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Revue de Presse du Mois Mois de Janvier 2003

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HEPATOLOGY

Table of Contents for January 2003 · Volume 37 · Number 1

Viral Hepatitis

Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease (*Human Study*)
Hajime Sumi, Osamu Yokosuka, Naohiko Seki, Makoto Arai, Fumio Imazeki, Tomoko Kurihara, Tatsuo Kanda, Kenichi Fukai, Masaki Kato, Hiromitsu Saisho
To investigate the hepatitis B virus (HBV) genotype-related differences in the progression of liver disease, 585 patients with chronic HBV infection including 258 with histologically verified chronic liver disease (CLD) and 74 with hepatocellular carcinoma (HCC) were examined. The mean ages of both patients with advanced fibrosis (F3 or F4) and with HCC were significantly older in genotype B than in genotype C patients (P = .018, P = .024, respectively). Both the hepatitis B e antigen (HBeAg) negativity rate at biopsy and the cumulative HBe seroconversion rate in patients with CLD were significantly higher in genotype B patients than genotype C patients (P < .01, P = .022, respectively). Multivariate analysis revealed that genotype B, presence of precore mutation, high ALT levels, and severe histologic activity were independent factors for HBe seroconversion. Among all the biopsy-proven CLD patients, the ratio of patients with advanced fibrosis in genotype B was significantly lower than that in genotype C (4/30 vs. 74/224, respectively; P = .034). This difference was more remarkable in younger patients (45 years; 1/25 vs. 47/180, respectively; P = .020), and there was no difference in older patients (>45 years). The distribution of each genotype between CLD and HCC was very similar (B and C: 11.2% and 87.0% vs. 10.8% and 89.2%, respectively). In conclusion, our results suggest that, although the patients with genotype B experience earlier HBe seroconversion, slower progression of liver fibrosis, and slower development of HCC, the life-long risk of progression to advanced fibrosis and development of HCC may not differ among genotypes B- and C-related chronic liver disease. (HEPATOLOGY 2003;37:19-26.)

Effect of the G1896A precore mutation on drug sensitivity and replication yield of lamivudine-resistant HBV in vitro
Robert Y. M. Chen, Ros Edwards, Tim Shaw, Danni Colledge, William E. Delaney, IV, Harriet Isom, Scott Bowden, Paul Desmond, Stephen A. Locarnini
Hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) is frequently caused by a mutation (G1896A) in the hepatitis B virus (HBV) precore (PC) reading frame that creates a stop codon, causing premature termination of the PC protein. During lamivudine treatment, drug resistance develops at a similar rate in HBeAg positive and HBeAg negative CHB. Lamivudine-resistant HBV mutants have been shown to replicate inefficiently in vitro in the absence of PC mutations, but it is unknown whether the presence of PC mutations affects replication efficiency or antiviral sensitivity. This study utilized the recombinant HBV baculovirus system to address these issues. HBV baculoviruses encoding the G1896A PC stop codon mutation were generated in wild-type (WT) and lamivudine-resistant (rtM204I and rtL180M + rtM204V) backgrounds, resulting in a panel of 6 related recombinant baculoviruses. In vitro assays were performed to compare the sensitivities of the PC mutant viruses with lamivudine and adefovir and to compare relative replication yields. The PC mutation did not significantly affect sensitivities to either adefovir or lamivudine. WT HBV and PC mutant HBV showed similar replication yields, whereas the replication yields of the lamivudine-resistant mutants were greatly reduced in HBeAg positive HBVs, confirming previous observations. However, the presence of the PC mutation was found to compensate for the replication deficiency in each of the lamivudine-resistant mutants, increasing the replication yields of each virus. In conclusion, the PC stop codon mutation appears to increase the replication efficacy of lamivudine-resistant virus but does not affect in vitro drug sensitivity. (HEPATOLOGY 2003;37:27-35.)

Development of antibody to hepatitis B surface antigen after liver transplantation for chronic hepatitis B (*Human Study*)
Chung-Mau Lo, James Tak-Kwan Fung, George Ka-Kit Lau, Chi-Leung Liu, Siu-Tim Cheung, Ching-Lung Lai, Sheung-Tat Fan, John Wong
Patients with chronic hepatitis B virus (HBV) infection have a defective HBV-specific immune response, and the spontaneous development of antibody against hepatitis B surface antigen (anti-HBs) after liver transplantation has not been observed. We report the spontaneous production of anti-HBs in 21 of 50 (42%) patients receiving lamivudine monoprophylaxis after liver transplantation. Seroconversion to anti-HBs status (>10 mIU/mL) was found at a median of 8 days (range, 1 to 43 days) after transplantation. In each case, serial serum samples showed a >100% increase in antibody titer as compared with that of day 7 after transplantation in the absence of any blood product transfusion. The anti-HBs titer increased to a maximum within 3 months, and the peak titer was <100 mIU/mL in 10 patients, 100 to 1000 mIU/mL in 5 patients, and >1,000 mIU/mL in 6 patients. In 12 patients, anti-HBs disappeared from serum at a median of 201 days (range, 24 to 414 days), whereas the other 9 patients remained positive for anti-HBs at a median of 221 days (range, 94 to 1,025 days) after transplantation. Patients in whom anti-HBs in serum developed had a more rapid clearance of serum hepatitis B surface antigen (HBsAg) (log rank test, P = .011). Using logistic regression analysis, the only predictor of anti-HBs production was an HBV-immune donor (odds ratio, 18.9; 95% confidence interval, 3.2 to 112.4; P = .001). In conclusion, patients who undergo liver transplantation for chronic hepatitis B using lamivudine prophylaxis may develop anti-HBs spontaneously. The antibody is likely to be of donor origin, suggesting the possibility of adoptive immunity transfer through a liver graft. (HEPATOLOGY 2003;37:36-43.)

Cost-effectiveness of hepatitis A vaccination in children, adolescents, and adults (*Human Study*)
Philip Rosenthal
Hepatitis A is a major public health problem in the United States and other developed countries, largely because decreased natural immunity allows for increased susceptibility. To evaluate the cost-effectiveness of routine vaccination of children, adolescents, and certain high-risk adults against hepatitis A, economic analyses of hepatitis A vaccination were identified through searches of MEDLINE, EMBASE, and BIOSIS (February, 1992, to December, 2001) for studies, reviews, editorials, and letters from peer-reviewed journals published in English, French, German, Italian, or Spanish. Experts were also contacted. Articles conforming to accepted standards of quality for health-economic studies were used to compile data on vaccination of children, and results were synthesized in a narrative review. This review of economic analyses of vaccine use in several developed countries shows cost-effectiveness comparable with that of other vaccines in children and within accepted boundaries for adolescents and high-risk adults. (HEPATOLOGY 2003;37:44-51.)

Hepatitis C virus­like particles combined with novel adjuvant systems enhance virus-specific immune responses
Ming Qiao, Kazumoto Murata, Anthony R. Davis, Sook-Hyang Jeong, T. Jake Liang
We have previously described the generation of hepatitis C virus­like particles (HCV-LPs) in insect cells and shown that immunization with HCV-LPs elicited both humoral and cellular immune responses in mice. To further characterize the HCV-LPs as a vaccine candidate, we evaluated the effects of adjuvant AS01B (monophosphoryl lipid A [MPL] and QS21), CpG 10105, and the combination of the 2 adjuvants on the immunogenicity of HCV-LPs in AAD mice (transgenic for HLA-A2.1). All HCV-LP­immunized mice (with or without adjuvant) developed high titers of anti-HCV E1/E2 antibodies after 4 injections intramuscularly. However, antibody titers in mice immunized with HCV-LP plus AS01B, plus CpG 10105, or plus the combination of AS01B and CpG 10105 were 4, 3, and 10 times higher, respectively, than that of HCV-LP alone. Isotype analysis of the induced anti-envelope antibodies showed that HCV-LP alone induced a predominant immunoglobulin (Ig) G1 response. In contrast, when the 2 adjuvants AS01B and CpG 10105 were combined, the response became predominantly IgG2a whereas HCV-LP plus AS01B or CpG 10105 gave a mixed IgG1 and IgG2a response, indicating that AS01B and CpG 10105 promote a more T-helper type 1 (Th1) response and that combining the 2 adjuvants results in an additive or synergistic interaction. These observations were further confirmed by the results of CD4+ enzyme-linked immunospot assay for interferon (IFN)- and interleukin (IL)-4 and intracellular cytokine staining of IFN- producing CD8+ cells. In conclusion, HCV-LP is a promising vaccine candidate against HCV infection and the adjuvants used are potent immune enhancers for this approach. (HEPATOLOGY 2003;37:52-59.)

Spontaneous viral clearance in patients with acute hepatitis C can be predicted by repeated measurements of serum viral load (*Human Study*)
Harald Hofer, Thomas Watkins-Riedel, Oskar Janata, Edward Penner, Heidemarie Holzmann, Petra Steindl-Munda, Alfred Gangl, Peter Ferenci
Early interferon (IFN) therapy prevents viral persistence in acute hepatitis C, but in view of the resulting costs and morbidity patients who really need therapy have to be identified. Twelve consecutive patients with acute hepatitis C (9 women, 3 men, mean age: 39.5 ± 18.8 y, genotype 1: 7, genotype 3a: 3, 2 could not be genotyped) were studied. The sources of infection were medical procedures in 6, sexual transmission in 3, and intravenous drug abuse in 3 patients. Viral load was measured by Cobas Amplicor HCV Monitor v2.0 (Roche Diagnostic Systems, Branchburg, NY). The time from infection to clinical symptoms was 43.3 ± 8.6 (mean ± SD) days. Eight patients cleared hepatitis C virus (HCV) spontaneously and remained HCV-RNA negative with a follow-up of 9.0 ± 3.9 months. In these patients viral load declined fast and continuously. The time from exposure to HCV-RNA negativity was 77.4 ± 25.3 and from the first symptoms was 34.7 ± 22.1 days. In 4 patients HCV-RNA levels remained high or even increased. Two of them became sustained responders to treatment initiated after a 6-week observation period. The 2 remaining patients were not treated (one because of contraindications for IFN, the other declined therapy) and are still HCV-RNA positive. In conclusion, patients with acute icteric hepatitis C have a high rate of spontaneous viral clearance within the first month after the onset of symptoms. IFN therapy appears only needed in patients who fail to clear the virus within 35 days after onset of symptoms. By this approach, IFN therapy was not necessary in two thirds of patients with acute hepatitis C. (HEPATOLOGY 2003;37:60-64.)

Interleukin-1 gene polymorphisms associated with hepatocellular carcinoma in hepatitis C virus infection (*Human Study*)
Yue Wang, Naoya Kato, Yujin Hoshida, Hideo Yoshida, Hiroyoshi Taniguchi, Tadashi Goto, Masaru Moriyama, Motoyuki Otsuka, Shuichiro Shiina, Yasushi Shiratori, Yoichi Ito, Masao Omata
Hepatitis C virus (HCV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), a life-threatening sequel. However, the factors that affect disease progression to HCC have not been thoroughly elucidated. Genetic polymorphisms in proinflammatory cytokines, the interleukin 1 (IL-1) family (IL-1 and IL-1ra) and tumor necrosis factor- (TNF-), were studied in 274 Japanese patients with chronic HCV infection and 55 healthy individuals using standard polymerase chain reaction-based genotyping techniques. The association between these polymorphisms and disease status was evaluated while controlling for confounding clinical variables. The proportion of patients with HCC in the IL-1-31 T/T (55%, odds ratio to C/C was 2.63, P = .009) genotype was higher than in the T/C (44%, odds ratio to C/C was 1.64, P = .149) and C/C genotypes (35%). The IL-1-31 and -511 loci were in near complete linkage disequilibrium, and the IL-1-511/-31 haplotype C-T was significantly associated with the presence of HCC (odds ratio of 1.51, P = .02). Polymorphisms in the TNF- gene were not associated with disease. A multivariate analysis revealed that the IL-1-31 T/T genotype, -fetoprotein >20 µg/L, presence of cirrhosis, male sex, and age >60 years were associated with the presence of HCC at odds ratios of 3.73 (T/T vs. C/C), 4.12, 4.03, 3.89, and 3.27, respectively. In conclusion, the IL-1-31 genotype T/T or the IL-1-511/-31 haplotype C-T is associated with the presence of HCC in Japanese patients with chronic HCV infection. (HEPATOLOGY 2003;37:65-71.)

