HEPATOLOGY
Volume 45, Issue 2, February 2007
Original Article
A randomized controlled trial of licartin for preventing hepatoma recurrence after liver transplantation (p 269-276)
Jing Xu, Zhong-Yang Shen, Xin-Guo Chen, Qing Zhang, Hui-Jie Bian, Ping Zhu, Hui-Yun Xu, Fei Song, Xiang-Min Yang, Li Mi, Qing-Chuan Zhao, Rong Tian, Qiang Feng, Si-He Zhang, Yu Li, Jian-Li Jiang, Ling Li, Xiao-Ling Yu, Zheng Zhang, Zhi-Nan Chen
Orthotopic liver transplantation (OLT) is the only curative therapy of HCC with underlying cirrhosis, but due to HCC metastasis and recurrence, its benefit is limited to a small population who meet the strict selection criteria. We previously reported that Licartin ([131I]mAb HAb18G/CD147) was safe and effective in treating HCC patients, and its antigen, HAb18G/CD147, was closely related to HCC invasion and metastasis. Here, we reported a randomized controlled trial to assess the post-OLT antirecurrence efficacy of Licartin in advanced HCC patients. We randomized 60 post-OLT patients with HCC, who were at tumor stage 3/4 and outside the Milan criteria before OLT, into 2 groups. Three weeks after OLT, the treatment group received 15.4 MBq/kg of Licartin, while the control group received placebo intravenously for 3 times with an interval of 28 days. At 1-year follow-up, the recurrence rate significantly decreased by 30.4% (P = 0.0174) and the survival rate increased by 20.6% (P = 0.0289) in the treatment group, compared with those in the control group. For the control group versus the treatment group, the hazard ratio for recurrence was 3.60 (95% confidence interval [CI], 1.50-8.60) and that for death was 3.87 (95% CI, 1.23-12.21). Licartin treatment also resulted in an earlier decreased AFP level and a longer time of normal AFP level than placebo (P = 0.0016). No Licartin-related toxic effects were observed. Conclusion: Licartin is a promising drug for preventing post-OLT tumor recurrence in advanced HCC patients excluded by the currently strict criteria for OLT. HAb18G/CD147 can be a good drug target.
Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma (p 277-285)
Teresa Pollicino, Giuseppina Raffa, Lucy Costantino, Antonella Lisa, Cesare Campello, Giovanni Squadrito, Massimo Levrero, Giovanni Raimondo
Occult HBV infection is characterized by the persistence of HBV DNA in the liver of individuals negative for HBV surface antigen (HBsAg). Occult HBV may exist in the hepatocytes as a free genome, although the factors responsible for the very low viral replication and gene expression usually observed in this peculiar kind of infection are mostly unknown. Aims of this study were to investigate whether the viral genomic variability might account for the HBsAg negativity and the inhibition of the viral replication in occult HBV carriers, and to verify in vitro the replication capability of occult HBV strains. We studied liver viral isolates from 17 HBV patients, 13 with occult infection and 4 HBsAg-positive. Full-length HBV genomes from each case were amplified and directly sequenced. Additionally, full-length HBV DNA from eight occult-HBV and two HBsAg-positive cases were cloned and sequenced. Finally, three entire, linear HBV genomes from occult cases were transiently transfected in HuH7 cells. Direct sequencing showed the absence of mutations capable of interfering with viral replication and gene expression in the major viral population of each case. Cloning experiments showed highly divergent HBV strains both in HBsAg-positive and HBsAg-negative individual cases (range of divergence 1.4%-7.1%). All of the 3 transfected full-length HBV isolates showed normal patterns of replication in vitro. Conclusion: Multiple viral variants accumulate in the liver of occult HBV-infected patients. Occult HBV strains are replication-competent in vitro, suggesting that host, rather than viral factors are responsible for cryptic HBV infection.
Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model (p 286-296)
Chih-Peng Chang, Ming-Cheng Yang, Hsiao-Sheng Liu, Yee-Shin Lin, Huan-Yao Lei
Concanavalin A (ConA), a lectin with mannose specificity that can induce acute hepatic inflammation, was tested for its therapeutic effect against hepatoma. ConA is cytotoxic or inhibitory to hepatoma cells, which is mediated by the autophagic pathway through mitochondria. Once it was bound to cell membrane glycoproteins, the ConA was internalized and preferentially localized onto the mitochondria. The mitochondria membrane permeability changed, and an autophagic pathway including LC3-II generation, double-layer vesicle, BNIP3 induction, and acidic vesicular organelle formation was induced. Either 3-MA or siRNA for BNIP3 and LC3, but neither beclin-1 nor ATG 5, partially inhibited the ConA-induced cell death. In addition to the autophagy induction, ConA is known to be a T cell mitogen. Using an in situ hepatoma model, ConA can exert an anti-hepatoma therapeutic effect, inhibiting tumor nodule formation in the liver and prolonging survival. Conclusion: ConA can be considered as an anti-hepatoma agent therapeutically because of its autophagic induction and immunomodulating activity. This dual function of ConA provides a novel mechanism for the biological effect of lectin.
Fibroindex, a practical index for predicting significant fibrosis in patients with chronic hepatitis C (p 297-306)
Masahiko Koda, Yoshiko Matunaga, Manri Kawakami, Yukihiro Kishimoto, Takeaki Suou, Yoshikazu Murawaki
Diagnosis of the stage of liver fibrosis in chronic hepatitis C is essential for making a prognosis and deciding on antiviral therapy. In the present study a simple model consisting of routine laboratory tests was constructed and then validated in cross-sectional and longitudinal investigations. Consecutive treatment-naive patients with chronic hepatitis C who had undergone liver biopsy were divided into 2 cohorts: an estimation set (n = 240) and a validation set (n = 120). A longitudinal set consisted of 30 patients who had undergone a liver biopsy twice, before and after IFN treatment. The FibroIndex was derived from the platelet count, AST, and gamma globulin measurements in the estimation set. The areas under the ROC curves of the FibroIndex for predicting significant fibrosis were 0.83 and 0.82 for the validation set, better than those of the Forns index and the aminotransferase-to-platelet ratio index (APRI). Using the best cutoff values, whether significant fibrosis was present was diagnosed with high positive predictive values, and 35% of patients could avoid liver biopsy. In the longitudinal set, there was a significant decrease in the FibroIndex of 14 patients whose fibrosis stage improved, and a significant increase in that of 5 patients whose fibrosis stage deteriorated. Change in the FibroIndex correlated significantly with variation in fibrosis stage. There was no such correlation with the Forns index or the APRI. Conclusion: The FibroIndex is a simple and reliable index for predicting significant fibrosis in chronic hepatitis C and could also be used as a surrogate marker during antifibrotic treatment for chronic hepatitis C.
Adding-on versus switching-to adefovir therapy in lamivudine-resistant HBeAg-negative chronic hepatitis B (p 307-313)
Irene Rapti, Evangelini Dimou, Panayota Mitsoula, Stephanos J. Hadziyannis
We studied the long-term efficacy of adefovir dipivoxil (ADV) treatment in 42 HBeAg-negative patients with chronic hepatitis B (CHB) who had developed genotypical lamivudine (LAM) resistance with virological and clinical breakthroughs under long-term LAM treatment. Patients were allocated in 2 treatment groups. In the first (n = 14), LAM was switched to ADV monotherapy whereas in the second (n = 28) ADV was added to LAM. The two groups did not differ in patients' characteristics, all of them having HBV genotype D infection with the precore stop codon mutation. Within 12 months from start of ADV treatment, serum HBV DNA became nondetectable and ALT normalized in 71% and 90% of patients, respectively, with no difference between the 2 arms. Patients with baseline HBV DNA levels less than 10[7] copies/ml experienced a significantly earlier and more frequent decline in serum HBV DNA to nondetectable levels as compared with patients with greater than 10[7] HBV DNA copies/ml at baseline (P = 0.0013) This response has hitherto been maintained (median treatment duration 40 months) in all patients with ADV added to LAM, whereas virological and biochemical breakthroughs due to development of ADV signature resistance mutations occurred in 3 of 14 patients (21%) on ADV monotherapy 15 to 18 months from start of treatment (P = 0.0174). Conclusion: Adding ADV to LAM in HBeAg-negative CHB patients with LAM resistance effectively suppresses HBV replication in most of them and induces biochemical remission that can be maintained in all of them at least for 3 years without any evidence of development of resistance to ADV.
AMA production in primary biliary cirrhosis is promoted by the TLR9 ligand CpG and suppressed by potassium channel blockers (p 314-322)
Yuki Moritoki, Zhe-Xiong Lian, Heike Wulff, Guo-Xiang Yang, Ya-Hui Chuang, Ruth Y. Lan, Yoshiyuki Ueno, Aftab A. Ansari, Ross L. Coppel, Ian R. Mackay, M. Eric Gershwin
We previously reported that peripheral blood mononuclear cells (PBMCs) from patients with primary biliary cirrhosis (PBC) produce significantly higher levels of polyclonal IgM than controls after exposure to CpG. Furthermore, the prevalence and unusually high levels of antimitochondrial antibodies (AMAs) in patients with PBC suggest a profound loss of B cell tolerance. We have addressed the issue of whether CpG will promote the production of AMAs and whether new experimental agents that inhibit the lymphocyte potassium channels Kv1.3 and KCa3.1 can suppress CpG-mediated B cell activation and AMA production. PBMCs were stimulated with and without CpG and were subsequently analyzed for phenotype, including expression of TLR9, CD86, and KCa3.1 concurrent with measurements of AMA and responses to a control antigen, tetanus toxoid, in supernatants. Additionally, K+ channel expression on B cells from PBC patients and controls was studied using whole-cell patch-clamp technology. In patients with PBC, CpG induces secretion of AMAs in PBMCs and also up-regulates B cell expression of TLR9, CD86, and KCa3.1. Additionally, K+ channel blockers suppress secretion of AMA without a reduction of CpG-B-enhanced IgM production. Furthermore, there is diminished up-regulation of TLR9 and CD86 without affecting proliferation of B cells, B cell apoptosis, or viability. Conclusion: These data suggest that the hyperresponsiveness of B cells in PBC accelerates B cell-mediated autoimmunity.
Bile duct proliferation in liver-specific Jag1 conditional knockout mice: Effects of gene dosage (p 323-330)
Kathleen M. Loomes, Pierre Russo, Matthew Ryan, Anthony Nelson, Lara Underkoffler, Curtis Glover, Hong Fu, Thomas Gridley, Klaus H. Kaestner, Rebecca J. Oakey
The Notch signaling pathway is involved in determination of cell fate and control of cell proliferation in multiple organ systems. Jag1 encodes a ligand in the Notch pathway and has been identified as the disease-causing gene for the developmental disorder Alagille syndrome. Evidence from the study of human disease and mouse models has implicated Jag1 as having an important role in the development of bile ducts. We have derived a conditional knockout allele (Jag1loxP) to study the role of Jag1 and Notch signaling in liver and bile duct development. We crossed Jag1loxP mice with a transgenic line carrying Cre recombinase under the control of the albumin promoter and -fetoprotein enhancer to ablate Jag1 in hepatoblasts. The liver-specific Jag1 conditional knockout mice showed normal bile duct development. To further decrease Notch pathway function, we crossed the Jag1 conditional knockout mice with mice carrying the hypomorphic Notch2 allele, and bile duct anatomy remained normal. When Jag1 conditional mice were crossed with mice carrying the Jag1 null allele, the adult progeny exhibited striking bile duct proliferation. Conclusion: These results indicate that Notch signaling in the liver is sensitive to Jag1 gene dosage and suggest a role for the Notch pathway in postnatal growth and morphogenesis of bile ducts.
Mutual changes of thioredoxin and nitrosothiols during biliary cirrhosis: Results from humans and cholestatic rats (p 331-339)
Ignazio Grattagliano, Piero Portincasa, Vincenzo O. Palmieri, Giuseppe Palasciano
Cholestasis is associated with changes in NO metabolism and thiol oxidation. Thioredoxin contributes to regulate vascular tone and intracellular redox status by cleaving nitrosothiols and maintaining -SH groups. This study investigated the changes in circulating thioredoxin and nitrosothiols and the relationship with protein sulfhydryls (PSH), hepatic concentrations, hyaluronate, and histology in patients with primary biliary cirrhosis (PBC) and in rats with bile duct ligation (BDL). PSH in erythrocytes were significantly decreased in stage III and IV PBC and at day 10 after BDL. Compared with controls, erythrocyte thioredoxin levels were higher in stage I through III PBC and lower in stage IV patients. Serum thioredoxin levels were significantly higher in PBC stages I and II and lower in stages III and IV. Serum nitrosothiols were higher in all PBC patients and inversely related to thioredoxin and hyaluronate. In rats, serum, hepatic, and mitochondrial thioredoxin had initially increased after BDL (day 1-3) and then decreased. After day 7 BDL, nitrosothiols were 10-fold increased in serum and liver, and even higher in mitochondria. In the liver, thioredoxin was inversely related to both nitrosothiols and PSH. In rats, the difference in time average changes from baseline among serum, hepatic, and erythrocyte thioredoxin suggests that most of circulating thioredoxin originates from the liver. Conclusion: Our findings indicate that cholestasis is associated with significant mutual and interrelated changes between circulating and hepatic thioredoxin and nitrosothiols. The increase of hepatic, mitochondrial, and circulating nitrosothiols with ongoing cholestasis suggests an active participation of NO in both liver injury and extrahepatic changes.
