p15INK4B and p16INK4A in cirrhosis and p16INK4A in HCC. In conclusion, a stepwise progression of methylating events is a feature of the sequence cirrhosis­HCC and contributes to the process of hepatic carcinogenesis with potential clinical implications. (HEPATOLOGY 2002;36:427-432.)

Underexpression of mRNA in human hepatocellular carcinoma focusing on eight loci (*Human Study*)
Moritoshi Kinoshita, Masahiko Miyata
Genetic alterations associated with human hepatocellular carcinoma (HCC) have been reported previously, but are not sufficient to specify differences of HCCs from precancerous diseases of the liver, such as hepatitis, hepatic fibrosis, and cirrhosis. In the present study, we performed differential gene display analysis (DGDA) to clarify the specific genetic alterations associated with gene expression changes in the course of development of HCC from chronic viral hepatitis. Four pairs of surgically resected HCCs and hepatitis tissues were investigated. We found 1,028 expression sequence tags (ESTs) that were decreased or increased in HCC tissues compared with hepatitis tissues in the same patient. Nucleotide sequencing showed that they included 55 EST clones in the GenBank database, which were considered candidates for specific messenger RNA (mRNA) expression alterations in HCCs. After excluding 9 ESTs that code mitochondrial DNA, we performed quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) for the 46 remaining EST clones. We found 8 mRNAs underexpressed in primary HCC tissues in 20 patients in higher percentages than found in previous studies, including 18 cases (90%) for aldolase B (ALDOB), 15 cases (75%) for carbamyl phosphate synthetase 1 (CPS1), albumin (ALB), plasminogen (PLG), and EST 51549, 13 cases (65%) for cytochrome P450 subfamily 2E1 (CYP2E1), 12 cases (60%) for human retinol-binding protein 4 (RBP4), and 11 cases (55%) for human organic anion transporter C (OATP-C) gene. In conclusion, underexpression of key gene products may be important in the development and/or progression of HCC. (HEPATOLOGY 2002;36:433-438.)

ERBB-2 overexpression and cyclooxygenase-2 up-regulation in human cholangiocarcinoma and risk conditions (*Human Study*)
Kanenori Endo, Byung-IL Yoon, Chawalit Pairojkul, Anthony J. Demetris, Alphonse Sirica
Quantitative immunohistochemistry of ERBB-2 and MET receptor proteins and of cyclooxygenase 2 (COX-2) was undertaken to determine if there is a positive correlation between overexpression of either ERBB-2 or MET and up-regulation of COX-2 in human cholangiocarcinogenesis. ERBB-2, MET, and COX-2 immunoreactivities were measured in cancerous parenchyma of 71 archival cases of human cholangiocarcinoma (ChC) compared with hyperplastic small biliary ducts in surrounding nonneoplastic liver and with bile ducts of normal adult human liver. ERBB-2, MET, and COX-2 immunoreactivities were also assessed in both large and small hyperplastic biliary ducts (HBDs) in 27 archival cases of hepatolithiasis and 20 archival cases of primary sclerosing cholangitis (PSC), both of which are risk conditions for human cholangiocarcinogenesis. There was a strong positive correlation between increased ERBB-2, but not MET, and COX-2 immunoreactivity measured in the tumors and risk conditions. Enhanced immunoreactivity for ERBB-2 and COX-2 also correlated directly with tumor differentiation and was highest in well-differentiated tumors. Interestingly, some but not all cases of hepatolithiasis and most cases of PSC showed increased ERBB-2 and COX-2 immunostaining in the large but not small HBDs, whereas strong MET immunostaining was detected in both the large and small ducts. In conclusion, overexpression of ERBB-2 and COX-2 may herald an early carcinogenic event in the human hepatic biliary tract and one that is consistent with a frequent anatomic site of origin of the tumors. The results also suggest ERBB-2 and COX-2 as potentially important targets relevant to chemoprevention or adjunct therapy of ChC. (HEPATOLOGY 2002;36:439-450.)

Incidence of drug-induced hepatic injuries: A French population-based study (*Human Study*)
Catherine Sgro, François Clinard, Kader Ouazir, Henry Chanay, Christian Allard, Christian Guilleminet, Claude Lenoir, Alain Lemoine, Patrick Hillon
The incidence of hepatic adverse drug reactions (ADRs) remains unknown in the general population. The goal of this population-based study was to assess the incidence and seriousness of hepatic ADRs. All new cases of symptomatic drug-induced hepatic injuries were collected by 139 trained physicians (general practitioners [GPs] and specialists) between November 1997 and November 2000 in an area containing 81,301 inhabitants who could not go elsewhere for medical care. Over 3 years, 34 cases of hepatic ADRs were collected, 82% of them in outpatients. Global crude annual incidence rate was 13.9 ± 2.4 per 100,000 inhabitants; corresponding standardized annual global rate was 8.1 ± 1.5. There was no difference between urban and rural areas. Standardized incidence female/male ratio was 0.86 (0.26-2.90) until 49 years of age and 2.62 (1.00-6.92) after this age. Diagnosis was carried out by GPs in half of the cases. The outcome was recovery for 32 patients and death for 2. The main drugs implicated were anti-infectious, psychotropic, hypolipidemic agents, and nonsteroidal anti-inflammatory drugs (NSAIDs). Our results suggest that the number of hepatic ADRs in the French population would be 16 times greater than the number noted by spontaneous reporting to French regulatory authorities. In conclusion, the incidence and seriousness of drug-induced hepatitis are largely underestimated in the general population. These results may be useful for further evaluation of drug-induced hepatotoxicity. (HEPATOLOGY 2002;36:451-455.)

Viral Hepatitis

Apolipoprotein E-4 protects against severe liver disease caused by hepatitis C virus
Matthew A. Wozniak, Ruth F. Itzhaki, E. Brian Faragher, Martin W. James, Steven D. Ryder, William L. Irving, on Behalf of the Trent HCV Study Group
The outcome of infection with hepatitis C virus (HCV) varies greatly. The virus associates with serum lipoproteins, including those containing apolipoprotein E (apoE) and apolipoprotein B (apoB), and may enter cells via the low-density lipoprotein receptor (LDLR). ApoE genotypes can affect the extent of damage in diseases caused by 2 other viruses-herpes simplex virus type 1 (HSV1; in Alzheimer's disease and herpes labialis) and human immunodeficiency virus (HIV). We therefore investigated whether specific apoE and apoB alleles were associated with different outcomes of HCV infection. A total of 156 anti­HCV-positive patients and 104 non­HCV-infected patients were studied. Liver biopsy specimens from patients with chronic HCV infection (n = 111) were assessed for disease severity by the Knodell system. ApoE and apoB genotypes were determined by standard polymerase chain reaction (PCR) methods. There was no significant difference among the apoE genotypes of HCV-infected subjects compared with previously published population data, or between HCV-RNA positive or negative patients. However, chronically HCV-infected subjects with mild liver disease (n = 65) had a significantly higher apoE-4 allele frequency (20.0%) than those (n = 46) with severe disease (6.5%). ApoB alleles alone or in combination with apoE were not associated with mild or severe disease. The overall apoE allele frequencies of patients with liver disease not caused by HCV were similar to those of the total HCV group and in contrast to the HCV patients, the apoE allele frequencies were similar in those patients with no or mild fibrosis as compared with those with bridging fibrosis or cirrhosis. In conclusion, carriage of an apoE-4 allele may be protective against liver damage caused by HCV, but not against damage due to various nonviral causes. This is yet another case in which apoE may determine the severity of a viral disease. (HEPATOLOGY 2002;36:456-463.)

Resistance surveillance in chronic hepatitis B patients treated with adefovir dipivoxil for up to 60 weeks (*Human Study*)
Huiling Yang, Christopher E. Westland, William E. Delaney, IV, Elizabeth J. Heathcote, Victoria Ho, John Fry, Carol Brosgart, Craig S. Gibbs, Michael D. Miller, Shelly Xiong
Current therapies for chronic hepatitis B virus (HBV) infection do not provide adequate long-term control of viral replication in the majority of patients. Monotherapy with nucleoside analogs, such as lamivudine and famciclovir, is effective for short periods but results in the emergence of drug-resistant HBV in a substantial number of patients within 1 year of therapy. Adefovir dipivoxil (ADV) has demonstrated clinical activity against wild-type and lamivudine-resistant HBV, but it is unclear whether resistance mutations will emerge after long-term therapy with this drug. To determine whether extended treatment with ADV led to the emergence of drug-resistant populations of HBV, we analyzed virus isolated from patients currently enrolled in a long-term open-label study. The reverse transcriptase domain of HBV polymerase was amplified and sequenced from patients that had received a cumulative exposure of up to 60 weeks of ADV. During our analyses, several previously unreported amino acid substitutions were observed in the reverse transcriptase domain of HBV. Importantly, none of the observed mutations occurred in more than 1 patient, nor were they associated with an adefovir-resistant phenotype in vitro. Furthermore, none of the patients from whom these mutant viruses were isolated had evidence of virologic rebound. In conclusion, these results, although based on a limited number of patients, suggest that treatment with ADV does not lead to the emergence of resistant virus after up to 60 weeks of therapy. (HEPATOLOGY 2002;36:464-473.)