Nucleolar hypertrophy correlates with hepatocellular carcinoma development in cirrhosis due to HBV infection
Davide Trerè, Mauro Borzio, Alberto Morabito, Franco Borzio, Massimo Roncalli, Massimo Derenzini
Patients with cirrhosis are at significant risk for hepatocellular carcinoma (HCC). The aim of the present study was to evaluate the relationship between the percentage of hepatocytes showing nucleolar hypertrophy and the development of HCC in cirrhosis of different causes. A total of 111 cirrhotic patients were studied, with a mean follow-up period of 83.3 months. Histologic sections from liver biopsy specimens were silver stained for selective visualization of the nucleolus; the nucleolar area was measured by image cytometry. Nucleoli with a size of 7 µm2 or greater were considered to be hypertrophic. The nucleolar index was obtained by calculating the percentage of hepatocytes disclosing a nucleolar area of 7 µm2 or greater. During the observation time, HCC was diagnosed in 39 of 111 patients. The incidence rate of HCC was greater in patients with nucleolar indexes of 2.5 or greater than in patients with nucleolar indexes of less than 2.5 (16.49%/y vs. 3.41%/y, respectively; P < .0001). The capacity of the nucleolar index to predict HCC development was separately tested in groups of patients divided by etiology, and it was found to be particularly relevant in hepatitis B virus (HBV)-related cirrhosis (P = .0006). Among patients with hepatitis C virus (HCV) infection, high nucleolar-index values were associated with a greater risk for HCC development, but the difference in the incidence rate of HCC between groups with a nucleolar index of 2.5 or greater and less than 2.5 was not statistically significant (P = .0944). In conclusion, our results have shown that high percentages of hepatocytes showing nucleolar hypertrophy significantly predict HCC development in patients with HBV infection, whereas their predictive value in HCV-related cirrhosis seems to be lower. (HEPATOLOGY 2003;37:72-78.)

Liver Biology and Pathobiology

Triiodothyronine enhances the regenerative capacity of the liver following partial hepatectomy
Raza Malik, Neil Mellor, Clare Selden, Humphrey Hodgson
This study investigates the effects of administering a primary mitogen, triiodothyronine (T3), at the time of 70% partial hepatectomy (PH) in the rat, thus combining the 2 distinct pathways of liver growth: direct hyperplasia and compensatory regeneration. T3 enhances the proliferative response of hepatocytes within the liver following PH. Flash bromodeoxyuridine (BrdU) labeling showed a cell proliferation index 24 hours after PH alone of 26.5% ± 2.8%; when T3 was administered at PH, it increased to 39.5% ± 5.0% (P < .01 compared with PH alone). Continuous BrdU labeling performed every 6 hours between 15 and 72 hours following surgery showed an index of 84.0% ± 4.0% when T3 was administered at PH compared with 71.0% ± 4.0% with PH alone (P < .01 compared with PH alone). This increase in cell proliferation resulted in a larger liver mass at 4 days in rats receiving T3 at PH compared with PH alone (P < .05 compared with PH alone). The difference in liver mass was matched with corresponding increases in total DNA and total protein levels as well as cell division, as confirmed by the frequent demonstration of twin daughter cells on histology. In conclusion, this study shows that a single dose of T3 enhances the regenerative capacity of the liver following PH. The ability to enhance cell proliferation during compensatory hyperplasia following PH could be therapeutically valuable if applicable to humans. (HEPATOLOGY 2003;37:79-86.)

Activated stellate cells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediated apoptosis
Pavel Taimr, Hajime Higuchi, Eva Kocova, Richard A. Rippe, Scott Friedman, Gregory J. Gores
Apoptosis has emerged as an important mechanism to reduce numbers of activated stellate cells during the resolution phase of hepatic fibrosis. These observations suggest that activated stellate cells may be more susceptible to apoptotic stimuli than their quiescent counterparts. Because other activated cell types are more sensitive than their quiescent phenotypes to apoptosis by tumor necrosis factor­related apoptosis-inducing ligand (TRAIL), we examined the expression of TRAIL death receptors (DRs) and susceptibility to TRAIL cytotoxicity in stellate cells undergoing progressive activation. A spontaneously immortalized human stellate cell line, LX-2, was analyzed during 14 days of progressive activation following plating, during which time ­smooth muscle actin (-SMA) and a -crystallin (markers of stellate cell activation) messenger RNA (mRNA) increased 7-fold and 5-fold, respectively. During this same interval, TRAIL-R1/DR4 and TRAIL-R2/DR5 mRNA expression increased 18-fold and 17.6-fold, although TRAIL-R2/DR5 expression was quantitatively 103-fold greater than TRAIL-R1/DR4; parallel changes occurred in TRAIL/DR5 protein expression and cellular susceptibility to TRAIL-mediated apoptosis. Similar findings were observed in primary murine stellate cells undergoing activation on a plastic surface. In conclusion, stellate cells show activation-dependent TRAIL-R2/DR5 expression and TRAIL-mediated apoptosis. Because TRAIL-R2/DR5 is not expressed by hepatocytes, TRAIL/DR5 agonists may be useful in reducing fibrosis by inducing stellate cell apoptosis. (HEPATOLOGY 2003;37:87-95.)

Cytochrome P450 2E1 responsiveness in the promoter of glutamate-cysteine ligase catalytic subunit
Natalia Nieto, Montserrat Marí, Arthur I. Cederbaum
Previous studies have shown cytochrome P450 2E1 (CYP2E1)-dependent transcriptional up-regulation of glutamate-cysteine ligase (GCL). To identify sequences mediating constitutive and induced expression of the catalytic subunit of GCL (GCLC), a series of deletion mutants from the 5'-flanking region (­3,802 to +465) were transfected into control (C34) and CYP2E1-overexpressing (E47) HepG2 cells. Increased luciferase expression, both basal (2- to 3-fold) and following exposure to ethanol, arachidonic acid (AA), or AA plus iron, was detected in E47 cells with the full-length but not shorter reporter vectors. Basal induction was blocked by CYP2E1 inhibitors and catalase. Basal and inducible luciferase expression in E47 cells was blunted by the full-length construct mutated in the ARE4 site. Catalase and diallyl sulfide prevented basal and AA-induced messenger RNA (mRNA) levels of GCLC and the modulatory subunit of GCL (GCLM). Preincubation with low doses of AA increased glutathione (GSH) levels as well as GCLC and GCLM mRNAs, and this protected against H2O2 and menadione toxicity. Primary hepatocytes from pyrazole-injected rats with high levels of CYP2E1 showed an increase in GSH levels as well as GCLC and GCLM mRNAs compared with saline controls, and this was prevented by diallyl sulfide. In conclusion, redox-sensitive elements directing constitutive and induced expression of the GCLC in CYP2E1-expressing cells are present in the ARE4 distal portion of the 5'-flanking region, between positions ­3,802 and ­2,752, perhaps a reflection of metabolic adaptation to CYP2E1-generated oxidative stress. (HEPATOLOGY 2003;37:96-106.)

Foxf1 +/­ mice exhibit defective stellate cell activation and abnormal liver regeneration following CCl4 injury
Vladimir V. Kalinichenko, Dibyendu Bhattacharyya, Yan Zhou, Galina A. Gusarova, Wooram Kim, Brian Shin, Robert H. Costa
Previous studies have shown that haploinsufficiency of the splanchnic and septum transversum mesoderm Forkhead Box (Fox) f1 transcriptional factor caused defects in lung and gallbladder development and that Foxf1 heterozygous (+/­) mice exhibited defective lung repair in response to injury. In this study, we show that Foxf1 is expressed in hepatic stellate cells in developing and adult liver, suggesting that a subset of stellate cells originates from septum transversum mesenchyme during mouse embryonic development. Because liver regeneration requires a transient differentiation of stellate cells into myofibroblasts, which secrete type I collagen into the extracellular matrix, we examined Foxf1 +/­ liver repair following carbon tetrachloride injury, a known model for stellate cell activation. We found that regenerating Foxf1 +/­ liver exhibited defective stellate cell activation following CCl4 liver injury, which was associated with diminished induction of type I collagen, ­smooth muscle actin, and Notch-2 protein and resulted in severe hepatic apoptosis despite normal cellular proliferation rates. Furthermore, regenerating Foxf1 +/­ livers exhibited decreased levels of interferon-inducible protein 10 (IP-10), delayed induction of monocyte chemoattractant protein 1 (MCP-1) levels, and aberrantly elevated expression of transforming growth factor 1. In conclusion, Foxf1 +/­ mice exhibited abnormal liver repair, diminished activation of hepatic stellate cells, and increased pericentral hepatic apoptosis following CCl4 injury. (HEPATOLOGY 2003;37:107-117.)

Low-dose TNF- protects against hepatic ischemia-reperfusion injury in mice: Implications for preconditioning
Narci Teoh, Isabelle Leclercq, Aileen Dela Pena, Geoffrey Farrell
Tumor necrosis factor (TNF-) is implicated in the pathogenesis of hepatic ischemia reperfusion injury but can also prime hepatocytes to enter the cell cycle. Ischemic preconditioning protects against ischemia-reperfusion (IR) liver injury and is associated with activation of nuclear factor B (NF-B) and cell cycle entry. We examined the pattern of TNF- release during hepatic IR in the presence or absence of ischemic preconditioning, and we tested whether a single low-dose injection of TNF could mimic the biologic effects of ischemic preconditioning. In naïve mice, hepatic and plasma levels of TNF- rose during hepatic ischemia, reaching high levels after 90 minutes; values remained elevated during reperfusion until 44 hours. Following the ischemic preconditioning stimulus, there was an early rise in hepatic and serum TNF- levels, but, during a second prolonged ischemic interval peak, TNF- values were lower than in naïve mice and declined to negligible levels by 2 hours reperfusion. An injection with 1 µg or 5 µg/kg body weight TNF- 30 minutes prior to hepatic IR substantially reduced liver injury determined by liver histology and serum alanine aminotransferase (ALT) levels. As in ischemic preconditioning, TNF- pretreatment activated NF-B DNA binding, STAT3, cyclin D1, cyclin-dependent kinase 4 (cdk4) expression, and cell cycle entry, determined by proliferating cell nuclear antigen (PCNA) staining of hepatocyte nuclei. In conclusion, the hepatoprotective effects of "preconditioning" can be simulated by TNF- injection, which has identical downstream effects on cell cycle entry. We propose that transient increases in TNF- levels may substitute for, as well as, mediate the hepatoprotective effects of ischemic preconditioning against hepatic IR injury. (HEPATOLOGY 2003;37:118-128.)

Dominant negative MORT1/FADD rescues mice from CD95 and TNF-induced liver failure
Marcus Schuchmann, Eugene E. Varfolomeev, Frank Hermann, Felix Rueckert, Dennis Strand, Heinz Koehler, Susanne Strand, Ansgar W. Lohse, David Wallach, Peter R. Galle
Derangement of the apoptotic program is considered an important cause of liver disease. It became clear that receptor-mediated apoptosis is of specific interest in this context, and CD95 and CD120a, both members of the tumor necrosis factor (TNF) receptor superfamily, are the most prominent cell death receptors involved. The death signal is induced upon ligand binding by recruitment of caspases via the adapter molecule MORT1/FADD to the receptor and their subsequent activation. To investigate the role of MORT1/FADD in hepatocyte apoptosis, we generated transgenic mice expressing liver-specific dominant negative mutant. Mice looked grossly normal; breeding and liver development were not different compared with wild-type littermates. Expression of the transgene completely protected animals from liver failure induced by the anti-Fas antibody Jo2, whereas control animals died as expected 3 to 6 hours after i.p. injection of 15 µg antibody from acute hemorrhagic liver failure. Histology demonstrated only moderate inflammatory changes in the transgenic animals, whereas severe hemorrhagic hepatitis was observed in controls. Similar results were obtained in a model of TNF-mediated liver failure, in which transgenic animals survived significantly better than wild-type animals. In conclusion, our experiments provide evidence that MORT1/FADD is indispensable for Fas and TNF-mediated hepatic injury. This is not only of great importance for targeting future therapies for liver disease but might also serve as an intriguing model to study other causes of liver injury. (HEPATOLOGY 2003;37:129-135.)

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes
Shoichiro Shishido, Hironori Koga, Masaru Harada, Hiroto Kumemura, Shinichiro Hanada, Eitaro Taniguchi, Ryukichi Kumashiro, Hiromasa Ohira, Yukio Sato, Masayoshi Namba, Takato Ueno, Michio Sata
Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazone-treated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (m) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2',7'-dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electron-dense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the m per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity. (HEPATOLOGY 2003;37:136-147.)

