Table of Contents for September 2005 • Volume 42, Issue 3
Liver Biology and Pathobiology
The third gas: H2S regulates perfusion pressure in both the isolated and perfused normal rat liver and in cirrhosis (p 539-548)
Stefano Fiorucci, Elisabetta Antonelli, Andrea Mencarelli, Stefano Orlandi, Barbara Renga, Giovanni Rizzo, Eleonora Distrutti, Vijay Shah, Antonio Morelli
The regulation of sinusoidal resistance is dependent on the contraction of hepatic stellate cells (HSC) around sinusoidal endothelial cell (SEC) through paracrine cross-talk of vasoconstrictor and vasodilator agents. Hydrogen sulfide (H2S), a recently discovered gas neurotransmitter, is a putative vasodilator whose role in hepatic vascular regulation and portal hypertension is unexplored. Four-week bile duct-ligated (BDL) rats with cirrhosis and control rats were treated daily with NaHS (56 mol/kg) for 5 days. Isolated livers were perfused first with NaHS for 20 minutes and then with norepinephrine (NE) and the intrahepatic resistance studied. In normal rats and animals with cirrhosis, administration of NE resulted in a dose-dependent increase of portal pressure. This effect was attenuated by H2S treatment (P < .05). The H2S-induced relaxation of hepatic microcirculation was attenuated by glibenclamide, an adenosine triphosphate (ATP)-sensitive K+ channel inhibitor. L-Cysteine, a substrate of cystathionine-gamma-lyase (CSE), decreased vasoconstriction in normal rat livers (P < .05) but failed to do so in livers with cirrhosis. BDL resulted in a downregulation of CSE mRNA/protein levels and activity (P < .05). Our in vitro data demonstrate that CSE is expressed in hepatocytes, HSCs, but not in sinusoidal endothelial cells (SEC). HSC activation downregulates CSE mRNA expression, resulting in a defective production of H2S and abrogation of relaxation induced by L-cysteine. In conclusion, CSE-derived H2S is involved in the maintenance of portal venous pressure. The reduction of CSE expression in the liver with cirrhosis contributes to the development of increased intrahepatic resistance and portal hypertension.
VPAC1 expression is regulated by FXR agonists in the human gallbladder epithelium (p 549-557)
Nicolas Chignard, Martine Mergey, Véronique Barbu, Laetitia Finzi, Emmanuel Tiret, Annick Paul, Chantal Housset
Vasoactive intestinal peptide receptor-1 (VPAC1) is the high-affinity receptor of vasoactive intestinal peptide (VIP), a major regulator of bile secretion. To better define the level at which VPAC1 stimulates bile secretion, we examined its expression in the different cell types participating in bile formation (i.e., hepatocytes, bile duct, and gallbladder epithelial cells). Because VPAC1 expression was previously shown to be regulated by nuclear receptors, we tested the hypothesis that it may be regulated by the farnesoid X receptor (FXR). Quantitative RT-PCR and immunoblot analyses of cell isolates indicated that VPAC1 is expressed in all three cell types lining the human biliary tree, with predominant expression in the gallbladder. In primary cultures of human gallbladder epithelial cells, VIP induced cAMP production and chloride secretion. Analysis of the VPAC1 gene revealed the presence of potential FXR response element sequences, and both FXR and RXR expressions were detected in gallbladder epithelial cells. In these cells, the FXR pharmacological agonist GW4064 upregulated VPAC1 expression in a dose-dependent manner, and this effect was antagonized by the RXR ligand, 9-cis retinoic acid. Chenodeoxycholate activated endogenous FXR in gallbladder epithelial cells, as ascertained by electromobility shift assay and upregulation of the FXR target gene, small heterodimer partner. Chenodeoxycholate also provoked an increase in VPAC1 mRNA and protein content in these cells. In conclusion, FXR agonists may increase gallbladder fluid secretion through transcriptional activation of VPAC1, which may contribute to the regulation of bile secretion by bile salts and to a protective effect of FXR pharmacological agonists in gallstone disease.
Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells (p 558-567)
Yusuke Yamamoto, Takumi Teratani, Hanako Yamamoto, Gary Quinn, Sigenori Murata, Rieko Ikeda, Kenji Kinoshita, Kenichi Matsubara, Takashi Kato, Takahiro Ochiya
Hepatic differentiation at the molecular level is poorly understood, mainly because of the lack of a suitable model. Recently, using adherent monoculture conditions, we demonstrated the direct differentiation of hepatocytes from embryonic stem (ES) cells. In this study, we exploited the direct differentiation model to compare the gene expression profiles of ES cell-derived hepatocytes with adult mouse liver using DNA microarray technology. The results showed that the ES cell-derived hepatocyte gene expression pattern is very similar to adult mouse liver. Through further analysis of gene ontology categories for the 232 most radically altered genes, we found that the significant categories related to hepatic function. Furthermore, through the use of small interfering RNA technology in vitro, hepatocyte nuclear factor 3/FoxA2 was identified as having an essential role in hepatic differentiation. These results demonstrate that ES cell-derived hepatocytes recapitulate the gene expression profile of adult mouse liver to a significant degree and indicate that our direct induction system progresses via endoderm differentiation. In conclusion, our system closely mimics in vivo hepatic differentiation at the transcriptional level and could, therefore, be useful for studying the molecular basis of hepatocyte differentiation per se.
Role of adiponectin in the protective action of dietary saturated fat against alcoholic fatty liver in mice (p 568-577)
Min You, Robert V. Considine, Teresa C. Leone, Daniel P. Kelly, David W. Crabb
The protective effect of dietary saturated fatty acids against the development of alcoholic liver disease has long been known, but the underlying mechanism is not completely understood. We examined the involvement of the adipocyte hormone adiponectin. Circulating adiponectin levels were significantly elevated by chronic ethanol administration to mice consuming a diet high in saturated fat. The increase in circulating adiponectin was associated with the activation a set of hepatic signaling pathways mediated through AMP-activated protein kinase, PPAR-, and PPAR- coactivator , which in turn led to markedly increased rates of fatty acid oxidation, prevention of hepatic steatosis, and alleviation of liver enzyme changes. Furthermore, treatment of rat 3T3-L1 adipocytes with saturated fatty acids (palmitic or stearic acids) in the presence of ethanol increased secretion of adiponectin and enhanced activity of a mouse adiponectin promoter. In conclusion, the protective action of saturated fat against the development of alcoholic fatty liver in mice is partially mediated through induction of adiponectin. The present findings suggest a novel paradigm for dietary fatty acids in the pathogenesis of alcoholic liver disease and provide a promising therapeutic strategy - nutritional modulation of adiponectin - in treating human alcoholic fatty liver disease.
Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death in mice (p 578-587)
Zsolt Sarang, Péter Molnár, Tamás Németh, Szabolcs Gomba, Tamás Kardon, Gerry Melino, Susanna Cotecchia, László Fésüs, Zsuzsa Szondy
Tissue transglutaminase (TG2) is a protein cross-linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha-1b-adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti-Fas antibodies, which is sublethal for wild-type mice, kills all the TG2 knock-out mice within 20 hours. Although TG2-/- thymocytes exposed to anti-Fas antibodies die at the same rate as wild-type mice, TG2-/- hepatocytes show increased sensitivity toward anti-Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIPL (FLICE-inhibitory protein), or the rate of I-B degradation, but a decrease in the Bcl-xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl-xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand-induced hepatocyte proliferation with a simultaneous inhibition of the Fas-death pathway plays a determinant role.
Proteasome inhibition sensitizes hepatocellular carcinoma cells, but not human hepatocytes, to TRAIL (p 588-597)
Tom M. Ganten, Ronald Koschny, Tobias L. Haas, Jaromir Sykora, Min Li-Weber, Kerstin Herzer, Henning Walczak
TRAIL exhibits potent anti-tumor activity on systemic administration in mice. Because of its proven in vivo efficacy, TRAIL may serve as a novel anti-neoplastic drug. However, approximately half of the tumor cell lines tested so far are TRAIL resistant, and potential toxic side effects of certain recombinant forms of TRAIL on human hepatocytes have been described. Pretreatment with the proteasome inhibitor MG132 and PS-341 rendered TRAIL-resistant hepatocellular carcinoma (HCC) cell lines but not primary human hepatocytes sensitive for TRAIL-induced apoptosis. We investigated the different levels of possible MG132-induced interference with resistance to apoptotic signal transduction. Although proteasome inhibition efficiently suppressed nuclear factor-kappaB (NF-B) activity, specific suppression of NF-B by mutIB failed to sensitize TRAIL-resistant cell lines for TRAIL-induced apoptosis. In contrast to the previously reported mechanism of sensitization by 5-fluorouracil (5-FU), cellular FLICE-inhibitory protein (cFLIP)L and cFLIPS were markedly upregulated in the TRAIL death inducing signaling complex (DISC) by proteasome inhibitor pretreatment. Compared with 5-FU pretreatment, caspase-8 was more efficiently recruited to the DISC in MG132 pretreated cells despite the presence of fewer death receptors and more cFLIP in the DISC. But downregulation of cFLIP by short interference RNA (siRNA) further sensitized the HCC cell lines. In conclusion, these results show that otherwise chemotherapy-resistant tumor cells can be sensitized for TRAIL-induced apoptosis at the DISC level in the presence of high levels of cFLIP, which suggests the existence of an additional factor that modulates the interaction of FADD and the TRAIL death receptors. Of clinical relevance, proteasome inhibitors sensitize HCC cells but not primary human hepatocytes for TRAIL-induced apoptosis.
Glutathione depletion renders rat hepatocytes sensitive to nitric oxide donor-mediated toxicity (p 598-607)
Tracy Chen, Linda L. Pearce, Jim Peterson, Detcho Stoyanovsky, Timothy R. Billiar
Nitric oxide (NO) can be either cytoprotective or cytotoxic in hepatocytes, depending on conditions within the cell. We hypothesized that redox status is a determinant of NO effects on cell viability. To cause the disturbance of redox homeostasis in the hepatocytes, cells were treated with the following glutathione (GSH) depleting agents: (1) chronic depletion by 18 hours pretreatment with buthionine sulfoximine (BSO), which depletes GSH by blocking its biosynthesis; and (2) acute depletion by 1 hour pretreatment with diethyl maleate (DEM), which conjugates GSH by the GSH-S-transferase catalyzed reaction. S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, was added after removal of GSH-depleting agents. Individual treatment with either SNAP or GSH depletion did not appreciably affect viability. A significant increase of cytotoxicity in hepatocytes was observed with the combination of a concentration and time course regimen of SNAP and GSH depletion. SNAP treatment of GSH-depleted hepatocytes led to an increase in LDH release and oxidative stress, disruption of mitochondrial membrane potential, the presence of nitrotyrosine (an indicator of peroxynitrite (ONOO-) generation), and a decrease in adenosine triphosphate (ATP) content. The interference of mitochondrial respiratory enzymes, especially with the combination treatments, indicated different levels of disturbance of electron transfer, superoxide generation, and ATP production. Other commonly used NO donors were found to exhibit lower and slower toxicity in the setting of GSH depletion than that evident with SNAP. In conclusion, the disruption of cellular redox homeostasis by GSH depletion leads hepatocytes to be more susceptible to NO (especially S-nitrosothiols) and subsequent necrotic cell death.
Significance and therapeutic potential of prostaglandin E2 receptor in hepatic ischemia/reperfusion injury in mice (p 608-617)
Yukiyasu Kuzumoto, Masayuki Sho, Naoya Ikeda, Kaoru Hamada, Takashi Mizuno, Satoru Akashi, Yoshikazu Tsurui, Hisanori Kashizuka, Takeo Nomi, Atsushi Kubo, Hiromichi Kanehiro, Yoshiyuki Nakajima
Prostaglandin E2 (PGE2) mediates a variety of innate and adaptive immunity through four distinct receptors: EP1-EP4. It has been suggested that each EP plays a unique and pivotal role in various disease conditions. We investigated the pathophysiological role of EP receptors in hepatic ischemia/reperfusion (I/R) injury. In this study, a 70% hepatic ischemic model was used in male C57BL/6 mice. Selective EP agonists were used to clarify the function of each PGE2 receptor in I/R injury. Although all four receptors were expressed in the naïve liver, EP4 expression was significantly upregulated after hepatic I/R. Although EP1, 2, or 3 agonists did not show any protective effect on liver function, the EP4 agonist significantly inhibited hepatic I/R injury as determined by serological and histological analyses. Furthermore, the EP4 agonist downregulated the local expressions of several proinflammatory cytokines, chemokines, and adhesion molecules in the early phase of reperfusion. In contrast, it augmented the local expression of an anti-inflammatory cytokine, interleukin 10. Additionally, the neutrophil accumulation was also inhibited by EP4 agonist treatment. Finally, to confirm the therapeutic efficacy of the EP4 agonist in hepatic I/R injury, the nonischemic shunt liver was removed after 120 minutes of ischemia, resulting in the death of 86% of control mice within 48 hours. In sharp contrast, 80% of mice treated with the EP4 agonist survived. In conclusion, the PGE2-EP4 signaling pathway has an inhibitory role in hepatic I/R injury. An EP4 agonist effectively protects against ischemic injury.
