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1
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Lam AM, Mani N, Ardzinski A, Stever K, Cuconati A, Micolochick Steuer H, Thi EP, Graves IE, Espiritu CL, Mesaros E, Kultgen SG, Fan K, Cole AG, Harasym TO, Rijnbrand R, Brown J, Eley T, Varughese T, Gane E, Picchio G, Sims KD, Sofia MJ. Preclinical and clinical antiviral characterization of AB-836, a potent capsid assembly modulator against hepatitis B virus. Antiviral Res 2024; 231:106010. [PMID: 39326502 DOI: 10.1016/j.antiviral.2024.106010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2024] [Revised: 09/11/2024] [Accepted: 09/23/2024] [Indexed: 09/28/2024]
Abstract
HBV capsid assembly modulators (CAMs) target the core protein and inhibit pregenomic RNA encapsidation and viral replication. HBV CAMs also interfere with cccDNA formation during de novo infection, which in turn suppresses transcription and production of HBV antigens. In this report, we describe the antiviral activities of AB-836, a potent and highly selective HBV CAM. AB-836 inhibited viral replication (EC50 = 0.010 μM) in HepDE19 cells, and cccDNA formation (EC50 = 0.18 μM) and HBsAg production (EC50 = 0.20 μM) in HepG2-NTCP cells during de novo infection. AB-836 showed broad genotype coverage, remained active against variants resistant to nucleos(t)ide analogs, and demonstrated improved antiviral potency against core variants resistant to other CAMs. AB-836 also mediated potent inhibition of HBV replication in a hydrodynamic injection mouse model, reducing both serum and liver HBV DNA. In a Phase 1 clinical study, 28 days of once-daily AB-836 oral dosing at 50, 100, and 200 mg resulted in mean serum HBV DNA declines of 2.57, 3.04, and 3.55 log10 IU/mL from baseline, respectively. Neither on-treatment viral rebound nor the emergence of viral resistance was observed during the 28-day treatment period. Furthermore, HBV DNA sequence analysis of baseline samples from the Phase 1 study revealed that 51.4% of the chronic hepatitis B participants contained at least one core polymorphism within the CAM-binding pocket, suggesting that genetic variations exist at this site. While AB-836 was discontinued due to clinical safety findings, data from the preclinical and clinical studies could help inform future optimization of HBV CAMs.
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Affiliation(s)
| | | | | | - Kim Stever
- Arbutus Biopharma Inc., Warminster, PA, USA
| | | | | | | | | | | | | | | | - Kristi Fan
- Arbutus Biopharma Inc., Warminster, PA, USA
| | | | | | | | | | | | | | - Edward Gane
- Auckland Clinical Studies, Grafton, Auckland, New Zealand
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2
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Cole AG, Kultgen SG, Mani N, Fan KY, Ardzinski A, Stever K, Dorsey BD, Mesaros EF, Thi EP, Graves I, Tang S, Harasym TO, Lee ACH, Olland A, Suto RK, Sofia MJ. Rational Design, Synthesis, and Structure-Activity Relationship of a Novel Isoquinolinone-Based Series of HBV Capsid Assembly Modulators Leading to the Identification of Clinical Candidate AB-836. J Med Chem 2024; 67:16773-16795. [PMID: 39231272 DOI: 10.1021/acs.jmedchem.4c01568] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/06/2024]
Abstract
Inhibition of Hepatitis B Virus (HBV) replication by small molecules that modulate capsid assembly and the encapsidation of pgRNA and viral polymerase by HBV core protein is a clinically validated approach toward the development of new antivirals. Through definition of a minimal pharmacophore, a series of isoquinolinone-based capsid assembly modulators (CAMs) was identified. Structural biology analysis revealed that lead molecules possess a unique binding mode, exploiting electrostatic interactions with accessible phenylalanine and tyrosine residues. Key analogs demonstrated excellent primary potency, absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic properties, and efficacy in a mouse model of HBV. The optimized lead also displayed potent inhibition of capsid uncoating in HBV-infected HepG2 cells expressing the sodium-taurocholate cotransporting polypeptide (NTCP) receptor, affecting the generation of HBsAg and cccDNA establishment. Based on these results, isoquinolinone derivative AB-836 was advanced into clinical development. In Phase 1b trials, AB-836 demonstrated >3 log10 reduction in serum HBV DNA, however, further development was discontinued due to the observation of incidental alanine aminotransferase (ALT) elevations.
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Affiliation(s)
- Andrew G Cole
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Steven G Kultgen
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Nagraj Mani
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Kristi Yi Fan
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Andrzej Ardzinski
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Kim Stever
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Bruce D Dorsey
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Eugen F Mesaros
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Emily P Thi
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Ingrid Graves
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Sunny Tang
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Troy O Harasym
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Amy C H Lee
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
| | - Andrea Olland
- Xtal BioStructures Inc., 12 Michigan Drive, Natick, Massachusetts 01760, United States
| | - Robert K Suto
- Xtal BioStructures Inc., 12 Michigan Drive, Natick, Massachusetts 01760, United States
| | - Michael J Sofia
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, Pennsylvania 18974, United States
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3
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Cole AG, Kultgen SG, Mani N, Quintero JG, Yi Fan K, Ardzinski A, Stever K, Dorsey BD, Phelps JR, Lee ACH, Thi EP, Chiu T, Tang S, Horanyi PS, Mayclin SJ, Harasym TO, Sofia MJ. Design, synthesis, and structure-activity relationship of a bicyclic HBV capsid assembly modulator chemotype leading to the identification of clinical candidate AB-506. Bioorg Med Chem Lett 2023; 94:129456. [PMID: 37633618 DOI: 10.1016/j.bmcl.2023.129456] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2023] [Revised: 08/15/2023] [Accepted: 08/22/2023] [Indexed: 08/28/2023]
Abstract
Disruption of the HBV capsid assembly process through small-molecule interaction with HBV core protein is a validated target for the suppression of hepatitis B viral replication and the development of new antivirals. Through combination of key structural features associated with two distinct series of capsid assembly modulators, a novel aminochroman-based chemotype was identified. Optimization of anti-HBV potency through generation of SAR in addition to further core modifications provided a series of related functionalized aminoindanes. Key compounds demonstrated excellent cellular potency in addition to favorable ADME and pharmacokinetic profiles and were shown to be highly efficacious in a mouse model of HBV replication. Aminoindane derivative AB-506 was subsequently advanced into clinical development.
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Affiliation(s)
- Andrew G Cole
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA.
| | - Steven G Kultgen
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Nagraj Mani
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Jorge G Quintero
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Kristi Yi Fan
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Andrzej Ardzinski
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Kim Stever
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Bruce D Dorsey
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Janet R Phelps
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Amy C H Lee
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Emily P Thi
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Tim Chiu
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Sunny Tang
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Peter S Horanyi
- UCB Pharma, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | | | - Troy O Harasym
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
| | - Michael J Sofia
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA
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4
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Nakanishi A, Okumura H, Hashita T, Yamashita A, Nishimura Y, Watanabe C, Kamimura S, Hayashi S, Murakami S, Ito K, Iwao T, Ikeda A, Hirose T, Sunazuka T, Tanaka Y, Matsunaga T. Ivermectin Inhibits HBV Entry into the Nucleus by Suppressing KPNA2. Viruses 2022; 14:2468. [PMID: 36366568 PMCID: PMC9695645 DOI: 10.3390/v14112468] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Revised: 10/28/2022] [Accepted: 11/04/2022] [Indexed: 11/09/2022] Open
Abstract
Hepatitis B virus (HBV) specifically infects human hepatocytes and increases the risks of cirrhosis and liver cancer. Currently, nucleic acid analogs are the main therapeutics for chronic hepatitis caused by HBV infection. Although nucleic acid analogs can eliminate HBV DNA by inhibiting HBV reverse transcriptase, they cannot lead to negative conversion of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg). In this study, we revealed that the antifilarial drug ivermectin suppresses HBV production by a different mechanism from the nucleic acid analog entecavir or Na+ taurocholate co-transporting polypeptide-mediated entry inhibitor cyclosporin A. Ivermectin reduced the levels of several HBV markers, including HBsAg, in HBV-infected human hepatocellular carcinoma cells (HepG2-hNTCP-C4 cells) and humanized mouse hepatocytes (PXB hepatocytes). In addition, ivermectin significantly decreased the expression of HBV core protein and the nuclear transporter karyopherin α2 (KPNA2) in the nuclei of HepG2-hNTCP-C4 cells. Furthermore, depletion of KPNA1-6 suppressed the production of cccDNA. These results suggest that KPNA1-6 is involved in the nuclear import of HBV and that ivermectin suppresses the nuclear import of HBV by inhibiting KPNA2. This study demonstrates the potential of ivermectin as a novel treatment for hepatitis B.
