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Revue de Presse du Mois Mois de Janvier 2002

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HEPATOLOGY

January 2002 · Volume 35 · Number 1

Current and novel immunosuppressive therapy for autoimmune hepatitis
Michael A. Heneghan, Ian G. McFarlane

Corticosteroids alone or in conjunction with azathioprine is the treatment of choice in patients with autoimmune hepatitis (AIH) and results in remission induction in over 80% of patients. Sustained response to therapy may result in substantial regression of fibrosis even in advanced cases. The outcome of rapid withdrawal of immunosuppression is disease relapse in many patients. Consequently, the use of 2 mg/kg/d of azathioprine as a sole agent to maintain remission has been widely accepted in clinical practice. Persistent severe laboratory abnormalities or histologic abnormalities such as bridging necrosis or multilobular necrosis are absolute indications for treatment based on controlled clinical trials, but debate exists as to whether all patients with AIH need treatment. Examination of liver tissue remains the best method of evaluating both treatment response and need for treatment in patients who have little biochemical activity. Alternative strategies in patients who have failed to achieve remission on "standard therapy" of corticosteroids with or without azathioprine or patients with drug toxicity include the use of cyclosporine, tacrolimus, or mycophenolate mofetil. Liver transplantation is the treatment of choice in managing decompensated disease. In this review we examine current management strategies of AIH, and evaluate available data pertaining to the use of novel immunosuppressive agents in this condition. (HEPATOLOGY 2002;35:7-13.)

Liver Biology and Pathobiology

Contribution to antimitochondrial antibody production: Cleavage of pyruvate dehydrogenase complex-E2 by apoptosis-related proteases(*Human Study*)
Shuji Matsumura, Judy Van De Water, Hiroto Kita, Ross L. Coppel, Takao Tsuji, Kazuhide Yamamoto, Aftab A. Ansari, M. Eric Gershwin

Patients with PBC produce a directed, specific response to a single immunodominant autoepitope of PDC-E2 within the inner lipoyl domain. In contrast, immunized animals react to multiple epitopes and rarely recognize the inner lipoyl domain. In other autoimmune diseases, apoptosis plays a critical role in antigen presentation; the caspases and granzyme B are the key proteases in the generation of autoepitopes. To determine the specific cleavage pattern of full-length recombinant PDC-E2, we performed in vitro digestion with caspases-3, -6, -8 and granzyme B. The resulting fragments were immunoblotted and probed with an extensive panel of monoclonal anti-PDC-E2 antibodies and sera from patients with PBC. Interestingly, on granzyme B digestion, PDC-E2 lost reactivity, suggesting the destruction of the immunodominant epitope. Because this site contains the major epitope for both B cells and T cells, it suggests that granzyme B is unlikely to be involved in generation of autoepitopes in primary biliar cirrhosis (PBC). In contrast, following treatment with the caspase enzymes, immunoreactive fragments were generated. Indeed, by confocal microscopy, activated caspase-3 is found in the marginal hepatocytes and bile ducts. Moreover, caspase-3 staining was strongest in the small intrahepatic bile ducts, the major site of tissue destruction in PBC. In conclusion, these data suggest that following apoptosis, the caspase family of proteolytic enzymes have the potential to generate immunogenic fragments that contribute to the autoantigen reservoir and the production of antimitochondrial antibodies. These findings are also consistent with the generation of an autoimmune response against an intracellular antigen that evades catabolism during apoptosis. (HEPATOLOGY 2002;35:14-22.)

Oligoclonal expansion of T cell receptor V beta 2 and 3 cells in the livers of mice with graft-versus-host disease
Weiran Chen, Charles D. Howell

The nonsuppurrative destructive cholangitis lesions in the B10.D2 (donor) into BALB/c (host) mouse graft-versus-host disease (GVHD) model are dependent on CD4 T cells that use a T cell receptor- chain variable region (V) repertoire, which is heavily biased toward V2 and V3 usage. We hypothesized that liver V2+ and V3+ CD4 T cells originate from donor mice and recognize BALB/c minor histocompatibility alloantigens and BALB/c endogenous retroviral superantigen-6, respectively. To test this hypothesis, we determined the donor:host chimera status of infiltrating liver lymphocytes and the clonal states of liver V2+ and liver V3+ CD4 cells isolated from GVHD mice. A limited donor TCR V repertoire composed of V1+, 2+, 3+, 4+, 6+, and 8+ cells infiltrated the livers of GVHD mice on day 3. Consistent with a response to immunodominant host minor histocompatibility antigens, we detected oligoclonal liver V2+ T cells in 40% of GVHD mice studied on day 3 and in 100% of GVHD mice studied on day 14. Typical of superantigen stimulation, extremely polyclonal liver V3+ T cells were detected in 100% of GVHD mice studied on day 3 and 40% of GVHD mice studied on day 14. Yet, the liver V3+ T cells in 60% of the day 14 GVHD mice were oligoclonal, pointing to a response to minor histocompatibility antigens. (HEPATOLOGY 2002;35:23-29.)


Adenovirus-mediated increase in HNF-3 or HNF-3 shows differences in levels of liver glycogen and gene expression
Yongjun Tan, Douglas Hughes, Xinhe Wang, Robert H. Costa

We previously generated a transgenic mouse line (T-77) in which increased hepatic expression of the hepatocyte nuclear factor-3 (HNF-3) protein was used to assess its role in hepatocyte-specific gene transcription. The T-77 transgenic mice displayed elevated serum bile acid and bilirubin levels and a complete absence of hepatic glycogen storage. These postnatal liver defects were associated with diminished expression of hepatocyte genes involved in gluconeogenesis and bile acid transport as well as reduced levels of hepatocyte transcription factors. In this study, we show that mouse tail vein injections of adenovirus expressing the rat HNF-3 (AdHNF3) cDNA efficiently increased its levels throughout the liver lobule and recapitulated the T-77 transgenic liver phenotype within several days postinfection. Likewise, the AdHNF3-infected liver phenotype was associated with reduced hepatic expression of genes involved in glucose homeostasis, bile acid transport, and bilirubin conjugation, which were not found with control adenovirus infections. These studies show that adenovirus-mediated gene transfer is an effective method for rapid hepatic increases in transcription factor levels to determine in vivo target genes. In contrast, AdHNF3-infected liver displayed only a transient reduction in hepatic glycogen levels and was associated with less severe decreases in hepatic expression of gluconeogenic and bilirubin metabolism genes. Consistent with these findings, only T-77 transgenic and AdHNF3-infected liver exhibited diminished hepatic expression of the HNF-6 transcription factor, suggesting that reduced HNF-6 levels contribute to diminished HNF-3­specific transcriptional activity. (HEPATOLOGY 2002;35:30-39.)


Interleukin-6 from intrahepatic cells of bone marrow origin is required for normal murine liver regeneration
Xavier Aldeguer, Fotini Debonera, Abraham Shaked, Alyssa M. Krasinkas, Andrew E. Gelman, Xingyi Que, Gideon A. Zamir, Shungo Hiroyasu, Kellen K. Kovalovich, Rebecca Taub, Kim M. Olthoff

Interleukin-6 (IL-6) is required for normal liver regeneration, but the specific cellular source of this growth factor is unknown. We investigated whether this signal originates from the resident macrophage, the Kupffer cell. Using a murine model of bone marrow transplantation, we replaced recipient bone marrow­derived cells, including Kupffer cells, with cells of donor genetic phenotype. Recipients deficient in IL-6 (IL-6­/­) were lethally irradiated, then rescued with 107 donor bone marrow cells capable of expressing IL-6 (IL-6+/+). Conversely, IL-6+/+ recipients received IL-6­/­ marrow. Successful engraftment was measured by the presence of the Y chromosome SRY locus in the livers of female recipients receiving male marrow, in situ IL-6 expression by Kupffer cells, and up-regulation of IL-6 in splenocytes after activation with lipopolysaccharide (LPS). Kupffer cell isolation in IL-6­/­ females receiving IL-6+/+ male marrow clearly showed the presence of the SRY locus and IL-6 disrupted allele, whereas males receiving female marrow demonstrated no SRY or IL-6 signals, confirming the extent of replacement. Replacement of these cells in IL-6­/­ mice with IL-6+/+ bone marrow successfully restored the regenerative response after partial hepatectomy (PHx) as indicated by signal transduction and activator of transcription 3 (STAT3) activation and hepatocyte DNA replication. Alternatively, complete replacement of Kupffer cells in IL-6+/+ mice by transplantation with IL-6­/­ cells significantly inhibited liver regeneration and was partially restored by administration of IL-6. This investigation demonstrates a paracrine mechanism by which cells of bone marrow origin, most likely Kupffer cells, regulate the regenerative capacity of the hepatocyte through IL-6 expression. (HEPATOLOGY 2002;35:40-48.)


Differential regulation of TGF- signal in hepatic stellate cells between acute and chronic rat liver injury
Yoshiya Tahashi, Koichi Matsuzaki, Masataka Date, Katsunori Yoshida, Fukiko Furukawa, Yasushi Sugano, Masanori Matsushita, Yasuo Himeno, Yutaka Inagaki, Kyoichi Inoue

During chronic liver injury, transforming growth factor (TGF-) plays a prominent role in stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells (HSCs). On the other hand, Smad 7 was recently shown to antagonize the TGF-­induced activation of signal-transducing Smads (2 and 3). In this study, we investigated the regulatory mechanisms of the TGF- signals in rat HSCs during acute liver injury and myofibroblasts (MFBs) during chronic liver injury, focusing on the roles of Smad 2 and antagonistic Smad 7. In acute liver injury, HSC-derived TGF- increased plasminogen activator inhibitor type 1 (PAI-1) and 2(I) procollagen (COL1A2) transcripts. Smad 2 in HSCs during liver injury and primary cultured HSCs were activated by an autocrine mechanism, because high levels of Smad 2 phosphorylation and induction of PAI-1 transcript by TGF- were observed in HSCs. Thereafter, Smad 7 induced by TGF- negatively regulated the Smad 2 action. These results indicated that endogenous TGF­mediated Smad 7 in HSCs terminated the fibrotic signals mediated by signal-transducing Smads, and might be involved in the transient response to autocrine TGF- signal after acute liver injury. By contrast, Smad 7 was not induced by the autocrine TGF- signal, and constitutive Smad 2 activation was observed in MFBs throughout chronic liver injury, although Smad 7 could inhibit the TGF- signal requiring Smad 2 phosphorylation by activated TGF- receptor in cultured MFBs. This constitutive phosphorylation of Smad 2 by endogenous TGF- under a low level of Smad 7 could be involved in the progression of liver fibrosis. (HEPATOLOGY 2002;35:49-61.)


Stimulation and proliferation of primary rat hepatic stellate cells by cytochrome P450 2E1­derived reactive oxygen species
Natalia Nieto, Scott L. Friedman, Arthur I. Cederbaum

The alcohol-inducible cytochrome P450 2E1 (CYP2E1) is expressed mainly in hepatocytes and generates reactive oxygen species (ROS). To better understand how hepatic stellate cells (HSC) become activated in the presence of oxidative stress and evaluate whether CYP2E1-derived ROS activate stellate cells, we coincubated primary stellate cells with HepG2 cells, which do (E47 cells) or do not (C34 cells) express CYP2E1. Morphologic changes and loss of lipid droplets were more apparent in the stellate cells cocultured with E47 cells. There was a more pronounced increase in -smooth muscle actin (-sma), intracellular and secreted collagen type I protein, and intra- and extracellular H2O2 and lipid peroxidation products in stellate cells coincubated with E47 cells. Expression of collagen in stellate cells did not change when cocultured with HepG2 cells expressing a different P450, CYP3A4. Stellate cells cultured on Matrigel expressed increased -sma and collagen when incubated with E47 cells. The increase in collagen production by coculture with E47 cells was prevented by antioxidants, by CYP2E1 inhibitors, and by transfected antisense CYP2E1. The addition of arachidonic acid plus ferric nitrilotriacetate (Fe-NTA), agents that potentiate oxidative stress, further induced collagen protein in the E47 coculture. Stellate cell proliferation was greater in the E47 coculture, and this was partially abrogated by catalase and vitamin E. These results show that hepatocytes containing CYP2E1 release diffusible mediators including ROS, which can activate HSC. Thus, besides perturbing the homeostasis of hepatocytes, CYP2E1-derived diffusible oxidants may also interact with stellate cells and contribute to hepatic fibrosis. (HEPATOLOGY 2002;35:62-73.)