Human and rat bile acid-CoA:amino acid N-acyltransferase are liver-specific peroxisomal enzymes: Implications for intracellular bile salt transport (p 340-348)
Antonella Pellicoro, Fiona A. J. van den Heuvel, Mariska Geuken, Han Moshage, Peter L. M. Jansen, Klaas Nico Faber
Bile acid-coenzyme A:amino acid N-acyltransferase (BAAT) is the sole enzyme responsible for conjugation of primary and secondary bile acids to taurine and glycine. Previous studies indicate a peroxisomal location of BAAT in peroxisomes with variable amounts up to 95% detected in cytosolic fractions. The absence or presence of a cytosolic pool of BAAT has important implications for the intracellular transport of unconjugated/deconjugated bile salts. We used immunofluorescence microscopy and digitonin permeabilization assays to determine the subcellular location of endogenous BAAT in primary human and rat hepatocytes. In addition, green fluorescent protein (GFP)-tagged rat Baat (rBaat) and human BAAT (hBAAT) were transiently expressed in primary rat hepatocytes and human fibroblasts. Catalase and recombinant GFP-SKL and DsRed-SKL were used as peroxisomal markers. Endogenous hBAAT and rBaat were found to specifically localize to peroxisomes in human and rat hepatocytes, respectively. No significant cytosolic fraction was detected for either protein. GFP-tagged hBAAT and rBaat were efficiently sorted to peroxisomes of primary rat hepatocytes. Significant amounts of GFP-tagged hBAAT or rBaat were detected in the cytosol only when coexpressed with DsRed-SKL, suggesting that hBAAT/rBaat and DsRed-SKL compete for the same peroxisomal import machinery. When expressed in fibroblasts, GFP-tagged hBAAT localized to the cytosol, confirming earlier observations. Conclusion: hBAAT and rBaat are peroxisomal enzymes present in undetectable amounts in the cytosol. Unconjugated or deconjugated bile salts returning to the liver need to shuttle through the peroxisome before reentering the enterohepatic circulation.
The role of chrysin and the Ah receptor in induction of the human UGT1A1 gene in vitro and in transgenic UGT1 mice (p 349-360)
Jessica A. Bonzo, Alain Bélanger, Robert H. Tukey
The flavonoid chrysin is an important dietary substance and induces UGT1A1 protein expression in cell culture. As a representative of the class of dietary flavonoids, clinical investigations have been considered as a means of inducing hepatic UGT1A1 expression. We demonstrate the necessity of a xenobiotic response element (XRE) in support of chrysin induction of UGT1A1 in the human hepatoma cell line HepG2. Receptor binding assays confirm that chrysin is a ligand for the Ah receptor by competition with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, key differences in Ah receptor recognition and activation of UGT1A1 by chrysin exist when compared with classical mechanisms of UGT1A1 induction by TCDD. Ah receptor degradation, an indicator of Ah receptor activation, does not occur after chrysin treatment, and chrysin cannot transactivate the Ah receptor in a TCDD-dependent fashion. Knock-down of the Ah receptor by siRNA indicates that chrysin uses the Ah receptor in conjunction with other factors through MAP kinase signaling pathways to maximally induce UGT1A1. Most importantly, oral treatment of chrysin to transgenic mice that express the human UGT1 locus is unable to induce UGT1A1 expression in either the small intestine or liver. Conclusion: Although the implications for chrysin as an atypical agonist of the Ah receptor are intriguing at the molecular level, the relevance of chrysin-induced transcription for the purpose of clinical therapies or to regulate phase 2-dependent glucuronidation is questionable given the lack of in vivo regulation of human UGT1A1 by chrysin in a transgenic animal model.
Liver-specific loss of -catenin results in delayed hepatocyte proliferation after partial hepatectomy (p 361-368)
Shigeki Sekine, Pedro J. A. Gutiérrez, Billy Yu-Ang Lan, Sandy Feng, Matthias Hebrok
Recent studies have suggested that -catenin is involved in the regulation of hepatocyte proliferation in multiple contexts, including organ development and tumorigenesis. We explored the role of -catenin during liver regeneration using T cell factor/lymphoid enhancer factor (TCF/LEF)-reporter mice (TOPGal mice) and liver-specific -catenin knockout mice. Liver-specific -catenin knockout mice showed a delayed onset of DNA synthesis after hepatectomy, whereas recovery of liver mass was not affected. Among putative -catenin target genes examined, the induction of Ccnd1 expression was reduced, whereas the expression of Myc and Egfr was unaffected. Furthermore, cyclin D1 protein levels were not induced, and the expression of cyclins A, E, and proliferating cell nuclear antigen was delayed. Intriguingly, the analysis of TOPGal mice showed that hepatocytes with active TCF/LEF transcription are confined to the pericentral zone and are not increased in number during regeneration, indicating an uncoupling between -catenin/TCF signaling activity and hepatocyte proliferation. Conclusion: Our results indicate that -catenin is critical for the proper regulation of hepatocyte proliferation during liver regeneration; however, the activity of -catenin/TCF signaling does not correlate with hepatocyte proliferation, suggesting that this regulation might be indirect/secondary.
Platelets and platelet-derived serotonin promote tissue repair after normothermic hepatic ischemia in mice (p 369-376)
Antonio Nocito, Panco Georgiev, Felix Dahm, Wolfram Jochum, Michael Bader, Rolf Graf, Pierre-Alain Clavien
Hepatic ischemia and reperfusion (I/R) leads to the formation of leukocyte-platelet aggregates. Upon activation, platelets generate reactive oxygen species and release proapoptotic and proinflammatory mediators as well as growth factors. In cold hepatic ischemia, adhesion of platelets to endothelial cells mediates sinusoidal endothelial cell apoptosis. Furthermore, platelet-derived serotonin mediates liver regeneration. We hypothesized that platelets may contribute to reperfusion injury and repair after normothermic hepatic ischemia. The aim of this study was to assess the impact of platelets in normothermic hepatic I/R injury using models of impaired platelet function and immune thrombocytopenia. Inhibition of platelet function in mice was achieved via clopidogrel feeding. Immune thrombocytopenia was induced via intraperitoneal injection of anti-CD41 antibody. Platelet-derived serotonin was investigated using mice lacking tryptophan hydroxylase 1. Mice were subjected to 60 minutes of partial hepatic ischemia and various time points of reperfusion. Hepatic injury was determined via AST and histological analysis of the necrotic area as well as leukocyte infiltration. Liver regeneration was determined via proliferating cell nuclear antigen and Ki67 immunohistochemistry. Neither inhibition of platelet function nor platelet depletion led to a reduction of I/R injury. Liver regeneration and repair were significantly impaired in platelet-depleted animals. Mice lacking peripheral serotonin were deficient in hepatocyte proliferation, but otherwise displayed normal tissue remodeling. Conclusion: Platelets have no direct impact on the pathogenesis of normothermic I/R injury. However, they mediate tissue repair and liver regeneration. Furthermore, platelet-derived serotonin is a mediator of hepatocyte proliferation in the postischemic liver, but has no impact on tissue remodeling.
A national French survey on the use of growth factors as adjuvant treatment of chronic hepatitis C (p 377-383)
Thierry Thévenot, Jean-François Cadranel, Vincent Di Martino, Alexandre Pariente, Xavier Causse, Christophe Renou, Hervé Hagege, Jacques Denis, Françoise Lunel-Fabiani
We conducted a national retrospective survey on hospital practitioners to evaluate the magnitude of erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF) prescriptions in patients treated for chronic hepatitis C. Four hundred seventy-one questionnaires were sent, and 274 practitioners (58.2%) responded. Forty-six percent of practitioners used EPO, and 31% used G-CSF. The total number of HCV-infected patients receiving antiviral therapy per year was estimated at 6,630 patients, of whom 8.8% and 4% received EPO and G-CSF, respectively. EPO- was the main EPO molecule prescribed at a median dose of 30,000 IU/wk (range: 2,000-80,000). The indications for prescribing EPO varied greatly, including fragile patients (34%), low Hb level (8-11 g/dL) (19%), rapid decline in Hb level (2-5 g/dL during the first month of therapy) (12%), and symptomatic anemic patients (7%). G-CSF was mainly prescribed for a low level of neutrophils ranging from 400 to 750 neutrophils/mm3. In multivariate analysis, independent predictors of EPO and G-CSF prescription were age of practitioner less than 45 years (EPO: OR = 1.96, P = 0.03; G-CSF: OR = 2.27, P = 0.004), practice in university hospital (EPO: OR = 5.89, P < 0.0001; G-CSF: OR = 2.39, P = 0.003), and the high number of CHC treated/year (EPO: OR = 6.18, P < 0.0001; G-CSF: OR = 2.58, P = 0.002). Conclusion: Our survey reveals an important rate of EPO and G-CSF prescriptions but with considerable disparity in the schedule of injections, the molecules used, and above all the indications. The suitable role of EPO and G-CSF as complements to HCV therapy urgently needs to be clarified.
A genomewide DNA microsatellite association study of Japanese patients with autoimmune hepatitis type 1 (p 384-390)
Shuichi Yokosawa, Kaname Yoshizawa, Masao Ota, Yoshihiko Katsuyama, Shigeyuki Kawa, Tetsuya Ichijo, Takeji Umemura, Eiji Tanaka, Kendo Kiyosawa
Genetic predisposition to type 1 autoimmune hepatitis (AIH) is linked mainly to HLA class II genes. We previously searched the whole HLA region for AIH susceptibility genes using microsatellite markers and found only HLA-DR/DQ to be a candidate region for this suspected multifactorial disease. As such, the aim of this study was to broaden our search and screen the whole genome for additional genes that might contribute to type 1 AIH susceptibility. Eighty-one patients with type 1 AIH (15 men, 66 women, average age 55.9) and 80 healthy sex- and age-matched Japanese controls were enrolled in this study. We performed a case-control association study using 400 polymorphic microsatellite markers with an average spacing of 10.8 cM distributed throughout the whole genome. Two markers, one on chromosome 11 (D11S902, Pc = 0.013) and one on chromosome 18 (D18S464, Pc = 0.008), were revealed to have statistically significant associations with AIH. An additional 7 markers (D2S367, D6S309, D9S273, D11S1320, D16S423, D17S938, and D18S68) were also found to be candidate susceptibility regions. In addition, our results showed there were 17 regions that may contain genes of resistance to AIH. No specific markers were detected in HLA-DR4-negative patients, and no differences were seen in the clinical courses of patients (severe versus mild to moderate). Conclusion: This first genomewide scan of Japanese AIH patients revealed at least 26 candidate AIH susceptibility or resistance regions other than HLA class II loci. These results also suggested that the products of several genes interact to determine heritable susceptibility to AIH.
Nonalcoholic fatty liver sensitizes rats to carbon tetrachloride hepatotoxicity (p 391-403)
Shashikiran Donthamsetty, Vishakha S. Bhave, Mayurranjan S. Mitra, John R. Latendresse, Harihara M. Mehendale
This study tested whether hepatic steatosis sensitizes liver to toxicant-induced injury and investigated the potential mechanisms of hepatotoxic sensitivity. Male Sprague-Dawley rats were fed a methionine- and choline-deficient diet for 31 days to induce steatosis. On the 32nd day, administration of a nonlethal dose of CCl4 (2 mL/kg, intraperitoneally) yielded 70% mortality in steatotic rats 12-72 hours after CCl4administration, whereas all nonsteatotic rats survived. Neither CYP2E1 levels nor covalent binding of [14C]CCl4-derived radiolabel differed between the groups, suggesting that increased bioactivation is not the mechanism for this amplified toxicity. Cell division and tissue repair, assessed by [3H]thymidine incorporation and proliferative cell nuclear antigen assay, were inhibited in the steatotic livers after CCl4administration and led to progressive expansion of liver injury culminating in mortality. The hypothesis that fatty hepatocytes undergo cell cycle arrest due to (1) an inability to replenish ATP due to overexpressed uncoupling protein-2 (UCP-2) or (2) induction of growth inhibitor p21 leading to G1/S phase arrest was tested. Steatotic livers showed 10-fold lower ATP levels due to upregulated UCP-2 throughout the time course after CCl4 administration, leading to sustained inhibition of cell division. Western blot analysis revealed an up-regulation of p21 due to overexpression of TGF 1 and p53 and down-regulation of transcription factor Foxm1b in steatotic livers leading to lower phosphorylated retinoblastoma protein. Thus, fatty hepatocytes fail to undergo compensatory cell division, rendering the liver susceptible to progression of liver injury. Conclusion: Impaired tissue repair sensitizes the steatotic livers to hepatotoxicity.
Maid (GCIP) is involved in cell cycle control of hepatocytes (p 404-411)
Eva Sonnenberg-Riethmacher, Torsten Wüstefeld, Michaela Miehe, Christian Trautwein, Dieter Riethmacher
The function of Maid (GCIP), a cyclinD-binding helix-loop-helix protein, was analyzed by targeted disruption in mice. We show that Maid function is not required for normal embryonic development. However, older Maid-deficient mice - in contrast to wild-type controls - develop hepatocellular carcinomas. Therefore, we studied the role of Maid during cell cycle progression after partial hepatectomy (PH). Lack of Maid expression after PH was associated with a delay in G1/S-phase progression as evidenced by delayed cyclinA expression and DNA replication in Maid-deficient mice. However, at later time points liver mass was restored normally. Conclusion: These results indicate that Maid is involved in G1/S-phase progression of hepatocytes, which in older animals is associated with the development of liver tumors.
Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury (p 412-421)
Calivarathan Latchoumycandane, Catherine W. Goh, Michie M.K. Ong, Urs A. Boelsterli
Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Leflunomide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP)-catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH2-terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-XL inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepatotoxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabilization.
Activation of LXRs prevents bile acid toxicity and cholestasis in female mice (p 422-432)
Hirdesh Uppal, Simrat P.S. Saini, Antonio Moschetta, Ying Mu, Jie Zhou, Haibiao Gong, Yonggong Zhai, Songrong Ren, George K. Michalopoulos, David J. Mangelsdorf, Wen Xie
Liver X receptors (LXRs) have been identified as sterol sensors that regulate cholesterol and lipid homeostasis and macrophage functions. In this study, we found that LXRs also affect sensitivity to bile acid toxicity and cholestasis. Activation of LXR in transgenic mice confers a female-specific resistance to lithocholic acid (LCA)-induced hepatotoxicity and bile duct ligation (BDL)-induced cholestasis. This resistance was also seen in wild-type female mice treated with the synthetic LXR ligand TO1317. In contrast, LXR double knockout (DKO) mice deficient in both the and isoforms exhibited heightened cholestatic sensitivity. LCA and BDL resistance in transgenic mice was associated with increased expression of bile acid-detoxifying sulfotransferase 2A (Sult2a) and selected bile acid transporters, whereas basal expression of these gene products was reduced in the LXR DKO mice. Promoter analysis showed that the mouse Sult2a9 gene is a transcriptional target of LXRs. Activation of LXRs also suppresses expression of oxysterol 7-hydroxylase (Cyp7b1), which may lead to increased levels of LXR-activating oxysterols. Conclusion: We propose that LXRs have evolved to have the dual functions of maintaining cholesterol and bile acid homeostasis by increasing cholesterol catabolism and, at the same time, preventing toxicity from bile acid accumulation.