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Affiliation(s)
- Anna Nakanishi
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Hiroki Okumura
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Tadahiro Hashita
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Aya Yamashita
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Yuka Nishimura
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Chihiro Watanabe
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Sakina Kamimura
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Sanae Hayashi
- Department of Virology and Liver Unit, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
- Department of Gastroenterology and Hepatology, Kumamoto University, Kumamoto 860-8556, Japan
| | - Shuko Murakami
- Department of Virology and Liver Unit, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
| | - Kyoko Ito
- Department of Virology and Liver Unit, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
| | - Takahiro Iwao
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Akari Ikeda
- Ōmura Satoshi Memorial Institute, Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan
| | - Tomoyasu Hirose
- Ōmura Satoshi Memorial Institute, Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan
| | - Toshiaki Sunazuka
- Ōmura Satoshi Memorial Institute, Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
- Department of Gastroenterology and Hepatology, Kumamoto University, Kumamoto 860-8556, Japan
| | - Tamihide Matsunaga
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
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Kayesh MEH, Hashem MA, Sanada T, Kitab B, Rashid MHO, Akter L, Ezzikouri S, Murakami S, Ogawa S, Tanaka Y, Kohara M, Tsukiyama-Kohara K. Characterization of innate immune response to hepatitis B virus genotype F acute infection in tree shrew (Tupaia belangeri) model. FRONTIERS IN VIROLOGY 2022; 2. [DOI: 10.3389/fviro.2022.926831] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/11/2025]
Abstract
Hepatitis B virus (HBV) infection is a global public health problem. The clinical outcomes of HBV infections are influenced by host as well as viral factors, including viral genotypes and subgenotypes. The interplay between HBV and host innate immunity remains unclear because of the lack of a suitable small animal model. Tree shrews (Tupaia belangeri) have been utilized as a useful animal model for hepatitis viruses such as hepatitis B and C viruses. In this study, we characterized acute infections by HBV genotype F (HBV-F) wild type (Wt) and mutant type (Mt) viruses in adult tree shrews. Serum alanine aminotransferase levels were measured before and post- infection 7 and 14 dpi. Both HBV-F-Wt and Mt were detected in the HBV-F-infected tree shrew serum and liver tissue at 7 and 14 dpi. We examined the intrahepatic expression patterns of Toll-like receptors (TLRs) (TLR1–9 mRNAs), cGAS, several transcription factors such as STAT1, STAT2, IRF7, HNF4, PD-L1, and cytokines, including IFN-β, IFN-γ, IL-6, and TNF-α in HBV-F Wt/Mt-infected tree shrews. When compared with uninfected animal group, significant suppression of TLR8 in HBV-F-Wt infected animals and significant suppression of PD-L1 in both HBV-F-Wt and Mt infected animals were observed. Thus, tree shrew can be a useful animal model to characterize HBV-F pathogenesis.
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6
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Saitta C, Pollicino T, Raimondo G. Occult Hepatitis B Virus Infection: An Update. Viruses 2022; 14:v14071504. [PMID: 35891484 PMCID: PMC9318873 DOI: 10.3390/v14071504] [Citation(s) in RCA: 50] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2022] [Revised: 06/28/2022] [Accepted: 06/29/2022] [Indexed: 11/16/2022] Open
Abstract
Occult hepatitis B virus (HBV) infection (OBI) refers to a condition in which replication-competent viral DNA is present in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for the HBV surface antigen (HBsAg). In this peculiar phase of HBV infection, the covalently closed circular DNA (cccDNA) is in a low state of replication. Many advances have been made in clarifying the mechanisms involved in such a suppression of viral activity, which seems to be mainly related to the host's immune control and epigenetic factors. OBI is diffused worldwide, but its prevalence is highly variable among patient populations. This depends on different geographic areas, risk factors for parenteral infections, and assays used for HBsAg and HBV DNA detection. OBI has an impact in several clinical contexts: (a) it can be transmitted, causing a classic form of hepatitis B, through blood transfusion or liver transplantation; (b) it may reactivate in the case of immunosuppression, leading to the possible development of even fulminant hepatitis; (c) it may accelerate the progression of chronic liver disease due to different causes toward cirrhosis; (d) it maintains the pro-oncogenic properties of the "overt" infection, favoring the development of hepatocellular carcinoma.
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Affiliation(s)
- Carlo Saitta
- Division of Medicine and Hepatology, University Hospital of Messina, 98124 Messina, Italy;
- Department of Clinical and Experimental Medicine, University of Messina, 98124 Messina, Italy
| | - Teresa Pollicino
- Department of Human Pathology, University Hospital of Messina, 98124 Messina, Italy;
| | - Giovanni Raimondo
- Division of Medicine and Hepatology, University Hospital of Messina, 98124 Messina, Italy;
- Department of Clinical and Experimental Medicine, University of Messina, 98124 Messina, Italy
- Correspondence: ; Tel.: +39-(0)-902212392
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7
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Cole AG, Kultgen SG, Mani N, Ardzinski A, Fan KY, Thi EP, Dorsey BD, Stever K, Chiu T, Tang S, Daly O, Phelps JR, Harasym T, Olland A, Suto RK, Sofia MJ. The identification of highly efficacious functionalised tetrahydrocyclopenta[ c]pyrroles as inhibitors of HBV viral replication through modulation of HBV capsid assembly. RSC Med Chem 2022; 13:343-349. [PMID: 35434625 PMCID: PMC8942244 DOI: 10.1039/d1md00318f] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Accepted: 01/17/2022] [Indexed: 01/21/2023] Open
Abstract
Disruption of the HBV viral life cycle with small molecules that prevent the encapsidation of pregenomic RNA and viral polymerase through binding to HBV core protein is a clinically validated approach to inhibiting HBV viral replication. Herein we report the further optimisation of clinical candidate AB-506 through core modification with a focus on increasing oral exposure and oral half-life. Maintenance of high levels of anti-HBV cellular potency in conjunction with improvements in pharmacokinetic properties led to multi-log10 reductions in serum HBV DNA following low, once-daily oral dosing for key analogues in a preclinical animal model of HBV replication.
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Affiliation(s)
- Andrew G. Cole
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | | | - Nagraj Mani
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | | | - Kristi Yi Fan
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Emily P. Thi
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Bruce D. Dorsey
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Kim Stever
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Tim Chiu
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Sunny Tang
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Owen Daly
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Janet R. Phelps
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Troy Harasym
- Arbutus Biopharma, Inc.701 Veterans CircleWarminsterPA 18974USA
| | - Andrea Olland
- Xtal BioStructures Inc.12 Michigan DriveNatickMA 01760USA
| | - Robert K. Suto
- Xtal BioStructures Inc.12 Michigan DriveNatickMA 01760USA
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8
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Mani N, Cole AG, Phelps JR, Ardzinski A, Burns R, Chiu T, Cuconati A, Dorsey BD, Evangelista E, Fan K, Guo F, Harasym TO, Kadhim S, Kowalski R, Kultgen SG, Lee ACH, Li AH, Majeski SA, Miller A, Pasetka C, Reid SP, Rijnbrand R, Micolochick Steuer HM, Stever K, Tang S, Teng X, Wang X, Sofia MJ. Preclinical characterization of AB-506, an inhibitor of HBV replication targeting the viral core protein. Antiviral Res 2021; 197:105211. [PMID: 34826506 DOI: 10.1016/j.antiviral.2021.105211] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2021] [Revised: 11/19/2021] [Accepted: 11/22/2021] [Indexed: 02/06/2023]
Abstract
AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC50 of 0.077 μM, CC50 > 25 μM) and in vivo (HBV mouse model: ∼3.0 log10 reductions in serum HBV DNA compared to the vehicle control). Binding of AB-506 to HBV core protein accelerates capsid assembly and inhibits HBV pgRNA encapsidation. Furthermore, AB-506 blocks cccDNA establishment in HBV-infected HepG2-hNTCP-C4 cells and primary human hepatocytes, leading to inhibition of viral RNA, HBsAg, and HBeAg production (EC50 from 0.64 μM to 1.92 μM). AB-506 demonstrated activity across HBV genotypes A-H and maintains antiviral activity against nucleos(t)ide analog-resistant variants in vitro. Evaluation of AB-506 against a panel of core variants showed that T33N/Q substitutions results in >200-fold increase in EC50 values, while L30F, L37Q, and I105T substitutions showed an 8 to 20-fold increase in EC50 values in comparison to the wild-type. In vitro combinations of AB-506 with NAs or an RNAi agent were additive to moderately synergistic. AB-506 exhibits good oral bioavailability, systemic exposure, and higher liver to plasma ratios in rodents, a pharmacokinetic profile supporting clinical development for chronic hepatitis B.
