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Zarnegar G, Farhadi A, Seyyedi N, Aboualizadeh F, Ranjbaran R, Farzanfar E, Nejabat N, Behzad-Behbahani A. In Vitro Replication of HCV RNA in Peripheral Blood Mononuclear Cells Isolated from Patients Undergoing Treatment for Hepatitis C Virus Infection. HEPATITIS MONTHLY 2020; 20. [DOI: 10.5812/hepatmon.108070] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/06/2025]
Abstract
Background: Although the liver is the main site for the Hepatitis C Virus (HCV) replication, there is still an essential debate about extrahepatic HCV reservoirs. Objectives: It has been proposed that Peripheral Blood Mononuclear Cells (PBMCs) could be the possible virus replication sites. Therefore, PBMCs may be candidates for recurrent HCV infection after achieving Sustained Virologic Response (SVR). In this study, we designed a lymphocyte culture to explore more about virus replication in PBMCs collected from patients with chronic hepatitis C. Methods: Plasma and PBMC samples were collected from 16 randomly selected seropositive patients for the anti-HCV antibody. Four out of 16 (25%) patients received combination therapy with alpha interferon and ribavirin. PBMCs were isolated from whole blood. Between 106-107 cells were cultured with optimized concentrations of IL-2 (10 mg/ml) and phytohemagglutinin A (5 mg/ml). Total RNA was extracted from the first collected sera and harvested lymphocytes. Constructed plasmids containing the NCR coding region were used to plot the standard curve for the relative quantification of SYBR green real-time PCR. The sensitivity and specificity of the detection were established by using plasmids containing cDNA. Results: With this plasmid containing the NCR coding region, the Limit of Detection (LOD) of in-house-developed real-time RT-PCR sensitivity was 2×101 copies. Using primers for the NCR region, 10 out of 16 (62.5 %) PBMCs were positive for negative-strand HCV RNA. Among the four samples collected from patients with SVR, negative-strand HCV RNA was found in two patient samples. Conclusions: Our results indicated that cultured lymphoid cells from patients with chronic hepatitis, even with SVR, in the presence of IL-2 and PHA, markedly enhanced the detection of HCV RNA replica-tive strands. Therefore, PBMCs may be reservoirs for recurrent hepatitis infection after SVR and antiviral treatment. However, more clinical samples and control groups (lymphocyte culture without mitogen) should be examined to support the data presented in this study.
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Mahmoudvand S, Shokri S, Azaran A, Seyedian SS, Makvandi M, Mirzaei H, Sheikhrobat SB. Seronegative occult hepatitis C infection among hemodialysis patients: A prevalence study. Ther Apher Dial 2020; 25:218-224. [PMID: 32510846 DOI: 10.1111/1744-9987.13535] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2020] [Revised: 05/24/2020] [Accepted: 06/04/2020] [Indexed: 11/29/2022]
Abstract
The aim of this study was to investigate the prevalence of occult hepatitis C virus (HCV) infection (OCI) among HD patients. Blood samples were taken from 79 HD patients and their sera were evaluated for the presence of anti-HCV. Both the sera and peripheral blood mononuclear cells (PBMCs) were then checked for HCV RNA by nested reverse transcriptase-polymerase chain reaction. Anti-HCV was positive among 4/79 (5.1%) of the patients. From 75 patients who were negative for anti-HCV, 71 (94.7%) patients were also negative for HCV RNA in sera samples but five of them were positive for HCV RNA in PBMCs. Totally, out of 79 patients, HCV RNA was detected in PBMCs of five (6.3%) patients, indicating that these patients had OCI. No significant difference was observed between the frequency of OCI and gender (P-value = .6). HCV genotype in all five cases of OCI was genotype 3a. Our study showed prevalence rate of 6.3% OCI infection in HD patients. Regarding the serious complications and the clinical importance of OCI in HD patients, sensitive diagnostic methods for identifying HCV RNA in the PBMCs should be implemented for all HD patients.
