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Bian Y, Zhao S, Zhu S, Zeng J, Li T, Fu Y, Wang Y, Zheng X, Zhang L, Wang W, Yang B, Zhou Y, Allain JP, Li C. Significance of monoclonal antibodies against the conserved epitopes within non-structural protein 3 helicase of hepatitis C virus. PLoS One 2013; 8:e70214. [PMID: 23894620 PMCID: PMC3722154 DOI: 10.1371/journal.pone.0070214] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2013] [Accepted: 06/18/2013] [Indexed: 12/16/2022] Open
Abstract
Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope 1231PTGSGKSTK1239 (EP05) or core motif 1373IPFYGKAI1380 (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.
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Affiliation(s)
- Yixin Bian
- Department of Transfusion Medicine, Southern Medical University, Guangzhou, China
| | - Shuoxian Zhao
- Department of Transfusion Medicine, Southern Medical University, Guangzhou, China
| | - Shaomei Zhu
- Department of Transfusion Medicine, Southern Medical University, Guangzhou, China
| | | | - Tingting Li
- Department of Transfusion Medicine, Southern Medical University, Guangzhou, China
| | | | - Yuanzhan Wang
- Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Xin Zheng
- Shenzhen Blood Center, Shenzhen, China
| | - Ling Zhang
- Department of Transfusion Medicine, Southern Medical University, Guangzhou, China
| | - Wenjing Wang
- Department of Transfusion Medicine, Southern Medical University, Guangzhou, China
| | | | - Yuanping Zhou
- Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Jean-Pierre Allain
- Department of Transfusion Medicine, Southern Medical University, Guangzhou, China
- Department of Hematology, University of Cambridge, Cambridge, United Kingdom
| | - Chengyao Li
- Department of Transfusion Medicine, Southern Medical University, Guangzhou, China
- * E-mail:
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Hepatitis C virus-induced mitochondrial dysfunctions. Viruses 2013; 5:954-80. [PMID: 23518579 PMCID: PMC3705306 DOI: 10.3390/v5030954] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2013] [Revised: 03/15/2013] [Accepted: 03/20/2013] [Indexed: 12/15/2022] Open
Abstract
Chronic hepatitis C is characterized by metabolic disorders and a microenvironment in the liver dominated by oxidative stress, inflammation and regeneration processes that lead in the long term to hepatocellular carcinoma. Many lines of evidence suggest that mitochondrial dysfunctions, including modification of metabolic fluxes, generation and elimination of oxidative stress, Ca2+ signaling and apoptosis, play a central role in these processes. However, how these dysfunctions are induced by the virus and whether they play a role in disease progression and neoplastic transformation remains to be determined. Most in vitro studies performed so far have shown that several of the hepatitis C virus (HCV) proteins localize to mitochondria, but the consequences of these interactions on mitochondrial functions remain contradictory, probably due to the use of artificial expression and replication systems. In vivo studies are hampered by the fact that innate and adaptive immune responses will overlay mitochondrial dysfunctions induced directly in the hepatocyte by HCV. Thus, the molecular aspects underlying HCV-induced mitochondrial dysfunctions and their roles in viral replication and the associated pathology need yet to be confirmed in the context of productively replicating virus and physiologically relevant in vitro and in vivo model systems.
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Rahbin N, Frelin L, Aleman S, Hultcrantz R, Sällberg M, Brenndörfer ED. Non-structural 3 protein expression is associated with T cell protein tyrosine phosphatase and viral RNA levels in chronic hepatitis C patients. Biochem Biophys Res Commun 2013; 433:31-5. [PMID: 23454379 DOI: 10.1016/j.bbrc.2013.02.075] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2013] [Accepted: 02/14/2013] [Indexed: 12/20/2022]
Abstract
The hepatitis C virus (HCV) non-structural 3 (NS3) protein plays key roles in both the viral life cycle and in the modulation of intrahepatic signaling and immunity. We recently showed that NS3 cleaves the T cell protein tyrosine phosphatase (TCPTP). To better understand the inactivation of TCPTP in HCV-infected humans, we investigated whether there is an association between TCPTP cleavage, NS3 protein levels and clinical parameters in hepatitis C patients. Liver biopsies were obtained from 69 HCV RNA positive patients with confirmed chronic HCV infection and 16 control patients. Hepatic NS3 and TCPTP protein levels were determined and correlated to viral load or clinical parameters for the severity of liver disease. We found a positive correlation between the viral load and the intrahepatic NS3 protein levels in patients infected with HCV. HCV-infected patients had significantly lower intrahepatic TCPTP levels than non-infected control patients. In HCV-infected patients both intrahepatic NS3 expression and the viral load were inversely correlated with the intrahepatic TCPTP protein levels. Detection of NS3 did not associate with any other clinical parameters such as liver damage, the grade of liver inflammation or fibrosis stage. This is the first study reporting a detailed analysis of HCV NS3 and TCPTP protein levels in the liver. It demonstrates a clear link between HCV viral load, NS3 expression in the liver and intrahepatic TCPTP levels. Thus, the association between TCPTP cleavage and viral replication may have important consequences for the HCV life cycle and HCV-induced liver diseases.
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Affiliation(s)
- Nogol Rahbin
- Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, 14186 Stockholm, Sweden
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Clinicopathological features of hepatitis C virus disease after living donor liver transplantation: relationship with in situ hybridisation data. Pathology 2011; 43:156-60. [PMID: 21233678 DOI: 10.1097/pat.0b013e32834317ed] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
AIMS Recurrent hepatitis is a significant complication after liver transplantation for hepatitis C virus (HCV) disease. To evaluate responsiveness to treatment of HCV disease after liver transplantation, in situ hybridisation (ISH) was employed. METHODS Sense and anti-sense probes for HCV were synthesised, and ISH studies were performed on 19 liver biopsy specimens from 19 recipients who had undergone living donor liver transplantation for HCV disease. ISH positive cells and total hepatocytes were counted, and the percentage of positive cells was calculated. Other clinical findings were compared retrospectively with the ISH results. RESULTS The subjects were divided into three groups: recurrent HCV hepatitis (RHC, n = 11), acute cellular rejection (ACR, n = 5), and recurrent HCV hepatitis with ACR (MIX, n = 3). The percentage of ISH positive cells was almost the same degree (10-20%) in the three groups. The RHC group was subdivided into two sets of patients in whom serum HCV titres decreased (group D, n = 7) or did not decrease (group ND, n = 3) after 1 month of IFN therapy. The percentage of ISH positive cells in group D was significantly lower than that in group ND (p < 0.05) CONCLUSIONS ISH for the recipients with HCV may be useful for predicting the response to interferon therapy.
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Naas T, Ghorbani M, Soare C, Scherling N, Muller R, Ghorbani P, Diaz-Mitoma F. Adoptive transfer of splenocytes to study cell-mediated immune responses in hepatitis C infection using HCV transgenic mice. COMPARATIVE HEPATOLOGY 2010; 9:7. [PMID: 20727132 PMCID: PMC2936292 DOI: 10.1186/1476-5926-9-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/30/2009] [Accepted: 08/20/2010] [Indexed: 01/12/2023]
Abstract
Background Hepatitis C virus (HCV) is a major cause of chronic hepatitis and a health problem affecting over 170 million people around the world. We previously studied transgenic mice that express HCV Core, Envelope 1 and Envelope 2 proteins predominantly in the liver, resulting in steatosis, liver and lymphoid tumors, and hepatocellular carcinoma. Herein, the immune-mediated cell response to hepatitis C antigens was evaluated by adoptive transfers of carboxyfluorescein succinimidyl ester (CFSE) labelled splenocytes from HCV immunized mice into HCV transgenic mice. Results In comparison to non-transgenic mice, there was a significant decrease in the percentage of CFSE-labeled CD4+ and CD8+ T cells in transgenic mouse peripheral blood receiving adoptive transfers from immunized donors. Moreover, the percentage of CFSE-labeled CD4+ and CD8+ T cells were significantly higher in the spleen of transgenic and non-transgenic mice when they received splenocytes from non-immunized than from immunized mice. On the other hand, the percentages of CD4+ and CD8+ T cells in the non-transgenic recipient mouse lymph nodes were significantly higher than the transgenic mice when they received the adoptive transfer from immunized donors. Interestingly, livers of transgenic mice that received transfers from immunized mice had a significantly higher percentage of CFSE labeled T cells than livers of non-transgenic mice receiving non-immunized transfers. Conclusions These results suggest that the T cells from HCV immunized mice recognize the HCV proteins in the liver of the transgenic mouse model and homed to the HCV antigen expression sites. We propose using this model system to study active T cell responses in HCV infection.
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Affiliation(s)
- Turaya Naas
- Infectious Disease and Vaccine Research Centre, Children's Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada.