Hepatitis E virus superinfection in patients with chronic liver disease (*Human Study*)
Saeed S. Hamid, Muslim Atiq, Farooq Shehzad, Aneela Yasmeen, Tayyabun Nissa, Abdul Salam, Anwar Siddiqui, Wasim Jafri
Infection with hepatitis A virus (HAV) can cause severe illness in adult patients with chronic liver disease (CLD) caused by hepatitis C. In endemic areas such as South Asia, however, most adult patients already have been exposed to HAV but could still be susceptible to hepatitis E virus (HEV) infection. We document that HEV superinfection in 4 of our CLD patients caused severe liver decompensation. We then determined the seroprevalence of HAV and HEV in 233 patients with stable CLD, with the goal of defining the need for protection against these viruses in these patients. Overall, 41 (17.5%) of 233 CLD patients were HEV antibody immunoglobulin G (IgG)­positive, and 228 of 233 (97.8%) were HAV IgG­positive. As controls, we tested 90 age- and sex-matched healthy volunteer blood donors for HAV and HEV antibodies IgG. There was no difference in the percentage of CLD patients and blood donors positive for HEV antibody IgG (17.7% vs. 17.5%) or for HAV IgG (97.8% vs. 94%). No differences were observed in the severity of liver disease between previously HEV-exposed and -nonexposed patients. In conclusion, superinfection with HEV in patients with underlying CLD can cause severe hepatic decompensation leading to increased morbidity and mortality. The large majority of adult CLD patients in endemic countries are vulnerable to infection with HEV, but are protected against hepatitis A, and are ideal candidates for an HEV vaccine. (HEPATOLOGY 2002;36:474-478.)

GASTROENTEROLOGY

Table of Contents for August 2002 · Volume 123 · Number 2

Randomized trial of medical or endoscopic therapy to prevent recurrent ulcer hemorrhage in patients with adherent clots
D. M. Jensen, T. O. G. Kovacs, R. Jutabha, G. A. Machicado, I. M. Gralnek, T. J. Savides, J. Smith, M. E. Jensen, G. Alofaituli, J. Gornbein
Background & Aims: Treatment of high-risk patients with nonbleeding adherent clots on ulcers is controversial. In a previous randomized trial, there was no benefit to endoscopic therapies compared with medical therapy for prevention of ulcer rebleeding. Our purpose was to test the hypothesis that patients treated with combination endoscopic therapy would have significantly lower rebleeding rates than those treated with medical therapy.
Methods: In this randomized, controlled trial, 32 high-risk patients with severe ulcer hemorrhage and nonbleeding adherent clots resistant to target irrigation were randomized to medical therapy or to combination endoscopic therapy (with epinephrine injection, shaving down the clot with cold guillotining, and bipolar coagulation on the underlying stigmata). Physicians blinded to the endoscopic therapy managed all patients.
Results: Patients were similar at study entry, except for older age in the medical group and lower platelet count in the endoscopic group. By hospital discharge, significantly more medically treated patients (6/17; 35.3%) than endoscopically treated patients (0/15; 0%) rebled (P = 0.011). There were no complications of endoscopic treatment.
Conclusions: Combination endoscopic therapy of nonbleeding adherent clots significantly reduced early ulcer rebleeding rates in high-risk patients compared with medical therapy alone. This endoscopic treatment was safe.

Importance of Helicobacter pylori oipA in clinical presentation, gastric inflammation, and mucosal interleukin 8 production
Y. Yamaoka, S. Kikuchi, H. M. T. El-Zimaity, O. Gutierrez, M. S. Osato, D. Y. Graham
Background & Aims: Disease-associated virulence factors of Helicobacter pylori may not be independent of one another. The aim was to determine which H. pylori virulence factor(s) was the most important predictor of severity of gastric inflammation or clinical outcome.
Methods: cag Pathogenicity island (PAI), vacA babA2, and iceA status were determined by polymerase chain reaction (PCR). oipA functionality was based on switch status determined by PCR-based sequencing. A backward stepwise multiple regression analysis was performed to determine which factor(s) was the most discriminating for clinical outcome as well as the relationship to mucosal histology (H. pylori density, neutrophil infiltration, intestinal metaplasia, and gastric atrophy) and mucosal interleukin 8 (IL-8) production.
Results: H. pylori were obtained from 247 patients (86 with gastritis, 86 with duodenal ulcer, and 75 with gastric carcinoma). Although oipA status was closely linked to specific cag PAI, vacA, and babA2 genotypes, only oipA status remained in the final model to discriminate duodenal ulcer from gastritis (adjusted odds ratio [OR] = 5 and 95% confidence interval [CI] = 2.1­11.9). Among the factors, only a functional oipA was significantly associated with high H. pylori density, severe neutrophil infiltration, and high mucosal IL-8 levels (P < 0.001). oipA status had no relationship to gastric atrophic changes.
Conclusions: oipA functional status was related to clinical presentation, H. pylori density, and gastric inflammation. cag PAI, babA2, or vacA status appear important only as surrogate markers for a functional oipA gene.

Serotonin-transporter polymorphism pharmacogenetics in diarrhea-predominant irritable bowel syndrome
M. Camilleri, E. Atanasova, P. J. Carlson, U. Ahmad, H. J. Kim, B. E. Viramontes, S. McKinzie, R. Urrutia
Background & Aims: A serotonin (5-HT)3 receptor antagonist relieves symptoms in women with diarrhea-predominant irritable bowel syndrome (D-IBS). 5-HT undergoes reuptake by a transporter protein (SERT). Polymorphisms in the promoter for synthesis of SERT (SERT-P) influence response to serotonergic medications in depression. Our hypothesis is that polymorphisms of the promoter region for the SERT influence colonic transit in response to treatment with alosetron in D-IBS.
Methods: Thirty patients (15 men, 15 women) with D-IBS received 1 mg twice a day alosetron for 6 weeks; colonic transit was measured by scintigraphy at baseline and at the end of treatment. Twenty-three patients consented to provide blood DNA samples. Long, short, and heterozygous SERT polymorphisms were identified by polymerase chain reaction­based restriction fragment length polymorphisms and confirmed by direct sequencing. We sought pharmacogenomic association of long, short, and heterozygote polymorphisms with a change in colonic transit and with an a priori­defined, clinically meaningful change in transit at 24 hours (>1.1 colonic regions).
Results: SERT polymorphisms tended to be associated with colonic transit response (P = 0.075); there was a greater response in those with long homozygous than heterozygous polymorphisms (P = 0.039). Slowing of transit by >1.1 colonic region was observed in 9 women and 3 men and was more frequent in long homozygous than heterozygous patients (P = 0.024). Age, gender, and duration of IBS were not significantly different in the 3 groups.
Conclusions: Genetic polymorphisms at the SERT promoter influence response to a 5-HT3 antagonist in D-IBS and may influence benefit-risk ratio with this class of compounds

Mechanisms of diarrhea in collagenous colitis
N. Bürgel, C. Bojarski, J. Mankertz, M. Zeitz, M. Fromm, J.-D. Schulzke
Background & Aims: Collagenous colitis is an inflammatory disease of unknown etiology with diarrhea as the leading symptom. The aim of this study was to examine the pathogenic mechanisms of this disease.
Methods: Biopsy specimens of the sigmoid colon were obtained endoscopically. Short-circuit current and 22Na and 36Cl fluxes were measured in miniaturized Ussing chambers. Alternating current impedance analysis discriminated epithelial from subepithelial resistance. Tight junction proteins occludin and claudin 1­5 were characterized in membrane fractions by Western blotting. Apoptotic ratio was determined by DAPI and TUNEL staining.
Results: In collagenous colitis, net Na+ flux decreased from 8.8 ± 1.8 to 0.2 ± 1.5 and net Cl­ flux from 11.2 ± 3.0 to ­3.0 ± 2.7 µmol · h­1 · cm­2, indicating a pronounced decrease in NaCl absorption. The fact that short-circuit current increased from 1.5 ± 0.4 to 3.9 ± 0.8 µmol · h­1 · cm­2, together with the negative net Cl­ flux, points to activation of active electrogenic chloride secretion. Subepithelial resistance increased from 7 ± 1 to 18 ± 2 · cm2 due to subepithelial collagenous bands of 48 ± 8­µm thickness. Epithelial resistance was diminished from 44 ± 3 to 29 ± 2 · cm2, and this was accompanied by a decrease in occludin and claudin-4 expression. Neither mucosal surface area nor apoptotic ratio was altered in collagenous colitis.
Conclusions: Reduced net Na+ and Cl­ absorption is the predominant diarrheal mechanism in collagenous colitis, accompanied by a secretory component of active electrogenic chloride secretion. The subepithelial collagenous band as a significant diffusion barrier is a cofactor. Down-regulation of tight junction molecules but not epithelial apoptoses is a structural correlate of barrier dysfunction contributing to diarrhea by a leak flux mechanism.

