Volume 39, Issue 2 - February 2004
Viral Hepatitis
SARS-associated viral hepatitis caused by a novel coronavirus: Report of three cases (p 302-310)
Tai-Nin Chau, Kam-Cheong Lee, Hung Yao, Tak-Yin Tsang, Tat-Chong Chow, Yiu-Cheong Yeung, Kin-Wing Choi, Yuk-Keung Tso, Terence Lau, Sik-To Lai, Ching-Lung Lai
Liver impairment is commonly reported in up to 60% of patients who suffer from severe acute respiratory syndrome (SARS). Here we report the clinical course and liver pathology in three SARS patients with liver impairment. Three patients who fulfilled the World Health Organization case definition of probable SARS and developed marked elevation of alanine aminotransferase were included. Percutaneous liver biopsies were performed. Liver specimens were examined by light and electron microscopy, and immunohistochemistry. Reverse-transcriptase polymerase chain reaction (RT-PCR) using enhanced real-time PCR was applied to look for evidence of SARS-associated coronavirus infection. Marked accumulation of cells in mitosis was observed in two patients and apoptosis was observed in all three patients. Other common pathologic features included ballooning of hepatocytes and mild to moderate lobular lymphocytic infiltration. No eosinophilic infiltration, granuloma, cholestasis, fibrosis, or fibrin deposition was noted. Immunohistochemical studies revealed 0.5% to 11.4% of nuclei were positive for proliferative antigen Ki-67. RT-PCR showed evidence of SARS-associated coronavirus in the liver tissues, but not in the sera of all 3 patients. However, electron microscopy could not identify viral particles. No giant mitochondria, micro- or macro-vesicular steatosis was observed. In conclusion, hepatic impairment in patients with SARS is due to SARS-associated coronavirus infection of the liver. The prominence of mitotic activity of hepatocytes is unique and may be due to a hyperproliferative state with or without disruption of cell cycle by the coronavirus. With better knowledge of pathogenesis, specific therapy may be targeted to reduce viral replication and modify the disease course. (H EPATOLOGY 2004;39:302-310.)
Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine (p 311-317)
Ulrich A. Walker, Jochen Bäuerle, Montse Laguno, Javier Murillas, Stefan Mauss, Günther Schmutz, Bernhard Setzer, Rosa Miquel, José M. Gatell, Josep Mallolas
The D drug HIV reverse-transcriptase inhibitors zalcitabine, didanosine, and stavudine are relatively strong inhibitors of polymerase-gamma compared with the non-D drugs zidovudine, lamivudine, and abacavir. D drugs deplete mitochondrial DNA (mtDNA) in cultured hepatocytes. This mtDNA depletion is associated with an increased in vitro production of lactate. To investigate the origin of hyperlactatemia in HIV-infected patients and the effects of antiretroviral therapy on liver mtDNA, we biopsied liver tissue from 94 individuals with chronic hepatitis C virus (HCV) infection. Eighty subjects were coinfected with HIV. Serum lactate was measured at the time of biopsy. Hepatic mtDNA and liver histology were centrally assessed. Liver mtDNA content of HIV-infected patients receiving D drugs at the time of biopsy (n = 34) was decreased by 47% ( P<.0001) compared with those without D drugs (n = 35). Aside from a possible association between HCV genotype I status and mtDNA depletion in multivariate analysis, there were no other virologic, immunologic, histologic, demographic or treatment-related variables that could explain the mtDNA depletion. Lactate was above the upper limit of normal in only three patients, all of whom were treated with D drugs. The mtDNA in each of them was lower than in any non-D drug patient and significantly ( P= .017) depleted compared with D drug patients with normal lactate. In conclusion, D drug treatment is associated with decreased hepatic mtDNA in HIV-infected patients with chronic HCV infection. Moderate mtDNA depletion in liver does not necessarily lead to hyperlactatemia, but more pronounced decreases in hepatic mtDNA may be an important contributor to lactate elevation. (H EPATOLOGY 2004;39:311-317.)
HBsAg and HBx knocked into the p21 locus causes hepatocellular carcinoma in mice (p 318-324)
Youliang Wang, Fang Cui, Yaxin Lv, Cuiling Li, Xiaoling Xu, Chuxia Deng, Dongping Wang, Yansong Sun, Gengxi Hu, Zhenwei Lang, Cuifen Huang, Xiao Yang
Hepatocellular carcinoma (HCC) affects males in a significantly higher proportion than females and is one of the human cancers etiologically related to viral factors. Many studies provide strong evidence of the direct role that hepatitis B virus (HBV) plays in hepatic carcinogenesis, but the functions of HBV surface antigen ( HBsAg ) and X protein ( HBx ) in hepatocarcinogenesis through direct or indirect mechanisms are still being debated. We generated two HBV gene knock-in transgenic mouse lines by homologous recombination. HBsAg and HBx genes were integrated into the mouse p21 locus. Both male and female p21-HBx transgenic mice developed HCC after the age of 18 months; however, male p21-HBsAg transgenic mice began to develop HCC 3 months earlier. The expression of a number of genes related to metabolism and genomic instability largely resembled the molecular changes during the development of HCC in humans. ER- (estrogen receptor- ) was extremely up-regulated only in tumor tissues of male p21-HBsAg mice, providing genetic evidence that HBsAg might be the major risk factor affecting the gender difference in the causes of HCC. In conclusion, these mice might serve as good models for studying the different roles of HBsAg and HBx in early events of HBV-related hepatocarcinogenesis. (H EPATOLOGY 2004;39:318-324.)
Epidemiology and risk factors for hepatitis C in Alaska Natives (p 325-332)
Brian J. McMahon, Thomas W. Hennessy, Carol Christensen, Dana Bruden, Daniel G. Sullivan, Chriss Homan, Heike Deubner, Michael G. Bruce, Stephen Livingston, James Williams, David R. Gretch
Large cohorts of persons infected with hepatitis C virus (HCV) that include patients with multiple risk exposures and behaviors have been rarely reported. We herein describe a population-based cohort of 759 Alaska Natives (AN) with HCV who were recruited into a long-term follow-up study. History of injection drug use (IDU) was reported by 60.1% and blood transfusion by 14.0%. The most common genotype was 1a (42.0%), followed by 1b (20.3%), 2b (14.7%), 3a (14.3%), and 2a (7.8%). By multivariable analysis, risk exposures (blood transfusion vs. other; P< 0.01; odds ratio [OR], 2.87; 95% confidence interval [CI], 1.51-5.45) and year of infection ( P< 0.01; OR, 3.47; 95% CI, 1.34-8.96) were significantly associated with HCV RNA-positivity. Having an RNA concentration 2 million copies/mL was associated with male gender (OR, 1.94) and genotype ( P< 0.01 overall; 1a vs. 3a: OR, 1.92; 2b vs. 3a: OR, 3.17) by multivariable analysis. In conclusion, the two principal risk exposures for AN infected with HCV (IDU and blood transfusion) are the same as the overall U.S. population. Persons with a history of blood transfusion were more likely to be HCV RNA positive than those without such history. Higher RNA levels found in males may explain the more severe disease previously reported in this group. (H EPATOLOGY 2004;39:325-332.)
Effect of peginterferon alfa-2a on liver histology in chronic hepatitis C: A meta-analysis of individual patient data (p 333-342)
Calogero Cammà, Danilo Di Bona, Filippo Schepis, E. Jenny Heathcote, Stefan Zeuzem, Paul J. Pockros, Patrick Marcellin, Luis Balart, Alfredo Alberti, Antonio Craxì
Multicenter randomized trials have shown that once-weekly pegylated interferon (peginterferon) alfa-2a is more efficacious than conventional interferon alfa-2a (IFN) in patients with chronic hepatitis C. We performed a meta-analysis of 1,013 previously untreated patients (from 3 randomized trials) with pretreatment and post-treatment liver biopsies to assess the differences between peginterferon alfa-2a and IFN in terms of their effects on liver histology. Reported values were standardized mean differences (SMD) between patients receiving peginterferon alfa-2a and those receiving IFN (post-treatment value minus baseline value for each group). We used a random-effects model to quantify the average effect of peginterferon alfa-2a on liver histology. Peginterferon alfa-2a significantly reduced fibrosis compared with IFN (SMD, -0.14; 95% CI: -0.27, -0.01; P= .04). A reduction in fibrosis was observed among sustained virologic responders (SMD, -0.59; 95% CI: -0.89, -0.30; P< .0001) and patients with recurrent disease (SMD, -0.34; 95% CI: -0.54, -0.14; P= .0007), whereas no significant reduction was observed among nonresponders (SMD, -0.13; 95% CI: -0.32, 0.05; P= .15). Logistic regression analysis indicated that patients with sustained virologic responses (SVRs) had an odds ratio (OR) of 1.61 (95% CI: 1.14, 2.29) for reduction in fibrosis compared with patients without SVRs, whereas obese patients (body mass index [BMI] > 30 kg/m 2) had an OR of 0.56 (95% CI: 0.35, 0.90) compared with normal-weight (BMI < 25 kg/m 2) and overweight patients (BMI, 25-30 kg/m 2). In conclusion, in patients with chronic hepatitis C with or without cirrhosis, peginterferon alfa-2a (relative to IFN) significantly reduced fibrosis. The beneficial effects of peginterferon on liver histology are closely related to virologic response. (H EPATOLOGY 2004;39:333-342.)
Liver Biology & Pathobiology
Stereological measurement of porto-central gradients in gene expression in mouse liver (p 343-352)
Jan M. Ruijter, Roben G. Gieling, Marry M. Markman, Jaco Hagoort, Wouter H. Lamers
The liver is thought to consist of lobules, numerous repeating, randomly oriented units. Within these lobules, genes are expressed in gradients along the porto-central axis, which spans the distance between portal and central veins. We have developed a robust stereological method to map all points in an image to their position on this porto-central axis. This approach is based on the distribution of well-characterized periportal and pericentral enzymes, which are visualized on sections preceding and following the section of interest. Because expression of the model genes phosphoenolpyruvate carboxykinase and ornithine aminotransferase declines gradually with increasing distance from the portal vein and central vein, respectively, these genes can be used to prepare images with topographical information without any assumption about the shape of the hepatic unit, or about the direction or shape of the gradient to be determined. The relative distance image is a 2-dimensional image that accurately maps the relative position of hepatocytes on the porto-central axis in 3-dimensional space. It is superimposed on the serial section under investigation to relate local staining density to position on the porto-central axis and obtain the gene expression gradient. The method was used to determine the expression gradient of 2 periportal and 2 pericentral enzymes and their response to fasting. The total distance image was used to measure the length of the porto-central axis, which was approximately 210 m in mice and found to decrease 13% after 1 day of starvation. The method can be applied to any tissue component that can be stained quantitatively. (H EPATOLOGY 2004;39:343-352.)
Altered gene expression in acute systemic inflammation detected by complete coverage of the human liver transcriptome (p 353-364)
Cédric Coulouarn, Grégory Lefebvre, Céline Derambure, Thierry Lequerre, Michel Scotte, Arnaud Francois, Dominique Cellier, Maryvonne Daveau, Jean-Philippe Salier
The goal of the current study was to provide complete coverage of the liver transcriptome with human probes corresponding to every gene expressed in embryonic, adult, and/or cancerous liver. We developed dedicated tools, namely, the Liverpool nylon array of complementary DNA (cDNA) probes for approximately 10,000 nonredundant genes and the LiverTools database. Inflammation-induced transcriptome changes were studied in liver tissue samples from patients with an acute systemic inflammation and from control subjects. One hundred and fifty-four messenger RNAs (mRNA) correlated statistically with the extent of inflammation. Of these, 134 mRNA samples were not associated previously with an acute-phase (AP) response. The hepatocyte origin and proinflammatory cytokine responsiveness of these mRNAs were confirmed by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) in cytokine-challenged hepatoma cells. The corresponding gene promoters were enriched in potential binding sites for inflammation-driven transcription factors in the liver. Some of the corresponding proteins may provide novel blood markers of clinical relevance. The mRNAs whose level is most correlated with the AP extent ( P< .05) were enriched in intracellular signaling molecules, transcription factors, glycosylation enzymes, and up-regulated plasma proteins. In conclusion, the hepatocyte responded to the AP extent by fine tuning some mRNA levels, controlling most, if not all, intracellular events from early signaling to the final secretion of proteins involved in innate immunity. Supplementary material for this article can be found on the H EPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ). (H EPATOLOGY 2004;39:353-364.)
PAR 1antagonism protects against experimental liver fibrosis. Role of proteinase receptors in stellate cell activation (p 365-375)
Stefano Fiorucci, Elisabetta Antonelli, Eleonora Distrutti, Beatrice Severino, Roviezzo Fiorentina, Monia Baldoni, Giuseppe Caliendo, Vincenzo Santagada, Antonio Morelli, Giuseppe Cirino
In fibroblasts, thrombin induces collagen deposition through activation of a G-protein-coupled receptor, proteinase-activated receptor 1 (PAR 1). In the current study, we examined whether PAR 1antagonism inhibits hepatic stellate cell (HSC) activation in vitro and whether it protects against fibrosis development in a rodent model of cirrhosis. A rat HSC line was used for in vitro studies whereas cirrhosis was induced by bile duct ligation (BDL). The current results demonstrated that HSCs express PAR 1, as well as proteinase-activated receptors 2 (PAR 2) and 4 (PAR 4), and that all three PARs were up-regulated in response to exposure to growth factor in vitro . Exposure to thrombin and to SFLLRN-(SF)-NH 2, a PAR 1agonist, and GYPGKF (GY)-NH 2, a PAR 4agonist, triggered HSC proliferation and contraction, as well as monocyte chemotactic protein-1 (MCP-1) production and collagen I synthesis and release. These effects were inhibited by the PAR 1antagonist. Administration of this antagonist, 1.5 mg/kg/d, to BDL rats reduced liver type I collagen messenger RNA (mRNA) expression and surface collagen by 63%, as measured by quantitative morphometric analysis. Similarly, hepatic and urinary excretion of hydroxyproline was reduced significantly by the PAR 1antagonist. In conclusion, PAR sregulates HSC activity; development of PAR antagonists might be a feasible therapeutic strategy for protecting against fibrosis in patients with chronic liver diseases. (H EPATOLOGY 2004;39:365-375.)