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Affiliation(s)
- Nagraj Mani
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA.
| | - Andrew G Cole
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Janet R Phelps
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Andrzej Ardzinski
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Robbin Burns
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Tim Chiu
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Andrea Cuconati
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Bruce D Dorsey
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Ellen Evangelista
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Kristi Fan
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Fang Guo
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Troy O Harasym
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Salam Kadhim
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Roseann Kowalski
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Steven G Kultgen
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Amy C H Lee
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Alice H Li
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Sara A Majeski
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Angela Miller
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Chris Pasetka
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Stephen P Reid
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Rene Rijnbrand
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | | | - Kim Stever
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Sunny Tang
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Xiaowei Teng
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Xiaohe Wang
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Michael J Sofia
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
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9
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Kayesh MEH, Amako Y, Hashem MA, Murakami S, Ogawa S, Yamamoto N, Hifumi T, Miyoshi N, Sugiyama M, Tanaka Y, Mizokami M, Kohara M, Tsukiyama-Kohara K. Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA. Virus Res 2020; 290:198191. [PMID: 33049308 DOI: 10.1016/j.virusres.2020.198191] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2020] [Revised: 10/03/2020] [Accepted: 10/05/2020] [Indexed: 02/07/2023]
Abstract
Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.
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Affiliation(s)
- Mohammad Enamul Hoque Kayesh
- Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Department of Microbiology and Public Health, Patuakhali Science and Technology University, Bangladesh
| | - Yutaka Amako
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Md Abul Hashem
- Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
| | - Shuko Murakami
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Shintaro Ogawa
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Naoki Yamamoto
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Tatsuro Hifumi
- Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Department of Veterinary Histopathology, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
| | - Noriaki Miyoshi
- Department of Veterinary Histopathology, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
| | - Masaya Sugiyama
- Genome Medical Sciences Project, National Center for Global Health and Medicine, Chiba, Japan
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Department of Gastroenterology and Hepatology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Masashi Mizokami
- Genome Medical Sciences Project, National Center for Global Health and Medicine, Chiba, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Kyoko Tsukiyama-Kohara
- Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
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10
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Zhang Y, Wen Z, Shi X, Liu YJ, Eriksson JE, Jiu Y. The diverse roles and dynamic rearrangement of vimentin during viral infection. J Cell Sci 2020; 134:134/5/jcs250597. [PMID: 33154171 DOI: 10.1242/jcs.250597] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Epidemics caused by viral infections pose a significant global threat. Cytoskeletal vimentin is a major intermediate filament (IF) protein, and is involved in numerous functions, including cell signaling, epithelial-mesenchymal transition, intracellular organization and cell migration. Vimentin has important roles for the life cycle of particular viruses; it can act as a co-receptor to enable effective virus invasion and guide efficient transport of the virus to the replication site. Furthermore, vimentin has been shown to rearrange into cage-like structures that facilitate virus replication, and to recruit viral components to the location of assembly and egress. Surprisingly, vimentin can also inhibit virus entry or egress, as well as participate in host-cell defense. Although vimentin can facilitate viral infection, how this function is regulated is still poorly understood. In particular, information is lacking on its interaction sites, regulation of expression, post-translational modifications and cooperation with other host factors. This Review recapitulates the different functions of vimentin in the virus life cycle and discusses how they influence host-cell tropism, virulence of the pathogens and the consequent pathological outcomes. These insights into vimentin-virus interactions emphasize the importance of cytoskeletal functions in viral cell biology and their potential for the identification of novel antiviral targets.
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Affiliation(s)
- Yue Zhang
- The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China.,University of Chinese Academy of Sciences, Yuquan Road No. 19(A), Shijingshan District, Beijing 100049, China
| | - Zeyu Wen
- The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China.,University of Chinese Academy of Sciences, Yuquan Road No. 19(A), Shijingshan District, Beijing 100049, China
| | - Xuemeng Shi
- The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yan-Jun Liu
- Shanghai Institute of Cardiovascular Diseases, and Institutes of Biomedical Sciences, Zhongshan Hospital, Fudan University, Shanghai 200032, China
| | - John E Eriksson
- Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University, Turku FI-20520, Finland .,Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku FI-20520, Finland
| | - Yaming Jiu
- The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China .,University of Chinese Academy of Sciences, Yuquan Road No. 19(A), Shijingshan District, Beijing 100049, China
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11
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Occult hepatitis B infection and hepatocellular carcinoma: Epidemiology, virology, hepatocarcinogenesis and clinical significance. J Hepatol 2020; 73:952-964. [PMID: 32504662 DOI: 10.1016/j.jhep.2020.05.042] [Citation(s) in RCA: 132] [Impact Index Per Article: 26.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/18/2020] [Revised: 05/21/2020] [Accepted: 05/25/2020] [Indexed: 12/12/2022]
Abstract
Occult hepatitis B infection (OBI) refers to a condition where replication-competent HBV DNA is present in the liver, with or without HBV DNA in the blood, in individuals with serum HBsAg negativity assessed by currently available assays. The episomal covalently closed circular DNA (cccDNA) in OBI is in a low replicative state. Viral gene expression is mediated by epigenetic control of HBV transcription, including the HBV CpG island methylation pathway and post-translational modification of cccDNA-bound histone, with a different pattern from patients with chronic HBV infection. The prevalence of OBI varies tremendously across patient populations owing to numerous factors, such as geographic location, assay characteristics, host immune response, coinfection with other viruses, and vaccination status. Apart from the risk of viral reactivation upon immunosuppression and the risk of transmission of HBV, OBI has been implicated in hepatocellular carcinoma (HCC) development in patients with chronic HCV infection, those with cryptogenic or known liver disease, and in patients with HBsAg seroclearance after chronic HBV infection. An increasing number of prospective studies and meta-analyses have reported a higher incidence of HCC in patients with HCV and OBI, as well as more advanced tumour histological grades and earlier age of HCC diagnosis, compared with patients without OBI. The proposed pathogenetic mechanisms of OBI-related HCC include the influence of HBV DNA integration on the hepatocyte cell cycle, the production of pro-oncogenic proteins (HBx protein and mutated surface proteins), and persistent low-grade necroinflammation (contributing to the development of fibrosis and cirrhosis). There remain uncertainties about exactly how, and in what order, these mechanisms drive the development of tumours in patients with OBI.
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12
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Construction of complete Tupaia belangeri transcriptome database by whole-genome and comprehensive RNA sequencing. Sci Rep 2019; 9:12372. [PMID: 31451757 PMCID: PMC6710255 DOI: 10.1038/s41598-019-48867-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2018] [Accepted: 08/13/2019] [Indexed: 01/02/2023] Open
Abstract
The northern tree shrew (Tupaia belangeri) possesses high potential as an animal model of human diseases and biology, given its genetic similarity to primates. Although genetic information on the tree shrew has already been published, some of the entire coding sequences (CDSs) of tree shrew genes remained incomplete, and the reliability of these CDSs remained difficult to determine. To improve the determination of tree shrew CDSs, we performed sequencing of the whole-genome, mRNA, and total RNA and integrated the resulting data. Additionally, we established criteria for the selection of reliable CDSs and annotated these sequences by comparison to the human transcriptome, resulting in the identification of complete CDSs for 12,612 tree shrew genes and yielding a more accurate tree shrew genome database (TupaiaBase: http://tupaiabase.org). Transcriptome profiles in hepatitis B virus infected tree shrew livers were analyzed for validation. Gene ontology analysis showed enriched transcriptional regulation at 1 day post-infection, namely in the “type I interferon signaling pathway”. Moreover, a negative regulator of type I interferon, SOCS3, was induced. This work, which provides a tree shrew CDS database based on genomic DNA and RNA sequencing, is expected to serve as a powerful tool for further development of the tree shrew model.