The role of hemochromatosis susceptibility gene mutations in protecting against iron deficiency in celiac disease
J. R. Butterworth, B. T. Cooper, W. M. C. Rosenberg, M. Purkiss, S. Jobson, M. Hathaway, D. Briggs, W. M. Howell, G. M. Wood, D. H. Adams, T. H. Iqbal
Background & Aims: Celiac disease and hereditary hemochromatosis are common HLA-defined conditions in northwestern Europe. We sought to determine whether there is a genetic relationship between the 2 diseases and if hemochromatosis susceptibility gene (HFE) mutations are protective against iron deficiency in celiac disease.
Methods: Polymerase chain reaction amplification using sequence-specific primers capable of identifying the 2 HFE gene mutations (H63D and C282Y) and the HLA class I and II alleles was used to type 145 white patients with celiac disease and 187 matched controls. Hemoglobin and fasting serum iron levels in celiac patients were measured at diagnosis.
Results: HFE gene mutations, H63D or C282Y, were identified in 70 celiac patients (48.3%) and 61 controls (32.6%) (P = 0.004). The C282Y mutation was associated with HLA-A*03 and B*07 alleles in controls and with A*01, A*03, B*08, and DRB1*0301 alleles in celiac patients; the H63D mutation was associated with HLA-A*25 and DRB1*03 alleles in controls and A*29 and DRB1*03 alleles in celiac patients. At diagnosis, celiac patients with the C282Y mutation had higher mean hemoglobin and fasting serum iron levels compared with the HFE wild type (P = 0.0002 and 0.006, respectively). This was not observed with the H63D mutation.
Conclusions: In celiac disease, HFE gene mutations are common and are in linkage disequilibrium with different HLA alleles compared with controls. A disease-specific haplotype that carries C282Y and DQB1*02 is suggested. We propose that HFE gene mutations provide a survival advantage by ameliorating the iron deficiency seen in celiac patients.

Use of surrogate markers of inflammation and Rome criteria to distinguish organic from nonorganic intestinal disease
J. A. Tibble, G. Sigthorsson, R. Foster, I. Forgacs, I. Bjarnason
Background & Aims: Differentiating symptoms of irritable bowel syndrome (IBS) from those of organic intestinal disease is a familiar problem for physicians. The aim of this study was to assess the sensitivity, specificity, and odds ratios (ORs) of fecal calprotectin, small intestinal permeability, Rome I criteria, and laboratory markers of inflammation (erythrocyte sedimentation rate [ESR], C-reactive protein [CRP], blood count) in distinguishing organic from nonorganic intestinal disease.
Methods: A total of 602 new referrals to a gastroenterology clinic who had symptoms suggestive of IBS or organic intestinal disease were studied for these parameters. All patients underwent invasive imaging (barium/endoscopic examination) and other investigations as appropriate, with physicians blinded to the results of fecal calprotectin and intestinal permeability.
Results: A total of 263 patients were diagnosed with organic disease and 339 with IBS. At 10 mg/L, the sensitivity and specificity of calprotectin for organic disease were 89% and 79%, respectively, and that of intestinal permeability for small intestinal disease were 63% and 87%, respectively. Sensitivity of positive Rome criteria for IBS was 85% with a specificity of 71%. An abnormal calprotectin test had an OR for disease of 27.8 (95% confidence interval [CI], 17.6­43.7; P < 0.0001) compared with ORs of 4.2 (95% CI, 2.9­6.1; P < 0.0001) and 3.2 (95% CI, 2.2­4.6; P < 0.0001) for elevated CRP and ESR values. An abnormal permeability test gave an OR of 8.9 (95% CI, 5.8­14.0; P < 0.0001) for small intestinal disease. The OR for IBS with positive Rome criteria was 13.3 (95% CI, 8.9­20.0).
Conclusions: Fecal calprotectin, intestinal permeability, and positive Rome I criteria provide a safe and noninvasive means of helping differentiate between patients with organic and nonorganic intestinal disease.

Prevalence of Barrett's esophagus in asymptomatic individuals
L. B. Gerson, K. Shetler, G. Triadafilopoulos
Background & Aims: The incidence of esophageal adenocarcinoma in the western world has been linked to chronic heartburn, regurgitation, and the development of the premalignant epithelium of Barrett's esophagus (BE). However, up to 40% of esophageal adenocarcinomas occur in patients without prior reflux symptoms. We prospectively screened for the presence of BE in asymptomatic subjects older than 50 years of age undergoing screening sigmoidoscopy for colorectal cancer.
Methods: Subjects undergoing sigmoidoscopy for colorectal cancer (CRC) screening were invited to undergo upper endoscopy. Exclusion criteria included symptoms of gastroesophageal reflux disease (GERD) more than once a month, use of medications for GERD, or previous endoscopy. BE was classified as long-segment BE (LSBE), short-segment BE (SSBE), and microscopic specialized intestinal metaplasia of the esophagogastric junction (SIM-EGJ).
Results: Of 408 potential study candidates, 110 subjects were screened; 9 were women. The mean (±SD) age was 61 ± 9.3 (range, 50­80) years, most of them (73%) Caucasian. Intestinal metaplasia (IM) extending above the EGJ was detected in 27 (25%) subjects; 8 (7%) had LSBE, and 19 (17%) had SSBE. Patients with BE were no more likely to be obese, consumers of tobacco or alcohol, report a family history of GERD, show association with toxic exposure, or use antacids more than once a month, compared with those without BE.
Conclusions: BE was detected in 25% of asymptomatic male veterans older than 50 years of age undergoing screening sigmoidoscopy for CRC.

Long-term fracture risk in patients with Crohn's disease: A population-based study in Olmsted County, Minnesota
E. V. Loftus, Jr., C. S. Crowson, W. J. Sandborn, W. J. Tremaine, W. M. O'Fallon, L. J. Melton, III
Background & Aims: Osteoporosis is common in patients with Crohn's disease, but less is known about their risk of actual fractures.
Methods: The medical records of all 238 Olmsted County, Minnesota, residents diagnosed with Crohn's disease between 1940 and 1993 were reviewed for evidence of subsequent fractures compared with a control group of county residents matched by age and sex. The risk ratio of fracture in patients relative to controls was estimated using the Cox proportional hazards regression model. The cumulative incidence of fracture following diagnosis was estimated using the Kaplan-Meier method.
Results: Sixty-three patients had 117 different fractures. The cumulative incidence of any fracture from the time of diagnosis onward was 36% at 20 years versus 32% in controls (P = 0.792). Compared with controls, the overall risk ratio for any fracture was 0.9 (95% confidence interval [CI], 0.6­1.4), whereas the relative risk for an osteoporotic fracture was 1.4 (95% CI, 0.7­2.7). The risk ratio for thoracolumbar vertebral fracture was 2.2 (95% CI, 0.9­5.5). Cox proportional hazards regression identified only age as a significant clinical predictor of fracture risk (hazard ratio per 10-year increase in age, 1.3; 95% CI, 1.1­1.5). Specifically, use of corticosteroids and surgical resection did not predict risk of fracture among these unselected patients with Crohn's disease from the community.
Conclusions: In this population-based inception cohort of patients with Crohn's disease, the risk of fracture was not elevated relative to age- and sex-matched controls.

Clinical ­ Liver, Pancreas, and Biliary Tract

Psychiatric disorders among veterans with hepatitis C infection
H. B. El-Serag, M. Kunik, P. Richardson, L. Rabeneck
Background & Aims: The presence of psychiatric, drug-, and alcohol-use disorders in hepatitis C virus (HCV)-infected patients may influence their management and prognosis. The frequency and the risk for these disorders among HCV-infected patients are unknown.
Methods: We identified all HCV-infected veteran patients who were hospitalized during 1992­1999 and searched the inpatient and outpatient computerized files for predefined psychiatric, drug-, and/or alcohol-use disorders. We then performed a case-control study among Vietnam veterans; controls without HCV were randomly chosen from hospitalized patients.
Results: We identified 33,824 HCV-infected patients, in whom 86.4% had at least one past or present psychiatric, drug-, or alcohol-use disorder recorded. However, only 31% had active disorders as defined by hospitalization to psychiatric or drug-detoxification bed sections. There were 22,341 HCV-infected patients from the Vietnam period of service (cases) who were compared with 43,267 patients without HCV (controls). Cases were more likely to have depressive disorders (49.5% vs. 39.1%), posttraumatic stress disorder (PTSD) (33.5% vs. 24.5%), psychosis (23.7% vs. 20.9%), bipolar disorder (16.0% vs. 12.6%), anxiety disorders (40.8% vs. 32.9%), alcohol (77.6% vs. 45.0%), and drug-use disorders (69.4% vs. 31.1%). In multivariable regression analyses that adjust for age, sex, and ethnicity, drug use, alcohol-use, depression, PTSD, and anxiety remained strongly associated with HCV.
Conclusions: Several psychiatric, drug-, and alcohol-use disorders are commonly found among HCV-infected veterans compared with those who are not infected. At least one third of these patients have active disorders. A multidisciplinary approach to the management of HCV-infected patients is needed.