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Affiliation(s)
- Shahab Mahmoudvand
- Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.,Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Somayeh Shokri
- Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Azarakhsh Azaran
- Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.,Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Seyed S Seyedian
- Alimentary Tract Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Manoochehr Makvandi
- Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Habibollah Mirzaei
- Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Sheida B Sheikhrobat
- Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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Hanno AFF, Mohiedeen KM, Alshayeb AF, Deghedy A. HCV RNA in peripheral blood mononuclear cells (PBMCs) as a predictor of the response to antiviral therapy in chronic hepatitis C. ALEXANDRIA JOURNAL OF MEDICINE 2019. [DOI: 10.1016/j.ajme.2013.05.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Affiliation(s)
| | | | | | - Akram Deghedy
- Clinical and Chemical Pathology, Alexandria Faculty of Medicine , Egypt
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Bolen CR, Robek MD, Brodsky L, Schulz V, Lim JK, Taylor MW, Kleinstein SH. The blood transcriptional signature of chronic hepatitis C virus is consistent with an ongoing interferon-mediated antiviral response. J Interferon Cytokine Res 2013; 33:15-23. [PMID: 23067362 PMCID: PMC3539252 DOI: 10.1089/jir.2012.0037] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2012] [Accepted: 08/13/2012] [Indexed: 01/27/2023] Open
Abstract
Blood transcriptional profiling is a powerful tool for understanding global changes after infection, and may be useful for prognosis and prediction of drug treatment responses. This study characterizes the effects of chronic hepatitis C virus (HCV) infection on gene expression by analyzing blood samples from 10 treatment-naïve HCV patients and 6 healthy volunteers. Differential expression analysis of microarray data from peripheral blood mononuclear cells (PBMCs) identified a 136-gene signature, including 66 genes elevated in infected individuals. Most of the upregulated genes were associated with interferon (IFN) activity (including members of the OAS and MX families, ISG15, and IRF7), suggesting an ongoing immune response. This HCV signature was also found to be consistently enriched in many other viral infection and vaccination datasets. These genes were validated using a second cohort composed of 5 HCV patients and 5 healthy volunteers, confirming the upregulation of the IFN signature. In summary, this is the first study to directly compare blood transcriptional profiles from HCV patients with healthy controls. The results show that chronic HCV infection has a pronounced effect on gene expression in PBMCs of infected individuals, and significantly elevates the expression of a subset of IFN-stimulated genes.
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Affiliation(s)
- Christopher R. Bolen
- Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut
| | - Michael D. Robek
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut
| | - Leonid Brodsky
- Institute of Evolution, University of Haifa, Haifa, Israel
| | - Vincent Schulz
- Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut
| | - Joseph K. Lim
- Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut
| | | | - Steven H. Kleinstein
- Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut
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5
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Liu S, Hao Q, Peng N, Yue X, Wang Y, Chen Y, Wu J, Zhu Y. Major vault protein: a virus-induced host factor against viral replication through the induction of type-I interferon. Hepatology 2012; 56:57-66. [PMID: 22318991 DOI: 10.1002/hep.25642] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/21/2011] [Accepted: 01/25/2012] [Indexed: 12/21/2022]
Abstract
UNLABELLED Major vault protein (MVP) is the major constituent of vaults and is involved in multidrug resistance, nucleocytoplasmic transport, and cell signaling. However, little is known about the role of MVP during viral infections. In this study, high levels of MVP were found in peripheral blood mononuclear cells, sera, and liver tissue from patients infected with hepatitis C virus (HCV) relative to healthy individuals. HCV infections resulted in elevated levels of MVP messenger RNA (mRNA) and protein expression in the hepatocyte cell lines Huh7.5.1 and Huh7. Further studies demonstrated that the nuclear factor kappa B (NF-κB) and Sp1 pathways are involved in the induction of MVP expression by HCV. Interestingly, MVP expression suppressed HCV replication and protein synthesis by way of induction of type-I interferon mRNA expression and protein secretion. Upon investigating the mechanisms behind this event, we found that MVP enhanced the expression of interferon regulatory factor 7 (IRF7), but not IRF3. Translocation of activated IRF7 and NF-κB from the cytosol to the nucleus was involved in this process. Furthermore, vesicular stomatitis virus, influenza A virus, and enterovirus 71 also induced MVP production, and MVP in turn hampered viral replication and production. CONCLUSION MVP is a novel virus-induced host factor and its expression up-regulates type-I interferon production, leading to cellular antiviral responses.