Hepatic HNF4 deficiency induces periportal expression of glutamine synthetase and other pericentral enzymes (p 433-444)
Vesna S. Stanulovi, Irene Kyrmizi, Marianna Kruithof-de Julio, Maarten Hoogenkamp, Jacqueline L. M. Vermeulen, Jan M. Ruijter, Iannis Talianidis, Theodorus B. M. Hakvoort, Wouter H. Lamers
In liver, most genes are expressed with a porto-central gradient. The transcription factor hepatic nuclear-factor4 (HNF4) is associated with 12% of the genes in adult liver, but its involvement in zonation of gene expression has not been investigated. A putative HNF4-response element in the upstream enhancer of glutamine synthetase (GS), an exclusively pericentral enzyme, was protected against DNase-I and interacted with a protein that is recognized by HNF4-specific antiserum. Chromatin-immunoprecipitation assays of HNF4-deficient (H4LivKO) and control (H4Flox) livers with HNF4 antiserum precipitated the GS upstream enhancer DNA only from H4Flox liver. Identical results were obtained with a histone-deacetylase1 (HDAC1) antibody, but antibodies against HDAC3, SMRT and SHP did not precipitate the GS upstream enhancer. In H4Flox liver, GS, ornithine aminotransferase (OAT) and thyroid hormone-receptor 1 (TR1) were exclusively expressed in pericentral hepatocytes. In H4LivKO liver, this pericentral expression remained unaffected, but the genes were additionally expressed in the periportal hepatocytes, albeit at a lower level. The expression of the periportal enzyme phosphoenolpyruvate carboxykinase had declined in HNF4-deficient hepatocytes. GS-negative cells, which were present as single, large hepatocytes or as groups of small cells near portal veins, did express HNF4. Clusters of very small GS- and HNF4-negative, and PCNA- and OV6-positive cells near portal veins were contiguous with streaks of brightly HNF4-positive, OV6-, PCNA-, and PEPCK-dim cells. Conclusion: Our findings show that HNF4 suppresses the expression of pericentral proteins in periportal hepatocytes, possibly via a HDAC1-mediated mechanism. Furthermore, we show that HNF4 deficiency induces foci of regenerating hepatocytes.
Murine liver plasmacytoid dendritic cells become potent immunostimulatory cells after Flt-3 ligand expansion (p 445-454)
T. Peter Kingham, Umer I. Chaudhry, George Plitas, Steven C. Katz, Jesse Raab, Ronald P. DeMatteo
The liver has unique immunological properties. Although dendritic cells (DCs) are central mediators of immune regulation, little is known about liver DCs. Plasmacytoid DCs (pDCs) are a recently identified subtype of murine liver DC. We sought to define the function of freshly isolated murine liver pDCs. We found that normal liver pDCs were weak in stimulating T cells, yet they possessed a proinflammatory cytokine profile with high tumor necrosis factor- and low IL-10 secretion. To facilitate the investigation of murine liver pDCs, we expanded them in vivo with fms-like tyrosine kinase 3 ligand (Flt3L). After Toll-like receptor-9 ligation, expanded liver pDCs secreted high levels of IFN- and were able to stimulate NK cells, NKT cells, and antigen-specific CD8+ T cells in vitro. In addition, Flt3L expansion alone generated pDCs capable of activating antigen-specific CD8+ T cells in vivo. Conclusion: Unstimulated liver pDCs exist in a latent state with the potential to become potent activators of the innate and adaptive immune systems through their interactions with other immune effectors. Our findings have implications for understanding the role of the liver in tolerance and immunity.
Hypoxic conformance of metabolism in primary rat hepatocytes: A model of hepatic hibernation (p 455-464)
Ram M. Subramanian, Navdeep Chandel, G. R. Scott Budinger, Paul T. Schumacker
Following prolonged hypoxia, mammalian cells invoke adaptive mechanisms to enhance oxygen delivery and promote energy conservation. We previously reported that hepatocytes subjected to prolonged moderate hypoxia (PO2 = 20-50 mmHg for >3 hours) demonstrated a reversible inhibition of cellular respiration with maintenance of cell viability, associated with a decrease in mitochondrial adenosine triphosphate (ATP) synthesis; acute hypoxia (similar PO2 for <30 minutes) did not induce a similar suppression of respiration and ATP synthesis. In the current study, using an in vitro model of primary rat hepatocytes, we measured the changes in metabolic demand for ATP during hypoxic conformance, and tested whether viability is maintained by preferentially suppressing nonessential processes while sustaining processes essential for maintaining cell homeostasis. In addition, the rate of recovery of oxygen consumption and ATP concentrations following reoxygenation after prolonged and acute hypoxia/anoxia was compared. Oxygen consumption and ATP concentrations decreased during prolonged hypoxia compared with acute hypoxia. However, ouabain-inhibitable respiration did not decrease during prolonged hypoxia, indicating that membrane Na+/K+ ATPase activity, an essential process for cell viability, was maintained. In contrast, ATP-dependent glucuronidation and sulfation of acetaminophen, deemed non-essential processes, were decreased significantly compared with normoxic cells. After reoxygenation, cells exposed to prolonged moderate hypoxia demonstrated a more rapid recovery of respiration compared to acute hypoxia/anoxia. Conclusion: This hepatic hibernation during prolonged moderate hypoxia may represent an anticipatory adaptation that seeks to maintain cell viability while delaying or preventing the onset of lethal hypoxia, and facilitates rapid recovery after the resumption of normoxia.
Activation of vascular adhesion protein-1 on liver endothelium results in an NF-B-dependent increase in lymphocyte adhesion (p 465-474)
Patricia F. Lalor, Phoebe Jun Sun, Chris J. Weston, Azucena Martin-Santos, Michael J. O. Wakelam, David H. Adams
Vascular adhesion protein-1 (VAP-1) is an adhesion molecule and amine oxidase that is expressed at high levels in the human liver. It promotes leukocyte adhesion to the liver in vivo and drives lymphocyte transmigration across hepatic sinusoidal endothelial cells in vitro. We report that in addition to supporting leukocyte adhesion, provision of specific substrate to VAP-1 results in hepatic endothelial cell activation, which can be abrogated by treatment with the enzyme inhibitor semicarbazide. VAP-1-mediated activation was rapid; dependent upon nuclear factor-B, phosphatidylinositol-3 kinase, and mitogen-activated protein kinase pathways; and led to upregulation of the adhesion molecules E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 and secretion of the chemokine CXCL8. This response resulted in enhanced lymphocyte adhesion, was restricted to hepatic endothelial cells that expressed VAP-1, and was not observed in human umbilical vein endothelial cells. Conclusion: We propose that as well as directly promoting adhesion via interactions with the as yet unknown ligand, binding of enzyme substrate to VAP-1 can indirectly promote inflammatory cell recruitment via upregulation of adhesion molecules and chemokines. This response is likely to be important for the recruitment of leukocytes to the liver and suggests that VAP-1 inhibitors have therapeutic potential for treating chronic inflammatory liver disease.
IL-10, regulatory T cells, and Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice (p 475-485)
Annette Erhardt, Markus Biburger, Thomas Papadopoulos, Gisa Tiegs
The liver appears to play an important role in immunological tolerance, for example, during allo-transplantation. We investigated tolerance mechanisms in the model of concanavalin A (ConA)-induced immune-mediated liver injury in mice. We found that a single injection of a sublethal ConA dose to C57BL/6 mice induced tolerance toward ConA-induced liver damage within 8 days. This tolerogenic state was characterized by suppression of the typical Th1 response in this model and increased IL-10 production. Tolerance induction was fully reversible in IL-10-/- mice and after blockade of IL-10 responses by anti-IL10R antibody. Co-cultures of CD4+CD25+ regulatory T cells (Tregs) and CD4+CD25- responder cells revealed Treg from ConA-tolerant mice being more effective in suppressing polyclonal T cell responses than Treg from control mice. Moreover, Treg from tolerant but not from control mice were able to augment in vitro IL-10 expression. Depletion by anti-CD25 monoclonal antibody (MAb) indicated a functional role of Tregs in ConA tolerance in vivo. Cell depletion studies revealed Tregs and Kupffer cells (KC) to be crucial for IL-10 expression in ConA tolerance. Studies with CD1d-/- mice lacking natural killer T (NKT) cells disclosed these cells as irrelevant for the tolerogenic effect. Finally, cellular immune therapy with CD4+CD25+ cells prevented ConA-induced liver injury, with higher protection by Treg from ConA-tolerized mice. Conclusion: The immunosuppressive cytokine IL-10 is crucial for tolerance induction in ConA hepatitis and is mainly expressed by CD4+CD25+ Treg and KC. Moreover, Tregs exhibit therapeutic potential against immune-mediated liver injury.
Opposing roles of gp130-mediated STAT-3 and ERK-1/2 signaling in liver progenitor cell migration and proliferation (p 486-494)
George C. T. Yeoh, Matthias Ernst, Stefan Rose-John, Barbara Akhurst, Christine Payne, Sarah Long, Warren Alexander, Ben Croker, Dianne Grail, Vance B. Matthews
Gp130-mediated IL-6 signaling may play a role in oval cell proliferation in vivo. Levels of IL-6 are elevated in livers of mice treated with a choline-deficient ethionine-supplemented (CDE) diet that induces oval cells, and there is a reduction of oval cells in IL-6 knockout mice. The CDE diet recapitulates characteristics of chronic liver injury in humans. In this study, we determined the impact of IL-6 signaling on oval cell-mediated liver regeneration in vivo. Signaling pathways downstream of gp130 activation were also dissected. Numbers of A6+ve liver progenitor oval cells (LPCs) in CDE-treated murine liver were detected by immunohistochemistry and quantified. Levels of oval cell migration and proliferation were compared in CDE-treated mouse strains that depict models of gp130-mediated hyperactive ERK-1/2 signaling (gp130STAT), hyperactive STAT-3 signaling (gp130Y757F and Socs-3-/Alb) or active ERK-1/2 as well as active STAT-3 signaling (wild-type). The A6+ve LPC numbers were increased with IL-6 treatment in vivo. The gp130Y757F mice displayed increased A6+ve LPCs numbers compared with wild-type and gp130STAT mice. Numbers of A6+ve LPCs were also increased in the livers of CDE treated Socs-3-/Albmice compared with their control counterparts. Lastly, inhibition of ERK-1/2 activation in cultured oval cells increased hyper IL-6-induced cell growth. For the first time, we have dissected the gp130-mediated signaling pathways, which influence liver progenitor oval cell proliferation. Conclusion: Hyperactive STAT-3 signaling results in enhanced oval cell numbers, whereas ERK-1/2 activation suppresses oval cell proliferation.
Vascular dysfunction in human and rat cirrhosis: Role of receptor-desensitizing and calcium-sensitizing proteins (p 495-506)
Martin Hennenberg, Jonel Trebicka, Erwin Biecker, Michael Schepke, Tilman Sauerbruch, Jörg Heller
In cirrhosis, vascular hypocontractility leads to vasodilation and contributes to portal hypertension. Impaired activation of contractile pathways contributes to vascular hypocontractility. Angiotensin II type 1 receptors (AT1-Rs) are coupled to the contraction-mediating RhoA/Rho-kinase pathway and may be desensitized by phosphorylation through G-protein-coupled receptor kinases (GRKs) and binding of -arrestin-2. In the present study, we analyzed vascular hypocontractility to angiotensin II in cirrhosis. Human hepatic arteries were obtained during liver transplantation. In rats, cirrhosis was induced by bile duct ligation (BDL). Contractility of rat aortic rings was measured myographically. Protein expression and phosphorylation were analyzed by Western blot analysis. Immunoprecipitation was performed with protein A-coupled Sepharose beads. Myosin light chain (MLC) phosphatase activity was assessed as dephosphorylation of MLCs. Aortas from BDL rats were hyporeactive to angiotensin II and extracellular Ca2+. Expression of AT1-R and Gq/11,12,13 remained unchanged in hypocontractile rat and human vessels, whereas GRK-2 and -arrestin-2 were up-regulated. The binding of -arrestin-2 to the AT1-R was increased in hypocontractile rat and human vessels. Inhibition of angiotensin II-induced aortic contraction by the Rho-kinase inhibitor Y-27632 was pronounced in BDL rats. Basal phosphorylation of the ROK-2 substrate moesin was reduced in vessels from rats and patients with cirrhosis. Analysis of the expression and phosphorylation of Ca2+-sensitizing proteins (MYPT1 and CPI-17) in vessels from rats and patients with cirrhosis suggested decreased Ca2+ sensitivity. Angiotensin II-stimulated moesin phosphorylation was decreased in aortas from BDL rats. MLC phosphatase activity was elevated in aortas from BDL rats. Conclusion: Vascular hypocontractility to angiotensin II in cirrhosis does not result from changes in expression of AT1-Rs or G-proteins. Our data suggest that in cirrhosis-induced vasodilation, the AT1-R is desensitized by GRK-2 and -arrestin-2 and that changed patterns of phosphorylated Ca2+-sensitizing proteins decrease Ca2+ sensitivity.
Copyright © 2007 by the American Association for the Study of Liver Diseases. All rights reserved.