Characterization of cell types during rat liver development
Henning C. Fiegel, Jonas J. H. Park, Michael V. Lioznov, Andreas Martin, Stefan Jaeschke-Melli, Peter M. Kaufmann, Boris Fehse, Axel R. Zander, Dietrich Kluth
Hepatic stem cells have been identified in adult liver. Recently, the origin of hepatic progenitors and hepatocytes from bone marrow was demonstrated. Hematopoietic and hepatic stem cells share the markers CD 34, c-kit, and Thy1. Little is known about liver stem cells during liver development. In this study, we investigated the potential stem cell marker Thy1 and hepatocytic marker CK-18 during liver development to identify putative fetal liver stem cell candidates. Livers were harvested from embryonic and fetal day (ED) 16, ED 18, ED 20, and neonatal ED 22 stage rat fetuses from Sprague-Dawley rats. Fetal livers were digested by collagenase-DNAse solution and purified by percoll centrifugation. Magnetic cell sorting (MACS) depletion of fetal liver cells was performed using OX43 and OX44 antibodies. Cells were characterized by immunocytochemistry for Thy1, CK-18, and proliferating cell antigen Ki-67 and double labeling for Thy1 and CK-18. Thy1 expression was found at all stages of liver development before and after MACS in immunocytochemistry. Thy1 positive cells were enriched after MACS only in early developmental stages. An enrichment of CK-18 positive cells was found after MACS at all developmental stages. Cells coexpressing Thy1 and CK-18 were identified by double labeling of fetal liver cell isolates. In conclusion, hepatic progenitor cells (CK-18 positive) in fetal rat liver express Thy1. Other progenitors express only CK-18. This indicates the coexistence of different hepatic cell compartments. Isolation and further characterization of such cells is needed to demonstrate their biologic properties. (HEPATOLOGY 2003;37:148-154.)

An optimal therapeutic expression level is crucial for suicide gene therapy for hepatic metastatic cancer in mice
Yasuhiro Terazaki, Shojiro Yano, Kentaro Yuge, Satoshi Nagano, Mari Fukunaga, Z. Sheng Guo, Setsuro Komiya, Kazuo Shirouzu, Ken-ichiro Kosai
The most serious problem in current gene therapy is discrepancies between experimental data and actual clinical outcomes, which may be due to insufficient analyses and/or inappropriate animal models. We have explored suicide gene therapy by using various clinically relevant animal models and doubt the clinical use of maximal suicide gene expression, which has been generally recommended. To explore this subject further, we studied what expression level of suicide gene and what promoter led to the maximal clinical benefit in the case of hepatic metastatic cancer in mice. Therapeutic and adverse side effects of 4 adenoviral vectors that express herpes simplex virus thymidine kinase (HSV-tk) under different promoters were scrupulously investigated in 2 mouse models of hepatic metastasis of gastric cancer that possess clinical characteristics. Surprisingly, increases in HSV-tk expression beyond a certain point, achieved by the Rous sarcoma virus long terminal repeat promoter, not only enhanced the adverse side effects of lethal hepatotoxicity and ganciclovir-independent cytotoxicity but also failed to further increase therapeutic potential. Moreover, the carcinoembryonic antigen (CEA) tumor-specific promoter, the therapeutic potential of which had been underestimated, was much more useful-even in the case of low CEA-producing cancer-than had been previously reported. In conclusion, the optimal therapeutic expression level of a suicide gene is a novel concept and a crucial factor for successful cancer gene therapy. The present results, which contradict those of previous studies, alert researchers about possible problems with ongoing and future clinical trials that lack this concept. (HEPATOLOGY 2003;37:155-163.)

Liver Failure And Liver Disease

Clinical profile of autosomal dominant polycystic liver disease (*Human Study*)
Qi Qian, Airong Li, Bernard F. King, Patrick S. Kamath, Donna J. Lager, John Huston, III, Clarence Shub, Sonia Davila, Stefan Somlo, Vicente E. Torres
Most reports on the natural history, manifestations, and treatment of polycystic liver disease are based on the disease as it manifests in patients with autosomal dominant polycystic kidney disease (ADPKD). The purpose of this study was to develop a clinical profile of isolated autosomal dominant polycystic liver disease (ADPLD) using nonaffected family members as controls. The study included 146 probands, known affected relatives, and first-degree relatives of affected individuals. Participants underwent a formalized medical history interview and physical examination, ultrasonographic examination of the liver and kidneys, magnetic resonance angiography of the brain, and echocardiography. Thirty-eight of the 49 individuals diagnosed with polycystic liver disease before participation in the study were or had been symptomatic. Of 97 previously undiagnosed at-risk individuals, 23 were affected, 39 were unaffected, and 35 were indeterminate. Compared with patients with a negative or indeterminate diagnosis, those with polycystic liver disease had slightly higher levels of serum alkaline phosphatase and total bilirubin and lower levels of total cholesterol and triglycerides. Female patients had a significantly higher mean cyst score than male patients. The cysts were found to arise from the dilatation of biliary microhamartomas and from peribiliary glands. Structural mitral leaflet abnormalities were detected more frequently in affected than in indeterminate or nonaffected individuals. A vascular phenotype was detected in 5.6% of the patients with isolated ADPLD diagnosed clinically and/or by linkage analysis but in none of the unaffected patients. In conclusion, isolated ADPLD is underdiagnosed and genetically distinct from polycystic liver disease associated with ADPKD but with similar pathogenesis, manifestations, and management. (HEPATOLOGY 2003;37:164-171.)

Cyclooxygenase-derived products modulate the increased intrahepatic resistance of cirrhotic rat livers
Mariona Graupera, Joan-Carles García-Pagán, Juan G. Abraldes, Carmen Peralta, Mireia Bragulat, Helena Corominola, Jaume Bosch, Juan Rodés
In cirrhotic livers, increased resistance to portal flow, in part due to an exaggerated response to vasoconstrictors, is the primary factor in the pathophysiology of portal hypertension. Our aim was to evaluate the response of the intrahepatic circulation of cirrhotic rat livers to the 1-adrenergic vasoconstrictor methoxamine and the mechanisms involved in its regulation. A portal perfusion pressure dose-response curve to methoxamine was performed in control and cirrhotic rat livers preincubated with vehicle, the nitric oxide synthase blocker NG-nitro-L-arginine (L-NNA), indomethacin cyclooxygenase (COX) inhibitor, L-NNA + indomethacin, or the thromboxane (TX) A2 receptor blocker SQ 29,548. TXA2 production, COX-1 and COX-2 mRNA expression, and immunostaining for TXA2 synthase were evaluated. Cirrhotic livers exhibited a hyperresponse to methoxamine associated with overexpression of COX-2 and TXA2 synthase as well as with increased production of TXA2. The hyperresponse to methoxamine of cirrhotic livers disappeared by COX inhibition with indomethacin but not after NO inhibition. SQ 29,548 also corrected the hyperresponse of cirrhotic livers to methoxamine. In conclusion, COX-derived prostanoids, mainly TXA2, play a major role in regulating the response of cirrhotic livers to methoxamine. (HEPATOLOGY 2003;37:172-181.)

A vasopressin receptor antagonist (VPA-985) improves serum sodium concentration in patients with hyponatremia: A multicenter, randomized, placebo-controlled trial (*Human Study*)
Florence Wong, Andres T. Blei, Laurence M. Blendis, Paul J. Thuluvath, for The North American VPA-985 Study Group
Hyponatremia in advanced cirrhosis and ascites or congestive heart failure (CHF) is the result of an inappropriate increase in vasopressin secretion, which acts through activation of specific V2 receptors in the distal renal nephron to increase water reabsorption. This study investigates the efficacy and safety of 3 different doses of the V2 receptor antagonist, VPA-985, in correcting hyponatremia over a 7-day inpatient study period. Forty-four hospitalized patients (33 patients with cirrhosis, 6 with CHF, and 5 with syndrome of inappropriate antidiuretic hormone (SIADH) were studied on a constant sodium intake, with VPA doses of 25, 125, and 250 mg twice daily or placebo. Serum sodium measurements were repeated after every daily dose, and the next dose withheld for excessive serum sodium rises. Fluid intake was adjusted according to previous 24-hour urinary outputs. Adverse events were based on clinical signs of dehydration or encephalopathy. VPA-985 produced a significant overall aquaretic response compared with placebo, with significant dose related increases in free water clearance (P < .05) and serum sodium (P < .05), without significant changes in orthostatic blood pressure or serum creatinine levels. Five patients (50%) on 250 mg twice daily had to have medication withheld on multiple occasions. End-of-study plasma vasopressin levels increased significantly in the 2 larger dose groups. In conclusion, VPA-985 appears effective and safe in appropriate doses in correcting abnormal renal water handling and hyponatremia in conditions associated with water retention. Higher doses of VPA-985 may produce significant dehydration and will require close monitoring with their use. (HEPATOLOGY 2003;37:182-191.)

Prospective evaluation of outcomes and predictors of mortality in patients with hepatopulmonary syndrome undergoing liver transplantation (*Human Study*)
Miguel R. Arguedas, Gary A. Abrams, Michael J. Krowka, Michael B. Fallon
The hepatopulmonary syndrome (HPS) occurs in a subgroup of patients with cirrhosis and results from intrapulmonary vasodilatation, which may cause significant hypoxemia. Liver transplantation has emerged as a therapeutic option for patients with HPS based on retrospective case series and reports. However, morbidity and mortality appear to be increased after transplantation for HPS, and no prospective studies evaluating clinical features that may predict poor surgical outcome are available. Therefore, we prospectively evaluated the utility of the degree of hypoxemia, the arterial oxygen response to 100% oxygen administration, and the macroaggregated albumin (MAA) scan quantification of intrapulmonary shunting as predictors for outcome after liver transplantation. Our cohort consisted of 24 patients with cirrhosis and HPS who underwent liver transplantation over a 5-year period at 2 transplant centers who were followed at least 1 year after transplantation. All patients underwent preoperative evaluation for HPS with standardized methods. Seven patients (29%) died postoperatively, 5 of cardiorespiratory complications. All deaths occurred within 10 weeks after transplantation. A preoperative arterial oxygen tension (PaO2) of 50 mm Hg alone or in combination with a MAA shunt fraction 20% were the strongest predictors of postoperative mortality. In conclusion, we found that mortality is increased after liver transplantation for HPS, particularly in patients with more severe hypoxemia and significant intrapulmonary shunting. Preoperative testing for the severity of HPS can be used to stratify patients according to the risk for postoperative mortality. (HEPATOLOGY 2003;37:192-197.)

Expression profiling in multistage hepatocarcinogenesis: Identification of HSP70 as a molecular marker of early hepatocellular carcinoma
Makoto Chuma, Michiie Sakamoto, Ken Yamazaki, Tsutomu Ohta, Misao Ohki, Masahiro Asaka, Setsuo Hirohashi
Hepatocellular carcinoma (HCC) associated with chronic liver disease evolves from precancerous lesions and early HCC to a progressed form. Nodule-in-nodule­type HCC (progressed HCC within early HCC) represents the transition from early to progressed HCC and, therefore, is useful in molecular genetic analysis of HCC progression during multistage carcinogenesis. We compared expression profiles among 7 early components and 7 progressed components of nodule-in-nodule­type HCCs and their corresponding noncancerous liver tissues with oligonucleotide array. Of the approximately 12,600 genes that were analyzed, a set of 95 genes provided a molecular signature that distinguished between early HCC components and their noncancerous liver tissues, and a set of 92 genes distinguished between progressed and early HCC components. Of these genes, the most abundantly up-regulated gene in early HCC components (P < .001) was heat-shock protein 70 (HSP70). Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed this finding. Further immunohistochemical examination of HSP70 revealed its significant overexpression in early HCC compared with precancerous lesions (P < .001) and in progressed HCC compared with early HCC (P < .001). In conclusion, molecular signatures were clearly different in noncancerous liver tissue as compared with the early and progressed components of nodule-in-nodule­type HCC. Moreover, HSP70 could be a sensitive marker for the differential diagnosis of early HCC from precancerous lesion or noncancerous liver, a difficult distinction for pathologists due to very well differentiated histology with little atypia in early HCC. (HEPATOLOGY 2003;37:198-207.)

Increased lipopolysaccharide binding protein in cirrhotic patients with marked immune and hemodynamic derangement
Agustín Albillos, Antonio de la Hera, Mónica González, Jose-Luis Moya, Jose-Luis Calleja, Jorge Monserrat, Luis Ruiz-del-Arbol, Melchor Alvarez-Mon
Intestinal bacterial overgrowth and translocation, both common in cirrhosis with ascites, may lead to the activation of monocytes and lymphocytes, increased levels of proinflammatory cytokines, and enhanced synthesis of nitric oxide present in cirrhosis. Bacterial endotoxin promotes the synthesis of lipopolysaccharide (LPS)-binding protein (LBP), and forms a LPS-LBP complex that binds to CD14. This study was designed to evaluate LBP levels and their correlation to the immune response and the hemodynamic status in cirrhotic patients. Plasma LBP, endotoxin, soluble CD14 (sCD14), cytokines, renin, nitrites, and systemic vascular resistance were determined before and 4 weeks after norfloxacin or placebo in 102 cirrhotic patients and 30 controls. LBP was elevated in 42% of ascitic cirrhotic patients (15.7 ± 0.7 versus 6.06 ± 0.5 µg/mL, P < .01). In 60% of high LBP patients, endotoxin was within normal range. Among ascitic patients, those with high LBP showed greater (P < .05) levels of sCD14, tumor necrosis factor (TNF-), interleukin 6 (IL-6), nitrites + nitrates (NOx)/creatinine, and renin, and lower vascular resistance. In the cirrhotic patients with high LBP, norfloxacin normalized (P < .01) LBP (from 16.6 ± 0.5 to 5.82 ± 0.8 µ g/mL) and sCD14; reduced the level of cytokines, NOx/creatinine, and renin; and increased vascular resistance; but lacked effect in patients with normal LBP. Portal pressure was unchanged after norfloxacin in another group of 18 cirrhotic patients with high and 19 with normal LBP. In conclusion, the subset of ascitic cirrhotic patients with marked immune and hemodynamic derangement is identified by increased LBP levels. Amelioration of these abnormalities by norfloxacin suggests the involvement of enteric bacteria or their products in the triggering of the process. (HEPATOLOGY 2003;37:208-217.)