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Affiliation(s)
- Shi Liu
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, China
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6
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Blackard JT, Ma G, Welge JA, Martin CM, Sherman KE, Taylor LE, Mayer KH, Jamieson DJ. Analysis of a non-structural gene reveals evidence of possible hepatitis C virus (HCV) compartmentalization. J Med Virol 2012; 84:242-52. [PMID: 22170544 DOI: 10.1002/jmv.22269] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Viral diversity is a hallmark of hepatitis C virus (HCV) infection; however, only limited data are available regarding HCV variability in extrahepatic sites, and none have systematically compared diversity in non-structural and structural genomic regions. Therefore, HCV diversity in the NS5B and envelope 1 (E1) hypervariable region 1 (HVR1) genes was evaluated in matched sera and peripheral blood mononuclear cells (PBMCs) obtained from 13 HCV-infected women. Multiple clonal sequences were compared to evaluate quasispecies diversity and viral compartmentalization in PBMCs. Genetic distances were higher for E1/HVR1 compared to NS5B in both the sera and PBMCs (P = 0.0511 and 0.0284). Genetic distances were higher in serum NS5B compared to PBMC NS5B (P = 0.0003); however, they were not different when comparing E1/HVR1 in sera to PBMCs. By phylogenetic analysis of NS5B, evidence of possible PBMC compartmentalization was observed for one woman, while statistical methods were consistent with PBMC compartmentalization for six women. Evidence of compartmentalization within a non-structural genomic region may suggest that viral adaptation to a unique extracellular microenvironment(s) may be required for efficient replication and could contribute to HCV persistence.
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Affiliation(s)
- Jason T Blackard
- Division of Digestive Diseases, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
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Natarajan V, Kottilil S, Hazen A, Adelsberger J, Murphy AA, Polis MA, Kovacs JA. HCV in peripheral blood mononuclear cells are predominantly carried on the surface of cells in HIV/HCV co-infected individuals. J Med Virol 2011; 82:2032-7. [PMID: 20981790 DOI: 10.1002/jmv.21906] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
HCV replication in extra-hepatic reservoirs has been suggested to occur in many tissues including PBMCs. A recent study showed evidence for compartmentalization and evolution of HCV in PBMCs. However, the cells that support HCV replication in PBMCs have not been identified. In this study we have fractionated the PBMC from HIV/HCV co-infected patients into T, monocytes, B and NK cells, and most of the HCV was located in CD3-cell fractions. Protease treatment of PBMCs to remove cell surface receptors resulted in the loss of HCV RNA suggesting that most of the HCV is present on the cell surface. PBMCs were treated by freeze-thaw nuclease method that would protect the HCV RNA in the virus but not the intracellular viral RNA. Data from this analysis support the conclusion that most of HCV is present on the cell surface. Even though the presence of minus strand RNA in PBMCs suggests that a low level HCV replication takes place within the PBMCs of HIV/HCV co-infected individuals, HCV in PBMC is present mainly on the surface of non-T cells, mostly on NK, monocytes and B cells. These results suggest a unique pathogenic role of NK, monocyte and B cells as carriers of HCV.