GASTROENTEROLOGY
Volume 132, Issue 2, February 2007
Clinical - Alimentary Tract
Peptic Ulcer and Bleeding Events Associated With Rofecoxib in a 3-Year Colorectal Adenoma Chemoprevention Trial, 14 November 2006
Lanas A, Baron JA, Sandler RS, Horgan K, Bolognese J, Oxenius B, Quan H, Watson D, Cook TJ, Schoen R, Burke C, Loftus S, Niv Y, Ridell R, Morton D, Bresalier R
Background & Aims: Our aim was to establish the incidence of symptomatic upper gastrointestinal ulcers, ulcer perforation, ulcer obstruction, or bleeding episodes (PUBs) associated with the use of selective cyclooxygenase-2 inhibitors at standard clinical doses compared with placebo. We report here on the PUB outcomes associated with the use of rofecoxib 25 mg in a 3-year, multicenter, double-blind, placebo-controlled trial designed to determine the effect of rofecoxib on the risk of recurrent neoplastic polyps of the colon. Methods: A total of 2587 patients with a history of colorectal adenomas underwent randomization to 25 mg/day of rofecoxib or to placebo. Investigator-reported PUBs were adjudicated by an external blinded committee. Kaplan–Meier and Cox proportional hazards techniques were used to estimate incidence and relative risks of PUBs in an intention-to-treat analysis. Results: Patients assigned to rofecoxib had a higher incidence of confirmed PUBs than those randomized to placebo (.88 vs .18 events per 100 patient-years; relative risk, 4.9; 95% confidence interval, 1.98–14.54). The incidence of confirmed complicated PUBs (ulcer perforation, obstruction, or bleeds) was low, but was numerically higher in the rofecoxib than in the placebo group (.23 vs .06 events per 100 patient-years; relative risk, 3.8; 95% confidence interval, .72–37.46; P = .14). Rofecoxib increased the incidence of confirmed PUBs vs placebo in both low-dose aspirin users and nonusers. Conclusions: Among patients with a history of colorectal adenomas, the long-term use of 25 mg/day of rofecoxib was associated with an increased risk of clinically relevant upper gastrointestinal events when compared with placebo.
Risk of Upper Gastrointestinal Complications Among Users of Traditional NSAIDs and COXIBs in the General Population, 6 December 2006
García Rodríguez LA, Barreales Tolosa L
Background & Aims: Traditional nonaspirin, nonsteroidal anti-inflammatory drugs (tNSAIDs) have been associated with a 3- to 5-fold increased risk in upper gastrointestinal complications (UGIC). Whether use of selective inhibitors of cyclooxygenase-2 (COXIBs) will translate into a clinically relevant reduced toxicity has not been widely investigated in the general population. Methods: We conducted a nested case control study using The Health Improvement Network Database identifying 1561 cases of UGIC between January 2000 and 2005. A random sample of 10,000 controls was frequency matched to the cases by age, sex, and calendar year. Results: The adjusted relative risk (RR) of UGIC associated with current use was 3.7 (95% CI: 3.1–4.3) for tNSAIDs and 2.6 (95% CI: 1.9–3.6) for COXIBs. Daily dose was a predictor of increased risk for both tNSAIDs and COXIBs. Users of tNSAIDs with a prolonged plasma half-life or slow release formulations had an augmented risk of UGIC. Overall, the estimate of RR associated with COXIBs was 0.8 (95% CI: 0.6–1.1) compared with current use of tNSAIDs, and, among nonusers of aspirin, the corresponding estimate of RR associated with COXIBs was 0.6 (95% CI: 0.4–0.9). Conclusions: COXIBs present a better upper gastrointestinal safety than tNSAIDs, although the risk of UGIC for an individual drug is determined by its daily dose and plasma drug exposure in addition to its selectivity for cyclooxygenase-2. Also, concomitant use of aspirin is a strong effect modifier of COXIBs that negates the superior gastrointestinal safety over tNSAIDs in the absence of aspirin use.
Low Colectomy Rates in Ulcerative Colitis in an Unselected European Cohort Followed for 10 Years, 20 November 2006
Hoie O, Wolters FL, Riis L, Bernklev T, Aamodt G, Clofent J, Tsianos E, Beltrami M, Odes S, Munkholm P, Vatn M, Stockbrügger RW, Moum B, European Collaborative Study Group of Inflammatory Bowel Disease
Background & Aims: The colectomy rate in ulcerative colitis (UC) is related to morbidity and to treatment decisions made during disease course. The aims of this study were to determine the colectomy risk in UC in the first decade after diagnosis and to identify factors that may influence the choice of surgical treatment. Methods: In 1991-1993, 781 UC patients from 9 centers located in 7 countries in northern and southern Europe and in Israel were included in a prospective inception cohort study. After 10 years of follow-up, 617 patients had complete medical records, 73 had died, and 91 had been lost to follow-up. Results: There were no significant differences in age, sex, or disease extent at diagnosis between patients followed for 10 years and those lost to follow-up. The 10-year cumulative risk of colectomy was 8.7%: 10.4% in the northern and 3.9% in the southern European centers (P < .001). Colectomy was more likely in extensive colitis than in proctitis, with an adjusted hazard ratio (HR) of 4.1 (95% CI: 2.0–8.4). Compared with the southern centers, the adjusted HR was 2.7 (95% CI: 1.3–5.6) for The Netherlands and Norway together and 8.2 (95% CI: 3.6–18.6) for Denmark. Age at diagnosis, sex, and smoking status at diagnosis had no statistically significant influence on colectomy rates. Conclusions: The colectomy rate was found to be lower than that in previous publications, but there was a difference between northern and southern Europe. Colectomy was associated with extensive colitis, but the geographic variations could not be explained.
A Meta-Analysis of the Placebo Rates of Remission and Response in Clinical Trials of Active Ulcerative Colitis, 30 December 2006
Su C, Lewis JD, Goldberg B, Brensinger C, Lichtenstein GR
Background & Aims: Knowledge of the placebo outcomes and understanding specific study features that influence these outcomes is important for designing future clinical trials evaluating therapy of ulcerative colitis (UC). The aims of this study were to estimate the placebo rates of remission and response in placebo-controlled, randomized clinical trials for active UC and to identify factors influencing these rates. Methods: We performed a systematic review and meta-analysis of placebo-controlled, randomized clinical trials evaluating therapies for active UC identified from MEDLINE from 1966 through 2005. Results: Forty studies met the inclusion criteria. The pooled estimates of the placebo rates of remission and response were 13% (95% confidence interval, 9%–18%; range, 0%–40%; median, 12%) and 28% (95% confidence interval, 23%–33%; range, 0%–67%; median, 30%), respectively, both with significant heterogeneity. Studies that used more stringent definitions of outcomes had lower placebo rates of remission and response. Study duration, number of study visits, disease duration, baseline composite and rectal bleeding scores of the disease activity index, and inclusion of endoscopic mucosal healing as the remission definition all were associated with the placebo remission rate. Conclusions: Rates of remission in the placebo arm of UC clinical trials ranges from 0% to 40%. The placebo remission rates are influenced by the trial length, number of study visits, use of stricter remission definitions, and design features that enroll patients with more active disease. These factors should be considered when designing future placebo-controlled clinical trials in patients with active UC.
Homozygous PMS2 Deletion Causes a Severe Colorectal Cancer and Multiple Adenoma Phenotype Without Extraintestinal Cancer, 6 December 2006
Will O, Carvajal-Carmona LG, Gorman P, Howarth KM, Jones AM, Polanco-Echeverry GM, Chinaleong JA, Günther T, Silver A, Clark SK, Tomlinson I
Background & Aims: We report a patient of Indian descent with parental consanguinity, who developed 10 carcinomas and 35 adenomatous polyps at age 23 and duodenal adenocarcinoma at age 25. He also had dysmorphic features, mental retardation, and café-au-lait spots but no brain tumor. We aimed to establish his molecular diagnosis. Methods: Germ-line screening for APC and MYH/MUTYH mutations was normal as was immunohistochemistry for MLH1 and MSH2 proteins. Investigation by array-comparative genomic hybridization revealed deletion of a small region on chromosome 7. Using polymerase chain reaction, this region was refined to a 400-kilobase deletion, which included exons 9–15 of the PMS2 gene, and all coding regions of oncomodulin, TRIAD3, and FSCN1. Results: The deletion was confirmed as homozygous, and both parents were carriers. Immunohistochemistry showed absent PMS2 expression in all tumors and normal tissue. Most tumors showed microsatellite instability, more marked at dinucleotide than mononucleotide repeats. The tumors harbored no somatic mutations in APC, BRAF, AXIN2, or ?-catenin, but KRAS2 and TGFBR2 mutations were found. Conclusions: Our patient represents a novel phenotype for homozygous PMS2 mutation and perhaps the most severe colorectal cancer phenotype—in terms of numbers of malignancies at an early age—described to date. PMS2 mutations—and perhaps other homozygous mismatch repair mutations—should be considered in any patient presenting with multiple gastrointestinal tumors, since our patient could not be distinguished clinically from cases with attenuated familial adenomatous polyposis or MUTYH-associated polyposis.
Clinical - Liver, Pancreas, and Biliary Tract
Quantification of Liver Glucose Metabolism by Positron Emission Tomography: Validation Study in Pigs, 30 December 2006
Iozzo P, Jarvisalo MJ, Kiss J, Borra R, Naum GA, Viljanen A, Viljanen T, Gastaldelli A, Buzzigoli E, Guiducci L, Barsotti E, Savunen T, Knuuti J, Haaparanta–Solin M, Ferrannini E, Nuutila P
Background & Aims: The liver is inaccessible to organ balance measurements in humans. To validate [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography (PET) in the quantification of hepatic glucose uptake (HGU), we determined [18F]FDG modeling parameters, lumped constant (LC), and input functions (single arterial versus dual). Methods: Anesthetized pigs were studied during fasting (n = 6), physiologic (n = 4), and supraphysiologic (n = 4) hyperinsulinemia. PET was performed with C15O (blood pool) and [18F]FDG (glucose uptake). 6,6-Deuterated glucose ([2H]G) was coinjected with [18F]FDG and blood collected from the carotid artery and portal and hepatic veins to compute LC as ratio between tracers fractional extraction. HGU was estimated from PET images and ex vivo from high-performance liquid chromatography measurements of liver [18F]FDG versus [18F]FDG-6-phosphate and [18F]-glycogen. Endogenous glucose production was measured with [2H]G and hepatic blood flow by flowmeters. Results: HGU was increased in hyperinsulinemia versus fasting (P < .05). Fractional extraction of [18F]FDG and [2H]G was similar (not significant), intercorrelated (r = 0.98, P < .0001), and equally higher during hyperinsulinemia than fasting (P ≤ .05), with an LC of 0.98 ± 0.10 and 1.18 ± 0.26, respectively. [18F]FDG-PET modeling provided HGU values that did not differ from, and were correlated with, those from ex vivo measurements (r = 0.61, P ≤ .02); proportional estimates of liver perfusion and endogenous glucose production were also obtained. Single and dual input functions produced strongly intercorrelated results (r > 0.95, P < .0001), with a modest underestimation of HGU by the former. Conclusions: [18F]FDG-PET–derived parameters provide accurate quantification of HGU and estimates of liver perfusion and glucose production. In the liver, LC of [18F]FDG is nearly unitary. Using a single arterial input introduces only a small error in estimation of HGU.
Identification of a Hepatitis B Virus S Gene Mutant in Lamivudine-Treated Patients Experiencing HBsAg Seroclearance, 6 December 2006
Hsu CW, Yeh CT, Chang ML, Liaw YF
Background & Aims: Seroclearance of hepatitis B virus (HBV) surface antigen (HBsAg) is a rare event in chronic hepatitis B patients receiving lamivudine therapy. It is generally believed to be a benevolent sign, implicating clearance of viremia. The aim of this study is to examine the authenticity of this dogma. Methods: In a 5-year period, 11 patients treated with lamivudine experienced seroclearance of HBsAg. The clinical data were examined. The HBV S gene sequences derived from the patient’s serum samples before and after seroclearance of HBsAg were analyzed. Results: Serum HBV-DNA could be detected by nested polymerase chain reaction (PCR) in all 11 patients, by 1-step PCR in 8, and by Cobas Amplicor HBV-DNA test (>200 copies/mL) in 5. A mutation hot spot, P120A in the S gene, was identified in 6 of the 11 patients. Site-directed mutagenesis experiments indicated that the Ausria-II RIA test failed to detect this mutant. Decreased sensitivity of detection was also observed when other monoclonal antibodies were applied. Conclusions: Seroclearance of HBsAg during lamivudine therapy may not indicate viral clearance. Specifically, it may be caused by a point mutation in the S gene, which results in detection failure. In such patients, further verification and follow-up using a sensitive HBV-DNA test are advised.
Basic - Alimentary Tract
Extracellular Superoxide Production by Enterococcus faecalis Promotes Chromosomal Instability in Mammalian Cells, 6 December 2006
Wang X, Huycke MM
Background & Aims: We investigated whether Enterococcus faecalis, a Gram-positive intestinal commensal that produces extracellular superoxide, could promote chromosomal instability (CIN) in mammalian cells. Methods: We measured the ability of E faecalis to promote CIN using hybrid hamster cells (ALN) containing human chromosome 11. Results: E faecalis promoted CIN in ALN cells with average mutant fractions per 105 survivors (±SD) of 72.3 ± 6.7 at 1 ? 109 cfu mL?1 compared with 22.2° ± 4.5 for the no bacteria control. ?-Irradiation at 2 Gray similarly resulted in 74.7 ± 5.7 mutant clones per 105 survivors. Deletions in chromosome 11 consistent with CIN were verified in 80% of mutant clones. E faecalis-treated ALN cells were protected from CIN by superoxide dismutase, ?-tocopherol, and cyclooxygenase-2 (COX-2) inhibitors. In a dual-chamber tissue culture model designed to mimic stromal-epithelial cell interactions, macrophages pretreated with E faecalis grown on permeable supports increased mutant fractions 2.5-fold for ALN cells. COX-2 was up-regulated by superoxide from E faecalis and mutant fractions decreased when COX-2 was silenced using short interfering RNA. Escherichia coli, a Gram-negative commensal that produces negligible extracellular superoxide, only modestly promoted CIN in this model. Conclusions: These findings indicate that macrophage COX-2 is induced by superoxide from E faecalis and promotes CIN in mammalian cells through diffusible factors. This mechanism links the oxidative physiology of E faecalis to propagation of genomic instability through a bystander effect, and offers a novel theory for the role of commensal bacteria in the etiology of sporadic colorectal cancer.