Activation of NF- B and STAT3 in rat oval cells during 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration (p 376-385)
Aránzazu Sánchez, Valentina M. Factor, Insa S. Schroeder, Peter Nagy, Snorri S. Thorgeirsson
Proliferation and differentiation of hepatic stem cell progenies ( i.e. , oval cells) sustain liver regeneration when the replicative and functional capacity of hepatocytes is impaired. The signaling pathways that control stem cell activation remain poorly understood. In this study, we investigated the involvement of nuclear factor-kappa B (NF- B) and signal transducer and activator of transcription 3 (STAT3) in oval cell-mediated liver regeneration induced by 2-acetylaminofluorene/partial hepatectomy (AAF/PH) protocol. Using OV1 as a marker for identification and sorting of oval cells, we established that both NF- B and STAT3 were highly activated in the OV1 +cell population. Three distinct subpopulations of oval cells were defined as OV1 low , OV1 medium , and OV1 high , based on the intensity of OV1 staining. Quantitative polymerase chain reaction analysis revealed that they represent different stages of oval cell differentiation along hepatocyte lineage. OV1 low cells displayed the least differentiated phenotype as judged by high expression of c-kit and lack of hepatocytic differentiation markers, whereas OV1 high cells lost c-kit expression, were more proliferative, and acquired more mature hepatocytic phenotype. Notably, NF- B was activated uniformly in all three subpopulations of oval cells. In contrast, phosphorylation of STAT3 was detected only in OV1 high cells. In conclusion, transcriptional activity supported by NF- B and STAT3 is required for oval cell activation, expansion, and differentiation. The differential induction of NF- B and STAT3 point to a distinct role for these transcription factors at different stages of hepatic stem cell differentiation. (H EPATOLOGY 2004;39:376-385.)
Uncoupling protein-2 deficiency promotes oxidant stress and delays liver regeneration in mice (p 386-392)
Masayoshi Horimoto, Péter Fülöp, Zoltán Derdák, Jack R. Wands, György Baffy
The control of liver regeneration remains elusive. Because reactive oxygen species (ROS) are able to mediate cell growth arrest and activate proteins that inhibit the cell cycle, ROS production may have a negative impact on liver regeneration. We examined how liver regeneration is affected by uncoupling protein-2 (UCP2), an inner mitochondrial membrane carrier that senses and negatively regulates superoxide production. Liver regeneration was monitored up to 5 days and was found to be significantly delayed in UCP2 -/- mice after partial hepatectomy. Apoptosis rates in UCP2 +/+ and UCP2 - /- liver remnants were similar, while parameters of cell proliferation indicated a diminished response in UCP2 - /- mice with corresponding changes in the expression of key cell cycle regulatory proteins and prolonged activation of stress-responsive protein kinase p38. Levels of malondialdehyde, a marker of ROS generation and oxidant stress, were elevated in UCP2 - /- livers at every examined time point. Liver remnants of UCP2 + /+ mice 48 hours post-hepatectomy showed a fourfold increase in the expression of UCP2 protein primarily detected in hepatocytes. In conclusion, our results suggest that absent or insufficient UCP2 function in the regenerating liver results in increased ROS production and negatively modulates the control of cell cycle. (H EPATOLOGY 2004;39:386-392.)
Extracellular ATP activates c-jun N-terminal kinase signaling and cell cycle progression in hepatocytes (p 393-402)
Sundararajah Thevananther, Hongdan Sun, Duo Li, Vijaya Arjunan, Samir S. Awad, Samuel Wyllie, Tracy L. Zimmerman, John A. Goss, Saul J. Karpen
Partial hepatectomy leads to an orchestrated regenerative response, activating a cascade of cell signaling events necessary for cell cycle progression and proliferation of hepatocytes. However, the identity of the humoral factors that trigger the activation of these pathways in the concerted regenerative response in hepatocytes remains elusive. In recent years, extracellular ATP has emerged as a rapidly acting signaling molecule that influences a variety of liver functions, but its role in hepatocyte growth and regeneration is unknown. In this study, we sought to determine if purinergic signaling can lead to the activation of c-jun N-terminal kinase (JNK), a known central player in hepatocyte proliferation and liver regeneration. Hepatocyte treatment with ATP S, a nonhydrolyzable ATP analog, recapitulated early signaling events associated with liver regeneration - that is, rapid and transient activation of JNK signaling, induction of immediate early genes c-fos and c-jun , and activator protein-1 (AP-1) DNA-binding activity. The rank order of agonist preference, UTP>ATP>ATP S, suggests that the effects of extracellular ATP is mediated through the activation of P2Y2 receptors in hepatocytes. ATP S treatment alone and in combination with epidermal growth factor (EGF) substantially increased cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expression and hepatocyte proliferation in vitro. Extracellular ATP as low as 10 nM was sufficient to potentiate EGF-induced cyclin D1 expression. Infusion of ATP by way of the portal vein directly activated hepatic JNK signaling, while infusion of a P2 purinergic receptor antagonist prior to partial hepatectomy inhibited JNK activation. In conclusion, extracellular ATP is a hepatic mitogen that can activate JNK signaling and hepatocyte proliferation in vitro and initiate JNK signaling in regenerating liver in vivo . These findings have implications for enhancing our understanding of novel factors involved in the initiation of regeneration, liver growth, and development. (H EPATOLOGY 2004;39:393-402.)
Oval cells compensate for damage and replicative senescence of mature hepatocytes in mice with fatty liver disease (p 403-411)
Shiqi Yang, Ayman Koteish, Huizhi Lin, Jiawen Huang, Tania Roskams, Valina Dawson, Anna Mae Diehl
Hepatic steatosis may have a generally benign prognosis, either because most hepatocytes are not significantly injured or mechanisms to replace damaged hepatocytes are induced. To determine the relative importance of these mechanisms, we compared hepatocyte damage and replication in ethanol-fed and ob/ob mice with very indolent fatty liver disease to that of healthy control mice and PARP-1 -/- mice with targeted disruption of the DNA repair enzyme, poly(ADP-ribose) polymerase. Compared to the healthy controls, both groups with fatty livers had significantly higher serum alanine aminotransferase values, hepatic mitochondrial H 2O2production, and hepatocyte oxidative DNA damage. A significantly smaller proportion of the hepatocytes from fatty livers entered S phase when cultured with mitogens. Moreover, this replicative senescence was not reversed by treating cultured hepatocytes with agents ( i.e ., betaine or leptin) that improve liver disease in intact ethanol-fed or leptin-deficient mice. Hepatocytes from PARP1 -/- mice also had more DNA damage and reduced DNA synthesis in response to mitogens. However, neither mice with fatty livers nor PARP-1-deficient mice had atrophic livers. All of the mice with senescent mature hepatocytes exhibited hepatic accumulation of liver progenitor (oval) cells and oval cell numbers increased with the demand for hepatocyte replacement. Therefore, although hepatic oxidant production and damage are generally increased in fatty livers, expansion of hepatic progenitor cell populations helps to compensate for the increased turnover of damaged mature hepatocytes. In conclusion, these results demonstrate that induction of mechanisms to replace damaged hepatocytes is important for limiting the progression of fatty liver disease. (H EPATOLOGY 2004;39:403-411.)
Dual role of tumor necrosis factor- in hepatic ischemia-reperfusion injury: Studies in tumor necrosis factor- gene knockout mice (p 412-421)
Narci Teoh, Jacqueline Field, Jaim Sutton, Geoffrey Farrell
Although hepatic ischemia-reperfusion (IR) injury is partially mediated by tumor necrosis factor- (TNF), we recently found that low-dose TNF before IR is hepatoprotective. We examined the seemingly conflicting roles of TNF in mediating liver injury in a partial hepatic IR model using TNF gene knockout ( TNF ko) mice to allow TNF replacement at specified times. Compared with wild-type mice, TNF ko mice exhibit minimal alanine aminotransferase release and few hepatonecrotic lesions during the early (time, 2 hours) and late (time, 24 hours) phases of IR. TNF ko mice differed from wild-type mice in that TNF ko mice exhibited no activation or induction of nuclear factor- B, p38, cyclin D1, or proliferating cell nuclear antigen after IR. A single low-dose TNF injection 1 minute before the onset of hepatic ischemia restored hepatic IR injury in TNF ko mice. To clarify the importance of TNF for hepatoprotection, preconditioning (10 minutes of ischemia and 10 minutes of reperfusion) was performed before the onset of IR for TNF ko mice whose capacity to undergo IR injury had been restored by TNF replacement. Ischemic preconditioning failed to protect these mice from TNF-augmented IR injury; however, following the administration of intravenous TNF (1 g per kg body weight, which mimics the early increase in hepatic and plasma TNF levels that is mobilized by ischemic preconditioning), significant hepatoprotection against both the early and late phases of TNF-augmented IR injury was observed. In conclusion, TNF appears to mediate both the early and late phases of liver injury in hepatic IR, but it also is an essential mediator of hepatoprotective effects brought about by ischemic preconditioning. (H EPATOLOGY 2004;39:412-421.)
Blockade of receptor for advanced glycation end product (RAGE) attenuates ischemia and reperfusion injury to the liver in mice (p 422-432)
Shan Zeng, Nikki Feirt, Michael Goldstein, James Guarrera, Nikalesh Ippagunta, Udeme Ekong, Hao Dun, Yan Lu, Wu Qu, Ann Marie Schmidt, Jean C. Emond
Hepatic ischemia/reperfusion (I/R) injury associated with liver transplantation and hepatic resection is characterized by hepatocellular damage and a deleterious inflammatory response. In this study, we examined whether receptor for advanced glycation end product (RAGE) activation is linked to mechanisms accentuating inflammation on I/R in a murine model of total hepatic ischemia. Animals treated with soluble RAGE (sRAGE), the extracellular ligand-binding domain of RAGE, displayed increased survival after total hepatic I/R compared with vehicle treatment. TUNEL assay and histologic analysis revealed that blockade of RAGE was highly protective against hepatocellular death and necrosis on I/R; in parallel, proliferating cell nuclear antigen was enhanced in livers of mice treated with sRAGE. Rapid activation of p38, p44/42, stress-activated protein kinase and c-Jun N-terminal kinase mitogen-activated protein kinases, signal transducer and activator of transcription-3, and nuclear translocation of activator protein-1 was evident at early times on I/R. In the remnants of sRAGE-treated livers, however, activation of each of these signaling and transcription factor pathways was strikingly decreased. sRAGE-treated remnants displayed enhanced activation of nuclear factor B, in parallel with increased transcripts for the proregenerative cytokine, tumor necrosis factor- . In conclusion, these data suggest that RAGE modulates hepatic I/R injury, at least in part by activation of key signaling pathways linked to proinflammatory and cell death-promoting responses. We propose that blockade of this pathway may represent a novel strategy to attenuate injury in hepatic I/R and to facilitate regeneration. (H EPATOLOGY 2004;39:422-432.)
Chronic liver disease in murine hereditary tyrosinemia type 1 induces resistance to cell death (p 433-443)
Arndt Vogel, Inge E.T. van den Berg, Muhsen Al-Dhalimy, John Groopman, Ching-Nan Ou, Olga Ryabinina, Mihail S. Iordanov, Milton Finegold, Markus Grompe
The murine model of hereditary tyrosinemia type 1 (HT1) was used to analyze the relationship between chronic liver disease and programmed cell death in vivo . In healthy fumarylacetoacetate hydrolase deficient mice ( Fah -/- ), protected from liver injury by the drug 2-(2- nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), the tyrosine metabolite homogentisic acid (HGA) caused rapid hepatocyte death. In contrast, all mice survived the same otherwise lethal dose of HGA if they had preexisting liver damage induced by NTBC withdrawal. Similarly, Fah -/- animals with liver injury were also resistant to apoptosis induced by the Fas ligand Jo-2 and to necrosis-like cell death induced by acetaminophen (APAP). Molecular studies revealed a marked up-regulation of the antiapoptotic heat shock proteins (Hsp) 27, 32, and 70 and of c-Jun in hepatocytes of stressed mice. In addition, the p38 and Jun N-terminal kinase (JNK) stress-activated kinase pathways were markedly impaired in the cell-death resistant liver. In conclusion, these results provide evidence that chronic liver disease can paradoxically result in cell death resistance in vivo . Stress-induced failure of cell death programs may lead to an accumulation of damaged cells and therefore enhance the risk for cancer as observed in HT1 and other chronic liver diseases. (H EPATOLOGY 2004;39:433-443.)
CYP2E1 overexpression alters hepatocyte death from menadione and fatty acids by activation of ERK1/2 signaling (p 444-455)
Jörn M. Schattenberg, Yongjun Wang, Raina M. Rigoli, Dennis R. Koop, Mark J. Czaja
Chronic oxidative stress induced by overexpression of the cytochrome P450 isoform 2E1 (CYP2E1) has been implicated in hepatocyte injury and death. However, the mechanism by which CYP2E1 overexpression may promote cell death is unknown. Acute oxidative stress activates mitogen-activated protein kinases (MAPK), suggesting that chronic oxidant generation by CYP2E1 may regulate cellular responses through these signaling pathways. The effect of CYP2E1 overexpression on MAPK activation and their function in altering death responses of CYP2E1-overexpressing hepatocytes were investigated. Chronic CYP2E1 overexpression led to increased extracellular signal-regulated kinase 1/2 (ERK1/2) activation constitutively and in response to oxidant stress from the superoxide generator menadione. CYP2E1-overexpressing cells were resistant to menadione toxicity through an ERK1/2-dependent mechanism. Similar to menadione, the polyunsaturated fatty acid (PUFA) arachidonic acid (AA) induced an increased activation of ERK1/2 in hepatocytes that overexpressed CYP2E1. However, CYP2E1-overexpressing cells were sensitized to necrotic death from AA and the PUFA -linolenic acid, but not from saturated or monounsaturated fatty acids. Death from PUFA resulted from oxidative stress and was blocked by inhibition of ERK1/2, but not p38 MAPK or activator protein-1 signaling. CYP2E1 expression induced ERK1/2 activation through increased epidermal growth factor receptor (EGFR)/c-Raf signaling. Inhibition of EGFR signaling reversed CYP2E1-induced resistance to menadione and sensitization to AA toxicity. In conclusion, chronic CYP2E1 overexpression leads to sustained ERK1/2 activation mediated by EGFR/c-Raf signaling. This adaptive response in hepatocytes exposed to chronic oxidative stress confers differential effects on cellular survival, protecting against menadione-induced apoptosis, but sensitizing to necrotic death from PUFA. (H EPATOLOGY 2004;39;444-445.)