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Affiliation(s)
- Ven Natarajan
- Laboratory of Molecular Cell Biology, SAIC-Frederick, Inc., Frederick, Maryland, USA
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8
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Hepatitis C virus RNA quantitation in venous and capillary small-volume whole-blood samples. J Clin Microbiol 2009; 47:3231-40. [PMID: 19692560 DOI: 10.1128/jcm.00925-09] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Quantitation of hepatitis C virus (HCV) RNA in plasma and serum samples is a costly procedure in both time and reagents. Additionally, cell-associated viral RNA may not be detected. This study evaluated the accuracy of HCV RNA quantitation in small-volume whole-blood (WB) samples, which would be appropriate for point-of-care diagnostic devices. HCV RNA was extracted from 222 clinical plasma and WB samples of 82 patients with chronic hepatitis C by a specific locked nucleic acid-mediated capture method and quantified by real-time reverse transcription-PCR. The results were compared to the reference plasma viral load determined with the COBAS AmpliPrep/TaqMan (CAP/CTM) HCV test. This assay had an analytical sensitivity of 9 IU per 10-microl sample (95% limit of detection [95% LOD]), a linearity range of 500 to 5 x 10(6) IU/ml, and was accurate in testing 10 HCV subtypes (<0.22 log10 unit) in plasma. The assay was matrix equivalent for plasma and WB samples (coefficient of determination [R2] of 0.943) and had a specificity of 100% (n = 20) in WB samples. The HCV RNA concentration in clinical WB samples exceeded the estimated hematocrit-corrected plasma viral loads by 0.22 log10 unit, but absolute quantitation results in plasma and WB samples were identical (95% confidence interval, -0.06 to 0.04 log10 unit). The sensitivity in WB samples was 100% (n = 141) for plasma concentrations above the 95% LOD. Quantitation results in 10-microl WB samples correlated linearly with the CAP/CTM HCV plasma test results (R2 = 0.919; n = 140) and did not differ between capillary and venous samples (R2 = 0.960; n = 40). This study shows that HCV RNA quantitation in 10-microl WB samples is appropriate for monitoring viral loads of >900 IU/ml, although the use of WB does not increase the diagnostic sensitivity.
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9
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Mahmood-ur-Rahman, Ali I, Husnain T, Riazuddin S. RNA interference: The story of gene silencing in plants and humans. Biotechnol Adv 2008; 26:202-9. [DOI: 10.1016/j.biotechadv.2007.12.002] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2006] [Revised: 10/22/2007] [Accepted: 12/04/2007] [Indexed: 01/27/2023]
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Abstract
As the survival of HIV-infected patients has been lengthening over the past 10 years as a consequence of effective antiretroviral therapy, hepatitis C virus (HCV) coinfection has emerged as a major cause of morbidity and mortality in this population. HCV/HIV coinfection is associated with accelerated progression of liver disease, untoward effects on the immunologic and virologic response to antiretroviral medications, and possibly with a more aggressive course of HIV disease. The results of major trials of combination therapy for HCV in coinfected patients have clearly established the combination of pegylated interferon-alpha with ribavirin as the treatment of choice in this population. However, the effectiveness and tolerability of this regimen remains suboptimal, particularly in patients with genotype 1 HCV infection. This paper reviews the impact of HCV coinfection in HIV-infected patients, outlines current concepts on management and antiviral treatment, and discusses some of the newer agents, currently in the therapeutic pipeline, that are directed against novel molecular targets.
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Affiliation(s)
- Benigno Rodriguez
- Division of Infectious Diseases, University Hospitals of Cleveland, 2061 Cornell Road, Suite 401, Cleveland, OH 44106, USA.