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13
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Preclinical Profile of AB-423, an Inhibitor of Hepatitis B Virus Pregenomic RNA Encapsidation. Antimicrob Agents Chemother 2018; 62:AAC.00082-18. [PMID: 29555628 DOI: 10.1128/aac.00082-18] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2018] [Accepted: 03/10/2018] [Indexed: 12/11/2022] Open
Abstract
AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC50] = 0.08 to 0.27 μM; EC90 = 0.33 to 1.32 μM) with no significant cytotoxicity (50% cytotoxic concentration > 10 μM). Addition of 40% human serum resulted in a 5-fold increase in the EC50s. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants in vitro Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a de novo infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein. In vitro dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the in vitro combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.
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14
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Kawagishi N, Suda G, Onozawa M, Kimura M, Maehara O, Ohara M, Izumi T, Umemura M, Ito J, Nakai M, Sho T, Natsuizaka M, Morikawa K, Ogawa K, Sakamoto N. Comparing the risk of hepatitis B virus reactivation between direct-acting antiviral therapies and interferon-based therapies for hepatitis C. J Viral Hepat 2017. [PMID: 28632923 DOI: 10.1111/jvh.12737] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) reactivation has been reported during antihepatitis C treatment in patients with hepatitis C virus (HCV) and HBV co-infection. We aimed to evaluate the frequency and risk factors of HBV reactivation during anti-HCV therapy and compared those between interferon (IFN)-free direct-acting antiviral (DAA) therapies and IFN-based therapies. Three hundred and twenty-two patients with HCV infection receiving anti-HCV therapy were retrospectively screened. The baseline HBV infection statuses of all eligible patients and the HBV-DNA level of all patients with current or previous HBV infection were examined at the end of treatment. In patients with baseline anti-HBs positivity, changes in anti-HBs titre were evaluated. Of 287 patients who met the inclusion criteria, 157 had current (n=4) or previous (n=153) HBV infection; 85 were treated with IFN-free DAA therapies and 72 were treated with IFN-based therapies. Six patients experienced HBV reactivation (n=2) or HBV reappearance (n=4) after IFN-free DAA therapies, while no patient developed HBV reactivation after IFN-based therapies. The risk factors of HBV reactivation or reappearance were DAA therapies and a reduction in anti-HBs titre to <12 mIU mL-1 by the end of treatment. The decline changes of anti-HBs titre were significantly higher in patients treated with DAA therapies. Although HBV reactivation hepatitis was not observed, three of four patients with HBV reactivation or reappearance after achieving HCV eradication had viremia 8 weeks after completion of therapy. A significant proportion of patients develop HBV reactivation or reappearance without hepatitis after IFN-free DAA therapies. Low levels of anti-HBs and their decrease to <12 mIU mL-1 after treatment are significant risk factors for HBV reactivation or reappearance.
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Affiliation(s)
- N Kawagishi
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - G Suda
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - M Onozawa
- Department of Hematology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - M Kimura
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - O Maehara
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - M Ohara
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - T Izumi
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - M Umemura
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - J Ito
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - M Nakai
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - T Sho
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - M Natsuizaka
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - K Morikawa
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - K Ogawa
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
| | - N Sakamoto
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan
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15
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Sato Y, Matsui H, Yamamoto N, Sato R, Munakata T, Kohara M, Harashima H. Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticle-associated toxicity leading to the safe and efficacious decrease in the hepatitis B virus. J Control Release 2017; 266:216-225. [DOI: 10.1016/j.jconrel.2017.09.044] [Citation(s) in RCA: 80] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2017] [Revised: 09/12/2017] [Accepted: 09/30/2017] [Indexed: 12/12/2022]
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16
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Rosa AS, Araujo OC, Savassi-Ribas F, Fernandes CA, Coelho HS, Niel C, Villela-Nogueira CA, Araujo NM. Prevalence of occult hepatitis B virus infection and Torque teno virus infection and their association with hepatocellular carcinoma in chronic hepatitis C patients. Virus Res 2017; 242:166-172. [PMID: 28966070 DOI: 10.1016/j.virusres.2017.09.022] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2017] [Revised: 09/27/2017] [Accepted: 09/28/2017] [Indexed: 02/07/2023]
Abstract
BACKGROUND The role of occult hepatitis B virus (HBV) infection and Torque teno virus (TTV) infection in the development of hepatocellular carcinoma (HCC) in chronic hepatitis C patients is still uncertain. AIM The aim of the present study was to investigate the prevalence and significance of OBI and TTV infection, and to examine the genetic diversity of these viruses, in chronic hepatitis C patients with and without HCC. METHODS Sera from 151 hepatitis C virus (HCV)-infected patients (49 patients with HCC and 102 without HCC) negative for HBV surface antigen (HBsAg) were tested for the presence of OBI and TTV infection by semi-nested and group-specific multiplex PCR assays, respectively. Nucleotide sequencing of HBV S region was further performed. RESULTS OBI and TTV infection were detected in 5 (3.3%) and 68 (45%) patients, respectively. HBV isolates were classified into genotypes A (4/5, 80%) and D (1/5, 20%), and no HBsAg escape mutation was observed. TTV phylogenetic group 3 was the most prevalent among both HCC and non-HCC patients. OBI and TTV infection were significantly more frequent in patients with HCC than patients without HCC (p=0.003, and p=0.009, respectively). Moreover, TTV infection was associated with HCC (OR=2.23, 95%CI=1.04-4.80, p=0.040), independently of liver cirrhosis. CONCLUSIONS A low prevalence of OBI was observed in patients with HCV-related chronic liver disease, and TTV infection was an independent factor associated with the occurrence of HCC. Whether TTV influences the progression of liver disease in chronic hepatitis C patients remains to be elucidated.
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Affiliation(s)
- Agatha S Rosa
- Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil
| | - Oscar C Araujo
- Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil
| | - Flavia Savassi-Ribas
- Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil; Department of Microbiology and Parasitology, Fluminense Federal University, Niteroi, RJ, Brazil
| | - Carlos A Fernandes
- Hepatitis Division, Central Public Health Laboratory Noel Nutels, Rio de Janeiro, RJ, Brazil
| | - Henrique S Coelho
- Hepatology Division, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
| | - Christian Niel
- Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil
| | - Cristiane A Villela-Nogueira
- Hepatology Division, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
| | - Natalia M Araujo
- Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
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17
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Yamamoto N, Sato Y, Munakata T, Kakuni M, Tateno C, Sanada T, Hirata Y, Murakami S, Tanaka Y, Chayama K, Hatakeyama H, Hyodo M, Harashima H, Kohara M. Novel pH-sensitive multifunctional envelope-type nanodevice for siRNA-based treatments for chronic HBV infection. J Hepatol 2016; 64:547-555. [PMID: 26505121 DOI: 10.1016/j.jhep.2015.10.014] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2015] [Revised: 10/02/2015] [Accepted: 10/12/2015] [Indexed: 12/12/2022]
Abstract
BACKGROUND & AIMS Antiviral agents including entecavir (ETV) suppress the replication of the hepatitis B virus (HBV) genome in human hepatocytes, but they do not reduce the abundance of viral proteins. The present study focused on effectively reducing viral protein levels. METHODS We designed siRNAs (HBV-siRNA) that target consensus sequences in HBV genomes. To prevent the emergence of escaped mutant virus, we mixed three HBV-siRNAs (HBV-siRNAmix); the mixture was encapsulated in a novel pH-sensitive multifunctional envelope-type nanodevice (MEND), a hepatocyte-specific drug delivery system. Coagulation factor 7 siRNA was used to assess delivery and knockdown efficiencies of MEND/siRNA treatments in mice. The potency of MEND/HBV-siRNAmix was evaluated in primary human hepatocytes and in chimeric mice with humanized liver persistently infected with HBV. RESULTS Effective knockdown of targets, efficient delivery of siRNA, and liver-specific delivery were each observed with MEND. MEND/HBV-siRNA caused efficient reduction of HBsAg and HBeAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg or HBeAg when compared with a single MEND/HBV-siRNAmix treatment. Furthermore, the suppressive effects of a single dose of MEND/HBV-siRNAmix persisted for 14days in vitro and in vivo. CONCLUSION We demonstrated that MEND/HBV-siRNA controlled HBV more efficiently than did ETV. Furthermore, the effect of a single dose of MEND/HBV-siRNA persisted for a long time. These results indicated that MEND/HBV-siRNA may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors.
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Affiliation(s)
- Naoki Yamamoto
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
| | - Yusuke Sato
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan
| | - Tsubasa Munakata
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
| | - Masakazu Kakuni
- PhoenixBio Co., Ltd., 3-4-1, Kagamiyama, Hiroshima 739-0046, Japan
| | - Chise Tateno
- PhoenixBio Co., Ltd., 3-4-1, Kagamiyama, Hiroshima 739-0046, Japan
| | - Takahiro Sanada
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
| | - Yuichi Hirata
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
| | - Shuko Murakami
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan
| | - Kazuaki Chayama
- Department of Medicine and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8551, Japan
| | - Hiroto Hatakeyama
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan
| | - Mamoru Hyodo
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan
| | - Hideyoshi Harashima
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.