-Glutamyl transpeptidase overexpression increases metastatic growth of B16 melanoma cells in the mouse liver
Elena Obrador, Julian Carretero, Angel Ortega, Ignacio Medina, Vicente Rodilla, José A. Pellicer, José M. Estrela

B16 melanoma (B16M) cells with high glutathione (GSH) content show rapid proliferation in vitro and high metastatic activity in the liver in vivo. -Glutamyl transpeptidase (GGT)-mediated extracellular GSH cleavage and intracellular GSH synthesis were studied in vitro in B16M cells with high (F10) and low (F1) metastatic potential. GGT activity was modified by transfection with the human GGT gene (B16MF1/Tet-GGT cells) or by acivicin-induced inhibition. B16MF1/Tet-GGT and B16MF10 cells exhibited higher GSH content (35 ± 6 and 40 ± 5 nmol/106 cells, respectively) and GGT activity (89 ± 9 and 37 ± 7 mU/106 cells, respectively) as compared (P < .05) with B16MF1 cells (10 ± 3 nmol GSH and 4 mU GGT/106 cells). Metastasis (number of foci/100 mm3 of liver) increased in B16MF1 cells pretreated with GSH ester (~3-fold, P < .01), and decreased in B16MF1/Tet-GGT and B16MF10 cells pretreated with the GSH synthesis inhibitor L-buthionine (S,R)-sulphoximine (~5-fold and 2-fold, respectively, P < .01). Liver, kidney, brain, lung, and erythrocyte GSH content in B16MF1/Tet-GGT- or B16MF10-bearing mice decreased as compared with B16MF1- and non­tumor-bearing mice. Organic anion transporting polypeptide 1­independent sinusoidal GSH efflux from hepatocytes increased in B16MF1/Tet-GGT­ or B16MF10-bearing mice (~2-fold, P < .01) as compared with non­tumor-bearing mice. Our results indicate that tumor GGT activity and an intertissue flow of GSH can regulate GSH content of melanoma cells and their metastatic growth in the liver. (HEPATOLOGY 2002;35:74-81.)


Regulation of the -fetoprotein gene by the isoforms of ATBF1 transcription factor in human hepatoma(*Human Study*)
Toshiaki Ninomiya, Koichiro Mihara, Kazuo Fushimi, Yoshitake Hayashi, Tomoko Hashimoto-Tamaoki, Taiki Tamaoki

We investigated mechanisms regulating expression of -fetoprotein (AFP) in 3 human hepatoma cell lines, HuH-7, HepG2, and huH-1, producing high, medium, and low levels of AFP, respectively. The silencer, a negative cis-acting element of the AFP gene, was highly activated in huH-1 and HepG2 to repress AFP enhancer activity by 91%, whereas only 26% repression was observed in HuH-7. To account for the difference in AFP production between HepG2 and huH-1, we investigated the roles of two isoforms of the AT motif-binding factor 1 (ATBF1) transcription factor, ATBF1-A and -B. Cotransfection assays showed that the ATBF1 isoforms regulated the AFP gene differently in HepG2 and huH-1. In huH-1 and HuH-7, both ATBF1 isoforms suppressed strongly enhancer activity and slightly promoter activity. In HepG2, on the other hand, ATBF1-A suppressed the enhancer and promoter activities, but surprisingly, ATBF1-B was found to stimulate enhancer activity while showing no effect on the promoter. Levels of ATBF1-A mRNA were similar in all 3 cell lines, whereas the expression ATBF1-B mRNA varied greatly, with the highest level seen in HepG2 followed by huH-1 and HuH-7. These results suggest that, in HepG2, ATBF1-B may have a dominant negative effect to relieve the transcriptional repression caused by its isoform. In support of this view, we found that the N-terminal region specific to the ATBF1-A molecule possessed transcriptional repressor activity. Thus, the use of the ATBF1 variants as well as the silencer may provide a unique mechanism that contributes to the determination of AFP levels in human hepatoma cell lines. (HEPATOLOGY 2002;35:82-87.)


The atrial natriuretic peptide and cGMP: Novel activators of the heat shock response in rat livers
Alexandra K. Kiemer, Alexander L. Gerbes, Manfred Bilzer, Angelika M. Vollmar

Preischemic treatment with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury of the rat liver via cyclic guanosine monophosphate (cGMP). The attenuated activation of nuclear factor B (NF-B) seems to contribute to this effect. The aim of this study was to determine whether heat shock proteins are involved in these molecular pathways. Livers of male Sprague-Dawley rats were continuously perfused with Krebs-Henseleit (KH) buffer with or without ANP or 8-Br-cGMP. In different experiments livers were perfused with or without ANP for 20 minutes, kept in cold storage solution for 24 hours, and reperfused. Activation of heat shock transcription factor (HSF) (by electrophoretic mobility shift assay), heat shock protein 70 (HSP70), and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), as well as HSP70 (by Western blot) were investigated in freeze-clamped liver samples. During continuous perfusion ANP as well as 8-Br-cGMP activated HSF, HSP70 protein concentrations paralleled HSF-activation. ANP pretreated livers exhibited elevated HSF after 24 hours of ischemia and elevated HSP70 mRNA levels during reperfusion. ANP prevented the marked decrease of HSP70 protein during reperfusion. Coimmunoprecipitation studies showed increased binding of HSP70 to inhibitory factor B (IB) in ANP-treated livers. In conclusion, we showed the cGMP-mediated activation of HSF by ANP, which resulted in elevated HSP70 mRNA and protein concentrations and correlated with enhanced binding of HSP70 to IB. This could be an important mechanism of ANP-mediated prevention of hepatic preservation damage. (HEPATOLOGY 2002;35:88-94.)


Correction of CFTR malfunction and stimulation of Ca2+-activated Cl­ channels restore HCO secretion in cystic fibrosis bile ductular cells(*Human Study*)
Ákos Zsembery, Wolfgang Jessner, Gerlinde Sitter, Carlo Spirlí, Mario Strazzabosco, Jürg Graf

In view of the occurrence of hepatobiliary disorders in cystic fibrosis (CF) this study addresses the role of the cystic fibrosis transmembrane conductance regulator (CFTR) and of Ca2+-activated Cl­ channels in promoting HCO secretion in bile ductular cells. Human cholangiocytes were isolated from control livers and from 1 patient with CF (F508/G542X mutations). Single channel and whole cell currents were analyzed by patch clamp techniques, and HCO secretion was determined by fluorometric analysis of the rate of recovery of intracellular pH following alkaline loading. In control cholangiocytes, both cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) catalytic subunit, activated CFTR Cl­ channels that exhibited a nonrectifying conductance of 8 pS and appeared in clusters. Activation of Cl­ current by cAMP was associated with an increase in the rate of HCO secretion. The basal rate of HCO secretion was lower in CF than in control cholangiocytes. In both control and CF cholangiocytes, raising intracellular Ca2+ concentrations with ionomycin led to a parallel activation of Cl­ current and HCO secretion. Consistent with reports that premature stop codon mutations (class I; e.g., G542X) can be read over by treatment with aminoglycoside antibiotics, exposure of CF cholangiocytes to gentamicin restored activation by cAMP of Cl­ current and HCO secretion. The observation that activation of Ca2+-dependent Cl­ channels can substitute for cystic fibrosis transmembrane conductance regulator (CFTR) in supporting HCO secretion and the efficacy of gentamicin in restoring CFTR function and HCO secretion in class I mutations are of potential clinical interest. (HEPATOLOGY 2002;35:95-104.)

Liver Failure and Liver Disease

Obesity and its effect on survival in patients undergoing orthotopic liver transplantation in the United States (*Human Study*)
Satheesh Nair, Sumita Verma, Paul J. Thuluvath

Studies assessing morbidity and mortality in obese patients undergoing orthotopic liver transplantation (OLT) have produced conflicting results, mainly because of the small sample size. The objective of our study was to determine graft and patient survival in obese adults receiving OLT in the U.S. between 1988 through 1996 using the United Network for Organ Sharing (UNOS) database. Among the 23,675 transplantations performed during the 9-year study period, 18,172 (75%) patients fulfilled the inclusion criteria. Of these, 8,382 (46%) were nonobese (body mass index [BMI] < 25 kg/m2), 5,913 (33%) were overweight (BMI, 25.1-30 kg/m2), 2,611 (14%) were obese (BMI, 30.1-35 kg/m2), 911 (5%) were severely obese (BMI, 35.1-40 kg/m2), and 355 (2%) were morbidly obese (BMI, 40.1-50 kg/m2). The outcome measures assessed were immediate (30-day), 1-, 2-, and 5-year patient survival. Obese groups had a higher proportion of women, a greater prevalence of cryptogenic cirrhosis (P < .05) and diabetes (P < .05), and a higher serum creatinine. Primary graft nonfunction, and immediate, 1-year, and 2-year mortality were significantly higher in the morbidly obese group (P < .05). Five-year mortality was significantly higher both in the severely and morbidly obese subjects (P < .05), mostly as a result of adverse cardiovascular events. Kaplan-Meier survival was significantly lower in morbidly obese patients, and morbid obesity was an independent predictor of mortality. Obesity is associated with a significant increase in long-term mortality, mostly as a result of cardiovascular events. Weight loss should be recommended for all patients awaiting a liver transplantation, especially if their BMI is more than 35 kg/m2. (HEPATOLOGY 2002;35:105-109.)


High frequency of epithelial chimerism in liver transplants demonstrated by microdissection and STR-analysis (*Human Study*)
Wolfram Kleeberger, Thomas Rothämel, Sabine Glöckner, Peer Flemming, Ulrich Lehmann, Hans Kreipe

It has recently been shown that epithelial cells derived from stem cells originating outside the liver are integrated into liver allografts. Whether epithelial intragraft chimerism protects transplants from rejection or chronic transplant dysfunction, and whether it interferes with recurrence of primary liver disease, is not known. Twenty-seven sequential biopsies derived from 9 liver-transplant recipients were studied for chimerism of hepatocytes and cholangiocytes. The target cells were isolated by laser microdissection after cytokeratin immunolabeling and genotyped using DNA analysis of a highly polymorphic short tandem repeat. Irrespective of whether early (up to 4 weeks) or late (more than 12 months) posttransplantation biopsies were studied, cholangiocyte chimerism was almost constantly found in 91% of the samples. No significant differences occurred between samples derived from patients with chronic organ dysfunction (n = 3), recurrent hepatitis (n = 3), or mild, unspecific changes (n = 3). By contrast, hepatocyte chimerism tended to occur later (55% vs. 22%) and appeared to be associated with recurrent hepatitis (67% vs. 27%). In this respect, chronic organ dysfunction did not differ from mild, unspecific changes. While cholangiocyte chimerism represents a constant and early phenomenon in liver transplantations, an enhanced chimeric integration of recipient-derived hepatocytes can be observed in recurrent hepatitis, supporting the concept of an increased recruitment of extrahepatic progenitor cells to the liver in chronic hepatitis. (HEPATOLOGY 2002;35:110-116.)


Accuracy of bile duct changes for the diagnosis of chronic liver allograft rejection: Reliability of the 1999 Banff schema (*Human Study*)
Mylène Sebagh, Karin Blakolmer, Bruno Falissard, Bruno Roche, Jean-Francois Emile, Henri Bismuth, Didier Samuel, Michel Reynès

Chronic rejection (CR) after liver transplantation is thought to be a dynamic and potentially reversible process. The Banff working group has developed recommendations for its histopathologic staging. The 1999 Banff classification of CR (i.e., bile duct dystrophy >50% and/or bile duct loss >20%) was applied to: 1) biopsies from patients retransplanted for CR (N = 19) and pathologies other than CR (N = 21) to evaluate its specificity and sensitivity, especially of the early stage lesions of CR; and 2) biopsies from nonretransplanted patients (N = 21) to evaluate the evolution of CR lesions. Atypical forms of CR were also described. Including an early stage into the definition of CR has resulted in a much higher sensitivity for its diagnosis, as compared with the former classification (i.e., bile duct loss >50%) (89% vs. 33%; P = .0001), while keeping an acceptable specificity (74% vs. 100%; P = .03). In 55% of the nonretransplanted patients, CR lesions were reversible. No histologic feature reliably predicted CR outcome. Transient lobular hepatitis, unrelated to viral infection, and veno-occlusive disease were seen significantly more often in the CR group (P = .04 and P = .03, respectively). We conclude that the application of the 1999 Banff classification is superior to the previous classification for the diagnosis of CR. However, limited information can be drawn regarding the outcome of CR based on histology alone. Transient lobular hepatitis, unrelated to viral infection and veno-occlusive disease, may be an unusual expression of CR. (HEPATOLOGY 2002;35:117-125.)


Genetic association of vitamin D receptor polymorphisms with primary biliary cirrhosis and autoimmune hepatitis (*Human Study*)
Arndt Vogel, Christian P. Strassburg, Michael P. Manns

Autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are immune-mediated chronic inflammatory diseases of the liver of unknown etiology. Genetic factors appear to be involved in the pathogenesis of both diseases. 1,25-Dihydroxyvitamin D3 has been implicated as an immunomodulator, which acts through its own receptor (VDR). Polymorphisms of the VDR have been linked to a variety of autoimmune diseases. In this study VDR polymorphisms were analyzed in 123 patients with AIH, 74 patients with PBC, and 214 controls. VDR polymorphisms were assessed by BsmI, TaqI, ApaI, and Fok endonuclease digestion after specific polymerase chain reaction (PCR) amplification. We found a significant association between the BsmI polymorphisms in PBC patients in comparison with controls (2 = 9.49, P = .009). Furthermore we detected a significant association of the Fok polymorphims in AIH patients in comparison to controls (2 = 9.71, P = .008) indicating a genetic link of VDR polymorphisms to autoimmune liver diseases such as PBC and AIH in German patients. These findings contribute to the knowledge of the complex events determining immunologic tolerance in the liver. Further studies are needed to elucidate the mechanisms by which the vitamin D receptor contributes to the development of autoimmune diseases. (HEPATOLOGY 2002;35:126-131.)