Interferon therapy prolonged life expectancy among chronic hepatitis C patients
H. Yoshida, Y. Arakawa, M. Sata, S. Nishiguchi, M. Yano, S. Fujiyama, G. Yamada, O. Yokosuka, Y. Shiratori, M. Omata
Background & Aims: The effects of interferon therapy in chronic hepatitis C patients on survival are unclear. Our objective was to analyze survival among a large cohort of chronic hepatitis C patients.
Methods: We used a retrospective cohort study design in the setting of 7 university hospitals and 1 regional core hospital in Japan. Our study included 2889 patients with histological-proven chronic hepatitis C: 2430 patients received interferon therapy, and 459 patients were untreated. For intervention, the median dose and duration of interferon administration was 480 million units and 137 days, respectively. For measurements, survival status was confirmed by medical records or direct questionnaires. The effect of interferon therapy on survival was assessed by standardized mortality ratio (SMR) based on published mortality among the Japanese general population and by risk ratio calculated by proportional hazards regression.
Results: Thirty of 459 untreated patients, 7 of 817 virologic sustained responders, and 49 of 1613 nonresponders died in 5.4-years follow-up. Fifty-eight (67%) of 86 patient deaths were due to liver diseases (39 to hepatocellular carcinoma). Compared with the general population, overall mortality was high among untreated patients (SMR: 1.9; CI: 1.3­2.8) but not among interferon-treated patients (SMR: 0.9; CI: 0.7­1.1). The risk of death was reduced, compared with untreated patients, among interferon-treated patients (risk ratio for overall death: 0.367; CI: 0.236­0.596; for liver-related death: 0.284; CI: 0.164­0.494) and among sustained responders (risk ratios: 0.148; CI: 0.064­0.343 and 0.050; CI: 0.012­0.216). The risk of liver-unrelated deaths remained unchanged.
Conclusions: Interferon therapy improved survival of chronic hepatitis C patients by preventing liver-related deaths

Basic ­ Alimentary Tract

Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice
K.-P. Janssen, F. El Marjou, D. Pinto, X. Sastre, D. Rouillard, C. Fouquet, T. Soussi, D. Louvard, S. Robine
Background & Aims: Ras oncoproteins are mutated in about 50% of human colorectal cancers, but their precise role in tumor initiation or progression is still unclear.
Methods: This study presents transgenic mice that express K-rasV12G, the most frequent oncogenic mutation in human tumors, under control of the murine villin promoter in epithelial cells of the large and small intestine.
Results: More than 80% of the transgenic animals displayed single or multiple intestinal lesions, ranging from aberrant crypt foci (ACF) to invasive adenocarcinomas. Expression of K-rasV12G caused activation of the MAP kinase cascade, and the tumors were frequently characterized by deregulated cellular proliferation. Unexpectedly, we obtained no evidence of inactivating mutations of the tumor suppressor gene Apc, the "gatekeeper" in colonic epithelial proliferation. However, spontaneous mutation of the tumor-suppressor gene p53, a frequent feature in the human disease, was found in 3 of 7 tumors that were tested.
Conclusions: This animal model recapitulates the stages of tumor progression as well as a part of the genetic alterations found in human colorectal cancer. Furthermore, it indicates that activation of K-ras in concert with mutations in p53 may constitute a route to digestive tumor formation and growth, underlining the fact that the pathway to intestinal cancer is not necessarily a single road.

Corticotropin-releasing hormone antagonists possess anti-inflammatory effects in the mouse ileum
M. Wlk, C. C. Wang, M. Venihaki, J. Liu, D. Zhao, P. M. Anton, A. Mykoniatis, A. Pan, J. Zacks, K. Karalis, C. Pothoulakis
Background & Aims: Corticotropin-releasing hormone (CRH) released at local sites of inflammation promotes inflammation in the periphery. We investigated its effects in the intestinal responses caused by toxin A from Clostridium difficile, the causative agent of antibiotic-associated colitis.
Methods: Ileal loops were injected with 10 µg of toxin A, and enterotoxic responses were measured at various time points.
Results: Pretreatment of mice with 2.5 µg/kg of the CRH receptor antagonist -helical CRH(9­41) that blocks both CRH receptor subtypes reduced toxin A­mediated ileal secretion, epithelial cell damage, mucosal edema, neutrophil infiltration, and mucosal content of interleukin 1 and tumor necrosis factor . Pretreatment with the specific CRH1 receptor antagonist antalarmin (20 mg/kg, IP) also inhibited toxin A­induced fluid secretion and toxin A­associated histologic changes. CRH messenger RNA and protein were increased in mouse ileum 30 minutes after intraluminal toxin A administration. In situ hybridization and immunohistochemistry demonstrated that toxin A at 1 hour caused a substantial increase in the expression of both CRH receptor subtypes in the ileal mucosa.
Conclusions: Peripheral CRH may play a proinflammatory role in toxin A­induced intestinal secretion and inflammation and that CRH1 receptor, at least in part, is important in the mediation of these responses.

Deletion of functional gastrin gene markedly increases colon carcinogenesis in response to azoxymethane in mice
S. Cobb, T. Wood, L. Tessarollo, M. Velasco, R. Given, A. Varro, N. Tarasova, P. Singh
Background & Aims: We recently reported that transgenic mice overexpressing progastrin were at a higher risk for developing colon cancers in response to azoxymethane (AOM), whereas mice overexpressing gastrin-17 were at a reduced risk. To examine further the role of gastrins in colon carcinogenesis, we generated gastrin gene knockout mice (GAS-KO).
Methods: The height and proliferative index (PI) of colonic crypts were similar in GAS-KO and wild-type (WT) mice, suggesting that the absence of gastrins in GAS-KO mice did not significantly affect the growth of colonic mucosa. GAS-KO and WT mice were treated with AOM for 3­4 weeks; control mice received saline.
Results: Colonic proliferation in response to AOM was significantly increased in GAS-KO vs. WT mice. Aberrant crypt foci (ACFs) were similarly increased significantly by ~2­5-fold in GAS-KO vs. WT mice after 2 weeks of AOM treatment. Female GAS-KO mice developed adenomas (Ads) and adenocarcinomas (AdCAs) at earlier times (~10 months) than the male GAS-KO mice and the male and female WT mice (~12 months). The total numbers of Ads and AdCAs were significantly higher in GAS-KO than in WT mice.
Conclusions: These results suggest the novel possibility that loss of gastrin expression (and hence amidated gastrins) significantly increases susceptibility to colon carcinogenesis in response to AOM. Previous studies with FVB/N transgenic mice similarly suggested a protective role of amidated gastrins against colon carcinogenesis, which supports the present findings of an increase in colon carcinogenesis in GAS-KO mice lacking normal physiological levels of amidated gastrins.

An alternate pathway of cAMP-stimulated Cl­ secretion across the NKCC1-null murine duodenum
N. M. Walker, M. Flagella, L. R. Gawenis, G. E. Shull, L. L. Clarke
Background & Aims: Adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated anion secretion across the duodenal epithelium requires the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane and anion uptake proteins in the basolateral membrane. NKCC1, the epithelial Na+/K+/2Cl­ cotransporter, is the major protein responsible for Cl­ uptake. In this study, we evaluate the role of NKCC1 in determining the relative rates of transepithelial Cl­ and HCO3­ secretion during cAMP stimulation of the duodenum.
Methods: Bicarbonate and chloride secretion across duodenal mucosa was measured in Ussing chambers by pH stat and 36Cl flux methods using mice with either gene-targeted deletion of NKCC1 (NKCC1­/­) or bumetanide blockade of NKCC1.
Results: Total anion secretion stimulated by forskolin treatment of NKCC1-null duodenum resulted from approximately equivalent rates of electrogenic chloride, electrogenic bicarbonate, and electroneutral bicarbonate secretion. Evaluation of the alternate chloride secretory pathway indicated chloride uptake by a basolateral membrane anion exchange process with characteristics consistent with the anion exchanger isoform AE2.
Conclusions: Chloride uptake by basolateral anion exchanger activity (AE2) supports intracellular cAMP­stimulated chloride secretion in the NKCC1-null duodenum. A model for the alternate chloride secretion pathway is proposed whereby chloride uptake via AE2 is coupled to basolateral NaHCO3 cotransport to support CFTR-mediated chloride and bicarbonate secretion.