GASTROENTEROLOGY

Table of Contents for January 2003 · Volume 124 · Number 1
   
Clinical­Alimentary Tract

The safety of 6-mercaptopurine for childbearing patients with inflammatory bowel disease: A retrospective cohort study
A. Francella, A. Dyan, C. Bodian, P. Rubin, M. Chapman, D. H. Present
Background & Aims: 6-mercaptopurine/azathioprine is effective in IBD patients. However, data regarding toxicity associated with pregnancy are lacking, raising both patients' and physicians' concerns and sometimes resulting in elective abortion.
Methods: To evaluate potential toxicity of 6-mercaptopurine (6-MP), we reviewed the records of 485 patients who had received the drug. We contacted 462, of whom 155 had conceived at least 1 pregnancy after developing IBD. Pregnancies were analyzed as to whether the patient had taken 6-MP before, or at the time of, conception. These were compared with IBD patients who had their pregnancies before taking 6-MP. We collected data on live births, spontaneous abortions, prematurity, abortions secondary to birth defects, major and minor congenital birth defects, infections, and neoplasia. Outcomes were analyzed comparing pregnancies from men and women who had taken or were currently taking 6-MP to controls.
Results: There was no statistical difference in conception failures (defined as a spontaneous abortion), abortion secondary to a birth defect, major congenital malformations, neoplasia, or increased infections among male or female patients taking 6-MP compared with controls (RR = 0.85 [0.47­1.55], P = 0.59).
Conclusions: 6-MP use before or at conception or during pregnancy appears to be safe. Discontinuation of the drug before and during pregnancy is not indicated.

The prostaglandin E2 receptor-1 (EP-1) mediates acid-induced visceral pain hypersensitivity in humans
S. Sarkar, A. R. Hobson, A. Hughes, J. Growcott, C. J. Woolf, D. G. Thompson, Q. Aziz
Background & Aims: Central sensitization, an activity-dependent increase in spinal cord neuronal excitability, has been shown to contribute to esophageal pain hypersensitivity. Prostaglandin E2 (PGE2) is a mediator in both peripheral and central sensitization, in part via the prostaglandin E2 receptor-1 (EP-1), and may be a potential target for treating visceral pain. The purpose of this study was to determine whether acid-induced pain hypersensitivity within the non­acid-exposed esophagus (secondary hyperalgesia) is mediated by PGE2 activation of the EP-1 receptor.
Methods: Twelve healthy male subjects participated in a randomized, placebo-controlled crossover study. Upper esophageal pain thresholds (PTs) to electrical stimulation were determined, and either the EP-1 antagonist ZD6416 or a placebo was orally administered. One-hour after dosing, acid or saline (0.15 mol/L) was infused into the lower esophagus for 30 minutes. Upper esophageal PT was monitored for 120 minutes after infusion.
Results: Except in 1 subject (who was excluded), the pH in the upper esophagus remained above 5 throughout all studies. In 8 subjects, ZD6416 attenuated the reduction in PT in the upper esophagus normally induced by acid infusion into the lower esophagus (area under curve [AUC]: ­11.9 ± 2.5 and 6.4 ± 6.7 for placebo and ZD6416, respectively; P < 0.01). After saline infusion, the effects of ZD6416 and placebo were similar (AUC: 9.9 ± 6 and 4.1 ± 2, respectively; P = 0.8). Three subjects had no reduction in PT to acid infusion with placebo and were excluded at post hoc analysis.
Conclusions: The attenuation of secondary esophageal hyperalgesia by ZD6416 suggests that PGE2, via the EP-1 receptor, contributes to human visceral pain hypersensitivity.

Association between the C3435T MDR1 gene polymorphism and susceptibility for ulcerative colitis
M. Schwab, E. Schaeffeler, C. Marx, M. F. Fromm, B. Kaskas, J. Metzler, E. Stange, H. Herfarth, J. Schoelmerich, M. Gregor, S. Walker, I. Cascorbi, I. Roots, U. Brinkmann, U. M. Zanger, M. Eichelbaum
Background & Aims: The human multidrug resistance 1 (MDR1) gene product P-glycoprotein is highly expressed in intestinal epithelial cells, where it constitutes a barrier against xenobiotics. The finding that mdr1a knockout mice develop a form of colitis that is similar to ulcerative colitis, which can be prevented by antibiotics, indicates a barrier function for P-glycoprotein against the invasion of bacteria or toxins. Because the MDR1 single nucleotide polymorphism C3435T is associated with lower intestinal P-glycoprotein expression, we tested whether this polymorphism predisposes to development of ulcerative colitis.
Methods: Allele frequencies and genotype distributions of the C3435T single nucleotide polymorphism were investigated in 149 patients with ulcerative colitis, 126 patients with Crohn's disease, and sex-matched healthy controls.
Results: Significantly increased frequencies of the 3435T allele and the 3435TT genotype were observed in patients with ulcerative colitis compared with controls (3435T: P = 0.049; odds ratio, 1.4; 95% confidence interval, 1.02­1.94; 3435TT: P = 0.045; odds ratio, 2.03; 95% confidence interval, 1.04­3.95). In contrast, frequencies of the T allele and the TT genotype were the same in patients with Crohn's disease as in controls (P = 0.66 and P = 0.59, respectively). In comparison to 998 non­sex-matched controls, the effect for the TT genotype in ulcerative colitis patients was more pronounced (P = 0.0055; odds ratio, 2.1).
Conclusions: The higher frequency of the 3435TT genotype in patients with ulcerative colitis corroborates the findings from the mdr1a knockout mice. The results support the notion that P-glycoprotein plays a major role in the defense against intestinal bacteria or toxins. Impairment of barrier function in 3435TT subjects could render this genotype more susceptible to the development of ulcerative colitis.

Intestinal glucose transport: Evidence for a membrane traffic­based pathway in humans
R. Santer, G. Hillebrand, B. Steinmann, J. Schaub
Background & Aims: The presence of glucose transporter 2 (GLUT2) molecules in the basolateral membrane of enterocytes has long been considered to be of major importance for intestinal glucose absorption. The aim of this study was to reevaluate the role of GLUT2 in a patient with congenital GLUT2 deficiency (Fanconi-Bickel syndrome, FBS).
Methods: Oral mono- and disaccharide tolerance tests including gaschromatographic determination of breath hydrogen concentrations were performed in an FBS patient. For comparison, a patient with a microsomal carbohydrate transport defect, glucose-6-phosphate translocase 1 (G6PT1) deficiency, and a control individual were investigated.
Results: No increase in breath hydrogen concentration was found in the GLUT2-deficient patient after a glucose load. In G6PT1 deficiency, basal hydrogen concentrations were repeatedly found to be elevated.
Conclusions: From the fact that a GLUT2-deficient patient does not show any impairment of intestinal monosaccharide transport measurable by the hydrogen breath test, we conclude that mechanisms other than facilitative glucose transport by GLUT2 must be involved in the transport of monosaccharides at the basolateral membrane of enterocytes. When relating this observation to the high intestinal expression of human hexokinase, G6PT1, and glucose-6-phosphatase and to our results of oral carbohydrate tolerance tests in a G6PT1-deficient patient, there is evidence that a microsomal membrane traffic-based transport pathway, as recently suggested for GLUT2-deficient animals, also plays a major role in transcellular monosaccharide transport of the human intestine.

Appendectomy is followed by increased risk of Crohn's disease
R. E. Andersson, G. Olaison, C. Tysk, A. Ekbom
Background & Aims: Appendectomy is associated with a low risk of subsequent ulcerative colitis. This study analyzes the risk of Crohn's disease after appendectomy.
Methods: We followed-up 212,218 patients with appendectomy before age 50 years and a cohort of matched controls, identified from the Swedish Inpatient Register and the nationwide Census, for any subsequent diagnosis of Crohn's disease.
Results: An increased risk of Crohn's disease was found for more than 20 years after appendectomy, with incidence rate ratio 2.11 (95% confidence interval [CI], 1.21­3.79) after perforated appendicitis, 1.85 (95% CI, 1.10­3.18) after nonspecific abdominal pain, 2.15 (95% CI, 1.25­3.80) after mesenteric lymphadenitis, 2.52 (95% CI, 1.43­4.63) after other diagnoses. After nonperforated appendicitis, there was an increased risk among women but not among men (incidence rate ratio 1.37; 95% CI, 1.03­1.85, respectively, 0.89, 95% CI, 0.64­1.24). Patients operated on before age 10 years had a low risk (incidence rate ratio 0.48, 95% CI, 0.23­0.97). Crohn's disease patients with a history of perforated appendicitis had a worse prognosis.
Conclusions: Appendectomy is associated with an increased risk of Crohn's disease that is dependent on the patient's sex, age, and the diagnosis at operation. The pattern of associations suggests a biologic cause.

Protective association of aspirin/NSAIDs and esophageal cancer: A systematic review and meta-analysis
D. A. Corley, K. Kerlikowske, R. Verma, P. Buffler
Background & Aims: Esophageal carcinomas have high fatality rates, making chemoprevention agents desirable. We performed a systematic review with meta-analysis of observational studies evaluating the association of aspirin/nonsteroidal anti-inflammatory drug (NSAID) use and esophageal cancer.
Methods: We evaluated the MEDLINE, BIOSIS, and Web of Science electronic databases (1980­2001); manually reviewed the literature; and consulted with experts. Studies were included if they: (1) evaluated exposure to NSAIDs, aspirin, or both; (2) evaluated esophageal cancer; and (3) reported relative risks or odds ratios or provided data for their calculation. Data were independently abstracted by 2 investigators. The primary and sensitivity analyses used both fixed and random-effects models.
Results: Nine studies (2 cohort, 7 case control) containing 1813 cancer cases were identified. All primary summary estimates were homogeneous. Statistical pooling showed a protective association between any use of aspirin/NSAID and esophageal cancer (odds ratio [OR] = 0.57; 95% confidence interval [CI], 0.47­0.71). Both intermittent (OR = 0.82; CI, 0.67­0.99) and frequent medication use were protective (OR = 0.54; CI, 0.43­0.67), with greater protection with more frequent use. Stratified by medication type, aspirin use was protective (OR = 0.5; CI, 0.38­0.66), and NSAIDs had a borderline protective association (OR = 0.75; CI, 0.54­1.0). Any use was protective against both esophageal adenocarcinoma (OR = 0.67; CI, 0.51­0.87) and squamous cell carcinoma (OR = 0.58; CI, 0.43­0.78).
Conclusions: Pooled results support a protective association between aspirin and NSAIDs and esophageal cancer (of both histological types) and provide evidence for a dose effect. These findings support evaluating these agents in clinical trials of high-risk patients.

Expression of the leukotriene D4 receptor CysLT1, COX-2, and other cell survival factors in colorectal adenocarcinomas
J. F. öhd, C. Kamp Nielsen, J. Campbell, G. Landberg, H. Löfberg, A. Sjölander
Background & Aims: The effects of leukotriene (LT) D4 on intestinal epithelial cells govern events that are involved in cell survival and colon cancer, notably increased expression of cyclooxygenase (COX)-2 and enhanced production of prostaglandin E2. We investigated possible correlations between distribution of the recently described LTD4 receptor CysLT1R and factors previously shown to be up-regulated by LTD4 as well as clinicopathologic traits.
Methods: Immunohistochemistry and in situ hybridization were performed on tissue arrays, which were made using colorectal cancer samples from 84 patients.
Results: CysLT1R was significantly correlated to COX-2, 5-lipoxygenase, and Bcl-xL. Male subjects more often exhibited high levels of this receptor relative to female subjects, and Dukes' B patients with elevated CysLT1R expression showed markedly poorer survival than those with low-level expression. Furthermore, this was paralleled by an increased viability of CysLT1R-overexpressing cells in a colon cancer cell line.
Conclusions: Our results further implicate the involvement of LTs in colorectal carcinoma. Based on our present and earlier findings, we propose that LT/CysLT1R signaling facilitates survival of colon cancer cells, which may affect disease outcome. Like COX-2, LTs are accessible targets for pharmacologic treatment.