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18
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Sanada T, Tsukiyama-Kohara K, Yamamoto N, Ezzikouri S, Benjelloun S, Murakami S, Tanaka Y, Tateno C, Kohara M. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes. Biochem Biophys Res Commun 2016; 469:229-235. [PMID: 26654952 DOI: 10.1016/j.bbrc.2015.11.121] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2015] [Accepted: 11/26/2015] [Indexed: 01/25/2023]
Abstract
The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV.
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Affiliation(s)
- Takahiro Sanada
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan
| | - Kyoko Tsukiyama-Kohara
- Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima-city, Kagoshima 890-0065, Japan; Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima, Kagoshima 890-0065, Japan.
| | - Naoki Yamamoto
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan
| | - Sayeh Ezzikouri
- Viral Hepatitis Laboratory, Virology Unit, Institut Pasteur du Maroc, 1, Louis Pasteur, Casablanca 20360, Morocco
| | - Soumaya Benjelloun
- Viral Hepatitis Laboratory, Virology Unit, Institut Pasteur du Maroc, 1, Louis Pasteur, Casablanca 20360, Morocco
| | - Shuko Murakami
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya, Aichi 467-8601, Japan
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya, Aichi 467-8601, Japan
| | - Chise Tateno
- PhoenixBio Co. Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
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19
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Tateno C, Kawase Y, Tobita Y, Hamamura S, Ohshita H, Yokomichi H, Sanada H, Kakuni M, Shiota A, Kojima Y, Ishida Y, Shitara H, Wada NA, Tateishi H, Sudoh M, Nagatsuka SI, Jishage KI, Kohara M. Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice. PLoS One 2015; 10:e0142145. [PMID: 26536627 PMCID: PMC4633119 DOI: 10.1371/journal.pone.0142145] [Citation(s) in RCA: 101] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2015] [Accepted: 10/19/2015] [Indexed: 11/18/2022] Open
Abstract
We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression—not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.
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Affiliation(s)
- Chise Tateno
- PhoenixBio Co., Ltd., Higashihiroshima, Hiroshima, Japan
- Liver Research Project Center, Hiroshima University, Hiroshima, Japan
- * E-mail: (CT); (M. Kohara)
| | - Yosuke Kawase
- Chugai Research Institute for Medical Science, Inc., Gotemba, Shizuoka, Japan
| | - Yoshimi Tobita
- Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | | | - Hiroki Ohshita
- PhoenixBio Co., Ltd., Higashihiroshima, Hiroshima, Japan
| | | | - Harumi Sanada
- PhoenixBio Co., Ltd., Higashihiroshima, Hiroshima, Japan
| | | | - Akira Shiota
- PhoenixBio Co., Ltd., Higashihiroshima, Hiroshima, Japan
| | - Yuha Kojima
- PhoenixBio Co., Ltd., Higashihiroshima, Hiroshima, Japan
| | - Yuji Ishida
- PhoenixBio Co., Ltd., Higashihiroshima, Hiroshima, Japan
- Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Hiroshi Shitara
- Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Naoko A. Wada
- Chugai Research Institute for Medical Science, Inc., Gotemba, Shizuoka, Japan
| | - Hiromi Tateishi
- Chugai Research Institute for Medical Science, Inc., Gotemba, Shizuoka, Japan
| | - Masayuki Sudoh
- Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka, Japan
| | | | | | - Michinori Kohara
- Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
- * E-mail: (CT); (M. Kohara)
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Ezzikouri S, Kimura K, Sunagozaka H, Kaneko S, Inoue K, Nishimura T, Hishima T, Kohara M, Tsukiyama-Kohara K. Serum DHCR24 Auto-antibody as a new Biomarker for Progression of Hepatitis C. EBioMedicine 2015; 2:604-612. [PMID: 26288822 PMCID: PMC4535309 DOI: 10.1016/j.ebiom.2015.04.007] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2015] [Revised: 04/10/2015] [Accepted: 04/10/2015] [Indexed: 02/02/2023] Open
Abstract
BACKGROUND New biomarkers are needed to identify the stage of hepatitis C virus (HCV)-infected diseases in order to reduce the mortality rates. Herein, we investigated whether serum 3β-hydroxysterol Δ24-reductase antibody (DHCR24 Ab) may serve as a prognostic marker for hepatitis C infection progression to hepatocellular carcinoma (HCC). METHODS Serum DHCR24 Abs from 395 HCV-positive patients, including 133 chronic hepatitis (CHC), 85 liver cirrhosis (LCC), and 177 HCC (HCC-C) patients; 232 hepatitis B virus (HBV)-positive patients, including 103 chronic hepatitis (CHB), 56 liver cirrhosis (LCB), and 73 HCC (HCC-B) patients; and 24 healthy controls, were measured using enzyme-linked immunosorbent assay. RESULTS The serum DHCR24 Ab levels were significantly higher in patients with CHC than in healthy controls, in LCC than in CHC, and in LCC than in HCC-C (P < 0.0001 for all). The concentration of serum DHCR24 Ab in HCC-B patients showed no significant difference compared to CHB and LCB patients (P = 0.1247). The DHCR24 Ab levels were significantly higher in early HCC-C than CHC or LCC patients and in late HCC-C compared to early HCC-C patients. The sensitivity of the DHCR24 Ab for HCC-C detection (70.6%) was higher than that of alpha-fetoprotein (AFP; 54.8%) and protein induced by vitamin K absence or antagonist-II (PIVKA-II; 42 · 5%). Moreover, DHCR24 was up-regulated in HCV-positive, but not HBV-positive tissues or HBV-negative, HCV-negative HCC specimens. CONCLUSIONS DHCR24 auto-antibody represents a potential noninvasive biomarker for HCV-related liver disease and may facilitate the diagnosis of PIVKA-II and AFP-negative HCC.
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Affiliation(s)
- Sayeh Ezzikouri
- Virology Unit, Viral Hepatitis Laboratory, Pasteur Institute of Morocco, Casablanca, Morocco
- Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
- Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
| | - Kiminori Kimura
- Division of Hepatology, The Tokyo Metropolitan Komagome Hospital, Tokyo, Japan
| | - Hajime Sunagozaka
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan
| | - Shuichi Kaneko
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan
| | - Kazuaki Inoue
- Department of Gastroenterology, Showa University, Fujigaoka Hospital, Kanagawa, Japan
| | | | - Tsunekazu Hishima
- Division of Pathology, The Tokyo Metropolitan Komagome Hospital, Tokyo, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan
| | - Kyoko Tsukiyama-Kohara
- Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
- Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
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Song K, Han C, Dash S, Balart LA, Wu T. MiR-122 in hepatitis B virus and hepatitis C virus dual infection. World J Hepatol 2015; 7:498-506. [PMID: 25848473 PMCID: PMC4381172 DOI: 10.4254/wjh.v7.i3.498] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/16/2014] [Revised: 09/06/2014] [Accepted: 12/17/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of chronic liver diseases and hepatocelluar carcinomas. Over the past few years, the liver-enriched microRNA-122 (miR-122) has been shown to differentially regulate viral replication of HBV and HCV. It is notable that the level of miR-122 is positively and negatively regulated by HCV and HBV, respectively. Consistent with the well-documented phenomenon that miR-122 promotes HCV accumulation, inhibition of miR-122 has been shown as an effective therapy for the treatment of HCV infection in both chimpanzees and humans. On the other hand, miR-122 is also known to block HBV replication, and HBV has recently been shown to inhibit miR-122 expression; such a reciprocal inhibition between miR-122 and HBV suggests an intriguing possibility that miR-122 replacement may represent a potential therapy for treatment of HBV infection. As HBV and HCV have shared transmission routes, dual infection is not an uncommon scenario, which is associated with more advanced liver disease than either HBV or HCV mono-infection. Thus, there is a clear need to further understand the interaction between HBV and HCV and to delineate the role of miR-122 in HBV/HCV dual infection in order to devise effective therapy. This review summarizes the current understanding of HBV/HCV dual infection, focusing on the pathobiological role and therapeutic potential of miR-122.