Helicobacter pylori impairs DNA mismatch repair in gastric epithelial cells
J. J. Kim, H. Tao, E. Carloni, W. K. Leung, D. Y. Graham, A. R. Sepulveda
Background & Aims: Helicobacter pylori infection is a major gastric cancer risk factor. H. pylori gastritis occurs more frequently in individuals with microsatellite instability­positive than those with microsatellite instability­negative gastric cancers, raising the possibility that H. pylori infection affects DNA mismatch repair (MMR). The aim of this study was to determine the effect of H. pylori on the expression of DNA MMR proteins and RNA in gastric epithelial cells.
Methods: Gastric cancer cell lines were cocultured with H. pylori, bacterial extracts, and Campylobacter jejuni or Escherichia coli. MutS (hMSH2 and hMSH6) and MutL (hMLH1, hPMS2, and hPMS1) DNA MMR protein and RNA levels were determined.
Results: All cell lines examined showed decreased levels of MutS and MutL DNA MMR proteins in a dose-dependent manner after coculture with H. pylori strains. The reduction in DNA MMR protein levels was caused by heat-sensitive H. pylori products. The levels of DNA MMR proteins were affected by C. jejuni but not by E. coli. RNA levels of hMSH2 and hMSH6 were also reduced after exposure to H. pylori.
Conclusions: H. pylori infection of gastric epithelial cells leads to a decrease in DNA MMR proteins that is at least in part related to an H. pylori­induced decrease in messenger RNA levels of repair genes. These data suggest that H. pylori infection might lead to a deficiency of DNA MMR in gastric epithelial cells that may increase the risk of mutation accumulation in gastric mucosa cells and the risk of gastric cancer during chronic H. pylori infection.

Experimental murine colitis is regulated by two genetic loci, including one on chromosome 11 that regulates IL-12 responses
G. Bouma, A. Kaushiva, W. Strober
Background & Aims: Immunogenetic analysis of experimental colitis may contribute to the further unraveling of the complex genetic basis of the inflammatory bowel diseases, Crohn's disease and ulcerative colitis.
Methods: Genetic regions associated with susceptibility to trinitrobenzene sulfonic acid (TNBS)-induced colitis were identified in a genome-wide linkage analysis in F2 progeny of colitis-susceptible SJL/J and -resistant C57BL/6 mice. An immunogenetic approach was then used to further study the pathophysiologic role of one of the identified loci.
Results: We identified susceptibility loci on chromosomes 9 (Tnbs1) and 11 (Tnbs2). Tnbs2 harbors the interleukin (IL)-12 p40 gene, a likely candidate gene because IL-12 is a known central mediator for both experimental colitis and human Crohn's disease. We therefore tested the ability of colitis-susceptible and -resistant strains to mount IL-12 responses to lipopolysaccharide (LPS), a strong inducer of IL-12 that is abundantly present in the intestine. We observed a remarkably higher serum IL-12 response to LPS in susceptible SJL/J mice. Subsequently, we showed that the genetic region regulating the IL-12 response to LPS colocalizes with Tnbs2.
Conclusions: These data strongly suggest that the tendency to mount a high LPS-induced IL-12 response and susceptibility to TNBS-induced colitis are related and that in fact a genetically determined high IL-12 response is involved in the immunologic basis of susceptibility to colitis.

Nitric oxide regulates the release of somatostatin from cultured gastric rabbit primary D-cells
N. Arebi, Z. V. Healey, P. W. Bliss, M. Ghatei, S. Van Noorden, R. J. Playford, J. Calam
Background & Aims: Neuronal nitric oxide synthase (nNOS) is present in gastric D-cells. Mucosal somatostatin is diminished in H. pylori gastritis, where production of nitric oxide (NO) is increased. Therefore, we investigated the role of NO in D-cell function and the effects of prolonged exposure of D-cells to NO.
Methods: Rabbit gastric D-cells were cultured. Somatostatin-14 was measured after 2 hours to examine the effects of arginine, nitric oxide sythase (NOS) inhibitors, and NO donors. Some cells were preincubated with a slow releasing NO donor for 12 hours. Results are expressed as percentage of total cell content. Nitrate content was measured by chemiluminescent assay.
Results: L-arginine increased somatostatin-14 release in the presence of CCK8 from 4.4% ± 0.5% to 6.4% ± 0.4% (P < 0.02), and this was accompanied by NO release from 27 ± 7 µmol/L to 86 ± 12 µmol/L (P = 0.001). D-arginine and L-lysine had no effect. NOS inhibitors LNNA, SMT, and 7NI significantly attenuated the stimulatory response to L-arginine. NO donors sodium nitroprusside (SNP), 1 mmol/L, and S-nitroso-N-acetyl-D-L-penicillamine, 0.1 mmol/L, significantly increased basal and cholecystokinin-8 (CCK8) stimulated somatostatin release. Oxyhemoglobin attenuated the effect of SNP but not of L-arginine. Neither cyclic guanosine monophosphate nor guanylate cyclase were involved in the response to NO. However, inhibition of adenosine diphosphate (ADP) ribosyltransferase significantly decreased the response to L-arginine. Preincubation for 12 hours with 150 µmol/L (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; IP3, inositol triphosphate decreased the 2-hour cellular response to CCK8 and SNP.
Conclusions: NO regulates rabbit D-cells. Acute exposure stimulates somatostatin mediated by ADP ribosylation, whereas long-term exposure reduces cellular responses to stimuli. The latter pathway may be responsible for the suppression of somatostatin in H. pylori gastritis.

Differentiation of gastric ECL cells is altered in CCK2 receptor­deficient mice
D. Chen, C.-M. Zhao, W. Al-Haider, R. Håkanson, J. F. Rehfeld, A. S. Kopin
Background & Aims: Gastrin stimulation of the type 2 cholecystokinin (CCK2) receptor results in ECL cell proliferation and histamine secretion. This report describes the effects of targeted disruption of the CCK2 receptor gene on ECL cell morphology and function.
Methods: The ECL cells in the oxyntic mucosa of CCK2 receptor­deficient (knockout [KO]) vs. wild-type (WT) mice were investigated by immunocytochemical and biochemical approaches, as well as by electron microscopy.
Results: Immunocytochemistry demonstrates similar numbers (cells per millimeter of horizontal length of mucosa) of pancreastatin- or vesicle monoamine transporter-2 (VMAT-2)­immunoreactive cells in WT mice and KO mice. However, only WT mice harbor histamine-immunoreactive ECL cells. The mucosal histamine content in KO mice (likely originating from mast cells) is only a minute fraction of that present in WT animals. The activity of the histamine forming enzyme, histidine decarboxylase (a marker of ECL cells), was undetectable in the oxyntic mucosa of KO mice yet was readily apparent in the mucosa from WT animals. Electron microscopy revealed numerous ECL cells in WT mice. In KO animals, these cells were replaced by an "ECL-like" cell type, characterized by a lack of secretory vesicles (a hallmark feature of normal ECL cells) and the presence of dense-core granules and microvesicles in numbers comparable to those found in WT ECL cells. Based on ultrastructural features, the ECL-like cells in KO mice can be readily distinguished from other gastric endocrine cells, including A-like cells and D cells.
Conclusions: Absence of a single gene product, the CCK2 receptor, alters the differentiation and function of gastric ECL cells.

Lipopolysaccharide preconditioning and cross-tolerance: The induction of protective mechanisms for rat intestinal ileus
N. T. Schwarz, B. Engel, M. K. Eskandari, J. C. Kalff, J. R. Grandis, A. J. Bauer
Background & Aims: Endotoxin elicits an inflammatory response within the intestinal muscularis and causes intestinal muscle dysfunction. Our aims were to investigate intestinal muscle recovery after a single or repeated lipopolysaccharide (LPS) injections. We also investigated the ability of LPS to induce cross-tolerance to postoperative ileus.
Methods: Motility was measured in vivo and in vitro by transit and organ-bath techniques. Nuclear factor kappa-B, nuclear factor interleukin 6, and signal transducer and activator of transcription were quantified by using electrophoretic mobility shift assay, and tumor necrosis factor alpha, interleukin 6, inducible nitric oxide synthase, and cyclooxygenase 2 were measured with reverse-transcription polymerase chain reaction. Myeloperoxidase histochemistry for neutrophils was performed in jejunal muscularis whole mounts.
Results: Endotoxin-induced suppression of in vitro muscle contractility temporally recovered over 7 days with a similar profile whether after a single dose or during the continuous daily injection of LPS. Functional adaptation to continuous LPS was reflected in a significant blunting of transcription factor activation and cytokine messenger RNA up-regulation compared with the naive LPS-stimulated muscularis. Preconditioning of the muscularis showed significant cross-tolerance to the functional, molecular, and leukocytic sequelae of intestinal manipulation.
Conclusions: The muscularis externa recovered and developed tolerance to endotoxin over 7 days, which conferred cross-tolerance to intestinal manipulation. Thus, preconditioning induces protective mechanisms to a subsequent insult within the muscularis externa.