HIP/PAP accelerates liver regeneration and protects against acetaminophen injury in mice (p 618-626)
Hanh-Tu Lieu, Frédéric Batteux, Marie-Thérèse Simon, Alexandre Cortes, Carole Nicco, Flora Zavala, Alain Pauloin, José Guilherme Tralhao, Olivier Soubrane, Bernard Weill, Christian Bréchot, Laurence Christa
Human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein HIP/PAP is a secreted C-type lectin belonging to group VII, according to Drickamer's classification. HIP/PAP is overexpressed in liver carcinoma; however, its functional role remains unclear. In this study, we demonstrate that HIP/PAP is a paracrine hepatic growth factor promoting both proliferation and viability of liver cells in vivo. First, a low number of implanted hepatocytes deriving from HIP/PAP-transgenic mice (<1:1,000) was sufficient to stimulate overall recipient severe combined immunodeficiency liver regeneration after partial hepatectomy. After a single injection of HIP/PAP protein, the percentages of bromodeoxyuridine-positive nuclei and mitosis were statistically higher than after saline injection, indicating that HIP/PAP acts as a paracrine mitogenic growth factor for the liver. Comparison of the early events posthepatectomy in control and transgenic mice indicated that HIP/PAP accelerates the accumulation/degradation of nuclear phospho-signal transducer activator transcription factor 3 and tumor necrosis factor level, thus reflecting that HIP/PAP accelerates liver regeneration. Second, we showed that 80% of the HIP/PAP-transgenic mice versus 25% of the control mice were protected against lethal acetaminophen-induced fulminate hepatitis. A single injection of recombinant HIP/PAP induced a similar cytoprotective effect, demonstrating the antiapoptotic effect of HIP/PAP. Comparison of Cu/Zn superoxide dismutase activity and glutathione reductase-like effects in control and transgenic liver mice indicated that HIP/PAP exerts an antioxidant activity and prevents reactive oxygen species-induced mitochondrial damage by acetaminophen overdose. In conclusion, the present data offer new insights into the biological functions of C-type lectins. In addition, HIP/PAP is a promising candidate for the prevention and treatment of liver failure.
Liver Failure and Liver Disease
A randomized unblinded pilot study comparing albumin versus hydroxyethyl starch in spontaneous bacterial peritonitis (p 627-634)
Javier Fernández, Joan Monteagudo, Xavier Bargallo, Wladimiro Jiménez, Jaume Bosch, Vicente Arroyo, Miguel Navasa
The administration of albumin improves circulatory function, prevents hepatorenal syndrome, and reduces hospital mortality in patients with cirrhosis and spontaneous bacterial peritonitis. This randomized unblinded pilot study compared the effect of albumin (10 patients) and the synthetic plasma expander hydroxyethyl starch 200/0.5 (10 patients) on the systemic hemodynamics of patients with spontaneous bacterial peritonitis. Baseline measurements were performed within 12 hours after diagnosis of infection. Patients then received 2 doses of the volume expander (1.5 g/kg body weight after baseline measurements and 1 g/kg body weight on day 3). Measurements were repeated after infection resolution. Treatment with albumin was associated with a significant increase in arterial pressure and a suppression of plasma renin activity, indicating an improvement in circulatory function. This occurred in the setting of a significant expansion of central blood volume (increase in cardiopulmonary pressures and atrial natriuretic factor) and an increase in systolic volume and systemic vascular resistance. In contrast, no significant changes were observed in these parameters in patients treated with hydroxyethyl starch. Von Willebrand-related antigen plasma levels significantly decreased in patients treated with albumin but not in those treated with hydroxyethyl starch. Serum nitrates and nitrites increased in patients treated with hydroxyethyl starch but not in those treated with albumin. These data suggest an effect of albumin on endothelial function. In conclusion, albumin but not hydroxyethyl starch improves systemic hemodynamics in patients with spontaneous bacterial peritonitis. This effect is due not only to volume expansion but also to an action on the peripheral arterial circulation.
Quality of life in refractory ascites: Transjugular intrahepatic portal-systemic shunting versus medical therapy (p 635-640)
Mical S. Campbell, Colleen M. Brensinger, Arun J. Sanyal, Chris Gennings, Florence Wong, Kris V. Kowdley, Timothy McCashland, K. Rajender Reddy
Uncontrolled studies suggest that transjugular intrahepatic portal-systemic shunting (TIPS) may improve quality of life in patients with refractory ascites. We hypothesized that any improvement of quality of life in patients with TIPS would be matched in controls due to the competing effects of improved ascites and worsened hepatic encephalopathy. Thus, an analysis of quality of life was performed using original data from the North American Study for the Treatment of Refractory Ascites, a multicenter trial of 109 patients randomized to TIPS or repeated large volume paracentesis (LVP) for refractory ascites. Short form 36 (SF-36) surveys were completed at baseline and at 6- and 12-month follow-up. Variables analyzed were: randomization group, number of LVP performed, cumulative volume from LVP, shortness of breath, abdominal distention, abdominal pain, diuretic usage, confusion, hospitalizations, and emergency room visits. Outcomes were changes in physical component scale (PCS) and mental component scale (MCS) of SF-36 results. We constructed multivariable, mixed effects models, including randomization group and baseline MCS and PCS. Changes in PCS and MCS from baseline were similar between the two randomization groups. In multivariate analysis, PCS improvement was associated with lack of confusion, improved ascites, and lack of hospitalizations, but not directly with randomization group. Improvement in MCS was associated with randomization to TIPS and lack of confusion. In conclusion, patients with refractory ascites randomized to TIPS or repeated LVP had similar changes in quality of life. Competing effects of hepatic encephalopathy, requirement for repeated LVP, and need for hospitalizations explain similar changes in quality of life between the two groups.
Histopathology of pediatric nonalcoholic fatty liver disease (p 641-649)
Jeffrey B. Schwimmer, Cynthia Behling, Robert Newbury, Reena Deutsch, Caroline Nievergelt, Nicholas J. Schork, Joel E. Lavine
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are common in children and adolescents. However, standard histological criteria for pediatric NAFLD and NASH are undeveloped. We reviewed consecutive patients ages 2 to 18 years with biopsy-proven NAFLD diagnosed between 1997 and 2003. Biopsies were evaluated by two pathologists for individual features of steatohepatitis. Agglomerative hierarchical cluster analysis demonstrated two different forms of steatohepatitis. Type 1 was characterized by steatosis, ballooning degeneration, and perisinusoidal fibrosis; type 2 was characterized by steatosis, portal inflammation, and portal fibrosis. The study included 100 children with NAFLD. Simple steatosis was present in 16% of subjects, and advanced fibrosis was present in 8%. Type 1 NASH was present in 17% of subjects, and type 2 NASH was present in 51%. Boys were significantly (P < .01) more likely to have type 2 NASH and less likely to have type 1 NASH than girls. The NASH type differed significantly (P < .001) by race and ethnicity. Type 1 NASH was more common in white children, whereas type 2 NASH was more common in children of Asian, Native American, and Hispanic ethnicity. In cases of advanced fibrosis, the pattern was generally that of type 2 NASH. In conclusion, type 1 and type 2 NASH are distinct subtypes of pediatric NAFLD, and type 2 is the most common pattern in children. NASH subtypes should be considered when interpreting liver biopsies and planning studies of the pathophysiology, genetics, natural history, or response to treatment in pediatric NAFLD.
Spectrum of NAFLD and diagnostic implications of the proposed new normal range for serum ALT in obese women (p 650-656)
Sachin S. Kunde, Audrey J. Lazenby, Ronald H. Clements, Gary A. Abrams
The upper limit of normal for ALT activity has been recommended to be lowered to 30 U/L in men and 19 U/L in women. These changes have been suggested to be diagnostically useful in subjects with nonalcoholic fatty liver disease (NAFLD). Our aim was to investigate the prevalence and spectrum of NAFLD with regard to the new ALT guidelines in 233 women with class II/III obesity. We compared our prior reference range for ALT (ULN 30 U/L in women) with the new standard. Our study demonstrates that only 86 patients (36.9%) would be classified as having normal ALT levels compared with 169 patients (72.5%) by the new and old standards, respectively. In patients with normal ALT activity (new vs. old standard), the prevalence of fatty liver (FL: 39.5% vs 40.2%), portal fibrosis, and steatosis (IPF: 37.2% vs. 33.7%) and nonalcoholic steatohepatitis (NASH: 23.3% vs. 26%) were similar. In comparison, newly defined patients with elevated ALT levels (>19 U/L) demonstrated an increased prevalence of FL (36%) and IPF (11.6%) but a 23.8% decrease in the prevalence of NASH as compared with the old standard. The sensitivity and specificity for NASH were 42% and 80% (ALT > 30 U/L) compared with 74% and 42% (ALT > 19 U/L). In conclusion, a significant increase in the prevalence of FL and IPF is detected in subjects with elevated ALT levels with the application of the new standard. However, the diagnostic utility for ALT to identify NASH or IPF remains poor, and significant healthcare expenditures may be incurred if this standard is adopted.
Characterization of intrahepatic cholangiocarcinoma of the intraductal growth-type and its precursor lesions (p 657-664)
Ta-Sen Yeh, Jeng-Hwei Tseng, Tse-Ching Chen, Nai-Jen Liu, Cheng-Tang Chiu, Yi-Yin Jan, Miin-Fu Chen
A cohort of patients with intraductal growth-type intrahepatic cholangiocarcinoma (IG-ICC) and its precursor lesions, collectively termed intraductal papillary neoplasm of the liver (IPNL), was characterized with respect to demographics, clinical manifestations, perioperative management, long-term survival, and molecular features associated with carcinogenesis. A total of 122 patients with IPNL types 1 through 4, 108 patients with non-IG-ICC and 210 patients with hepatolithiasis alone were studied. Expression of CDX2, TFF1, MUC1, MUC2, MUC5AC, EGFR, and p53 was determined by using immunohistochemistry. Females predominated in those with hepatolithiasis alone and IPNL. The mean age of patients with hepatolithiasis alone was 6 to 8 years younger than that of those with IPNL. The association with hepatolithiasis in patients with IPNL types 1 and 2, IPNL types 3 and 4, and non-IG-ICC was 100%, 79%, and 64%, respectively. Mucobilia, anemia, and elevated serum carcinoembryonic antigen levels were helpful in distinguishing IG-ICC and its precursor lesions. The mean survival of patients with IPNL type 3, IPNL type 4, and non-IG-ICC was 55.5 months, 36.9 months, and 15.8 months, respectively. The incidence of expression of CDX2 and TFF1 was maximal in IPNL type 3. Expression and cellular distribution of MUC2 and CDX2 were similar. MUC5AC was strongly expressed in all patients with IPNL; EGFR and p53 were rarely expressed in patients with IPNL. In conclusion, hepatolithiasis appears to be a precipitating factor in the development of IPNL. Signs of mucobilia were specific for the diagnosis of IPNL. Expression of CDX2 and MUC2 are helpful in differentiating IPNL and non-IG-ICC. Significant differences in survival associated with the various lesions studied warrants a more aggressive surgical strategy in their management.
A genomic and proteomic study of the spectrum of nonalcoholic fatty liver disease (p 665-674)
Zobair M. Younossi, Ancha Baranova, Katharine Ziegler, Luca Del Giacco, Karen Schlauch, Timothy L. Born, Hazem Elariny, Francesco Gorreta, Amy VanMeter, Abraham Younoszai, Janus P. Ong, Zachary Goodman, Vikas Chandhoke
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and some of its forms are progressive. This study describes the profiling of hepatic gene expression and serum protein content in patients with different subtypes of NAFLD. Liver biopsy specimens from 98 bariatric surgery patients were classified as normal, steatosis alone, steatosis with nonspecific inflammation, and nonalcoholic steatohepatitis (NASH). Microarray hybridizations were performed in triplicate and the microarray expression levels of a selected group of genes were confirmed using real-time quantitative reverse-transcriptase polymerase chain reaction. Serum protein profiles of the same patients were determined by SELDI-TOF mass spectrometry. Of 98 obese patients, 91 were diagnosed with NAFLD (12 steatosis alone, 52 steatosis with nonspecific inflammation, and 27 NASH), and 7 patients without NAFLD served as obese controls. Each group of NAFLD patients was compared with the obese controls, and 22 genes with more than twofold differences in expression levels were revealed. Proteomics analyses were performed for the same group comparisons and revealed twelve significantly different protein peaks. In conclusion, this genomic/proteomic analysis suggests differential expression of several genes and protein peaks in patients within and across the forms of NAFLD. These findings may help clarify the pathogenesis of NAFLD and identify potential targets for therapeutic intervention.
Viral Hepatitis
LKM1 autoantibodies in chronic hepatitis C infection: A case of molecular mimicry? (p 675-682)
Gabriel Marceau, Pascal Lapierre, Kathie Béland, Hugo Soudeyns, Fernando Alvarez
Anti-liver-kidney microsome type 1 (LKM1) autoantibodies directed against the cytochrome P450 2D6 (CYP2D6) are considered specific markers of type 2 autoimmune hepatitis, but are also found in 5% of sera from patients chronically infected by hepatitis C virus (HCV). Molecular mimicry between HCV proteins and CYP2D6 has been proposed to explain the emergence of these autoantibodies. Anti-LKM1 autoantibodies from hepatitis C-infected patients were affinity-purified against immobilized CYP2D6 protein and used to screen a phage display library. CYP2D6 conformational epitopes were identified using phage display analysis and the identification of statistically significant pairs (SSPs). Cross-reactivity between CYP2D6 and HCV protein candidates was tested by immunoprecipitation. Nineteen different clones were isolated, and their sequencing resulted in the mapping of a conformational epitope to the region of amino acids 254-288 of CYP2D6. Candidate HCV proteins for molecular mimicry included: core, E2, NS3 and NS5a. Affinity-purified autoantibodies from HCV+/LKM1+ patients immunoprecipitated either NS3, NS5a, or both, and these reactivities were specifically inhibited by immobilized CYP2D6. In conclusion, HCV+/LKM1+ sera recognize a specific conformational epitope on CYP2D6 between amino acids 254 to 288, the region that contains the major linear epitope in type 2 autoimmune hepatitis patients. Cross-reactivity due to molecular mimicry at the B-cell level was shown between the CYP2D6 and the HCV NS3 and NS5a proteins and could explain the presence of anti-LKM1 in patients chronically infected with HCV. Further investigation of the role played by this molecular mimicry in HCV-infected patients may lead to more specific strategies for diagnosis and treatment.
