Thus, HBx could up-regulate over 35-fold the expression of a luciferase reporter gene driven by the HBV Enhancer I and associated core promoter in human hepatoma HepG2 cells, in which HBx enhances HBV replication8, 9, 27, 29 (Fig. 1A). HBx also exhibited activity when expressed from an HBV genomic plasmid or at very low levels from a chromosomally integrated construct (Supporting Fig. S1). Vemurafenib nmr The woodchuck WHx protein

showed comparable transactivation potential, in accordance with previous studies (Fig. 1A).8 Activation by HBx and WHx decreased upon overexpression of the paramyxovirus SV5-V protein, which competitively inhibits HBx binding to DDB1,23 and this occurred only when HBx and WHx expression was low (Fig. 1B and data not shown). Furthermore, the HBx(R96E) point mutant that is impaired in its DDB1-binding ability14, 23 is essentially inactive in this assay (Fig. 1A). However, the mutant regains CP-868596 order full activity when covalently fused to wildtype DDB1, a situation that forces interaction between the two proteins (Fig. 1C).23 This is not the case when mutations are introduced into DDB1 to block its incorporation into the E3 ligase complex, or further

compromise the HBx-DDB1 interaction (Fig. 1C).14, 23 This suggests that HBx(R96E) is impaired solely in DDB1 binding and that HBx requires DDB1 to function as a subunit of the E3 ligase 上海皓元 complex to carry out its stimulatory activity. We conclude that HBx and WHx can efficiently stimulate transient reporter gene activity and that they likely do so by a conserved mechanism involving the DDB1 E3 ligase. We then examined whether HBx would exhibit the same strong activation potential on luciferase reporter constructs placed under control of other,

unrelated promoter and enhancer elements. Figure 2 shows that this is indeed the case; HBx showed a similarly strong effect on expression of an SV40 promoter-driven construct, regardless of the presence or absence of a downstream SV40 enhancer (Fig. 2A), and on expression of an interferon-regulated promoter construct (Fig. 2A). HBx also increased activity of a synthetic NF-κB responsive promoter (Fig. 2B), and basal activity of a tetracycline-inducible promoter even in cells producing no tetracycline-regulated activator (Fig. 2C). In all cases, the DDB1-binding HBx(R96E) point mutant failed to transactivate, suggesting that the stimulatory function requires interaction of HBx with the DDB1 E3 ligase at all promoter types tested. This suggests that HBx functions by a common mechanism regardless of the nature of the cis-regulatory elements. An obvious common feature of reporter constructs tested by transient transfection is the extrachromosomal nature of the DNA template.

2 These findings are not characteristic of mice genetically null for Mrp4 or Mrp312, 13, 20 and highlight the importance of ileal Ostα-Ostβ as a regulator of normal bile acid homeostasis. As might be expected with such a small bile acid pool, PDGFR inhibitor the Ostα−/− mice show less accumulation of hepatic bile acids after BDL, especially of polyhydroxylated forms. However, because obstructive cholestasis in these animals prevents bile acids from entering the intestine, there is a loss of signaling from Fgf15 and a lowering of the elevated liver levels of Shp

and FgfR4 mRNA that otherwise occur in wild-type BDL mice. Thus, Cyp7a1 and Bsep are up-regulated and the bile acid pool is increased. Fxr, Car, and Pxr are all key nuclear receptors

that participate in the adaptive response to cholestatic injury.21, 22 Car and Pxr play important roles in bile acid–detoxifying enzymes in mice and in the regulation of Mrp4 and Sult2a1.23–25 However, unlike Fxr or Pxr, we find that sham-operated and BDL Ostα-deficient mice have a significant increase in Car mRNA compared to the wild-type controls, suggesting that this nuclear receptor may play a more important regulatory role in detoxification in these mice. Our data are consistent with Car-induced Phase I (Cyp3a11, Cyp2b10) and Phase II (Sult2a1, Ugt1a1) detoxification enzymes.24, 25 Furthermore, they support the BI 6727 concentration concept that this nuclear receptor can induce expression of the Phase III transporters Mrp3 and Mrp4, and provide alternative pathways for bile acid export from the liver.24 Another particularly

novel finding in this study is that in the absence of Ostα, obstructive cholestasis leads to a further increase in urinary excretion of bile acids than otherwise occurs in cholestasis. This has also been shown in mice treated with Car agonists and subjected to 24-hour BDL.24 We show that adaptive regulation of key membrane transporters in the kidney could be responsible for this change. First, in the absence MCE of Ostα-Ostβ in the proximal tubule, Ostα-deficient mice cannot reabsorb the increase in urinary filtration of bile acids that occurs after BDL. Second, the renal apical uptake transporter Asbt is further decreased, and the renal apical export transporters Mrp2 and Mrp4 are both increased. Thus, bile acids are blocked from being transported back to the systemic circulation, and the limited amount that are taken up into the proximal tubule are effectively exported back out the apical membrane into the urine. This conclusion is also supported by the finding of increased urinary excretion of the Ostα-Ostβ substrates [3H]estrone 3-sulfate and [3H]dehydroepiandrosterone sulfate when administered to Ostα−/− mice.1 In summary, liver injury is attenuated in Ostα−/− mice following BDL.