TIPS is a useful long-term derivative therapy for patients with Budd-Chiari syndrome uncontrolled by medical therapy (*Human Study*)
Antonia Perelló, Juan Carlos García-Pagán, Rosa Gilabert, Yanette Suárez, Eduardo Moitinho, Francisco Cervantes, Juan Carlos Reverter, Angels Escorsell, Jaume Bosch, Juan Rodés

Patients with Budd-Chiari syndrome (BCS) may require treatment with portal decompressive surgery or liver transplantation. Transjugular intrahepatic portosystemic shunt (TIPS) represents a new treatment alternative, but its long-term effect on BCS outcome has not been evaluated. Twenty-one patients with BCS consecutively admitted to our unit were evaluated. The mean follow-up was 4 ± 3 years. Seven patients had nonprogressive forms and were successfully controlled with medical therapy; 1 case, with a short-length hepatic vein stenosis was successfully treated by angioplasty. All 8 patients are alive and asymptomatic. The remaining 13 patients, had a TIPS because of clinical deterioration (in one of them, because early TIPS thrombosis a successful side-to-side portacaval shunt [SSPCS] was performed) followed by an improvement in clinical condition. However, a patient with fulminant liver failure before TIPS insertion, died 4 months later and another patient with cirrhosis at diagnosis had liver transplantation 2 years later. The remaining 11 patients are alive and free of ascites. In 3 of these patients TIPS is patent after 3, 6, and 12 months. The remaining 8 patients developed late TIPS dysfunction. In two of these cases, after angioplasty and restenting, TIPS is patent after a follow-up of 9 and 80 months. In 5 other patients, recurring TIPS occlusion was not further corrected because no signs of portal hypertension were present. In conclusion, in patients with BCS uncontrolled with medical therapy, TIPS is a highly effective technique that is associated with long-term survival. (HEPATOLOGY 2002;35:132-139.)


Bacterial infections in cirrhosis: Epidemiological changes with invasive procedures and norfloxacin prophylaxis(*Human Study*)
Javier Fernández, Miquel Navasa, Juliá Gómez, Jordi Colmenero, Jordi Vila, Vicente Arroyo, Juan Rodés

The extensive use of invasive procedures and of long-term norfloxacin prophylaxis in the management of cirrhotic patients may have influenced the epidemiology of bacterial infections in cirrhosis. We conducted a prospective evaluation of all bacterial infections diagnosed in patients with cirrhosis in a Liver Unit between April 1998 and April 2000. A total of 405 patients presented 572 bacterial infections in 507 admissions. Spontaneous bacterial peritonitis was the most frequent infection (138 cases). Gram-positive cocci were responsible for 53% of total bacterial infections in the study, being the main bacteria isolated in nosocomial infections (59%). Patients requiring treatment in an intensive care unit and those submitted to invasive procedures presented a higher rate of infections caused by gram-positive cocci (77% vs. 48%, P < .001 and 58% vs. 40%, P < .02, respectively). Fifty percent of culture-positive spontaneous bacterial peritonitis in patients on long-term norfloxacin administration (n = 93) and 16% in patients not receiving this therapy (n = 414) were caused by quinolone-resistant gram-negative bacilli, P = .01. The rate of culture-positive spontaneous bacterial peritonitis caused by trimethoprim-sulfamethoxazole­resistant gram-negative bacilli was also very high in patients on long-term norfloxacin administration (44% vs. 18%, P = .09). In conclusion, infections caused by gram-positive cocci have markedly increased in cirrhosis. This phenomenon may be related to the current high degree of instrumentation of cirrhotic patients. Quinolone-resistant spontaneous bacterial peritonitis constitutes an emergent problem in patients on long-term norfloxacin prophylaxis, with trimethoprim-sulfamethoxazole not being a valid alternative. (HEPATOLOGY 2002;35:140-148.)


Interleukin-1 receptor type I gene-deficient bile duct­ligated mice are partially protected against endotoxin
Miguel E. Sewnath, Tom Van Der Poll, Fiebo J. W. Ten Kate, Cornelis J. F. Van Noorden, Dirk J. Gouma

Cholestatic liver injury is associated with an increased susceptibility toward endotoxin-induced toxicity. To determine the role of interleukin 1 (IL-1) herein, extrahepatic cholestasis was induced by bile duct ligation (bdl) in IL-1 receptor type I gene-deficient (IL-1R­/­) mice, which are unresponsive to IL-1 and IL-1, and normal IL-1R+/+ mice. Bdl elicited increases in hepatic IL-1 and IL-1 messenger RNA (mRNA) and protein. Hepatocellular injury at 2 weeks after bdl was similar in IL-1R­/­ and IL-1R+/+ mice as shown by clinical chemistry and histopathology. Administration of endotoxin to cholestatic mice at 2 weeks after bdl was associated with enhanced cytokine release, more severe liver damage, and occurrence of death when compared with sham-operated mice. Endotoxin effects in sham-operated IL-1R­/­ and IL-1R+/+ mice were largely similar, but cholestatic IL-1R­/­ mice were better protected against toxic effects of endotoxin, as reflected by lowered cytokine release, less profound liver injury, and reduced mortality. These data indicate that IL-1 and IL-1 are produced in the liver after bdl, but that these cytokines do not play a significant role in cholestatic liver damage; however, endogenous IL-1 activity is an important denominator of enhanced endotoxin sensitivity that is observed during cholestasis induced by bdl. (HEPATOLOGY 2002;35:149-158.)


Hyperdynamic circulation in portal-hypertensive rats is dependent on central c-fos gene expression
Daisheng Song, Hongqun Liu, Keith A. Sharkey, Samuel S. Lee

Portal hypertension is associated with hyperdynamic circulation, but the pathogenesis remains unclear. To clarify the role of central cardiovascular regulatory mechanisms, several protocols were conducted in rats with portal hypertension due to portal vein stenosis (PVS). Neuronal activation was quantified by immunohistochemical staining for Fos, the protein product of the c-fos gene. Fos expression in several brain nuclei with cardiovascular-regulatory roles was examined at 1, 3, 5, and 10 days following PVS surgery. This was correlated with development of cardiovascular changes measured at the same time points. Finally, Fos expression in the nucleus tractus solitarius (NTS) was blocked by local microinjection of c-fos antisense oligonucleotides twice daily for 5 days following PVS. The results showed that Fos-positive neurons were significantly increased in the paraventricular nucleus of hypothalamus, supraoptic nucleus, ventrolateral medulla, and NTS, detectable at day 1 and persistently increased at every day examined in the PVS rats. However, the hyperdynamic circulation developed between days 3 to 5. Administration of c-fos antisense oligonucleotides eliminated the hyperdynamic circulation in PVS rats, but had no effect on sham-operated controls. We conclude that the activation of central cardiovascular-regulatory nuclei, through a c-fos­dependent pathway, is necessary for development of hyperdynamic circulation in portal-hypertensive rats. (HEPATOLOGY 2002;35:159-166.)


Decreases in portal flow trigger a hepatorenal reflex to inhibit renal sodium and water excretion in rats: Role of adenosine
Zhi Ming, Donald D. Smyth, W. Wayne Lautt

The regulation of renal sodium and water excretion through a hepatorenal reflex activated by the changes in hemodynamics of the portal circulation has been suggested. We hypothesize that the changes in intrahepatic blood flow and flow-related intrahepatic adenosine are involved in the control of renal water and sodium excretion by triggering a hepatorenal reflex. Anesthetized rats were instrumented to monitor the systemic, hepatic, and renal circulation. A vascular shunt connecting the portal vein and central vena cava was established to allow for control of the portal venous blood flow (PVBF). Urine was collected from the bladder. The effects of decreased PVBF on renal water and sodium excretion were compared in normal and hepatic denervated rats. Decreasing intrahepatic PVBF by half for 30 minutes decreased urine flow by 38% (12.1 ± 1.1 vs. 7.5 ± 0.7 µL · min­1) and urine sodium excretion by 44% (1.11 ± 0.30 vs. 0.62 ± 0.17 µmol · min­1). Renal arterial blood flow (RABF) and creatinine clearance were also reduced by the decreases in intrahepatic PVBF. Hepatic denervation, or intrahepatic administration of an adenosine receptor antagonist, 8-phenyltheophylline (8-PT), abolished the effects of decreasing PVBF on urine flow and sodium excretion. The data suggest that the decrease in intrahepatic PVBF triggers a hepatorenal reflex through the activation of adenosine receptors within the liver, thereby inhibiting renal water and sodium excretion. The water and sodium retention commonly seen in the hepatorenal syndrome may be related to intrahepatic adenosine accumulation resulting from the associated decrease in intrahepatic portal flow. (HEPATOLOGY 2002;35:167-175.)

Viral Hepatitis

Failure of a reinforced triple course of hepatitis B vaccination in patients transplanted for HBV-related cirrhosis(*Human Study*)
Mario Angelico, Daniele Di Paolo, Massimo O. Trinito, Alessandra Petrolati, Antonio Araco, Settimio Zazza, Raffaella Lionetti, Carlo U. Casciani, Giuseppe Tisone

Long-term immunoprophylaxis with anti-HBs immunoglobulins (HBIg) is used to prevent hepatitis B (HBV) reinfection after liver transplantation for HBV-related cirrhosis. This approach is highly expensive. A recent report proposed posttransplant HBV vaccination with a reinforced schedule as an alternative strategy to allow HBIg discontinuation. We investigated the efficacy of a reinforced triple course of HBV vaccination in 17 patients transplanted for HBsAg-positive cirrhosis 2 to 7 years earlier. The first cycle consisted of 3 double intramuscular doses (40 µg) of recombinant vaccine at month 0, 1, and 2, respectively. This was followed, in nonresponders, by a second cycle of 6 intradermal 10 µg doses every 15 days. All nonresponders then received a third cycle identical to the first one. Vaccination started 4.5 months after HBIg discontinuation, and lamivudine (100 mg/day) was given throughout the study. All patients were seronegative for HBsAg and HBV-DNA (by PCR) and positive for anti-HBe, and 7 were positive for anti-HDV. After the first cycle one patient (#5, 53 years old, male) developed an anti-HBs titer of 154 IU/L, another (#12) reached a titer of 20 IU/L and the remainder had titers <10 IU/L. At month 7, patient #5 reached a titer of 687 IU/L. After the second cycle only one additional patient (#9) had a slight response (an anti-HBs titer of 37 IU/L). After the third cycle patient #9 rose to an anti-HBs titer of 280 IU/L, patient #12 dropped to 10 IU/L, and no other patient responded. In conclusion, a highly reinforced HBV vaccination program is effective only in a few patients who had liver transplants for HBV-related cirrhosis. (HEPATOLOGY 2002;35:176-181.)


Hepatotoxicity associated with nevirapine or efavirenz-containing antiretroviral therapy: Role of hepatitis C and B infections (*Human Study*)
Mark S. Sulkowski, David L. Thomas, Shruti H. Mehta, Richard E. Chaisson, Richard D. Moore

Hepatologists are frequently asked to evaluate human immunodeficiency virus (HIV)-infected patients with abnormal liver enzymes and to assess the causal role of medications, such as antiretroviral drugs. Recently, the use of HIV-1 specific non-nucleoside reverse transcriptase inhibitors (NNRTIs), including nevirapine (NVP) and efavirenz (EFV), has been associated with severe hepatic injury. We prospectively studied the incidence of severe hepatotoxicity (grade 3 or 4 change in alanine or aspartate transaminase levels) among 568 patients receiving NNRTI-containing antiretroviral therapy, including 312 and 256 patients prescribed EFV and NVP, respectively. Hepatitis C virus (HCV) and hepatitis B virus (HBV) were detected in 43% and 7.7% of patients, respectively. Severe hepatotoxicity was observed in 15.6% of patients prescribed NVP and 8.0% of those prescribed EFV, but only 32% of NVP and 50% of EFV-associated episodes were detected during the first 12-weeks of therapy. The risk was significantly greater among persons with chronic viral hepatitis (69% of cases) and those prescribed concurrent protease inhibitors (PIs) (82% of cases). Nonetheless, 84% of patients with chronic HCV or HBV did not experience severe hepatotoxicity. Severe hepatotoxicity occurs throughout the course of NNRTI therapy and is more common among patients prescribed nevirapine, those coinfected with HCV or HBV, and those coadministered protease inhibitors. (HEPATOLOGY 2002;35:182-189.)