Intrafamilial transmission of hepatitis C in Egypt (p 683-687)
Mostafa K. Mohamed, Mohamed Abdel-Hamid, Nabiel N. Mikhail, Fatma Abdel-Aziz, Ahmed Medhat, Laurence S. Magder, Alan D. Fix, G. Thomas Strickland
The incidence of hepatitis C (HCV) infection and associated risk factors were prospectively assessed in a cohort of 6,734 Egyptians from 2 rural villages who were negative for antibodies to HCV (anti-HCV). Initial and follow-up sera were tested for anti-HCV by enzyme immunoassay (EIA), and possible incident cases were confirmed by using the microparticle enzyme immunoassay (MEIA) and tested for HCV RNA. All follow-up serum samples converting from negative to positive without detectable HCV-RNA were further tested by recombinant immunoblot assay. Over an average of 1.6 years, asymptomatic anti-HCV seroconversion occurred in 33 people (3.1/1,000 person-years [PY]), including 28 (6.8/1,000 PY) in the Nile Delta village (AES), where prevalence was 24% and 5 (0.8/1,000 PY) in the Upper Egypt village (baseline prevalence of 9%). The strongest predictor of incident HCV was having an anti-HCV-positive family member. Among those that did, incidence was 5.8/1,000 PY, compared (P < .001) with 1.0/1,000 PY; 27 of 33 incident cases had an anti-HCV-positive family member. Parenteral exposures increased the risk of HCV but were not statistically significant; 67% of seroconverters were younger than 20 years of age, and the highest incidence rate (14.1/1,000 PY) was in children younger than 10 who were living in AES households with an anti-HCV-positive parent. In conclusion, young children would especially benefit from measures reducing exposures or preventing infection with HCV.
Susceptibility to hepatitis A in patients with chronic liver disease due to hepatitis C virus infection: Missed opportunities for vaccination (p 688-695)
Michael Shim, Inessa Khaykis, James Park, Edmund J. Bini
Hepatitis A virus (HAV) superinfection is associated with a high risk of liver failure and death in patients with underlying chronic liver disease. Although HAV vaccination is recommended for all patients with chronic hepatitis C virus (HCV) infection, little is known about adherence to these recommendations in clinical practice. The aims of this study were to determine the frequency of HAV testing and vaccination among patients with chronic HCV infection. We conducted a retrospective cohort study of 1,193 patients diagnosed with chronic HCV infection over a 1-year period. During 1,646 person-years of follow-up, patients were seen by their primary care provider a median of 10.0 times (interquartile range, 4.0-20.0). HAV antibody testing was performed in 640 subjects (53.6%), and 317 (49.5%) of those tested were susceptible (HAV antibody negative). Only 94 of the 1,193 patients (7.9%) received the HAV vaccine, including 26.8% of the 317 susceptible patients, 0.9% of the 323 patients who were already immune to HAV, and 1.1% of the 553 subjects who were never tested. Among the 94 vaccinated patients, 45 received only one dose of the vaccine. Three of the unvaccinated patients developed acute HAV infection during follow-up, and 1 of them died of acute liver failure. In conclusion, despite published recommendations to vaccinate against HAV in patients with chronic HCV infection, we found that HAV testing and vaccination rates were low in clinical practice. Public health programs to increase awareness about HAV vaccination in patients with chronic liver disease are needed.
The potential of angiogenesis soluble markers in chronic hepatitis C (p 696-701)
Xamila Salcedo, Jesús Medina, Paloma Sanz-Cameno, Luisa García-Buey, Samuel Martín-Vilchez, María J. Borque, Manuel López-Cabrera, Ricardo Moreno-Otero
Angiogenesis, the formation of new vessels, has been reported to play a significant pathogenic role in liver damage-associated hepatitis C virus infection. Most of our current knowledge derives from immunohistochemical studies of hepatic biopsy samples obtained from chronic hepatitis C (CHC) patients. We evaluated whether CHC is associated with elevated serum levels of angiogenesis markers and whether these are modulated by therapy. Vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and soluble Tie-2 (sTie-2) were determined in the serum of 36 CHC patients, before and after receiving antiviral combination therapy with pegylated interferon alpha-2b plus ribavirin, and in 15 healthy controls. CHC patients showed elevated baseline VEGF and Ang-2 levels. After treatment, both factors were decreased, whereas antiangiogenic sTie-2 was increased, indicating a shift toward an anti-angiogenic profile of serum markers in CHC patients. In conclusion, this suggests that serum VEGF, Ang-2, and sTie-2 levels could be useful as noninvasive, mechanistically based markers of response to therapy and disease progression in CHC.
Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector (p 702-710)
Karla J. Helbig, Daryl T.-Y. Lau, Ljiljana Semendric, Hugh A. J. Harley, Michael R. Beard
Interferon (IFN) inhibits hepatitis C virus (HCV) replication both clinically and in vitro; however, the complete spectrum of interferon-stimulated genes (ISGs) expressed in the HCV-infected liver or the genes responsible for control of HCV replication have not been defined. To better define ISG expression in the chronically infected HCV liver, DNA microarray analysis was performed on 9 individuals with chronic hepatitis C (CHC). A total of 232 messenger RNAs were differentially regulated in CHC compared with nondiseased liver controls. A significant proportion of these were potential ISGs that were transcriptionally elevated, suggesting an ongoing response to endogenous IFN and/or double-stranded RNA. One ISG significantly elevated in all patients was viperin, an evolutionary conserved ISG that has antiviral activity against human cytomegalovirus. Stimulation of Huh-7 and HepG2 cells with IFN- or - revealed viperin is predominantly a type I ISG. Furthermore, viperin expression could also be induced following transfection of Huh-7 cells with either poly(I:C) or HCV RNA. Transient expression of viperin in cells harboring the HCV genomic replicon resulted in a significant decrease in HCV replication, suggesting that viperin has anti-HCV activity. In conclusion, even in the face of a persistent HCV infection, there is an active ISG antiviral cellular response, highlighting the complexity of the host viral relationship. Furthermore, ISG viperin has anti-HCV activity in vitro; we postulate that viperin, along with other ISGs, acts to limit HCV replication.
Modeling the current and future disease burden of hepatitis C among injection drug users in Scotland (p 711-723)
Sharon J. Hutchinson, Sheila M. Bird, David J. Goldberg
Quantitative estimates of the current and future burden of hepatitis C virus (HCV) disease are required to plan a public health response to the HCV epidemic with regard to both prevention and treatment. A forward projection model was used to estimate the numbers of both current and former injecting drug users (IDUs) who acquired HCV and progressed to moderate and severe disease in Glasgow and Scotland during 1960-2030. The model was designed to synthesize information on the incidence and cessation of injecting drug use, the incidence of HCV infection among IDUs, the rate of HCV disease progression, and the annual number of IDUs developing HCV-related decompensated cirrhosis. During 2003, a total of 17,400 and 42,900 HCV-infected IDUs were estimated in Glasgow and Scotland, respectively; this compares with approximately 5,000 and 13,900 diagnosed, respectively, and 13,200 and 32,200 with chronic HCV, respectively. The number of IDUs developing HCV-related decompensated cirrhosis in Scotland is estimated to double between 2000 and 2020. As many as 16% and 27% of former IDUs in 2005 aged 30-39 and 40-49 years, respectively, were estimated to have moderate disease, which highlights the potential benefit of targeting HCV testing at former IDUs who belong to these age groups. In conclusion, the identification and treatment of a larger proportion of former IDUs with HCV disease and education about the importance of minimal alcohol consumption are needed to help achieve a greater impact on the future morbidity and mortality of this disease.
Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection (p 724-731)
Yves Horsmans, Thomas Berg, Jean-Pierre Desager, Tobias Mueller, Eckart Schott, Simon P. Fletcher, Kevin R. Steffy, Lisa A. Bauman, Bradley M. Kerr, Devron R. Averett
Immune-based therapy is the mainstay treatment for chronic hepatitis C virus (HCV) infection but causes multiple side effects and achieves durable viral clearance in only approximately 50% of patients. Most new investigational anti-HCV compounds are direct-acting antivirals for which durability of response and risk of viral mutations and resistance are not yet known. Therefore, continuing discovery and development of new immune-based treatments is desirable. Toll-like receptors (TLRs) are pathogen recognition receptors that initiate the innate immune response. The responsiveness of HCV or other ongoing chronic systemic infections to treatment with a selective TLR agonist has not been reported. Isatoribine is a selective agonist of TLR7. In a proof-of-concept study, we found that once-daily 7-day treatment with intravenous isatoribine 800 mg caused a significant (P = .001) reduction of plasma HCV RNA (mean, -0.76; range, -2.85 to +0.21 log10 units) in otherwise untreated patients (n = 12) who were chronically infected with HCV. Viral load reduction occurred in patients infected with genotype 1 as well as non-genotype 1 HCV. The reduction of viral load was correlated with induction of markers of a heightened immune antiviral state, including 2-, 5- oligoadenylate synthetase levels in whole blood. This treatment was well tolerated, with a low frequency of mild to moderate adverse events. In conclusion, systemic administration of the selective TLR7 agonist isatoribine resulted in dose-dependent changes in immunologic biomarkers and a statistically significant antiviral effect with relatively few and mild side effects.
Copyright © 2005 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for September 2005 • Volume 129 • Number 3
Clinical–alimentary tract
Limited Efficiency of Prolyl-Endopeptidase in the Detoxification of Gliadin Peptides in Celiac Disease
Matysiak–Budnik T, Candalh C, Cellier C, Dugave C, Namane A, Vidal–Martinez T, Cerf–Bensussan N, Heyman M
Background & Aims: The resistance of prolamines to digestive enzymes is thought to be a key contributor to the pathogenesis of celiac disease by promoting the intestinal entrance of peptides able to trigger inflammation in at-risk individuals. Oral administration of a bacterial prolyl-endopeptidase (PEP) therefore was proposed as a treatment for celiac disease. To delineate the feasibility of this treatment, the effect of PEP on gliadin peptides was assessed in vitro, and ex vivo during their transport across intestinal biopsy specimens of active celiac disease patients. Methods: In vitro degradation by PEP of 3H-labeled gliadin peptides 56–88 (33-mer) and 31–49, was analyzed by radio–reverse-phase high-performance liquid chromatography and mass spectrometry. For ex vivo studies, PEP and 3H-peptides were added together onto the mucosal side of duodenal biopsy specimens mounted in Ussing chambers, and peptide transport and digestion were assessed by radio–reverse-phase high-performance liquid chromatography. Results: Gliadin peptides were degraded partly by 20 mU/mL PEP both in vitro and ex vivo. This concentration of PEP decreased the amount of intact peptides 31–49 and 56–88 crossing the intestinal biopsy specimens of celiac disease patients, but could not prevent the intestinal passage of toxic or immunostimulatory metabolites. PEP concentrations of at least 500 mU/mL for 3 hours were required to achieve full detoxification of peptides and to prevent intestinal transport of active fragments. Conclusions: After prolonged exposure to high concentrations of PEP, the amount of immunostimulatory gliadin peptides reaching the local immune system in celiac patients is decreased. These results provide a basis to establish whether such conditions are achievable in vivo.
Natural Variation in Toxicity of Wheat: Potential for Selection of Nontoxic Varieties for Celiac Disease Patients
Spaenij–Dekking L, Kooy–Winkelaar Y, van Veelen P, Wouter Drijfhout J, Jonker H, van Soest L, Smulders MJM, Bosch D, Gilissen LJWJ, Koning F
Background & Aims: Celiac disease (CD) is an intestinal disorder caused by T-cell responses to peptides derived from the gluten proteins present in wheat. Such peptides have been found both in the gliadin and glutenin proteins in gluten. The only cure for CD is a lifelong gluten-free diet. It is unknown, however, if all wheat varieties are equally harmful for patients. We investigated whether wheat varieties exist with a natural low number of T-cell–stimulatory epitopes. Methods: Gluten proteins present in public databases were analyzed for the presence of T-cell–stimulatory sequences. In addition, wheat accessions from diploid (AA, SS/BB, and DD genomes), tetraploid (AABB), and hexaploid (AABBDD) Triticum species were tested for the presence of T-cell–stimulatory epitopes in gliadins and glutenins by both T-cell and monoclonal antibody–based assays. Results: The database analysis readily identified gluten proteins that lack 1 or more of the known T-cell–stimulatory sequences. Moreover, both the T-cell– and antibody-based assays showed that a large variation exists in the amount of T-cell–stimulatory peptides present in the wheat accessions. Conclusions: Sufficient genetic variation is present to endeavor the selection of wheat accessions that contain low amounts of T-cell–stimulatory sequences. Such materials may be used to select and breed wheat varieties suitable for consumption by CD patients, contributing to a well-balanced diet and an increase in their quality of life. Such varieties also may be useful for disease prevention in individuals at risk.