Clinical­Liver, Pancreas, and Biliary Tract

Determinants of the association of overweight with elevated serum alanine aminotransferase activity in the United States
C. E. Ruhl, J. E. Everhart
Background & Aims: In the absence of other causes, overweight and obesity increase the risk of liver disease. We examined whether central adiposity and metabolic markers explain the association of body mass index (BMI as kg/m2) with abnormal serum alanine aminotransferase (ALT) activity in a national, population-based study.
Methods: Adult participants (5724) in the third U.S. National Health and Nutrition Examination Survey (1988­1994) underwent anthropometric measures and phlebotomy after an overnight fast. Participants with excessive alcohol consumption, hepatitis B, hepatitis C, iron overload, or known diabetes were excluded.
Results: Elevated ALT levels were found in 2.8% of the population. In univariate analysis, factors associated with elevated ALT levels (P < 0.05) included younger age, male sex, Mexican-American ethnicity, and higher BMI, waist-to-hip circumference ratio (WHR), and fasting serum leptin, triglyceride, insulin, and glucose concentrations. The proportion of elevated ALT activity due to overweight and obesity (BMI 25 kg/m2) was 65%. In multivariate logistic regression analysis, control for WHR, demographic factors, and glucose concentration diminished but did not eliminate the association of higher BMI with elevated ALT activity. After adding leptin and insulin concentrations, abnormal ALT activity was most strongly associated with higher WHR (odds ratio [OR], 1.32; 95% confidence interval [CI], 1.12­1.56) and leptin (OR, 1.12; 95% CI, 1.01­1.24) and insulin (OR, 1.27; 95% CI, 1.01­1.60) concentrations, whereas BMI was not independently related.
Conclusions: In this large, national, population-based study, central adiposity, hyperleptinemia, and hyperinsulinemia were the major determinants of the association of overweight with elevated serum ALT activity.

A prospective study characterizing full-length hepatitis B virus genomes during acute exacerbation
C.­J. Liu, P.­J. Chen, M.­Y. Lai, J.­H. Kao, C.­F. Chang, H.­L. Wu, W.­Y. Shau, D.­S. Chen
Background & Aims: Hepatitis B virus (HBV) evolves rapidly in patients with chronic hepatitis B, and HBV variation may trigger acute exacerbation. To study this relationship, we investigated full-length viral sequences before, during, and after exacerbation.
Methods: We prospectively studied 14 patients with exacerbation of hepatitis B, either spontaneously (n = 4) or after receiving various medical interventions (n = 10), and measured their serum alanine aminotransferase (ALT) and HBV DNA levels monthly. Full-length HBV genomes at baseline, at the peak of serum viral load, at ALT peak, and after ALT peak were obtained by polymerase chain reaction, sequenced, and compared. Replication activities of serial HBV variants were assayed by in vitro transfection.
Results: Serum viral load was increased in all exacerbations. Viral peak preceded ALT peak in 13 (93%) of the 14 patients. At virologic peak, 12 patients (86%) harbored viral genome identical to the corresponding baseline genome. At and after ALT peak, 9 (64%) and 7 (50%) of the viral genomes remained identical to baseline, respectively. Mean nucleotide change per genome was 0.2 at virologic peak but increased to 4.4 and 8.1 at and after ALT peak, respectively. The replication potential of the viral variant that emerged during or after exacerbation was equivalent to that at baseline.
Conclusions: Most exacerbations were preceded by an upsurge of serum HBV identical to the preexisting HBV strain. After exacerbation, about half of the patients were repopulated by a different viral variant, which was likely a result of immune selection.

Model for end-stage liver disease (MELD) and allocation of donor livers
R. Wiesner, E. Edwards, R. Freeman, A. Harper, R. Kim, P. Kamath, W. Kremers, J. Lake, T. Howard, R. M. Merion, R. A. Wolfe, R. Krom, the United Network For Organ Sharing Liver Disease Severity Score Committee
Background & Aims: A consensus has been reached that liver donor allocation should be based primarily on liver disease severity and that waiting time should not be a major determining factor. Our aim was to assess the capability of the Model for End-Stage Liver Disease (MELD) score to correctly rank potential liver recipients according to their severity of liver disease and mortality risk on the OPTN liver waiting list.
Methods: The MELD model predicts liver disease severity based on serum creatinine, serum total bilirubin, and INR and has been shown to be useful in predicting mortality in patients with compensated and decompensated cirrhosis. In this study, we prospectively applied the MELD score to estimate 3-month mortality to 3437 adult liver transplant candidates with chronic liver disease who were added to the OPTN waiting list at 2A or 2B status between November, 1999, and December, 2001.
Results: In this study cohort with chronic liver disease, 412 (12%) died during the 3-month follow-up period. Waiting list mortality increased directly in proportion to the listing MELD score. Patients having a MELD score <9 experienced a 1.9% mortality, whereas patients having a MELD score 40 had a mortality rate of 71.3%. Using the c-statistic with 3-month mortality as the end point, the area under the receiver operating characteristic (ROC) curve for the MELD score was 0.83 compared with 0.76 for the Child-Turcotte-Pugh (CTP) score (P < 0.001).
Conclusions: These data suggest that the MELD score is able to accurately predict 3-month mortality among patients with chronic liver disease on the liver waiting list and can be applied for allocation of donor livers.

Progression of fibrosis in chronic hepatitis C
M. G. Ghany, D. E. Kleiner, H. Alter, E. Doo, F. Khokar, K. Promrat, D. Herion, Y. Park, T. J. Liang, J. H. Hoofnagle
Background & Aims: Fibrosis is the hallmark of hepatic cirrhosis, worsening of which is probably the best surrogate marker for progression of chronic liver disease. We evaluated a large cohort of patients with chronic hepatitis C (CHC) using liver histology to assess the rate and predictors of progression of fibrosis.
Methods: The cohort consisted of 123 patients with CHC who underwent 2 liver biopsies 4­212 months (mean, 44 months) apart without intervening treatment. Liver histology was graded using the histology activity index (score, 0­18) and fibrosis staged using a scoring system of 0 (no fibrosis) to 6 (cirrhosis).
Results: Among 123 patients, 48 (39%) showed progression in fibrosis scores, 46 (37%) showed no change, and 29 (24%) showed improvement. Of those with worsening fibrosis, 75% had a 1-point increase and 25% a 2-point or greater increase in scores, and 9% showed progression to cirrhosis. The overall rate of progression was 0.12 fibrosis units per year, a rate that predicts progression to cirrhosis in 50 years if progression was linear. The rate of fibrosis progression was variable, and it was higher among older patients, those with higher serum alanine and aspartate aminotransferase levels, and those with the most extensive periportal necrosis on initial liver biopsy.
Conclusions: The best predictors of fibrosis progression in CHC are the extent of serum aminotransferase elevations and the degree of hepatocellular necrosis and inflammation on liver biopsy. These findings support the recommendation that patients with normal aminotransferase levels and mild liver histology can safely defer treatment.

Histological outcome during long-term lamivudine therapy
J. L. Dienstag, R. D. Goldin, E. J. Heathcote, H. W. L. Hann, M. Woessner, S. L. Stephenson, S. Gardner, D. F. Gray, E. R. Schiff
Background & Aims: One year of lamivudine for chronic hepatitis B results in histologic improvement. We aimed to assess the histological impact of longer-term treatment.
Methods: Sets of 3 liver biopsies, from 63 patients before and after 1 year of randomized lamivudine treatment and after 2 years of further open-label treatment, were assigned Histologic Activity Index scores under code.
Results: At the end of year 1, 36/63 (57%) showed 2 point improvement and 24/63 (38%) no change in necroinflammatory activity; after 2 additional years of lamivudine, 38/63 (60%) remained stable and 12/63 (19%) continued to improve. Worsening occurred in similar proportions of patients with and without YMDD (tyrosine, methionine, aspartate, aspartate) variants. After all 3 years of lamivudine treatment, 35/63 (56%) of patients showed improvement, 21/63 (33%) no change, and 7/63 (11%) worsening. Those without, compared with those with, YMDD variants were more likely to improve (17/22 [77%] vs. 18/41 [44%]) and less likely to deteriorate (1/22 [5%] vs. 6/41 [15%]). Patients with YMDD variants for >2 years were least likely to improve (8/22 [36%]). Bridging fibrosis improved by 1 level in 12/19 (63%), and cirrhosis improved (score of 4 to 3) in 8/11 (73%). Only 1/52 [2%]) showed progression to cirrhosis, and 3/34 (9%) showed progression to bridging fibrosis (all with YMDD variants).
Conclusions: Three years of lamivudine therapy reduces necroinflammatory activity and reverses fibrosis (including cirrhosis) in most patients. The emergence of YMDD variants blunts histologic responses; therefore, extended-duration YMDD variants may require additional therapies to maintain the histological benefit of treatment.

Basic­Alimentary Tract

Regulation of intestinal nuclear factor-B activity and E-selectin expression during sepsis: A role for peroxynitrite
C. W. Lush, G. Cepinskas, P. R. Kvietys
Background & Aims: During sepsis, the production of both nitric oxide and superoxide are increased. Furthermore, NO and O2­ may interact to produce peroxynitrite. The major aim of the present study was to assess the relative roles of NO, O2­, and ONOO­ in the regulation of nuclear factor B (NF-B) activity and subsequent E-selectin expression during the early stages of sepsis.
Methods: Mice were administered 5 µg lipopolysaccharide (LPS) intraperitoneally, and NF-B activity and E-selectin expression in the small intestine were assessed 3 hours later.
Results: In wild-type mice, increased levels of NF-B in nuclear extracts were noted. By contrast, in both inducible nitric oxide synthase­deficient and transgenic Cu/Zn superoxide dismutase­overexpressing mice, NF-B mobilization to the nucleus was diminished. Pretreatment with a ONOO­ decomposition catalyst (5,10,15,20-tetrakis[4-sulfonatophenyl]porphyrinato iron [III] [FeTPPS]) resulted in a diminished impact of LPS on NF-B activation. LPS increased vascular E-selectin expression in wild-type mice. E-selectin expression was diminished in inducible nitric oxide synthase­deficient mice after LPS challenge, but E-selectin expression remained elevated in both Cu/Zn superoxide dismutase transgenic mice and wild-type mice pretreated with FeTPPS.
Conclusions: Our observations suggest that NO, O2­, and ONOO­ production are all important mediators in the induction of NF-B activity during endotoxemia and that, in vivo, E-selectin expression on endothelium may not always be associated with whole-organ NF-B activation.

Localization of orexin-1 receptors to vagal afferent neurons in the rat and humans
G. Burdyga, S. Lal, D. Spiller, W. Jiang, D. Thompson, S. Attwood, S. Saeed, D. Grundy, A. Varro, R. Dimaline, G. J. Dockray
Background & Aims: Orexin-A and -B are brain-gut peptides that stimulate food intake via orexin-R1 and -R2 receptors. Cholecystokinin (CCK) inhibits food intake via CCKA receptors expressed on vagal afferent neurons. The purpose of the study was to determine whether vagal afferent neurons express OX-R1 and OX-R2 and whether orexin-A inhibits responses to CCK.
Methods: OX-R1 and -R2 expression by rat and human nodose ganglia was examined by reverse-transcriptase polymerase chain reaction (RT-PCR). Receptor localization was determined by immunohistochemistry. Responses of rat jejunal afferent fibers were examined by electrophysiology.
Results: Both rat and human nodose ganglia expressed OX-R1 as detected by RT-PCR, and humans also expressed OX-R2. The identity of the products was confirmed by sequencing. Immunohistochemistry indicated expression of OX-R1 in both species in neurons that also expressed CCKA and leptin receptors. In human ganglia there was also expression in glial cells that was absent in rats. Orexin-A had no effect on the resting discharge of afferent nerve fibers but inhibited responses to CCK.
Conclusions: OX-R1 and CCKA receptors are expressed by human and rat vagal afferent neurons. Orexin inhibits responses to CCK suggesting a role in modulation of gut to brain signaling.

Crohn's disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan
D. K. Bonen, Y. Ogura, D. L. Nicolae, N. Inohara, L. Saab, T. Tanabe, F. F. Chen, S. J. Foster, R. H. Duerr, S. R. Brant, J. H. Cho, G. Nuñez
Background & Aims: The NOD2 variants R702W, G908R, and L1007fsinsC are strongly associated with Crohn's disease (CD) in both European and American populations, but whether this susceptibility extends to all ethnic groups remains unknown. Except for the L1007fsinsC mutation, which produces a truncated NOD2 protein, the functional activity of the major CD-associated variants G908R and R702W is unknown.
Methods: Individuals were genotyped for R702W, G908R, and L1007fsinsC. The ability of G908R, R702W, and L1007fsinsC variants in the presence and absence of P268S to confer responsiveness to lipopolysaccharide (LPS) and peptidoglycan (PGN) was determined in HEK293T kidney cells.
Results: G908R and L1007fsinsC, but not R702W, were associated with disease susceptibility in Ashkenazi Jews. Ashkenazi Jews with CD had significantly higher allele frequency carriage of G908R and lower carriage of R702W compared with non-Jewish whites with CD. Functional studies revealed that the G908R, R702W, and L1007fsinsC variants in the presence and absence of P268S are defective in their ability to respond to bacterial LPS and PGN, whereas P268S alone exhibited wild-type activity.
Conclusions: R702W is not associated with susceptibility to CD in Ashkenazi Jews. The G908R, R702W, and L1007fsinsC variants share a common signaling defect in response to bacterial components, providing evidence for a unifying molecular mechanism whereby NOD2 mutations contribute to disease susceptibility.