Frequencies of HCV-specific effector CD4+ T cells by flow cytometry: Correlation with clinical disease stages (*Human Study*)
Hugo R. Rosen, Camette Miner, Anna W. Sasaki, David M. Lewinsohn, Andrew J. Conrad, Antony Bakke, H. G. Archie Bouwer, David J. Hinrichs

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, affecting approximately 2% of the world's population. The immune mechanisms responsible for the highly variable natural history in a given individual are unknown. We used a multiparameter flow cytometric technique to functionally and phenotypically characterize HCV-specific effector T cells in the peripheral blood of 32 individuals with different stages of hepatitis C disease (resolved, mild chronic, advanced chronic) and normal controls. We found the highest frequencies of virus-specific effector cells with an activated memory phenotype (CD45RO+CD69+) in subjects who had resolved HCV infection, either spontaneously or with antiviral therapy. Effector cells from patients with resolved infection produced Th1 type cytokines following stimulation with nonstructural antigens (NS3 and NS4), whereas effector cells from chronically infected patients produced Th1 type cytokines predominantly following stimulation with the HCV core antigen. Stimulation with superantigen staphylococcal enterotoxin (SEB) induced the same levels of cytokine production in the different patient groups. Among the HCV-seropositive patients, viral load inversely correlated with the Th1 effector cell response to NS3. Interleukin (IL)-4 was produced only in response to the control antigens, but not in response to the HCV recombinant proteins. Taken together, these findings suggest that a vigorous HCV-specific CD4+ Th1 response, particularly against the nonstructural proteins of the virus, may be associated with viral clearance and protection from disease progression. Prospective studies using this new flow cytometric assay will be required to determine whether antiviral therapy modifies the frequency, specificity, and function of these virus-specific effector cells. (HEPATOLOGY 2002;35:190-198.)


Inhibition of internal ribosomal entry site­directed translation of HCV by recombinant IFN- correlates with a reduced La protein (*Human Study*)
Takeo Shimazaki, Masao Honda, Shuichi Kaneko, Kenichi Kobayashi

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5'-nontranslated region (5'NTR). We investigated the effect of interferon alfa (IFN-) on the IRES-directed translation of HCV, using two stably transformed cell lines, RCF-1 and RCF-26, of Huh7 cells derived from human hepatocellular carcinoma that express dicistronic reporter proteins, Renilla luciferase (RL) and firefly luciferase (FL), separated by HCV-IRES. After the administration of IFN- or poly(I)-poly(C), HCV-IRES­directed translation was inhibited in a dose-dependent manner. The relative HCV-IRES activity (F/L) decreased to 60% at 5,000 IU/mL of IFN- and 45% at 40 µg/mL of poly(I)-poly(C). Thus, IFN- or poly(I)-poly(C) inhibited HCV-IRES­directed translation more efficiently than a cellular cap­dependent translation. 2',5'­oligoadenylate synthetase (2',5'AS) protein level in cells analyzed significantly increased after the administration of IFN-, but not upon poly(I)-poly(C). Overexpression of double-stranded RNA-activated protein kinase (PKR) gene did not mimic the selective inhibition of HCV-IRES­directed translation in the transformant cells, suggesting that neither the 2',5'AS nor the PKR system are involved in this selective inhibition. Interestingly, the expression of the autoantigen, La, which has been reported to enhance HCV-IRES­directed translation, was significantly reduced after the administration of IFN- and poly(I)-poly(C) in a dose-dependent manner. Transient expression of La protein completely restored the selective inhibition of HCV-IRES­directed translation by IFN- and poly(I)-poly(C). These findings suggested a new antiviral mechanism induced by IFN- in that IFN- or poly(I)-poly(C) selectively inhibited HCV-IRES­directed translation compared with the eukaryotic cap-dependent translation through the reduction of La protein. (HEPATOLOGY 2002;35:199-208.)


Cis-preferential recruitment of duck hepatitis B virus core protein to the RNA/polymerase preassembly complex
Fritz von Weizsäcker, Josef Köck, Stefan Wieland, Jürgen Beck, Michael Nassal, Hubert E. Blum

Hepadnaviral replication requires the concerted action of the polymerase and core proteins to ensure selective packaging of the RNA pregenome into nucleocapsids. Virus assembly is initiated by cis-preferential binding of polymerase to the encapsidation signal , present on pregenomic RNA. Using the duck hepatitis B virus (DHBV) model, we analyzed how core protein is recruited to the RNA/polymerase preassembly complex. Two sets of trans-complementation assays were performed in cotransfected hepatoma cells. First, a replication-competent DHBV construct was tested for its ability to rescue replication of genomes bearing mutations within the core region. Self-packaging of wild-type pregenomes was more efficient than cross-packaging of core-deficient pregenomes, and this bias was strongly enhanced if mutant pregenomes coded for self-assembly­competent, but packaging-deficient, core proteins. Second, the site of wild-type core protein translation, i.e., pregenomic RNA (cis) or separate messenger RNA (trans), was analyzed for its effect on the phenotype of a previously described dominant-negative (DN) DHBV core protein mutant. This mutant forms chimeric nucleocapsids with wild-type core proteins and blocks reverse transcription within most, but not all, mixed particles. Strikingly, suppression of viral DNA synthesis by the mutant increased 100-fold when wild-type core protein was provided in trans. Our results suggest that recruitment of core protein to the DHBV preassembly complex occurs in a cis-preferential manner. This mechanism may account for the leakiness of DN DHBV core protein mutants targeting reverse transcription. (HEPATOLOGY 2002;35:209-216.)


Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation
Maura Dandri, Martin R. Burda, Alexander Bürkle, David M. Zuckerman, Hans Will, Charles E. Rogler, Heimer Greten, Joerg Petersen

Chronic infection with hepatitis B virus (HBV) is associated with an increased risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Although clonal HBV DNA integrations are detected in nearly all HCCs the role of these integrations in hepatocarcinogenesis is poorly understood. We have used a cloning protocol that allows studying the frequency and the natural history of HBV DNA integrations in cell culture. Southern blot analysis of the genomic DNA of HepG2 2.2.15 subclones, which replicate HBV, enabled us to detect new HBV DNA integrations in approximately 10% of the HepG 2.2.15 subclones over 4 rounds of sequential subcloning, whereas no loss of any preexisting HBV DNA integrations was observed. Treatments of HepG2 cells with H2O2, designed to increase DNA damage, increased the frequency of HBV integrations to approximately 50% of the subclones and treatments designed to inhibit DNA repair, by inhibiting Poly(ADP-ribosyl)ation, also increased the frequency of HBV integration to 50%. These findings suggest that DNA strand breaks induced by oxidative stress during persistent HBV infection in humans may increase HBV DNA integration events, whereas PARP-1 activity may function to limit the occurrence of de novo HBV DNA integrations. (HEPATOLOGY 2002;35:217-223.)

GASTROENTEROLOGY

1 January 2002; Vol. 122, No. 1

RAPID COMMUNICATIONS

Inhibition of Stress-Activated MAP Kinases Induces Clinical Improvement in Moderate to Severe Crohn's Disease
DAAN HOMMES, BERNT VAN DEN BLINK, TERRY PLASSE, JOEP BARTELSMAN, CUIPING XU, BRET MACPHERSON, GUIDO TYTGAT, MAIKEL PEPPELENBOSCH, and SANDER VAN DEVENTER

Background & Aims: We investigated if inhibition of mitogen-activated protein kinases (MAPKs) was beneficial in Crohn's disease. Methods: Inhibition of JNK and p38 MAPK activation with CNI-1493, a guanylhydrazone, was tested in vitro. Twelve patients with severe Crohn's disease (mean baseline, CDAI 380) were randomly assigned to receive either 8 or 25 mg/m2 CNI-1493 daily for 12 days. Clinical endpoints included safety, Crohn's Disease Activity Index (CDAI), Inflammatory Bowel Disease Questionnaire, and the Crohn's Disease Endoscopic Index of Severity. Results: Colonic biopsies displayed enhanced JNK and p38 MAPK activation. CNI-1493 inhibition of both JNK and p38 phosphorylation was observed in vitro. Treatment resulted in diminished JNK phosphorylation and tumor necrosis factor production as well as significant clinical benefit and rapid endoscopic ulcer healing. No serious adverse events were noted. A CDAI decrease of 120 at week 4 (P = 0.005) and 146.5 at week 8 (P = 0.005) was observed. A clinical response was seen in 67% of patients at 4 weeks and 58% at 8 weeks. Clinical remission was observed in 25% of patients at week 4 and 42% at week 8. Endoscopic improvement occurred in all but 1 patient. Response was seen in 3 of 6 infliximab failures, 2 of whom showed remission. Fistulae healing occurred in 4 of 5 patients, and steroids were tapered in 89% of patients. Conclusions: Inflammatory MAPKs are critically involved in the pathogenesis of Crohn's disease and their inhibition provides a novel therapeutic strategy.

Gastroenterology 2001;122 7-14, Published online 17 December 2001

CLINICAL RESEARCH

Ulcerative Colitis: Female Fecundity Before Diagnosis, During Disease, and After Surgery Compared With a Population Sample
KASPER ORDING OLSEN, SVEND JUUL, INA BERNDTSSON, TOM ORESLAND, and SOREN LAURBERG

Background & Aims: Women with ulcerative colitis generally have normal fertility. The aim of this study was to compare patients' fecundability before and after restorative proctocolectomy with ileal pouch-anal anastomosis with the fecundability of the general population. Methods: Historical follow-up was performed on 343 consecutive female patients aged 10.6-40.5 years at surgery and a reference population of 1200 women aged 25-40 years. A total of 290 (85%) patients and 661 (55%) women in the reference population agreed to participate in a structured telephone interview concerning reproductive behavior and waiting times to pregnancy. Cox regression and Kaplan-Meier plots were used for analysis. Results: Surgery significantly reduced the ratio of patient to reference population fecundability, which decreased to 0.20 (P < 0.0001). Before diagnosis and from diagnosis until colectomy, the fecundability of the patients was similar to that of the reference population. Conclusions: Female patients with ulcerative colitis have normal fecundity before surgical treatment. Surgery severely reduces female fecundity. Information about this reduction in fecundity should be given before surgery, and if a woman has an unfulfilled wish for pregnancy after surgery, early referral to a gynecologist is recommended.

Gastroenterology 2002;122 15-19, Published online 7 January 2002

Budesonide in Collagenous Colitis: A Double-Blind Placebo-Controlled Trial With Histologic Follow-Up
FILIP BAERT, ALAIN SCHMIT, GEERT D'HAENS, FRANCESKA DEDEURWAERDERE, EDOUARD LOUIS, MARC CABOOTER, MARTINE DE VOS, FERNAND FONTAINE, SERGE NAEGELS, PIET SCHURMANS, HEDWIG STALS, KAREL GEBOES, and PAUL RUTGEERTS

Background & Aims: Collagenous colitis (CC) is a well-described entity causing chronic diarrhea and characteristic histologic findings. Several treatment options have been suggested, but no controlled data are available. We conducted a placebo-controlled trial to show the clinical and histologic effects of budesonide in CC. Methods: Twenty-eight patients were randomly assigned to receive placebo (n = 14) or budesonide 9 mg daily (n = 14) for 8 weeks. Patients were evaluated clinically, and blinded biopsy specimens were analyzed from fixed locations at weeks 0 and 8. Clinical response was defined as a decrease of at least 50% in the disease activity score (number of bowel movements in the last 7 days). At week 8, nonresponders received open-label budesonide for the next 8-week period; responders discontinued treatment and were followed up. Results: Three patients discontinued the study prematurely. Intention-to-treat analysis showed clinical response in 8 of 14 patients in the budesonide group compared with 3 of 14 responders for placebo (P = 0.05) after 8 weeks of blinded therapy, together with improved stool consistency. Histologically, there was no change in the mean thickness of the collagen band but a significant decrease of the lamina propria infiltrate in the budesonide group (P < 0.001). Conclusions: Budesonide is efficacious in inducing short-term clinical response in CC with significant reduction of the histologic infiltrate in the lamina propria.

Gastroenterology 2002;122 20-25, Published online 7 January 2002

Preoperative Prevalence of Barrett's Esophagus in Esophageal Adenocarcinoma: A Systematic Review
GARETH S. DULAI, SUSHOVAN GUHA, KATHERINE L. KAHN, JEFFREY GORNBEIN, and WILFRED M. WEINSTEIN

Background & Aims: The public health impact of past screening and surveillance practices on the outcomes of Barrett's related cancers has not previously been quantified. Our purpose was to determine the prior prevalence of Barrett's esophagus in reported cases of incident adenocarcinoma undergoing resection, as an indirect measure of impact. Methods: We performed a systematic review of the literature from 1966 to 2000. Studies were included if they reported: (1) the number of consecutive adenocarcinomas resected, and (2) the number of those resected who had a previously known diagnosis of Barrett's. We generated summary estimates using a random effects model. Results: We identified and reviewed 752 studies. Twelve studies representing a total of 1503 unique cases of resected adenocarcinomas met inclusion criteria. Using a random effects model, the overall percentage of patients undergoing resection who had a prior diagnosis of Barrett's was 4.7% ± 2.9%. Conclusions: The low prior prevalence (~5%) of Barrett's esophagus in this study population provides indirect evidence to suggest that recent efforts to identify patients with Barrett'swhether through endoscopic screening or evaluation of symptomatic patientshave had minimal public health impact on esophageal adenocarcinoma outcomes. The potential benefits of endoscopic surveillance seem to have been limited to only a fraction of those individuals at risk. These data thus provide a clear and compelling rationale for the development of effective screening strategies to identify patients with Barrett's esophagus.