A Randomized, Placebo-Controlled Trial of Certolizumab Pegol (CDP870) for Treatment of Crohn’s Disease
Schreiber S, Rutgeerts P, Fedorak RN, Khaliq–Kareemi M, Kamm MA, Boivin M, Bernstein CN, Staun M, Thomsen OØ, Innes A
Background & Aims: To investigate the efficacy and safety of certolizumab pegol (a polyethylene-glycolated Fab? fragment of anti–tumor necrosis factor, CDP870) in Crohn’s disease. Methods: In a placebo-controlled, phase II study, 292 patients with moderate to severe Crohn’s disease received subcutaneous certolizumab 100, 200, or 400 mg or placebo at weeks 0, 4, and 8. The primary end point was the percentage of patients with a clinical response at week 12 (a Crohn’s Disease Activity Index decrease of ≥ 100 points or remission [Crohn’s Disease Activity Index ≤ 150 points]) in the intent-to-treat population. Results: All certolizumab doses produced significant clinical benefit over placebo at week 2 (placebo, 15.1%; certolizumab 100 mg, 29.7% [P = .033]; 200 mg, 30.6% [P = .026]; 400 mg, 33.3% [P = .010]). At all time points, the clinical response rates were highest for certolizumab 400 mg, greatest at week 10 (certolizumab 400 mg, 52.8%; placebo, 30.1%; P = .006) but not significant at week 12 (certolizumab 400 mg, 44.4%; placebo, 35.6%; P = .278). Patients with baseline C-reactive protein levels of 10 mg/L or greater (n = 119) showed clearer separation between active treatment and placebo (week 12 clinical response: certolizumab 400 mg, 53.1%; placebo, 17.9%; P = .005; post hoc analysis) owing to a lower placebo response rate than patients with C-reactive protein levels of less than 10 mg/L. Adverse events were similar among groups. Conclusions: Certolizumab 400 mg may be effective and is well tolerated in patients with active Crohn’s disease. High placebo response rates in the large patient subgroup with low C-reactive protein levels may have obscured statistical separation between certolizumab and placebo. Ongoing phase III trials are necessary to establish the clinical efficacy of certolizumab.
Increased Risk for Demyelinating Diseases in Patients With Inflammatory Bowel Disease
Gupta G, Gelfand JM, Lewis JD
Background & Aims: Reports of multiple sclerosis (MS), demyelination, and optic neuritis (ON) associated with anti–tumor necrosis factor ? therapy resulted in warnings on prescribing instructions for infliximab, etanercept, and adalimumab. However, the underlying relationship between IBD and these neurologic conditions has not been established. Methods: We performed a retrospective cohort study and a retrospective cross-sectional study using 1988 to 1997 data from the General Practice Research Database. A total of 7988 Crohn’s disease and 12,185 ulcerative colitis patients were matched for age, sex, and primary care practice to 80,666 randomly selected controls. In the cohort study, incident cases of MS, demyelination, and/or ON (MS/D/ON) had to occur at least 1 year after registration with the physician and after the diagnosis of IBD. In the cross-sectional study, the diagnosis of MS/D/ON could either precede or follow the IBD diagnosis. Results: In the cohort study, the incidence of MS/D/ON was higher in patients with Crohn’s disease and ulcerative colitis compared with their matched controls, reaching statistical significance for ulcerative colitis (ulcerative colitis incidence rate ratio [IRR], 2.63; 95% confidence interval, 1.29–5.15; Crohn’s disease IRR, 2.12; 95% confidence interval, .94–4.50). In the cross-sectional study, MS/D/ON was more prevalent in patients with Crohn’s disease and ulcerative colitis compared with their matched controls (Crohn’s disease odds ratio, 1.54; 95% confidence interval, 1.03–2.32; ulcerative colitis odds ratio, 1.75; 95% confidence interval, 1.28–2.39). Conclusions: Demyelinating diseases occur more commonly among patients with IBD than among non-IBD patients. Future studies should clarify whether treatment with tumor necrosis factor ? blockers results in further increased incidence of MS/D/ON among IBD patients.
The Clustering of Other Chronic Inflammatory Diseases in Inflammatory Bowel Disease: A Population-Based Study
Bernstein CN, Wajda A, Blanchard JF
Background & Aims: We aimed to discern the relative risk for several chronic inflammatory conditions in patients with ulcerative colitis (UC) and Crohn’s disease. Methods: We used the population-based University of Manitoba IBD Database that includes longitudinal files on all patients from all health system contacts identified by International Classification of Diseases, 9th revision, Clinical Modification codes for visit diagnosis. From the provincial database we extracted a control cohort matching the IBD patients 10:1 by age, sex, and geography. We considered a potential comorbid disease to be present if the patient had 5 or more health system contacts for that diagnosis. The comorbid disease period prevalence was analyzed separately for patients with UC and Crohn’s disease and a prevalence ratio was calculated comparing the IBD populations with the matched cohort. Results: There were 8072 cases of IBD from 1984 to 2003, including UC (n = 3879) and Crohn’s disease (n = 4193). There was a mean of approximately 16 person-years of coverage for both patients and control patients. Both UC and Crohn’s disease patients had a significantly greater likelihood of having arthritis, asthma, bronchitis, psoriasis, and pericarditis than population controls. An increased risk for chronic renal disease and multiple sclerosis was noted in UC but not Crohn’s disease patients. The most common nonintestinal comorbidities identified were arthritis and asthma. Conclusions: The finding of asthma as the most common comorbidity increased in Crohn’s disease patients compared with the general population is novel. These may be diseases with common causes or complications of one disease that lead to the presentation with another. Studies such as this should encourage further research into the common triggers in the organ systems that lead to autoimmune diseases.
Evaluation of a Large, Population-Based Sample Supports a CpG Island Methylator Phenotype in Colon Cancer
Samowitz WS, Albertsen H, Herrick J, Levin TR, Sweeney C, Murtaugh MA, Wolff RK, Slattery ML
Background & Aims: The concept of a CpG island methylator phenotype (CIMP), especially in microsatellite stable colon cancer, is not accepted universally. We therefore evaluated a large population-based sample of individuals with colon cancer and used univariate and multivariate analyses of CIMP with clinicopathologic variables and tumor mutations to determine the biologic relevance of this phenotype. Methods: A total of 864 tumors from individuals with colon cancer from Utah and Northern California were evaluated by methylation-specific polymerase chain reaction of CpG islands in hMLH1, methylated in tumors (MINT) 1, MINT 2, MINT 31, and CDKN2A (p16). CIMP high was defined as methylation at 2 or more of these loci. The BRAF V600E mutation was determined by sequencing. Microsatellite instability had been determined previously. Results: In a multivariate analysis of microsatellite stable tumors, CIMP high was related significantly to the V600E BRAF mutation (odds ratio, 39.52; 95% confidence interval, 11.44–136.56), KRAS2 mutations (odds ratio, 2.22; 95% confidence interval, 1.48–3.34), older age (P trend = .03), and increased stage (P trend = .03), and these tumors were less likely to be located in the distal colon (odds ratio, .42; 95% confidence interval, .27–.65). CIMP-high unstable tumors also were more likely to have the V600E BRAF mutation, be located proximally, and occur in older individuals (in univariate analyses). However, CIMP-high unstable tumors were significantly more likely than their stable counterparts to be KRAS2 wild type, TP53 wild type, poorly differentiated, proximally located, occur at lower stages, and have the BRAF V600E mutation (64.1% vs 17.6%). Conclusions: The evaluation of a large, population-based sample strongly supports the biologic relevance of CIMP in colon cancer. However, the presence or absence of microsatellite instability has a major effect on the expression of this phenotype.
Characterization of hMLH1 and hMSH2 Gene Dosage Alterations in Lynch Syndrome Patients
Baudhuin LM, Ferber MJ, Winters JL, Steenblock KJ, Swanson RL, French AJ, Butz ML, Thibodeau SN
Background & Aims: A significant proportion of Lynch syndrome cases are believed to be due to large genomic alterations in the mismatch repair genes hMLH1 and hMSH2. However, previous studies have not adequately identified the frequency and scope of such mutations, and routine clinical Lynch syndrome testing often does not include analysis for these mutations. Our aim was to characterize hMLH1 and hMSH2 genomic rearrangements in a large population of suspected Lynch syndrome patients. Methods: A total of 365 samples from probands referred for genetic testing for Lynch syndrome were analyzed for the presence of large genomic alterations in hMLH1 or hMSH2 by using a combination of techniques. Samples with a deletion in exons 1–6 in hMSH2 were further characterized by polymerase chain reaction to establish the presence of the hMSH2 American founder deletion. Results: An hMLH1 or hMSH2 mutation was identified in 153 cases, and, of these, 12 of 67 (17.9%) and 39 of 86 (45.3%) had a large genomic alteration in hMLH1 and hMSH2, respectively. Overall, 6 different hMLH1 and 12 different hMSH2 deletions/duplications, including 10 novel mutations, were identified. Analysis of the hMSH2 exon 1–6 deletion samples showed that 13 of 18 (72.2%) had the American founder deletion. Conclusions: These data show a high frequency and diverse spectrum of large genomic alterations in hMLH1 and hMSH2 in suspected Lynch syndrome patients. Thus, a comprehensive mutation identification strategy that includes the ability to detect large genomic rearrangements is imperative for the clinical genetic identification of Lynch syndrome patients and families.
Endoscopic Therapy Versus Medical Therapy for Bleeding Peptic Ulcer With Adherent Clot: A Meta-analysis
Kahi CJ, Jensen DM, Sung JJY, Bleau BL, Jung HK, Eckert G, Imperiale TF
Background & Aims: The optimal management of bleeding peptic ulcer with adherent clot is controversial and may include endoscopic therapy or medical therapy. Methods: We searched MEDLINE, BIOSIS, EMBASE, and the Cochrane Library to identify all randomized controlled trials comparing the 2 interventions. Outcomes evaluated in the meta-analysis were recurrent bleeding, need for surgical intervention, length of hospitalization, transfusion requirement, and mortality. Results: Six studies were identified that included 240 patients from the United States, Hong Kong, South Korea, and Spain. Patients in the endoscopic therapy group underwent endoscopic clot removal and treatment of the underlying lesion with thermal energy, electrocoagulation, and/or injection of sclerosants. Rebleeding occurred in 5 of 61 (8.2%) patients in the endoscopic therapy group, compared with 21 of 85 (24.7%) in the medical therapy group (P = .01), for a pooled relative risk of 0.35 (95% confidence interval, 0.14–0.83; number needed to treat, 6.3). There was no difference between endoscopic therapy and medical therapy in length of hospital stay (mean, 6.8 vs 5.6 days; P = .27), transfusion requirement (mean, 3.0 vs 2.8 units of packed red blood cells; P = .75), or mortality (9.8% vs 7%; P = .54). Patients in the endoscopic therapy group were less likely to undergo surgery (pooled relative risk, 0.43; 95% confidence interval, 0.19–0.98; number needed to treat, 13.3); however, this outcome became nonsignificant when only peer-reviewed studies were considered. Conclusions: Endoscopic therapy is superior to medical therapy for preventing recurrent hemorrhage in patients with bleeding peptic ulcers and adherent clots. The interventions are comparable with respect to the need for surgical intervention, length of hospital stay, transfusion requirement, and mortality.
Randomized, Placebo-Controlled, Esophageal Squamous Cell Cancer Chemoprevention Trial of Selenomethionine and Celecoxib
Limburg PJ, Wei W, Ahnen DJ, Qiao Y, Hawk ET, Wang G, Giffen CA, Wang G, Roth MJ, Lu N, Korn EL, Ma Y, Caldwell KL, Dong Z, Taylor PR, Dawsey SM
Background & Aims: Esophageal squamous cell carcinoma remains a leading cause of cancer death worldwide. Squamous dysplasia, the accepted histological precursor for esophageal squamous cell carcinoma, represents a potentially modifiable intermediate end point for chemoprevention trials in high-risk populations. Methods: We conducted a randomized, controlled trial of selenomethionine 200 ?g daily and/or celecoxib 200 mg twice daily (2 ? 2 factorial design) among residents of Linxian, People’s Republic of China. Subjects had histologically confirmed mild or moderate esophageal squamous dysplasia at baseline. Esophagogastroduodenoscopy was performed before and after a 10-month intervention. Per-subject change (regression, stable, or progression) in the worst dysplasia grade was defined as the primary end point. Results were compared by agent group (selenomethionine vs placebo; celecoxib vs placebo). Results: Two hundred sixty-seven subjects fulfilled all eligibility criteria, and 238 (89%) completed the trial. Overall, selenomethionine resulted in a trend toward increased dysplasia regression (43% vs 32%) and decreased dysplasia progression (14% vs 19%) compared with no selenomethionine (P = .08). In unplanned stratified analyses, selenomethionine favorably affected a change in dysplasia grade among 115 subjects with mild esophageal squamous dysplasia at baseline (P = .02), but not among 123 subjects with moderate esophageal squamous dysplasia at baseline (P = 1.00). Celecoxib status did not influence changes in dysplasia grade overall (P = .78) or by baseline histology subgroup. Conclusions: After a 10-month intervention, neither selenomethionine nor celecoxib inhibited esophageal squamous carcinogenesis for all high-risk subjects. However, among subjects with mild esophageal squamous dysplasia at baseline, selenomethionine did have a protective effect. Although it is based on unplanned stratified analyses, this finding is the first report of a possible beneficial effect for any candidate esophageal squamous cell carcinoma chemopreventive agent in a randomized controlled trial.