Basic­Liver, Pancreas, and Biliary Tract

Liver fibrosis: Insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors
C. Yang, M. Zeisberg, B. Mosterman, A. Sudhakar, U. Yerramalla, K. Holthaus, L. Xu, F. Eng, N. Afdhal, R. Kalluri
Background & Aims: In liver fibrosis, alterations within the space of Disse microenvironment occur and facilitate further progression of chronic liver disease. The normal basement membrane­like matrix present within the space of Disse converts to a matrix rich in fibril-forming collagens during fibrosis.
Methods: To further understand the pathogenesis of liver fibrosis, we modified an in vitro Boyden chamber system to partially mimic in vivo conditions of hepatic stellate cells (HSCs) during health and disease.
Results: Stimulation of HSCs with platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-1, and/or epithelial growth factor (EGF) resulted in an increase in their migratory capacity and up-regulated matrix metalloproteinase (MMP)-2 activity. Migration induced by PDGF-BB was associated with increased proliferation, whereas TGF-1/EGF­induced migration was proliferation independent. COL-3, an inhibitor of MMP-2 and MMP-9, inhibited migration of HSCs induced by direct activation of PDGF-BB or TGF-1 but had no effect on migration induced by chemotactic stimuli without direct contact, suggesting 2 distinct MMP-dependent and MMP-independent mechanisms of PDGF-BB­ or TGF-1­induced migration. Additionally, we show that type I collagen by itself induced migration of HSCs. Migration induced by PDGF-BB, TGF-1, and collagen I could be inhibited by 1- and/or 2-integrin blocking antibodies, collectively suggesting an integrin-dependent, MMP-2­mediated migration of HSCs.
Conclusions: Basement membrane matrix integrity, composition, and cell-matrix interactions play an important role in anchoring HSCs and preventing them from spreading within the space of Disse and potentially elsewhere in the liver. Additionally, our data provide strong evidence for MMPs in regulation of HSCs migration.

Induction of hepatic ABC transporter expression is part of the PPAR­mediated fasting response in the mouse
T. Kok, H. Wolters, V. W. Bloks, R. Havinga, P. L. M. Jansen, B. Staels, F. Kuipers
Background & Aims: Fatty acids are natural ligands of the peroxisome proliferator-activated receptor (PPAR). Synthetic ligands of this nuclear receptor, i.e., fibrates, induce the hepatic expression of the multidrug resistance 2 gene (Mdr2), encoding the canalicular phospholipid translocator, and affect hepatobiliary lipid transport. We tested whether fasting-associated fatty acid release from adipose tissues alters hepatic transporter expression and bile formation in a PPAR­dependent manner.
Methods: A 24-hour fasting/48-hour refeeding schedule was used in wild-type and Ppar(­/­) mice. Expression of genes involved in the control of bile formation was determined and related to secretion rates of biliary components.
Results: Expression of Ppar, farnesoid X receptor, and liver X receptor genes encoding nuclear receptors that control hepatic bile salt and sterol metabolism was induced on fasting in wild-type mice only. The expression of Mdr2 was 5-fold increased in fasted wild-type mice and increased only marginally in Ppar(­/­) mice, and it normalized on refeeding. Mdr2 protein levels and maximal biliary phospholipid secretion rates were clearly increased in fasted wild-type mice. Hepatic expression of the liver X receptor target genes ATP binding cassette transporter a1 (Abca1), Abcg5, and Abcg8, implicated in hepatobiliary cholesterol transport, was induced in fasted wild-type mice only. However, the maximal biliary cholesterol secretion rate was reduced by approximately 50%.
Conclusions: Induction of Mdr2 expression and function is part of the PPAR­mediated fasting response in mice. Fasting also induces expression of the putative hepatobiliary cholesterol transport genes Abca1, Abcg5, and Abcg8, but, nonetheless, maximal biliary cholesterol excretion is decreased after fasting.

New method of delivering gene-altered Kupffer cells to rat liver: Studies in an ischemia-reperfusion model
M. Froh, M. D. Wheeler, O. Smutney, Z. Zhong, B. U. Bradford, R. G. Thurman
Background & Aims: Kupffer cells play a major role in the pathogenesis of several diseases. They release physiologically active substances that often lead to localized tissue injury. Therefore, the aim of this study was to establish a model to protect the liver through supplementation of Kupffer cells that have been transduced by recombinant adenovirus.
Methods: Optimal conditions for intravenous injection in rats were established using carbon-labeled Kupffer cells. Adenoviral-transduced Kupffer cells encoding the Cu/Zn-SOD gene (Ad.SOD1) or -galactosidase reporter gene (Ad.LacZ) were transplanted into recipient rats. Twenty-four hours after transplantation, 70% hepatic ischemia-reperfusion was used to induce hepatic oxidative stress, and liver injury was determined 8 or 24 hours later.
Results: In initial experiments, 10%­20% of the injected carbon-labeled cells were localized in the host liver after 24 hours, representing ~1% of the total population of Kupffer cells. Pretreatment of the recipient with a single dose of cyclosporin A maximized Kupffer cell reseeding up to 4%­10% of the total Kupffer cell population, suggesting that efficiency is limited by host immune response. Moreover, reseeded Kupffer cells were retained in host livers for up to 14 days after transplant. In livers of animals injected with Kupffer cells transduced with Ad.LacZ, transgene expression was observed, indicating Kupffer cell functional integrity. Injection of Kupffer cells transduced with Ad.SOD1 significantly blunted the increase in serum transaminases and liver injury because of ischemia-reperfusion compared with controls.
Conclusions: This novel approach allows delivery of transduced Kupffer cells in rats, which can be used as an investigative tool as well as a therapeutic strategy against inflammatory liver diseases.

Peroxisome proliferator-activated receptor- signaling contributes to enhanced proliferation of hepatic stellate cells
K. Hellemans, L. Michalik, A. Dittie, A. Knorr, K. Rombouts, J. De Jong, C. Heirman, E. Quartier, F. Schuit, W. Wahli, A. Geerts
Background & Aims: The peroxisome proliferator-activated nuclear receptors (PPAR-, PPAR-, and PPAR-), which modulate the expression of genes involved in energy homeostasis, cell cycle, and immune function, may play a role in hepatic stellate cell activation. Previous studies focused on the decreased expression of PPAR- in hepatic stellate cell activation but did not investigate the expression and role of the PPAR- and - isotypes. The aim of this study was to evaluate the expression of the different PPARs during hepatic stellate cell activation in vitro and in situ and to analyze possible factors that might contribute to their expression. In a second part of the study, the effect of a PPAR- agonist on acute liver injury was evaluated.
Methods: The effects of PPAR isotype-specific ligands on hepatic stellate cell transition were evaluated by bromodeoxyuridine incorporation, gel shifts, immunoprecipitation, and use of antisense PPAR- RNA­expressing adenoviruses. Tumor necrosis factor ­induced PPAR- phosphorylation and expression was evaluated by metabolic labeling and by using specific P38 inhibitors.
Results: Hepatic stellate cells constitutively express high levels of PPAR-, which become further induced during culture activation and in vivo fibrogenesis. No significant expression of PPAR- or - was found. Stimulation of the P38 mitogen-activated protein kinase pathway modulated the expression of PPAR-. Transcriptional activation of PPAR- by L165041 enhanced hepatic stellate cell proliferation. Treatment of rats with a single bolus of CCl4 in combination with L165041 further enhanced the expression of fibrotic markers.
Conclusions: PPAR- is an important signal-transducing factor contributing to hepatic stellate cell proliferation during acute and chronic liver inflammation.

-catenin antisense studies in embryonic liver cultures: Role in proliferation, apoptosis, and lineage specification
S. P. S. Monga, H. K. Monga, X. Tan, K. Mulé, P. Pediaditakis, G. K. Michalopoulos
Background & Aims: Wnt/-catenin pathway activation occurs during liver growth in hepatoblastomas, hepatocellular cancers, and liver regeneration. The aim of this study was to investigate the role of -catenin, a key component of the Wnt pathway, in liver development as well as its normal distribution in developing liver.
Methods: Embryonic liver cultures and -catenin antisense phosphorodiamidate morpholino oligomer (PMO) were used to elucidate the role of -catenin in liver development. Livers from embryos at 10 days of gestational development were cultured in the presence of antisense or control PMO for 72 hours and analyzed.
Results: -Catenin shows stage-specific localization and distinct distribution compared with known markers in developing liver. A substantial decrease in -catenin protein was evident in the organs cultured in the presence of antisense. -Catenin inhibition decreased cell proliferation and increased apoptosis in these organ cultures. Presence of antisense resulted in loss of CK19 immunoreactivity of the bipotential stem cells. -Catenin inhibition also promoted c-kit immunoreactivity of the hepatocytes.
Conclusions: We conclude that the PMO antisense to -catenin effectively inhibits synthesis of its protein. -Catenin modulates cell proliferation and apoptosis in developing liver. It may play a significant role in early biliary lineage commitment of the bipotential stem cells and also seems to be important in hepatocyte maturation.

Case Reports

Liver disease caused by failure to racemize trihydroxycholestanoic acid: Gene mutation and effect of bile acid therapy
K. D. R. Setchell, J. E. Heubi, K. E. Bove, N. C. O'Connell, T. Brewsaugh, S. J. Steinberg, A. Moser, R. H. Squires, Jr
Background & Aims: Inborn errors of bile acid metabolism may present as neonatal cholestasis and fat-soluble vitamin malabsorption or as late onset chronic liver disease. Our aim was to fully characterize a defect in bile acid synthesis in a 2-week-old African-American girl presenting with coagulopathy, vitamin D and E deficiencies, and mild cholestasis and in her sibling, whose liver had been used for orthotopic liver transplantation (OLT).
Methods: Bile acids were measured by mass spectrometry in urine, bile, serum, and feces of the patient and in urine from the unrelated recipient.
Results: Liver biopsy specimens showed neonatal hepatitis with giant cell transformation and hepatocyte necrosis; peroxisomes were reduced in number. High concentrations of (25R)3,7,12-trihydroxy-5-cholestanoic acid in the urine, bile, and serum established a pattern similar to that of Zellweger syndrome and identical to the Alligator mississippiensis. Serum phytanic acid was normal, whereas pristanic acid was markedly elevated. Biochemical, MRI, and neurologic findings were inconsistent with a generalized defect of peroxisomal function and were unique. Analysis of the urine from the recipient of the deceased sibling's liver confirmed the same bile acid synthetic defect. A deficiency in 2-methylacyl-CoA racemase, which is essential for conversion of (25R)THCA to its 25S-isomer, the substrate to initiate peroxisomal -oxidation to primary bile acids, was confirmed by DNA analysis revealing a missense mutation (S52P) in the gene encoding this enzyme. Long-term treatment with cholic acid normalized liver enzymes and prevented progression of symptoms.
Conclusions: This genetic defect further highlights bile acid synthetic defects as a cause of neonatal cholestasis.

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JOURNAL OF HEPATOLOGY

Table of Contents for Volume 38, Issue 1, January 2003

Biliary Tract and Cholestasis

Combined polymorphisms in UDP-glucuronosyltransferases 1A1 and 1A6: implications for patients with Gilbert's syndrome
Wilbert H.M. Peters, Rene H.M. te Morsche and Hennie M.J. Roelofs
Background/Aims: UDP-glucuronosyltransferases (UGTs) are important enzymes involved in glucuronidation of various exogenous and endogenous compounds. Studies were undertaken on the variability of three UGT enzyme activities in human livers. Enzyme activities were associated with genetic polymorphisms in UGT1A1 (UGT1A1*28) and UGT1A6 (UGT1A6*2). UGT1A1*28 is associated with Gilbert's syndrome, a deficiency in glucuronidation of bilirubin leading to mild hyperbilirubinemia, whereas UGT1A6*2 may result in low glucuronidation rates of several drugs. Methods: Enzyme activities and genetic polymorphisms were assessed in 39 human liver samples, and polymorphisms were also assessed in blood of 253 healthy controls. Results: Associations were found between UGT enzyme activities of bilirubin (B) and 4-nitrophenol (NP; r=0.47, P=0.0024), B and 4-methylumbelliferone (MUB; r=0.54, P=0.0003), and NP and MUB (r=0.89, P<0.0001). In addition to the association between B-UGT enzyme activity and UGT1A1*28 (r=0.45, P=0.0034) as reported earlier, an association between B-UGT and UGT1A6*2 (r=0.43, P=0.007) was found. In 253 Dutch Caucasian controls, co-occurrence of UGT1A1*28 and UGT1A6*2 was found (r=0.9, P<0.0001). Conclusions: Most patients with Gilbert's syndrome, in addition to their reduced B-UGT enzyme activity, may have abnormalities in the glucuronidation of aspirin or coumarin- and dopamine-derivatives, due to this combination of UGT1A1*28 and UGT1A6*2 genotypes.