Bile acids enhance the activity of the insulin receptor and glycogen synthase in primary rodent hepatocytes (p 456-463)
Song Iy Han, Elaine Studer, Seema Gupta, Youwen Fang, Liang Qiao, Weiqun Li, Steven Grant, Philip B. Hylemon, Paul Dent
Previously, we demonstrated that deoxycholic acid (DCA)-induced ERK1/2 and AKT signaling in primary hepatocytes is a protective response. In the present study, we examined the regulation of the phosphatidylinositol 3 (PI3) kinase/AKT/glycogen synthase (kinase) 3 (GSK3)/glycogen synthase (GS) pathway by bile acids. In primary hepatocytes, DCA activated ERBB1 (the epidermal growth factor receptor), ERBB2, and the insulin receptor, but not the insulin-like growth factor 1 (IGF-1) receptor. DCA-induced activation of the insulin receptor correlated with enhanced phosphorylation of insulin receptor substrate 1, effects that were both blocked by the insulin receptor inhibitor AG1024 and by expression of the dominant negative IGF-1 receptor (K1003R), which inhibited in trans . Expression of the dominant negative IGF-1 receptor (K1003R) also abolished DCA-induced AKT activation. Bile acid-induced activation of AKT and phosphorylation of GSK3 were blunted by the ERBB1 inhibitor AG1478 and abolished by AG1024. Bile acids caused activation of GS to a similar level induced by insulin (50 nM); both were blocked by inhibition of insulin receptor function and the PI3 kinase/AKT/GSK3 pathway. In conclusion, these findings suggest that bile acids and insulin may cooperate to regulate glucose storage in hepatocytes. (H EPATOLOGY 2004;39:456-463.)
Liver Failure & Liver Disease
The effect of hypertonic sodium chloride on intracranial pressure in patients with acute liver failure (p 464-470)
Nicholas Murphy, Georg Auzinger, William Bernel, Julia Wendon
Acute liver failure (ALF) is a rare condition characterized by the development of encephalopathy in the absence of chronic liver disease. Cerebral edema occurs in up to 80% of patients with Grade IV encephalopathy. In the current prospective randomized controlled clinical trial, we examined the effect of induced hypernatremia on the incidence of intracranial hypertension (IH) in patients with ALF. Thirty patients with ALF and Grade III or IV encephalopathy were randomized. Patients in Group 1 ( n= 15) received the normal standard of care. Patients in Group 2 ( n= 15) received standard care and hypertonic saline (30%) via infusion to maintain serum sodium levels of 145-155 mmol/L. Intracranial pressure (ICP) was monitored in all patients with a subdural catheter (Camino Systems, San Diego, CA) for up to 72 hours after inclusion. Serum sodium levels became significantly different from the levels observed in the control group at 6 hours ( P< .01). Over the first 24 hours, norepinephrine dose increased relative to baseline in the control group ( P< .001; 13 patients) but not in the treatment group. ICP decreased significantly relative to baseline over the first 24 hours in the treatment group ( P= .003; 13 patients) but not in the control group. The incidence of IH, defined as a sustained increase in ICP to a level of 25 mm Hg or greater, was significantly higher in the control group ( P= .04). In conclusion, induction and maintenance of hypernatremia can reduce the incidence and severity of IH in patients presenting with ALF. (H EPATOLOGY 2004;39:464-470.)
Worsening of cerebral hyperemia by the administration of terlipressin in acute liver failure with severe encephalopathy (p 471-475)
Debbie L. Shawcross, Nathan A. Davies, Rajeshwar P. Mookerjee, Peter C. Hayes, Roger Williams, Alistair Lee, Rajiv Jalan
There is increasing evidence that terlipressin is useful in patients with cirrhosis and hepatorenal syndrome, but there are no data of its use in patients with acute liver failure (ALF) in whom hepatorenal syndrome is common. Although terlipressin produces systemic vasoconstriction, it produces cerebral vasodilatation and may increase cerebral blood flow (CBF). Increased CBF contributes to intracranial hypertension in patients with ALF. The aim of this study was to evaluate the safety of terlipressin in patients with ALF with respect to cerebral hemodynamics. Six successive patients with ALF were ventilated electively for grade IV hepatic encephalopathy. Patients were monitored invasively and CBF was measured (Kety-Schmidt technique). Measurements were made before and at 1, 3, and 5 hours after intravenous (single bolus) administration of terlipressin (0.005 mg/kg), median, 0.25 mg (range, 0.2-0.3 mg). There was no significant change in heart rate, mean arterial pressure, or cardiac output. CBF and jugular venous oxygen saturation both increased significantly at 1 hour ( P= 0.016). Intracranial pressure increased significantly at 1 hour ( P= 0.031), returning back to baseline values at 2 hours. In conclusion, administration of terlipressin, at a dose that did not alter systemic hemodynamics, resulted in worsening of cerebral hyperemia and intracranial hypertension in patients with ALF and severe hepatic encephalopathy. These data suggest the need to exercise extreme caution in the use of terlipressin in these patients in view of its potentially deleterious consequences on cerebral hemodynamics. (H EPATOLOGY 2004;39:471-475.)
Chronic liver disease mortality in the United States, 1990-1998 (p 476-483)
Sirenda Vong, Beth P. Bell
In 1998, chronic liver disease (CLD) was the tenth leading cause of death in the U.S. Alcohol and hepatitis C are thought to be important etiologies. However, traditional methods for calculating CLD mortality rates from death certificates may underestimate hepatitis C-related CLD mortality. We studied patterns of CLD deaths reported from 1990 through 1998, using an expanded definition that included death certificates where CLD, viral hepatitis, or CLD-related sequelae were reported as the underlying cause. We calculated overall age-specific and age-adjusted mortality rates, and according to demographic characteristics and recorded causes, and evaluated trends using linear regression modeling. CLD mortality declined 5% overall from 1990 through 1994 (12.1 to 11.6/100,000; P= 0.002), but remained unchanged from 1995 through 1998 ( P= 0.366). Decreases were similar for all causes except hepatitis C, for which rates increased 220% from 1993 to 1998 (0.57 to 1.67/100,000). Rates declined in all racial-ethnic groups except American Indians and Alaska Natives (AI/AN), among whom rates were unchanged. Of 30,933 CLD deaths in 1998, 39% were coded as alcohol related, 15% as hepatitis C, 4% as hepatitis B, and 44% had no recorded cause. Age-adjusted rates were higher among males (47.6/100,000) than females (32.2/100,000) and among Hispanics (19.1/100,000) compared with non-Hispanics (10.8/100,000). Rates among AI/AN (28.7/100,000) were more than twice those of African Americans and whites (12.9/100,000 and 11.5/100,000, respectively). In conclusion, 1998 CLD deaths and the proportion attributable to viral hepatitis increased by 23% and 19%, respectively, compared with traditional methods. Mortality declines of the early 1990s were not sustained after 1994. Large disparities in CLD mortality remain, particularly among American Indians and Alaska Natives. (H EPATOLOGY 2004;39:476-483.)
A sequential study of serum bacterial DNA in patients with advanced cirrhosis and ascites (p 484-491)
Rubén Francés, Susana Benlloch, Pedro Zapater, José M. González, Beatriz Lozano, Carlos Muñoz, Sonia Pascual, Juan A. Casellas, Francisco Uceda, José M Palazón, Fernando Carnicer, Miguel Pérez-Mateo, José Such
Bacterial translocation is currently considered the main pathogenic mechanism leading to spontaneous bacterial peritonitis in patients with advanced cirrhosis and ascites. However, to the authors' knowledge there is no information regarding the characteristics of this process in humans. The goals of the current study were to pursue partially identified bacterial DNA in blood (what the authors consider molecular evidence of bacterial translocation) through its relative quantification in a 72-hour study period by using real-time polymerase chain reaction (PCR). A consecutive series of 17 patients with advanced cirrhosis and culture-negative, nonneutrocytic ascites were studied. Therapeutic paracentesis was performed at the time of admission, and blood samples were obtained at baseline and every 8 hours in a 3-day period. Bacterial DNA was detected by a PCR-based method, relatively quantified by real-time PCR, and identified by automated nucleotide sequencing. Seven of 17 patients demonstrated the simultaneous presence of bacterial DNA in blood and ascitic fluid at the time of admission. After therapeutic paracentesis was performed, bacterial DNA persisted in the blood for a minimum of 24 hours, and was reported to last as long as 72 hours in some patients. In addition, different patterns of bacterial DNA appearance and clearance from the blood were identified. The nucleotide sequencing process demonstrated that bacteria detected in the first sample were identical to those noted in subsequent detections over time. In conclusion, bacterial translocation is a single-species, dynamic process that appears to develop in a subgroup of patients with advanced cirrhosis. (H EPATOLOGY 2004;39:484-491.)
Increased duodenal expression of divalent metal transporter 1 and iron-regulated gene 1 in cirrhosis (p 492-499)
Katherine Anne Stuart, Gregory Jon Anderson, David Michael Frazer, Therese Luna Murphy, Lawrie William Powell, Linda Maria Fletcher, Darrell Henry Crawford
Hepatic hemosiderosis and increased iron absorption are common findings in cirrhosis. It has been proposed that a positive relation exists between intestinal iron absorption and the development of hepatic hemosiderosis. The current study investigated the duodenal expression of the iron transport molecules divalent metal transporter 1 ( DMT1 [IRE ]), iron-regulated gene 1 ( Ireg1 [ferroportin ]), hephaestin , and duodenal cytochrome b ( Dyctb ) in 46 patients with cirrhosis and 20 control subjects. Total RNA samples were extracted from duodenal biopsy samples and the expression of the iron transport genes was assessed by ribonuclease protection assays. Expression of DMT1 and Ireg1 was increased 1.5 to 3-fold in subjects with cirrhosis compared with iron-replete control subjects. The presence of cirrhosis per se and serum ferritin (SF) concentration were independent factors that influenced the expression of DMT1 . However, only SF concentration was independently associated with Ireg1 expression. In cirrhosis, the expression of DMT1 and Ireg1 was not related to the severity of liver disease or cirrhosis type. There was no correlation between the duodenal expression of DMT1 and Ireg1 and the degree of hepatic siderosis. In conclusion, the presence of cirrhosis is an independent factor associated with increased expression of DMT1 but not Ireg1 . The mechanism by which cirrhosis mediates this change in DMT1 expression has yet to be determined. Increased expression of DMT1 may play an important role in the pathogenesis of cirrhosis-associated hepatic iron overload. (H EPATOLOGY 2004;39:492-499.)
Determinants of survival and the effect of portosystemic shunting in patients with Budd-Chiari syndrome (p 500-508)
Sarwa Darwish Murad, Dominique-Charles Valla, Piet C. de Groen, Guy Zeitoun, Judith A.M. Hopmans, Elizabeth B. Haagsma, Bart van Hoek, Bettina E. Hansen, Frits R. Rosendaal, Harry L.A. Janssen
Budd-Chiari syndrome (BCS) is a rare disorder that is characterized by hepatic venous outflow obstruction. The aim of this study was to assess determinants of survival and to evaluate the effect of portosystemic shunting. In this international multicenter study, 237 patients with BCS, diagnosed between 1984 and 2001, were investigated. Univariate, multivariate, and time-dependent Cox regression analyses were performed. Overall survival at 1, 5, and 10 years was 82% (95% CI, 77%-87%), 69% (95% CI, 62%-76%), and 62% (95% CI, 54%-70%), respectively. Encephalopathy, ascites, prothrombin time, and bilirubin were independent determinants of survival. A prognostic classification combining these factors could identify three classes of patients (classes I-III). The 5-year survival rate was 89% (95% CI, 79%-99%) for class I, 74% (95% CI, 65%-83%) for class II, and 42% (95% CI, 28%-56%) for class III. Anticoagulants were administered to 72%; only for patients in class I was this associated with a trend toward improved survival (relative risk [RR], 0.14; 95% CI, 0.02-1.21). Portosystemic shunting was performed in 49% of the patients (n = 117); only for patients in class II, time-dependent analyses suggested an improved survival (RR, 0.63; 95% CI, 0.26-1.49). In conclusion, at the time of diagnosis, patients with BCS can be classified into good (I), intermediate (II), and poor (III) prognostic classes, according to simple baseline clinical and laboratory parameters. Our results suggest an improved survival after surgical portosystemic shunting for patients with an intermediate prognosis (class II). (H EPATOLOGY 2004;39:500-508.)