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Huang X, Hollinger FB. Occult hepatitis B virus infection and hepatocellular carcinoma: a systematic review. J Viral Hepat 2014; 21:153-62. [PMID: 24438677 DOI: 10.1111/jvh.12222] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2013] [Accepted: 12/08/2013] [Indexed: 12/13/2022]
Abstract
Occult hepatitis B (OHB) infection has been reported to play an important role in the development of hepatocellular carcinoma (HCC). In this systematic review, a significantly higher prevalence of OHB was observed in patients with HCC in the presence or absence of HCV infection when compared with control populations without HCC. Correspondingly, among adequately designed prospective studies, the cumulative probability of developing HCC was significantly greater among patients with OHB than among HBV DNA-negative patients in the presence or absence of HCV infection. Study design, inclusion criteria, treatment options, methodology and potential confounding variables were evaluated, and immunopathogenic mechanisms that could be involved in OHB as a risk factor in HCC were reviewed. From this analysis, we conclude that although OHB is an independent risk factor in HCC development in anti-HCV-negative patients, a synergistic or additive role in the occurrence of HCC in HCV-coinfected patients is more problematic due to the HCC risk attributable to HCV alone, especially in patients with advanced fibrosis and cirrhosis.
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Affiliation(s)
- X Huang
- Department of Blood Transfusion, The General Hospital of Jinan Military Command, Jinan, China
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24
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Nakagawa SI, Hirata Y, Kameyama T, Tokunaga Y, Nishito Y, Hirabayashi K, Yano J, Ochiya T, Tateno C, Tanaka Y, Mizokami M, Tsukiyama-Kohara K, Inoue K, Yoshiba M, Takaoka A, Kohara M. Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection. PLoS One 2013; 8:e59611. [PMID: 23555725 PMCID: PMC3610702 DOI: 10.1371/journal.pone.0059611] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2012] [Accepted: 02/15/2013] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND & AIMS The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). METHODS This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. RESULTS Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. CONCLUSIONS These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.
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Affiliation(s)
- Shin-ichiro Nakagawa
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan
- Discovery Research Laboratories, Nippon Shinyaku Co., Ltd., Tsukuba, Ibaraki, Japan
| | - Yuichi Hirata
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan
- Division of Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, Kanagawa, Japan
| | - Takeshi Kameyama
- Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan
| | - Yuko Tokunaga
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan
| | - Yasumasa Nishito
- Center for Microarray Analysis, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan
| | - Kazuko Hirabayashi
- Discovery Research Laboratories, Nippon Shinyaku Co., Ltd., Tsukuba, Ibaraki, Japan
| | - Junichi Yano
- Discovery Research Laboratories, Nippon Shinyaku Co., Ltd., Tsukuba, Ibaraki, Japan
| | - Takahiro Ochiya
- Division of Molecular and Cellular Medicine, Japanese National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan
| | - Chise Tateno
- PhoenixBio Co., Ltd., Higashi-Hiroshima, Hiroshima, Japan
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Aichi, Japan
| | - Masashi Mizokami
- Research Center for Hepatitis and Immunology, International Medical Center of Japan Konodai Hospital, Ichikawa, Chiba, Japan
| | - Kyoko Tsukiyama-Kohara
- Transboundary Animal Diseases Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
| | - Kazuaki Inoue
- Division of Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, Kanagawa, Japan
| | - Makoto Yoshiba
- Division of Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, Kanagawa, Japan
| | - Akinori Takaoka
- Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan
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Covolo L, Pollicino T, Raimondo G, Donato F. Occult hepatitis B virus and the risk for chronic liver disease: a meta-analysis. Dig Liver Dis 2013; 45:238-44. [PMID: 23146778 DOI: 10.1016/j.dld.2012.09.021] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2012] [Revised: 09/12/2012] [Accepted: 09/30/2012] [Indexed: 02/08/2023]
Abstract
BACKGROUND The role of occult hepatitis B virus infection as a cause of liver disease is still debated although many studies found a higher prevalence of this condition in subjects than those without liver disease compared. A recent meta-analysis showed an increased risk of hepatocellular carcinoma for occult hepatitis B virus infection. AIMS We carried out a meta-analysis of observational studies to summarize the existing evidence and assess quantitatively the association between occult hepatitis B virus infection and chronic liver disease. METHODS We searched the available literature on this issue published up to May 2012 using PubMed and EMBASE. All articles that provided enough information to estimate the chronic liver disease risk associated with occult hepatitis B virus infection were selected. Fourteen studies were retrieved. RESULTS A total of 1503 subjects with (cases) and 2052 without chronic liver disease (controls) were included. The summary odds ratio for chronic liver disease from all studies was 8.9 (95% confidence interval: 4.1-19.5). The meta-analysis restricted to 7 studies with more precise effect estimate (wt%>8%) provided a lower odds ratio estimate (odds ratio=3.9; 95% confidence interval: 1.7-9.0). CONCLUSIONS These findings suggest a relevant association between occult hepatitis B virus infection and chronic liver disease, confirming the hypothesis that hepatitis B virus may play a pathogenic role even in the "occult" status.
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Affiliation(s)
- Loredana Covolo
- Institute of Hygiene, Epidemiology and Public Health, University of Brescia, Brescia, Italy.
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Demonstration of hepatitis C virus RNA with in situ hybridization employing a locked nucleic Acid probe in humanized liver of infected chimeric mice and in needle-biopsied human liver. Int J Hepatol 2013; 2013:249535. [PMID: 23853723 PMCID: PMC3703335 DOI: 10.1155/2013/249535] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2012] [Revised: 05/21/2013] [Accepted: 05/23/2013] [Indexed: 11/18/2022] Open
Abstract
Background. In situ hybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.
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Sato Y, Tsurumi T. Genome guardian p53 and viral infections. Rev Med Virol 2012; 23:213-20. [PMID: 23255396 DOI: 10.1002/rmv.1738] [Citation(s) in RCA: 72] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2012] [Revised: 11/18/2012] [Accepted: 11/20/2012] [Indexed: 01/07/2023]
Abstract
Because virus infections elicit various cellular responses that inhibit viral replication and growth, viruses must intervene to attenuate antiviral measures in order to thrive. The genome guardian p53 plays a central part not only in DNA damage responses, inducing cell cycle arrest or apoptosis, but also in the innate host immune control of viral infections by orchestrating diverse signaling pathways originating from many different cellular receptors and sensors. Many viruses have acquired sophisticated mechanisms to regulate p53 functions by deploying subversive proteins and modulating its post-transcriptional status. In this review, we overview the mechanisms by which DNA and RNA viruses manage p53 signaling in favor of their continued survival.
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Affiliation(s)
- Yoshitaka Sato
- Division of Virology, Aichi Cancer Center Research Institute, Nagoya, 464-8681, Japan
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Shi Y, Wu YH, Wu W, Zhang WJ, Yang J, Chen Z. Association between occult hepatitis B infection and the risk of hepatocellular carcinoma: a meta-analysis. Liver Int 2012; 32:231-40. [PMID: 21745272 DOI: 10.1111/j.1478-3231.2011.02481.x] [Citation(s) in RCA: 109] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
BACKGROUND The association between occult hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) remains controversial. AIMS We conducted a meta-analysis of prospective studies and retrospective studies to examine whether occult HBV infection increases the risk of HCC. METHODS Two independent reviewers searched databases for eligible studies published in English or Chinese dated from 1966 to 6 April 2010. The odds ratios or the relative risks (RRs) of each study were considered respectively. RESULTS We identified 16 eligible studies. A significantly increased risk of HCC was found in subjects with occult HBV infection in comparison with non-infected controls in both retrospective [OR(unadjusted) =6.08, 95% confidence interval (CI)=3.45-10.72] and prospective studies (RR(adjusted) =2.86, 95% CI=1.59-4.13), and occult HBV increased the risk for HCC in both hepatitis C virus (HCV)-infected populations (summary RR=2.83, 95% CI=1.56-4.10) and in non-infected populations (OR(unadjusted) =10.65, 95% CI=5.94-19.08). A higher prevalence of occult HBV was observed in individuals who were positive for anti-HBs and anti-HBc (OR(unadjusted) =1.81, 95% CI=1.06, 3.09). CONCLUSION Our findings suggest that occult HBV infection was associated with an increased risk of HCC. Occult HBV may serve as a cofactor in the development of HCV-related HCC, and it may also play a direct role in promoting Non-B and Non-C HCC growth. Suggestive evidence indicates that individuals with a concomitant presence of anti-HBs and anti-HBc had an increased risk of occult HBV infection. However, further studies are needed to clarify these observations.