Gene-Expression Profiling Predicts Recurrence in Dukes’ C Colorectal Cancer
Arango D, Laiho P, Kokko A, Alhopuro P, Sammalkorpi H, Salovaara R, Nicorici D, Hautaniemi S, Alazzouzi H, Mecklin J, Järvinen H, Hemminki A, Astola J, Schwartz Jr S, Aaltonen LA
Background & Aims: Although approximately 50% of Dukes’ C colorectal cancer patients are surgically cured, it is currently not possible to distinguish these patients from those at high risk of recurrence. The recent advent of routine adjuvant chemotherapy for these patients has greatly complicated the identification of new markers predicting the response to surgery, which is now reliant on archived materials. Microarray analysis allows fine tumor classification but cannot be used with paraffin-embedded archival samples. Methods: We used microarray analysis of a unique set of fresh-frozen tumor samples from Dukes’ C patients who had surgery as the only form of treatment to identify molecular signatures that characterize tumors from patients with good and bad prognosis. Results: Unsupervised hierarchical clustering and a K-nearest neighbors–based classifier identified groups of patients with significantly different survival (P = .019 and P = .0001). Expression profiling outperformed previously reported genetic markers of prognosis such as TP53 and K-RAS mutational status and allelic imbalance in chromosome 18q, which were of limited prognostic power in this study. Functional categories significantly enriched in gene-expression differences included protein transport and folding. The prognostic potential of the RAS homologue RHOA, one of the most differentially expressed genes, was further investigated using immunohistochemistry and a tissue microarray containing 137 independent Dukes’ C tumor samples. Reduced RHOA expression was associated with significantly shorter survival (P = .01). Conclusions: This study shows that gene-expression profiling of surgical tumor samples can predict recurrence in Dukes’ C patients. Therefore, this approach could be used to guide decisions concerning the clinical management of these patients.
Clinical–liver, pancreas, and biliary tract
Keratins as Susceptibility Genes for End-Stage Liver Disease
Ku N, Lim JK, Krams SM, Esquivel CO, Keeffe EB, Wright TL, Parry DAD, Omary M B
Background & Aims: Keratins 8 and 18 protect the liver from stress. Keratin 8 and 18 variants in 17 of 467 liver disease explants and 2 of 349 blood bank controls were previously reported in 5 analyzed exonic regions. We asked whether mutations were present in the remaining 10 exons of keratins 8 and 18. Methods: Exonic regions were polymerase chain reaction–amplified from genomic DNA, isolated from the above-mentioned 2 cohorts, and analyzed for the presence of mutations. Mutant keratins were also studied biochemically. Results: We identified 10 novel keratin 8 and 18 heterozygous variants in 44 of 467 explants and 11 of 349 controls: keratin 18 deletion (?64–71), a keratin 8 frameshift that truncates the last 14 amino acids; 8 missense keratin 8 and 18 alterations; and several new polymorphisms. The most common variant, keratin 8 R340H, at the highly conserved R340 was found in 30 of 467 explants and 10 of 349 controls (P = .02) and was confirmed in the diseased livers by generation of an R340H-specific antibody. Germline transmission and variant protein expression were verified. The mutations involved a variety of liver diseases, and some variants had an ethnic background preponderance. Mutations that introduced disulfide bonds (keratin 8 G61C or R453C) decreased keratin solubility, particularly after oxidative stress, whereas others decreased keratin 8 phosphorylation (keratin 8 G433S). Conclusions: The overall frequency of keratin 8 and 18 variants was 12.4% in 467 liver disease explants and 3.7% in 349 blood bank controls (P < .0001). Variants can alter keratin solubility or phosphorylation and may render individuals susceptible to end-stage liver disease, depending on their genetic background and exposure to other insults, such as alcohol or viral infection.
Efficacy and Safety of Ursodeoxycholic Acid Versus Cholestyramine in Intrahepatic Cholestasis of Pregnancy
Kondrackiene J, Beuers U, Kupcinskas L
Background & Aims: Treatment of intrahepatic cholestasis of pregnancy with ursodeoxycholic acid appears promising, but data are limited so far. The aim of this randomized study was to evaluate the efficacy and safety of ursodeoxycholic acid in comparison with cholestyramine. Methods: Eighty-four symptomatic patients with intrahepatic cholestasis of pregnancy were randomized to receive either ursodeoxycholic acid, 8–10 mg/kg body weight daily (n = 42), or cholestyramine, 8 g daily (n = 42), for 14 days. The primary end point was a reduction of pruritus by more than 50% after 14 days of treatment as evaluated by a pruritus score. Secondary end points were outcome of pregnancy, reduction of serum aminotransferase activities and serum bile acid levels, and drug safety. Intention-to-treat analysis was applied. Results: Pruritus was more effectively reduced by ursodeoxycholic acid than cholestyramine (66.6% vs 19.0%, respectively; P < .005). Babies were delivered significantly closer to term by patients treated with ursodeoxycholic acid than those treated with cholestyramine (38.7 ± 1.7 vs 37.4 ± 1.5 weeks, respectively, P < .05). Serum alanine and aspartate aminotransferase activities were markedly reduced by 78.5% and 73.8%, respectively, after ursodeoxycholic acid, but by only 21.4%, each, after cholestyramine therapy (P < .01 vs ursodeoxycholic acid). Endogenous serum bile acid levels decreased by 59.5% and 19.0%, respectively (P < .02). Ursodeoxycholic acid, but not cholestyramine was free of adverse effects. Conclusions: Ursodeoxycholic acid is safe and more effective than cholestyramine in intrahepatic cholestasis of pregnancy.
Basic–alimentary tract
Epithelial Barrier Function In Vivo Is Sustained Despite Gaps in Epithelial Layers
Watson AJM, Chu S, Sieck L, Gerasimenko O, Bullen T, Campbell F, McKenna M, Rose T, Montrose MH
Background & Aims: Epithelial cells of the small intestine migrate to the tip of the villus at which they are shed. It is not understood how the intestinal barrier is maintained during this high cell turnover. The aim of this study was to use high-resolution in vivo light microscopy to investigate the mechanism of epithelial shedding and the site of the permeability barrier during cell shedding. Methods: A laparotomy was performed on anesthetized mice, and a segment of small intestine was opened. The exposed epithelial surface of the intestine was imaged by multiphoton microscopy. Nuclei, cytosol, and cell membranes were imaged using the dyes Hoescht 33258, BCECF, a transgenically expressed fluorescent protein, and the membrane dye DiI. The fluorescent caspase substrate PhiPhiLux was used to detect apoptosis. Results: In the epithelial monolayer, gaps were observed that lacked nuclei or cytosol but appeared to be filled with an impermeable substance. Studies with membrane impermeant fluorophores (Lucifer Yellow and Alexa-dextran) showed that the impermeable substance completely fills the void left by the absent cell. Only a fraction of gaps have either ZO-1 staining or cytoplasmic extensions from neighboring cells at the basal pole. Time-lapse studies reveal that cell shedding results in genesis of a gap and that shedding usually occurs prior to detectable cellular activation of caspase 3 or nuclear condensation. Conclusions: Results suggest that epithelial barrier function is sustained at the apical pole of the epithelial layer, despite discontinuities in the cellular layer.
CpG Motifs of Bacterial DNA Essentially Contribute to the Perpetuation of Chronic Intestinal Inflammation
Obermeier F, Dunger N, Strauch UG, Hofmann C, Bleich A, Grunwald N, Hedrich HJ, Aschenbrenner E, Schlegelberger B, Rogler G, Schölmerich J, Falk W
Background & Aims: Recently, we demonstrated a proinflammatory effect of cytosin-guanosin dinucleotide (CpG)-oligodeoxynucleotide (ODN) treatment in established dextran sulphate sodium (DSS)-induced colitis. Here, we investigated whether DNA derived from luminal bacteria plays a role in the perpetuation of chronic intestinal inflammation. Methods: Toll-like receptor (TLR9)-deficient and wild-type (wt) control mice were used for the induction of chronic DSS colitis. Moreover, mice with established chronic colitis using different experimental models were treated with adenoviral ODN (AV-ODN) known to block CpG effects. Colonic inflammation was scored and cytokine production was quantified both in colonic tissue and draining mesenteral lymph node cells (MLC). Results: Eight weeks after induction of chronic DSS colitis in TLR9-deficient mice, intestinal inflammation was significantly lower (?68%), and proinflammatory cytokine production was drastically reduced. Treatment of wt mice with chronic DSS-induced colitis with AV-ODN resulted in a significant amelioration of disease with a reduced histologic score (?43%) and reduced cytokine production of MLC (interleukin [IL]-6: ?68%; interferon [IFN]-?: ?48%) and RNA expression of the T helper (Th)1-specific transcription factor T-bet (?62%) in colonic tissue. Qualitatively, the same results were obtained in the severe combined immunodeficiency disease (SCID) transfer model of colitis and in spontaneous colitis in IL-10–deficient mice. Conclusions: Bacterial DNA derived from luminal bacteria contributes significantly to the perpetuation of chronic intestinal inflammation. Inhibition of the immune-stimulating properties of bacterial DNA using AV-ODN may offer a novel and specific tool for the treatment of inflammatory bowel disease.
CD23-Mediated IgE Transport Across Human Intestinal Epithelium: Inhibition by Blocking Sites of Translation or Binding
Tu Y, Salim S, Bourgeois J, Di Leo V, Irvine E J, Marshall JK, Perdue MH
Background & Aims: In previous studies in rodent models of food allergy, we identified that sensitization induces expression of CD23 on intestinal epithelial cells and results in enhanced IgE-dependent transepithelial antigen uptake; further studies in CD23?/? mice provided evidence that CD23 is involved in protected transport of antigen into the body. Little information exists in humans on receptor-mediated immunoglobulin (Ig)E transport across epithelia. The present study was designed to examine expression of CD23 by human epithelial cells, determine its isoform and regulation by interleukin (IL) 4, and identify the role of CD23 in transepithelial IgE transport. Methods: Epithelial expression of CD23 was studied in cell lines, ileal biopsy specimens, and explanted fetal intestine. Bidirectional transport of IgE was measured across filter-grown cells, either normal cells or those transfected with antisense CD23 oligonucleotides, or in the presence of blocking antibody. Results: Expression of the low-affinity IgE receptor was demonstrated in cultured epithelial cells as well as in situ cells in human intestine. CD23b was the isoform expressed by HT29, T84, and Caco-2 cells. IL-4 up-regulated the expression of epithelial CD23. IgE was transported in both the basal-to-apical direction and the apical-to-basal direction across filter-grown epithelial cells, a process that was inhibited by transfection of cells with CD23 antisense oligonucleotides or pretreatment with nonspecific IgE or anti-CD23 antibody. Conclusions: These findings provide evidence that CD23 encodes a functional IgE receptor on human intestinal epithelial cells and that this epithelial receptor is likely to play an important role in food allergies.
Fatty Acid Amide Hydrolase Controls Mouse Intestinal Motility In Vivo
Capasso R, Matias I, Lutz B, Borrelli F, Capasso F, Marsicano G, Mascolo N, Petrosino S, Monory K, Valenti M, Di Marzo V, Izzo AA
Background & Aims: Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis both of the endocannabinoids (which are known to inhibit intestinal motility) and other bioactive amides (palmitoylethanolamide, oleamide, and oleoylethanolamide), which might affect intestinal motility. The physiologic role of FAAH in the gut is largely unexplored. In the present study, we evaluated the possible role of FAAH in regulating intestinal motility in mice in vivo. Methods: Motility was measured by evaluating the distribution of a fluorescent marker along the small intestine; FAAH messenger RNA (mRNA) levels were analyzed by reverse-transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by isotope-dilution, liquid chromatography, mass spectrometry. Results: Motility was inhibited by N-arachidonoylserotonin (AA-5-HT) and palmitoylisopropylamide, 2 selective FAAH inhibitors, as well as by the FAAH substrates palmitoylethanolamide, oleamide, and oleoylethanolamide. The effect of AA-5-HT was reduced by the CB1 receptor antagonist rimonabant and by CB1 deficiency in mice but not by the vanilloid receptor antagonist 5?-iodoresiniferatoxin. In FAAH-deficient mice, pharmacologic blockade of FAAH did not affect intestinal motility. FAAH mRNA was detected in different regions of the intestinal tract. Conclusions: We conclude that FAAH is a physiologic regulator of intestinal motility and a potential target for the development of drugs capable of reducing intestinal motility.
Gastrin Suppresses Growth of CCK2 Receptor Expressing Colon Cancer Cells by Inducing Apoptosis In Vitro and In Vivo
Müerköster S, Isberner A, Arlt A, Witt M, Reimann B, Blaszczuk E, Werbing V, Fölsch UR, Schmitz F, Schäfer H
Background & Aims: The role of amidated gastrin17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis is still a controversial issue. Here, we investigated the effect of G17 on proliferation and apoptosis of CCK2 receptor-expressing human colon cancer cell lines in vitro and in vivo. Methods: Proliferation was determined by cell counting and cell cycle analysis. Apoptosis was analyzed by annexin V staining, TUNEL staining, caspase-3/7 assay, and JC1 (??) assay. Signal-transduction pathways were analyzed by Western blotting and gel-shift and luciferase assays. An in vivo tumor model with subcutaneously inoculated colon cancer cells in SCID mice was used, and systemic hypergastrinemia was induced by omeprazole. Results: In Colo320 cells stably transfected with the wild-type CCK2 receptor (Colo320wt) or in Lovo cells endogenously expressing CCK2 receptors, G17 treatment inhibited proliferation along with a G2/M cell cycle arrest. Furthermore, the administration of G17 significantly augmented apoptosis of CCK2 receptor-expressing cells. In contrast, G17 had no effect on proliferation and apoptosis in Colo320 cells stably transfected with a tumor-derived CCK2 receptor mutant (Colo320mut) or in cells lacking CCK2 receptor expression. Systemic hypergastrinemia in severe combined immunodeficiency (SCID) mice suppressed the growth of Colo320wt tumors accompanied by enhanced apoptosis as compared with untreated tumors. In contrast, omeprazole did not affect Colo320mut tumors reflecting a loss-of-function state of the CCK2(mut) receptor. This is supported by the observation that, in Colo320wt cells, but not in Colo320mut cells, G17 treatment induced the MAPK/ERK/AP-1 pathway and inhibited the activity of NF-?B. Conclusions: G17 exerts an antiproliferative and proapoptotic effect on human colon cancer cells expressing the wild-type CCK2 receptor. This supports the view that amidated gastrin prevents rather than promotes colorectal carcinogenesis.