Structured treatment interruption in patients with alveolar echinococcosis (p 509-517)
Stefan Reuter, Andreas Buck, Burkhard Manfras, Wolfgang Kratzer, Hanns Martin Seitz, Kassa Darge, Sven Norbert Reske, Peter Kern
In human alveolar echinococcosis (AE), benzimidazoles are given throughout life because they are only parasitostatic. It has been a longstanding goal to limit treatment, and recent reports suggest that, in selected cases, benzimidazoles may be parasitocidal. Previously, we showed that positron -emission tomography (PET) using [ 18 F]fluoro-deoxyglucose discriminates active from inactive lesions in AE. We have now performed a 3-year prospective study in 23 patients and conducted a structured treatment interruption in those without signs of PET activity. Disease progression was further assessed by ultrasound, computerized tomography, laboratory parameters, and clinical examination.We found PET-negative lesions in 15 of 23 patients and benzimidazoles were discontinued in these patients. After 18 months, patients were reevaluated, and, of the 15 initially PET-negative patients, 8 showed either new activity on PET (n = 6) or signs of clinical progression (n = 2). Reinitiation of benzimidazoles halted parasite growth again. No further progression was detected after 36 months. PET had a sensitivity of 91% for the detection of active lesions. In conclusion, despite successful suppression of metabolic activity, in most cases benzimidazoles do not kill the parasite. PET is a reliable tool for assessing metabolic activity and for timely detection of relapses. Neither duration of treatment, kind of treatment, lesion size, calcifications, or regressive changes reliably indicate parasite death. We discourage the discontinuation of benzimidazoles in inoperable AE even after many years of treatment. However, patients with a poor compliance of benzimidazole intake or patients suffering from side effects to benzimidazoles might be assessed for PET negativity. If permanent discontinuation of benzimidazoles is attempted, the course of disease should be followed by PET. (H EPATOLOGY 2004;39:509-517.)
Cancer-associated molecular signature in the tissue samples of patients with cirrhosis (p 518-527)
Jin Woo Kim, Qinghai Ye, Marshonna Forgues, Yidong Chen, Anuradha Budhu, Jessica Sime, Lorne J. Hofseth, Rashmi Kaul, Xin Wei Wang
Several types of aggressive cancers, including hepatocellular carcinoma (HCC), often arise as a multifocal primary tumor. This suggests a high rate of premalignant changes in noncancerous tissue before the formation of a solitary tumor. Examination of the messenger RNA expression profiles of tissue samples derived from patients with cirrhosis of various etiologies by complementary DNA (cDNA) microarray indicated that they can be grossly separated into two main groups. One group included hepatitis B and C virus infections, hemochromatosis, and Wilson's disease. The other group contained mainly alcoholic liver disease, autoimmune hepatitis, and primary biliary cirrhosis. Analysis of these two groups by the cross-validated leave-one-out machine-learning algorithms revealed a molecular signature containing 556 discriminative genes ( P< .001). It is noteworthy that 273 genes in this signature (49%) were also significantly altered in HCC ( P< .001). Many genes were previously known to be related to HCC. The 273-gene signature was validated as cancer-associated genes by matching this set to additional independent tumor tissue samples from 163 patients with HCC, 56 patients with lung carcinoma, and 38 patients with breast carcinoma. From this signature, 30 genes were altered most significantly in tissue samples from high-risk individuals with cirrhosis and from patients with HCC. Among them, 12 genes encoded secretory proteins found in sera. In conclusion, we identified a unique gene signature in the tissue samples of patients with cirrhosis, which may be used as candidate markers for diagnosing the early onset of HCC in high-risk populations and may guide new strategies for chemoprevention. Supplementary material for this article can be found on the H EPATOLOGY website
Hepatic expression of ANG2 RNA in metastatic colorectal cancer (p 528-539)
Minoru Ogawa, Hirofumi Yamamoto, Hiroaki Nagano, Yasuhiro Miyake, Yurika Sugita, Taishi Hata, Byung-no Kim, Chew Yee Ngan, Bazarragchaa Damdinsuren, Masakazu Ikenaga, Masataka Ikeda, Masayuki Ohue, Shoji Nakamori, Mitsugu Sekimoto, Masato Sakon, Nariaki Matsuura, Morito Monden
We examined the RNA content of the gene encoding angiopoietin (Ang)-2, a modifier of angiogenesis, in hepatic metastases of colorectal cancer (CRC) to explore the role of this protein in neovascularization of metastatic foci. Metastatic CRC exhibited notable blood flow and tumor vessel formation at tumor frontiers. Reverse-transcription polymerase chain reaction assays indicated that the ANG2 RNA content was greater in metastatic CRC than in primary CRC. Investigation of metastatic foci using laser capture microdissection revealed that the RNA content of ANG2 , but not ANG1 , increased from the bordering liver region to the periphery of the metastatic disease, and also from the periphery to the intermediate portion of the metastatic lesion; immunohistochemical analysis confirmed that there was a corresponding gradual increase in Ang-2 protein expression. Tie-2, a receptor for angiopoietins, was preferentially expressed in the bordering liver region rather than in metastatic CRC. Vascular endothelial growth factor (VEGF) also exhibited an expression pattern similar to that of Ang-2, and there was a significant correlation between the RNA content of ANG2 and that of VEGF in dissected samples ( P= .002). Western blot analysis suggested that expression of Ang-1, Ang-2, Tie-2, and VEGF may be regulated at a transcriptional level. The increase in ANG2 RNA content from the peripheral portion of the tumor to the intermediate portion, coinciding with the decrease in recruitment of periendothelial supporting cells around the vascular endothelial cells, suggests that Ang-2 may play a role in the immaturity of tumor vessels. In conclusion, the current study suggests that Ang-2 and VEGF may cooperate to enhance the formation of new blood vessels in metastases of CRC to the liver. (H EPATOLOGY 2004;39:528-539.)
Detection and identification of tumor-associated protein variants in human hepatocellular carcinomas (p 540-549)
Evelyn Zeindl-Eberhart, Sibylle Haraida, Sibylle Liebmann, Peter Roman Jungblut, Stephanie Lamer, Doris Mayer, Gundula Jäger, Stephen Chung, Hartmut Manfred Rabes
The proteomic approach is a valuable tool to detect and identify proteins that are associated with cancer. In previous investigations on experimentally induced rat hepatomas, we detected aldose reductase-like protein (ARLP) as a highly significant marker protein. Our present study was intended to look for the presence of similar tumor-associated marker proteins on human hepatocellular carcinomas (HCC). We found several novel tumor-associated protein variants that represent members of the aldo-keto reductase (AKR) superfamily. Human aldose reductase-like protein-1 (hARLP-1) was the most prominent tumor-associated AKR member detected in HCC by 2-dimensional electrophoresis (2-DE) and identified by mass spectrometric fingerprinting. The enzyme was found in 4 distinct forms (hARLP-1, 36/7.4 (kd/pI); hARLP-2, 36/7.2; hARLP-3, 36/6.4; and hARLP-4, 33/7.35). In addition, a human aldose reductase-like protein (hARLP-5, 36/7.6) was identified that differed from hARLP-1 by 1 amino acid (D313N), indicating 2 allelic forms of the human aldose reductase-like gene. A novel antibody directed against common parts of the hARLPs revealed hARLP reactivity in human HCC by immunohistochemistry. Furthermore, aldose reductase (AR) was identified and characterized as a tumor-associated variant. In conclusion, in all investigated human HCCs at least one of the various types of the described tumor-associated proteins of the AKR superfamily was clearly present. Of these HCC samples, 95% were positive for hARLPs as proven by 2-DE analysis and/or by use of the antibody directed against hARLP. Thus, hARLP is a strong candidate for use as an immunohistochemical diagnostic marker of human HCC. (H EPATOLOGY 2004;39:540-549.)
Save Article Copyright © 2004 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for February 2004 , Volume 126, Number 2
Rapid Communication
Use of 5-fluorouracil and survival in patients with microsatellite-unstable colorectal cancer
John M. Carethers, E. Julieta Smith, Cynthia A. Behling, Lanchinh Nguyen, Akihiro Tajima, Ryan T. Doctolero, Betty L. Cabrera, Ajay Goel, Christian A. Arnold, Katsumi Miyai, C. Richard Boland
Background & Aims :5-Fluorouracil improves mortality in stage III colorectal cancer patients. In vitro studies suggest that microsatellite instability influences cell survival after 5-fluorouracil treatment. We investigated the survival influence of 5-fluorouracil in patients with microsatellite instability-high tumors. Methods :We collected data and tumors on 204 consecutive stage II and III colorectal cancer patients from registries at the University of California and Veterans Administration hospitals in San Diego, California, from 1982 to 1999. Archival DNA was extracted, and microsatellite instability was assessed by National Cancer Institute-recommended markers. Cox proportional hazard modeling was used to determine survival associations for microsatellite instability and 5-fluorouracil treatment status. Results :We identified 36 microsatellite instability-high (17.6%) and 168 non-microsatellite instability-high tumors (82.4%). Microsatellite instability-high tumors were significantly associated with proximal colon location, presence of mucin, and surrounding lymphoid reaction. Univariate and multivariate analyses showed no survival difference between microsatellite instability-high and non-microsatellite instability-high groups (hazard ratio, 1.04; P= 0.88). Dichotomized by use of 5-fluorouracil, there was increased risk of death in patients who received no adjuvant chemotherapy (hazard ratio, 2.02; P= 0.02). However, the benefit of 5-fluorouracil was different between microsatellite instability-high and non-microsatellite instability-high groups. Patients with non-microsatellite instability-high tumors who received 5-fluorouracil had better survival compared with patients who were not treated ( P< 0.05). Conversely, patients with microsatellite instability-high tumors who were treated with 5-fluorouracil had no survival difference compared with patients without treatment ( P= 0.52). Conclusions :There is improved survival in patients with non-microsatellite instability-high tumors after 5-fluorouracil-based chemotherapy that does not extend to patients with microsatellite instability-high tumors. The microsatellite instability status of a patient’s colorectal cancer may indicate differences in 5-fluorouracil-based chemosensitivity; this is consistent with in vitro studies.
Comparison of scheduled and episodic treatment strategies of infliximab in Crohn’s disease
Paul Rutgeerts, Brian G. Feagan, Gary R. Lichtenstein, Lloyd F. Mayer, Stefan Schreiber, Jean Frederic Colombel, Daniel Rachmilewitz, Douglas C. Wolf, Allan Olson, Weihang Bao, Stephen B. Hanauer
Background & aims : This analysis of Crohn’s disease patients treated with infliximab in ACCENT I compared episodic and scheduled treatment strategies under conditions that simulate clinical practice. Methods : After 5 mg/kg infliximab at week 0, 573 patients were randomized to infusions at weeks 2 and 6 and every 8 weeks until week 46 of placebo (episodic), infliximab 5 mg/kg at weeks 2 and 6 followed by 5 mg/kg (5 mg/kg scheduled) every 8 weeks, or infliximab 5 mg/kg at weeks 2 and 6 followed by 10 mg/kg (10 mg/kg scheduled) every 8 weeks. At or after week 14, treatment could be given with a dose of infliximab 5 mg/kg higher upon loss of response. Results : The efficacy of scheduled infliximab therapy was better than episodic treatment. Crohn’s Disease Activity Index (CDAI) scores were consistently significantly better in the 10 mg/kg scheduled maintenance group from weeks 10 to 54, and response and remission rates (combined scheduled) were significantly higher from weeks 10 to 30. A greater proportion of patients achieved complete mucosal healing at week 54 ( P= 0.041). A lower proportion developed antibodies to infliximab in the scheduled groups than in the episodic group (9% [5 mg/kg], 6% [10 mg/kg], 28% [episodic], respectively). Scheduled strategy patients had fewer Crohn’s disease-related hospitalizations ( P= 0.014) and surgeries ( P= 0.01) than episodic strategy patients. Conclusions : The scheduled infliximab groups, particularly the 10 mg/kg group, had better CDAI and Inflammatory Bowel Disease Questionnaire (IBDQ) responses than those in the episodic group. Both scheduled groups had fewer hospitalizations, higher rates of mucosal healing, and fewer developed antibodies than those in the episodic group, with no increase in side effects.
Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease
William S. Mow, Eric A. Vasiliauskas, Ying-Chao Lin, Phillip R. Fleshner, Konstantinos A. Papadakis, Kent D. Taylor, Carol J. Landers, Maria T. Abreu-Martin, Jerome I. Rotter, Huiying Yang, Stephan R. Targan
Background & Aims : Crohn’s disease patients can be characterized by antibody responses against Crohn’s disease-related bacterial sequence, Escherichia coli outer membrane porin C, Saccharomyces cerevisiae (oligomannan), and neutrophil nuclear antigens. Our aim was to determine whether expression of antibodies against Crohn’s disease-related bacterial sequence and Escherichia coli outer membrane porin C is associated with distinct phenotypic manifestations. Methods : Sera from 303 patients were tested for antibodies to the Crohn’s disease-related bacterial sequence (I2), anti- Escherichia coli outer membrane porin C, anti- Saccharomyces cerevisiae, and perinuclear antineutrophil cytoplasmic antibodies and for 3 Crohn’s disease-associated variants of the NOD2 gene (R702W, G908R, and 1007fs) and compared with clinical data. Results : Patients expressing I2 were more likely to have fibrostenosing Crohn’s disease (64.4% vs. 40.7%; P< 0.001) and to require small bowel surgery (62.2% vs. 37.4%; P< 0.001). Patients with anti- Escherichia coli outer membrane porin C were more likely to have internal perforating disease (50.0% vs. 30.7%; P= 0.001) and to require small bowel surgery (61.4% vs. 44.2%; P= 0.003). Anti-Crohn’s disease-related bacterial sequence was independently associated with fibrostenosis ( P= 0.027) and small bowel surgery ( P= 0.01), whereas anti- Escherichia coli outer membrane porin C was independently associated with internal perforations ( P< 0.006). Patients positive for I2, anti- Escherichia coli outer membrane porin C, and anti- Saccharomyces cerevisiae were the most likely to have undergone small bowel surgery (72.0%; odds ratio, 8.6; P< 0.001) compared with patients without reactivity (23.0%). When the presence and magnitude of antibody responses were considered, 90% of patients with small bowel disease who required surgery had high levels of I2, Escherichia coli outer membrane porin C, and oligomannan antibodies, compared with only 18.2% with low-titer responses ( P< 0.001). Conclusions : I2 and anti- Escherichia coli outer membrane porin C are associated with Crohn’s disease phenotypes, and patients with the highest level of serum reactivity toward an increasing number of microbiota have the greatest frequency of strictures, internal perforations, and small bowel surgery.