Gastroenterology 2002;122 26-33, Published online 7 January 2002

Diagnosis and Patient Management of Intraductal Papillary-Mucinous Tumor of the Pancreas by Using Peroral Pancreatoscopy and Intraductal Ultrasonography
TARO HARA, TAKETO YAMAGUCHI, TAKESHI ISHIHARA, TOSHIO TSUYUGUCHI, FUKUO KONDO, KAZUKI KATO, TAKEHIDE ASANO, and HIROMITSU SAISHO

Background & Aims: Intraductal papillary-mucinous tumor (IPMT) of the pancreas has attracted increasing interest because of its unique presentation. The differential diagnosis between malignant and benign tumors is extremely important in the determination of the therapy for IPMT. The aims of this study are to determine the usefulness of peroral pancreatoscopy (POPS) and intraductal ultrasonography (IDUS) in IPMT for the differentiation of malignant from benign disease, and to evaluate the significance of these techniques as new preoperative examinations. Methods: Sixty histopathologically confirmed patients with IPMT underwent POPS and/or IDUS preoperatively. POPS was perfomed in all patients, and IDUS in 40. Findings of POPS and IDUS were compared with histopathology of resected specimens. The postoperative follow-up data were analyzed. Results: Protruding lesions were detected by POPS in 40 patients. They were classified into 5 groups. Fish-egg-like type with vascular images, villous type, and vegetative type were considered to be malignant. By IDUS, lesions protruding 1 mm or more were observed in 36 patients. Of the lesions protruding 4 mm or more, 88% were malignant. Combination of POPS and IDUS improved the differential diagnosis between benign and malignant IPMT. The 3-year cumulative survival rate and disease-free survival rate were extremely high at 95% and 93%, respectively. Conclusions: The combination of POPS and IDUS results in a considerably improved differential diagnosis between malignant and benign IPMT and is useful for determining an effective therapeutic approach. These new techniques can contribute to improvements in postoperative results.

Gastroenterology 2002;122 34-43, Published online 7 January 2002

Mucosal Flora in Inflammatory Bowel Disease
ALEXANDER SWIDSINSKI, AXEL LADHOFF, ANNELIE PERNTHALER, SONJA SWIDSINSKI, VERA LOENING-BAUCKE, MARIANNE ORTNER, JUTTA WEBER, UWE HOFFMANN, STEFAN SCHREIBER, MANFRED DIETEL, and HERBERT LOCHS

Background & Aims: Microorganisms that directly interact with the intestinal mucosa are obscured by fecal flora and poorly characterized. Methods: We investigated the mucosal flora of washed colonoscopic biopsies of 305 patients with bowel inflammation and 40 controls. The microbial cultures were validated by quantitative polymerase chain reaction with subsequent cloning and sequencing, fluorescence in-situ hybridization, and electron microscopy. Results: We found high concentrations of mucosal bacteria in patients with bowel inflammation, but not in controls. The concentrations of mucosal bacteria increased progressively with the severity of disease, both in inflamed and non-inflamed colon. In patients with >10,000 cfu/µL, a thick bacterial band was attached to the intact mucosa without signs of translocation. Patients with inflammatory bowel disease (IBD) and concentrations of mucosal bacteria >50,000 cfu/µL had characteristic inclusions of multiple polymorphic bacteria within solitary enterocytes located next to the lamina propria, without or having no contact with the fecal stream. The identified bacteria were of fecal origin. Conclusions: Our findings suggest that the changes in the mucosal flora in IBD are not secondary to inflammation, but a result of a specific host response. We hypothesize that the healthy mucosa is capable of holding back fecal bacteria and that this function is profoundly disturbed in patients with IBD.

Gastroenterology 2002;122 44-54, Published online 7 January 2002

Gastroesophageal Reflux Disease in Monozygotic and Dizygotic Twins
ALAN J. CAMERON, JESPER LAGERGREN, CHRISTER HENRIKSSON, OLOF NYREN, G.RICHARD LOCKE, III, and NANCY L. PEDERSEN

Background & Aims: Gastroesophageal reflux disease (GERD) interferes with the quality of life and carries an increased risk for esophageal adenocarcinoma. We investigated genetic influence in the development of reflux. Methods: We compared concordance for reflux in monozygotic (MZ) and dizygotic (DZ) twins. All twins age 55 and older in the nationwide Swedish Twin Registry were invited to participate. Data were collected by computer-assisted telephone interviews. Reflux disease was defined by symptomatic heartburn or acid regurgitation occurring at least weekly. Results: A total of 2178 monozygotic, 3219 same-sex dizygotic, and 3014 unlike-sex dizygotic twin pairs provided information. Overall, 15.3% of the twins had reflux. In men, the intraclass correlation for reflux was 0.29 (95% confidence interval [CI], 0.15-0.43) for monozygotic and 0.13 (95% CI, 0.02-0.25) for dizygotic pairs. In women, the correlation was 0.33 (95% CI, 0.22-0.44) for monozygotic and 0.14 (95% CI, 0.04-0.24) for dizygotic pairs. For unlike-sex dizygotic pairs, the correlation was 0.06 (95% CI, 0.01 to 0.14). Concordance for reflux was not caused by inherited obesity or alcohol use; inherited smoking may be a minor factor. Conclusions: The increased concordance for reflux in monozygotic pairs, compared with dizygotic pairs, indicates genetic rather than shared environmental effects. Heritability accounted for 31% (23%-39%) of the liability to reflux disease in this population.

Gastroenterology 2002;122 55-59, Published online 7 January 2002

Oncogenic beta -Catenin and MMP-7 (Matrilysin) Cosegregate in Late-Stage Clinical Colon Cancer
ANDREI V. OUGOLKOV, KANAME YAMASHITA, MASAYOSHI MAI, and TOSHINARI MINAMOTO

Background & Aims: Recent in vitro studies showed that -catenin translocated into the tumor cell nucleus functions as an oncogene by transactivating oncogenes, including MMP-7. We conducted a large-scale analysis of -catenin and MMP-7 expression in human colon cancer to determine the potential clinical importance of these molecules. Methods: In 202 colon cancer patients with known postoperative outcomes, we determined the expression of -catenin and MMP-7 in the tumors immunohistochemically and correlated the findings with the patients' clinicopathological characteristics and survival. Results: We found 2 distinct patterns of -catenin nuclear accumulation (NA) in the colon cancers: diffuse NA (NAd) in 89 cases (44%) and selective NA at the invasion front (NAinv) in 18 cases (9%). The presence of the NAinv pattern was significantly correlated with advanced Dukes' stage (P = 0.0187) and tumor recurrence (P = 0.0005) as well as with MMP-7 expression in the tumor invasion front (P = 0.0025), resulting in extremely unfavorable clinical outcomes. A multivariate analysis determined that the NAinv expression pattern and Dukes' C stage were independent prognostic factors. Conclusions: Oncogenic activation of -catenin in the tumor invasion front, as represented by its NAinv pattern of expression, may be an independent and reliable indicator of membership in a subset of colon cancer patients who are highly susceptible to tumor recurrence and have a less favorable survival rate.

Gastroenterology 2002;122 60-71, Published online 7 January 2002

Epstein-Barr Virus-Positive Lymphoma in Patients With Inflammatory Bowel Disease Treated With Azathioprine or 6-Mercaptopurine
GERALD A. DAYHARSH, EDWARD V. LOFTUS, Jr., WILLIAM J. SANDBORN, WILLIAM J. TREMAINE, ALAN R. ZINSMEISTER, THOMAS E. WITZIG, WILLIAM R.MACON, and LAWRENCE J. BURGART

Background & Aims: The use of azathioprine and 6-mercaptopurine for inflammatory bowel disease increased in the early 1990s. We sought to determine the effect of this change in therapy on the risk of lymphoma in patients with inflammatory bowel disease. Methods: All patients with inflammatory bowel disease at a single tertiary care medical center who developed lymphoma between 1985-2000 were identified and the pathologic features of the lymphoma including presence of Epstein- Barr virus were determined. The patients were divided into two 8-year periods (1985-1992, 1993-2000) corresponding with the introduction of azathioprine and 6-mercaptopurine in 1993. Results: Eighteen patients with lymphoma were identified, 6 between 1985-1992 and 12 between 1993-2000. Six of 18 lymphomas occurred in patients treated with azathioprine or 6-mercaptopurine, all between 1993-2000. Seven patients developed Epstein-Barr virus-positive lymphoma (1 from 1985-1992, 6 from 1993-2000). Five of 7 Epstein-Barr virus-positive lymphomas occurred in patients treated with azathioprine or 6-mercaptopurine compared with 1 of 11 Epstein-Barr virus-negative lymphomas (P = 0.01). Approximately 1200 patients with inflammatory bowel disease were treated with these agents between 1993-2000. Conclusions: Treatment of inflammatory bowel disease with azathioprine or 6-mercaptopurine appears to be associated with a small increased risk of Epstein-Barr virus-positive lymphoma.

Gastroenterology 2002;122 72-77, Published online 7 January 2002

Chemoprevention of Colorectal Cancer by Aspirin: A Cost-effectiveness Analysis
SAUD SULEIMAN, DOUGLAS K. REX, and AMNON SONNENBERG

Background & Aims: The aim of the study is to compare the cost-effectiveness of aspirin and colonoscopy in the prevention of colorectal cancer. Methods: A Markov process is used to follow a hypothetical cohort of 100,000 subjects aged 50 years until death. Four strategies are compared: (1) no intervention, (2) colonoscopy once per 10 years and every 3 years in subjects with polyps, (3) chemoprevention with 325 mg of daily aspirin, and (4) combination of the second and third strategies. The various strategies are compared calculating incremental cost-effectiveness ratios (ICERs). Results: The expected number of colorectal cancers is 5904 per 100,000 subjects. Colonoscopy prevents 4428 colorectal cancers and saves 7951 life-years at an ICER of $10,983 per life-year saved compared with no intervention. Aspirin prevents 2952 colorectal cancers and saves 5301 life-years at an ICER of $47,249 per life-year saved compared with no intervention. The cost of aspirin therapy plus management of aspirin-related complications was reported to be $172 per year per patient. Varying the annual aspirin-related costs between $50 and $200 results in ICER changes between $4617 and $57,080, with the 2 strategies breaking even at $70. Applying aspirin chemoprevention plus colonoscopy screening concomitantly yields an ICER of $227,607 per life-year saved compared with screening colonoscopy alone. Conclusion: As compared with colonoscopy once per 10 years, the use of aspirin to prevent colorectal cancer saves fewer lives at higher costs. The high complication cost and the lower efficacy of aspirin render screening colonoscopy a more cost-effective strategy to prevent colorectal cancer.

Gastroenterology 2002;122 78-84, Published online 7 January 2002

BASIC RESEARCH

Endogenous Cannabinoids: A New System Involved in the Homeostasis of Arterial Pressure in Experimental Cirrhosis in the Rat
JOSEFA ROS, JOAN CLARIA, JORDI TO-FIGUERAS, ANNA PLANAGUMA, PILAR CEJUDO-MARTIN, GUILLERMO FERNANDEZ-VARO, RAUL MARTIN-RUIZ, VICENTE ARROYO, FRANCISCA RIVERA, JUAN RODES, and WLADIMIRO JIMENEZ

Background & Aims: Recent studies have described the existence of endogenous cannabinoids with vasodilator activity because of their interaction with peripheral CB1 receptors, anandamide being the most extensively investigated. The study investigated whether endogenous cannabinoids are involved in the pathogenesis of the cardiovascular disturbances in experimental cirrhosis. Methods: Arterial pressure, cardiac output, and total peripheral resistance were measured before and after the administration of a cannabinoid CB1 receptor antagonist to cirrhotic rats with ascites and to control rats. Blood pressure was also assessed in normotensive recipient rats after the intravenous administration of blood cells or isolated monocytes obtained from cirrhotic and control rats. Moreover, the endogenous content of anandamide was measured in circulating monocytes of cirrhotic and control rats by gas chromatography/mass spectrometry. Results: CB1 receptor blockade did not modify systemic hemodynamics in control rats, but significantly increased arterial pressure and peripheral resistance in cirrhotic animals. Blood cell suspension or monocytes from cirrhotic animals, but not from controls, induced arterial hypotension in recipient rats. Finally, anandamide was solely detected in monocytes of cirrhotic animals. Conclusions: Monocytes of cirrhotic rats with ascites are activated to produce anandamide and this substance contributes to arterial hypotension in experimental cirrhosis.