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11
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Piccioli D, Tavarini S, Nuti S, Colombatto P, Brunetto M, Bonino F, Ciccorossi P, Zorat F, Pozzato G, Comar C, Abrignani S, Wack A. Comparable functions of plasmacytoid and monocyte-derived dendritic cells in chronic hepatitis C patients and healthy donors. J Hepatol 2005; 42:61-7. [PMID: 15629508 DOI: 10.1016/j.jhep.2004.09.014] [Citation(s) in RCA: 87] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2004] [Revised: 09/14/2004] [Accepted: 09/21/2004] [Indexed: 12/24/2022]
Abstract
BACKGROUND/AIMS Dendritic cells (DCs) play a key role in immune responses through antigen presentation and cytokine secretion. Hepatitis C virus (HCV) is able to escape elimination by the immune system and often establishes a chronic infection. To investigate whether DC dysfunction is involved in this process, we have studied monoycte-derived DCs (Mo-DCs) and plasmacytoid DCs (pDCs), which produce large amounts of IFN-alpha, from chronic HCV patients and healthy donors. METHODS We have assessed TNF-alpha and IFN-alpha production by pDCs using intracellular staining after total PBMCs stimulation with unmethylated CG dinucleotides (CpGs). The induction of allogeneic T cell proliferation by immature Mo-DCs was measured using the MLR assay. The up-regulation of maturation markers and the production of TNF-alpha in response to LPS were analyzed using flow cytometry and ELISA, respectively. RESULTS We have detected comparable frequencies of pDCs producing TNF-alpha and IFN-alpha in both chronic HCV patients and healthy donors and we have found that immature Mo-DCs from both patients and donors similarly induce allogeneic T cell proliferation and mature and secrete TNF-alpha in response to LPS. CONCLUSIONS Our results demonstrate that both pDC and Mo-DCs are not impaired in HCV infected patients.
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Affiliation(s)
- Diego Piccioli
- Immunology and Virology Department, Chiron Vaccines Research Center, via Fiorentina 1, 53100 Siena, Italy
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Shire N, Koziel MJ. Prediction of Relapse following Treatment for Hepatitis C: Is Whole Blood More than the Sum of Its Parts? Clin Infect Dis 2004; 39:1761-3. [PMID: 15578396 DOI: 10.1086/425619] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2004] [Accepted: 08/19/2004] [Indexed: 12/20/2022] Open
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Peng SH, Deng H, Feng DY, Zheng H. Expression of HSP 70 and caspase 3 and their significance in hepatocellular carcinoma tissues. Shijie Huaren Xiaohua Zazhi 2004; 12:782-784. [DOI: 10.11569/wcjd.v12.i4.782] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the expression of heat shock protein 70 (HSP70) and caspase 3 protein and their clinical significance in hepatocellular carcinomas and surrounding liver tissues.
METHODS: The expression of HSP70 and caspase 3 protein were detected by immunohistochemistry in hepatocellular carcinomas (HCC) and their surrounding liver tissues.
RESULTS: The positive rate and intensity of HSP70 in HCCs were significantly higher than those in pericarcinomatous liver tissues (68.6% vs 31.4%, P < 0.01), and these of caspase protein were significantly lower (17.1% vs 35.7%, P < 0.01). The expression level of HSP70 and caspase protein in HCCs was remarkably related to differentiation degree and tumor size of HCCs, and the poorer differentiation, the stronger the expression of HSP70 (F = 5.219 and 5.421 respectively, P < 0.01), the weaker the expression of caspase 3 protein (F = 5.944 and 4.571 respectively, P < 0.01). The correlation analysis indicated that there was a negative relationship between expression of HSP70 and caspase protein in HCC and their surrounding liver tissues (r = 0.4 126 and 0.5 237 respectively, P < 0.01).
CONCLUSION: The expression of HSP70 may make uncontrolled growth and unceasingly increased malignant degree of HCC by accelerating cell transformation and proliferation and inhibiting apoptosis. HSP70 may be an important marker for evaluation of prognosis in patients with HCC.
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Fu LJ, Lü SL, Cheng XF, Wang XY. Alterations of HCV in peripheral blood mononuclear cells from IFN-treated patients with chronic HCV RNA infection. Shijie Huaren Xiaohua Zazhi 2004; 12:610-613. [DOI: 10.11569/wcjd.v12.i3.610] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study HCV RNA and HCV protein expression in peripheral blood mononuclear cells (PBMC) and to evaluate the therapeutic efficacy of alpha 2b interferon on the treatment of HCV RNA in PBMC of patients with chronic hepatitis C.