Basic ­ Liver, Pancreas, and Biliary Tract

 Organ-specific alterations in RAR:RXR abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis
L. A. Denson, A. Bohan, M. A. Held, J. L. Boyer
Background & Aims: Obstructive cholestasis is associated with adaptive changes in expression of hepatocyte transport proteins. These include a significant reduction in hepatic expression of Mrp2 (Abcc2), the principal canalicular multispecific organic anion transporter. Renal Mrp2 expression is preserved under these conditions. We have recently reported that the rat Mrp2 promoter is activated by RAR:RXR, and that interleukin 1 (IL-1) repressed promoter activity via this element. We hypothesized that cytokines, which are up-regulated in obstructive cholestasis, would reduce nuclear RAR:RXR levels, and that this would be associated with suppression of hepatic Mrp2 expression.
Methods: Male Sprague-Dawley rats were subjected to bile duct ligation (BDL) or sham surgery, and liver and kidney RNA and protein were isolated. Primary rat hepatocytes were treated with bile acids, retinoids, or cytokines, and RNA and protein were isolated. Mrp2 and RAR:RXR protein abundance and activity were assessed by using electrophoretic mobility shift assays (EMSA) and immunoblots. IL-1 abundance was determined by enzyme-linked immunosorbent assay. RAR, RXR, and Mrp2 RNA levels were determined by using ribonuclease protection assays (RPA).
Results: Mrp2 down-regulation and IL-1 up-regulation were observed in liver after BDL. This was temporally associated with down-regulation of liver RAR:RXR nuclear protein levels and binding to the Mrp2 promoter cis element. Renal RAR:RXR and Mrp2 expression were preserved under these conditions. IL-1 treatment of primary hepatocytes reduced Mrp2 and RXR expression.
Conclusions: Organ-specific regulation of Mrp2 expression in obstructive cholestasis is associated with cytokine-dependent alterations in RAR:RXR nuclear receptors. Preservation of renal Mrp2 expression may permit urinary excretion of toxic organic anions and xenobiotics under conditions in which biliary excretion is impaired.

Gene therapy by intrahepatic and intratumoral trafficking of p53-VP22 induces regression of liver tumors
L. Zender, R. Köck, M. Eckhard, B. Frericks, T. Gösling, T. Gebhardt, S. Drobek, M. Galanski, F. Kühnel, M. Manns, S. Kubicka
Background & Aims: VP22-mediated intercellular transport provides an approach to deliver functional chimeric proteins into a high percentage of target cells. The aim of this study was to evaluate the efficacy of p53/VP22 fusion protein in gene therapy of liver tumors.
Methods: Expression vectors of N- and C-terminal fusion proteins of p53 and VP22 were subcloned and transcriptional properties of chimeric proteins were assessed by luciferase assays. Adenoviral vectors expressing p53 wild type (AdGFP/p53wt) and p53-VP22 (AdGFP/p53-VP22) were generated to investigate the VP22-mediated spreading in normal liver and in liver tumors in vivo by green fluorescent protein fluorescence and p53 immunohistochemistry. Gene therapy was investigated in subcutaneous and preclinical orthotopic animal tumor models after subcutaneous and intra-arterial administration of the adenoviruses, and tumor growth was assessed by direct calibration and magnetic resonance imaging.
Results: p53-VP22 showed enhanced transcriptional activity compared with p53 wild type. VP22-mediated intercellular transport of p53 could be observed in the normal liver and in liver tumors in vivo and was correlated with increased antitumor efficacy of gene therapy and improved survival of the animals.
Conclusions: Fusion of VP22 to p53 strongly improves the results of p53 replacement gene therapy. Furthermore, the demonstrated VP22-mediated intercellular transport in the liver could be important for other strategies in liver gene therapy, providing a tool for enhancing the effect of gene therapy in liver diseases such as metabolic disorders or viral hepatitis.

Deficiency of survivin in transgenic mice exacerbates Fas-induced apoptosis via mitochondrial pathways
E. M. Conway, S. Pollefeyt, M. Steiner-Mosonyi, W. Luo, A. DeVriese, F. Lupu, F. Bono, N. Leducq, F. Dol, P. Schaeffer, D. Collen, J.-M. Herbert
Background & Aims: Survivin is an inhibitor of apoptosis protein (IAP), which also is crucial for mitosis and cell cycle progression. IAPs participate in regulating Fas ligand-induced hepatic apoptosis. The aim was to study the contribution of survivin to hepatic apoptosis by generating transgenic mice lacking survivin.
Methods: The survivin gene was inactivated in mice by homologous recombination in embryonic stem cells. Survivin+/­ and survivin+/+ mice were generated and injected with the Fas agonistic antibody Jo2.
Results: In 3 genetic backgrounds, survivin­/­ embryos died before 4.5 days post coitum. Survivin+/­ mice appeared normal, but liver lysates revealed aseline low-level activation of procaspase-8, Bid, procaspase-9, and procaspase-3, with accumulation of Bax, and release of cytochrome c, indicating a proapoptotic state. Intraperitoneal injection of low-dose Jo2 had no effect on survivin+/+ mice at 2 hours. However, in survivin+/­ mice, Jo2 caused hemorrhagic necrosis of the liver, associated with prominent activation of the apoptotic pathway via the mitochondria, and up-regulation of hepatocellular expression of survivin in the cytosol, nuclei, and mitochondria. Isolated mitochondria from survivin+/­ livers had more defects in oxidative phosphorylation after C2-ceramide exposure.
Conclusions: Absence of survivin is incompatible with life. Although Jo2 induces expression of survivin, diminished baseline levels render the liver more sensitive to Fas, possibly due to functional effects on the mitochondria. This is the first in vivo documentation that survivin modulates caspase activation and that Fas-mediated hepatic apoptosis is regulated by survivin via mitochondrial pathways.

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Journal of Hepatology

Volume 37, Issue 2, August 2002

Biliary Tract and Cholestasis

Koichi Tsuneyama et al.
Damaged interlobular bile ducts in primary biliary cirrhosis show reduced expression of glutathione-S-transferase-pi and aberrant expression of 4-hydroxynonenal
Background/Aims: Chronic inflammation induces oxidative stress by producing reactive oxygen species. We investigated how the oxidative stress associated with chronic cholangitis induce bile duct damages in primary biliary cirrhosis. Methods: The intracellular status of lipid peroxidation due to oxidative stress and that of glutathione, an endogenous cytoprotective molecule, were examined in primary biliary cirrhosis and controls by immunostaining of 4-hydroxynonenal and glutathione-S-transferase-pi. The former is a by-product of lipid peroxidation, and the latter is involved in the formation of intracellular glutathione. Results: In the damaged bile ducts of primary biliary cirrhosis, glutathione-S-transferase-pi expression was markedly reduced, reflecting reduction of intracellular glutathione, and perinuclear expression of 4-hydroxynonenal was frequent, reflecting active lipid peroxidation associated with biliary epithelial damages. There was diffuse/luminal expression of 4-hydroxynonenal in the bile ducts frequent in primary biliary cirrhosis and controls, likely reflecting absorption of 4-hydroxynonenal, also a component of oxidized low-density lipoprotein, from bile via scavenger receptor class B type 1 on biliary epithelium. Conclusions: The data suggest that lipid peroxidation in the bile ducts with reduced expression of glutathione-S-transferase-pi, may be an important pathologic process leading to the bile duct damage of primary biliary cirrhosis.

Biliary Tract and Cholestasis

Laura Fouassier et al.
Contribution of mrp2 in alterations of canalicular bile formation by the endothelin antagonist bosentan
Background/Aims: Bosentan, a dual endothelin ETA/B receptor antagonist, may cause dose-dependent reversible cholestatic liver injury. We herein tested whether bosentan or metabolites, both eliminated in bile, induce alterations in bile secretion. Methods: Bile flow and output of bile constituents were monitored in pentobarbital-anesthetized rats with biliary fistulas. Normal and TR rats with a genetic defect in mrp2, received bosentan intravenous injections. Results: Bosentan bolus intravenous injections of 0.1-10mg/kg triggered a dose-dependent increase in biliary bilirubin excretion. In addition, doses (10mg/kg) caused a sustained increase in canalicular bile salt-independent bile flow, combined with significant increases in the concentration and output of glutathione and of bicarbonate in bile. In rats receiving bosentan (10mg/kg), both under basal conditions and under intravenous taurocholate perfusion (2µmol/min/kg), phospholipid and cholesterol secretions were profoundly inhibited and uncoupled from bile salt secretion. In TR rats, the choleretic effect of bosentan was reduced to non-significant levels. The stimulation of bilirubin secretion and the uncoupling of phospholipid from bile salt secretion were absent, whereas that of cholesterol was maintained. Conclusions: Bosentan alters canalicular bile formation in major part via mrp2-mediated mechanisms. Intermittent uncoupling of lipid from bile salt secretion may contribute to bosentan hepatic adverse reaction.