Gastroenterology 2002;122 85-93, Published online 7 January 2002

Distinct Inflammatory Mechanisms Mediate Early Versus Late Colitis in Mice
DAVID M. SPENCER, GEERTRUIDA M. VELDMAN, SUBHASHIS BANERJEE, JOSEPH WILLIS, and ALAN D. LEVINE

Background & Aims: Progression from the acute to chronic phase of inflammatory bowel disease cannot be easily evaluated in patients and has not been characterized in animal models. We report a longitudinal study investigating changes in the mucosal immune response in an experimental model of colitis. Methods: Severity of colitis, body mass, stool consistency and blood content, serum amyloid A, and tissue histology were examined in interleukin (IL)-10-deficient mice over 35 weeks. The corresponding production of IL-12, IL-18, interferon , tumor necrosis factor , IL-4, and IL-13 by lamina propria mononuclear cells in the inflamed intestine was measured. Administration of neutralizing antibody to IL-12 at distinct times during disease progression permitted evaluation of its therapeutic potential. Results: The clinical manifestations and intestinal inflammation delineated an early phase of colitis (10-24 weeks), characterized by a progressive increase in disease severity, followed by a late phase (>25 weeks), in which chronic inflammation persisted indefinitely. Lamina propria mononuclear cells from mice with early disease synthesized progressively greater quantities of IL-12 and interferon gamma, whereas production of both cytokines dramatically declined and returned to pre-disease levels in the late phase of colitis. Consistent with this pattern, neutralizing antibody to IL-12 reversed early, but not late, disease. In contrast, IL-4 and IL-13 production increased progressively from pre- to early to late disease. Conclusions: Colitis that develops in IL-10-deficient mice evolves into 2 distinct phases. IL-12 plays a pivotal role in early colitis, whereas its absence and the synthesis of IL-4 and IL-13 in late disease indicate that other immune mechanisms sustain chronic inflammation.

Gastroenterology 2002;122 94-105, Published online 7 January 2002

Ethanol Metabolism and Transcription Factor Activation in Pancreatic AcinarCells in Rats
ANNA S. GUKOVSKAYA, MICHELLE MOURIA, ILYA GUKOVSKY, CHRISTOPHER N. REYES, VLADIMIR N. KASHO, LARRY D. FALLER, and STEPHEN J. PANDOL

Background & Aims: Ethanol metabolism by pancreatic acinar cells and the role of its metabolites in ethanol toxicity to the pancreas remain largely unknown. Here, we characterize ethanol metabolism in pancreatic acinar cells and determine the effects of ethanol metabolites on nuclear factor B (NF-B) and activator protein (AP)-1, transcription factors that are activated in pancreatitis and mediate expression of inflammatory molecules critical for this disease. Methods: We measured activities of fatty acid ethyl ester (FAEE) synthase and alcohol dehydrogenase (ADH), as well as accumulation of ethanol metabolites. We measured the effects of ethanol and its metabolites on NF-B and AP-1 activation by using a gel shift assay. Results: Pancreas metabolizes ethanol via both oxidative and nonoxidative pathways. Acinar cells are the main source of ethanol metabolism in the pancreas. Compared with the liver, FAEE synthase activity in the pancreas is greater, whereas that of ADH is much less. FAEEs activated NF-B and AP-1, whereas acetaldehyde inhibited NF-B activation. Ethanol decreased NF-B binding activity in acinar cells, which was potentiated by cyanamide. Conclusion: Oxidative and nonoxidative ethanol metabolites regulate transcription factors differently in pancreatic acinar cells. Ethanol may regulate NF-B and AP-1 positively or negatively, depending on which metabolic pathway's effect predominates. These regulatory mechanisms may play a role in ethanol toxicity to the pancreas.

Gastroenterology 2002;122 106-118, Published online 7 January 2002

Genetic or Chemical Hypochlorhydria Is Associated With Inflammation That Modulates Parietal and G-Cell Populations in Mice
YANA ZAVROS, GABRIELE RIEDER, AMY FERGUSON, LINDA C. SAMUELSON, and JUANITA L. MERCHANT

Background & Aims: Reduced gastric acid predisposes the stomach to colonization by bacteria and inflammation. Therefore, we investigated how the chronic gastritis in mice made hypochlorhydric by either gastrin deficiency or omeprazole treatment modulates epithelial cell function. Methods: The gastric pathology of 16-week-old wild-type gastrin-expressing (G+/+) and gastrin-deficient (G/) mice maintained in conventional housing was compared. G/ mice were then treated with antibiotics for 20 days. In a separate experiment, G+/+ mice were treated with omeprazole for 2 months or treated with omeprazole and antibiotics. Results: Compared with the G+/+ animals, the hypochlorhydric G/ mice showed significant inflammation that resolved after 20 days of antibiotic treatment and correlated with a decrease in bacterial overgrowth. Elevated G- and parietal-cell numbers in the G/ mice, quantified by flow cytometry, normalized after antibiotic treatment. G+/+ mice treated with omeprazole had increased bacteria and mucosal lymphocytes that resolved after antibiotic therapy. Quantitation of the gastric cells in these omeprazole-treated mice revealed a significant increase in G- and parietal-cell numbers. On resolution of the gastritis, a decrease in parietal and gastrin-expressing (G) cells was observed despite sustained hypochlorhydria in the presence of omeprazole. Conclusions: Genetic or chemical hypochlorhydria predisposes the stomach to bacterial overgrowth resulting in inflammation. The specific changes in parietal and G cells correlate with the presence of inflammation and not directly with gastric acid. Thus, the normal stomach responds to inflammation by increasing the number and function of cell types that are able to maximize gastric acid output.

Gastroenterology 2002;122 119-133, Published online 7 January 2002

Role of Tumor Necrosis Factor Receptor 2 (TNFR2) in Colonic Epithelial Hyperplasia and Chronic Intestinal Inflammation in Mice
EMIKO MIZOGUCHI, ATSUSHI MIZOGUCHI, HIDETOSHI TAKEDATSU, ELKE CARIO, YPE P. DE JONG, CHOON JIN OOI, RAMNIK J. XAVIER, COX TERHORST, DANIEL K. PODOLSKY, and ATUL K. BHAN

Background & Aims: Tumor necrosis factor (TNF) induces multiple effects including cell proliferation and death by ligation with TNF receptor type II (TNFR2). We studied the role of TNFR2 in chronic inflammation-induced colonic epithelial alteration. Methods: TNFR2 expression in colonic epithelial cells (CECs) was assessed by ribonuclease protection assay (RPA) and immunohistochemistry (IHC) in patients with inflammatory bowel disease (IBD) and murine colitis models. TNFR2 expression was also analyzed using COLO205 cells. The role of TNFR2 in colonic epithelial homeostasis was examined by generating interleukin 6-deficient TCRKO (IL-6DKO) or TNFR2-deficient TCR (TNFR2DKO) mice. Results: TNFR2 expression was up-regulated in CEC in both human ulcerative colitis and Crohn's disease. In vitro studies showed that TNFR2 expression was up-regulated by a cooperative effect of key proinflammatory cytokines. By RPA, the increased expression of TNFR2 was detectable in TCRKO mice with colitis compared with TCRKO mice without colitis or wild-type mice. In IL-6DKO mice, TNFR2 expression, proliferation, and nuclear factor kappa B activation of CECs were markedly reduced compared with TCRKO mice. TNFR2 mice also showed significantly less colonic epithelial proliferation compared with TCRKO mice. Conclusions: Expression of TNFR2 is consistently increased on CECs in both murine colitis models as well as patients with IBD. TNFR2 may play an important role in colonic inflammation-associated alteration in the intestinal epithelium.

Gastroenterology 2002;122 134-144, Published online 7 January 2002

Gastric Acid Secretion in L-Histidine Decarboxylase-Deficient Mice
SATOSHI TANAKA, KIYOMI HAMADA, NOBORU YAMADA, YUKO SUGITA, SHUNSUKE TONAI, BELA HUNYADY, MIKLOÒS PALKOVITS, ANDRAS FALUS, TAKEHIKO WATANABE, SUSUMU OKABE, HIROSHI OHTSU, ATSUSHI ICHIKAWA, and ANDRAS NAGY

Background & Aims: Histamine, gastrin, and acetylcholine are known to be the primary secretagogues of gastric acid secretion, but how the roles are shared among these secretagogues remains to be fully clarified. To evaluate the cooperation between histamine and the other secretagogues, acid secretion responses induced by each secretagogue were measured in L-histidine decarboxylase (HDC)-deficient mice. Methods: Acid secretion was measured by the titration of acid under anesthesia. The expression of selected genes involved in acid secretion was determined by Northern blot and/or immunoblot analysis. Histamine-2 (H2) receptor binding in the gastric mucosa was investigated using [3H]tiotidine. Results: HDC-deficient mice showed low basal and high exogenous histamine-stimulated acid secretion. The mutant mice showed hypergastrinemia and did not undergo acid secretion upon treatment with exogenous gastrin. However, carbachol stimulated weak and transient acid secretion in the mutants. The Bmax values for H2 and the expression of Gs in gastric mucosal membranes were higher in the mutants than in the wild-type mice. Conclusions: This study confirms the concept that histamine production is essential for gastric acid secretion induced by gastrin, but not for that induced by carbachol. HDC-deficient mice should be a suitable model for further functional analyses of the correlation between histamine and the other acid secretagogues.
Gastroenterology 2002;122 145-155, Published online 7 January 2002

Thermal Stress-Induced HSP70 Mediates Protection Against Intrapancreatic Trypsinogen Activation and Acute Pancreatitis in Rats
LAKSHMI BHAGAT, VIJAY P. SINGH, ALBERT M. SONG, GIJS J. D. VAN ACKER, SUDHIR AGRAWAL, MICHAEL L. STEER, and ASHOK K. SALUJA

Background & Aims: Prior thermal stress induces heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue-induced pancreatitis, but it is not clear that this thermal stress-induced protection is actually mediated by HSP70 since thermal stress may have other, non-HSP related, effects. Methods: In the present study, we have administered antisense (AS) oligonucleotides, which prevent pancreatic expression of HSP70 to rats, in vivo, to evaluate this issue. In a separate series of experiments, designed to examine the role of pancreatitis-induced HSP70 expression in modulating the severity of pancreatitis, rats not subjected to prior thermal stress were given AS-HSP70 before cerulein administration, and trypsinogen activation as well as the severity of pancreatitis were evaluated. Results: Hyperthermia induced HSP70 expression, prevented intrapancreatic trypsinogen activation, and protected against cerulein-induced pancreatitis. Administration of AS-HSP70 but not sense-HSP70 reduced the thermal stress-induced HSP70 expression, restored the ability of supramaximal cerulein stimulation to cause intrapancreatic trypsinogen activation, and abolished the protective effect of prior thermal stress against pancreatitis. In nonthermally stressed animals, pretreatment with AS-HSP70 before the induction of pancreatitis exacerbated all the parameters associated with pancreatitis. Conclusions: These findings lead us to conclude that HSP70 induction, rather than some other thermal stress-related phenomenon, mediates the thermal stress-induced protection against pancreatitis and that it protects against pancreatitis by preventing intrapancreatic activation of trypsinogen. The worsening of pancreatitis, which occurs when nonthermally stressed animals are given AS-HSP70 before cerulein, suggests that cerulein-induced HSP70 expression in nontreated animals acts to limit the severity of pancreatitis.

Gastroenterology 2002;122 156-165, Published online 7 January 2002

Fractalkine-Mediated Signals Regulate Cell-Survival and Immune-Modulatory Responses in Intestinal Epithelial Cells
STEPHAN BRAND, TAKANORI SAKAGUCHI, XIUBIN GU, SEAN P. COLGAN, and HANS-CHRISTIAN REINECKER

Background & Aims: In this study, we determined the signal transduction and functional consequences after ligand-specific activation of the fractalkine receptor CX3CR1 in human intestinal epithelial cells. Methods: CX3CR1 expression in human colonic tissues and intestinal epithelial cell lines was determined by immunohistochemistry, immunoblotting, and reverse-transcription polymerase chain reaction. The regulation of mitogen-activated protein kinase (MAPK) activation was assessed by immunoblotting. Regulation of chemokine messenger RNA (mRNA) expression was determined by Northern blotting. NF-B and p53 activation was assessed by electromobility shift assays. Results: Fractalkine mediated the MEK-1 and Gi-dependent but phosphatidylinositol-3-kinase-independent activation of extracellular signal-regulated kinase-MAPK. Fractalkine activated NF-B and p53 resulting in interleukin 8 and fractalkine mRNA expression. CX3CR1-mediated activation of intestinal epithelial cells was able to induce migration of human neutrophils into but not through the intestinal epithelial cell monolayer. Conclusions: CX3CR1 mediates distinct functional responses in intestinal epithelial cells, which include the autocrine regulation of cell-survival signals and activation of immune modulators, indicating a role of CX3CR1 in host defense mechanisms originating from the intestinal epithelium.

Gastroenterology 2002;122 166-177, Published online 7 January 2002

Sodium Current in Human Jejunal Circular Smooth Muscle Cells
ADRIAN N. HOLM, ADAM RICH, STEVEN M. MILLER, PETER STREGE, YIJUN OU, SIMON J. GIBBONS, MICHAEL G. SARR, JOSEPH H. SZURSZEWSKI, JAMES L. RAE, and GIANRICO FARRUGIA

Background & Aims: Sodium channels are key regulators of neuronal and muscle excitability. However, sodium channels have not been definitively identified in gastrointestinal smooth muscle. The aim of the present study was to determine if a Na+ current is present in human jejunal circular smooth muscle cells. Methods: Currents were recorded from freshly dissociated cells using patch-clamp techniques. Complementary DNA (cDNA) libraries constructed from the dissociated cells were screened to determine if a message for  subunits of Na+ channels was expressed. Smooth muscle cells were also collected using laser-capture microdissection and screened. Results: A tetrodotoxin-insensitive Na+ channel was present in 80% of cells patch-clamped. Initial activation was at 65 mV with peak inward current at 30 mV. Steady-state inactivation and activation curves revealed a window current between 75 and 60 mV. The Na+ current was blocked by lidocaine and internal and external QX314. A cDNA highly homologous to SCN5A, the  subunit of the cardiac Na+ channel, was present in the cDNA libraries constructed from dissociated cells and from smooth muscle cells collected using laser-capture microdissection. Conclusions: Human jejunal circular smooth muscle cells express a tetrodotoxin-insensitive Na+ channel, probably SCN5A. Whether SCN5A plays a role in the pathophysiology of human gut dysmotilities remains to be determined.