Prostanoids, ornithine decarboxylase, and polyamines in primary chemoprevention of familial adenomatous polyposis
Francis M. Giardiello, Robert A. Casero, Jr, Stanley R. Hamilton, Linda M. Hylind, Jill D. Trimbath, Deborah E. Geiman, Katharine R. Judge, Walter Hubbard, G. Johan A. Offerhaus, Vincent W. Yang
Background & Aims : Familial adenomatous polyposis because of germline mutation of the adenomatous polyposis coli gene is characterized by development of colorectal adenomas and, ultimately, colorectal cancer. The usefulness of colorectal mucosal compounds to predict the effect on adenoma development of primary chemoprevention with the nonsteroidal anti-inflammatory drug sulindac was evaluated. Methods : A randomized, double-blind, placebo-controlled study of 41 subjects genotypically affected with familial adenomatous polyposis but phenotypically unaffected was conducted. Patients received either sulindac or placebo for 48 months, and development of new adenomas was evaluated. The levels of 5 prostanoids, ornithine decarboxylase, and polyamines were measured serially in normal-appearing rectal mucosa. Results : There were no statistically significant differences between treatment groups in baseline levels of prostanoids, ornithine decarboxylase, or polyamines. At conclusion of the study, 4 of 5 prostaglandin levels were statistically significantly lower in the sulindac group than in the placebo group. Among the subset of patients taking sulindac, 3 of 5 prostaglandin levels were statistically significantly lower in patients who were polyp free than in those who developed polyps. By contrast, there were no statistically significant differences in ornithine decarboxylase or polyamines between treatment groups or in those on sulindac who were polyp free compared with those who developed polyps. Conclusions : Colorectal mucosal prostaglandin levels, but not ornithine decarboxylase or polyamines, may be valuable biomarkers to assess appropriate drug dosage and medication compliance in patients undergoing primary chemoprevention therapy with sulindac. Reduction of mucosal prostaglandin levels may be necessary to achieve chemopreventive benefit from this agent.
Independent influences of body mass and gastric volumes on satiation in humans
Silvia Delgado-aros, Filippo Cremonini, Janet E. Castillo, Heather J. Chial, Duane D. Burton, Irene Ferber, Michael Camilleri
Background & Aims : We assessed the association of body mass and gastric volumes (fasting and postprandial) with satiation and postprandial symptoms. Methods : Healthy obese and nonobese subjects underwent measurement of caloric intake at maximum satiation; postprandial symptoms were measured with visual analogue scales 30 minutes after a meal. Gastric volume during fasting and after 300 mL of Ensure was measured with technetium-99m single-photon emission computed tomography imaging. We used multiple regression analysis to assess the associations among variables. Results : Among 134 participants (81 women and 53 men), the median age was 26 years (range, 1258 years), and the median body mass index was 24 kg/m 2(range, 1748 kg/m 2). Increased body mass index, but not height, was associated with delayed satiation ( P< 0.003, adjusted for sex). Overweight and obese subjects ingested, on average, 225 ± 57 more kilocalories (945 ± 239 kJ) at maximum satiation compared with normal weight individuals. Increased fasting gastric volume was not associated with body mass index or height, but it was significantly associated with delayed satiation ( P= 0.001, adjusted for body mass index and sex). An increase of 50 mL in the fasting gastric volume was associated with 114 ± 32 kcal (479 ± 134 kJ) more ingested at maximum satiation. Increased body mass index was associated with lower fullness scores 30 minutes after a meal ( P= 0.0012, adjusted for sex and volume of Ensure ingested). In contrast, scores of postprandial bloating and pain were higher with increased body mass index (both P< 0.05, adjusted for sex and volume of Ensure ingested). Conclusions : Greater body mass index and fasting gastric volume are associated with reduced satiation. Increased body mass index or height was not associated with greater gastric volumes.
Addition of a second endoscopic treatment following epinephrine injection improves outcome in high-risk bleeding ulcers
Xavier Calvet, Mercedes Vergara, Enric Brullet, Javier P. Gisbert, Rafel Campo
Background & Aims : Endoscopic therapy reduces the rebleeding rate, the need for surgery, and the mortality in patients with peptic ulcer and active bleeding or visible vessel. Injection of epinephrine is the most popular therapeutic method. Guidelines disagree on the need for a second hemostatic procedure immediately after epinephrine; although it seems to reduce further bleeding, its effects on morbidity, surgery rates, and mortality remain unclear. The aim of this study was to perform a systematic review and meta-analysis to determine whether the addition of a second procedure improves hemostatic efficacy and/or patient outcomes after epinephrine injection. Methods : An extensive search for randomized trials comparing epinephrine alone vs. epinephrine plus a second method was performed in MEDLINE and EMBASE and in the abstracts of the AGA Congresses between 1990 and 2002. Selected articles were included in a meta-analysis. Results : Sixteen studies including 1673 patients met inclusion criteria. Adding a second procedure reduced the further bleeding rate from 18.4% to 10.6% (Peto odds ratio 0.53, 95% CI: 0.400.69) and emergency surgery from 11.3% to 7.6% (OR: 0.64, 95% CI: 0.460.90). Mortality fell from 5.1% to 2.6% (OR: 0.51, 95% CI: 0.310.84). Subanalysis showed that the risk of further bleeding decreased regardless of which second procedure was applied. In addition, the risk was reduced in all subgroups, although reduction was more evident in high-risk patients and when no scheduled follow-up endoscopies were performed. Conclusions : Additional endoscopic treatment after epinephrine injection reduces further bleeding, need for surgery, and mortality in patients with bleeding peptic ulcer.
Severity of inflammation is a risk factor for colorectal neoplasia in ulcerative colitis
Matthew Rutter, Brian Saunders, Kay Wilkinson, Steve Rumbles, Gillian Schofield, Michael Kamm, Christopher Williams, Ashley Price, Ian Talbot, Alastair Forbes
Background & Aims : Patients with ulcerative colitis are at increased risk of colorectal cancer. It is widely believed that this is secondary to colonic inflammation. However, the severity of colonic inflammation has never been shown to be a risk factor. Methods : We devised a case-control study of patients with long-standing extensive ulcerative colitis to examine various potential risk factors for neoplasia. All cases of colorectal neoplasia detected from our surveillance program between January 1, 1988, and January 1, 2002, were studied (n = 68). Each patient was matched with 2 control patients from the same surveillance population (n = 136). Matching was for sex, colitis extent, age at onset, duration of colitis, and year of index surveillance colonoscopy. Segmental colonoscopic and histological inflammation was recorded by using a simple score (0, normal; 1, quiescent/chronic inflammation; and 2, 3, and 4, mild, moderate, and severe active inflammation, respectively). Other data collected included history of primary sclerosing cholangitis, family history of colorectal cancer, and smoking and drug history (mesalamine 5-aminosalicylic acid, azathioprine, and folate). Results : Univariate analysis showed a highly significant correlation between the colonoscopic (odds ratio, 2.5; P= 0.001) and histological (odds ratio, 5.1; P< 0.001) inflammation scores and the risk of colorectal neoplasia. No other factors reached statistical significance. On multivariate analysis, only the histological inflammation score remained significant (odds ratio, 4.7; P< 0.001). Conclusions : In long-standing extensive ulcerative colitis, the severity of colonic inflammation is an important determinant of the risk of colorectal neoplasia. Endoscopic and histological grading of inflammation could allow better risk stratification for surveillance programs.
Clinical-liver, Pancreas, and Biliary Tract
Diabetes increases the risk of chronic liver disease and hepatocellular carcinoma
Hashem B. El-serag, Thomas Tran, James E. Everhart
Background & Aims : An association between diabetes and chronic liver disease has been reported. However, the temporal relationship between these conditions remains unknown. Methods : We identified all patients with a hospital discharge diagnosis of diabetes between 1985 and 1990 using the computerized records of the Department of Veterans Affairs. We randomly assigned 3 patients without diabetes for every patient with diabetes. We excluded patients with concomitant liver disease. The remaining cohort was followed through 2000 for the occurrence of chronic nonalcoholic liver disease (CNLD) and hepatocellular carcinoma (HCC). Hazard rate ratios (HRR) were determined in Cox proportional hazard survival analysis. Results : The study cohort comprised 173,643 patients with diabetes and 650,620 patients without diabetes. Most were men (98%). Patients with diabetes were older (62 vs. 54 years) than patients without diabetes. The incidence of chronic nonalcoholic liver disease was significantly higher among patients with diabetes (incidence rate: 18.13 vs. 9.55 per 10,000 person-years, respectively, P< 0.0001). Similar results were obtained for HCC (incidence rate: 2.39 vs. 0.87 per 10,000 person-years, respectively, P< 0.0001). Diabetes was associated with an HRR of 1.98 (95% CI: 1.88 to 2.09, P< 0.0001) of CNLD and an HRR of 2.16 (1.86 to 2.52, P< 0.0001) of hepatocellular carcinoma. Diabetes carried the highest risk among patients with longer than 10 years of follow-up. Conclusions : Among men with diabetes, the risk of CNLD and HCC is doubled. This increase in risk is independent of alcoholic liver disease, viral hepatitis, or demographic features.
Improved clinical outcome using polytetrafluoroethylene-coated stents for tips: Results of a randomized study
Christophe Bureau, Juan carlos Garcia-Pagan, Philippe Otal, Gilles Pomier-Layrargues, Valérie Chabbert, Carlos Cortez, Pierre Perreault, Jean marie Péron, Juan G. Abraldes, Louis Bouchard, José Ignacio Bilbao, Jaume Bosch, Hervé Rousseau, Jean pierre Vinel
Background & Aims : A 50% dysfunction rate at 1 year is one of the main drawbacks of the transjugular intrahepatic portosystemic shunt procedure. Preliminary experimental and clinical studies suggest that the use of stents covered with polytetrafluoroethylene could tremendously decrease this risk. Methods : Eighty patients with cirrhosis and uncontrolled bleeding (n = 23), recurrent bleeding (n = 25), or refractory ascites (n = 32) were randomized to be treated by transjugular intrahepatic portosystemic shunts with either a polytetrafluoroethylene-covered stent (group 1; 39 patients) or a usual uncovered prosthesis (group 2; 41 patients). Follow-up Doppler ultrasound was scheduled at day 7, at 1 month, and then every 3 months for 2 years. Angiography and portosystemic pressure gradient measurements were performed 6, 12, and 24 months after the transjugular intrahepatic portosystemic shunt procedure and whenever dysfunction was suspected. Dysfunction was defined as a >50% reduction of the lumen of the shunt at angiography or a portosystemic pressure gradient >12 mm Hg. Results : After a median follow-up of 300 days, 5 patients (13%) in group 1 and 18 (44%) in group 2 experienced shunt dysfunction ( P< 0.001). Clinical relapse occurred in 3 patients (8%) in group 1 and 12 (29%) in group 2 ( P< 0.05). Actuarial rates of encephalopathy were 21% in group 1 and 41% in group 2 at 1 year (not significant). Estimated probabilities of survival were 71% and 60% at 1 year and 65% and 41% at 2 years in groups 1 and 2, respectively (not significant). Conclusions : The use of polytetrafluoroethylene-covered prostheses improves transjugular intrahepatic portosystemic shunt patency and decreases the number of clinical relapses and reinterventions without increasing the risk of encephalopathy.
Basic-alimentary Tract
Altered control of gastric acid secretion in gastrin-cholecystokinin double mutant mice
Duan Chen, Chun-Mei Zhao, Rolf Håkanson, Linda C. Samuelson, Jens F. Rehfeld, Lennart Friis-Hansen
Background & Aims : Three pathways control gastric acid secretion: the gastrin-enterochromaffin-like (ECL) cell axis, the vagus-parietal cell axis, and the cholecystokinin (CCK)-D cell axis. Mice lacking gastrin or both gastrin and CCK were examined to determine the role of the hormones. Methods : Acid was measured after pylorus ligation, and biopsies from gastrin knockout (KO), gastrin-CCK double-KO, and wild-type (WT) mice were collected for biochemical, immunocytochemical, and electron-microscopic examination. Results : The ECL cells were inactive in both groups of mutant mice but the cell number was unaffected. Both parietal cell number and level of H +/K +-ATPase messenger RNA (mRNA) were reduced in the mutant strains, but gastrin-CCK double-KO mice displayed more active parietal cells and larger acid output than the gastrin KO mice. The acid response to histamine in double-KO mice was unchanged whereas that to gastrin was diminished, but it could be restored by infusion of gastrin. Oxyntic D-cell density was the same in both mutant strains, but the D cells were more active in the gastrin KO than in the double-KO mice. CCK infusion in gastrin-CCK double-KO mice raised the somatostatin mRNA level and inhibited acid secretion to the level seen in gastrin KO mice. Vagotomy and atropine abolished acid secretion in all 3 groups of mice. Conclusions : Lack of gastrin impairs the gastrin-ECL axis, whereas lack of gastrin and CCK impairs both hormonal pathways. In the gastrin-CCK double-KO mice, acid secretion is only controlled by cholinergic vagal stimulation, which normalizes the acid output.
High-molecular-weight polyethylene glycol prevents lethal sepsis due to intestinal Pseudomonas aeruginosa
Licheng Wu, Olga Zaborina, Alex Zaborin, Eugene B. Chang, Mark Musch, Christopher Holbrook, James Shapiro, Jerrold R. Turner, Guohui Wu, Ka yee C. Lee, John C. Alverdy
Background & Aims : During stress, erosion of protective intestinal mucus occurs in association with adherence to and disruption of the intestinal epithelial barrier by invading opportunistic microbial pathogens. The aims of this study were to test the ability of a high-molecular-weight polyethylene glycol compound, polyethylene glycol 1520, to protect the intestinal epithelium against microbial invasion during stress. Methods : The ability of polyethylene glycol 1520 to protect the intestinal epithelium against the opportunistic pathogen Pseudomonas aeruginosa was tested in cultured Caco-2 cells. Bacterial virulence gene expression, bacterial adherence, and transepithelial electrical resistance were examined in response to apical inoculation of P. aeruginosa onto Caco-2 cells. Complementary in vivo studies were performed in a murine model of lethal sepsis due to intestinal P. aeruginosa in which surgical stress (30% hepatectomy) was combined with direct inoculation of P. aeruginosa into the cecum. Results : High-molecular-weight polyethylene glycol (polyethylene glycol 1520) conferred complete protection against the barrier-dysregulating effects of P. aeruginosa in Caco-2 cells. Intestinal application of polyethylene glycol 1520 in stressed mice protected against the lethal effects of intestinal P. aeruginosa . Mechanisms of this effect seem to involve the ability of polyethylene glycol 1520 to distance P. aeruginosa from the intestinal epithelium and render it completely insensate to key environmental stimuli that activate its virulence. Conclusions : High-molecular-weight polyethylene glycol has the potential to function as a surrogate mucin within the intestinal tract of a stressed host by inhibiting key interactive events between colonizing microbes and their epithelial cell targets.