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Affiliation(s)
- Yu Shi
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
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Inoue K. Reply to “Significance of a Single-Nucleotide Primer Mismatch in Hepatitis B Virus Real-Time PCR Diagnostic Assays”
†. J Clin Microbiol 2011; 49:4420-4420. [PMCID: PMC3232993 DOI: 10.1128/jcm.05516-11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Affiliation(s)
- Kazuaki Inoue
- Department of MedicineDivision of GastroenterologyShowa University Fujigaoka Hospital1-30 Fujigaoka, Aoba-kuYokohama 227-8501, Japan
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Nuriya H, Inoue K, Tanaka T, Hayashi Y, Hishima T, Funata N, Kaji K, Hayashi S, Kaneko S, Kohara M. Detection of hepatitis B and C viruses in almost all hepatocytes by modified PCR-based in situ hybridization. J Clin Microbiol 2010; 48:3843-3851. [PMID: 20739486 PMCID: PMC3020860 DOI: 10.1128/jcm.00415-10] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2010] [Revised: 04/22/2010] [Accepted: 08/16/2010] [Indexed: 02/07/2023] Open
Abstract
Although PCR-based in situ hybridization (PCR-ISH) can be used to determine the distribution and localization of pathogens in tissues, this approach is hampered by its low specificity. Therefore, we used a highly specific and sensitive PCR-ISH method to reveal the lobular distribution and intracellular localization of hepatitis B virus (HBV) and HCV in chronic liver disease and to clarify the state of persistent HBV and HCV infection in the liver. HBV genomic DNA was detected in almost all hepatocytes, whereas HBV RNA or protein was differentially distributed only in a subset of the HBV DNA-positive region. Further, HCV genomic RNA was detected in almost all hepatocytes and was localized to the cytoplasm. HCV RNA was also detected in the epithelium of the large bile duct but not in endothelial cells, portal tracts, or sinusoidal lymphocytes. In patients with HBV and HCV coinfection, HCV RNA was localized to the noncancerous tissue, whereas HBV DNA was found only in the cancerous tissue. Using this novel PCR-ISH method, we could visualize the staining pattern of HBV and HCV in liver sections, and we obtained results consistent with those of real-time detection (RTD)-PCR analysis. In conclusion, almost all hepatocytes are infected with HBV or HCV in chronic liver disease; this finding implies that the viruses spreads throughout the liver in the chronic stage.
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Affiliation(s)
- Hideko Nuriya
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Kazuaki Inoue
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Takeshi Tanaka
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Yukiko Hayashi
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Tsunekazu Hishima
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Nobuaki Funata
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Kyosuke Kaji
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Seishu Hayashi
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Shuichi Kaneko
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan, Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30 Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan, Liver Unit, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Pathology, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Ishikawa 920-8641, Japan
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31
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Nishimura T, Kohara M, Izumi K, Kasama Y, Hirata Y, Huang Y, Shuda M, Mukaidani C, Takano T, Tokunaga Y, Nuriya H, Satoh M, Saito M, Kai C, Tsukiyama-Kohara K. Hepatitis C virus impairs p53 via persistent overexpression of 3beta-hydroxysterol Delta24-reductase. J Biol Chem 2009; 284:36442-36452. [PMID: 19861417 DOI: 10.1074/jbc.m109.043232] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3beta-hydroxysterol Delta24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.
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Affiliation(s)
- Tomohiro Nishimura
- Department of Experimental Phylaxiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Kumamoto 860-8556, Japan; Chemo-Sero-Therapeutic Research Institute, Kikuchi Research Center, Kyokushi, Kikuchi, Kumamoto 869-1298, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 1-6 Kamikitazawa 2-chome, Setagaya-ku, Tokyo 156-8506, Japan
| | - Kosuke Izumi
- Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
| | - Yuri Kasama
- Department of Experimental Phylaxiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Kumamoto 860-8556, Japan
| | - Yuichi Hirata
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 1-6 Kamikitazawa 2-chome, Setagaya-ku, Tokyo 156-8506, Japan
| | - Ying Huang
- Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
| | - Masahiro Shuda
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 1-6 Kamikitazawa 2-chome, Setagaya-ku, Tokyo 156-8506, Japan
| | - Chise Mukaidani
- Study Service Department, PhoenixBio Company, Ltd., 3-4-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan
| | - Takashi Takano
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 1-6 Kamikitazawa 2-chome, Setagaya-ku, Tokyo 156-8506, Japan
| | - Yuko Tokunaga
- Department of Experimental Phylaxiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Kumamoto 860-8556, Japan
| | - Hideko Nuriya
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 1-6 Kamikitazawa 2-chome, Setagaya-ku, Tokyo 156-8506, Japan
| | - Masaaki Satoh
- Department of Experimental Phylaxiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Kumamoto 860-8556, Japan
| | - Makoto Saito
- Department of Experimental Phylaxiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Kumamoto 860-8556, Japan
| | - Chieko Kai
- Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
| | - Kyoko Tsukiyama-Kohara
- Department of Experimental Phylaxiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Kumamoto 860-8556, Japan.
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32
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Saitou K, Mizumoto K, Nishimura T, Kai C, Tsukiyama-Kohara K. Hepatitis C virus-core protein facilitates the degradation of Ku70 and reduces DNA-PK activity in hepatocytes. Virus Res 2009; 144:266-71. [DOI: 10.1016/j.virusres.2009.05.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2009] [Revised: 05/14/2009] [Accepted: 05/17/2009] [Indexed: 10/20/2022]
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33
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Nitahara-Kasahara Y, Fukasawa M, Shinkai-Ouchi F, Sato S, Suzuki T, Murakami K, Wakita T, Hanada K, Miyamura T, Nishijima M. Cellular vimentin content regulates the protein level of hepatitis C virus core protein and the hepatitis C virus production in cultured cells. Virology 2008; 383:319-27. [PMID: 19013628 DOI: 10.1016/j.virol.2008.10.009] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2008] [Revised: 09/03/2008] [Accepted: 10/06/2008] [Indexed: 01/17/2023]
Abstract
Hepatitis C virus (HCV) core protein is essential for virus particle formation. Using HCV core-expressing and non-expressing Huh7 cell lines, Uc39-6 and Uc321, respectively, we performed comparative proteomic studies of proteins in the 0.5% Triton X-100-insoluble fractions of cells, and found that core-expressing Uc39-6 cells had much lower vimentin content than Uc321 cells. In experiments using vimentin-overexpressing and vimentin-knocked-down cells, we demonstrated that core protein levels were affected by cellular vimentin content. When vimentin expression was knocked-down, there was no difference in mRNA level of core protein; but proteasome-dependent degradation of the core protein was strongly reduced. These findings suggest that the turnover rate of core protein is regulated by cellular vimentin content. HCV production was also affected by cellular vimentin content. Our findings together suggest that modulation of hepatic vimentin expression might enable the control of HCV production.
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Affiliation(s)
- Yuko Nitahara-Kasahara
- Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, Japan
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34
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Adachi S, Shibuya A, Miura Y, Takeuchi A, Nakazawa T, Saigenji K. Impact of occult hepatitis B virus infection and prior hepatitis B virus infection on development of hepatocellular carcinoma in patients with liver cirrhosis due to hepatitis C virus. Scand J Gastroenterol 2008; 43:849-56. [PMID: 18584524 DOI: 10.1080/00365520801935459] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
OBJECTIVE Although hepatitis B virus (HBV) DNA can be detected in liver or sera of patients without serum hepatitis B surface antigen (HBsAg), its clinical relevance in hepatocarcinogenesis remains controversial. This observational cohort study was conducted to clarify the risk factors, including the presence of serum HBV DNA and hepatitis B core antibody (anti-HBc), for hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV)-related liver cirrhosis (LC). MATERIAL AND METHODS The study comprised 123 patients with LC due to HCV, and negative for HBsAg. The risk factors for HCC development were analyzed by univariate and multivariate analysis. Serum samples were assayed for HBV DNA using real-time polymerase chain reaction. RESULTS Serum HBV DNA was detectable in 14 patients (11.4%) and serum anti-HBc in 96 (78.0%). During the follow-up period (mean 53.3 months), 80 patients (65.0%) developed HCC. The cumulative HCC development rate was significantly higher in the anti-HBc-positive group than in the anti-HBc-negative group (p=0.0039), but did not differ between the serum HBV DNA-positive and -negative groups (p=0.8570). The multivariate analysis indicated that male gender, alpha-fetoprotein (AFP) 20 ng/ml or greater, average serum alanine aminotransferase (ALAT) 80 IU/l or greater and the presence of anti-HBc were independent risk factors for development of HCC (p=0.038, p=0.013, p=0.020 and p=0.001, respectively). CONCLUSIONS Serum anti-HBc, which indicates a previous HBV infection, has clinical significance in hepatocarcinogenesis in patients with HCV-related LC, but serum HBV DNA does not. Therefore, anti-HBc in serum is a significant predictor for HCC.