Gastroenterology 2002;122 178-187, Published online 7 January 2002

Matrix Metalloproteinase-9 Promotes Neutrophil Migration and Alveolar Capillary Leakage in Pancreatitis-Associated Lung Injury in the Rat
TOBIAS KECK, JAMES H. BALCOM, IV, CARLOS FERNANDEZ-DEL CASTILLO, BOZENA A. ANTONIU, and ANDREW L. WARSHAW

Background & Aims: In pancreatitis-associated lung injury, neutrophils (PMN) access the lung by migration through endothelial basement membranes. We hypothesize that degeneration of the basement membrane by specific PMN-produced matrix metalloproteinases (MMPs) may facilitate this process. Methods: Mild or severe pancreatitis was induced in rats and the consequent pulmonary injury characterized. MMP-2 and MMP-9 activity in supernatant of PMN cultures and homogenates of lungs were assessed by zymography and Western blot. Congruence of PMN and MMP expression in lung tissue was evaluated by neutrophil depletion and fluorescent immunohistochemistry (IHC). The contribution of MMPs to PMN transmigration and lung injury was tested with the MMP inhibitor batimastat (BB-94) in vitro (PMN transmigration across matrigel chambers) and in vivo (myeloperoxidase activity and Evans blue in broncho-alveolar lavage fluid). Results: MMP-9 was highly expressed in lungs and supernatant of neutrophil cultures in severe pancreatitis, and, to a lesser degree, in mild pancreatitis. Lung IHC showed colocalization of MMP-9 and PMN. PMN depletion simultaneously reduced neutrophil infiltration and MMP-9 levels in lung tissue. Trypsin, interleukin 1, and tumor necrosis factor (TNF)- all potently stimulated MMP-9 release from PMN. BB-94 significantly reduced TNF--induced PMN transmigration across matrigel and ameliorated transendothelial PMN migration and protein leak in severe pancreatitis. Conclusions: MMP-9 secretion by PMN can be stimulated by trypsin and proinflammatory cytokines and increases in pancreatitis in proportion to its severity. MMP inhibition reduces PMN transmigration and reduces resultant alveolar-capillary leakage. These findings suggest an important role for MMP-9 from PMN in the pathogenesis of pancreatitis-associated lung injury.

Gastroenterology 2002;122 188-201, Published online 7 January 2002

Tumor Necrosis Factor alpha , But Not Fas, Mediates Hepatocellular Apoptosis in the Murine Ischemic Liver
HANNES A. RUDIGER and PIERRE-ALAIN CLAVIEN

Background & Aims: Apoptosis of hepatocytes is a central feature of ischemic injury in the liver. The aim of this study was to identify extracellular inducers of apoptosis in the murine ischemic liver. Methods: Involvement of tumor necrosis factor (TNF)- and Fas signaling was evaluated using various knockout mice (TNF-receptor 1 [TNF-R1]/, Fas[lpr]/, and Fas ligand[gld]/) and wild-type mice pretreated with pentoxifylline, an inhibitor of TNF- synthesis. Results: Expression of TNF- was increased after ischemia and reperfusion in wild-type mice and TNF-R1-deficient mice when compared with sham-operated animals. Pentoxifylline prevented up-regulation of TNF- expression. Inhibition of TNF- resulted in significant decrease of serum aspartate aminotransferase levels and prolonged animal survival. Markers of apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, cytochrome C release, and caspase 3 activity) were consistently decreased, and animal survival was prolonged after blocking TNF-. In contrast, inhibition of Fas signaling did not alter parameters of tissue injury or apoptosis, and animal survival remained unchanged. Conclusions: We identify TNF- as a crucial inducer of apoptotic cell death in the ischemic liver. A role for Fas could not be identified. These findings may lead to novel strategies to prevent ischemic injury of the liver.

Gastroenterology 2002;122 202-210, Published online 7 January 2002

Functional Analysis of hMLH1 Variants and HNPCC-Related Mutations Using a Human Expression System
JOERG TROJAN, STEFAN ZEUZEM, ANN RANDOLPH, CHRISTINE HEMMERLE, ANGELA BRIEGER, JOCHEN RAEDLE, GUIDO PLOTZ, JOSEF JIRICNY, and GIANCARLO MARRA

Background & Aims: Germline mutations in the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 are associated with susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Because a significant proportion of hMLH1 mutations are missense, the assessment of their pathogenic role may be difficult. To date, functional analysis of missense mutations has been performed primarily in Saccharomyces cerevisiae. The aim of this study was to examine the biochemical properties of hMLH1 protein variants in a human expression system. Methods: The HNPCC-related hMLH1 mutations T117M, V185G, R217C, G244D, R265C, V326A, and K618T, the polymorphisms I219V and R265H, and a hMLH1 splicing variant lacking exon 9 and 10 (hMLH19/10) were cloned. On transfection of these constructs into human 293T cells, which do not express hMLH1 because of promoter hypermethylation, the hMLH1 protein variants were analyzed by Western blotting and in a MMR assay. Results: Transfection was successful for all hMLH1 constructs. As anticipated, the mutations K618T and T117M, which affect the highly conserved domains of hMLH1 that are necessary for interaction with hPMS2 or for adenosine triphosphate (ATP) binding, respectively, affected protein stability or its ability to complement MMR-deficient 293T-cell extracts. The V185G, G244D, and 9/10 variants were also unable to complement MMR in 293T cells, whereas hMLH1 proteins carrying the I219V, R265H, R265C, R217C, and V326A mutations were MMR competent. Conclusions: These data show that the pathogenic role of hMLH1 missense mutations and splicing variants can be assessed by analyzing the biochemical properties of their protein products in a homologous expression system.

Gastroenterology 2002;122 211-219, Published online 7 January 2002

CASE REPORTS

Esophageal Stenosis in Childhood: Dystrophic Epidermolysis Bullosa Without Skin Blistering Due to Collagen VII Mutations
KLAUS-PETER ZIMMER, HAUKE SCHUMANN, SABINE MECKLENBECK, and LEENA BRUCKNER-TUDERMAN

We report a 9-year-old girl who experienced recurrent dysphagia since infancy. Crohn's disease was suspected because she had aphthous ulcers of the mouth and anal dermatitis with hematochezia. After bougienages of esophageal stenoses and medication for inflammatory bowel disease proved unsuccessful, interdisciplinary re-examination revealed the cause of the symptoms to be an extracutaneous form of dystrophic epidermolysis bullosa, a genetic skin fragility disorder. Dystrophic epidermolysis bullosa is caused by mutations in the COL7A1 gene encoding collagen VII, a protein of the epidermal attachment complex, and typically manifests with trauma-induced skin blistering, scarring, nail dystrophy, and, in some cases, mucosal involvement. The present proband never developed skin blisters but had nail dystrophy and erosions of the oral, esophageal, and genitoanal mucosa, which healed with slight scarring. Mutation analysis disclosed compound heterozygosity for recessive mutations in the COL7A1 gene. The paternal mutation 425 AG caused abnormal splicing resulting in a premature stop codon. The maternal mutation G2775S led to the substitution of a glycine by a serine in the triple helical domain of collagen VII. This case shows that mucosal disease and esophageal strictures in childhood are not always acquired, but can also represent a genetic defect of dermal-epidermal adhesion, even in the absence of skin blistering.

Gastroenterology 2002;122 220-225, Published online 7 January 2002

BRITISH MEDICAL JOURNAL

19 January 2002 Volume 324, Issue 7330

Obstetric cholestasis
Piotr Milkiewicz, Elwyn Elias, Catherine Williamson, and Judith Weaver
BMJ 2002; 324: 123-124. [Full text]

Obstetric cholestasis (or intrahepatic cholestasis of pregnancy) remains widely disregarded as an important clinical problem, with many obstetricians still considering its main symptom, pruritus, a natural association of pregnancy. Obstetric cholestasis is associated with cholesterol gallstones. It may be extremely stressful for the mother but also carries risks for the baby.

Clinical studies clearly show that when obstetric cholestasis complicates pregnancies it may lead to premature births in up to 60%, fetal distress in up to 33%, and intrauterine death in up to 2% of patients.1 The cause of fetal death is acute anoxia.2 The incidence of obstetric cholestasis varies from 0.1% to 1.5% of pregnancies in Europe and 9.2%-15.6% in South American countries such as Bolivia or Chile. It is particularly high in the native Araucanian population in Chile, where the proportion of affected pregnancies reaches nearly 28%.3 The low quoted incidence of obstetric cholestasis in Europe may reflect an underestimation of the problem, and growing awareness of the condition will probably increase the numbers. For example, early studies from North America, published in 1962, showed its incidence to be less than 0.1%, whereas a study published 17 years later estimated the incidence to be around 0.7%.1

The aetiology of obstetric cholestasis is undoubtedly multifactorial, with genetic, environmental, and hormonal factors having important roles. Historically obstetric cholestasis has been associated with the cholestatic effect of oestradiol metabolites, in particular 17- oestradiol glucuronide. Progesterone metabolites, however, play an even more important part in its pathogenesis. Patients with obstetric cholestasis have a significantly increased ratio of 3  to 3  hydroxysteroids and large amounts of mono or disulphated progesterone metabolites excreted in their urine.4 Excess of these metabolites in the urine in obstetric cholestasis may be related to malfunction of biliary canalicular transporters normally responsible for their secretion from hepatocytes into bile. Several of these transporters have recently been characterised. These include proteins responsible for bile canalicular secretion of bile acids (bile salt export pump), organic anions (multidrug resistant protein 2) or phospholipids (multidrug resistance protein 3). ............

12 January 2002 Vol. 324, No. 7329

Excess winter mortality: influenza or cold stress? Observational study
G C Donaldson and W R Keatinge BMJ 2002;324 89-90

Epidemics of influenza are associated with increases in mortality and morbidity.1 Health professionals and the media, therefore, have often focused their attention on influenza as a cause of increased mortality and demands on health services in winter. Cold weather alone causes striking short term increases in mortality, mainly from thrombotic and respiratory disease.2 Non-thermal seasonal factors such as diet may also affect mortality.3 The increases in mortality are greater in London than in regions surveyed in continental Europe.4 We used multiple regression to assess the proportion of excess winter mortality that was attributable to influenza in south east England. [Full text]  

NEW ENGLAND JOURNAL

Volume 346 January 31, 2002 Number 5

Detection of APC Mutations in Fecal DNA from Patients with Colorectal Tumors
Giovanni Traverso, B.A., Anthony Shuber, M.S., Bernard Levin, M.D., Constance Johnson, R.N., M.S., Louise Olsson, M.D., David J. Schoetz, Jr., M.D., Stanley R. Hamilton, M.D., Kevin Boynton, B.S., Kenneth W. Kinzler, Ph.D., and Bert Vogelstein, M.D.

Background Noninvasive methods for detecting colorectal tumors have the potential to reduce morbidity and mortality from this disease. The mutations in the adenomatous polyposis coli (APC) gene that initiate colorectal tumors theoretically provide an optimal marker for detecting colorectal tumors. The purpose of our study was to determine the feasibility of detecting APC mutations in fecal DNA with the use of newly developed methods. Methods We purified DNA from routinely collected stool samples and screened for APC mutations with the use of a novel approach called digital protein truncation. Many different mutations could potentially be identified in a sensitive and specific manner with this technique. Results Stool samples from 28 patients with nonmetastatic colorectal cancers, 18 patients with adenomas that were at least 1 cm in diameter, and 28 control patients without neoplastic disease were studied. APC mutations were identified in 26 of the 46 patients with neoplasia (57 percent; 95 percent confidence interval, 41 to 71 percent) and in none of the 28 control patients (0 percent; 95 percent confidence interval, 0 to 12 percent; P<0.001). In the patients with positive tests, mutant APC genes made up 0.4 to 14.1 percent of all APC genes in the stool. Conclusions APC mutations can be detected in fecal DNA from patients with relatively early colorectal tumors. This feasibility study suggests a new approach for the early detection of colorectal neoplasms.

Volume 346 January 3, 2002 Number 1

Helicobacter pylori Infection and the Development of Gastric Cancer
 
Uemura, N. To the Editor: Uemura et al. (Sept. 13 issue)1 make an important contribution to our understanding of the complications of Helicobacter pylori infection, but I have a question regarding the ethics of their study. The authors enrolled 1526 patients, of whom 1246 had H. pylori infection. Of the H. pylori­positive patients, 297 had gastric ulcers and 275 had duodenal ulcers. Hence, 572 patients had a condition for which treatment to eradicate the H. pylori infection is indicated. The remaining H. pylori­positive patients had gastric polyps and nonulcer dyspepsia, conditions for which eradication treatment is not currently indicated.