Leptin reduces the development of the initial precancerous lesions induced by azoxymethane in the rat colonic mucosa
Thomas Aparicio, Sandra Guilmeau, Hélène Goiot, Annick Tsocas, Jean-Pierre Laigneau, André Bado, Iradj Sobhani, Thérèse Lehy
Background & Aims :Recent studies suggest that leptin, a hormone involved in food intake regulation, released into the circulation and gastrointestinal juice, may be a growth factor for intestine and may be involved in carcinogenesis; however, data are contradictory. This study investigates in rat colonic mucosa (1) the effects of hyperleptinemia on epithelial cell proliferation and development of aberrant crypts, earliest preneoplastic lesions, and (2) whether luminal leptin affects cell proliferation. Methods :Leptin (1 mg/kg/d) or vehicle was administered systemically by miniosmotic pump in Fischer 344 rats either for 7 days (BrdU-labeling indices study) or 23 days (azoxymethane-induced colonic lesions study). The effects of injections or continuous infusion of leptin into the colon were also studied. Results :In systemic leptin-treated rats, plasma leptin levels were 4- to 5-fold increased ( P< 0.008 to P< 0.001); labeling indices were higher in proximal colon than in pair-fed control rats ( P= 0.006) but unaffected in distal colon. Unexpectedly, in azoxymethane-treated rats, leptin significantly inhibited aberrant crypt foci formation in the middle and distal colon compared with controls ( P= 0.006). Under these conditions, plasma insulin levels were reduced by 41%58%, but gastrin levels were unchanged. In controls, luminal immunoreactive leptin reached the colon. A 3.6-fold increase in intraluminal leptin had no effect on epithelial cell proliferation. Conclusions :This study provides the first evidence that leptin reduces the development of chemically induced precancerous lesions in colon, perhaps through decreased insulinemia, and thus does not support an important role for leptin in carcinogenesis promotion. Moreover, the study indicates that leptin is not a potent growth factor for normal intestine.
Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor
Jay R. Thiagarajah, Talmage Broadbent, Emily Hsieh, Alan S. Verkman
Background & Aims : The cystic fibrosis transmembrane conductance regulator (CFTR) provides an important apical route for Cl secretion across intestinal epithelia. A thiazolidinone-type CFTR blocker (CFTR inh -172) reduced cholera toxin-induced fluid accumulation in mouse intestinal loops. Here, we characterize the efficacy and pharmacodynamics of CFTR inh -172 in blocking cAMP and cGMP induced Cl /fluid secretion in rodent and human intestine. Methods & Results : CFTR inh -172 inhibited cAMP and cGMP agonist induced short-circuit current by >95% in T84 colonic epithelial cells (K I~ 3 µmol/L) and in mouse and human intestinal sheets (K I~ 9 µmol/L). A single intraperitoneal injection of CFTR inh -172 (200 µg) blocked intestinal fluid secretion in a rat closed-loop model by >90% for cholera toxin and >70% for STa Escherichia coli toxin. In mice, CFTR inh -172 (20 µg) inhibited cholera toxin-induced intestinal fluid secretion by 90% (persistence t 1/2 ~10 hours, K I~ 5 µg) and STa toxin by 75% (K I~ 10 µg). Tissue distribution and pharmacokinetic studies indicated intestinal CFTR inh -172 accumulation facilitated by enterohepatic circulation. An oral CFTR inh -172 preparation reduced fluid secretion by >90% in a mouse open-loop cholera model. Conclusions : A small molecule CFTR blocker markedly reduced intestinal ion and fluid secretion caused by cAMP/cGMP-dependent bacterial enterotoxins. CFTR inhibition may thus reduce fluid secretion in infectious secretory diarrheas.
Clinical-liver, Pancreas, and Biliary Tract
Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis
Daniel Rachmilewitz, Kyoko Katakura, Fanny Karmeli, Tomoko Hayashi, Constantin Reinus, Bernard Rudensky, Shizuo Akira, Kiyoshi Takeda, Jongdae Lee, Kenji Takabayashi, Eyal Raz
Background & Aims : We tested whether the attenuation of experimental colitis by live probiotic bacteria is due to their immunostimulatory DNA, whether toll-like receptor (TLR) signaling is required, and whether nonviable probiotics are effective. Methods : Methylated and unmethylated genomic DNA isolated from probiotics (VSL-3), DNAse-treated probiotics and Escherichia coli (DH5 ) genomic DNA were administered intragastricly (i.g.) or subcutaneously (sc) to mice prior to the induction of colitis. Viable or -irradiated probiotics were administered i.g. to wild-type mice and mice deficient in different TLR or in the adaptor protein MyD88, 10 days prior to administration of dextran sodium sulfate (DSS) to their drinking water and for 7 days thereafter. Results : ntragastric and sc administration of probiotic and E. coli DNA ameliorated the severity of DSS-induced colitis, whereas methylated probiotic DNA, calf thymus DNA, and DNase-treated probiotics had no effect. The colitis severity was attenuated to the same extent by i.g. delivery of nonviable -irradiated or viable probiotics. Mice deficient in MyD88 did not respond to -irradiated probiotics. The severity of DSS-induced colitis in TLR2 and TLR4 deficient mice was significantly decreased by i.g. administration of -irradiated probiotics, whereas, in TLR9-deficient mice, -irradiated probiotics had no effect. Conclusions : The protective effects of probiotics are mediated by their own DNA rather than by their metabolites or ability to colonize the colon. TLR9 signaling is essential in mediating the anti-inflammatory effect of probiotics, and live microorganisms are not required to attenuate experimental colitis because nonviable probiotics are equally effective.
Basic-liver, Pancreas, and Biliary Tract
Hepatitis C virus core and nonstructural proteins induce fibrogenic effects in hepatic stellate cells
Ramó Bataller, Yong-han Paik, Jeffrey N. Lindquist, John J. Lemasters, David A. Brenner
Background & Aims : The mechanisms by which hepatitis C virus (HCV) induces liver fibrosis are unknown. Hepatocytes secrete HCV proteins, which may interact with hepatic stellate cells (HSCs). Our aims were to investigate whether HCV proteins induce fibrogenic effects on HSCs. Methods & Results : Human-activated HSCs expressed messenger RNA (mRNA) for the putative HCV receptors CD81, LDL receptor, and C1q receptor as assessed by RT-PCR. Incubation of activated but not quiescent human HSCs with recombinant core and NS3 protein increased intracellular calcium concentration and reactive oxygen species production, as well as stimulated intracellular signaling pathways. Adenoviruses encoding core and nonstructural proteins (NS3-NS5) were used to express HCV proteins in HSCs. Expression of core protein increased cell proliferation in a Ras/ERK and PI3K/AKT dependent manner. In contrast, NS3-NS5 protein expression preferentially induced proinflammatory actions, such as increased chemokine secretion and expression of intercellular cell adhesion molecule type 1 (ICAM-1) through the NF- B and c-Jun N-terminal kinase pathways. These effects were attenuated by antioxidants. Infection of freshly isolated rat HSCs with adenovirus-encoding core protein resulted in accelerated cell activation, as assessed by -smooth muscle actin expression. Moreover, adenovirus-encoding core and NS3-NS5 proteins increased the secretion of bioactive TGF 1 and the expression of procollagen 1(I) in early cultured rat HSCs, as assessed by ELISA and RNase protection assay, respectively. Conclusions : HCV core and nonstructural proteins regulate distinct biologic functions in HSCs. A direct interaction between HCV proteins and HSCs may contribute to HCV-induced liver fibrosis.
Trafficking of the bile salt export pump from the Golgi to the canalicular membrane is regulated by the p38 MAP kinase
Ralf Kubitz, Gerrit Sütfels, Thomas Kühlkamp, Ralf Kölling, Dieter Häussinger
Background & Aims : Bile secretion depends on the delivery and removal of transporter proteins to and from the canalicular membrane. Trafficking of the bile salt export pump (BSEP) to the canalicular membrane was investigated in HepG2 cells and rat hepatocytes. Methods : Subcellular localization of BSEP was determined by confocal laser scanning microscopy using different BSEP antibodies. Results : Ten percent of untreated HepG2 cells developed pseudocanaliculi, but only 15% of these pseudocanaliculi contained BSEP, which largely colocalized with the Golgi marker GM130. Cycloheximide, an inhibitor of protein translation, induced a microtubule- and p38 MAP kinase-dependent decrease of Golgi-associated BSEP, accompanied by a more than 2-fold increase in BSEP-positive pseudocanaliculi. Also, tauroursodeoxycholate (TUDC), which activates p38 MAP kinase (p38 MAPK , increased BSEP-positive pseudocanaliculi by more than 50% in rat sodium taurocholate cotransporting peptide (Ntcp)-transfected but not in untransfected HepG2 cells. The TUDC-dependent increase was sensitive to inhibitors of p38 MAPK and microtubules and involved Ca 2+ -independent protein kinase C isoforms as suggested by its sensitivity to Gö6850 but insensitivity to Gö6976. In isolated rat hepatocytes with intact bile secretion, no colocalization of rat isoforms of the bile salt export pump (Bsep) and Golgi was found, but colocalization occurred after inhibition of p38 MAPK and PKC, suggesting that Bsep trafficking to the canalicular membrane depends on the basal activity of these kinases in polarized cells. Conclusions : p38 MAPK regulates BSEP trafficking from the Golgi to the canalicular membrane, and the Golgi may serve as a BSEP pool in certain forms of cholestasis or when p38 MAPK activity is inhibited. Activation of p38 MAPK by TUDC can recruit Golgi-associated BSEP in line with its choleretic action.
Phosphatidylinositide 3-kinase regulates key pathologic responses to cholecystokinin in pancreatic acinar cells
Ilya Gukovsky, Jason H. Cheng, Kyung J. Nam, Oliver T. Lee, Aurelia Lugea, Lars Fischer, Josef M. Penninger, Stephen J. Pandol, Anna S. Gukovskaya
Background & Aims : Early events in the pancreatic acinar cell critical for development of pancreatitis include activation of the transcription factor nuclear factor B (NF- B), abnormal Ca 2+ responses, and trypsinogen activation. Mechanisms underlying these responses, which can be studied in isolated pancreatic acini stimulated with supraphysiologic doses of cholecystokinin (CCK-8), remain poorly understood. We here report that these responses are regulated by phosphatidylinositide 3-kinase (PI3K) .Methods : To inactivate PI3K, we used mice deficient in the catalytic PI3K subunit p110 as well as the PI3K inhibitors LY294002 and wortmannin. We measured Ca 2+ responses by using Fura-2, NF- B-binding activity by electromobility shift assay, I B degradation by Western blotting, and trypsinogen activation by fluorogenic assay. Results : CCK-induced intracellular Ca 2+ mobilization, Ca 2+ influx, trypsinogen, and NF- B activation were all diminished in pancreatic acini isolated from p110 / mice. Both in mouse and rat acini, these responses were inhibited by the PI3K inhibitors. The Ca 2+ signal and trypsinogen activation were similarly reduced in acini isolated from p110 / and p110 +/ mice compared with wild-type mice. By contrast, NF- B activation was inhibited in p110 / acini but not in p110 +/ acini. These differences indicate that the mechanism of NF- B regulation by PI3K differs from those for the Ca 2+ and trypsinogen responses. CCK-induced responses in p110 / acini were all further inhibited by LY294002, indicating the involvement of other PI3K isoform(s), in addition to PI3K .Conclusions : The results show that key pathologic responses of the pancreatic acinar cell are regulated by PI3K and suggest an important role for this PI3K isoform in pancreatitis.
Gastrointestinal cancers and neurofibromatosis type 1 features in children with a germline homozygous MLH 1 mutation
Steven Gallinger, Melyssa Aronson, Katayoon Shayan, Elyanne M. Ratcliffe, Justin T. Gerstle, Patricia C. Parkin, Heidi Rothenmund, Marina Croitoru, Ewa Baumann, Peter R. Durie, Rosanna Weksberg, Aaron Pollett, Robert H. Riddell, Bo Y. Ngan, Ernest Cutz, Alain E. Lagarde, Helen S.L. Chan
Heterozygous germline DNA mismatch repair gene mutations are typically associated with hereditary nonpolyposis colorectal cancer. The molecular hallmark of this syndrome is high-frequency microsatellite instability in the tumors. Rare childhood cases with homozygous or compound heterozygous DNA mismatch repair gene mutations have a described predisposition to leukemia, lymphoma, and brain tumors but not to gastrointestinal cancer. We have now characterized a family in which 2 children with a homozygous germline DNA mismatch repair gene mutation developed early-onset gastrointestinal cancers. The 11-year-old proband had café-au-lait macules and developed metastatic duodenal adenocarcinoma that arose in a tubulovillous adenoma. His 9-year-old sister with café-au-lait macules and axillary freckling presented with malignant colon polyps. A 6-year-old sister with café-au-lait macules, hairy nevi, and a plexiform neurofibroma of the tongue has no malignancies to date. The family history did not fulfill the Amsterdam criteria for hereditary nonpolyposis colorectal cancer, but 2 relatives in their 60s had gastric cancer and colorectal cancer, whereas the parents, who are first cousins, remain cancer free. The proband’s metastatic duodenal cancer and his sister’s malignant colon polyps had high-frequency microsatellite instability but had detectable MLH1, MSH2, and MSH6 proteins by immunohistochemistry. Because some germline DNA mismatch repair gene deficiencies are associated with apparently intact immunohistochemical DNA mismatch repair gene expression in tumors, we proceeded to DNA sequencing, which showed that all 3 children had a germline homozygous MLH1 missense mutation (exon 18, codon 687, CGG TGG), whereas both parents were heterozygous for this mutation.