Cell Biology, Metabolism and Transport

Bacterial lipopolysaccharide decreases thrombomodulin expression in the sinusoidal endothelial cells of rats - a possible mechanism of intrasinusoidal microthrombus formation and liver dysfunction
Masane Kume et al.
Background/Aims: To elucidate the mechanism of liver dysfunction occurring in patients with sepsis, we evaluated the effect of bacterial lipopolysaccharide (LPS) on the expression of thrombomodulin (TM) in rat sinusoidal endothelial cells (SECs) and the therapeutic efficacy of exogenous recombinant TM. Methods: We induced endotoxemia in rats by bolus intraperitoneal injection of LPS. TM antigen levels within tissues were assessed by immunohistochemistry. We measured TM in cultured SECs by enzyme immunoassay, functional analysis and real-time polymerase chain reaction (PCR). Results: TM antigen and activity levels were significantly decreased in SECs isolated from LPS-treated rats after 3 and 6 h treatment, and recovered after 12 h treatment, correlating with immunohistochemical observations. In contrast, TM messenger RNA was decreased after 6 and 12 h treatment, and slightly recovered after 24 h treatment. TM expression in cultured SECs isolated from normal rats was also reduced after treatment with LPS and tumor necrosis factor (TNF)- in vitro. The increased levels of serum fibrin degradation products (FDP), fibrin deposition within liver sinusoids, injury of SECs and liver dysfunction induced by LPS in our rat model was improved by recombinant TM treatment. Conclusions: Decreased TM expression in SECs of LPS-treated rats may result in intrasinusoidal microthrombus formation and subsequent liver dysfunction during sepsis.

Glucose intolerance and serum aminotransferase activities in Japanese men
Yumi Miyake et al.
Background/Aims: Elevated activities of serum aminotransferase are commonly observed in patients with diabetes mellitus. Few studies have addressed the relation between glucose intolerance and serum activities of aminotransferase in free-living populations. Methods: Using a 75 g oral glucose tolerance test, we examined the association of impaired fasting glycemia (IFG), impaired glucose tolerance (IGT), and type 2 diabetes mellitus with serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT) among 4621 men aged 49-59 years of the Japan Self-Defense Forces. Statistical adjustment was made for body mass index, waist-hip ratio, and other possible confounding factors. Results: Proportions of an elevated ALT (>50 IU/l) in men with normal glucose tolerance, IFG, IGT, and newly diagnosed diabetes mellitus were 3.5%, 9.5%, 7.7%, and 18.0%, respectively. Adjusted odds ratios of an elevated ALT for IFG, IGT, and newly diagnosed diabetes mellitus were 2.2 (95% confidence interval 1.1-4.3), 1.7 (1.2-2.4), and 4.4 (3.0-6.6), respectively. IGT and diabetes mellitus were also significantly positively associated with elevated AST (>40 IU/l) and GGT (>50 IU/l). Conclusions: Glucose intolerance is associated with elevated serum aminotransferase independent of obesity, but even a mildly elevated ALT is relatively uncommon in free-living men with glucose intolerance.

Chronic Liver Diseases

Extracorporeal liver support with molecular adsorbents recirculating system in patients with severe acute alcoholic hepatitis
Rajiv Jalan et al.
Background/Aims: The mortality of patients with severe acute alcoholic hepatitis (AH) remains high, leading to interest in the use of extracorporeal liver support. The molecular adsorbents recirculating system (MARS) is a liver support device based upon a hollow fibre module in which the patient's blood is dialyzed across an albumin-impregnated membrane. The aim of this paper is to assess the safety, efficacy and feasibility of using MARS in patients with severe AH. Methods: Eight patients (all encephalopathic; hepatorenal syndrome: Type 1, five patients; Type 2, two patients) were treated with MARS. Clinical, biochemical and haemodynamic assessments were done. Results: Five patients were discharged from hospital, and four are alive at 3 months of follow-up, compared with an estimated survival of about 20%. There were significant improvement in serum bilirubin (P=0.008), creatinine (P=0.02), prothrombin time (P=0.04), and grade of encephalopathy (P=0.05). Sustained improvements in mean arterial pressure, systemic vascular resistance and cardiac output were observed. Thrombocytopaenia was the only MARS-related adverse event observed. Conclusions: MARS resulted in improved liver biochemistry, cardiovascular haemodynamics, renal function and encephalopathy in patients with severe AH, with an apparent reduction in mortality. On this basis, a multi-centre, randomized clinical trial has been initiated.

Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease
Yoshio Sumida et al.
Background/Aims: Thioredoxin (TRX) is a stress-inducible thiol-containing protein. The aim of this study was to evaluate the clinical significance of serum TRX in patients with nonalcoholic steatohepatitis (NASH) or simple steatosis. Methods: Serum TRX levels were determined using an enzyme-linked immunosorbent assay kit in 25 patients with NASH, 15 patients with simple steatosis, and 17 healthy volunteers. Results: Serum TRX levels (medians and (ranges), ng/ml) were significantly elevated in patients with NASH (60.3 (17.6-104.7)), compared to those in patients with simple steatosis (24.6 (16.6-69.7), P=0.0009) and in healthy controls (23.5 (1.3-50.7), P<0.0001). Serum ferritin levels in patients with NASH were also significantly higher than the levels in patients with simple steatosis. The receiver operating characteristic curve confirmed that serum TRX and ferritin levels were predictors for distinguishing NASH from simple steatosis. Higher grades of histological iron staining were observed in NASH than in simple steatosis. Serum TRX tended to increase in accordance with hepatic iron accumulation and the histological severity in patients with NASH. Conclusions: The pathogenesis of NASH may be associated with iron-related oxidative stress. The serum TRX level is a parameter for discriminating NASH from simple steatosis as well as a predictor of the severity of NASH.

Polycystic liver disease is genetically heterogeneous: clinical and linkage studies in eight Finnish families
Pia Tahvanainen et al.
Background/Aims: Polycystic liver disease (PCLD), a dominantly inherited condition separate from polycystic kidney disease (PKD), has recently been found to be linked to a locus on chromosome 19p13.2-13.1 in two North American families. Our aim was to study whether there is clinical or genetic heterogeneity within PCLD families. Methods: We collected clinical data of eight Finnish PCLD families and performed both linkage analysis and an extended admixture test. We used genetic markers located on chromosome 19p13.2-13.1 and, in addition, on the three known PKD loci on chromosomes 4q21-q23 (PKD2), 6p21 (ARPKD) and 16p13.3-p13.12 (PKD1). Results: There were a total of 33 PCLD patients among which the severity of the disease varied greatly even within families. Seven patients had disabling symptoms requiring cyst decompression while ten patients were found only when the symptomless family members were studied by abdominal ultrasound. When genetic homogeneity was assumed, the PCLD locus on chromosome 19p13.2-13.1 was excluded but when genetic heterogeneity was allowed, five families out of seven showed linkage to that locus. All three PKD loci were excluded. Conclusions: Most Finnish PCLD families are linked to chromosome 19p13.2-13.1 but there exists also a second PCLD locus yet to be found.

Cirrhosis and its Complications

Nitric oxide mediates the reduced vasoconstrictor response to angiotensin II in patients with preascitic cirrhosis
Ahmed Helmy et al.
Background/Aims: Altered vascular responses to vasopressor agents contribute to the pathogenesis of the circulatory dysfunction in cirrhosis. This study aims to assess the role of endogenous nitric oxide (NO) in the reduced vascular responsiveness to angiotensin II (ANG-II) in eight patients with preascitic cirrhosis compared with eight age- and sex-matched healthy controls. Methods: Forearm blood flow (FBF) responses to sub-systemic, locally-active intra-brachial infusions of ANG-II were measured using venous occlusion plethysmography before and during the application of an `NO-clamp', a balanced co-infusion of L-NG-monomethyl-arginine (a selective NO synthase inhibitor) and sodium nitroprusside (an exogenous NO donor) to block endogenous NO production and restore normal NO-mediated basal blood flow, respectively. Results: Before applying the `NO-clamp', ANG-II caused dose-dependent reductions of FBF in both groups (P<0.001) that were significantly attenuated in the cirrhotic patients (P=0.012). In the presence of the `NO-clamp', the ANG-II-mediated vasoconstriction was enhanced in cirrhotic patients (P<0.01), unchanged in controls, and now similar in both groups. Conclusions: This study confirms that vasoconstriction to ANG-II is reduced in patients with preascitic cirrhosis, and suggests that this is principally due to enhanced NO generation mediated by ANG-II.

Comparison of rifaximin and lactitol in the treatment of acute hepatic encephalopathy: results of a randomized, double-blind, double-dummy, controlled clinical trial
Antoni Mas et al.
Background/Aims: The efficacy and safety of rifaximin in comparison with lactitol in the treatment of acute hepatic encephalopathy was assessed in a prospective randomized, double-blind, double-dummy, controlled trial. Methods: A total of 103 patients with grade I-III acute hepatic encephalopathy were randomized to receive rifaximin (50 patients, 1200 mg/day) or lactitol (53 patients, 60 g/day) for 5-10 days. Changes in the portal-systemic encephalopathy (PSE) index on entry and at the end of the study were used to evaluate the efficacy of the two therapies. Results: Both groups were comparable before treatment with regard to demographic data and characteristics of the hepatic encephalopathy episode. The global efficacy of both therapies was similar: 81.6% in the rifaximin group and 80.4% in the lactitol group showed improvement or total regression of the episode. A significantly better evolution of the PSE index was observed in the rifaximin group, due to a greater effect of rifaximin in two components of the index: EEG abnormalities and ammonia levels. No serious adverse events related to either treatment were found during the study. Conclusions: Rifaximin may be considered a useful and safe alternative therapy to lactitol in the treatment of acute hepatic encephalopathy in cirrhosis.

Liver Cell Injury and Liver Failure

Ischemic preconditioning protects from hepatic ischemia/reperfusion-injury by preservation of microcirculation and mitochondrial redox-state
Matthias Glanemann et al.
Background/Aims: Ischemic preconditioning (IP) is known to protect hepatic tissue from ischemia-reperfusion injury. However, the mechanisms involved are not fully understood yet. Methods: Using intravital multifluorescence microscopy in the rat liver, we studied whether IP exerts its beneficial effect by modulating postischemic Kupffer cell activation, leukocyte-endothelial cell interaction, microvascular no-reflow, mitochondrial redox state, and, thus, tissue oxygenation. Results: Portal triad cross-clamping (45 min) followed by reperfusion induced Kupffer cell activation, microvascular leukocyte adherence, sinusoidal perfusion failure (no-reflow) and alteration of mitochondrial redox state (tissue hypoxia) (P<0.05). This resulted in liver dysfunction and parenchymal injury, as indicated by decreased bile flow and increased serum glutamate dehydrogenase (GLDH) levels (P<0.05). IP (5 min ischemia and 30 min intermittent reperfusion) was capable to significantly reduce Kupffer cell activation (P<0.05), which was associated with a slight attenuation of leukocyte adherence. Further, IP markedly ameliorated sinusoidal perfusion failure (P<0.05), and, thereby, preserved adequate mitochondrial redox state (P<0.05). As a consequence, IP prevented the decrease of bile flow (P<0.05) and the increase in serum GLDH levels (P<0.05). Conclusions: IP may exert its beneficial effects on hepatic ischemia-reperfusion injury by preserving mitochondrial redox state, which is guaranteed by the prevention of reperfusion-associated Kupffer cell activation and sinusoidal perfusion failure.

Viral Hepatitis

Liver-infiltrating lymphocytes in end-stage hepatitis C virus: Subsets, activation status, and chemokine receptor phenotypes
Judie Boisvert et al.
Background: Hepatitis C virus (HCV) is a leading cause of chronic liver disease, yet little is known about the intrahepatic immune response in end-stage patients. Chemokines and their receptors are important regulators of immunity, particularly in the migration and localization of circulating leukocytes within peripheral tissues. Aims: This report provides a comprehensive comparison of the chemokine receptor and activation phenotype of the major leukocyte subsets present in end-stage HCV-infected and non-HCV infected livers. Methods: Lymphocytes were purified from homogenized explant liver tissue and analyzed by flow cytometry. Results: NK cells are the predominant cell type, followed by T cells, B cells and NK-T cells, independent of HCV status. T cells displayed a memory phenotype and low levels of activation markers. CCR5, CXCR3 and CXCR6 were expressed on a large fraction of activated cells, while moderate to low expression of CCR2, CCR6 and CX3CR1 was observed. Several other tissue-specific and inflammatory chemokine receptors were absent from infiltrating lymphocytes. Conclusions: These results identify the chemokine receptors present on infiltrating lymphocytes during end-stage liver disease and suggest that such infiltration is predominantly controlled by non-tissue-specific inflammatory chemokines, a situation that may be distinct from liver homing pathways under normal conditions.