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Hiramatsu N, Oze T, Yakushijin T, Inoue Y, Igura T, Mochizuki K, Imanaka K, Kaneko A, Oshita M, Hagiwara H, Mita E, Nagase T, Ito T, Inui Y, Hijioka T, Katayama K, Tamura S, Yoshihara H, Imai Y, Kato M, Yoshida Y, Tatsumi T, Ohkawa K, Kiso S, Kanto T, Kasahara A, Takehara T, Hayashi N. Ribavirin dose reduction raises relapse rate dose-dependently in genotype 1 patients with hepatitis C responding to pegylated interferon alpha-2b plus ribavirin. J Viral Hepat 2009; 16:586-94. [PMID: 19552664 DOI: 10.1111/j.1365-2893.2009.01106.x] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
The impact of ribavirin exposure on virologic relapse remains controversial in combination therapy with pegylated interferon (Peg-IFN) and ribavirin for patients with chronic hepatitis C (CH-C) genotype 1. The present study was conducted to investigate this. Nine hundred and eighty-four patients with CH-C genotype 1 were enrolled. The drug exposure of each medication was calculated by averaging the dose actually taken. For the 472 patients who were HCV RNA negative at week 24 and week 48, multivariate logistic regression analysis showed that the degree of fibrosis (P = 0.002), the timing of HCV RNA negativiation (P < 0.001) and the mean doses of ribavirin (P < 0.001) were significantly associated with relapse, but those of Peg-IFN were not. Stepwise reduction of the ribavirin dose was associated with a stepwise increase in relapse rate from 11% to 60%. For patients with complete early virologic response (c-EVR) defined as HCV RNA negativity at week 12, only 4% relapse was found in patients given > or = 12 mg/kg/day of ribavirin and ribavirin exposure affected the relapse even after treatment week 12, while Peg-IFN could be reduced to 0.6 microg/kg/week after week 12 without the increase of relapse rate. Ribavirin showed dose-dependent correlation with the relapse. Maintaining as high a ribavirin dose as possible (> or = 12 mg/kg/day) during the full treatment period can lead to suppression of the relapse in HCV genotype 1 patients responding to Peg-IFN alpha-2b plus ribavirin, especially in c-EVR patients.
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Affiliation(s)
- N Hiramatsu
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
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Wang Y, Mao S, Li B, Tan P, Feng D, Wen J. Treatment of hepatitis C virus core-positive hepatocytes with the transfer of recombinant caspase-3 using the 2',5'-oligoadenylate synthetase gene promoter. Acta Biochim Biophys Sin (Shanghai) 2009; 41:554-60. [PMID: 19578719 DOI: 10.1093/abbs/gmp044] [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: 11/13/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a leading cause of liver-related morbidity and mortality throughout the world. There is no vaccine available and current therapy is only partially effective. Since HCV infects only a minority of hepatocytes, we hypothesized that induction of apoptosis might be a promising approach for the treatment of hepatitis C. In the present study, recombinant caspase-3 gene (re-caspase-3) was used because it has the ability to induce apoptosis that is independent of the initiator caspases. An HCV-specific promoter is required to regulate the cytotoxic caspase-3 expression in HCV-infected cells. It has been reported that HCV core protein can specifically activate the 2',5'-oligoadenylate synthetase (OAS) gene promoter in human hepatocytes. Therefore, we constructed an expression vector consisting of the re-caspase-3 under the OAS gene promoter (pGL3-OAS-re-caspase-3) and then investigated its effect on HCV core-positive liver cells. It was found that the pGL3-OAS-re-caspase-3 construct induced apoptosis in HCV core-positive liver cells, but not in normal liver cells. These results strongly suggested that the transfer of the re-caspase-3 gene under the OAS promoter was a novel targeting approach for the treatment of HCV infection.
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Affiliation(s)
- Ying Wang
- Department of Pathology, Basic Medical College, Central South University, Changsha 410078, China
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Sadamori H, Yagi T, Iwagaki H, Matsuda H, Shinoura S, Umeda Y, Ohara N, Yanai H, Ogino T, Tanaka N. Immunohistochemical staining of liver grafts with a monoclonal antibody against HCV-Envelope 2 for recurrent hepatitis C after living donor liver transplantation. J Gastroenterol Hepatol 2009; 24:574-80. [PMID: 19368635 DOI: 10.1111/j.1440-1746.2008.05638.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
AIM We evaluated the expression of hepatitis C virus (HCV) antigen on liver grafts by immunohistochemical staining (IHS) using IG222 monoclonal antibody (mAb) against HCV-envelope 2 (E2). METHODS The study material was 84 liver biopsy specimens obtained from 28 patients who underwent living donor liver transplantation (LDLT) for HCV infection. The biopsy samples were examined histopathologically, and by IHS using IG222 mAb against HCV-E2. Serum HCV-RNA level was measured in all patients. The IHS grades were compared among the three groups classified according to the time elapsed from LDLT (at 1-30, 31-179 and > or =180 days post-LDLT) and among four post-transplant conditions, including acute cellular rejection (ACR). RESULTS Immunoreactivity to IG222 was detected in 78.6% of the specimens obtained during the first month after LDLT, and there were no significant differences on the IHS grades between the three groups classified according to the time elapsed from LDLT. The IHS grades were significantly stronger in definite recurrent HCV (n = 12) and probable recurrent HCV (n = 7) than in definite ACR (n = 7) and other complications (n = 8). There were no significant differences in serum HCV-RNA levels among the four post-transplant conditions. There was no significant correlation between the IHS grades using IG222 mAb and serum HCV-RNA levels when data of 84 liver biopsy specimens were analyzed. CONCLUSIONS Constant HCV-E2 expression was observed in liver biopsy specimens obtained 1 month or longer. The strong HCV-E2 expression on liver grafts were associated with recurrent hepatitis C after LDLT, but the serum HCV-RNA levels were not.
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Affiliation(s)
- Hiroshi Sadamori
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata, Okayama 700-8558, Japan.
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Li H, McMahon BJ, McArdle S, Bruden D, Sullivan DG, Shelton D, Deubner H, Gretch DR. Hepatitis C virus envelope glycoprotein co-evolutionary dynamics during chronic hepatitis C. Virology 2008; 375:580-91. [PMID: 18343477 PMCID: PMC2488153 DOI: 10.1016/j.virol.2008.02.012] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2007] [Revised: 01/16/2008] [Accepted: 02/12/2008] [Indexed: 12/20/2022]
Abstract
Hepatitis C virus (HCV) envelope glycoprotein co-evolution was studied in 14 genotype 1-infected and treatment-naive subjects, including 7 with mild and 7 with severe liver disease. Cassettes encoding the envelope 1 gene (E1) and hypervariable region (HVR1) of the envelope 2 gene were isolated at 38 different time points over 81 follow-up years. There were no significant differences in age, gender, alcohol use, or viral load between the mild and severe disease groups. Virus from subjects with severe disease had significantly slower evolution in HVR1, and significant divergent evolution of E1 quasispecies, characterized by a preponderance of synonymous mutations, compared to virus from subjects with mild disease. Phylogenetic comparisons indicated higher similarity between amino acid sequences of the E1 and HVR1 regions with mild disease versus severe disease (r=0.44 versus r=0.17, respectively; P=0.01). In summary, HCV envelope quasispecies co-evolution differs during mild versus severe disease.
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Affiliation(s)
- Hui Li
- Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington
| | - Brian J. McMahon
- Arctic Investigations Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Anchorage, Alaska
- Liver Disease and Hepatitis Program, Alaska Native Medical Center, Anchorage, Alaska
| | - Susan McArdle
- Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington
| | - Dana Bruden
- Arctic Investigations Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Anchorage, Alaska
| | - Daniel G. Sullivan
- Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington
| | - Dave Shelton
- Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington
| | - Heike Deubner
- Department of Pathology, University of Washington Medical Center, Seattle, Washington
| | - David R. Gretch
- Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington
- Department of Medicine, University of Washington Medical Center, Seattle, Washington
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Reynolds GM, Harris HJ, Jennings A, Hu K, Grove J, Lalor PF, Adams DH, Balfe P, Hübscher SG, McKeating JA. Hepatitis C virus receptor expression in normal and diseased liver tissue. Hepatology 2008; 47:418-27. [PMID: 18085708 DOI: 10.1002/hep.22028] [Citation(s) in RCA: 86] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
UNLABELLED The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. CONCLUSION HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.
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Affiliation(s)
- Gary M Reynolds
- Liver Laboratories, Institute for Biomedical Research, University of Birmingham and University Hospital Birmingham NHS Foundation Trust, Birmingham, United Kingdom
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Abstract
Apoptosis is central for the control and elimination of viral infections. In chronic hepatitis C virus (HCV) infection, enhanced hepatocyte apoptosis and upregulation of the death inducing ligands CD95/Fas, TRAIL and TNFα occur. Nevertheless, HCV infection persists in the majority of patients. The impact of apoptosis in chronic HCV infection is not well understood. It may be harmful by triggering liver fibrosis, or essential in interferon (IFN) induced HCV elimination. For virtually all HCV proteins, pro- and anti-apoptotic effects have been described, especially for the core and NS5A protein. To date, it is not known which HCV protein affects apoptosis in vivo and whether the infectious virions act pro- or anti-apoptotic. With the availability of an infectious tissue culture system, we now can address pathophysiologically relevant issues. This review focuses on the effect of HCV infection and different HCV proteins on apoptosis and of the corresponding signaling cascades.
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Affiliation(s)
- Richard Fischer
- Department of Internal Medicine II, University of Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany.