Steatosis and hepatitis C virus: Mechanisms and significance for hepatic and extrahepatic disease
Amedeo Lonardo, Luigi E. Adinolfi, Paola Loria, Nicola Carulli, Giuseppe Ruggiero, Christopher P. Day
Nonalcoholic fatty liver disease (NAFLD) and hepatitis C virus (HCV)-related liver disease are common in the general population, but their concurrence is 2- to 3-fold higher than would be expected by chance alone. In patients with chronic HCV infection, steatosis is attributable to a variable combination of the mechanisms considered to play a role in the pathogenesis of NAFLDinsulin resistance in the obese and in the lean subjectalong with a direct effect of HCV on hepatic lipid metabolism that leads to triglyceride accumulation through inhibition of export proteins that are required for very low density lipoprotein (VLDL) assembly and secretion. Accumulating evidence suggests that steatosis contributes to the progression of fibrosis in HCV-related disease in a pattern similar to that observed in NAFLD. Potential mechanisms of this effect include the increased sensitivity of steatotic livers to oxidative stress and cytokine-mediated injury. Steatosis-related hepatic insulin resistance may also play a role through the profibrogenic effects of the compensatory hyperinsulinemia and provides a potential explanation for the association between HCV and type 2 diabetes mellitus. Indeed, an appreciation of the importance of fat in HCV has recently led to trials of adjuvant therapy for HCV directed at steatosis-associated disease mechanisms, with encouraging results reported for various modalities, including weight loss and antioxidants. Future therapy should be aimed at exploiting the interactions of HCV with host insulin and lipid metabolism, particularly in nonresponders to standard antiviral schedules.
Copyright © 2001-2004 by the American Gastroenterological Association. All rights reserved.
Table of Contents for Volume 40, Issue 2, February 2004
Biliary Tract and Cholestasis
Takeshi Saito et al.
Lack of evidence for reovirus infection in tissues from patients with biliary atresia and congenital dilatation of the bile duct
Background /Aims : To clarify the association between the reovirus infection of the hepatobiliary tree and the development of infantile obstructive cholangiopathy (IOC) including biliary atresia (BA) and congenital dilatation of the bile duct (CBD).
Methods : We designed reovirus common primers for nested RT-PCR based on the L3 gene segment. The spectrum and the sensitivity of common primers were evaluated with purified reoviral RNAs and reovirus mixed with stool samples. Then, nested RT-PCRs were performed with hepatobiliary and fecal samples obtained from patients with BA, CBD, and control diseases. Additionally, electron microscopy of stool samples was performed. Results : The L3 common primers could amplify cDNAs synthesized from RNAs of three prototypes of reovirus, and detect as much as 5.0?10 3plaque forming unit of serotype 3 Dearing strain in 100 mg of fecal samples. However, no amplification product was detected in 136 hepatobiliary tissues taken from 67 patients including 26 BAs and 28 CBDs, or in 65 fecal samples obtained from 15 patients including 10 BAs and 1 CBD. Additionally, viral particles were not found in any stool specimens by the electron microscope. Conclusions : These data do not suggest that reoviruses play a major role in the etiology of IOC or BA.
Keywords: Reovirus ;Biliary atresia ;Etiology ;RT-PCR ;Infantile obstructive cholangiopathy ;Stool ;Feces ;Electron microscope
Cell Biology, Metabolism and Transport
Stephan R. Vavricka et al.
The human organic anion transporting polypeptide 8 ( SLCO1B3 ) gene is transcriptionally repressed by hepatocyte nuclear factor 3 in hepatocellular carcinoma
Background/Aims : The organic anion transporting polypeptides (OATPs) mediate the uptake of numerous amphipathic compounds into hepatocytes. Our aim was to study the expression and regulation of OATP8 (OATP1B3, SLC21A8/SLCO1B3 ) and OATP-C (OATP1B1, SLC21A6/SLCO1B1 ) in hepatocellular carcinomas (HCC). Methods : RNA and protein levels in 13 paired HCC and adjacent non-tumor liver samples were quantified by real-time polymerase chain reaction or Western blot, respectively. The OATP8 and OATP-C gene promoters were characterized by luciferase reporter assays and electrophoretic mobility shift assays (EMSA). Results : The expression of OATP8 was decreased in 60% of HCC compared to surrounding non-tumor liver tissue, on both the mRNA and protein levels. Expression of the liver-enriched transcription factor hepatocyte nuclear factor 3 (HNF3 ) was increased in 70% of HCC and correlated inversely with OATP8 mRNA ( r=0.75, P<0.05) and protein. In contrast to OATP8, expression of OATP-C was not significantly decreased in HCC. In transfected Huh7 cells, OATP8 promoter activity was inhibited by 70% when HNF3 was cotransfected. An HNF3 binding site was located at nt 39/ 23 by EMSA. The OATP-C promoter was not inhibited by HNF3 . Conclusions : HNF3 represses transcription of the OATP8 but not the OATP-C gene, providing a mechanism for reduced expression of OATP8 in HCC.
Keywords: Human organic anion transporting polypeptide 8 ;Hepatocellular carcinoma ;Hepatocyte nuclear factor 3
Chronic Liver Diseases
Ion V. Deaciuc et al.
Large-scale gene profiling of the liver in a mouse model of chronic, intragastric ethanol infusion
Background/Aims : The mechanisms underlying alcohol-induced liver injury are not fully elucidated. An approach in this direction would consist of an all-inclusive assessment of gene expression in the liver. The purpose of this study was to perform a comprehensive analysis of gene expression in the livers of mice treated with ethanol by means of intragastric infusion. Methods : An ethanol- or glucose-enriched liquid diet was fed to animals for 4 weeks via a long-term gastrostomy catheter. The animals were killed and plasma alanine:2-oxoglutarate aminotransferase (ALT) assay, liver histology and total RNA analysis by microarray gene technology were performed. Results : Alcohol increased ALT, induced steatosis, necrosis and inflammation. A total of 12,423 genes were analyzed for expression out of which 4867 were expressed by the liver. Alcohol repressed expression of 11 genes, induced expression of 13 genes, and up- or down-regulated expression of 44 and 42 genes >2-fold, respectively. Gene expression analysis identified several genes that have not previously been tested for alcohol effects. Conclusions : This study: (i) expands the knowledge of mechanism(s) of action of ethanol; (ii) indicates novel pathways of ethanol action on the liver, and (iii) illustrates the utility of microarray gene analysis in hepatology research.
Keywords: Alcohol-induced liver injury ;Large-scale gene expression ;cDNA microarrays ;Steatohepatitis ;Necrosis ;Intragastric ethanol infusion ;Total RNA
Cirrhosis and its Complications
Mathias Plauth et al.
Weight gain after transjugular intrahepatic portosystemic shunt is associated with improvement in body composition in malnourished patients with cirrhosis and hypermetabolism
Background/Aims : To search for changes in body composition and energy metabolism associated with the repeatedly observed weight gain of cirrhotic patients after portosystemic shunting. Methods : Twenty-one patients were studied prospectively before and 6 and 12 months after transjugular intrahepatic portosystemic shunt (TIPS) to assess body cell mass by two independent methods (total body potassium counting: body cell mass determined by TBP, BCM TBP , bioelectric impedance analysis: body cell mass determined by BIA, BCM BIA ), muscle mass (anthropometry), resting energy expenditure (REE CALO ) by indirect calorimetry, and nutritional intake by dietary recall analysis. Results : Prior to TIPS patients were hypermetabolic in terms of measured vs. predicted REE (REE CALO median 1423 (range 1164-1838) vs. REE PRED 1279 (1067-1687) kcal; P<0.05) and their body cell mass was lower (19.1 (10.9-33.4) vs. 31.7 (16.8-47.1) kg; P=0.001). After TIPS body cell mass (BCM BIA ) increased to 23.5 (12.7-44.3) ( P<0.025) and 25.7 (14.2-39.7) kg ( P=0.05) at 6 and 12 months after TIPS and this was confirmed by total potassium counting (BCM TBP before TIPS: 18.8 (10.6-26.7) vs. 22.4 (12.9-28.5) kg at 6 months; P<0.01). Hypermetabolism persisted throughout the study period. Energy and protein intake increased significantly by 26 and 33%. Conclusions : An increase of prognostically relevant variables body cell and muscle mass contributes to the weight gain after TIPS in malnourished patients with cirrhosis and hypermetabolism.
Keywords: Body cell mass ;Nutritional state ;Encephalopathy ;Protein intake ;Bioelectrical impedance analysis
Hui-Chun Huang et al.
Chronic inhibition of nitric oxide increases the collateral vascular responsiveness to vasopressin in portal hypertensive rats
Background/Aims : Nitric oxide (NO), a potent vasodilator, plays a significant role in the vascular hyposensitivity to vasoconstrictors related to portal hypertension. Chronic NO inhibition ameliorates portal-systemic collaterals in portal hypertensive rats. This study investigated whether chronic NO inhibition by N G-nitro- L-arginine methyl ester (L-NAME) improves the portal-systemic collateral vascular responsiveness to arginine vasopressin (AVP) in portal hypertensive rats. Methods : Partially portal vein-ligated (PVL) rats received L-NAME in tap water (~25 mg/kg per day) or tap water only (control) since 2 days prior to until 7 days after PVL. Mean arterial pressure was measured on the 8th day. By in situ perfusion model, different concentrations of AVP (10 10 -10 7M) with a constant flow rate (20 ml/min) were applied to assess the perfusion pressure of collateral vessels. In another series, perfusion with different flow rates (5-30 ml/min) was used to obtain flow-pressure curves: the slopes represent collateral vascular resistances and higher resistances indicate less collaterals. Results : Mean arterial pressure was higher in the L-NAME-treated group than that of the control group ( P<0.05). As compared with the controls, L-NAME-treated rats achieved significantly higher perfusion pressures in response to AVP. In addition, chronic L-NAME treatment also induced an increase of collateral vascular resistance, suggesting the attenuation of portal-systemic shunting. Conclusions : Chronic NO inhibition ameliorates portal-systemic shunting and improves the collateral vascular responsiveness to AVP in portal hypertensive rats.
Keywords: Nitric oxide ;Portal hypertension ;Portal-systemic collaterals ;Vasopressin
Jens H. Henriksen, Flemming Bendtsen, Erik Feldager Hansen and Søren Møller
Acute non-selective -adrenergic blockade reduces prolonged frequency-adjusted Q-T interval (QT c) in patients with cirrhosis
Background /Aims : Earlier studies have shown a prolonged frequency-adjusted Q-T interval ( QT c>0.440s 1/2 ) in a substantial fraction of patients with cirrhosis. The effect of -blockade on QT cis unknown, and its determination was the aim of the study. Methods : Seventeen patients with cirrhosis received 80 mg propranolol orally during a haemodynamic investigation with measurements at baseline and 90 min after propranolol ingestion. Results :-Blockade reduced cardiac output ( 21%, P<0.001), heart rate ( 20%, P<0.001), and the hepatic venous pressure gradient (HVPG, 17%, P<0.02). The mean QT c=0.460s 1/2 was prolonged compared to 0.410 s 1/2 in age-matched controls ( P<0.01). Whereas QT cdecreased during -blockade in the cirrhotic patients (from 0.460 to 0.440 s 1/2 ,P<0.01), no effect was found in the subgroup with normal QT c(0.429 vs. 0.422 s 1/2 , ns), and a reduction was seen in the patients with prolonged QT c(from 0.488 to 0.456 s 1/2 ,P< 0.01). The percentage decrease in QT cwas related to the reduction in HVPG ( r=0.48, P=0.03) and cardiac output ( r=0.56, P=0.02). Conclusions : Acute non-selective -blockade reduces prolonged QT ctowards normal values in patients with cirrhosis. The clinical significance of QT creduction in arrhythmia is a topic for future research.
Keywords: -adrenergic blockade ;Cirrhosis ;Hyperdynamic circulation ;Prolonged QT cinterval
Debbie L. Shawcross, Nathan A. Davies, Roger Williams and Rajiv Jalan
Systemic inflammatory response exacerbates the neuropsychological effects of induced hyperammonemia in cirrhosis
Background/Aims Studies in acute liver failure show correlation between evidence of a systemic inflammatory response syndrome (SIRS) and progression of hepatic encephalopathy (HE). We tested the hypothesis that SIRS mediators, such as nitric oxide and proinflammatory cytokines, may exacerbate the neuropsychological effects of hyperammonemia in cirrhosis. Methods Ten patients with cirrhosis were studied, 24-36 h after admission with clinical evidence of infection, and following its resolution. Hyperammonemia was induced by oral administration of an amino-acid (aa) solution mimicking hemoglobin composition. Inflammatory mediators, nitrate/nitrite, ammonia, aa profiles and a battery of neuropsychological tests were measured. Results The hyperammonemia generated in response to the aa solution was similar prior to, and after resolution, of the inflammation ( P=0.77). With treatment of the infection there were significant reductions in white blood cell count (WBC), C-reactive protein (CRP), nitrate/nitrite, interleukin-6, interleukin-1 and tumour necrosis factor . Induced hyperammonemia resulted in significant worsening of the neuropsychological scores when patients showed evidence of SIRS but not after its resolution. Conclusions The significant deterioration of neuropsychological test scores following induced hyperammonemia during the inflammatory state, but not after its resolution, suggests that the inflammation and its mediators may be important in modulating the cerebral effect of ammonia in liver disease.
Keywords: Minimal hepatic encephalopathy ;Neuropsychological function ;Proinflammatory cytokines ;Amino-acid solution Inflammation and Fibrosis
Shinji Baba et al.