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Affiliation(s)
- Shigeru Adachi
- Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan.
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35
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De Mitri MS, Cassini R, Morsica G, Bagaglio S, Andreone P, Loggi E, Muratori P, Bernardi M. Virological analysis, genotypes and mutational patterns of the HBV precore/core gene in HBV/HCV-related hepatocellular carcinoma. J Viral Hepat 2006; 13:574-581. [PMID: 16907843 DOI: 10.1111/j.1365-2893.2006.00726.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
We investigated the replicative profile of hepatitis B (HBV) and hepatitis C (HCV) viruses and the mutational pattern of the HBV precore/core (pre-C/C) domain in hepatocellular carcinoma (HCC). Thirty-eight consecutive patients with HCC were included in the study - 18 of them with HBV/HCV co-infection and 20 with HBV single infection. Twenty-three additional patients with co-infection, without HCC were recruited as the control group. Replication activity was evaluated by detecting and quantitating both HBV and HCV genomes. The HBV pre-C/C region, encompassing the pregenome encapsidation signal involved in viral replication, was analysed by direct sequencing. HBV viraemia levels were significantly lower (P = 0.04) in patients with co-infection in comparison with single-infected HCC, whereas two different HBV viraemia profiles were detected in co-infection with or without circulating HCV. HBV genotype D was prevalent in the three groups and HCV genotype 1b was found to be the infecting strain in all patients. Lower variability in the pre-C/C region was found in co-infection in comparison with HBV single infection (P = 0.0004). A synonymous T1936C mutation was found in all co-infected HCC cases not related to the presence or absence of circulating HCV, and a hypermutated pre-C strain, characterized by the same mutational pattern, was identified in three HCC cases. The mutational pattern of the pre-C/C region was closely related to HBV replication efficiency, and specific HBV mutations selectively associated with HCV co-infection could be linked with accelerated HBV/HCV-related disease progression.
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Affiliation(s)
- M S De Mitri
- Department of Internal Medicine, Cardioangiology, Hepatology, University of Bologna, Bologna, Italy.
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36
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Keasler VV, Lerat H, Madden CR, Finegold MJ, McGarvey MJ, Mohammed EMA, Forbes SJ, Lemon SM, Hadsell DL, Grona SJ, Hollinger FB, Slagle BL. Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein. Virology 2006; 347:466-75. [PMID: 16427673 DOI: 10.1016/j.virol.2005.11.050] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2005] [Accepted: 11/30/2005] [Indexed: 12/13/2022]
Abstract
Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.
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Affiliation(s)
- Victor V Keasler
- Department of Molecular Virology and Microbiology, Baylor College of Medicine (BCM-385), One Baylor Plaza, Houston, TX 77030-3411, USA
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37
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Abstract
Although hepatitis B (HBV) and C viruses (HCV) are, individually, major causes of hepatocellular carcinoma, the interaction, if any, between the carcinogenic effects of the two viruses is uncertain. Equal numbers of published studies have reported no risk interaction or a synergistic risk interaction. These conflicting results are explained by the rarity of concurrent infection with HBV and HCV in individuals without clinically evident liver disease, which severely limits the ability to accurately estimate the hepatocarcinogenic risk of dual infection compared with that of either infection alone. In an attempt to circumvent this difficulty, two meta-analyses have been performed, one based on studies published from a number of countries and the other on studies confined to Chinese patients. Both analyses concluded that a synergistic carcinogenic interaction existed between the two viruses and that the increased risk was super-additive but not multiplicative. If confirmed, this risk interaction will occur against a background of negative confounding effects on viral replication between HBV and HCV, which may be reciprocal. The mechanisms responsible for the carcinogenic interaction between the viruses are unknown. One possibility is that the increased incidence of cirrhosis with concurrent HBV and HCV infections acts as an even more potent tumour promoter than occurs with either virus alone. Synergism between the direct hepatocarcinogenic effects of the two viruses is another possible mechanism, but proof will have to await a fuller understanding of the pathogenetic mechanisms involved with the individual viruses.
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Affiliation(s)
- M C Kew
- MRC/University Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa.
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38
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Momosaki S, Umemura T, Scudamore CH, Kojiro M, Alter HJ, Tabor E. SEN virus infection in patients with hepatocellular carcinoma. J Viral Hepat 2005; 12:435-8. [PMID: 15985016 DOI: 10.1111/j.1365-2893.2005.00618.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Although most cases of hepatocellular carcinoma (HCC) are associated with either the hepatitis B or C viruses (HBV, HCV), about 10-20% of HCCs occur in patients with chronic hepatitis that is aetiologically undefined. The aim of the present study was to determine the prevalence of the transfusion-transmitted SEN virus (SEN-V) in patients with HCC, including those patients who do not otherwise appear to be infected with HBV or HCV. Fragments of SEN-V subtypes D and H were amplified separately by PCR from the sera of 50 patients with HCC (31 from Canada and 19 from Japan) as well as from HCC and adjacent nontumourous liver tissues from eight of the Canadian patients. SEN-V DNA was found in the serum of 10 of 31 (32%) Canadian patients and eight of 19 (42%) Japanese patients [overall, 18 of 50 (36%) HCC patients]. SEN-V DNA was detected in the serum of 10 of 23 (43%) HCC patients with antibody to HCV (anti-HCV), six of 11 (55%) with hepatitis B surface antigen (HBsAg), and two of 16 (12%) without detectable anti-HCV or HBsAg. Twenty-three HCC patients in this study had 'silent HBV,' characterized by the detection of HBV DNA in the absence of HBsAg; eight of these (35%) also had SEN-V infections. SEN-V DNA was detected in HCC patients most typically in those with coexistent HBV or HCV infection. SEN-V was found in only one of seven HCC patients without HBV (without HBsAg or HBV DNA) or HCV and thus does not appear to be an important cause of 'cryptogenic' HCC.
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Affiliation(s)
- S Momosaki
- Division of Emerging and Transfusion Transmitted Diseases, Food and Drug Administration, Bethesda, MD, USA
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39
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Sagnelli E, Pasquale G, Coppola N, Marrocco C, Scarano F, Imparato M, Sagnelli C, Scolastico C, Piccinino F. Liver histology in patients with HBsAg negative anti-HBc and anti-HCV positive chronic hepatitis. J Med Virol 2005; 75:222-226. [PMID: 15602732 DOI: 10.1002/jmv.20260] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The liver histology of 68 consecutive anti-HCV/HCV-RNA positive chronic hepatitis patients who were HBsAg/anti-HBs negative, anti-HBc positive (Case bC group) was compared with that of 68 anti-HCV/HCV-RNA positive chronic hepatitis patients who were HBsAg/anti-HBc negative (control C group). The patients were pair-matched by age (+/-5 years), sex, and risk factors for the acquisition of parenteral infection. Case bC group showed a significantly higher mean fibrosis score (2.3 +/- 1.1) than control C group (1.5 +/- 1.1, P <0.001) and more histological evidence of cirrhosis (22% vs. 7.3%, P <0.05). In addition, the patients in Case bC group showed more severe inflammation of the portal tracts (3.5 +/- 0.8 vs. 3.0 +/- 1.1, P <0.005) and there was a higher prevalence of patients with rhomboid-shaped hepatocytes (26.4% vs. 2.7%, P <0.005), acidophilic bodies (33.8% vs. 1.4%, P <0.0001), sinusoidal inflammation (29.4% vs. 10.3%, P <0.01), lymphoid follicles in the portal tracts (72% vs. 44.1%, P <0.05), Kupffer cell proliferation (29.4% vs. 11.8%, P <0.05), bile duct damage (44.1% vs. 10.3%, P <0.0001), and ductular proliferation (30.9% vs. 2.7%, P <0.001) than in control C group. No difference in these histological features was observed between HBV-DNA negative and positive patients in Case bC group. The data suggest that anti-HBc positive patients with HCV chronic infection have a significantly higher degree of liver fibrosis, and that hepatocellular apoptosis, bile duct damage, and ductular proliferation correlate with the presence of this antibody in the serum.
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Affiliation(s)
- Evangelista Sagnelli
- Department of Public Medicine, Division of Infectious Diseases, Second University of Naples, San Sebastiano Hospital, Caserta, Italy.
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