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McMAHON R, YATES A, McLINDON J, BABBS C, LOVE E, WARNES T. The histopathological features of asymptomatic hepatitis C virus-antibody positive blood donors. Histopathology 2007. [DOI: 10.1111/j.1365-2559.1994.tb00569.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
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Abstract
HCV (hepatitis C virus) has a high propensity to persist and to cause chronic hepatitis C, eventually leading to cirrhosis. Since HCV itself is not cytopathic, liver damage in chronic hepatitis C is commonly attributed to immune-mediated mechanisms. HCV proteins interact with several pathways in the host's immune response and disrupt pathogen-associated pattern recognition pathways, interfere with cellular immunoregulation via CD81 binding and subvert the activity of NK (natural killer) cells as well as CD4(+) and CD8(+) T-cells. Finally, HCV-specific T-cells become increasingly unresponsive and apparently disappear, owing to several possible mechanisms, such as escape mutations in critical viral epitopes, lack of sufficient help, clonal anergy or expansion of regulatory T-cells. The role of neutralizing antibodies remains uncertain, although it is still possible that humoral immunity contributes to bystander damage of virally coated cells via antibody-dependent cellular cytotoxicity. Cytotoxic lymphocytes kill HCV-infected cells via the perforin/granzyme pathway, but also release Fas ligand and inflammatory cytokines such as IFNgamma (interferon gamma). Release of soluble effector molecules helps to control HCV infection, but may also destroy uninfected liver cells and can attract further lymphocytes without HCV specificity to invade the liver. Bystander damage of these non-specific inflammatory cells will expand the tissue damage triggered by HCV infection and ultimately activate fibrogenesis. A clear understanding of these processes will eventually help to develop novel treatment strategies for HCV liver disease, independent from direct inhibition of HCV replication.
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Affiliation(s)
- Ulrich Spengler
- Department of Internal Medicine 1, University of Bonn, Sigmund-Freud-Strasse 25, Bonn, Germany.
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el-Awady MK, Tabll AA, el-Abd YS, Bahgat MM, Shoeb HA, Youssef SS, Bader el-Din NG, Redwan ERM, el-Demellawy M, Omran MH, el-Garf WT, Goueli SA. HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro. World J Gastroenterol 2006; 12:4836-4842. [PMID: 16937465 PMCID: PMC4087617 DOI: 10.3748/wjg.v12.i30.4836] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/18/2005] [Revised: 12/23/2005] [Accepted: 01/24/2006] [Indexed: 02/06/2023] Open
Abstract
AIM To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. RESULTS The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.
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Affiliation(s)
- Mostafa K el-Awady
- Department of Biomedical Technology, National Research Center, Tahrir Street, PO 12622, Dokki, Cairo, Egypt.
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el-Awady MK, Tabll AA, el-Abd YS, Bahgat MM, Shoeb HA, Youssef SS, Bader el-Din NG, Redwan ERM, el-Demellawy M, Omran MH, el-Garf WT, Goueli SA. HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro. World J Gastroenterol 2006. [PMID: 16937465 DOI: 10.3748/wjg.v12.i30.4836.] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 09/29/2022] Open
Abstract
AIM To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. RESULTS The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.
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Affiliation(s)
- Mostafa K el-Awady
- Department of Biomedical Technology, National Research Center, Tahrir Street, PO 12622, Dokki, Cairo, Egypt.
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Pal S, Shuhart MC, Thomassen L, Emerson SS, Su T, Feuerborn N, Kae J, Gretch DR. Intrahepatic hepatitis C virus replication correlates with chronic hepatitis C disease severity in vivo. J Virol 2006; 80:2280-90. [PMID: 16474135 PMCID: PMC1395397 DOI: 10.1128/jvi.80.5.2280-2290.2006] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The role of viral factors in the pathogenesis of chronic hepatitis C is unknown. The objective of the present study was to characterize markers of hepatitis C virus (HCV) infection and replication in liver biopsy specimens obtained from 65 genotype 1-infected subjects, including 31 who were coinfected with human immunodeficiency virus (HIV), and to analyze associations between intrahepatic viral markers and hepatitis C disease severity. The percentages of liver cells harboring HCV genomes (%G) and replicative-intermediate RNAs (%RI) were evaluated using strand-specific in situ hybridization, while HCV core and NS3 antigens were assessed by immunocytochemistry. HIV-positive and HIV-negative subjects had similar mean grades and stages of liver disease and had similar indices of HCV infection and replication in liver, even though coinfected subjects had significantly shorter mean disease duration (P = 0.0003). Multivariate analysis showed that %G was not associated with grade or stage of liver disease (P = 0.5 and 0.4, respectively), while %RI was strongly associated with liver inflammation (P < 0.001), liver fibrosis (P < 0.001), and serum alanine aminotransferase levels (P = 0.01). NS3 antigen (but not core) was more frequently detected in HCV RI-positive versus RI-negative specimens (P = 0.028). These findings demonstrate a link between HCV proliferation and hepatitis C disease severity and suggest similar pathogenic mechanisms in HIV-positive and HIV-negative individuals.
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Affiliation(s)
- Sampa Pal
- Department of Laboratory Medicine, University of Washington Medical Center, Seattle, 98104, USA
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17
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Kanto T, Hayashi N. Immunopathogenesis of hepatitis C virus infection: multifaceted strategies subverting innate and adaptive immunity. Intern Med 2006; 45:183-91. [PMID: 16543687 DOI: 10.2169/internalmedicine.45.1530] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022] Open
Abstract
Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. The critical role of innate as well as adaptive immunity has been reported in HCV persistence and liver injury. In the early phase of acute infection, HCV continues to replicate in the liver, suggesting the HCV capability of inhibiting innate immunity. The sustained, vigorous and multiepitope-specific CD4+ and CD8+ T cell responses are essential for spontaneous HCV clearance. HCV-specific CD8+ T cells are primary elements for HCV clearance by inducing hepatocyte apoptosis, in which Fas/CD95 is fundamentally involved. However, once HCV persistency develops, HCV utilizes multifaceted arms to subvert various immune effectors. During IFNalpha-based therapy, the enhancement of HCV-specific CD4+ T cell response followed by HCV eradication has been reported, however, it remains obscure whether the therapeutic HCV clearance is able to restore the durable immune competency to HCV. Further investigation is still warranted to establish the means to direct HCV-specific immune responses in the desired way.
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Affiliation(s)
- Tatsuya Kanto
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Osaka
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18
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Yagi S, Mori K, Tanaka E, Matsumoto A, Sunaga F, Kiyosawa K, Yamaguchi K. Identification of novel HCV subgenome replicating persistently in chronic active hepatitis C patients. J Med Virol 2005; 77:399-413. [PMID: 16173026 DOI: 10.1002/jmv.20469] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
In an effort to clarify the life cycle of HCV, the HCV genome in liver biopsies taken from chronic active hepatitis C patients undergoing interferon treatment was investigated. Molecular cloning by long distance reverse-transcription polymerase chain reaction (RT-PCR) revealed that the HCV genome in two patients with high viral loads in the liver had in-frame deletions of approximately 2 kb between E1 and NS2, which encode the E1-NS2 fusion protein and six other HCV proteins: core, NS3, NS4A, NS4B, NS5A, and NS5B. Among the remaining 21 chronic active hepatitis C patients, these types of deletion were found in another two patients and in two hepatocellular carcinoma patients. Out-of-frame deletions in the structural region were isolated from the other five patients, but the dominant RT-PCR products were non-truncated genomes. Retrospective analysis of a series of serum samples taken from a patient carrying the subgenome with the in-frame deletion revealed that both the subgenome and the full genome persisted through the 2-year period of investigation, with the subgenome being predominant during this period. Sequence analysis of the isolated cDNA suggested that both the subgenome and the full genome evolved independently. Western blotting analysis of HCV proteins from the HCV subgenome indicated that they were processed in the same way as those from the full genome. HCV subgenomes thus appear to be involved in the HCV life cycle.
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Affiliation(s)
- Shintaro Yagi
- R&D Group, Advanced Life Science Institute, Inc., Saitama, Japan
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Kasprzak A, Seidel J, Biczysko W, Wysocki J, Spachacz R, Zabel M. Intracellular localization of NS3 and C proteins in chronic hepatitis C. Liver Int 2005; 25:896-903. [PMID: 15998442 DOI: 10.1111/j.1478-3231.2005.01109.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
AIMS AND METHODS The cellular localization of hepatitis C virus (HCV) proteins in chronic hepatitis C remains a debatable issue. Aim of the studies included cellular and subcellular localization of two HCV proteins, NS3 and C protein, in liver biopsies from 20 children with chronic hepatitis C employing commercially available monoclonal antibodies and a semiquantitative technique of the proteins expression. In the studies advantage was taken of (Strept)avidin-biotin peroxidase complex and ImmunoMax technique. RESULTS The cytoplasmic localization of both proteins dominated over nuclear localization. The total amount of NS3 protein exceeded that of C protein. At the ultrastructural level, we observed both proteins in endoplasmic reticulum membranes and mitochondria, but small amount of both proteins was seen also in cell nuclei. The amount of either protein did not correlate with liver histology in our patients. No correlation could be disclosed between content of both proteins and HCV RNA levels in serum. Content of NS3 demonstrated negative correlation with activity of alanine transaminase. CONCLUSION In conclusion, the new aspect in present studies involved localization of C and NS3 proteins at the levels of light and electron microscopy in children. For the first time also intramitochondrial localization of NS3 protein was demonstrated.
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Affiliation(s)
- Aldona Kasprzak
- Department of Histology and Embryology, Poznan University of Medical Sciences, Poznań, Poland.