Soluble Proteins Produced by Probiotic Bacteria Regulate Intestinal Epithelial Cell Survival and Growth, 23 November 2006
Yan F, Cao H, Cover TL, Whitehead R, Washington MK, Polk DB
Background & Aims: Increased inflammatory cytokine levels and intestinal epithelial cell apoptosis leading to disruption of epithelial integrity are major pathologic factors in inflammatory bowel diseases. The probiotic bacterium Lactobacillus rhamnosus GG (LGG) and factors recovered from LGG broth culture supernatant (LGG-s) prevent cytokine-induced apoptosis in human and mouse intestinal epithelial cells by regulating signaling pathways. Here, we purify and characterize 2 secreted LGG proteins that regulate intestinal epithelial cell antiapoptotic and proliferation responses. Methods: LGG proteins were purified from LGG-s, analyzed, and used to generate polyclonal antibodies for immunodepletion of respective proteins from LGG-conditioned cell culture media (CM). Mouse colon epithelial cells and cultured colon explants were treated with purified proteins in the absence or presence of tumor necrosis factor (TNF). Akt activation, proliferation, tissue injury, apoptosis, and caspase-3 activation were determined. Results: We purified 2 novel proteins, p75 (75 kilodaltons) and p40 (40 kilodaltons), from LGG-s. Each of these purified protein preparations activated Akt, inhibited cytokine-induced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. TNF-induced colon epithelial damage was significantly reduced by p75 and p40. Immunodepletion of p75 and p40 from LGG-CM reversed LGG-CM activation of Akt and its inhibitory effects on cytokine-induced apoptosis and loss of intestinal epithelial cells. Conclusions: p75 and p40 are the first probiotic bacterial proteins demonstrated to promote intestinal epithelial homeostasis through specific signaling pathways. These findings suggest that probiotic bacterial components may be useful for preventing cytokine-mediated gastrointestinal diseases.
NOD2 Variants and Antibody Response to Microbial Antigens in Crohn’s Disease Patients and Their Unaffected Relatives, 14 November 2006
Devlin SM, Yang H, Ippoliti A, Taylor KD, Landers CJ, Su X, Abreu MT, Papadakis KA, Vasiliauskas EA, Melmed GY, Fleshner PR, Mei L, Rotter JI, Targan SR
Background & Aims: The Cdcs1 locus of the C3Bir mouse confers severe colitis associated with a decrease in innate immune function and an increase in adaptive T-cell responses to commensal bacterial products. The aim of our study was to determine if defects in innate immunity are similarly associated with increased adaptive immune responses to microbial antigens in Crohn’s disease patients. Methods: Sera from 732 patients, 220 unaffected relatives, and 200 healthy controls were tested for antibodies to oligomannan, the Pseudomonas fluorescens–related protein, Escherichia coli outer membrane porin C, CBir1 flagellin, and DNA from the same subjects was tested for 3 Crohn’s disease–associated variants of the NOD2 gene, and 5 toll-like receptor (TLR) 2, 2 TLR4, and 2 TLR9 variants. The magnitude of responses to microbial antigens was examined according to variant status. Results: NOD2 variant carriage increased in frequency with increasing number of positive antibodies and increasing cumulative quantitative response as measured by quartile sum (P for trend, .0008 and .0003, respectively). Mean antibody and quartile sums were higher for patients carrying any NOD2 variant versus those carrying none (2.24 vs 1.92 and 10.60 vs 9.72; P = .0008 and P = 0.0003, respectively). The mean quartile sum was higher for unaffected relatives carrying any NOD2 variant versus those carrying none (10.67 vs 9.75, respectively; P = .02). No association was found between any TLR variant and the magnitude of response. Conclusions: Patients with Crohn’s disease and unaffected relatives carrying variants of the NOD2 gene have increased adaptive immune responses to microbial antigens.
Cell-Cell Contacts Prevent Anoikis in Primary Human Colonic Epithelial Cells, 20 November 2006
Hofmann C, Obermeier F, Artinger M, Hausmann M, Falk W, Schoelmerich J, Rogler G, Grossmann J
Background & Aims: Colonic epithelial cells (CECs) receive important survival signals from the extracellular matrix and undergo detachment-induced apoptosis (anoikis) as soon as they lose their cell-matrix anchorage. In contrast to the established role of cell-matrix contact, the role of cell-cell contacts as a physiologic survival factor for CECs is less clear. Methods: Intact CEC crypts gently centrifuged to form a cell aggregate in which cell-cell contacts were maintained. Induction of apoptosis was assessed by Western Blot analysis, colorimetric assays, DNA electrophoresis, 4?,6-diamidino-2-phenylindole staining, and flow cytometry. Activation of survival pathways was analyzed by Western blot. The role of mitogen-activated protein kinase/extracellular signal–regulated kinase (MEK)/extracellular signal–regulated kinase (Erk)1/2, epidermal growth factor receptor, phosphatidylinositol 3-kinase (PI3-K), and Src signaling was investigated using specific inhibitors. Results: Despite a complete loss of cell-matrix adhesion after CEC isolation, activation of caspases was blocked and anoikis was prevented when cell-cell contacts were preserved. CECs with preserved cell-cell contacts exhibited a rapid dephosphorylation of focal adhesion kinase. Aggregated CECs had stable levels of active ?-catenin and phosphorylated Akt, Erk1/2, and epidermal growth factor receptor, but CECs undergoing anoikis rapidly degraded ?-catenin and dephosphorylated Akt. Inhibition of Src- and PI3-K–dependent signaling reversed the antiapoptotic effect of cell-cell contact preservation, while inhibition of the MEK pathway had no effect. Conclusions: Integrity of cell-cell contacts compensates for the loss of cell-matrix contact-mediated survival signals in CECs and prevents apoptosis. Cell-cell contact-triggered CEC survival involves antiapoptotic signaling through ?-catenin-, Src-, and PI3-K/Akt- but not through MEK- and focal adhesion kinase–dependent pathways.
Adiponectin Deficiency Protects Mice From Chemically Induced Colonic Inflammation, 23 November 2006
Fayad R, Pini M, Sennello JA, Cabay RJ, Chan L, Xu A, Fantuzzi G
Background & Aims: Adiponectin (APN) is an adipokine that regulates insulin sensitivity and is anti-inflammatory in atherosclerosis. The goal of this study was to investigate the role of APN in intestinal inflammation. Methods: APN knockout (KO) mice and their wild-type (WT) littermates received dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce intestinal inflammation. Clinical and histologic scores and proliferation of epithelial cells were assessed. Cytokines and APN levels were measured. Expression of APN and heparin binding epidermal growth factor (HB-EGF) was analyzed by immunohistochemistry. Expression of APN and its receptors, HB-EGF, and basic fibroblast growth factor (bFGF) messenger RNA was assessed by reverse-transcription polymerase chain reaction. Association of serum APN with HB-EGF and bFGF was studied by coimmunoprecipitation. Results: APN KO mice are protected from chemically induced colitis; administration of APN restores inflammation. APN is expressed in the colon, luminal APN associates with colonic epithelial cells. In vitro, APN increases production of proinflammatory cytokines from colonic tissue. Expression of colonic APN overlaps with that of bFGF and HB-EGF, which play a protective role in colitis. Circulating APN binds to bFGF and HB-EGF, likely inhibiting their protective activity. Inhibition of EGF receptor signaling, which is required for biologic activity of HB-EGF, restores inflammation in APN KO mice. Conclusions: APN deficiency is associated with protection from chemically induced colitis. APN exerts proinflammatory activities in the colon by inducing production of proinflammatory cytokines and inhibiting bioactivity of protective growth factors. Thus, in colitis, APN exerts an opposite role compared with atherosclerosis.
The Vanilloid Receptor Initiates and Maintains Colonic Hypersensitivity Induced by Neonatal Colon Irritation in Rats, 14 November 2006
Winston J, Shenoy M, Medley D, Naniwadekar A, Pasricha PJ
Background & Aims: Robust chemical or mechanical irritation of the colon of neonatal rats leads to chronic visceral hypersensitivity. The clinical and physiologic relevance of such noxious stimulation in the context of human irritable bowel syndrome is questionable. The aims of this study were to determine whether mild chemical irritation of the colon of neonatal rats produced persistent changes in visceral sensitivity and to evaluate the role of transient receptor potential vanilloid 1 (TRPV1) in the initiation and maintenance of visceral hypersensitivity. Methods: Ten-day-old rat pups received an intracolonic infusion of 0.5% acetic acid in saline. TRPV1 inhibitors were administered 30 minutes before acetic acid sensitization. Sensitivity of the colon to balloon distention (CRD) in adults was measured by grading their abdominal withdrawal reflex and electromyographic responses. In adult rats, TRPV1 antagonist was injected intraperitoneally 30 minutes before CRD. Results: Neonatal acetic acid treatment resulted in higher sensitivity to CRD in adult rats compared with controls in the absence of histopathologic signs of inflammation. Treatment of colons of adult rats with acetic acid did not produce persistent sensitization. Antagonism of the TRPV1 before neonatal administration of acetic acid and after established visceral hypersensitivity attenuated sensitivity to CRD. TRPV1 expression was increased in dorsal root ganglia–containing colon afferent neurons. Conclusions: We have described a new model for persistent colonic sensory dysfunction following a transient noxious stimulus in the neonatal period and a potentially important role for TRPV1 in initiation and maintenance of persistent visceral hypersensitivity.
The Intestinal Wnt/TCF Signature, 23 August 2006
Van der Flier LG, Sabates–Bellver J, Oving I, Haegebarth A, De Palo M, Anti M, Van Gijn ME, Suijkerbuijk S, Van de Wetering M, Marra G, Clevers H
Background & Aims: In colorectal cancer, activating mutations in the Wnt pathway transform epithelial cells through the inappropriate expression of a TCF4 target gene program, which is physiologically expressed in intestinal crypts. Methods: We have now performed an exhaustive array-based analysis of this target gene program in colorectal cancer cell lines carrying an inducible block of the Wnt cascade. Independently, differential gene-expression profiles of human adenomas and adenocarcinomas vs normal colonic epithelium were obtained. Results Expression analyses of approximately 80 genes common between these data sets were performed in a murine adenoma model. The combined data sets describe a core target gene program, the intestinal Wnt/TCF signature gene set, which is responsible for the transformation of human intestinal epithelial cells. Conclusions The genes were invariably expressed in adenomas, yet could be subdivided into 3 modules, based on expression in distinct crypt compartments. A module of 17 genes was specifically expressed at the position of the crypt stem cell.
Activin Type 2 Receptor Restoration in MSI-H Colon Cancer Suppresses Growth and Enhances Migration With Activin, 20 November 2006
Jung BH, Beck SE, Cabral J, Chau E, Cabrera BL, Fiorino A, Smith EJ, Bocanegra M, Carethers JM
Background & Aims: Colon cancers with high-frequency microsatellite instability (MSI-H) develop frameshift mutations in tumor suppressors as part of their pathogenesis. ACVR2 is mutated at its exon 10 polyadenine tract in >80% of MSI-H colon cancers, coinciding with loss of protein. ACVR2 transmits the growth effects of activin via phosphorylation of SMAD proteins to affect gene transcription. The functional effect of activin in colon cancers has not been studied. We developed and characterized a cell model in which we studied how activin signaling affects growth. Methods: hMLH1 and ACVR2 mutant HCT116 cells were previously stably transferred with chromosome 2 (HCT116+chr2), restoring a single regulated copy of wild-type ACVR2 but not hMLH1. Both HCT116+chr2 and parental HCT116 cells (as well as HEC59 and ACVR2 and hMSH2 complemented HEC59+chr2 cells) were assessed for genetic complementation and biologic function. Results: HCT116+chr2 cells and HEC59+chr2 cells, but not ACVR2-mutant HCT116 or HEC59 cells, acquired wild-type ACVR2 as well as expression of ACVR2 wild-type messenger RNA. Complemented ACVR2 protein complexed with ACVR1 with activin treatment, generating nuclear phosphoSMAD2 and activin-specific gene transcription. ACVR2-restored cells showed decreased growth and reduced S phase but increased cellular migration following activin treatment. ACVR2 small interfering RNA reversed these effects in complemented cells. Conclusions: ACVR2-complemented MSI-H colon cancers restore activin-SMAD signaling, decrease growth, and slow their cell cycle following ligand stimulation but show increased cellular migration. Activin is growth suppressive and enhances migration similar to transforming growth factor ? in colon cancer, indicating that abrogation of the effects of activin contribute to the pathogenesis of MSI-H colon cancers.
A Simple Method for the Routine Detection of Somatic Quantitative Genetic Alterations in Colorectal Cancer, 6 December 2006
Killian A, Di Fiore F, Le Pessot F, Blanchard F, Lamy A, Raux G, Flaman J, Paillot B, Michel P, Sabourin J, Tuech J, Michot F, Kerckaert J, Sesboüé R, Frebourg T
Background & Aims: Several quantitative genetic alterations have been suggested to have in colorectal cancer (CRC) either a prognostic or a therapeutic predictive value. Routine detection of these alterations is limited by the absence of simple methods.
Methods: The somatic quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) is based on the simultaneous amplification under quantitative conditions of several dye-labeled targets both from tumor and nonmalignant tissues. For each patient, the resulting QMPSF fluorescent profiles are superimposed, and quantitative changes are simply detected by an increase or decrease of the corresponding fluorescent peaks. Two assays were developed and applied to 57 CRC: a “bar code” exploring several loci with known prognostic value and a “kinogram” studying the copy number change of kinase genes, against which inhibitors have been developed.
Results: The bar code revealed that the most frequent alterations were the gain of AURKA/20q13 (53%) and MYC/8q24 (39%) and heterozygous deletion of DCC/18q21.3 (39%) and TP53/17p13 (23%). The kinogram detected a gene copy number increase for AURKA, PTK2, MET, and EGFR in 53%, 37%, 33%, and 28% of the tumors, respectively. QMPSF results were validated by comparative genomic hybridization and multiplex real-time polymerase chain reaction on genomic DNA.