Interleukin-22, a Member of the IL-10 Subfamily, Induces Inflammatory Responses in Colonic Subepithelial Myofibroblasts
Andoh A, Zhang Z, Inatomi O, Fujino S, Deguchi Y, Araki Y, Tsujikawa T, Kitoh K, Kim–Mitsuyama S, Takayanagi A, Shimizu N, Fujiyama Y
Background & Aims: Interleukin (IL)-22, a member of the IL-10 subfamily, is a recently identified T-cell-derived cytokine. We investigated IL-22 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD) and analyzed its biologic activities in human colonic subepithelial myofibroblasts (SEMFs). Methods: Mucosal IL-22 expression was evaluated by immunohistochemical procedures. The effects of IL-22 on colonic SEMFs were investigated by cDNA microarrays, Northern blots, enzyme-linked immunosorbent assay, and electrophoretic gel mobility shift assays (EMSAs). Results: IL-22 was not detectable in normal colonic mucosa. In IBD mucosa, IL-22 expression was detectable in CD4-positive T cells. IL-22-positive cells were increased in ulcerative colitis and even more so in Crohn’s disease. IL-22 receptor expression colocalized with a marker of SEMFs. IL-22 did not modulate SEMF proliferation and collagen synthesis. cDNA microarray analyses demonstrated that, in colonic SEMFs, IL-22 increased the messenger RNA (mRNA) expression of inflammatory cytokines (IL-6, IL-8, IL-11, and leukemia inhibitory factor [LIF]), chemokines, and matrix metalloproteinases. IL-22 induced an activation of nuclear factor (NF)-?B and activating protein (AP)-1 within 1 hour, and a blockade of NF-?B and AP-1 activation markedly reduced IL-22 induction of IL-6, IL-8, IL-11, and LIF mRNA. MAP-kinase inhibitors (PD98059, U0216, and SB202190) significantly reduced IL-22 induction of cytokine secretion. The combination of either IL-17 plus IL-22 or IL-19 plus IL-22 additively up-regulated cytokine secretion. Conclusions: IL-22 derived from activated T cells acts on SEMFs to elicit expression of proinflammatory cytokines and matrix-degrading molecules indicating proinflammatory/remodeling roles in IBD.
Epicutaneous Antigen Exposure Primes for Experimental Eosinophilic Esophagitis in Mice
Akei HS, Mishra A, Blanchard C, Rothenberg ME
Background & Aims: Eosinophilic esophagitis (EE) is frequently associated with atopic disease, including dermatitis and asthma. Data are emerging that atopic skin may provide an early entry point for antigen sensitization. We aimed to test the hypothesis that epicutaneous exposure to antigen primes for subsequent respiratory antigen-induced EE. Methods: Wild-type and genetically engineered mice were subjected to epicutaneous antigen sensitization and the development of experimental EE, and immune responses were examined. Results: We show that exposure to antigen via the epicutaneous route primes for marked eosinophilic inflammation in the esophagus triggered by a single airway antigen challenge. The development of experimental EE is associated with significant skin eosinophilia, accelerated bone marrow eosinophilopoiesis, blood eosinophilia, and large increases in serum antigen-specific immunoglobulin G1/immunoglobulin E using ovalbumin or Aspergillus fumigatus as the epicutaneous antigen. Mechanistic analysis with gene-targeted mice showed that interleukin-5 was required for esophageal eosinophilia and that interleukin-4, interleukin-13, and STAT6 contributed to a lesser extent. Conclusions: These findings provide the first evidence that epicutaneous exposure to allergens potently primes for EE via a Th2-dependent mechanism.
Inhibition of Transient Lower Esophageal Sphincter Relaxation and Gastroesophageal Reflux by Metabotropic Glutamate Receptor Ligands
Frisby CL, Mattsson JP, Jensen JM, Lehmann A, Dent J, Blackshaw L A
Background & Aims: Transient lower esophageal sphincter relaxation (TLESR) is the major mechanism of gastroesophageal acid reflux. TLESR is mediated via vagal pathways, which may be modulated by metabotropic glutamate receptors (mGluRs). Group I mGluRs (mGluR1 and 5) have excitatory effects on neurons, whereas group II (mGluR2 and 3) and group III (mGluR4, 6, 7, and 8) are inhibitory. This study determined the effect of mGluRs on triggering of TLESR and reflux in an established conscious ferret model. Methods: Esophageal manometric/pH studies were performed in ferrets with chronic esophagostomies. TLESR were induced by a gastric load of 25 mL glucose (pH 3.5) and 30 mL air. Results: In control treated animals, gastric load induced 3.52 ± 0.46 TLESRs per 47-minute study, 89.7% of which were associated with reflux episodes (n = 16). The mGluR5 antagonist MPEP inhibited TLESR dose dependently, with maximal 71% ± 7% inhibition at 35 ?mol/kg (n = 9; P < .0001). MPEP also significantly reduced reflux episodes (P < .001) and increased basal lower esophageal sphincter pressure (P < .05). MPEP inhibited swallowing dose dependently, suggesting a common action on trigger mechanisms for swallowing and TLESR. The more selective analogue, MTEP, had more potent effects (90% ± 6% inhibition TLESR at 40 ?mol/kg; n = 8; P < .0001). In contrast, the group I agonist DHPG tended to increase TLESR. The group II agonist (2R, 4R)-APDC was ineffective, whereas the group III agonist L-(AP4 slightly reduced TLESR (33% at 11 ?mol/kg; P < .05). The selective mGluR8 agonist (S)-3, 4-DCPG inhibited TLESR by 54% at 15 ?mol/kg (P < .01). Conclusions: mGluR5 antagonists potently inhibit TLESR and reflux in ferrets, implicating mGluR5 in the mechanism of TLESR. mGluR5 antagonists are therefore promising as therapy for patients with GERD.
Differential Regulation of Gastric Tumor Growth by Cytokines That Signal Exclusively Through the Coreceptor gp130
Howlett M, Judd LM, Jenkins B, La Gruta NL, Grail D, Ernst M, Giraud AS
Background & Aims: We have shown that mice with a mutation in gp130 (gp130757F/F), the signal transducing receptor for interleukin (IL)-6 family cytokines, have chronic gastric inflammation and develop distal stomach tumors associated with deregulated phosphorylated STAT3 expression. This model recapitulates many characteristics of intestinal-type gastric cancer in humans. Methods: To evaluate the role of IL-6 and IL-11 as ligands regulating tumor growth and submucosal invasion, we compared tumor characteristics of gp130757F/F mice with gp130757F/F mice lacking IL-6 or mature T and B cells. Results: As a result of the gp130757F/F mutation, expression of IL-6 and IL-11 was greatly up-regulated concomitant with activation of STAT3 and development of tumors. However, the lack of IL-6 or T and B cells did not impact on tumor growth. While IL-6 did not regulate tumor growth or tumor vascularization, gp130757F/F/IL-6?/? mice showed ?10–20—fold more submucosal tumor invasion, reduced mononuclear inflammatory cell infiltrate, and greater IL-11 and matrix metalloproteinase (MMP)-13 and MMP-9 synthesis than gp130757F/F mice. Expression of MMP-13 was largely restricted to tumor-associated stroma, but MMP-9 was also expressed in polymorphonuclear cells and a subset of epithelial cells. In addition, treatment with recombinant IL-11 stimulated expression of MMP-13 and MMP-9 in stomachs of wild-type mice. Conclusions: Increased submucosal invasion in gp130757F/F/IL-6?/? mice could not be explained by increased vascularization or reduced immunosurveillance but was most likely facilitated by augmented metalloproteinase activity driven by elevated IL-11 levels.
Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin ?5?1 as a Novel CCN2 Receptor
Gao R, Brigstock DR
Background & Aims: Pancreatic stellate cells (PSCs) are proposed to play a key role in the development of pancreatic fibrosis. The aim of this study was to evaluate the production by rat activated PSCs of the fibrogenic protein, connective tissue growth factor (CCN2), and to determine the effects of CCN2 on PSC function. Methods: CCN2 production was evaluated by immunoprecipitation and promoter activity assays. Expression of integrin ?5?1 was examined by immunoprecipitation and Western blot. Binding between CCN2 and integrin ?5?1 was determined in cell-free systems. CCN2 was assessed for its stimulation of PSC adhesion, migration, proliferation, DNA synthesis, and collagen I synthesis. Results: CCN2 was produced by activated PSCs, and its levels were enhanced by transforming growth factor ?1 treatment. CCN2 promoter activity was stimulated by transforming growth factor ?1, platelet-derived growth factor, alcohol, or acetaldehyde. CCN2 stimulated integrin ?5?1–dependent adhesion, migration, and collagen I synthesis in PSCs. Integrin ?5?1 production by PSCs was verified by immunoprecipitation, while direct binding between integrin ?5?1 and CCN2 was confirmed in cell-free binding assays. Cell surface heparan sulfate proteoglycans functioned as a partner of integrin ?5?1 in regulating adhesion of PSCs to CCN2. PSC proliferation and DNA synthesis were enhanced by CCN2. Conclusions: PSCs synthesize CCN2 during activation and after stimulation by profibrogenic molecules. CCN2 regulates PSC function via cell surface integrin ?5?1 and heparan sulfate proteoglycan receptors. These data support a role for CCN2 in PSC-mediated fibrogenesis and highlight CCN2 and its receptors as potential novel therapeutic targets.
Suppression of Hepatitis C Virus Replication by Cyclosporin A Is Mediated by Blockade of Cyclophilins
Nakagawa M, Sakamoto N, Tanabe Y, Koyama T, Itsui Y, Takeda Y, Chen C, Kakinuma S, Oooka S, Maekawa S, Enomoto N, Watanabe M
Background & Aims: Cyclosporin A specifically suppresses hepatitis C virus (HCV) replication in vitro at clinically achievable concentrations. In this study, we investigated the mechanisms of action of cyclosporin A against HCV replication. Methods: The in vitro effects of cyclosporin A on HCV replication were analyzed using an HCV replicon system that expresses chimeric luciferase reporter protein. Results: The significant effects of cyclosporin A on expression of an HCV replicon and the absence of such effects of FK506, which shares mechanisms of action with cyclosporin A, suggested the involvement of intracellular ligands of cyclosporin A, the cyclophilins. Transient and stable knockdown of the expression of cytoplasmic cyclophilins A, B, and C by short hairpin RNA–expressing vectors suppressed HCV replication significantly. A cyclosporin analogue, cyclosporin D, which lacks immunosuppressive activity but exhibits cyclophilin binding, induced a similar suppression of HCV replication. Furthermore, cyclosporin A treatment of Huh7 cells induced an unfolded protein response exemplified by expression of cellular BiP/GRP78. Treatment of cells with thapsigargin and mercaptoethanol, which induce the unfolded protein responses, suppressed HCV replication, suggesting that the cyclosporin-induced unfolded protein responses might contribute to the suppression of HCV protein processing and replication. Conclusions: The anti-HCV activity of cyclosporin A is mediated through a specific blockade of cyclophilins, and these molecules may constitute novel targets for anti-HCV therapeutics.
Case report
Novel Germline Mutation of KIT Associated With Familial Gastrointestinal Stromal Tumors and Mastocytosis
Hartmann K, Wardelmann E, Ma Y, Merkelbach–Bruse S, Preussner LM, Woolery C, Baldus SE, Heinicke T, Thiele J, Buettner R, Longley B J
Gastrointestinal stromal tumors (GISTs) are often associated with activating KIT mutations, affecting regulatory domains of the KIT tyrosine kinase. Sporadic mastocytosis in adults is usually also caused by KIT mutations that, however, activate KIT by affecting the intracellular enzymatic site of the molecule. Most GISTs respond to KIT inhibitors that bind to the enzymatic site; in most cases of mastocytosis, however, the modified enzymatic site is not affected by these drugs. We present a kindred with both familial GISTs and mastocytosis that express a novel germline KIT mutation in exon 8, resulting in deletion of codon 419 and affecting the extracellular domain of KIT. This mutation activates KIT, and the mutant KIT is inhibited by the tyrosine kinase inhibitor imatinib mesylate. Our studies identify a new regulatory region in the KIT molecule and strongly suggest that patients with extracellular KIT mutations respond to tyrosine kinase inhibitors.
Copyright © 2001-2005 by the American Gastroenterological Association. All rights reserved.
Table of Contents for September 2005 (Vol. 43, Issue 3)
Viral Hepatitis
Experts' opinions on the role of liver biopsy in HCV infection: A Delphi survey by the Italian Association of Hospital Gastroenterologists (A.I.G.O.), 16 May 2005
Almasio PL, Niero M, Angioli D, Ascione A, Gullini S, Minoli G, Oprandi NC, Pinzello GB, Verme G, Andriulli A
Background/Aims
Liver biopsy represents the gold standard to establish a diagnosis in all liver patients, but its current position in chronic viral hepatitis is questioned. We aimed to create a consensus on best practice of use of liver biopsy in the management of chronic HCV infection.
Methods
We applied the Delphi method to 12 clinical scenarios of chronic HCV infection, to assess the extent of agreement (consensus measurement) and to resolve disagreement (consensus development) on the appropriateness of liver biopsy.