The large GTPase dynamin is required for hepatitis B virus protein secretion from hepatocytes
Ahmad S. Abdulkarim, Hong Cao, Bing Huang and Mark A. McNiven
Background/Aims: The hepatocellular transport pathways and cellular proteins utilized during the packaging and secretion of hepatitis B virus are poorly understood. In this study, we tested if the large GTPase dynamin, a protein involved in vesicle formation and secretion at the trans-Golgi network in hepatocytes, is also used by hepatitis B virus (HBV) in secreting viral proteins. Methods: Using HepG2.2.15 cells expressing the full-length HBV genome, we tested the effects of wild-type and mutant dynamin on the localization and secretion of two hepatitis B antigens, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Distribution of these two antigens was analyzed morphologically in cells transiently transfected with wild-type or mutant dynamin constructs, whereas secretion of the antigens was measured by testing for antigen levels in the media of transfected cells. Results: Mutant dynamin was found to induce a striking redistribution of HBsAg and HBeAg to a perinuclear compartment, as well as a decrease in the levels of HBsAg and HBeAg present in cell culture media indicating a reduction in viral protein secretion. At the electron microscopy level, cells expressing the mutant dynamin showed a marked accumulation of viral particles in dilated cisternae of an uncharacterized cellular compartment. Conclusions: Intact dynamin function is required for secretion of HBV proteins from hepatocytes through an uncharacterized cellular compartment.

Complete genome sequence of hepatitis B virus (HBV) from a patient with fulminant hepatitis without precore and core promoter mutations: comparison with HBV from a patient with acute hepatitis infected from the same infectious source
Yan Chen et al.
Background/Aims: There is a paucity of information regarding hepatitis B virus (HBV) from patients with fulminant hepatitis (FH) without precore (pre-C, nt 1896) and core promoter (CP, nt 1762, 1764) mutations. Methods: Pre-C and CP mutations were studied in eight patients with FH and 26 patients with acute hepatitis (AH) due to HBV. One patient with FH (FH1) was infected with HBV without these mutations. Interestingly, the sera of the infectious source (IS1) and of a patient with AH (AH1) infected from IS1 were available. Complete HBV genomes from these three patients were analyzed. Results: These mutations were found in seven of eight FH and five of 26 AH (P<0.01). HBV from FH1, IS1 and AH1 belonged to genotype D. Nucleotide difference between FH1 and AH1 was six of 3182 bases (nt 493, 998, 1173, 2928, 3067, and 3078). Two and five substitutions of deduced amino acid sequences were found in the pre-S1 and polymerase regions, respectively. The same nucleotide substitutions at nt 493, 1173, 2928 and 3067 were found in several patients with FH in our laboratory or GenBank. Conclusions: These six nucleotide substitutions of HBV DNA could be candidates of mutations relating to FH.

Long-term prognosis by lamivudine monotherapy for severe acute exacerbation in chronic hepatitis B infection: emergence of YMDD motif mutant and risk of breakthrough hepatitis - an open-cohort studyNorio Akuta et al.
Background/Aims: Comparison of long-term prognosis in patients with chronic hepatitis B treated with lamivudine, with or without severe acute exacerbation (SAE). Methods: In chronic hepatitis B HBeAg-positive patients on lamivudine monotherapy, 21 patients with SAE were retrospectively compared with 63 patients without SAE. Both groups were matched for age and sex. We investigated the efficacy and problems associated with monotherapy with respect to SAE. Results: In SAE and non-SAE, HBeAg seroconversion rates were 21.1 vs. 27.6%, 20.0 vs. 50.0%, and 14.3 vs. 66.7% at 1, 2, and 3 years, respectively. YMDD mutant emerged later in SAE than in non-SAE, but the emergence rates in SAE almost exceeded those of non-SAE from 2 years (rates of about 35%). DNA breakthrough (hepatitis B virus DNA becoming detectable after a period of negativity, accompanied by emergence of YMDD mutant) and breakthrough hepatitis (alanine aminotransferase becoming abnormal after a period of normalization, accompanied by DNA breakthrough) also appeared later in SAE than in non-SAE, but the rates in SAE exceeded those of non-SAE at 3 years. Conclusions: Our results suggest that Japanese genotype C-dominant hepatitis B patients with SAE seem to be at greater risk of re-exacerbation after temporary relief of the initial SAE by long-term lamivudine monotherapy, compared with those without SAE.

Precore and core promoter mutations of hepatitis B virus and hepatitis B e antigen-negative chronic hepatitis B in Korea
Byung Chul Yoo et al.
Background/Aims: The aims of this study were to determine the frequency of precore/core promoter mutations and hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (e-CHB) in Korea. Methods: Patients with chronic hepatitis B virus (HBV) infection were tested for HBeAg, anti-HBe, liver profile and HBV-DNA by a branched DNA (bDNA) assay. Serum HBV-DNA was amplified by a polymerase chain reaction and the precore/core promoter sequence was determined. Results: Among the 413 consecutive HBeAg-negative patients, 19.6% were bDNA-positive. Evidence of liver disease was found in 90.1% of bDNA-positive and 41.7% of bDNA-negative patients. Overall, 17.7% of HBeAg-negative patients had e-CHB. Precore mutation (A1896) was detected in 93.7% of HBeAg-negative bDNA-positive and 93.9% of HBeAg-negative bDNA-negative patients. In 59 HBeAg-positive patients, 78% had wild-type and 22% had a mixture of wild-type and A1896 mutant. Core promoter TA mutation was detected in 89.9% of HBeAg-negative bDNA-positive patients, 89.8% of HBeAg-negative bDNA-negative patients, and 74.6% of HBeAg-positive patients. No correlation was found between the presence of precore/core promoter mutations and HBV-DNA levels or disease severity. Conclusions: In Korean patients infected with HBV genotype C, precore mutation occurred almost invariably along with HBeAg seroconversion and core promoter TA mutation was frequent irrespective of viral replication levels or disease severity.

BRITISH MEDICAL JOURNAL

Table of Contents for 4 January 2003 - Volume 326 - Issue 7379

Alosetron for irritable bowel syndrome
James B D Palmer and Robert H Palmer
BMJ 2003; 326: 51.

EDITORAs the senior doctor at GlaxoWellcome and GlaxoSmithKline who participated in all of the key meetings that resulted in the withdrawal of Lotronex (alosetron) and the subsequent approval for reintroduction, I feel compelled to reply to both the article and editorial on alosetron. Moynihan (1) sees a complex web of intrigue where none existed, at least from the viewpoint of GlaxoWellcome and GlaxoSmithKline.1 Here are the key elements of the story from our position. We had a medical disagreement with the Food and Drug Administration about the aetiology of the serious adverse events occurring in patients taking alosetron. In November 2000 we had several meetings with the administration, which led to a meeting on 28 November where we realised we could not come to agreement on certain key matters. Reluctantly, we decided that we had no viable option but to withdraw the drug voluntarily. Having withdrawn the drug, we had no intention of reintroducing it. But thousands of patients contacted both us and the Food and Drug Administration to emphasise the benefits of the drug. It was these pleas that led GlaxoSmithKline and the Food and Drug Administration to try to find common ground to reintroduce the drug. This culminated in April 2002 in an advisory committee, where a full and transparent review of all available data was undertaken. Over the past 20 months we have worked with the Food and Drug Administration to find a way forward for this drug that will balance safety concerns and practicality in prescribing with the clear desires of patients to have an effective treatment for irritable bowel syndrome. People who were at the advisory committee could not fail to have been moved by testimonies from patients whose lives had been changed by alosetron. The implication that we were somehow in collusion with the Food and Drug Administration is not true. The pharmaceutical industry is highly regulated, and everyone in industry accepts and respects the Food and Drug Administration's authority. The articles by Moynihan and Lièvre (2) have clouded the subject of the reintroduction of alosetron by misrepresenting the facts. I hope this helps clarify what happenedat least from our point of view. Also, let's not forget who is the ultimate beneficiarythe patient with irritable bowel syndrome for whom alosetron is an effective treatment.

1. Moynihan R. Alosetron: a case study in regulatory capture, or a victory for patients' rights? BMJ 2002; 325: 592-595 [Free Full Text]. (14 September.)
2. Lièvre M. Alosetron for irritable bowel syndrome. BMJ 2002; 325: 555-556 [Free Full Text]. (14 September.)

NEW ENGLAND JOURNAL

Table of Contents for 9 January 2003 - Volume 348 - Number 2

Roles of Drinking Pattern and Type of Alcohol Consumed in Coronary Heart Disease in Men
Kenneth J. Mukamal, M.D., M.P.H., Katherine M. Conigrave, M.B., B.S., Ph.D., Murray A. Mittleman, M.D., Dr.P.H., Carlos A. Camargo, Jr., M.D., Dr.P.H., Meir J. Stampfer, M.D., Dr.P.H., Walter C. Willett, M.D., Dr.P.H., and Eric B. Rimm, Sc.D.

Background Although moderate drinking confers a decreased risk of myocardial infarction, the roles of the drinking pattern and type of beverage remain unclear.

Methods We studied the association of alcohol consumption with the risk of myocardial infarction among 38,077 male health professionals who were free of cardiovascular disease and cancer at base line. We assessed the consumption of beer, red wine, white wine, and liquor individually every four years using validated food-frequency questionnaires. We documented cases of nonfatal myocardial infarction and fatal coronary heart disease from 1986 to 1998.

Results During 12 years of follow-up, there were 1418 cases of myocardial infarction. As compared with men who consumed alcohol less than once per week, men who consumed alcohol three to four or five to seven days per week had decreased risks of myocardial infarction (multivariate relative risk, 0.68 [95 percent confidence interval, 0.55 to 0.84] and 0.63 [95 percent confidence interval, 0.54 to 0.74], respectively). The risk was similar among men who consumed less than 10 g of alcohol per drinking day and those who consumed 30 g or more. No single type of beverage conferred additional benefit, nor did consumption with meals. A 12.5-g increase in daily alcohol consumption over a four-year follow-up period was associated with a relative risk of myocardial infarction of 0.78 (95 percent confidence interval, 0.62 to 0.99).

Conclusions Among men, consumption of alcohol at least three to four days per week was inversely associated with the risk of myocardial infarction. Neither the type of beverage nor the proportion consumed with meals substantially altered this association. Men who increased their alcohol consumption by a moderate amount during follow-up had a decreased risk of myocardial infarction.

Table of Contents for 2 January 2003 - Volume 348 - Number 1

Natalizumab for Active Crohn's Disease
S. Ghosh and Others
 
Background In chronic inflammatory conditions such as Crohn's disease, the migration of leukocytes from the circulation into the parenchyma and their activation within inflammatory sites are mediated in part by 4 integrins. Methods We conducted a double-blind, placebo-controlled trial of the 4 integrin­specific humanized monoclonal antibody natalizumab in 248 patients with moderate-to-severe Crohn's disease. Patients were randomly assigned to receive one of four treatments: two infusions of placebo; one infusion of 3 mg of natalizumab per kilogram of body weight, followed by placebo; two infusions of 3 mg of natalizumab per kilogram; or two infusions of 6 mg of natalizumab per kilogram. Infusions were given four weeks apart. Outcomes included changes in scores for the Crohn's Disease Activity Index (higher scores indicate more severe disease), the health-related quality of life, and C-reactive protein levels. Results The group given two infusions of 6 mg of natalizumab per kilogram did not have a significantly higher rate of clinical remission (defined by a score of less than 150 on the Crohn's Disease Activity Index) than the placebo group at week 6 (the prospectively defined primary end point in the efficacy analysis). However, both groups that received two infusions of natalizumab had higher remission rates than the placebo group at multiple time points. Natalizumab also produced a significant improvement in response rates (defined by a reduction of at least 70 points in the score on the Crohn's Disease Activity Index). The highest remission rate was 44 percent and the highest response rate was 71 percent (at week 6 in the group given two infusions of 3 mg per kilogram). Overall, the two infusions of 6 mg of natalizumab per kilogram and of 3 mg per kilogram had similar effects. The quality of life improved in all natalizumab groups; C-reactive protein levels improved in groups receiving two infusions of natalizumab. The rates of adverse events were similar in all four groups. Conclusions Treatment with the selective adhesion-molecule inhibitor natalizumab increased the rates of clinical remission and response, improved the quality of life and C-reactive protein levels, and was well tolerated in patients with active Crohn's disease.

LANCET

Volume 361, Number 9351 04 January 2003

Edrecolomab in the adjuvant treatment of colorectal carcinoma (no free)
Carol Alliot

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