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“The Editors and Editorial

Board of HEPATOLOGY are grateful to the following referees for their contributions to the journal in 2010. Abarca, Jorge Abdelmalek, Manal Abdelmoneim, Soha Abergel, Armand Abraldes, Juan Abumrad, Nada Adams, David Adams, Leon Afdhal, Nezam Agnello, Vincent Ahn, Joseph Ahn, Sang Hoon Aithal, Guruprasad Aitken, Campbell Alavian, Seyed Moayed Albano, Emanuele Alberti, Alfredo Albillos, Agustin Albrecht, Jeffrey H. Alison, Malcolm Allain, Jean-Pierre Aloman, Costica Alonso, Estella M. Alpini, Gianfranco Alter, Harvey Alter, Miriam Amitrano, Lucio Anania, Frank Ananthanarayanan, Meenakshisundaram Anderson, Christopher Andrade, Raul Angel, Peter Angeli, Paolo Angulo, Paul Anstee, Quentin Anwer, Mohammed Aoyagi, Yutaka Arii, Shigeki Protein Tyrosine Kinase inhibitor Arrese, Marco Arteel, Gavin Asahina, Kinji Asrani, Sumeet Asselah, Tarik Avila, Matias Awad, Tahany Ayuso, Carmen Bacon, Bruce R. Baffet, Georges Baffy, Gyorgy Bahr, Matthias Bailey, Shannon Baiocchi, Leonardo Bajaj, Jasmohan Bambha, Kiran Banares, Rafael Banerjee, Atrayee Bansal, Meena Bantel, Heike Bartenschlager, Ralf Barton, James Barve, Shirish Bass, Nathan Bataller, Ramon Bauer, Michael Baumert, Thomas Beaugrand, Michel Bédossa, Pierre Behari, Jaideep Beier-Arteel, Juliane Belghiti, Jacques Beraza, Naiara Beretta, Laura Berg,

Peter Berg, Thomas Berg, Trond Bergheim, Ina Bernardi, Mauro Bernuau, Jacques Bertoletti, Antonio Bertolini, Francesco Bertolino, Patrick Beuers, Ulrich Bezerra, Jorge Biernacka, Joanna Biggins, Scott Z-VAD-FMK research buy Billadeau, Daniel Billiar, medchemexpress Timothy Bioulac-Sage, Paulette Bjorkhem, Ingemar Bjornsson, Einar Blechacz, Boris Blom, Daniel Bode, Johannes Bodenheimer, Henry Boelsterli, Urs Bogdanos, Dimitrios Boix, Loreto Boland, C. Richard Bonkovsky, Herbert L. Bonnetain, Franck Bortolotti, Flavia Bosca, Lisardo Bosch, Jaime Boucher, Eveline

Boyer, James Boyer, Thomas Braillon, Alain Brancatelli, Giuseppe Breitenstein, Stefan Brenner, David Brojer, Ewa Brouwer, Kim Brown, Kyle Bru, Concepcion Bruix, Jordi Brunetto, Maurizia Buchman, Alan Buendia, Marie-Annick Bugianesi, Elisabetta Burns, Peter Burra, Patrizia Burroughs, Andrew Burt, Alastair Buti, Maria Butt, Adeel Buttar, Navtej Caballeria, Juan Cabrera, Roniel Callea, Francesco Calvisi, Diego Camma, Calogero Canbay, Ali Cantz, Tobias Cao, Sheng Caramiel-Haggai, Michal Cardenas, Andres Cardinal, Jon Carlin, Cathleen Carrilho, Flair Jose Carrington, Mary Castéra, Laurent Cave, Matthew Cengiz, Cem Chalasani, Naga Chan, Henry Lik-Yuen Chang, Kyong-Mi Chapman, Roger Charlton, Michael Chatterjee, Suvro Chavin, Kenneth Chawla, Yogesh Chen, Chien-Jen Chen, Mingdao Chen, Pei-Jer Chen, Yao Cheung, Onpan Chevaliez, Stephane Chiang, John Chini, Eduardo Choi, Byung Ihn Choi, Steve Chow, Pierce K.H.