Conclusions: The somatic QMPSF is a simple method able to detect simultaneously on a routine basis several quantitative changes in tumors. Its flexibility will allow the integration of clinically relevant genes. This high throughput method should be a valuable complementary tool of fluorescent in situ hybrization and comparative genomic hybridization.
Basic-Liver, Pancreas, and Biliary Tract
Discordant Role of CD4 T-Cell Response Relative to Neutralizing Antibody and CD8 T-Cell Responses in Acute Hepatitis C, 6 December 2006
Kaplan DE, Sugimoto K, Newton K, Valiga ME, Ikeda F, Aytaman A, Nunes FA, Lucey MR, Vance BA, Vonderheide RH, Reddy KR, McKeating JA, Chang K
Background & Aims: Acute hepatitis C virus (HCV) infection becomes chronic in the majority of patients. Although HCV-specific CD4 T-cell response is associated with HCV clearance, less is known about virus-specific CD8 T-cell or neutralizing antibody (nAb) responses and the role of CD4 help in their induction during acute infection.
Methods: HCV-specific CD4, CD8, and HCV pseudoparticle (HCVpp) nAb responses were monitored in acutely HCV-infected patients to define their relative contributions to viral clearance.
Results: Our results show that the outcome of acute hepatitis C is associated with a functional hierarchy in HCV-specific CD4 T-cell response and the scope of virus-specific, total T-cell interferon-? response. HCV-specific CD8 T-cell response was readily detectable in acutely HCV-infected patients regardless of virologic outcome or virus-specific CD4 T-cell response. In contrast, HCVpp-specific nAbs were readily detected in patients with chronic evolution and impaired virus-specific CD4 T-cell response but not in patients who cleared infection with robust virus-specific CD4 T-cell response.
Conclusions: The outcome of acute hepatitis C is associated with efficient virus-specific CD4 T-cell response(s) without which HCV-specific CD8 T-cell and heterologous nAb responses may develop but fail to clear viremia. Furthermore, HCV-specific nAb responses may not be induced despite robust virus-specific CD4 T-cell response.
Hepatitis C Virus Continuously Escapes From Neutralizing Antibody and T-Cell Responses During Chronic Infection In Vivo, 6 December 2006
von Hahn T, Yoon JC, Alter H, Rice CM, Rehermann B, Balfe P, McKeating JA
Background & Aims: Broadly reactive neutralizing antibodies (nAbs) and multispecific T-cell responses are generated during chronic hepatitis C virus (HCV) infection and yet fail to clear the virus. This study investigated the development of autologous nAb and HCV-glycoprotein–specific T-cell responses and their effects on viral sequence evolution during chronic infection in order to understand the reasons for their lack of effectiveness.
Methods: Numerous E1E2 sequences were amplified and sequenced from serum samples collected over a 26-year period from patient H, a uniquely well-characterized, chronically infected individual. HCV pseudoparticles (HCVpp) expressing the patient-derived glycoproteins were generated and tested for their sensitivity to neutralization by autologous and heterologous serum antibodies.
Results: A strain-specific nAb response developed early in infection (8 weeks postinfection), whereas cross-reactive antibodies able to neutralize HCVpp-bearing heterologous glycoproteins developed late in infection (>33 wk postinfection). The humoral response continuously failed to neutralize viruses bearing autologous glycoprotein sequences that were present in the serum at a given time. The amplified glycoprotein sequences displayed high variability, particularly in regions corresponding to defined linear B-cell epitopes. Mutations in defined neutralizing epitopes were associated with a loss of recognition by monoclonal antibodies against these epitopes and with decreased neutralization of corresponding HCVpp. Viral escape from CD4 and CD8 T-cell responses also was shown for several novel epitopes throughout the glycoprotein region.
Conclusions: During chronic infection HCV is subjected to selection pressures from both humoral and cellular immunity, resulting in the continuous generation of escape variants.
Genetic Study of Variation in Normal Mouse Iron Homeostasis Reveals Ceruloplasmin as an HFE-Hemochromatosis Modifier Gene, 23 November 2006
Gouya L, Muzeau F, Robreau A, Letteron P, Couchi E, Lyoumi S, Deybach J, Puy H, Fleming R, Demant P, Beaumont C, Grandchamp B
Background & Aims: Genetic hemochromatosis is one of the most common genetic disorders, with progressive tissue iron overload leading to severe clinical complications. In Northern European populations, genetic hemochromatosis is usually caused by homozygosity for the C282Y mutation in the HFE protein. However, penetrance of this mutation is incomplete, suggesting that other genetic and environmental factors contribute to its differential biologic or clinical expression.
Methods: To identify genes modifying iron homeostasis, we screened the 27 recombinant congenic strains of the C3H/DiSnA-C57BL/10ScSnA/Dem series for tissue and serum iron indices and genotyped 18 microsatellite markers in (C3H/DiSnA ? HcB-2) F2 hybrid mice.
Results: We identified 1 locus encompassing the Ceruloplasmin (Cp) gene with a strong linkage with liver iron, serum iron, and transferrin levels but not with spleen iron. Sequencing of Cp showed an R435X nonsense mutation in exon 7 in C3H/DiSnA mice. To evaluate whether Cp might act as a modifier gene of genetic hemochromatosis, we intercrossed C3H Hfe?/? and C3HDiSnA CpR435X/R435X mice. As expected, we found that double-mutant mice deposited more iron in the liver than mice defective for either one or both genes. In contrast, Hfe?/? ? CpR435/R435X or CpR435X/R435X ? Hfe+/? showed 30% decrease in liver iron when compared with single mutant mice.
Conclusions: This study highlights the existence of complex interactions between Cp and HFE and represents the first example of a modifier gene with a protective effect, in which heterozygosity reduces the iron load in the context of HFE deficiency.
Hepatic Expression of Candidate Genes in Patients With Alcoholic Hepatitis: Correlation With Disease Severity, 30 December 2006
Colmenero J, Bataller R, Sancho–Bru P, Bellot P, Miquel R, Moreno M, Jares P, Bosch J, Arroyo V, Caballería J, Ginès P
Background & Aims: Alcoholic hepatitis (AH) is a form of acute-on-chronic liver failure for which current therapy is not fully effective. We investigated the hepatic expression of candidate genes in patients with AH to identify new targets for therapy. Methods: Hepatic expression of candidate genes (n = 46) was assessed by quantitative polymerase chain reaction in patients with AH (n = 23) and in normal livers (n = 6). Disease severity was assessed by the Maddrey’s discriminant function and the occurrence of clinical complications. Histologic analysis included the assessment of myofibroblasts (smooth muscle ?-actin), collagen deposition (Sirius red), and inflammatory infiltrate (CD43). Portal hypertension was assessed by hepatic venous pressure gradient. Predictive association between gene expression and disease severity was assessed by k-nearest neighbor analysis. Results: Patients with AH showed profound hepatocellular dysfunction advanced fibrosis, and severe portal hypertension. Livers with AH showed up-regulation of genes encoding extracellular matrix proteins (procollagen I), fibrogenesis mediators, inflammatory cytokines, and apoptosis regulators. Key components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were markedly up-regulated, whereas cytochrome p450 2E1 and angiotensinogen were down-regulated. The expression of tissue inhibitor of metalloproteinases-1, growth-related oncogene ?, and several components of NADPH oxidase (dual oxidases 1 and 2) correlated with histologic findings and parameters indicative of disease severity. Conclusions: Genes involved in hepatic fibrogenesis, inflammatory response, and oxidative stress are overexpressed in AH. Some candidate genes correlate with histologic findings and disease severity, suggesting that they may be potential targets for therapy.
Chronic Ethanol Consumption Impairs Cellular Immune Responses Against HCV NS5 Protein Due to Dendritic Cell Dysfunction, 14 November 2006
Aloman C, Gehring S, Wintermeyer P, Kuzushita N, Wands JR
Background & Aims: Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8+ cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. Methods: Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. Results: Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1? and IL-10 and decreased tumor necrosis factor ?, IL-12, interferon ?, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. Conclusions: Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.
Nitroflurbiprofen, a Nitric Oxide-Releasing Cyclooxygenase Inhibitor, Improves Cirrhotic Portal Hypertension in Rats, 30 December 2006
Laleman W, Van Landeghem L, Van der Elst I, Zeegers M, Fevery J, Nevens F
Background & Aims: We studied whether administration of nitroflurbiprofen (HCT-1026), a cyclooxygenase inhibitor with nitric oxide (NO)-donating properties, modulates the increased intrahepatic vascular tone in portal hypertensive cirrhotic rats. Methods: In vivo hemodynamic measurements (n = 8/condition) and evaluation of the increased intrahepatic resistance by in situ perfusion (n = 5/condition) were performed in rats with thioacetamide-induced cirrhosis that received either nitroflurbiprofen (45 mg/kg), flurbiprofen (30 mg/kg, equimolar concentration to nitroflurbiprofen), or vehicle by intraperitoneal injection 24 hours and 1 hour prior to the measurements. Additionally, we evaluated the effect of acute administration of both drugs (250 ?mol/L) on the intrahepatic vascular tone in the in situ perfused cirrhotic rat liver (endothelial dysfunction and hyperresponsiveness to methoxamine) and on hepatic stellate cell contraction in vitro. Typical systemic adverse effects of nonsteroidal anti-inflammatory drugs, such as gastrointestinal ulceration, renal insufficiency, and hepatotoxicity, were actively explored. Results: In vivo, nitroflurbiprofen and flurbiprofen equally decreased portal pressure (8 ± 0.8 and 8.4 ± 0.1 mm Hg, respectively, vs 11.8 ± 0.6 mm Hg) and reduced the total intrahepatic vascular resistance. Systemic hypotension was not aggravated in the different treatment groups (P = .291). In the perfused cirrhotic liver, both drugs improved endothelial dysfunction and hyperresponsiveness. This was associated with a decreased hepatic thromboxane A2-production and an increased intrahepatic nitrate/nitrite level. In vitro, nitroflurbiprofen, more than flurbiprofen, decreased hepatic stellate cells contraction. Flurbiprofen-treated rats showed severe gastrointestinal ulcerations (bleeding in 3/8 rats) and nefrotoxicity, which was not observed in nitroflurbiprofen-treated cirrhotic rats. Conclusions: Treatment with nitroflurbiprofen, an NO-releasing cyclooxygenase inhibitor, improves portal hypertension without major adverse effects in thioacetamide-induced cirrhotic rats by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperreactivity to vasoconstrictors.
Isolation of Mouse Pancreatic Ductal Progenitor Cells Expressing CD133 and c-Met by Flow Cytometric Cell Sorting, 23 November 2006
Oshima Y, Suzuki A, Kawashimo K, Ishikawa M, Ohkohchi N, Taniguchi H
Background & Aims: Islet transplantation has become available across the globe since a novel protocol was reported. However, because donors are in short supply, only a minority of patients benefit from this procedure. Pancreatic progenitor cells are a promising resource for regeneration of new islets, but whether progenitor cells reside in ductal epithelium is not clear. Methods: Mouse pancreas was examined by immunohistochemistry with cell surface markers specific for ductal cells. We developed an isolation method for ductal cells by flow cytometric cell sorting using a newly identified specific marker for ductal cells. By using an in vitro colony assay, we characterized their proliferative and multipotent capacity. Results: CD133 is expressed specifically in ductal epithelium. Flow cytometric analysis revealed that purified ductal cells are highly enriched in the CD133+CD34?CD45?Ter119? fraction. An analysis of clonal epithelial colonies formed by individual cells revealed that progenitor cells with multilineage differentiation capacity are present in neonatal ductal epithelium. Moreover, these progenitor cells express c-Met. In adult mice, progenitor cells that show a high proliferative capacity but appear committed to a ductal lineage are co-purified with CD133+CD34?CD45?Ter119? cells. Conclusions: We established a system for isolating and culturing mouse pancreatic ductal cells that relies on flow cytometric cell sorting. Clonal analysis revealed that a population of progenitor cells is present among CD133+ ductal cells. Isolation of these cells will facilitate future studies into the roles of pancreatic progenitor cells in regeneration and carcinogenesis.
Defective Hepatic Response to Interferon and Activation of Suppressor of Cytokine Signaling 3 in Chronic Hepatitis C, 6 December 2006
Huang Y, Feld JJ, Sapp RK, Nanda S, Lin JH, Blatt LM, Fried MW, Murthy K, Liang TJ
Background & Aims: Approximately half of hepatitis C virus (HCV)-infected patients do not respond to current interferon (IFN)-? combination therapy. To understand IFN-? resistance in vivo, we examined the dynamic responses to both type I and type II IFNs, human IFN (hIFN)-?, -?, and consensus IFN, in the chimpanzee model. Methods: Naive and HCV-infected chimpanzees were treated with 3 forms of hIFNs in vivo. Quantitative real-time polymerase chain reaction was performed to evaluate the expression of IFN-stimulated genes (ISGs) in both peripheral blood mononuclear cells and liver to compare the responses to hIFN between naive and infected chimpanzees. The hepatic expression of IFN signaling components and inhibitory regulators including suppressor of cytokine signaling 3 (SOCS3) were assessed. SOCS3 expression was also evaluated in the liver of HCV-infected patients undergoing IFN treatment. Results: The in vivo responses to all 3 hIFNs were much lower in the HCV-infected chimpanzees than those in the naive chimpanzees. This defect was particularly evident in the liver because induction of hepatic ISGs was barely detectable in the infected animals. Following IFN administration, the expression of SOCS3 was significantly up-regulated, possibly through induction of interleukin-6, in the liver of HCV-infected chimpanzees. HCV-infected humans also showed a differential pattern of hepatic SOCS3 expression in response to IFN that is associated with treatment response. Conclusions: Our data indicate a predominantly defective hepatic response to IFN in HCV-infected chimpanzees, which is probably mediated through the activation of SOCS3 and may explain the nonresponse of many HCV patients to IFN-based therapy.