Biliary Tract and Cholestasis
Emmanuelle Girodon et al.
Cystic fibrosis transmembrane conductance regulator (CFTR) gene defects in patients with primary sclerosing cholangitis
Background/Aims: Because biliary tract lesions that resemble those of primary sclerosing cholangitis (PSC) may occur in cystic fibrosis (CF), we examined the prevalence and influence of CF transmembrane conductance regulator (CFTR) gene mutations in PSC patients. Methods: Genomic DNA was analyzed in 29 consecutive PSC patients and in 115 healthy control individuals. A scanning method followed by direct DNA sequencing was used to scan the CFTR coding regions. Results: Four patients (13.8%) were heterozygous for a CFTR mutation, including a new putative severe CF-causing mutation (N782K), and three mild defects (L997F, D1270N, and S1235R). The comparison of PSC patients with healthy controls showed no significant difference in the frequency of CFTR mutations (P=0.415). In addition, two patients (6.9%) were heterozygous for the IVS8-5T allele, which is not significantly different from the 5-6%-prevalence in the general population. Unusual clinical features including a severe outcome in childhood, with a lethal outcome at age 22, and biliary aspergillosis were recorded in patients with a CFTR mutation. Conclusions: The proportion of CF carriers is not significantly higher in PSC patients than in the general population. The possibility that CFTR mutations may contribute to a severe clinical course in PSC patients is worth further examining

Cell Biology, Metabolism and Transport

Andreas Geier et al.
Hepatobiliary organic anion transporters are differentially regulated in acute toxic liver injury induced by carbon tetrachloride
Background/Aims: Hepatobiliary transporters are down-regulated in cholestasis, but their expression in acute, non-cholestatic, cytokine-mediated liver injury is unknown. Thus we studied the molecular mechanisms, by which sodium taurocholate cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, multidrug-resistance protein 2 (Mrp2) and bile salt export pump (Bsep) are regulated in liver injury induced by carbon tetrachloride (CCl4). Methods: mRNA and protein levels were determined in rats 24 and 72h after CCl4 injection. Transporter gene transcription and binding activities of Ntcp and Mrp2 transactivators were assessed by nuclear runoff and electrophoretic mobility shift assays. Results: mRNA levels significantly declined to 41±44% for Ntcp, 65±41% for Oatp1 and 64±28% for Oatp2, but remained unchanged for Oatp4, canalicular Mrp2 and Bsep. Protein levels declined only for Oatp4 (50±17%) and Ntcp (23±13%) at 24h. Reduced mRNA levels (Ntcp, Oatp1, Oatp2) were associated with decreased transcriptional activities. Binding activity of Ntcp transactivators (hepatocyte nuclear factor 1 (HNF1) and CAAT enhancer binding protein (C/EBP) were reduced by 24h, whereas retinoid X receptor (RXR):retinoid acid receptor (RAR) as transactivator of both Ntcp and Mrp2 remained unaltered. Recovery of acute hepatitis and changes in gene expression occurred after 72h. Conclusions: Acute liver injury results in down-regulation of basolateral organic anion transporters similar to liver regeneration after partial hepatectomy, but in contrast to endotoxin-induced cholestasis. Maintained binding activity of RXR:RAR may explain differences in Mrp2 expression.

Inflammation and Fibrosis

Isabelle A. Leclercq, Geoffrey C. Farrell, Rixt Schriemer and Graham R. Robertson
Leptin is essential for the hepatic fibrogenic response to chronic liver injury
Background/Aims: Obesity is associated with hyperleptinemia and is also a risk factor for fibrosis and severity of fibrosis in several chronic liver diseases. The correlation between increased leptin, obesity and hepatic fibrosis prompted us to hypothesise that leptin has profibrogenic effects on the liver. Methods: We analysed the role of leptin in liver fibrosis in leptin-deficient mice fed a diet which generates steatohepatitis, and in chronic carbon tetrachloride-induced hepatic injury. Results: Leptin-deficient mice failed to develop fibrosis during steatohepatitis or in response to chronic toxic liver injury, and failed to up-regulate collagen-I while developing similar hepatic injury as their genetic controls. Restitution of physiological levels of circulating leptin by injection of exogenous leptin, but not correction of the obese phenotype by dietary manipulation, restored liver fibrosis in leptin-deficient mice during chronic liver injury. These results confirmed the absolute requirement of leptin for hepatic fibrosis. We showed that leptin deficiency did not alter hepatic TNF regulation but that leptin is necessary for induction of bioactive transforming growth factor beta 1 (TGF1) protein in the context of chronic liver injury. Conclusions: These data establish that leptin is an essential mediator of hepatic fibrosis in response to chronic liver injury, whether metabolic or toxic in aetiology.

Naondo Sohara, Iya Znoyko, Miriam T. Levy, Maria Trojanowska and Adrian Reuben
Reversal of activation of human myofibroblast-like cells by culture on a basement membrane-like substrate
Background: Liver injury transforms hepatic stellate cells into myofibroblast (MFB)-like cells. With recovery from injury, MFBs undergo apoptosis, but it is unknown whether they can also revert to quiescence. Aim: To determine whether human (h)MFBs become quiescent if cultured on a basement membrane-like substrate (Matrigel(TM)). Methods: hMFBs obtained from cirrhotic liver were re-cultured on plastic or Matrigel(TM). Expression of genes of collagen metabolism was assayed before and after transforming growth factor (TGF) and Oncostatin M (OSM) stimulation. Results: hMFBs had typical MFB-like morphology, with abundant -smooth muscle actin (SMA) but no cytoplasmic lipid droplets. hMFBs re-cultured on Matrigel(TM) reverted to SMA-negative, lipid droplet-positive quiescent morphology. SMA, collagen 1(1) (COL1A1) and collagen 2(1) (COL1A2) messages were upregulated in hMFBs cultured on plastic, but suppressed by Matrigel(TM). The opposite was true for metalloproteinase-1 mRNA. OSM but not TGF reduced SMA mRNA by 30% while TGF but not OSM upregulated COL1A1 mRNA by 48%, in hMFBs on plastic. TGF and OSM stimulated COL1A1 gene expression in Matrigel(TM) by 50 and 60%, respectively. Conclusions: Matrigel(TM) culture de-activates hMFBs yet collagen gene expression still responds to fibrogenic cytokines. The responses of hMFB gene expression to TGF and OSM, are regulated differently by the extracellular matrix.

Liver Growth and Cancer

Antonio Sa Cunha et al.
Inhibition of rat hepatocellular carcinoma tumor growth after multiple infusions of recombinant Ad.AFPtk followed by ganciclovir treatment
Background/Aims: The antitumor efficiency of thymidine kinase (tk) in Herpes Simplex virus-tk-based gene therapy of rat hepatocellular carcinoma (HCC) was examined by specific transcriptional targeting of tk to tumor cells by the -fetoprotein (AFP) gene promoter and by multiple infusions of recombinant adenovirus Ad.AFPtk. Methods: We developed a surgical procedure that allows efficient, non-invasive delivery (during 2 months) of recombinant Ad via the intra-hepatic artery (IHA) route. Results: Treatment of tumor-bearing rats with either three or five doses of 5¥109pfu Ad.AFPtk, administered every 3 days, and followed by intra-peritoneal treatment with ganciclovir (GCV), resulted in tumor growth inhibition and apoptosis, when compared to untreated tumor-bearing rats or animals treated with Ad.AFPlacZ or buffered saline. No treatment-related toxicity was noted. Antitumor efficacy, based on tumor size and number of tumors, was demonstrated in more than 50% of Ad.AFPtk+GCV-treated rats, as compared to control rats (P<0.0005). Conclusions: Our results demonstrate the safety and potential of multiple Ad.AFPtk administrations by the IHA route to inhibit HCC tumor growth, and support further clinical investigation of Ad.AFPtk gene therapy for treatment of multifocal tumor lesions in most primary liver cancers.

Keiji Minouchi, Shuichi Kaneko and Kenichi Kobayashi
Mutation of p53 gene in regenerative nodules in cirrhotic liver
Background/Aims: Mutations of p53 gene have been detected in precancerous stages of several cancers, and the possible role in multistep carcinogenesis is suggested. The aim of this study was to examine the mutation profile of p53 gene in regenerative nodules in cirrhotic livers. Methods: Ninety eight tissue specimens of regenerative nodules obtained from 15 cases of cirrhosis were used for analysis. Twenty cases of chronic hepatitis and two cases of fatty liver were used as controls. DNA was extracted from each of manually demarcated regenerative nodules, and nucleotide sequence analysis was performed on p53 gene exon 5. Results: Direct sequencing detected p53 mutations in seven of 98 DNA samples (7.1%) from regenerative nodules in six cases of cirrhosis. Subcloning analysis revealed that mutation sites differed in each subclone and the incidences of the mutation varied from 7.7 to 58.8% depending on individual nodules. The mutation was not detected in any of chronic hepatitis and fatty liver. There were inconsistent p53 sequence with regenerative nodules and accompanied hepatocellular carcinomas in six cases. Conclusions: Mutations of p53 gene were frequently found in cirrhotic livers compared with livers of patients with chronic hepatitis (P<0.01), suggesting that p53 mutations at the stage of cirrhosis may be a causative factor that may potentially lead to hepatocellular carcinoma.