Results
Among 108 chosen hepatologists, 61 (56.5%) accepted to participate to the first-round survey. In four patients the majority of experts (from 61.4 to 86.2%) agreed not to perform liver biopsy; in two cases an equivalent opinion was found, and in the remaining six scenarios the majority of experts would have recommended a biopsy. No expert recommended liver biopsy in all cases, while most agreed for an histological evaluation of 4 to 8 cases. At the second round, 36 experts (59%) submitted ballots. Fifty-four out of 431 (12.6%) original judgments were changed with equal distribution among different scenarios.
Conclusions
Our survey showed a great divergence of management of similar patients and should provide a stimulus for an evidence-based evaluation of liver histology in chronic HCV infection.
GNB3 C825T polymorphism and response to interferon-alfa/ribavirin treatment in patients with hepatitis C virus genotype 1 (HCV-1) infection, 31 May 2005
Sarrazin C, Berg T, Weich V, Mueller T, Frey UH, Zeuzem S, Gerken G, Roggendorf M, Siffert W
Background/Aims
The outcome of infection with the hepatitis C virus (HCV) has been shown to be influenced by genetic host factors. The G protein ?3 subunit (GNB3) C825T polymorphism has been shown to determine immune cell functions in vitro. We investigated the association of GNB3 genotypes with treatment response in HCV-infected patients.
Methods
We genotyped 1781 HCV-free blood donors and 232 HCV-infected patients treated with interferon-alfa/ribavirin. Sustained virologic response (SVR) was defined by undetectable HCV-RNA 24 weeks after discontinuation of therapy. Non-response (NR) was defined by positive HCV-RNA at the end of at least 24 weeks of treatment. GNB3 genotypes were determined by DNA restriction enzyme analyses.
Results
Genotype distribution was not significantly different in healthy controls and HCV-infected patients. Only in HCV genotype 1-infected patients a significant correlation between GNB3 CC genotype and NR could be observed (6 TT, 42 TC, 54 CC) versus SVR (11 TT, 25 TC, 19 CC) patients (P=0.004). In a logistic regression analysis including biochemical and virologic characteristics, only GNB3 CC genotype was significantly associated with NR (OR 4.9; 95% CI=1.4–16.5; P=0.011).
Conclusions
The GNB3 825 CC genotype is associated with NR in HCV-1-infected patients.
Hepatitis C virus-specific reactivity of CD4+-lymphocytes in children born from HCV-infected women, 31 May 2005
Della Bella S, Riva A, Tanzi E, Nicola S, Amendola A, Vecchi L, Nebbia G, Longhi R, Zanetti AR, Villa ML
Background/Aims
T-lymphocyte reactivity against viral antigens may represent the only immunological marker of host contact with a virus. Aim of the present study was to investigate whether vertical exposure to hepatitis C virus (HCV) could activate HCV-specific T-cell responses that may represent a biomarker of previous contact with the virus, and possibly contribute to the low rate of vertical HCV transmission.
Methods
We studied 28 children born from chronically HCV-infected mothers. HCV-specific activation and proliferation of CD4+-lymphocytes and cytokine production were evaluated in cultures of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with HCV-peptides.
Results
HCV-specific CD4+-cell reactivity was observed in 20 out of the 28 children (71%). The proliferation of HCV-specific CD4+-cells was more frequent and vigorous in children than in their mothers. In children, but not in the mothers, activation of CD4+-cells upon stimulation with HCV-peptides was directly correlated with proliferation. Early upon stimulation with HCV-peptides, lymphocytes from children produced lower levels of IL-10 than lymphocytes from the mothers.
Conclusions
Vertical exposure to HCV induces the development of viral-specific CD4+-cell-mediated immune responses, possibly endowed with protective function against infection, which may contribute to the low rate of vertical HCV transmission.
Accumulation of 8-nitroguanine in the liver of patients with chronic hepatitis C, 31 May 2005
Horiike S, Kawanishi S, Kaito M, Ma N, Tanaka H, Fujita N, Iwasa M, Kobayashi Y, Hiraku Y, Oikawa S, Murata M, Wang J, Semba R, Watanabe S, Adachi Y
Background/Aims
Nucleic acid damage by reactive nitrogen and oxygen species may contribute to inflammation-related carcinogenesis. To investigate the extent of nucleic acid damage in hepatitis C virus infection and its change after interferon treatment, we measured 8-nitroguanine and 8-hydroxy-2?-deoxyguanosine (8-OHdG) in the liver of patients with chronic hepatitis C (CHC) before and after interferon therapy.
Methods
Hepatic accumulation of 8-nitroguanine and 8-OHdG was immunohistochemically evaluated in 20 CHC patients and 7 control patients with non-alcoholic fatty liver.
Results
Immunoreactivities of 8-nitroguanine and 8-OHdG were strongly detected in the liver from patients with CHC, but not in control livers. 8-Nitroguanine accumulation was found not only in infiltrating inflammatory cells, but also hepatocytes particularly in the periportal area. The accumulation of 8-nitroguanine and 8-OHdG increased with inflammatory grade (8-nitroguanine; P=0.0019, 8-OHdG; P=0.0009). In the sustained virological responder group after interferon therapy, 8-nitroguanine and 8-OHdG accumulation were markedly decreased in the liver (8-nitroguanine; P=0.018, 8-OHdG; P=0.018).
Conclusions
In this study, we demonstrated for the first time that 8-nitroguanine accumulated in the liver of patients with CHC. 8-Nitroguanine is a useful biomarker to evaluate the severity of HCV-induced chronic inflammation in relation to hepatocellular carcinoma.
Genotype C hepatitis B virus infection is associated with a higher risk of reactivation of hepatitis B and progression to cirrhosis than genotype B: A longitudinal study of hepatitis B e antigen-positive patients with normal aminotransferase levels at baseline, 26 May 2005
Chu CM, Liaw YF
Background/Aims
Longitudinal studies on the relationship between hepatitis B virus (HBV) genotypes and reactivation of hepatitis B and progression to cirrhosis were very rare.
Methods
Liver biochemistry, virological markers and ultrasound were monitored in 202 hepatitis B e antigen (HBeAg)-positive patients with normal alanine aminotransferase (ALT) at baseline for 3–20 (average 10.8) years, and the outcome was correlated with HBV genotypes.
Results
There were 150 genotype B and 52 genotype C patients. Hepatitis activity during the HBeAg-positive phase showed no significant difference. However, genotype B was associated with a significantly earlier and higher rate of HBeAg seroconversion. HBeAg seroconversion correlated with age at entry for genotype B and with ALT levels for genotype C. Reactivation of hepatitis B was significantly more common in genotype C patients. Five genotype B and 10 genotype C patients progressed to cirrhosis. Multivariate analysis revealed that genotype C (P=0.03) and reactivation of hepatitis B (P=0.0004) were independent factor predictive of cirrhosis.
Conclusions
Rate and factors of HBeAg seroconversion, and rate of reactivation of hepatitis B differed between genotype B and genotype C patients. Genotype C and reactivation of hepatitis B were associated with increased risk of cirrhosis.
Changing pattern of hepatitis C virus spread in rural areas of Egypt, 31 May 2005
Arafa N, Hoseiny ME, Rekacewicz C, Bakr I, El-Kafrawy S, Daly ME, Aoun S, Marzouk D, Mohamed MK, Fontanet A
To identify patterns of HCV spread in the Nile Delta of Egypt.
Methods
Residents in a Nile Delta village were invited to participate in a cohort study of HCV infection. Risk factors for past or current infection were identified at cohort intake using generalized estimated equations models. Attributable fractions were calculated for all independent risk factors.
Results
The prevalence of HCV antibodies increased from 2.7% in those <20 years of age to more than 40% in males aged 40–54 years. The peak in HCV prevalence in the 40–54 year age group corresponds to the aging of the cohort of children infected through schistosomiasis intravenous treatments in the 1960s-70s (accounting for 12.4% of all HCV infections observed today among adults). Following this initial founding event, the HCV epidemic has spread in the community through iatrogenic factors, and particularly injections (37.9% of the overall attributable fraction in adults). In children, however, no iatrogenic factors were associated with increased risk of infection, suggesting a change in the pattern of HCV spread.
Conclusions
While HCV infections in adults could be attributed to iatrogenic factors, and particularly injections, infections in children could not be explained by similar routes of transmission.
Predicting sustained virological responses in chronic hepatitis C patients treated with peginterferon alfa-2a (40KD)/ribavirin, 31 May 2005
Ferenci P, Fried MW, Shiffman ML, Smith CI, Marinos G, Gonçales Jr. FL, Häussinger D, Diago M, Carosi G, Dhumeaux D, Craxì A, Chaneac M, Reddy K R
Background/Aims
Prediction of sustained virological response (SVR) during treatment would allow clinicians to identify patients most likely to benefit from therapy.
Methods
Retrospective analysis of data from 1121 adults with chronic hepatitis C treated for 48 weeks with peginterferon alfa-2a (40KD) 180?g/week plus placebo or ribavirin (1000/1200mg/day), or interferon alfa-2b 3 MIU three times/week plus ribavirin in a randomized, multinational, study.
Results
67% of patients treated with peginterferon alfa-2a (40KD)/ribavirin with early virological responses (HCV RNA negative or ≥2 log10 decrease) at week 12 had SVRs at week 72 (HCV RNA <50IU/mL). The negative predictive value (NPV) was 97%. The probability of an SVR increased with the rapidity of HCV RNA suppression. The highest SVR rates were achieved in patients with rapid virological responses at week 4, but the corresponding NPV (74%) is too low for a decision criterion. In patients with early virological responses by week 12, the SVR rate was ≈20% lower in those who received <80% compared with patients who received ≥80% of the planned ribavirin dose.
Conclusions
Early, sustained suppression of HCV replication portends an SVR. Cessation of treatment may be contemplated in patients without a ≥2 log10 reduction in HCV RNA after 12 weeks.
Cirrhosis and its Complications
Serum alpha-fetoprotein levels in patients with advanced hepatitis C: Results from the HALT-C Trial, 26 May 2005
Di Bisceglie AM, Sterling RK, Chung RT, Everhart JE, Dienstag JL, Bonkovsky HL, Wright EC, Everson GT, Lindsay KL, Lok ASF, Lee WM, Morgan TR, Ghany MG, Gretch DR, the HALT-C Trial Group
Background/Aims
Alpha-fetoprotein (AFP) has been useful in the diagnosis of hepatocellular carcinoma (HCC) but lacks specificity. We assessed serum AFP among patients with chronic hepatitis C and advanced fibrosis to establish predictors of AFP elevations and changes with antiviral therapy.
Methods
Serum AFP was measured at baseline and on therapy in patients in the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis (HALT-C). AFP levels were correlated with patient demographic and clinical features.
Results
Baseline AFP was ≥20ng/mL in 191 of 1145 patients (16.6%). Mean AFP values were significantly higher in patients with cirrhosis than in those with bridging fibrosis (22.5 vs. 11.4ng/mL, P<0.0001). Factors independently associated with raised serum AFP in patients with cirrhosis were female gender, black race, decreased platelet count, increased serum AST/ALT ratio, serum ferritin, and Mallory bodies in liver biopsies. Serum AFP levels decreased significantly during therapy with pegylated interferon ?-2a and ribavirin. HCC was identified in six subjects, only three of whom had AFP>20ng/mL.
Conclusions
Among patients with advanced chronic hepatitis C, serum AFP values are frequently elevated, even in the absence of HCC. Factors associated with raised AFP include severity of liver disease, female gender and black race. Serum AFP levels decline during antiviral therapy.
Liver Failure, Growth and Cancer
Protein transduction with bacterial cytosine deaminase fused to the TLM intercellular transport motif induces profound chemosensitivity to 5-fluorocytosine in human hepatoma cells, 5 May 2005
Hillemann A, Brandenburg B, Schmidt U, Roos M, Smirnow I, Lemken ML, Lauer UM, Hildt E
Background/Aims
This study investigates the application of protein based therapeutic suicide enzyme/prodrug approaches providing novel means for both safe and effective local therapeutic regimes in solid tumors.
Methods
Employing a novel cell permeable peptide, known as the translocation motif (TLM) of hepatitis B virus, E. coli cytosine deaminase (BCDase) suicide fusion proteins were generated.
Results
TLM fusion proteins formed hexamers (as do parental wtBCDase proteins) and retained the specific enzymatic activity of cytosine conversion to uracil also being comparable to parental wtBCDase proteins. However, only BCDase-TLM fusion proteins, but not TLM-BCDase fusion nor parental wtBCDase proteins were found to be taken up to the cytoplasm of target cells as demonstrated both by confocal laser scanning microscopy and cell fractionation. Uptake of BCDase-TLM worked both efficiently and rapidly and was found to be independent from the endosomal pathway. Since BCDase-TLM fusion proteins completely retained their suicide enzymatic activity in the course of translocation across the plasma membrane their usage as profound inducers of chemo-sensitivity to 5-FC strongly is suggested.
Conclusions
Future therapeutic local application of cell-permeable BCDase-TLM fusion proteins together with a systemic 5-FC prodrug application could result in profound antitumor activities without apparent side effects.
In vivo quantification of liver dialysis: Comparison of albumin dialysis and fractionated plasma separation, 16 May 2005
Krisper P, Haditsch B, Stauber R, Jung A, Stadlbauer V, Trauner M, Holzer H, Schneditz D
Background/Aims
Artificial liver support represents a potentially useful option for the treatment of severe liver failure. A sufficient ‘dose’ might be crucial for such treatments to provide a survival benefit. The aim of this study was to compare in vivo efficiency and resulting delivered treatment dose of two commercially available devices that use different therapeutic principles: albumin dialysis (AD, MARS®) and fractionated plasma separation (FPS, Prometheus®).