© 2007 American Gastroenterological Association (AGA) Institute. Published by Elsevier Inc. All rights reserved.
JOURNAL OF HEPATOLOGY
Volume 46, Issue 2, February 2007
"Seuls les abstracts des articles Originaux sont mis sur cette page. Les autres articles sont en ligne en FREE sur le site du Journal"
Original Articles
Viral Hepatitis
Polymorphisms of type I interferon receptor 1 promoter and their effects on chronic hepatitis B virus infection, 20 October 2006
Zhou J, Lu L, Yuen MF, Lam TW, Chung CP, Lam CL, Zhang B, Wang S, Chen Y, Wu SH, Poon VK, Ng F, Chan CC, Jiang S, Yuen KY, Zheng BJ
Background/Aims
Exposure to HBV leads to a distinct clinical course which is partially pertained to host genetic variability. We aimed to study polymorphisms of type I interferon receptor 1 (IFNAR1) promoter and their potential effects on chronic HBV infection.
Methods
Polymorphisms of IFNAR1 promoter were identified in 320 chronic hepatitis B patients, 148 spontaneously recovered individuals, 148 healthy Chinese donors and 114 Caucasians. Their functional capability in driving reporter gene expression was analyzed.
Results
Four polymorphic alleles were identified at loci ?568, ?408, ?77 and ?3. Association analysis revealed that carriers of alleles ?568G, ?408C and their related haplotype I were less susceptible to chronic HBV infection whereas those of alleles ?568C, ?408T and related haplotype III were significantly associated with higher risk to chronic hepatitis B (P<0.01). In a reporter-driven system, the promoter variants with alleles ?408C and ?3C could drive higher expression of the reporter gene than those with alleles ?408T and ?3T (P<0.01). Interestingly, an allele with 9 GT repeats at ?77 that was rarely found in Chinese but prevalent in Caucasian exhibited the highest transcriptional ability.
Conclusions
Our results showed that polymorphisms of IFNAR1 promoter may affect, at least in part, the outcomes of HBV infection.
Peginterferon alfa-2b and ribavirin in patients with hepatitis C virus and decompensated cirrhosis: A controlled study, 23 October 2006
Iacobellis A, Siciliano M, Perri F, Annicchiarico BE, Leandro G, Caruso N, Accadia L, Bombardieri G, Andriulli A
Background/Aim
To evaluate long-term outcomes in decompensated HCV-related cirrhotic patients treated with antiviral therapy.
Methods
Of 129 eligible patients, 66 received peginterferon alfa-2b and ribavirin for 24 weeks, and 63 were controls. Survival and recurrence of liver failure events after therapy were main outcomes.
Results
Therapy was tolerated by 27 patients, dose reduced in 26 for toxicity, and discontinued in 13 for intolerance. End-of-therapy and sustained virological response (SVR) rates were 82.6% and 43.5% for HCV 2/3 patients, and 30.2% and 7.0% for HCV 1/4 patients. During therapy, odds ratios for severe infections or deaths due to infection were 2.95 (95% C.I. 0.93–9.3) and 1.97 (95% C.I. 0.40–9.51) in treated patients as compared with controls. During a follow-up of 30 months off-therapy, decompensated events occurred in 52, 33, and 3 of controls, non-responders, and SVR patients. Odds ratios for ascites, encephalopathy, and oesophageal bleeding in treated patients significantly decreased as compared with controls. Annualized incidence of death was 2.34, 1.91, and 0 per 1000 patient-years, respectively, in controls, non-responders, and SVR patients. Survival curves showed early separation of SVR patients from both non-responders and controls at approximately 6 months.
Conclusions
In decompensated cirrhotics, HCV clearance by therapy is life-saving and reduces disease progression.
Cirrhosis and its Complications
Effects of a 7-day treatment with midodrine in non-azotemic cirrhotic patients with and without ascites, 31 October 2006
Kalambokis G, Fotopoulos A, Economou M, Pappas K, Tsianos EV
Background/Aims
Splanchnic arterial vasodilatation has been causally related with hyperdynamic circulation and impaired natriuresis in advanced cirrhosis and has also been suggested to be responsible for the subtle sodium retention in pre-ascitic cirrhosis. This study evaluated the effects of a 7-day treatment with the ?1-adrenergic agonist midodrine in non-azotemic cirrhotic patients with and without ascites.
Methods
Thirty-nine cirrhotic patients were studied at baseline and 7 days after administration of oral midodrine 10mg, t.i.d. (11 without and 12 with ascites) or placebo (8 without and 8 with ascites).
Results
A significant increase in urine sodium excretion was noted after midodrine administration in patients without and with ascites, in line with significant increases in mean arterial pressure and systemic vascular resistance, and significant decreases in cardiac output and heart rate. Significant increases in glomerular filtration rate, filtration fraction, and urine volume and significant decreases in plasma renin activity and aldosterone were observed in patients with ascites. Placebo had no effect in any study group.
Conclusions
The administration of midodrine for 7 days improves systemic haemodynamics and sodium excretion in non-azotemic cirrhotic patients without or with ascites. In patients with ascites, but not in those without ascites, these effects are associated with a suppression of the activity of the renin–angiotensin–aldosterone system, suggesting that the increase in natriuresis is related to the improvement in the effective arterial blood volume.
Liver Failure, Growth and Cancer
Tannic acid synergizes the cytotoxicity of chemotherapeutic drugs in human cholangiocarcinoma by modulating drug efflux pathways, 6 October 2006
Naus PJ, Henson R, Bleeker G, Wehbe H, Meng F, Patel T
ackground/Aims
Tannic acid is an orally active plant polyphenol with potential for use as an anti-cancer agent for cholangiocarcinoma. To determine the potential use of tannic acid as an adjunct therapy, we sought to evaluate the interaction between tannic acid and chemotherapeutic agents.
Methods
Cytotoxicity was assessed in malignant human cholangiocytes. Interactions between tannic acid, mitomycin C, 5-fluorouracil and gemcitabine were quantitated by calculating the combination index and dose reduction index. Cellular efflux pathways were assessed by calcein retention assays, and expression of membrane pumps was assessed by Western blots and real-time PCR.
Results
Tannic acid and the three agents decreased growth of malignant cholangiocytes to a similar extent. Tannic acid had a synergistic effect to mitomycin C and 5-fluorouracil, but not gemcitabine. However, the structurally related polyphenol gallic acid did not have a synergistic interaction with any of the agents. Tannic acid decreased calcein efflux and the expression of PGP, MRP1 and MRP2 membrane efflux pumps.
Conclusions
Tannic acid has a synergistic effect with selected chemotherapeutic drugs by a mechanism involving modulation of drug efflux pathways. Thus, tannic acid will be a useful adjunct to improve the effectiveness of chemotherapeutic agents in the treatment of cholangiocarcinoma.
Prevention of severe toxic liver injury and oxidative stress in MCP-1-deficient mice, 23 October 2006
Zamara E, Galastri S, Aleffi S, Petrai I, Aragno M, Mastrocola R, Novo E, Bertolani C, Milani S, Vizzutti F, Vercelli A, Pinzani M, Laffi G, LaVilla G, Parola M, Marra F
Background/Aims
Administration of carbon tetrachloride determines liver injury, inflammation and oxidative stress, but the molecular mechanisms of damage are only partially understood. In this study, we investigated the development of acute toxic damage in mice lacking monocyte chemoattractant protein-1 (MCP-1), a chemokine which recruits monocytes and activated lymphocytes.
Methods
Mice with targeted deletion of the MCP-1 gene and wild type controls were administered a single intragastric dose of carbon tetrachloride. Serum liver enzymes, histology, expression of different chemokines and cytokines, and intrahepatic levels of oxidative stress-related products were evaluated.
Results
Compared to wild type mice, peak aminotransferase levels were significantly lower in MCP-1-deficient animals. This was paralleled by a delayed appearance of necrosis at histology. In addition, MCP-1-deficient mice showed a shift in the pattern of infiltrating inflammatory cells, with a predominance of polymorphonuclear leukocytes. Lack of MCP-1 was also accompanied by reduced intrahepatic expression of cytokines regulating inflammation and tissue repair. The increase in tissue levels of reactive oxygen species and 4-hydroxy-nonenal following administration of the hepatotoxin was also significantly lower in animals lacking MCP-1.
Conclusions
Lack of MCP-1 affords protection from damage and development of oxidative stress in a toxic model of severe acute liver injury.
Molecular and Cell Biology
Marked changes of the hepatic sinusoid in a transgenic mouse model of acute immune-mediated hepatitis, 23 October 2006
Warren A, Bertolino P, Benseler V, Fraser R, McCaughan GW, Le Couteur DG
Background/Aims
The liver sinusoidal endothelial cell (LSEC) is increasingly recognized as having an important role in hepatic immunity. However, the responses of LSECs and the hepatic sinusoid in immune-mediated hepatitis are poorly described.
Methods
We studied a transgenic mouse model of acute immune-mediated hepatitis: Met-Kb mice injected with T cells from Des-TCR mice.
Results
Hepatitis was characterized by lymphocyte infiltrates causing severe but transient liver damage. There were marked changes in the ultrastructure of the LSEC five days after injection of the T cells that coincided with the peak of the hepatitis. The porosity of fenestrations in the LSEC decreased and the endothelium became thickened. LSECs appeared to be markedly activated. These changes were associated with narrowing of the space of Disse, loss of hepatocellular microvilli and deposition of basal lamina. Lymphocytes were seen passing through fenestrations. Loss of fenestration in the LSEC prevented hepatitis induced by a second injection of lymphocytes on day 5.
Conclusions
Structural changes in the LSEC occur during the peak of a mouse model of immune-mediated hepatitis. These changes were associated with attenuation of subsequent liver damage, suggesting that they may influence immunological responses mediated by LSECs or the passage of lymphocytes through LSEC fenestrations.
Remodelling of calcium signalling during liver regeneration in the rat, 9 October 2006
Nicou A, Serrière V, Hilly M, Prigent S, Combettes L, Guillon G, Tordjmann T
Background/Aims
During liver regeneration, a network of cytokines and growth factors interact with hepatocytes, helping to restore the liver mass and functions after partial tissue loss. Agonists that trigger Ca2+ signals in the liver contribute to this process, although little is known about calcium signalling during liver regeneration.
Results
We observed two phases in which the hepatocyte response to calcium-mobilising agonists was greatly reduced versus control cells at 24h and five days after partial hepatectomy. We found that both phases of hepatocyte desensitisation involved the down-regulation of cell surface receptors and the type II InsP3 receptor. Single cell studies with flash photolysis of caged InsP3 revealed that InsP3-mediated Ca2+ release was slower in regenerating hepatocytes at 24, 48h and 5 days than in control cells. Also, the temporal pattern of vasopressin-elicited intracellular calcium oscillations studied on fura2-loaded cells was altered, with the duration of each Ca2+ peak being longer. Finally, we showed an association between hepatocyte desensitisation and progression through the cell cycle towards the S phase at 24h after hepatectomy.
Conclusions
Our study supports the remodelling of hepatocyte calcium signalling during liver regeneration, and that this change is partly linked with cell cycle progression.
The murine liver is a potential target organ for IL-19, IL-20 and IL-24: Type I Interferons and LPS regulate the expression of IL-20R2, 25 September 2006
Wegenka UM, Dikopoulos N, Reimann J, Adler G, Wahl C
Background/Aims
The biological functions of the recently discovered IL-10-related cytokines IL-19, IL-20, IL-22, IL-24 and their receptors IL-20R1, IL-20R2 and IL-22R are not clear. Therefore, the expression of these cytokines and their receptors in the hepatic acute phase response to LPS was analysed. Type I interferons have important immunomodulatory functions in bacterial infections. We investigated if they influence release and organ-specific expression of TNF, IL-6 and IL-10 and the responsiveness of liver to IL-10 related cytokines during the reaction to LPS in vivo.
Methods
B6 and congenic IFNAR?/? mice were intraperitoneally injected with 5mg/kg LPS. Systemic release of cytokines was quantified by ELISA. Organ-specific expression of cytokines and their receptors was evaluated by (semi quantitative or quantitative) RT-PCR.
Results
The cytokines IL-19, IL-22 and the IL-20R2 receptor subunit are up-regulated by LPS in the liver of normal mice. IFN?/? enhance the secretion and expression of IL-6 and IL-10 during the response to LPS, but also the up-regulation of IL-20R2 expression.
Conclusions
We show that the liver is a potential target for IL-19, IL-20 and IL-24. During an LPS response, IFN?/? influence cytokine secretion and expression and possibly the response to IL-19 and IL-24.
Overexpression of the gene for transmembrane 4 superfamily member 4 accelerates liver damage in rats treated with CCl4, 2 October 2006
Qiu J, Liu Z, Da L, Li Y, Xuan H, Lin Q, Li F, Wang Y, Li Z, Zhao M
Background/Aims
Transmembrane 4 superfamily member 4 (TM4SF4) is up-regulated in regenerating liver after partial hepatectomy in rats, but the in vivo functions of this protein are still largely unknown. Therefore, we investigated the role of TM4SF4 during liver injury.
Methods
Expression of TM4SF4 was analyzed by RT-PCR and Western blotting in normal and CCl4-injured rats. Overexpression or reduced expression of TM4SF4 in the liver was achieved by injection of sense or antisense TM4SF4 expression plasmids. Assessment of liver injury (histology, serum ALT and AST levels), apoptosis by TUNEL assay were performed. Expression of injury-related genes was analyzed by quantitative real-time PCR.
Results
Overexpression of TM4SF4 in rats after CCl4 treatment showed extensive liver damage and increased levels of serum ALT and AST. Decreased TM4SF4 gene expression showed minimal liver necrosis and depressed ALT and AST levels. Increased expression of TM4SF4 affected the expression levels of growth factors and receptors, such as TNF-?, TNFR1 and c-met. Furthermore, pro-apoptotic and anti-apoptotic gene expression was altered after TM4SF4 administration.
Conclusions
Rat TM4SF4 is overexpressed in acutely injured liver induced by CCl4 and plays a crucial role in accelerating liver injury, which may be mediated by the TNF-? and HGF/c-met signaling pathways.