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20
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Shiha GE, Zalata KR, Abdalla AF, Mohamed MK. Immunohistochemical identification of HCV target antigen in paraffin-embedded liver tissue: reproducibility and staining patterns. Liver Int 2005; 25:254-60. [PMID: 15780047 DOI: 10.1111/j.1478-3231.2005.01101.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND Immunohistochemical staining has been applied successfully to detect hepatitis C virus (HCV) antigen in fresh frozen tissue. In paraffin-embedded tissues, however, minimal trials with conflicting results have been reported. AIMS The present study is a trial to evaluate the identification of HCV antigen in paraffin-embedded liver biopsies using the anti-HCV monoclonal antibody (MAb) TORDJI-22. METHODS We applied immunohistochemical staining for HCV in 56 paraffin-embedded liver biopsy specimens, 46 from patients seropositive for HCV-RNA and 10 control liver biopsy specimens. The TORDJI-22 MAb was applied in dilution 1:40, with overnight incubation. RESULTS Reproducible staining patterns of HCV antigen in tissues were identified among the majority (42/46-91%) of HCV RNA seropositive cases. The staining pattern was cytoplasmic of hepatocytes, with occasional nuclear hue. It is mainly coarse granular with microvesicular pattern. Three staining patterns were identified: A, diffuse or membranous; B, patchy; and C, occasional paranuclear. None of the control samples showed a similar staining pattern. CONCLUSION Immunohistochemical identification of HCV antigen is easy to apply in paraffin-embedded liver biopsy specimens when the optimal detection techniques are applied. The staining pattern is reproducible, being mainly coarse granular cytoplasmic. Cross reactivity with hepatitis B virus antigens was not detected.
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Affiliation(s)
- Gamal E Shiha
- Department of Internal medicine, Mansoura University, Mansoura , 35516, Egypt.
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21
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Gremion C, Grabscheid B, Wölk B, Moradpour D, Reichen J, Pichler W, Cerny A. Cytotoxic T lymphocytes derived from patients with chronic hepatitis C virus infection kill bystander cells via Fas-FasL interaction. J Virol 2004; 78:2152-7. [PMID: 14747581 PMCID: PMC369426 DOI: 10.1128/jvi.78.4.2152-2157.2004] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The role of Fas-mediated lysis of hepatocytes in hepatitis C virus (HCV)-induced injury is frequently discussed. We therefore analyzed the effect of the number of HCV antigen-expressing cells, the mode of antigen presentation, and the number of cytotoxic T lymphocytes in a coculture system mimicking cellular components of the liver. Here, we show that endogenously processed HCV proteins are capable of inducing bystander killing. We further demonstrate that 0.8 to 1.5% of cells presenting HCV antigens suffice to induce lysis of 10 to 29% of bystander cells, suggesting that the mechanism may be operative at low fractions of infected versus uninfected hepatocytes in vivo. Our data underscore the role of the Fas pathway in HCV-related liver injury and support the exploration of Fas-based treatment strategies for patients with chronic hepatitis C virus infection.
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Affiliation(s)
- Christel Gremion
- Clinic for Rheumatology and Clinical Immunology/Allergology, University of Bern, CH-3010 Bern, Switzerland
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22
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Ohkawa K, Ishida H, Nakanishi F, Hosui A, Ueda K, Takehara T, Hori M, Hayashi N. Hepatitis C virus core functions as a suppressor of cyclin-dependent kinase-activating kinase and impairs cell cycle progression. J Biol Chem 2004; 279:11719-26. [PMID: 14711830 DOI: 10.1074/jbc.m308560200] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
We investigated how the hepatitis C virus (HCV) core protein affects the cell cycle profile and cell cycle-related molecules by using the HCV core-expressing stable transfectant. Analysis of the cell cycle profile showed that HCV core impaired G(1) to S transition. The E2F-mediated transcription, phosphorylation of the retinoblastoma protein, and cyclin-dependent kinase (CDK) 4 and CDK2 activities were suppressed in HCV core-expressing cells. The expression levels of G(1) phase-related CDKs/cyclins and various CDK inhibitors were not substantially affected by expression of HCV core. When influences of HCV core on CDK-activating kinase (CAK) were examined, the expression levels of the CAK components, CDK7, cyclin H, and MAT1, were not affected. However, formation of the ternary CAK complex, CAK activity, and the CDK2 level with activating phosphorylation were inhibited by expression of the HCV core. The direct effect of HCV core on CAK was further assessed in the cell-free system by adding the in vitro translated HCV core protein to the anti-CDK7 immunoprecipitate from the cell. The results showed that HCV core led to dissociation of MAT1 from the CAK complex and suppressed the CAK activity. Furthermore, the binding assay revealed that the HCV core was directed against CDK7. Their interaction occurred mainly in the nucleus by the immunostaining. In conclusion, the HCV core protein interacts with CAK and functions as an extrinsic suppressor of CAK. This may be the molecular basis of HCV core-mediated suppression of cell cycle progression. Our findings suggest a novel mechanism concerning HCV core-mediated alteration in the cell cycle machinery.
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Affiliation(s)
- Kazuyoshi Ohkawa
- Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
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23
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Sansonno D, Lauletta G, Dammacco F. Detection and quantitation of HCV core protein in single hepatocytes by means of laser capture microdissection and enzyme-linked immunosorbent assay. J Viral Hepat 2004; 11:27-32. [PMID: 14738555 DOI: 10.1046/j.1365-2893.2003.00474.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Immunohistochemistry provides valuable information concerning the localization and distribution of hepatitis C virus (HCV)-related proteins in histological sections of liver tissue, but does not readily permit their quantitation in individual cells and the staining intensity of cell immunodeposits cannot be calibrated with the current number of antigen molecules. We specifically detected and quantitated HCV core protein in single hepatocytes by coupling laser capture microdissection (LCM) with a sensitive enzyme-linked immunosorbent assay (ELISA). Quantitation of HCV core protein per cell was carried out on liver tissue cells obtained by LCM from fixed and stained frozen sections of 10 HCV-positive patients with chronic active hepatitis (CAH). Macromolecules from captured cells were solubilized in an extraction buffer and directly assayed for core protein using a sandwich ELISA. Calibration was achieved by developing a standard curve based on known concentrations of HCV core protein. Precision, linearity and sensitivity were verified for known numbers of microdissected tissue cells. In this study, the concentration of HCV core protein in single hepatocytes ranged from 7 to 56 pg/cell. Specificity was verified on 10 replicates of 10 HCV-negative liver tissues. Immunohistochemical staining of HCV core protein was compared with the results of the soluble immunoassay for the adjacent liver tissue sections. Independent scoring of HCV immunostaining failed to parallel the LCM quantitative immunoassay. LCM-based immunoassay significantly expands our ability to investigate function-related antigens in apparently pure cell populations in HCV infection.
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Affiliation(s)
- D Sansonno
- Section of Internal Medicine and Clinical Oncology, Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy
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24
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Falcón V, Acosta-Rivero N, Chinea G, Gavilondo J, de la Rosa MC, Menéndez I, Dueñas-Carrera S, Viña A, García W, Gra B, Noa M, Reytor E, Barceló MT, Alvarez F, Morales-Grillo J. Ultrastructural evidences of HCV infection in hepatocytes of chronically HCV-infected patients. Biochem Biophys Res Commun 2003; 305:1085-90. [PMID: 12767942 DOI: 10.1016/s0006-291x(03)00884-2] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.
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Affiliation(s)
- Viviana Falcón
- Biomedicine Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, Havana, Cuba.
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25
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Abstract
Immunohistochemistry is a strong tool in hepatopathologic diagnosis: the technique is relatively simple and inexpensive. New and very sensitive detection methods have been recently developed (e.g., the EnVision technique and the microwave antigen retrieval method). This article discusses the role of immunohistochemistry in differentiating chronic cholestatic diseases from chronic hepatitis and in characterizing infectious agents. Algorythms for the typing of lymphomas and for the differentiation of primary tumors versus metastases are proposed as well. The immunohistochemical criteria for the diagnosis of premalignant lesions are discussed.
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Affiliation(s)
- Tania Roskams
- Departments of Morphology and Molecular Pathology, Head Liver Research Unit, Medical School, University of Leuven, Belgium.
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26
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Abstract
AIM: To establish an experimental model for exploring the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma.
METHODS: Recombinant plasmid of HCV-core gene was constructed with molecular cloning technique and transfected into QBC939 cells with lipofection. After it was selected with G418, resistant colonies were obtained. The colonies were analysed by immunocytochemistry and Western blotting.The morphology was observed under transmission electron microscope(TEM) and microscope.
RESULTS: The recombinant plasmid was proved to carry the target gene by PCR and restriction enzymed mapping. Moreover, it could express HCV-C protein efficiently in QBC939 cells. The HCV-like particles were found in the cytoplasm by electron microscope, which were spherical with a diameter of 50 nm-80 nm possessing outer membrane.The transfected cells had lower differentiation and higher malignant degree under microscope.
CONCLUSION: Because HCV-core gene could express steadily in cholangiocarcinoma cells,the transfected tumor cells(QBC939-HCVC) could be used to study the effect of HCV in the development of cholangiocarcinoma.
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Affiliation(s)
- Xiao-Fang Liu
- Department of General Surgery of Tongji Hospital, 1095 Jiefang Road, Wuhan 430030, Hubei Province,China.