Commitment of bone marrow cells to hepatic stellate cells in mouse
Background/Aims : Recently, several cells found within the liver have been reported to derive from bone marrow (BM). This study sought to examine the commitment of BM cells to hepatic stellate cell (HSC) lineage in mouse liver. Methods : We transplanted BM cells from green fluorescent protein (GFP) transgenic mice into age-matched C57BL/J mice. Hepatic nonparenchymal cells were isolated from the livers of BM-transplanted mice using density gradient centrifugation with Nycodenz. The expression of lineage markers by the isolated cells was evaluated by RT-PCR and immunostaining. We then examined the histology of liver tissues obtained from BM-transplanted mice with and without carbon tetrachloride-induced injury. Results : GFP-expressing cells with intracytoplasmic lipid droplets comprised 33.4±2.3% of the cells isolated by density gradient centrifugation. These cells expressed the HSC lineage markers, such as desmin and glial fibrillary acidic protein (GFAP), by both RT-PCR and immunostaining. During a 7-day culture, GFP-positive cells began to express -smooth muscle actin, a marker of activated HSC. In the liver of BM-transplanted mice, GFP-positive nonparenchymal cells expressed GFAP and extended their process around hepatocytes. Upon liver injury, these cells also co-expressed desmin and -smooth muscle actin. Conclusions : Nonparenchymal cells, derived from transplanted BM, acquired HSC characteristics in both quiescent and activated states. Keywords: Hepatic stellate cells ;Bone marrow ;Green fluorescent protein ;Carbon tetrachloride
Inflammation and Fibrosis
David L. Suskind and Marcus O. Muench
Searching for common stem cells of the hepatic and hematopoietic systems in the human fetal liver: CD34 +cytokeratin 7/8 +cells express markers for stellate cells
Background/Aims The hematopoietic and hepatic systems are intertwined in the liver during fetal life. Cells expressing the hematopoietic stem cell marker CD34 and cytokeratin 7/8 (CK7/8) are hypothesized to be common stem cells for the hematopoietic and hepatic systems. Our aim was to determine if human fetal liver cells expressing CD34 and CK7/8 represent a common stem cell for both the hematopoietic and hepatic systems. Methods CD34 +CK7/8 +cells from midgestation livers were analyzed for the expression of various markers by flow cytometry and isolated based on their expression of CD34, nerve growth factor receptor (NGFR) and lack of CD45 expression. CD34 +CD38 hematopoietic stem cells were also isolated and cultured in the presence of various hepatopoietins. Results CD34 +CK7/8 +cells comprised 3.4-8.5% of the erythrocyte-depleted liver. CD34 +CK7/8 +cells had unique light-scatter properties compared to hematopoietic precursors and did not express most markers associated with hematopoietic cells. They did stain with CD13, CD59, NGFR, desmin and -smooth muscle actin. In culture, these cells had a stellate appearance. Cultured hematopoietic stem cells failed to generate hepatocytes. Conclusions CD34 +CK7/8 +cells are not common stem cells but rather appear to be hepatic stellate cells. A link between the hematopoietic and hepatic systems during fetal life requires further investigation.
Keywords: Human fetal liver ;Cytokeratins ;CD34 ;Nerve growth factor receptor ;Stellate cells ;Hematopoietic stem cells ;Hepatic stem cells Inflammation and Fibrosis
Liver Growth and Cancer
Kohei Ogawa et al.
Sodium butyrate enhances Fas-mediated apoptosis of human hepatoma cells
Background/Aims : Human hepatoma cells have been reported to be resistant to Fas-mediated apoptosis. Sodium butyrate (SB) induced apoptosis of several cancer cells. We investigated the effects of SB on Fas-mediated apoptosis of hepatoma cells. Methods : In hepatoma cells (HuH-6, HuH-7, Hep-G2, and PLC/PRF/5), susceptibility to Fas-mediated apoptosis and Fas expression were assessed. Caspase-3 activation and cell cycle progression were evaluated in HuH-6. A cDNA microarray assay was performed to screen the changes in the expression of mRNAs. Results : Pretreatment with SB caused an enhancement of the sensitivity to anti-Fas-mediated cytotoxicity, though it did not increase the expression of Fas. The cDNA microarray assay revealed up-regulation of pro-apoptotic Bik, Bak, Bid and c-Jun N-terminal protein kinase-1, and down-regulation of anti-apoptotic Bag-1 and cellular Fas-associated death domain-like interleukin-1 -converting enzyme inhibitor protein. In some molecules, expression of the proteins was confirmed by Western blotting. An increase in truncated-Bid accompanying the reduction in Bid was also observed. Conclusions : SB enhances the susceptibility of hepatoma cells to anti-Fas-mediated cytotoxicity by altering the mRNA and protein expression and/or the activation status of proteins that could be involved in the Fas signaling pathway. SB may have an important role in the elimination of hepatoma cells.
Keywords: Hepatoma ;Fas ;Apoptosis ;Sodium butyrate ;cDNA microarray assay ;Histone deacetylase inhibitor
Takashi Kato et al.
Involvement of natural killer T cells and granulocytes in the inflammation induced by partial hepatectomy
Background/Aims Natural killer T (NKT) cells are present in the liver of mice. We examined whether NKT cells and other leukocytes were associated with hepatic inflammation after partial hepatectomy. Methods Approximately 70% of the liver was removed from mice using the method described by Higgins and Anderson. Results Partial hepatectomy induced the expansion of NKT cells in the liver and the elevation of transaminase. These responses were completely suppressed by the administration of tacrolimus. NKT cell-deficient mice showed a decreased level of transaminase after partial hepatectomy. Perforin ( /) mice showed an elevation of transaminase while B6-gld/gld mice (Fas ligand ) showed a decreased elevation of transaminase. In TAP-1( /) mice which lacked CD8 +cytotoxic T cells, inflammation remained at a normal level after partial hepatectomy. Since NKT cell-deficient mice showed up to 50% decrease in the level of inflammation, we examined the association of other leukocytes with the remaining inflammation. The number and proportion of granulocytes were increased by partial hepatectomy. Conclusions Both NKT cells and granulocytes participated in the hepatic inflammation after partial hepatectomy. The function of NKT cells, but not of granulocytes, was found to be sensitive to the immunosuppressive effect of tacrolimus.
Keywords: Natural killer T cells ;Granulocytes ;Inflammation ;Partial hepatectomy
Rene Gerolami et al.
Herpes simplex virus thymidine kinase-mediated suicide gene therapy for hepatocellular carcinoma using HIV-1-derived lentiviral vectors
Background/Aims Gene therapy is a promising approach for treatment of hepatocellular carcinoma (HCC). However, transduction of non-tumoral hepatocytes may lead to severe hepatitis when using suicide gene therapy approaches. The aim of our study was to evaluate the gene transfer efficiency into HCC cells and normal hepatocytes using human immunodeficiency virus (HIV)-derived lentiviral vectors in vitro and in vivo. Methods Lentiviral vectors encoding for the Lac Z gene or the fusion gene HSV-Tk/GFP were tested in vitro in human HCC cells and human hepatocytes in primary culture and in vivo in a chemically induced rat model of HCC. Results We show that HIV-1-derived lentiviral vectors are efficient in transducing HCC cells in vitro and in vivo. No significant transduction of non-tumorous hepatocytes was observed in vivo whatever the route of administration used. Measurement of tumor growth following direct intratumoral injection of a lentiviral vector containing the HSV-Tk gene and GCV treatment showed a strong antitumoral efficacy in the absence of normal liver toxicity. Conclusions These observations suggest that lentiviral vectors allow an antitumoral effect with low liver toxicity when using suicide gene therapy approach and could be efficient tools for HCC gene therapy.
Keywords: Gene therapy ;Hepatocellular carcinoma ;Human immunodeficiency virus-derived lentiviral vectors Liver Growth and Cancer
Haeryoung Kim et al.
Primary liver carcinoma of intermediate (hepatocyte-cholangiocyte) phenotype
Background/Aims Recent evidence of hepatic progenitor cells with the bipotential to differentiate into hepatocytes and cholangiocytes gives rise to the suggestion that primary hepatic carcinomas with features intermediate between hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) may originate from hepatic progenitor cells. Methods Fifty-four cases of primary liver carcinomas were selected and an immunohistochemical analysis was performed using hepatocytic markers ( -fetoprotein, hepatocyte), cholangiocytic markers (carcinoembryonic antigen, cytokeratin 19) and progenitor cell marker ( c-kit ). Results Thirteen cases designated `intermediate' carcinomas demonstrated strands/trabeculae of small, uniform, round-to-oval cells with scanty cytoplasm and hyperchromatic nuclei embedded within a thick desmoplastic stroma. Six were designated transitional type combined hepatocellular-cholangiocarcinoma (CHC). Ten were named HCC small cell type, demonstrating similar features to typical HCC, but composed of smaller cells. Simultaneous expression of hepatocytic and cholangiocytic markers was demonstrated in 8/13 (61.5%), 4/6 (66.7%), and 3/10 (30%) cases of intermediate carcinomas, transitional CHCs, and HCC small cell type, respectively, and c-kit expression was noted in 10/13 (76.9%), 4/6 (66.7%) and 7/10 (70%) cases, in the same order. Conclusions Intermediate carcinoma may be a distinct type of primary liver carcinoma, morphologically and phenotypically intermediate between HCC and CC, which originates from transformed hepatic progenitor cells.
Keywords: Intermediate carcinoma ;Hepatocellular carcinoma ;Cholangiocarcinoma ;Combined hepatocellular-cholangiocarcinoma ;c-kit
Cholangiocyte Liver Growth and Cancer
Claudio A. Redaelli et al.
Effect of vascular endothelial growth factor on functional recovery after hepatectomy in lean and obese mice
Background/Aims Liver regeneration is dependent upon coordinated proliferation of hepatocytes and endothelial cells. Vascular endothelial growth factor (VEGF) promotes angiogenesis. Hepatic steatosis delays regeneration and increases liver resection morbidity. We hypothesized that VEGF overexpression stimulates hepatic regeneration. Methods Recombinant adenovirus expressing human VEGF165 or adenovirus control-vector (Lac Z) were administered before 2/3 hepatectomy in lean and ob/ob mice. Galactose elimination capacity, a quantitative liver function test, was repeatedly measured before and after hepatectomy. Expression of VEGF receptors (flt1, flk1), endoglin and hypoxia inducible factor-1 (HIF-1 ) was assessed by quantitative RT-PCR and for endoglin also by immunohistochemistry. Results After 2/3 hepatectomy, VEGF gene transfer increased galactose elimination capacity in lean and ob/ob mice. HIF-1 , endoglin and VEGF receptor mRNA increased during regeneration in lean but not in obese mice. Staining of endothelial cells by endoglin immunohistochemistry returned to baseline reactivity in lean mice by day 6 and remained decreased in ob/ob mice. VEGF treatment decreased HIF-1 and increased flk1 response in lean mice. Conclusions Hepatic resection elicits an angiogenic response in the remnant liver, which is impaired in case of steatosis. Adenovirus-mediated transfer of VEGF hastens functional hepatic recovery in lean, and more importantly also, in obese mice after partial hepatectomy.
Keywords: Angiogenesis ;Breath test ;Fatty liver ;Regeneration ;Steatosis Viral Hepatitis
Mohamed Elrefaei, Nabila El-sheikh, Karim Kamal and Huyen Cao
Analysis of T cell responses against hepatitis C virus genotype 4 in Egypt
Background/Aims : Hepatitis C virus (HCV) remains the most common cause of chronic liver disease in Egypt. Despite the high prevalence of HCV and Schistosoma mansoni (S. mansoni ) in Egypt, the effect of co-infection on the immune response against HCV genotype 4a has not been extensively examined. Methods : We evaluated the HCV 4a-specific responses against the core and non-structural 5B proteins in chronic HCV with or without S. mansoni co-infection in 38 volunteers from Egypt. Results : HCV 4a-specific responses were detected in 8/15 and 13/23 individuals with HCV alone or with concomitant schistosomiasis, respectively. Despite the alteration in the Th1 cytokine profile caused by schistosomiasis, the overall immune response rate against HCV was not affected ( P=0.11). Seven individuals demonstrated HCV-specific responses against conserved regions of the Core that were previously identified for genotypes 1, 2 and 3 despite differences in HLA class I distribution. Conclusions : Egyptian patients infected with HCV genotype 4 can mount HCV-specific T cell responses, both CD4 +and CD8 +T cell-mediated, despite the prevalence of concomitant schistosomiasis. These findings suggest that S. mansoni co-infection may not represent a major obstacle to developing an HCV vaccine in this population.
Keywords: Hepatitis C virus ;CD4 ;CD8 ;Immunity ;Schistosoma mansoni Viral Hepatitis
Sylvie Deuffic-Burban et al.
Comparing the public health burden of chronic hepatitis C and HIV infection in France
Background /Aims : In France, the prevalence of hepatitis C virus (HCV) exceeds that of HIV, but in the absence of treatment, HIV infection progresses more rapidly than HCV. More HIV-infected patients, however, have received treatment. Using reported public health data in France and natural history models, we applied the back-calculation method to project future mortality from HCV and HIV incorporating current therapies. Methods : The HCV model was based on literature data for the natural history of HCV and reports of hepatocellular carcinoma mortality. The HIV model used estimates from the French Hospital Database on HIV and reported AIDS cases and deaths. Results : Peak annual mortality from HIV at 5000 occurred in 1994 and was 1000 in 1998, but HCV mortality likely increased through the 1990s and reached 3000 in 1998. Considering only HCV infections occurring until 1998 and currently available therapy, our model suggested that annual HCV-related mortality would continue to rise and would reach 4500 deaths in 2022. In contrast, AIDS-related deaths began to decrease in 1997. Conclusions : The public health burden of HCV is likely on the rise, while the burden of HIV, given the fairly widespread use of effective medications, may be on the decline. These results may help health policymakers in planning their responses to these epidemics.
Keywords: AIDS ;Back-calculation ;Disease burden ;HCV ;Hepatocellular carcinoma ;Liver disease ;Mortality ;Predictions
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