Masami Kawai et al.
Mechanical stress-dependent secretion of interleukin 6 by endothelial cells after portal vein embolization: clinical and experimental studies
Background/Aims: Interleukin-6 (IL-6) is an essential early signal in liver regeneration, however, little is known about what triggers IL-6 release. Changes in portal hemodynamics after portal vein embolization (PVE) may contribute to IL-6 release, leading to regeneration of non-embolized lobe. Methods: In 22 patients who underwent right PVE, the diameters of the left portal branches, liver volumes, and serum concentrations of IL-6, tumor necrosis factor- (TNF-), and hepatocyte growth factor (HGF) were measured. We then studied endothelial cells cultured on an elastic silicone membrane and subjected to continuous uni-axial stretch. Supernatant cytokine concentrations were measured. Results: The diameters of the portal branches increased by 150% after PVE. Serum IL-6 concentrations increased within 3h after PVE. The concentrations of TNF- and HGF remained unchanged. The left lobe volume increased 2 weeks after PVE. The IL-6 concentrations in the supernatant of endothelial cells with stretch stress were higher than that in the non-stretched control group. Conclusions: These findings indicate that PVE dilates the portal branches in the non-embolized lobe, exposing hepatic vasculature to stretch stress. This hemodynamic change may act as a trigger for IL-6 release from endothelial cells and contribute to the activation of regenerative cascade in the non-embolized lobes.

Transplantation and Surgery

Bruna Lavezzo et al.
Treatment of recurrent hepatitis C in liver transplants: efficacy of a six versus a twelve month course of interferon alfa 2b with ribavirin
Background/Aims: Interferon (IFN) with ribavirin combination therapy (CT) was proposed for the treatment of hepatitis C recurring in liver transplants. We assessed the efficacy of two protocols of CT in transplanted patients with recurrent severe hepatitis C virus (HCV) hepatitis. Methods: Fifty-seven patients (68% genotype 1b) were treated with IFN alfa-2b 3 million units three times weekly and oral ribavirin 800mg/die for 6 or 12 months. Study end-points were the end of treatment (ETVR) and the 12-month post-therapy sustained virologic response (SVR; negative HCV-RNA). Results: ETVR was induced in 9/27 (33%) and in 7/30 patients (23%) treated, respectively, for 6 and 12 months (P=0.4); a SVR was induced in six (22%) of the former and five (17%) of the latter (P=0.4). HCV genotype non-1 patients responded better than genotype 1 (SVR: 43% in genotype non-1 versus 12% in genotype 1, P: 0.02). In ETV responders the hepatitis activity index improved by >2 points in biopsies taken after therapy compared to pre-therapy biopsies. Anemia and leukopenia required reduction of therapy in 51% of the patients. Conclusions: CT is efficacious in controlling HCV disease in about 20% of transplants with recurrent hepatitis C. Six months of therapy are as efficacious as 12 months.

Viral Hepatitis

Leonieke M.M. Wolters et al.
The influence of baseline characteristics on viral dynamic parameters in chronic hepatitis B patients treated with lamivudine
Background/Aims: Viral decline during lamivudine therapy in chronic hepatitis B patients is bi-phasic. We studied the influence of lamivudine dose and baseline characteristics on parameters obtained from a mathematical model. Methods: Chronic hepatitis B patients were randomized to receive 150 mg (group 1; n=11) or 600 mg (group 2; n=10) lamivudine daily for 4 weeks. Hepatitis B virus DNA was measured frequently with the Digene Hybrid Capture II test and the Roche PCR assay. Results: The description of viral decline in our closely monitored patients by means of the mixed-effects approach with both the bi-phasic model and a piecewise linear regression model resulted in a good fit. Baseline alanine aminotransferase (ALT) was significantly related to the slope of the second phase of viral decline. Previous lamivudine-treated patients showed a significant slower first phase than patients naive to lamivudine treatment. Conclusions: The initial observed difference in viral decline between 150 and 600 mg of lamivudine disappeared when baseline ALT was taken into account. This strengthens the hypothesis that the level of intrinsic activity is related to the turnover of infected hepatocytes. Moreover, reintroduction of lamivudine in previously lamivudine-treated patients should be considered carefully.

Toshihiko Kirishima et al.
Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment
Background/Aims: The emergence of lamivudine-resistant hepatitis B virus (HBV) was reported in patients with prolonged lamivudine administration. There was no report of the existence of tyrosine-methionine-aspartate-aspartate (YMDD) mutant in non-lamivudine treated chronic hepatitis B patients. In the present study, we developed a sensitive assay and applied it to the detection of YMDD mutant. Methods: We developed peptide nucleic acid (PNA) mediated polymerase chain reaction clamping for detecting mutations in a YMDD motif of the hepatitis B virus DNA polymerase gene. We studied YMDD mutants in a patient with HBV DNA breakthrough longitudinally and in non-lamivudine treated patients (36 patients). Results: We could detect as little as 0.01-0.001% of mutant viruses coexisting in 105-109 copies of wild-type viruses using this assay. YMDD mutant was detected 7 months before clinical breakthrough, which was 6 months earlier than using the conventional restriction fragment length polymorphism assay. YMDD mutants were also detected in four of 18 anti-HBe antibody positive untreated chronic hepatitis type B: YMDD+tyrosine-valine-aspartate-aspartate (YVDD) in two patients and YMDD+tyrosine-isoleucine-aspartate-aspartate (YIDD) in two patients, however, none in HBe antigen positive patients. Conclusions: We developed a highly sensitive assay for detecting YMDD mutants. This is an effective procedure for monitoring patients during or before lamivudine treatment and can provide more insights into the therapeutic strategies for chronic hepatitis B patients.

Cihan Yurdaydin et al.
Famciclovir treatment of chronic delta hepatitis
Background/Aims: Interferon is the only established therapy for chronic delta hepatitis and alternative treatment options are an urgent need. Since successful treatment of a case of post-transplant delta hepatitis with the nucleoside analogue famciclovir had been reported, a pilot study was undertaken to evaluate the use of famciclovir in the treatment of chronic delta hepatitis. Methods: A total of 15 adult patients, 13 men, two women, ages 20-52 years, with chronic delta hepatitis were treated with famciclovir, 500 mg, three times a day for 6 months and were then followed-up for 6 months posttreatment. All patients had compensated chronic liver disease, elevated liver enzymes and were hepatitis delta virus (HDV) RNA positive by polymerase chain reaction at baseline. Patients were monitored and tested for HBsAg, hepatitis B virus (HBV) DNA and HDV RNA levels. Liver biopsies were obtained before starting famciclovir and within 1 month of completion of treatment. Results: HBV DNA levels decreased in nine of the 15 patients and levels rose again after treatment (P<0.05). Famciclovir had no effect on alanine aminotransferase (ALT) and HBsAg levels or on serum HDV RNA and overall, there was no improvement in liver histology. Conclusions: Treatment of chronic delta hepatitis with famciclovir has no effect on disease activity and HDV RNA levels.

BRITISH MEDICAL JOURNAL

10 August 2002 ,Volume 325, Issue 7359

Mortality from liver disease in the West Midlands, 1993-2000: observational study
N C Fisher, J Hanson, A Phillips, J N Rao, and E T Swarbrick
BMJ 2002; 325: 312-313. [Full text]

The study was set in three adjacent metropolitan boroughs in the West Midlands with a total population of 837 000. Around 8.4% of residents are of south Asian origin (Indian, Pakistani, or Bangladeshi; 1991 census). Deaths from liver disease were identified from public health mortality files supplied by the Office for National Statistics, which we searched using ICD-9 (international classification of diseases, 9th revision) reference codes 570-573, and from files supplied by the registrar of the local health authority. South Asian origin and religion were identified from subjects' names. In cases of deaths from liver disease of unspecified cause (ICD 571.5 and related codes) we analysed case notes to search for underlying causative factors.
 

NEW ENGLAND JOURNAL

LANCET

Volume 360, Number 9331 10 August 2002

Oesophageal cancer: a common malignancy in young people of Bomet District, Kenya  [Full Text] 

Russell E White, Christian C Abnet, Caesar K Mungatana, Sanford M Dawsey

Oesophageal cancer is a common cancer with uneven geographical distribution. We reviewed all malignancies diagnosed at Tenwek Hospital (Bomet District, Kenya) between 1989 and 1998. Oesophageal cancer was the most common malignancy; 274 cases accounted for 19% of 1459 malignancies diagnosed, and for a steady rise in total cancer cases during this period. A striking feature of our study was the presence of a subset of very young patients. 26 (11%) patients were aged 30 years or less at diagnosis, and the youngest patient was 14 years old. This area of West Kenya seems to be a high-risk region for oesophageal cancer.

Lancet 2002; 360: 462-63

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