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27
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Lerat H, Honda M, Beard MR, Loesch K, Sun J, Yang Y, Okuda M, Gosert R, Xiao SY, Weinman SA, Lemon SM. Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology 2002; 122:352-65. [PMID: 11832450 DOI: 10.1053/gast.2002.31001] [Citation(s) in RCA: 338] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
BACKGROUND & AIMS The aim of this study was to determine whether expression of hepatitis C virus proteins alters hepatic morphology or function in the absence of inflammation. METHODS Transgenic C57BL/6 mice with liver-specific expression of RNA encoding the complete viral polyprotein (FL-N transgene) or viral structural proteins (S-N transgene) were compared with nontransgenic littermates for altered liver morphology and function. RESULTS FL-N transcripts were detectable only by reverse-transcription polymerase chain reaction, and S-N transcripts were identified in Northern blots. The abundance of viral proteins was sufficient for detection only in S-N transgenic animals. There was no inflammation in transgenic livers, but mice expressing either transgene developed age-related hepatic steatosis that was more severe in males. Apoptotic or proliferating hepatocytes were not significantly increased. Hepatocellular adenoma or carcinoma developed in older male animals expressing either transgene, but their incidence reached statistical significance only in FL-N animals. Neither was ever observed in age-matched nontransgenic mice. CONCLUSIONS Constitutive expression of viral proteins leads to common pathologic features of hepatitis C in the absence of specific anti-viral immune responses. Expression of the structural proteins enhances a low background of steatosis in C57BL/6 mice, while additional low level expression of nonstructural proteins increases the risk of cancer.
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Affiliation(s)
- Hervé Lerat
- Department of Microbiology & Immunology, The University of Texas Medical Branch, Galveston, Texas 77555-1019, USA
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28
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Abstract
AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro.
METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RT-PCR, in situ hybridization and immunohisto-chemistry respectively.
RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3, CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed.
CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.
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Affiliation(s)
- Z Q Song
- Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
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29
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Schaff Z, Lotz G, Schulte-Herman R. Pathomorphological Characteristics and Pathogenesis of Viral Hepatitis. Pathol Oncol Res 2001; 2:117-131. [PMID: 11173596 DOI: 10.1007/bf02903516] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Viral hepatitis (VH) is an inflammatory reaction of the liver to hepatotropic viruses. Acute VH can be classified according to the virus and type of necrosis. Chronic hepatitis (CH) might be active, persistent or lobular based on previous classification. More recently the grade (necroinflammatory activity) and stage (fibrosis and architectural distorsion) of CH have been distinguished and scored. Apoptosis and necrosis probably coexist in VH and contribute to hepatocyte death. Several "death factors", such as transforming growth factor b, Apo1/Fas and tumor necrosis factor play a role in the execution of cell death. Injury of hepatocytes during viral infection can occur as a direct effect of the virus or as a result of the host immune response. Expression of different viral antigens can be detected during VH and might be visualized. Phenotyping of the portal inflammatory cell infiltrate in CH has shown a T-cell zone comprised of CD4+ helper T cells and CD8+ supressor/cytotoxic T cells at the periphery of the lobules. The pathogenetic mechanisms responsible for the final outcome of viral infection depend on viral factors (such as genotype, mutation etc.), virus-host interaction, expression of viral protein, several cytokines etc. which finally lead to the well known histological alterations of viral hepatitis.
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Affiliation(s)
- Zsuzsa Schaff
- Semmelweis University of Medicine, 1st Institute of Pathology and Experimental Cancer Research, Budapest, Hungary
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30
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Lee AY, Manning WC, Arian CL, Polakos NK, Barajas JL, Ulmer JB, Houghton M, Paliard X. Priming of hepatitis C virus-specific cytotoxic T lymphocytes in mice following portal vein injection of a liver-specific plasmid DNA. Hepatology 2000; 31:1327-33. [PMID: 10827159 DOI: 10.1053/jhep.2000.7297] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The immunology of hepatitis C virus (HCV) infection should be studied in the context of HCV antigen expression in the liver, because HCV primarily infects this organ. Indeed, the nature, function, and fate of T cells primed after antigen expression in the liver might differ from those primed when antigens are expressed systemically or in other organs, because the nature of the antigen-presenting cells (APCs) involved may be different. In addition, the normal liver contains a resident population of lymphocytes that differ from those present at other sites. Thus, we investigated whether HCV-specific CD8(+) cytotoxic T cells (CTLs) could be elicited following portal vein (PV) injection of plasmid DNA in mice whose hepatic veins were transiently occluded. We show that PV injection of mice with "naked" DNA expressing the HCV-NS5a protein, under the control of a liver-specific enhancer/promoter, resulted in NS5a expression in the liver and the priming of HCV-specific CTLs. These results suggested that such a model might be relevant to the study of HCV-specific immune responses primed during natural infection.
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Affiliation(s)
- A Y Lee
- Chiron Corporation, Emeryville, CA 94608, USA
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31
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García-Monzón C, Majano PL, Zubia I, Sanz P, Apolinario A, Moreno-Otero R. Intrahepatic accumulation of nitrotyrosine in chronic viral hepatitis is associated with histological severity of liver disease. J Hepatol 2000; 32:331-8. [PMID: 10707875 DOI: 10.1016/s0168-8278(00)80080-x] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
BACKGROUND/AIMS The toxicity of nitric oxide is thought to be engendered, at least in part, by its reaction with superoxide yielding peroxynitrite, a potent oxidant that promotes the formation of nitrotyrosine within cells and tissue lesions. In this study we assessed the intrahepatic localization and distribution of the inducible nitric oxide synthase (iNOS) and nitrotyrosine (NTY) in patients with viral and non-viral liver disease. METHODS We carried out single and double immunostaining experiments on cryostat liver biopsy sections using monoclonal antibodies against iNOS and NTY. We also performed a comparative analysis between the intrahepatic immunostaining score of NTY and the histological activity index of chronic viral hepatitis. RESULTS We found a marked hepatocellular expression of iNOS with a diffuse lobular pattern in all liver samples from patients with viral liver disease, whereas NTY localization was mainly restricted to cellular foci consisting of hepatocytes and Kupffer cells. Interestingly, we demonstrated by means of double immunostaining experiments the existence of hepatocellular co-localization of iNOS and NTY in the majority of NTY-expressing liver cells. The amount of NTY was significantly higher in liver biopsies from viral liver disease than in non-viral liver disease. In addition, a statistically significant association between the intrahepatic amount of NTY and the severity of viral liver disease was found. CONCLUSIONS Nitric oxide-mediated nitration of hepatocellular proteins is markedly induced in the inflamed liver tissue from patients with chronic viral hepatitis, and appears to be associated with the histological severity of viral chronic liver disease.
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Affiliation(s)
- C García-Monzón
- Hepatology Unit, Hospital Universitario Santa Cristina, Madrid, Spain.
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32
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Chang M, Marquardt AP, Wood BL, Williams O, Cotler SJ, Taylor SL, Carithers RL, Gretch DR. In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease. J Virol 2000; 74:944-55. [PMID: 10623757 PMCID: PMC111615 DOI: 10.1128/jvi.74.2.944-955.2000] [Citation(s) in RCA: 47] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Liver failure from chronic hepatitis C is the leading indication for liver transplantation in the United States. However, the pathogenesis of liver injury resulting from chronic hepatitis C virus (HCV) infection is not well understood. To examine the relationship between HCV replication in liver tissue and hepatocellular injury, a strand-specific in situ hybridization procedure was developed. The sensitivity and specificity of digoxigenin-labeled riboprobes were optimized by analyzing Northern blots and cell lines expressing HCV RNAs. For the current study, both genomic (sense) and replicative-intermediate (antisense) HCV RNAs were detected and quantified in 8 of 8 liver tissue specimens from infected patients versus 0 of 11 liver tissue specimens from noninfected controls. The distribution pattern for HCV replicative-intermediate RNA in liver was different from that for HCV genomic RNA. HCV genomic RNA was variably distributed throughout infected livers and was located primarily in the cytoplasm of hepatocytes, with some signal in fibroblasts and/or macrophages in the surrounding fibroconnective tissue. However, HCV replicative-intermediate RNA showed a more focal pattern of distribution and was exclusively localized in the cytoplasm of hepatocytes. There was no significant relationship between the distribution pattern for HCV genomic RNA and any indices of hepatocellular injury. However, a highly significant correlation was observed between the percentage of cells staining positive for replicative-intermediate RNA and the degree of hepatic inflammatory activity (P, < 0.0001). Furthermore, the ratio of cells staining positive for HCV replicative-intermediate versus genomic RNA correlated with the histological severity of liver injury (P, 0. 0065), supporting the hypothesis that active replication of HCV in liver tissue may be a significant determinant of hepatocellular injury.
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Affiliation(s)
- M Chang
- Departments of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington 98195, USA
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33
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Abstract
Hepatitis C virus (HCV) NS3 is a multifunctional protein with both protease and helicase activities and has been shown to interact with host cell proteins. It is shown that NS3 is present in the hepatocytes from patients with chronic HCV infection by using anti-NS3 antisera. NS3 is detectable in approximately 4% of the hepatocytes from these patients. In most infected cells, NS3 is present in the cytoplasm; however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own are positive for NS3. The presence of NS3 in the nuclei of hepatocytes in chronically infected patients indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection.