Eradication treatment was administered to 253 patients, but the authors do not specify whether these patients were among the 572 who had gastric ulcers or duodenal ulcers. Assuming that they were, there remained at least 319 patients who had a clinical indication for eradication therapy yet did not receive this treatment. More important, it appears that these remaining patients were not offered this therapy.

No gastric cancer developed after the eradication of H. pylori in the 253 infected patients who received this treatment. Therefore, at least 319 patients were exposed to an increased risk of gastric cancer even though they had an indication for eradication therapy that, if successful, would have eliminated the risk.

LANCET

Volume 359, Number 9303 26 January 2002

Alcohol consumption and risk of dementia: the Rotterdam Study  [Full Text]
Annemieke Ruitenberg, John C van Swieten, Jacqueline C M Witteman, Kala M Mehta, Cornelia M van Duijn, Albert Hofman, Monique M B Breteler

Summary: Background Light-to-moderate alcohol consumption reduces the risk of coronary heart disease and stroke. Because vascular disease is associated with cognitive impairment and dementia, we hypothesised that alcohol consumption might also affect the risk of dementia. Methods We examined the relation between alcohol consumption and risk of dementia in individuals taking part in the Rotterdam Study--a prospective population-based study of 7983 individuals aged 55 years and older. We studied all participants who did not have dementia at baseline (1990-93) and who had complete data on alcohol consumption (n=5395). Through follow-up examinations in 1993-94 and 1997-99 and an extensive monitoring system, we obtained nearly complete follow-up (99·7%) until the end of 1999. We used proportional hazards regression analysis, adjusted for age, sex, systolic blood pressure, education, smoking, and body-mass index, to compare the risk of developing dementia between individuals who regularly consumed alcohol and individuals who did not consume alcohol. Findings The average follow-up was 6·0 years. During this period, 197 individuals developed dementia (146 Alzheimer's disease, 29 vascular dementia, 22 other dementia). The median alcohol consumption was 0·29 drinks per day. Light-to-moderate drinking (one to three drinks per day) was significantly associated with a lower risk of any dementia (hazard ratio 0·58 [95% CI 0·38-0·90]) and vascular dementia (hazard ratio 0·29 [0·09-0·93]). We found no evidence that the relation between alcohol and dementia varied by type of alcoholic beverage. Interpretation These findings suggest that light-to-moderate alcohol consumption is associated with a reduced risk of dementia in individuals aged 55 years or older. The effect seems to be unchanged by the source of alcohol. Lancet 2002; 359: 281-86

Effect of race on outcome of orthotopic liver transplantation: a cohort study  
Satheesh Nair, Joseph Eustace, Paul J Thuluvath

Summary: Background There is significant evidence to suggest that long-term survival after renal transplantation is significantly lower in African Americans than in other races. We aimed to establish whether there was a difference in survival in African Americans compared with other races after orthotopic liver transplantation (OLT) and whether race was an independent predictor of survival. Methods We collected data from the United Network of Organ Sharing transplant registry for all liver transplants done between 1988 and 1996 in the USA. We also recorded information on age, sex, race, blood group, and cause of death for the donors and recipients. Findings 2-year graft survival was significantly lower for African Americans (601 of 884, 68%) and Asians (266 of 416, 64%) compared with white Americans (8703 of 11 762, 74%) and Hispanics (878 of 1220, 72%). 2-year and 5-year patient survival were significantly lower for African Americans (654 of 884 [74%], 270 of 565 [48%]) and Asians (287 of 416 [69%], 92 of 252 [37%]) compared with white Americans (9786 of 11 762 [83%], 4357 of 7514 [58%]) and Hispanics (964 of 1220 [79%], 341 of 657 [52%]). Compared with white Americans, African American (hazard ratio 1·36, 95% CI 1·16-1·60, p<0·0001) and Asian (1·25, 1·01-1·56, p=0·03) race were independent predictors of poor survival at 2 years. Interpretation African Americans and Asians have a worse outcome after OLT compared with white Americans and Hispanics. The higher rate of chronic rejection in African Americans and a relatively worse outcome in other minority races merits further examination. Lancet 2002; 359: 287-93

Volume 359, Number 9302 19 January 2002

Penetrance of 845G*A (C282Y) HFE hereditary haemochromatosis mutation in the USA
Ernest Beutler, Vincent J Felitti, James A Koziol, Ngoc J Ho, Terri Gelbart

Summary: Background There has been much interest in screening populations for disease-associated mutations. A favoured candidate has been the HFE gene, mutations of which are the most common cause of haemochromatosis in the European population. About five people in 1000 are homozygotes for the 845G*A mutation, but little is known of how many have mutation-caused clinical manifestations. Methods We screened 41 038 individuals attending a health appraisal clinic in the USA for the 845G*A and 187C*G HFE mutations, and analysed laboratory data and data on signs and symptoms of haemochromatosis as elicited by questionnaire. Findings The most common symptoms of haemochromatosis, including poor general health, diabetes, arthropathies, arrhythmias, impotence, and skin pigmentation were no more prevalent among the 152 identified homozygotes than among the controls. The age distribution of homozygotes and compound heterozygotes did not differ significantly from that of controls: there was no measurable loss of such individuals from the population during ageing. However, there was a significantly increased prevalence of a history of hepatitis or "liver trouble" among homozygotes and in the proportion of homozygotes with increased concentrations of serum aspartate aminotransferase and collagen IV; these changes were not related to iron burden or to age. Only one of the 152 homozygotes had signs and symptoms that would suggest a diagnosis of haemochromatosis. Interpretation The normal age distribution of people with the haemochromatosis genotype, and the lack of symptoms in patients of all ages, indicate that the penetrance of hereditary haemochromatosis is much lower than generally thought. The clinical penetrance of a disorder is an essential consideration in screening for genetic disease; disorders with low penetrance are more expensive candidates for screening than disorders with high penetrance. Our best estimate is that less than 1% of homozygotes develop frank clinical haemochromatosis. Lancet 2002; 359: 211-18

Counting alleles to predict recurrence of early-stage colorectal cancers  [Full Text] 
Wei Zhou, Steven N Goodman, Gennaro Galizia, Eva Lieto, Francesca Ferraraccio, Carlo Pignatelli, Colin A Purdie, Juan Piris, Robert Morris, David J Harrison, Philip B Paty, Al Culliford, Katharine E Romans, Elizabeth A Montgomery, Michael A Choti, Kenneth W Kinzler, Bert Vogelstein

Summary: Background Chromosome imbalances occur in many cancers and represent important biological properties of tumours. However, measurements of such imbalances are difficult. We used a new, quantitative approach to investigate the prognostic value of chromosome imbalances in early-stage colorectal cancers. Methods We studied 180 patients with no evidence of lymph-node or distant metastases at the time of surgery. DNA from paraffin-embedded tumours was tested for imbalances of chromosome 8p and 18q by digital SNP (single-nucleotide polymorphism)--a technique in which each allele in a sample is directly counted. Surviving patients had median follow-up of 68 months, and disease recurrence was used as the clinical endpoint. Findings Tumours were divided into three groups: "L" tumours (n=93) had allelic imbalances of chromosomes 8p and 18q, "L/R" tumours (n=60) had allelic imbalances of either chromosome 8p or 18q but not both, and "R" tumours (n=27) retained allelic balance for both chromosomes. 5-year disease-free survival was 100% (95% CI 80-100) for patients with R tumours, 74% (61-87) for patients with L/R tumours, and 58% (47-69) for those with L tumours. These differences were significant (p<0·0001) and were independent of other variables--eg, Duke's stage A tumours of class L were much more likely to recur than Duke's stage B tumours of class R (p=0·002). Interpretation In patients without metastasis, allelic imbalance is a better predictor of prognosis than histopathological stage. Lancet 2002; 359: 219-25

Volume 359, Number 9301 12 Janvier 2002

Research letters: Association between hepatitis C virus seropositivity,carotid-artery plaque, and intima-media thickening
Nobukazu Ishizaka, Yuko Ishizaka, Eiko Takahashi, Ei-ichi Tooda, HidekiHashimoto, Ryozo Nagai, Minoru Yamakado

We investigated the relation between positivity for hepatitis C virus (HCV) and carotid-artery plaque and carotid intima-media thickening by analysing cross-sectional data of individuals undergoing a general health screening test. Of 4784 individuals enrolled, 104 (2·2%) were seropositive for HCV. After adjustment for confounding risk factors, HCV seropositivity was found to be associated with an increased risk of carotid-artery plaque (odds ratio 1·92 [95% CI 1·56-2·38], p=0·002) and carotid intima-media thickening (2·85 [2·28-3·57], p<0·0001). These findings suggest a possible role for chronic hepatitis C in the pathogenesis of carotid arterial remodelling.

Volume 359, Number 9300 05 Janvier 2002

Eradication of Helicobacter pylori and risk of peptic ulcers in patients starting long-term treatment with non-steroidal anti-inflammatory drugs: a randomised trial  [Full Text] 
Francis K L Chan, K F To, Justin C Y Wu, M Y Yung, W K Leung, Timothy Kwok, Y Hui, Henry L Y Chan, Cynthia S Y Chan, Elsie Hui, Jean Woo, Joseph J Y Sung

Summary: Background Whether Helicobacter pylori increases the risk of ulcers in patients taking non-steroidal anti-inflammatory drugs (NSAIDs) is controversial. We hypothesised that eradication of H pylori infection would reduce the risk of ulcers for patients starting long-term NSAID treatment. Methods Patients were enrolled if they were NSAID naïve, had a positive urea breath test, had dyspepsia or an ulcer history, and required long-term NSAID treatment. They were randomly assigned omeprazole triple therapy (eradication group) or omeprazole with placebo antibiotics (placebo group) for 1 week. All patients were given diclofenac slow release 100 mg daily for 6 months from randomisation. Endoscopy was done at 6 months or if severe dyspepsia or gastrointestinal bleeding occurred. The primary endpoint was the probability of ulcers within 6 months. Analyses were by intention to treat. Findings Of 210 arthritis patients screened, 128 (61%) were positive for H pylori. 102 patients were enrolled, and 100 were included in the intention-to-treat analysis. H pylori was eradicated in 90% of the eradication group and 6% of the placebo group. Five of 51 eradication-group patients and 15 of 49 placebo-group patients had ulcers. The 6-month probability of ulcers was 12·1% (95% CI 3·1-21·1) in the eradication group and 34·4% (21·1-47·7) in the placebo group (p=0·0085). The corresponding 6-month probabilities of complicated ulcers were 4·2% (1·3-9·7) and 27·1% (14·7-39·5; p=0·0026). Interpretation Screening and treatment for H pylori infection significantly reduces the risk of ulcers for patients starting long-term NSAID treatment. Lancet 2002; 359: 9-13

Role of Helicobacter pylori infection and non-steroidal anti-inflammatory drugs in peptic-ulcer disease: a meta-analysis  [Full Text] 

Summary Background The relation between H pylori infection and use of non-steroidal anti-inflammatory drugs (NSAIDs) in the pathogenesis of peptic-ulcer disease is controversial. We undertook a meta-analysis to address this issue. Methods By computer and manually we sought observational studies on the prevalence of peptic-ulcer disease in adult NSAID takers or the prevalence of H pylori infection and NSAID use in patients with peptic-ulcer bleeding. Summary odds ratios were calculated from the raw data. Tests for homogeneity were done. Findings Of 463 citations identified, 25 studies met inclusion criteria. In 16 studies of 1625 NSAID takers, uncomplicated peptic-ulcer disease was significantly more common in patients positive than in those negative for H pylori (341/817 [41·7%] vs 209/808 [25·9%]; odds ratio 2·12 [95% CI 1·68-2·67]). In five controlled studies, peptic-ulcer disease was significantly more common in NSAID takers (138/385 [35·8%]) than in controls (23/276 [8·3%]), irrespective of H pylori infection. Compared with H pylori negative individuals not taking NSAIDs, the risk of ulcer in H pylori infected NSAID takers was 61·1 (9·98-373). H pylori infection increased the risk of peptic-ulcer disease in NSAID takers 3·53-fold in addition to the risk associated with NSAID use (odds ratio 19·4). Similarly, in the presence of risk of peptic-ulcer disease associated with H pylori infection (18·1), use of NSAIDs increased the risk of peptic-ulcer disease 3·55-fold. H pylori infection and NSAID use increased the risk of ulcer bleeding 1·79-fold and 4·85-fold, respectively. However, the risk of ulcer bleeding increased to 6·13 when both factors were present. Interpretation Both H pylori infection and NSAID use independently and significantly increase the risk of peptic ulcer and ulcer bleeding. There is synergism for the development of peptic ulcer and ulcer bleeding between H pylori infection and NSAID use. Peptic-ulcer disease is rare in H pylori negative non-NSAID takers. Lancet 2002; 359: 14-22

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