During the current follow up time, one relapse of an inhibitor occurred, in patient number 4. Low inhibitory activity (1 BU mL−1) without FVIII recovery was observed 48 months after successful ITI. This was treated by increasing his prophylactic dose to 25 IU FVIII kg−1 every other day. Partial success was achieved after 1 month, and complete success after 11 months. After partial success, surgery was performed in 13 patients. Seven patients had one surgical intervention, four patients two, one patient three and one patient four. All were performed with FVIII, without any complications of bleeding. This study reports results of 26 years of low dose ITI in severe haemophilia A

high throughput screening patients with inhibitors, treated in a single large haemophilia see more centre. Low dose ITI comprised of 25–50 IU FVIII kg−1, twice a week to every other day. Low dose ITI was successful

in 18 of 21 patients (86%, 95%CI 71–100%). Success rate was higher and time to success was shorter in patients with a maximum inhibitor level titre below 40 BU mL−1. This effect was even stronger in patients with low titre inhibitors (<5 BU mL−1). Although patient characteristics in this study are not completely comparable to those of the previous report (the 1995-study) on low dose ITI, the success rate of this study (86%) is in accordance with the 1995-study, in which a success rate of 87% (95% CI 74–100%) was found [4]. An important difference between the present and the 1995-study is that in the 1995-study, FVIII infusions were discontinued in two-thirds of patients who were included, because of historical treatment policies. The median age at inhibitor development was also different 上海皓元医药股份有限公司 in both studies: 5 years (range of 1–23 years) and 19 months (range 13–28 months) respectively. In the 1995-study, complete success was achieved after 0.5–28 months, with a median of 1 year. In this study the median time to success was 6.6 months (range 1–42 months). In both

studies, time to complete success was related to a maximum inhibitor titre of <40 BU mL−1. The association with age at inhibitor development (<2.5 years) was only observed in the 1995-study. This may be explained by the earlier inhibitor development in the second cohort of patients. This study describes patients with predominantly low inhibitor titres. Both the median pre-ITI titre of 4.5 BU mL−1, and the maximum titre during ITI of 4.6 BU mL−1 are substantially lower, compared to other studies. The median of the maximum titre reported in the International Immune Tolerance Registry (IITR) was 54 BU mL−1 (mean 530, range 1–25 000) in 314 patients. In the North American Immune Tolerance Registry (NAITR), the mean historical peak titre of patients who achieved success was 130 BU mL−1 (range 5–4833) in 128 high responders (>5 BU mL−1) [6,7]. Unuvar et al. described a median pre-ITI historical peak titre of 80 BU mL−1 (range 6–517) in a case series of 21 patients.

This result effectively

ruled out the possibility that LDPCs could have originated from the initial nonhepatocyte cell population in culture. Next, we wanted to substantiate our PKH staining results by documenting the phenotypic changes taking place during the transformation of hepatocytes into LDPCs. To that end, we performed RT-PCR and IF analyses of hepatocyte- and LDPC-specific markers at predetermined time points during the culture period. On days 0, 4, 8, and 12, cultures were examined for expression of albumin, HNF-1α (hepatocyte specific), Erismodegib cell line CD45, and LMO2 (LDPC specific). RT-PCR studies showed that in the beginning, cells expressed albumin and HNF-1α and no identifiable CD45 and LMO2. By day 4, there was a rapid decline

in hepatocyte-specific markers, and LDPC-specific markers became detectable at low levels. Subsequently, on days 8 and 12, hepatocyte markers became undetectable, and LDPC markers were expressed EGFR inhibitor at increasingly higher levels (Fig. 3A). IF studies revealed a similar pattern of marker expression, further confirming our RT-PCR data (Fig. 3B). In addition to these four markers, we examined the expression pattern of several other highly relevant hepatic genes during the culture period to better characterize the transformation process. We looked at the expression of mature hepatocyte markers HepPar1 and HNF-4α, immature hepatocyte marker Liv2,24 biliary ductal/oval cell

marker CK19, and liver progenitor/embryonic liver marker Sall425 in a time-dependent manner. IF staining and quantitative analysis of the images revealed a pattern (Supporting Fig. 2A,B), which was consistent with rapid transformation of mature hepatocytes into cells with liver progenitor phenotype, thus supporting our findings shown in Fig. 3. Both the RT-PCR and IF studies correlated well with the morphological changes that took place in the cultures, including temporal appearance of LDPCs. Taken together, the rat studies 上海皓元 strongly suggested that LDPCs originated from mature hepatocytes by direct dedifferentiation. To gain further insight into the process of dedifferentiation of hepatocytes to LDPCs and to establish a stem/progenitor cell hierarchy, we examined the expression of several oval cell markers during the culture period. We considered the possibility that hepatocytes could be transitioning through an oval cell-like stage en route to becoming LDPCs. This was based on the phenotypic similarities between oval cells and LDPCs, suggesting a potential lineage relationship. Therefore, we studied the expression of OV-6, CK7, and GGT during the dedifferentiation of hepatocytes into LDPCs.