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Affiliation(s)
- W Errington
- Department of Medicine, Imperial College School of Medicine, London, England
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34
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Ou-Yang P, Chiang BL, Hwang LH, Chen YG, Yang PM, Chi WK, Chen PJ, Chen DS. Characterization of monoclonal antibodies against hepatitis C virus nonstructural protein 3: different antigenic determinants from human B cells. J Med Virol 1999; 57:345-50. [PMID: 10089044 DOI: 10.1002/(sici)1096-9071(199904)57:4<345::aid-jmv3>3.0.co;2-n] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
The nonstructural (NS3) region protein of hepatitis C virus (HCV) possesses major B-cell epitopes that induce antibodies after infection. To elucidate further the characteristics of these B cells and their role in the immune regulation of HCV infection, T9 (portion of NS3 region, amino acids [a.a.] 1188-1493)-specific monoclonal antibodies were derived and mapped for B-cell antigenic determinants with recombinant proteins. A total of 10 T9-specific hybridomas were generated and tested for B-cell antigenic determinants. To analyze the B-cell antigenic determinants, eight recombinant proteins including NS3-e (a.a. 1175-1334), NS3-a' (a.a. 1175-1250), NS3-a (a.a. 1251-1334), NS3-b (a.a. 1323-1412), NS3-c (a.a. 1407-1499), NS3-a/b (a.a. 1251-1412), NS3-bc (a.a. 1323-1499), and NS3-abc (a.a. 1251-1499) encoded by NS3-region internal clones were expressed and tested for immunoblotting. The data suggested IgG hybridomas recognized NS3-a, NS3-a', or NS3-b protein by immunoblotting. By contrast, the NS3-e protein bears the major antigenic determinant recognized by human sera. Half of the hybridomas were found to react with protein NS3-a', which is not a major B-cell antigenic determinant in humans. These data suggested that conformational epitopes in vivo may be important for B-cell recognition.
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Affiliation(s)
- P Ou-Yang
- Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei
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35
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Miura H, Itoh Y, Matsumoto Y, Tani M, Tanabe N, Isonokami M, Kurachi K, Kozuka T. Long-term administration of cyclosporin A to HCV-antibody-positive patients with dermatologic diseases. Int J Dermatol 1999; 38:310-4. [PMID: 10321952 DOI: 10.1046/j.1365-4362.1999.00690.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
BACKGROUND Cyclosporine A (CYA) is an immunosuppressive agent which is being used in the treatment of an increasingly wide range of dermatologic diseases, but its use has been avoided in carriers of hepatitis C virus (HCV). METHODS We administered small doses of CYA (maximum, 3 mg/kg/day) for a long time to treat dermatologic diseases in one HCV-antibody-positive patient with no HCV-RNA in the blood, one patient with a small amount of HCV-RNA in the blood, and two patients with large amounts of HCV-RNA in the blood. RESULTS Skin lesions improved in all patients, but recurred upon complete or partial withdrawal of CYA. In the absence of HCV-RNA in the blood, or when only a small quantity of HCV-RNA was present in the blood, HCV-RNA load showed no apparent change. In one patient with a large blood HCV-RNA load, CYA dosage reduction was followed by increases in alanine aminotransferase (ALT) levels and decreases in blood HCV-RNA. Aggravation of hepatitis due to immunologic reactivation was suspected in this patient. CONCLUSIONS The reduction of CYA dosage is a key element in the use of this agent for cutaneous diseases.
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Affiliation(s)
- H Miura
- Department of Dermatology and Allergology, Osaka National Hospital, Japan
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36
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Doughty AL, Painter DM, McCaughan GW. Nonspecificity of monoclonal antibody Tordji-22 for the detection of hepatitis C virus in liver transplant recipients with cholestatic hepatitis. LIVER TRANSPLANTATION AND SURGERY : OFFICIAL PUBLICATION OF THE AMERICAN ASSOCIATION FOR THE STUDY OF LIVER DISEASES AND THE INTERNATIONAL LIVER TRANSPLANTATION SOCIETY 1999; 5:40-5. [PMID: 9873091 DOI: 10.1002/lt.500050104] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Detection of hepatitis C virus (HCV) antigens in liver tissue provides important diagnostic and pathological information. Limited studies have been performed on tissue taken after liver transplantation for HCV. In this study, serial post-liver transplantation biopsy tissue from patients with recurrent HCV was tested, with particular interest in patients showing severe cholestatic hepatitis. HCV-related antigens were detected using the commercial monoclonal antibody, Tordji-22. Initial results were promising, showing intense positive staining, especially in areas of hepatocyte ballooning. HCV-negative donor tissue was consistently negative by staining. However, as a final control for the level of tissue damage, HCV negative posttransplantation biopsy tissue showing hepatocyte ballooning was examined. These tissues also showed positive staining. All attempts to eliminate this nonspecific interaction failed. In conclusion, Tordji-22 was associated with nonspecificity in this posttransplantation population, and care is warranted when using this monoclonal antibody.
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Affiliation(s)
- A L Doughty
- Department of Anatomical Pathology, Royal Prince Alfred Hospital, New South Wales, Australia
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37
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Guerrero RB, Batts KP, Germer JJ, Perez RG, Wiesner RH, Persing DH. Reverse transcriptase-polymerase chain reaction fails to detect peripheral-blood hepatitis C RNA in formalin-fixed liver tissue. LIVER TRANSPLANTATION AND SURGERY : OFFICIAL PUBLICATION OF THE AMERICAN ASSOCIATION FOR THE STUDY OF LIVER DISEASES AND THE INTERNATIONAL LIVER TRANSPLANTATION SOCIETY 1998; 4:455-60. [PMID: 9791155 DOI: 10.1002/lt.500040611] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Currently, one of the major indications for liver transplantation is infection with hepatitis C virus (HCV). Many studies have suggested that recurrent infection with HCV is universal after transplantation. Fastidious techniques, such as reverse transcriptase-polymerase chain reaction (RT-PCR), have proved to be highly sensitive for detecting HCV RNA in serum and in fresh-frozen and formalin-fixed paraffin-embedded (FFPE) liver tissue. In this study, we wanted to determine whether the identification of HCV RNA in liver tissue by RT-PCR might reflect the detection of circulating HCV RNA in blood within the tissue, rather than implying true tissue infection. We performed RT-PCR for HCV RNA in FFPE liver biopsy specimens taken from 14 donor allografts shortly before and immediately after implantation into recipients. The recipients were known to have HCV RNA in serum and explanted liver tissue, as determined by RT-PCR. We were unable to detect HCV RNA in any of the study samples, either before or after transplantation. In a related study, qualitative and quantitative HCV RNA analyses were performed by RT-PCR and branched DNA (bDNA) amplification, respectively, on serum samples collected pretransplantation and immediately posttransplantation from 10 other patients who underwent transplantation for hepatitis C. HCV RNA was detected in all serum samples before and after transplantation by RT-PCR; however, the bDNA assay detected HCV RNA in only 6 of 10 samples pre-orthotopic liver transplantation (OLT) and in none of the immediately post-OLT samples. In our system, despite the RT-PCR detection of HCV RNA in serum before and after the transplantation, HCV RNA is not detectable in the peripheral blood that accompanies formalin-fixed liver tissue. This implies that RT-PCR detection of HCV RNA in tissue reflects true liver infection, rather than contamination by HCV RNA in accompanying peripheral blood.
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Affiliation(s)
- R B Guerrero
- Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, MN, USA
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38
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Hall PD. Broadsheet number 47: Chronic hepatitis: an update with guidelines for histopathological assessment of liver biopsies. Board of Education of The Royal College of Pathologists of Australasia. Pathology 1998; 30:369-80. [PMID: 9839312 DOI: 10.1080/00313029800169656] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The liver biopsy remains the 'gold standard' for the diagnosis of chronic hepatitis, particularly since it is the only investigation that permits assessment of the severity (grade of histological activity and stage of fibrosis) of liver injury. As outlined below, the liver biopsy is invaluable for both diagnosis and the monitoring of therapy. To optimise the value of the liver biopsy, a standardised approach for assessment and reporting of chronic hepatitis is recommended.
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Affiliation(s)
- P D Hall
- Department of Pathology, Flinders University of South Australia, Australia
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39
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Adinolfi LE, Andreana A, Utili R, Zampino R, Ragone E, Ruggiero G. HCV RNA levels in serum, liver, and peripheral blood mononuclear cells of chronic hepatitis C patients and their relationship to liver injury. Am J Gastroenterol 1998; 93:2162-6. [PMID: 9820390 DOI: 10.1111/j.1572-0241.1998.00613.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
OBJECTIVE We sought to evaluate the relationship between HCV RNA levels in serum, liver, and peripheral blood mononuclear cells (PBMC) and the degree of liver injury in chronic hepatitis C (CHC) patients. METHODS Thirty-six consecutive CHC patients were included in the study. The liver damage was evaluated by the histological activity index (HAI) score. The HCV RNA levels in the three compartments studied were assessed by bDNA assay. Nineteen patients were treated with alpha-interferon 2b (IFN). RESULTS Serum and liver HCV RNA levels in CHC patients were significantly associated with an increasing HAI score irrespective of the HCV genotypes. Cirrhotic patients showed higher HCV RNA levels than the CHC patients with HAI score 1-4 (p < 0.05), but had lower levels than the group with HAI score > 8 (p < 0.03). Patients with HAI score 1-4 showed the lowest levels of HCV RNA in PBMC. There was a strong relation (r = 0.78; p < 0.001) between serum and liver HCV RNA levels, but not between either serum or liver HCV RNA levels and those of PBMC. Seven patients showed a response to IFN and three of these had a sustained response. Pretreatment levels of HCV RNA in PBMC of the IFN responder patients were lower than those of the nonresponder patients (p < 0.02). CONCLUSIONS The data indicate a relation between serum or liver HCV RNA levels and the degree of liver injury in CHC patients, and show that serum HCV RNA level mirrors the hepatic viral burden.