Methods
Eight patients with acute-on-chronic liver failure were treated alternately with AD and FPS. Thirty-two treatments at identical blood and dialysate flow rates were evaluated. Clearance and reduction ratio (a measure of delivered treatment dose) were compared for bilirubin subfractions, ammonia and urea.
Results
FPS achieved significantly higher clearance for all measured protein-bound and water-soluble markers. This resulted in significantly higher reduction ratios for FPS compared to AD. Unconjugated bilirubin, a marker for strongly albumin-bound toxins, was influenced only by FPS.
Conclusions
FPS provided a higher delivered treatment dose than a matching treatment with AD. Reduction ratios of bilirubin and urea should be reported in clinical studies on liver dialysis, since delivered dose is likely to be linked to the clinical effectiveness of extracorporeal liver support therapies.
Percutaneous ethanol injection for small hepatocellular carcinoma: Therapeutic efficacy based on 20-year observation, 24 June 2005
Ebara M, Okabe S, Kita K, Sugiura N, Fukuda H, Yoshikawa M, Kondo F, Saisho H
Background/Aims
To evaluate the therapeutic efficacy of percutaneous ethanol injection (PEI) for patients with ≤3 lesions of small (≤3cm diameter) hepatocellular carcinoma (HCC).
Methods
PEI was applied to 270 patients with small HCC as the first-line treatment option during a 20-year period.
Results
(1) There was no treatment-related deaths, and only 2.2% of severe complications; (2) PEI induced a complete response of all HCCs according to CT evaluation performed within one month after the procedure, and the local recurrence rate at 3 years was 10%; (3) the overall 3- and 5-year survival rates after treatment were 81.6 and 60.3%, respectively, but the rates were higher, 87.3 and 78.3%, in Child A patients with a solitary tumor ≤2cm in diameter; (4) factors significantly influencing survival were liver function (P=0.0033) and serum alpha-fetoprotein level (P=0.0014), and (5) the recurrence rate at remote sites in the liver was lower in patients with HCC ≤2cm (P=0.0395) and in those with a solitary HCC (P<0.0001) according to Cox's proportional hazard model. (6) Radiofrequency ablation would not have been performed in approximately 25% of these patients.
Conclusions
PEI is considered a reliable treatment for small HCC in terms of safety and efficacy.
Molecular and Cell Biology
Recombinant HBsAg inhibits LPS-induced COX-2 expression and IL-18 production by interfering with the NF?B pathway in a human monocytic cell line, THP-1, 6 May 2005
Cheng J, Imanishi H, Morisaki H, Liu W, Nakamura H, Morisaki T, Hada T
Background/Aims
Hepatitis B virus suppresses the human immune-system and HBsAg inhibits the induction of cytokines by LPS in human macrophages, but the mechanisms involved remain unclear. COX-2 and its product, PGE2, play a role in hepatits B and IL-18 has also been shown to inhibit HBV infection in vivo. We investigated whether rHBsAg affects induction of COX-2 and IL-18 by LPS and, if so, which signal pathways are involved in a human monocytic cell line, THP-1.
Methods
Cell culture, Western blotting for COX-2, ERK and IKB-?, immunofluorescence for HBsAg and NF?B protein and ELISA for PGE2, IL-18 and IL-12 were performed.
Results
rHBsAg inhibits LPS-induced COX-2 expression in a time- and dose-dependent manner by blocking the ERK and NF?B pathways. LPS-induced IL-18 production was also down-regulated by rHBsAg by interfering mainly with the NF?B pathway. PGE2 reversed the inhibition of LPS-induced IL-18 production by rHBsAg. rHBsAg was also found to inhibit the induction of IL-12 by LPS in THP-1 cells.
Conclusions
These results showed a novel anti-inflammatory property of rHBsAg which involves inhibition of COX-2 and suggested that hepatits B virus may regulate IFN-? production by inhibiting IL-18 and IL-12 production.
Developmental expression of canalicular transporter genes in human liver, 6 May 2005
Chen HL, Chen HL, Liu YJ, Feng CH, Wu CY, Shyu MK, Yuan RH, Chang MH
BSEP, MRP2, and MDR3 are major hepatic canalicular transporters mediating bile secretion. Their expression in human liver during development has not been reported.
Methods
Human liver samples from fetus at gestational age 14–20 weeks, adult livers and liver samples of infants with biliary atresia were tested for mRNA expression of BSEP, MDR3, MRP2, NTCP, FIC1, and FXR genes by using real-time RT-PCR. Immunohistochemical staining of BSEP, MDR3, and MRP2 were performed on fetal and adult livers.
Results
All the genes tested were expressed at mid-gestational age. MDR3 and NTCP showed significant lower levels in fetal livers compared to adults. In patients with biliary atresia, all the genes tested showed higher mean expression levels than adults except for NTCP, but not statistically significant. The immunohistochemical staining of MRP2 in fetal liver was canalicular, BSEP showed both intracellular and canalicular staining, and MDR3 staining was faint, only occasional canalicular pattern could be seen.
Conclusions
The major canalicular transporter genes are expressed at mid-gestational stage during human fetal development, but are different in expression level and targeting pattern, indicating differential regulation and maturation.
Long-term 17?-ethinyl estradiol treatment decreases cyclin E and cdk2 expression, reduces cdk2 kinase activity and inhibits S phase entry in regenerating rat liver, 23 May 2005
Koroxenidou L, Ohlson LCE, Porsch Hällström I
Background/Aims
The synthetic estrogen 17?-ethinyl estradiol (EE), a potent tumor promoter in rat liver, stimulates growth during short-term treatment but inhibits hepatocyte proliferation upon prolonged treatment. To identify the molecular targets of the mitoinhibitory effect of EE, the expression of proteins regulating G1- and S-progression were analyzed during the first cell cycle in EE-treated female Wistar rats.
Methods
Long-term (60 days) EE treatment. Immunohistochemical staining for proliferation cell nuclear antigen (PCNA) to detect cells in S phase and quantification of mitosis. Western blot to monitor protein expression. Cdk2 kinase assay to examine histone H1 phosphorylation.
Results
EE reduced the number of cells in S phase and mitosis by about 70%. Cyclin D1 and D3 were unaffected, while cdk4 was moderately decreased. Cyclin E and cdk2 were markedly decreased with concomitant marked reduction of cdk2 kinase activity. EE also decreased cyclin A and increased G1 levels of p53 and p21.
Conclusions
EE causes a cell cycle block before S-phase. The reduction of the cdk2 kinase activity, essential for G1/S-transition, might be involved in the cell cycle block. Also, EE treatment results in p53 activation and upregulation of the cdk inhibitor p21 that might contribute to the G1 arrest.
Cyclin D1 is up-regulated in hepatocytes in vivo following cell-cycle block induced by retrorsine, 3 June 2005
Pitzalis S, Doratiotto S, Greco M, Montisci S, Pasciu D, Porcu G, Pani P, Laconi S, Laconi E
Background/Aims
We reported massive liver repopulation by transplanted hepatocytes in rats given retrorsine (RS), a pyrrolizidine alkaloid which blocks proliferation of resident cells. In these studies, molecular alterations induced by RS on hepatocyte cell cycle were investigated.
Methods
Animals were treated according to the protocol for liver repopulation, i.e. two injections of RS (30mg/kg) followed by two-thirds partial hepatectomy (PH) and were sacrificed at various time points thereafter. Livers were analyzed for the expression of cell cycle-related genes.
Results
Prior to PH, increased cyclin D1 mRNA and protein levels were found in livers of RS-treated rats. Expression of PCNA was also increased; however, DNA synthesis was not significantly changed. Other cyclins, including cyclin B and cyclin E, were not induced. Cyclin D1 expression increased in controls post-PH and then declined by 48h, as expected. By contrast, no such modulation of cyclin D1 levels was seen in RS group receiving PH and expression remained high at 48h, without mitotic division.
Conclusions
Exposure to RS is able to block cell cycle progression after cyclin D1 and PCNA induction, but prior to S phase. Such persistent block outside the resting phase may contribute to the selective replacement of resident cells during liver repopulation.
Cholestasis and Autoimmune Liver Disease
Cholestatic liver disease modulates susceptibility to ischemia/reperfusion-induced arrhythmia, but not necrosis and hemodynamic instability: The role of endogenous opioid peptides, 16 May 2005
Hajrasouliha AR, Tavakoli S, Jabehdar-Maralani P, Ebrahimi F, Shafaroodi H, Mirkhani SH, Amanpour S, Dehpour AR
Background/Aims
Acute cholestasis is associated with cardiovascular complications, which mainly manifest during stressful conditions. The goal of this study is to evaluate susceptibility of 7-day bile duct-ligated rats to ischemia/reperfusion-induced injury.
Methods
Sham-operated and cholestatic rats, treated with daily normal saline, L-NAME (a non-selective NO synthase inhibitor) naltrexone, or both L-NAME and naltrexone were subjected to 30min of ischemia followed by 2h of reperfusion.
Results
Cholestatic rats demonstrated significant bradycardia, hypotension (P<0.01), and QT prolongation (P<0.001). The incidence of premature ventricular contractions (P<0.01), incidence and duration of ventricular tachycardia (P<0.05), but not ventricular fibrillation, were significantly lower in cholestatic rats. There was no significant difference in hemodynamic instability and infarct size between the groups. L-NAME corrected QT prolongation in cholestatic rats (P<0.05), with no effect on heart rate, blood pressure and arrhythmia. Naltrexone restored normal heart rate (P<0.05), blood pressure (P<0.05) and susceptibility to arrhythmia (P<0.05) in cholestatic animals, with no significant effect on QT interval. L-NAME and naltrexone co-administration corrected bradycardia (P<0.05), hypotension (P<0.05), QT prolongation (P<0.05) and abolished resistance of cholestatic rats against arrhythmia (P<0.05).
Conclusions
This study suggests that short-term cholestasis is associated with resistance against ischemia/reperfusion-induced arrhythmia, which depends on availability of endogenous opioids.
Genetic and Metabolic Liver Disease
Abnormal deposition of collagen around hepatocytes in Wilson's disease is associated with hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2, 31 May 2005
Vadasz Z, Kessler O, Akiri G, Gengrinovitch S, Kagan HM, Baruch Y, Izhak OB, Neufeld G
Background/Aims
Lysyl-oxidases catalyze the oxidation of lysine residues in collagen and elastin thereby promoting their polymerization. We have studied here the expression of four lysyl-oxidases in normal and diseased human liver.
Methods
The expression of the different lysyl-oxidases in paraffin embedded liver sections was studied using in-situ hybridization and immunohistochemistry. The enzymatic activity of lysyl-oxidase like protein-2 (Loxl2 or LOR-1) using a previously described lysyl-oxidase assay.
Results
We have found that the four lysyl-oxidases which we examined are not significantly expressed in the normal liver. By contrast, Wilson's disease and primary biliary cirrhosis (PBC) patients express lysyl-oxidase (Lox) and lysyl-oxidase like protein-2 (Loxl2 or LOR-1) in hepatocytes, and the expression is accompanied by collagen deposition around the hepatocytes. Lysyl-oxidases are also expressed in additional fibrotic liver diseases such as hepatitis B and C but in these diseases the expression is confined to the fibrotic lesions and collagen does not accumulate around hepatocytes. We have found that Loxl2 is able to oxidize lysine residues of collagen, and behaves in that respect similarly to Lox. The copper chelator D-penicillamine inhibits Loxl2 induced oxidation of collagen but the Lox inhibitor ?-aminopropionitrile did not inhibit the oxidation using a BAPN concentration at which Lox activity was completely inhibited. Loxl2 also catalyzed the oxidation of cell surface proteins on HepG2 hepatoblastoma cells and inhibited their proliferation.
Conclusions
Upregulation of Lox and Loxl2 in hepatocytes of Wilson's disease and PBC patients may contribute to liver damage by various mechanisms. The upregulation of Lox and Loxl2 in Wilson's disease could perhaps be utilized for diagnostic purposes since their expression is up-regulated in hepatocytes even before the onset of fibrosis.
Prevalence of and risk factors for fatty liver in a general population of Shanghai, China, 16 May 2005
Fan JG, Zhu J, Li XJ, Chen L, Li L, Dai F, Li F, Chen SY
To determine the prevalence and risk factors of fatty liver (FL) among Shanghai adults.
Methods
A cross-sectional ultrasonographic survey with randomized multistage stratified cluster sampling was used.
Results
The study included 3175 subjects (1218 men) with a mean age of 52 years. FL was found in 661 (20.82%) subjects. After adjustment by age and sex, FL prevalence was found to be 17.29%, and the prevalences of alcoholic, suspected alcoholic and nonalcoholic FL were determined to be 0.79, 1.15 and 15.35%, respectively. Generally, age, body mass index (BMI), waist circumference, blood pressure, and the prevalences of obesity, diabetes, hypertension and dyslipidemia were all significantly higher in FL patients than in controls; In contrast, the levels of high-density-lipoprotein cholesterol (HDL-C), education and physical activity were markedly lower. Multiple regression analyses showed that only nine factors (male, educational level, waist circumference, BMI, HDL-C, triglyceride, fasting plasma glucose, diabetes and hypertension) were closely related to FL. In excessive drinkers, obesity increased the risk for FL by 4.8-fold, but excessive drinking was associated with only a 1.5-fold increased risk in obese subjects.
Conclusions
FL in Shanghai is highly prevalent and mainly related to multiple metabolic disorders.
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