LPS exacerbates endothelin-1 induced activation of cytosolic phospholipase A2 and thromboxane A2 production from Kupffer cells of the prefibrotic rat liver, 30 November 2006
Miller AM, Masrorpour M, Klaus C, Zhang JX
Background/Aims
Thromboxane A2 (TXA2) has been suggested to play a significant role in the development of portal hypertension in fibrosis, and Kupffer cell (KC) derived TXA2 has been shown to mediate the hyperresponsiveness of the portal circulation to the vasoconstrictive actions of endothelin-1 (ET-1) during endotoxemia. The aim of this study was to determine whether the double stresses of prefibrotic changes and endotoxemia additively activate KC to increase release of TXA2 in response to ET-1, resulting in elevated portal resistance.
Methods
One week Bile duct ligation (BDL) rats and sham-operated controls were subjected to isolated liver perfusions following LPS or saline for 6h. In a separate experiment, KC were isolated from BDL or sham rats and incubated with LPS or saline for 6h before the ET-1 treatment.
Results
The double stresses of early fibrosis and LPS resulted in a greater sustained increase in portal pressure in response to ET-1 in BDL rats, and this increase correlated well with the much enhanced release of TXA2 in the perfusate. Media from the cultured KC showed significantly greater TXA2 release in response to ET-1 in BDL group than those in sham group, and LPS exacerbated this effect. Protein levels of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2, and thromboxane synthase were also significantly elevated in KC from BDL rats. ET-1 produced a marked increase in cPLA2 activation as measured by the phosphorylation of cPLA2 in KC of both BDL and sham groups. LPS greatly exacerbated the activation of cPLA2.
Conclusions
The data suggest that the double stresses additively activate KC with an upregulation of the key enzymes in the TXA2 biosynthesis and release increased amount of TXA2 via the augmented activation of cPLA2 in response to ET-1, which leads to the increased portal resistance and ultimately hepatic microcirculatory dysfunction.
Low molecular weight heparin prevents hepatic fibrogenesis caused by carbon tetrachloride in the rat, 25 October 2006
Abe W, Ikejima K, Lang T, Okumura K, Enomoto N, Kitamura T, Takei Y, Sato N
Background/Aims
In this study, we investigated the effect of dalteparin sodium, a low molecular weight (LMW)-heparin, on hepatic fibrogenesis caused by chronic carbon tetrachloride (CCl4) administration in the rat.
Methods
Female Wistar rats were given a single, or repeated intraperitoneal injections of CCl4 (1ml/kg, twice per week) and dalteparin (50IU/kg, daily) for 7 weeks.
Results
Dalteparin did not prevent acute CCl4-induced hepatic necrosis and elevation in serum aminotransferases levels; however, proliferating cell nuclear antigen (PCNA)-positive hepatocytes were dramatically increased 24h after simultaneous administration of CCl4 and dalteparin. Interestingly, serum hepatocyte growth factor (HGF) levels 12h after injection of CCl4 were almost doubled when dalteparin was given simultaneously. Hepatic fibrosis following 7-week CCl4 treatment was markedly ameliorated by daily co-administration of dalteparin. Indeed, dalteparin largely inhibited CCl4-induction of smooth muscle ?-actin expression, ?1(I)procollagen and transforming growth factor (TGF)-?1 mRNA levels in the liver. Further, dalteparin blunted platelet-derived growth factor (PDGF)-induced increases in 5-bromo-2?deoxyuridine (BrdU) uptake in 3-day cultured hepatic stellate cells (HSCs) in a dose-dependent manner.
Conclusions
Dalteparin enhances hepatic regeneration and minimizes hepatic fibrogenesis caused by chronic CCl4 treatment. The mechanism underlying these effects most likely involves both up-regulation of HGF and inhibition of HSC proliferation.
IFN-? abrogates profibrogenic TGF-? signaling in liver by targeting expression of inhibitory and receptor Smads, 2 November 2006
Weng H, Mertens PR, Gressner AM, Dooley S
Background/Aims
In a randomized open-labeled multicenter trial with patients suffering from chronic HBV infection, we recently identified a benefit of 9-month IFN-? treatment resulting in decreased fibrosis scores and a reduced number of ?-smooth muscle actin-positive hepatic stellate cells (HSCs). Approaches opposing profibrogenic activities of TGF-? may be amenable in chronic liver disease. According to experimental models, IFN-? counteracts several TGF-? effects.
Methods
The crosstalk of IFN-? and TGF-? signaling relevant for fibrogenesis was investigated in primary cultured rat HSCs and a cell line representing activated HSCs.
Results
In vitro studies with HSCs demonstrate that TGF-?-dependent activation of (CAGA)9-MLP-Luc, a Smad3/4 responsive reporter construct, was significantly decreased by IFN-?, indicating a TGF-? antagonizing function. IFN-? induced the activity of the Smad7 promoter and Smad7 protein expression via STAT-1 signaling. In contrast to TGF-?, IFN-? was able to induce Smad7 expression in activated HSCs providing increased protein levels for at least 12h. In addition, expression of Smad2/3 was reduced by IFN-? and activation of Smads2/3 was abrogated.
Conclusions
IFN-? displays antifibrotic effects in liver cells via STAT-1 phosphorylation, upregulation of Smad7 expression and impaired TGF-? signaling.
Genetic and Metabolic Liver Disease
NADPH oxidase is not an essential mediator of oxidative stress or liver injury in murine MCD diet-induced steatohepatitis, 2 November 2006
dela Peña A, Leclercq IA, Williams J, Farrell GC
Background/Aims
Hepatic oxidative stress is a key feature of metabolic forms of steatohepatitis, but the sources of pro-oxidants are unclear. The NADPH oxidase complex is critical for ROS generation in inflammatory cells; loss of any one component (e.g., gp91phox) renders NADPH oxidase inactive. We tested whether activated inflammatory cells contribute to oxidant stress in steatohepatitis.
Methods
gp91phox?/? and wildtype (wt) mice were fed a methionine and choline-deficient (MCD) diet. Serum ALT, hepatic triglycerides, histopathology, lipid peroxidation, activation of NF-?B, expression of NF-?B-regulated genes and macrophage chemokines were measured.
Results
After 10 days of MCD dietary feeding, gp91phox?/? and wt mice displayed equivalent hepatocellular injury. After 8 weeks, there were fewer activated macrophages in livers of gp91phox?/? mice than controls, despite similar mRNA levels for MCP and MIP chemokines, but fibrosis was similar. NF-?B activation and increased expression of ICAM-1, TNF-? and COX-2 mRNA were evident in both genotypes, but in gp91phox?/? mice, expression of these genes was confined to hepatocytes.
Conclusions
A functional NADPH oxidase complex does not contribute importantly to oxidative stress in this model and therefore is not obligatory for induction or perpetuation of dietary steatohepatitis.
Betaine attenuates alcoholic steatosis by restoring phosphatidylcholine generation via the phosphatidylethanolamine methyltransferase pathway, 26 October 2006
Kharbanda KK, Mailliard ME, Baldwin CR, Beckenhauer HC, Sorrell MF, Tuma DJ
ackground/Aims
Previous studies in our laboratory implicated ethanol-induced decreases in hepatocellular S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH) ratios in lowering the activity of phosphatidylethanolamine methyltransferase (PEMT), which is associated with the generation of steatosis. Further in vitro studies showed that betaine supplementation could correct these alterations in the ratio as well as attenuate alcoholic steatosis. Therefore, we sought to determine whether the protective effect of betaine is via its effect on PEMT activity.
Methods
Male Wistar rats were fed the Lieber DeCarli control or ethanol diet with or without 1% betaine supplementation for 4 weeks.
Results
We observed that ethanol feeding resulted in decreased phosphatidylcholine (PC) production by a PEMT-catalyzed reaction. Betaine supplementation corrected the ethanol-induced decrease in hepatic SAM:SAH ratios and by normalizing PC production via the PEMT-mediated pathway, significantly reduced fatty infiltration associated with ethanol consumption. This restoration of hepatocellular SAM:SAH ratio by betaine supplementation was associated with increases in the activity, enzyme mass and gene expression of the enzyme, betaine homocysteine methyltransferase (BHMT), that remethylates homocysteine.
Conclusions
Betaine, by virtue of promoting an alternate remethylation pathway, restores SAM:SAH ratios that, in turn, correct the defective cellular methylation reaction catalyzed by PEMT resulting in protection against the generation of alcoholic steatosis.
A novel mechanism for drug-induced liver failure: Inhibition of histone acetylation by hydralazine derivatives, 3 November 2006
Murata K, Hamada M, Sugimoto K, Nakano T
Background/Aims
The aim of this study was to investigate the precise mechanism of liver failure by hydralazine derivatives, with special reference to liver regeneration failure.
Methods
Histone acetylation and proliferation of hepatocytes were evaluated by immunohistochemistry with anti-acetylated histone H4 and proliferating cell nuclear antigen (PCNA). Inhibition of histone acetylation by drugs was determined by in vitro histone acetylation assay. Mice livers fed with todralazine for 1 or 4 months were subjected to immunohistochemistry and Western blotting. Todralazine-fed mice were challenged with anti-Fas to check liver regeneration failure.
Results
On immunohistochemistry, histone acetylation in the hepatocytes was significantly impaired in patients with hydralazine derivatives. In an in vitro acetyl transferase assay, histone acetylation was inhibited by hydralazine derivatives in a dose-dependent manner. Mice fed with todralazine (3mg/day) for 4 months showed impairment of histone acetylation in hepatocytes whereas no inhibition was observed in mice fed with todralazine for 1 month. Anti-Fas challenge to todralazine-fed mice resulted in impairment of liver regeneration in respect of liver weight loss with impairment of histone acetylation in hepatocytes.
Conclusions
Todralazine could inhibit catalysis of histone acetyltransferase and long-term administration of todralazine may impair histone acetylation of the hepatocytes, resulting in liver regeneration failure.
Repeated whiskey binges promote liver injury in rats fed a choline-deficient diet, 2 November 2006
Nieto N, Rojkind M
Background/Aims
Alcoholic liver disease is associated with nutritional deficiency and it may aggravate within the context of fatty liver. We investigated the relationship between alcohol intake (whiskey binge drinking) and a choline-deficient diet (CD) and assessed whether stellate cells could contribute to liver injury in this model.
Results
Rats fed the CD diet plus whiskey showed increased liver damage compared to rats fed the CD diet, as demonstrated by H&E staining, elevated transaminases, steatosis, TNF-? levels, enhanced CYP2E1 activity, impaired antioxidant defense, elevated lipid peroxidation, and protein carbonyls. The combined treatment triggered an apoptotic response as determined by elevated Bax, caspase-3 activity, cytochrome-c release, and decreased Bcl-2 and Bcl-XL. Stellate cells were activated as increased expression of ?-Sma was observed over that by the CD diet alone. The combined treatment shifted extracellular matrix remodeling towards a pro-fibrogenic response due to up-regulation of collagen I, TIMP1, and Hsp47 proteins, along with down-regulation of MMP13, MMP2, and MMP9 expression, proteases which degrade collagen I. These events were accompanied by increased phosphorylation of p38, a kinase that elevates collagen I.
Conclusions
Repeated alcohol binges in the context of mild steatosis may promote activation of stellate cells and contribute to liver injury.
Copyright © 2007 European Association for the Study of the Liver. All rights reserved.
BRITISH MEDICAL JOURNAL
Copyright © 2007 BMJ Publishing Group Ltd
NEW ENGLAND JOURNAL
Volume 356:676-684 - February 15 - 2007 - Number 7
Endoscopic versus Surgical Drainage of the Pancreatic Duct in Chronic Pancreatitis
Djuna L. Cahen, M.D., Dirk J. Gouma, M.D., Ph.D., Yung Nio, M.D., Erik A. J. Rauws, M.D., Ph.D., Marja A. Boermeester, M.D., Ph.D., Olivier R. Busch, M.D., Ph.D., Jaap Stoker, M.D., Ph.D., Johan S. Laméris, M.D., Ph.D., Marcel G.W. Dijkgraaf, Ph.D., Kees Huibregtse, M.D., Ph.D., and Marco J. Bruno, M.D., Ph.D.
Background For patients with chronic pancreatitis and a dilated pancreatic duct, ductal decompression is recommended. We conducted a randomized trial to compare endoscopic and surgical drainage of the pancreatic duct.
Methods All symptomatic patients with chronic pancreatitis and a distal obstruction of the pancreatic duct but without an inflammatory mass were eligible for the study. We randomly assigned patients to undergo endoscopic transampullary drainage of the pancreatic duct or operative pancreaticojejunostomy. The primary end point was the average Izbicki pain score during 2 years of follow-up. The secondary end points were pain relief at the end of follow-up, physical and mental health, morbidity, mortality, length of hospital stay, number of procedures undergone, and changes in pancreatic function.
Results Thirty-nine patients underwent randomization: 19 to endoscopic treatment (16 of whom underwent lithotripsy) and 20 to operative pancreaticojejunostomy. During the 24 months of follow-up, patients who underwent surgery, as compared with those who were treated endoscopically, had lower Izbicki pain scores (25 vs. 51, P<0.001) and better physical health summary scores on the Medical Outcomes Study 36-Item Short-Form General Health Survey questionnaire (P=0.003). At the end of follow-up, complete or partial pain relief was achieved in 32% of patients assigned to endoscopic drainage as compared with 75% of patients assigned to surgical drainage (P=0.007). Rates of complications, length of hospital stay, and changes in pancreatic function were similar in the two treatment groups, but patients receiving endoscopic treatment required more procedures than did patients in the surgery group (a median of eight vs. three, P<0.001).
Conclusions Surgical drainage of the pancreatic duct was more effective than endoscopic treatment in patients with obstruction of the pancreatic duct due to chronic pancreatitis.
The New England Journal of Medicine is owned, published, and copyrighted © 2007 Massachusetts Medical Society. All rights reserved.
LANCET
The Lancet, published, and copyrighted © 2007. All rights reserved.