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Affiliation(s)
- L E Adinolfi
- Institute of Medical Therapy, Faculty of Medicine, Second University of Naples, Italy
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40
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Cotler SJ, Gaur LK, Gretch DR, Wile M, Strong DM, Bronner MP, Carithers RL, Emond MJ, Perkins JD, Nelson KA. Donor-recipient sharing of HLA class II alleles predicts earlier recurrence and accelerated progression of hepatitis C following liver transplantation. TISSUE ANTIGENS 1998; 52:435-43. [PMID: 9864033 DOI: 10.1111/j.1399-0039.1998.tb03070.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Both direct viral cytopathic effects and host immune responses appear to be important in the pathogenesis of hepatitis C virus (HCV) infection. Liver transplantation provides a means to explore the role of the immune system in the development of HCV-related liver damage through comparing the natural history of HCV in patients with different degrees of donor-recipient human leukocyte antigen (HLA) matching. We evaluated 36 patients with recurrent hepatitis C viremia following liver transplantation to determine whether hepatocellular injury or progression to bridging fibrosis occur more rapidly when donor and recipients share HLA alleles. HLA typing for the HLA-A and HLA-B loci was performed by serological techniques and PCR-based oligotyping was used to type alleles of the DRB1, DRB3, DQA1, and DQB1 loci. A median of eight liver biopsies, obtained during a median follow-up of 36 months, were reviewed per patient. Donor-recipient sharing of alleles of HLA-DQB1 or DRB1 was associated with more rapid development of recurrent hepatitis by univariate analysis (chi2=5.7, P=0.02 and chi2=5.54, P=0.02 respectively). However, only sharing of HLA-DRB1 alleles was identified as an independent predictor of reduced time to recurrent histologic injury by multivariate analysis (chi2 =5.74, P=0.02). Furthermore, sharing of HLA-DRB3 and histologic evidence of rejection were associated with more rapid progression to bridging fibrosis both by univariate methods (chi2=4.12, P=0.04 and chi2=4.66, P=0.03 respectively), and by multivariate analysis (chi2=13.01, P=0.001). These findings suggest that HLA class II-restricted immune responses may contribute to the pathogenesis of HCV-related liver injury in liver transplant recipients.
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Affiliation(s)
- S J Cotler
- Department of Medicine, University of Washington Medical Center, Seattle, USA
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41
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Antonaci S, Schiraldi O. Costimulatory molecules and cytotoxic T cells in chronic hepatitis C: defence mechanisms devoted to host integrity or harmful events favouring liver injury progression? A review. Immunopharmacol Immunotoxicol 1998; 20:455-72. [PMID: 9805228 DOI: 10.3109/08923979809031510] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The recruitment of antigen-specific lymphocytes at liver site represents a prominent feature in patients chronically infected with hepatitis C virus (HCV). However, despite the strong and multispecific response, chronic infection leads in a significant number of cases to the development of cirrhosis and hepatocellular carcinoma. The finding that the expression of CD80 structure positively correlates with disease histological worsening points out a role for the costimulatory pathway in the progression of liver cell injury. On the other hand, the demonstration of CD95 and CD95-ligand positive cells in the context of periportal areas, a pattern which is not strictly associated to HCV tissue distribution, indicates the occurrence of either virus-infected or innocent bystander hepatocyte killing. Nonetheless, the persistence of HCV, in spite of cytotoxic T lymphocyte (CTL) liver recruitment, suggests a possible in-situ imbalance of cytotoxic activities, above all referred to perforin-granzyme-dependent necrosis. Altogether, these findings outline that several factors might be involved in HCV-driven immunopathogenesis. Therefore, the fully clarification of these mechanisms may offer a suitable therapeutical approach for the improvement of clinical outcome in chronic hepatitis C.
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Affiliation(s)
- S Antonaci
- Department of Internal Medicine, Immunology and Infectious Diseases, University of Bari Medical School, Italy
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42
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Czaja AJ, Carpenter HA, Santrach PJ, Moore SB. Host- and disease-specific factors affecting steatosis in chronic hepatitis C. J Hepatol 1998; 29:198-206. [PMID: 9722200 DOI: 10.1016/s0168-8278(98)80004-4] [Citation(s) in RCA: 169] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
BACKGROUND/AIM Steatosis is commonly present in chronic hepatitis C. Our aim was to evaluate host- and disease-specific factors associated with its occurrence. METHODS Histologic findings in 60 patients were correlated with body mass index, human leukocyte antigens, and other conventional parameters. Comparisons were made with 41 patients who had nonalcoholic steatohepatitis and 18 patients who had chronic hepatitis B. RESULTS Patients with chronic hepatitis C and steatosis had lower serum concentrations of gamma-globulin (p=0.01) and immunoglobulin G (p=0.05) than their counterparts without steatosis, and they had a lower frequency of antinuclear antibodies (19% versus 52%, p=0.01). They also had a higher mean body mass index (p=0.002) and a greater frequency of risk factors for steatosis (70% versus 34%, p=0.009). These risk factors, however, occurred more commonly in patients with nonalcoholic steatosis (p=0.007). Furthermore, fat deposition occurred more often in chronic hepatitis C than in chronic hepatitis B (52% versus 22%, p=0.03), despite comparable metabolic findings. The degree of steatosis in chronic hepatitis C was not associated with individual metabolic features. CONCLUSIONS Steatosis in chronic hepatitis C is mainly a viral effect, and host-dependent metabolic factors may potentiate the manifestation. Fat deposition is associated with less immunoreactivity and it may connote a distinctive pathogenic mechanism.
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Affiliation(s)
- A J Czaja
- Division of Gastroenterology and Hepatology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA
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43
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Luo JC, Hwang SJ, Lai CR, Lu CL, Li CP, Tsay SH, Wu JC, Chang FY, Lee SD. Relationships between serum aminotransferase levels, liver histologies and virological status in patients with chronic hepatitis C in Taiwan. J Gastroenterol Hepatol 1998; 13:685-90. [PMID: 9715418 DOI: 10.1111/j.1440-1746.1998.tb00714.x] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
In patients with chronic hepatitis C, the relationships between serum alanine aminotransferase (ALT) levels, histological liver injury and serum hepatitis C virus (HCV) RNA titres remain controversial. To evaluate these relationships, 93 Chinese patients with histological diagnosis of chronic hepatitis C were enrolled for this study. Serum ALT levels, HCV-RNA titres and HCV genotypes were examined. The histology was evaluated according to a modified histological activity score based on the degree of periportal necro-inflammation, intralobular necro-inflammation, portal inflammation, total necro-inflammation and fibrosis. The mean serum ALT level was significantly higher in patients with severe intralobular necro-inflammation activity than in patients with mild or no activity (P = 0.013). However, scores of intralobular activity were only weakly correlated with serum ALT levels (r = 0.27) and could not be used to adequately predict ALT values. Serum ALT levels showed no significant correlation with the scores of portal inflammation, periportal necro-inflammation, total necro-inflammation and fibrosis. Also, there was no significant difference in the mean serum ALT level among different serum HCV-RNA levels and HCV genotypes. Serum HCV-RNA titres and genotypes showed no significant correlation with liver histology and serum HCV-RNA titres were only weakly correlated with the total necro-inflammatory score (r = 0.27). In conclusion, although serum ALT levels were higher in patients with more severe intralobular necro-inflammatory activity, the correlation was not strong enough to adequately predict ALT values. Serum HCV-RNA titres and genotypes also showed no significant correlation with serum ALT levels and liver histologies.
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Affiliation(s)
- J C Luo
- Department of Medicine, Veterans General Hospital-Taipei, Taiwan
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44
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Vargas V, Krawczynski K, Castells L, Martinez N, Esteban J, Allende H, Esteban R, Guardia J. Recurrent hepatitis C virus infection after liver transplantation: immunohistochemical assessment of the viral antigen. LIVER TRANSPLANTATION AND SURGERY : OFFICIAL PUBLICATION OF THE AMERICAN ASSOCIATION FOR THE STUDY OF LIVER DISEASES AND THE INTERNATIONAL LIVER TRANSPLANTATION SOCIETY 1998; 4:320-7. [PMID: 9649647 DOI: 10.1002/lt.500040407] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
BACKGROUND The value of immunohistochemical methods to identify hepatitis C virus antigen (HCVAg) in liver tissue has not been established. We have evaluated the significance of HCVAg expression in livers of patients with transplants and recurrent hepatitis C virus (HCV) infection. METHODS Forty-two liver biopsy specimens from 32 liver-transplant recipients with recurrent HCV infection were tested for HCVAg using fluorescein isothiocyanate-labeled polyclonal, polyreactive human immunoglobulin. Histologic assessment of liver and quantitation of HCV RNA in sera were carried out in specimens obtained simultaneously with biopsies. RESULTS HCVAg was found in 33% of the liver specimens obtained during the first month after transplantation and in all liver specimens obtained between 1 and 18 months after transplantation. Amounts of the antigen were significantly greater in specimens obtained more than 1 month after transplantation. A statistically significant increase of the average HCV RNA level in serum was observed in samples tested after the first month after the transplantation, and some decrease in the HCV RNA level was found in those obtained between 6 and 18 months after transplantation. Larger amounts of HCVAg were observed in specimens corresponding to episodes of acute or chronic hepatitis than in those associated with minimal parenchymal evidence of rejection. CONCLUSIONS OBSERVATIONS of HCVAg expression in liver biopsy specimens indicated that the presence of viral antigens in hepatocytes is a constant finding in specimens obtained 1 month or longer after transplantation. Although large amounts of HCVAg correlated with acute or chronic hepatitis, the nature of this association with the development of pathologic changes remains to be established.
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Affiliation(s)
- V Vargas
- Liver Unit, Hospital General Universitari Vall d'Hebron, Barcelona, Spain
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45
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Walker FM, Dazza MC, Dauge MC, Boucher O, Bedel C, Henin D, Lehy T. Detection and localization by in situ molecular biology techniques and immunohistochemistry of hepatitis C virus in livers of chronically infected patients. J Histochem Cytochem 1998; 46:653-60. [PMID: 9562573 DOI: 10.1177/002215549804600510] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Hepatitis C virus (HCV) detection in the livers of chronically infected patients remains a debatable issue. We used immunohistochemistry, in situ hybridization (ISH) alone or after microwave heating with FITC-labeled probes, RT-PCR with unlabeled primers followed by ISH (RT-PCR-ISH), and in situ RT-PCR with FITC-labeled primers (in situ RT-PCRd) to localize the virus in 38 liver biopsy specimens from 21 chronically infected HCV patients treated with interferon-alpha (IFN-alpha). Biopsies were taken at the beginning and end of IFN-alpha treatment and 1 year later. Results were compared with that of HCV-PCR in serum. RT-PCR-ISH and in situ RT-PCRd showed HCV signal in all liver biopsies even in responders with seronegative HCV PCR. This signal was intranuclear, diffuse, or peripheral, in hepatocytes, bile ductule cells, and lymphocytes. Cytoplasmic signals were occasionally observed. Whereas the percentage of labeled hepatocytes remained constant, the number of labeled lymphoid follicles decreased after INF-alpha therapy. Immunohistochemistry resulted in the same pattern of positivity but it was weaker and inconstant. This study indicates the persistency of HCV latency in IFN-alpha responders 1 year after IFN-alpha treatment cessation, a finding that certainly deserves confirmation.
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Affiliation(s)
- F M Walker
- Department of Pathology, Hôpital Bichat-Claude Bernard, Paris, France
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46
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Abstract
The basic morphologic features of acute and chronic viral hepatitis C are similar to those of other hepatitides; however, hepatitis C is characterized by the histologic triad of lymphoid aggregates in portal tracts, epithelial damage of small bile ducts and microvesicular and macrovesicular steatosis of hepatocytes. Significant progress has been made in the demonstration of HCV in infected liver tissues by immunohistochemical and in situ hybridization techniques. The new classification of chronic hepatitis, based on etiology, grading (extent of necroinflammatory activity) and staging (extent of fibrosis) has been widely accepted and will lead to a better understanding of the variable course and response to therapy of this enigmatic disease.
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Affiliation(s)
- M A Gerber
- Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112-2699, USA
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47
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Huang SN, Chen TC, Tsai SL, Liaw YF. Histopathology and pathobiology of hepatotropic virus-induced liver injury. J Gastroenterol Hepatol 1997; 12:S195-217. [PMID: 9407339 DOI: 10.1111/j.1440-1746.1997.tb00502.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The present report concerns current knowledge regarding immunopathogenesis that can be applied in the interpretation of histopathological changes in acute and chronic viral hepatitis. The histopathological features of viral hepatitis have not been changed and light microscopic examination remains essential for making a diagnosis and classification of chronic hepatitis and for the provision of objective parameters on grading and staging. However, new understanding and knowledge of viral pathogenesis, host immune responses, the biological behaviour of the causative viral agents and, in particular, viral interference in multiple hepatotropic viral infections must be taken into consideration in the interpretation of histopathological and immunopathological findings of liver tissues. This report also presents some histopathological analyses on multiple hepatotropic viral infections. It can be concluded that the diagnostic histological criteria for acute hepatitis remain applicable in such settings. However, the cause of acute flare up in chronic hepatitis could not be determined without clinical, virological and serological information. Routine histopathology cannot distinguish a new infection from an acute exacerbation due to a high level of viral replication or mutant virus. A repertoire of immunocytochemical stainings for viral antigens is helpful, but caution must be exercised in suggesting a specific viral aetiology due to the fact that suppression of pre-existing viral antigens can be pronounced when the new or concurrent infection is hepatitis C virus related.
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Affiliation(s)
- S N Huang
- Department of Pathology, Sunnybrook Health Science Centre, University of Toronto, North York, Ontario, Canada
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48
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Ballardini G, Manzin A, Giostra F, Francesconi R, Groff P, Grassi A, Solforosi L, Ghetti S, Zauli D, Clementi M, Bianchi FB. Quantitative liver parameters of HCV infection: relation to HCV genotypes, viremia and response to interferon treatment. J Hepatol 1997; 26:779-786. [PMID: 9126789 DOI: 10.1016/s0168-8278(97)80242-5] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
BACKGROUND/AIMS This study aimed to evaluate the relation between the number of hepatocytes positive for HCV antigens and the amount of HCV RNA in the liver and to evaluate the relationship between the above parameters and viremia levels, HCV genotype and response to interferon treatment. METHODS This was a retrospective study on 31 consecutive patients with chronic HCV-related liver disease, selected on the basis of the availability of frozen liver tissue for both liver HCV antigens detection and liver HCV RNA quantitation. HCV antigens (immunohistochemistry), liver and plasma HCV RNA (competitive RT-PCR), and HCV genotype (commercial kit) were studied. RESULTS A significant correlation (p=0.0005) was found between the amount of liver HCV RNA (log 10 copy/microg of extracted RNA) and the number of HCV-infected hepatocytes (scored from 0 to 3). These parameters were not significantly correlated with viremia levels. The highest liver HCV RNA levels and HCV antigen scores were found in patients infected with genotype 1b. Liver HCV RNA (median 541 x 10(3) vs 118 x 10(3) copy number/microg, p=0.031) and liver HCV antigens (mean score 2.3 vs 1.3, p=0.018) but not plasma HCV RNA (median 14956 x 10(3) vs 2885 [correction of 2.885] x 10(3) copy number/ml, ns) were significantly higher in patients not responding to interferon treatment compared to responders. CONCLUSIONS The tissue parameters tested in this study were significantly correlated, shared the same clinical implications and predicted short-term response to interferon treatment better than viremia levels. We suggest that these tests should be included in the study protocol of patients under evaluation for interferon treatment, basing the choice on local facilities.
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Affiliation(s)
- G Ballardini
- Semeiotica Medica II, Azienda Ospedaliera, University of Bologna, Italy
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49
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Barba G, Harper F, Harada T, Kohara M, Goulinet S, Matsuura Y, Eder G, Schaff Z, Chapman MJ, Miyamura T, Bréchot C. Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc Natl Acad Sci U S A 1997; 94:1200-5. [PMID: 9037030 PMCID: PMC19768 DOI: 10.1073/pnas.94.4.1200] [Citation(s) in RCA: 499] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
There is now abundant evidence to substantiate an important role of hepatitis C virus (HCV) core protein in cellular gene expression as well as in the viral cycle. Thus the subcellular localization of this protein has important implications. However, several studies have shown controversial results: the HCV core has been, indeed, described as cytoplasmic or nuclear depending on the size of the protein or on the genotype analyzed. We have studied the localization of the HCV core protein in two different cell lines, one nonhepatic (CHO) and the other hepatic (HepG2). Double immunofluorescence staining using a nuclear membrane marker and confocal analysis showed the core protein pattern to be cytoplasmic and globular. This pattern is not cell cycle-regulated. Electron microscopy analysis revealed the nature of the globular staining observed in immunofluorescence. The HCV core protein accumulated at the surface of lipid droplets that were also the unique morphological feature of nonhepatic core transfected cells. The lipid droplets were isolated by sequential ultracentrifugation on the basis of their density; biochemical analysis revealed a prevalence of triglycerides. In addition the core protein colocalized with apolipoprotein AII at the surface of the lipid droplets as revealed by confocal microscopy. Moreover analysis of liver biopsies from chronically HCV-infected chimpanzees revealed that HCV core is cytoplasmic and localized on the endoplasmic reticulum and on lipid droplets. These results clearly define the subcellular localization of the HCV core protein and suggest a relationship between the expression of the HCV core protein and cellular lipid metabolism.
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Affiliation(s)
- G Barba
- Liver Cancer and Molecular Virology, Institut National de la Santé et de la Recherche Médicale, Unité 370, Paris, France
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Chang KM, Rehermann B, Chisari FV. Immunopathology of hepatitis C. SPRINGER SEMINARS IN IMMUNOPATHOLOGY 1997; 19:57-68. [PMID: 9266631 DOI: 10.1007/bf00945025] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Hepatitis C virus (HCV) infection becomes persistent in the majority of instances in the face of a humoral and cellular immune response, and persistent HCV infection is associated with chronic hepatitis. In particular, cytotoxic T lymphocytes (CTL), crucial in the eradication of virus-infected cells, have been observed in the liver and the peripheral blood of chronically infected patients, suggesting that CTL cannot completely eliminate the virus, and may contribute to chronic liver injury. In this review, the potential host and the viral factors involved in the pathogenesis of chronic HCV infection will be discussed with emphasis on the HLA-A2 restricted peripheral blood CTL response and its relationship to liver disease and viral load.
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Affiliation(s)
- K M Chang
- Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA
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