METHODS: Fluorescence quantitative RT-PCR was used to detect the quantitation of HCV RNA in PBMC and plasma, and immunohistochemistry assay was applied to identify HCV NS3 protein expression in PBMC from 20 patients with chronic hepatitis C; Eight patients with chronic hepatitis C received IFN therapy (5 MU/d, three times/week for 16 wks). HCV RNA load in plasma and PBMC was tested with a quantitative assay before and after treatment for 16 wks and compared with that of the control. The data were analyzed by Wilcoxon 2-sample test and Fisher's exact test.
RESULTS: HCV RNA in plasma was found in 15 of 20 (75%) chronic hepatitis C patients and in PBMC was found in 9 of 20 (45%) by fluorescence quantitative RT-PCR. HCV NS3 protein expression was found in 7 of 20 (35%) chronic hepatitis C patients by immunohistochemical assay. HCV RNA loads in PBMC was uncorrelated with those in plasma.In 8 patients, there were significant differences of the decreasing of HCV-RNA loads in plasma and PBMC between before and after interferon treatment for 16 wks (aP = 0.0 017, bP = 0.0 059).The loads of HCV in plasma and PBMC in patients were significantly lower than that of controls after interferon treatment for 16 wks (cP = 0.0 042, dP = 0.0 155).
CONCLUSION: HCV can infect, replicate and express in PBMC. HCV RNA loads in PBMC is uncorrelated with those in plasma. HCV RNA in PBMC can be cleaned out by alpha 2b interferon treatment.
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Abstract
RNA interference (RNAi) is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA. The term encompasses related pathways found in a broad range of eukaryotic organisms, including fungi, plants, and animals. RNA interference is part of a sophisticated network of interconnected pathways for cellular defense, RNA surveillance, and development and it may become a powerful tool to manipulate gene expression experimentally. RNAi technology is currently being evaluated not only as an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop specific dsRNA based gene-silencing therapeutics. Several laboratories have been interested in using RNAi to control viral infection and many reports in Nature and in Cell show that short interfering (si) RNAs can inhibit infection by HIV-1, polio and hepatitis C viruses in a sequence-specific manner. RNA-based strategies for gene inhibition in mammalian cells have recently been described, which offer the promise of antiviral therapy.
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Affiliation(s)
- Quan-Chu Wang
- The Center of Diagnosis and Treatment for Infectious Diseases of Chinese PLA, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China
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Zhang L, Zhao GZ, Shi LL, Cao L. Mutations in nonstructural 5A gene of hepatitis C virus and its response to interferon alfa. Shijie Huaren Xiaohua Zazhi 2003; 11:1135-1138. [DOI: 10.11569/wcjd.v11.i8.1135] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the relationship between NS5A2209-2248 sequences and response to interferon therapy, and whether there is an IFN sensitivity determining region(ISDR) in the region of NS5A2209-2248.
METHODS We analyzed 11 patients with chronic HCV 1b infection who had received interferon alfa therapy for six months. Pretreatment serum samples were analyzed. The amino acid sequence of NS5A2209-2248 was determined by direct sequencing of the HCV genome amplified by the polymerase chain reaction.
RESULTS Among the 11 patients, only 1 was intermediate type, all the others were wild type. 2 of wild type patients showed complete response. Others were nonresponders. There was no significant difference in nucleotide and amino acid sequences between the two groups.The nucleotide and amino acid sequences changed after IFN treatment in one nonresponder.
CONCLUSION The NS5A2209-2248 region was highly conservative. In Chinease patients with chronic HCV 1b infection, there was no correlation between response to interferon and mutations in the NS5A gene. HCV quasispecies changed after IFN therapy.
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Affiliation(s)
- Lin Zhang
- Department of Infectious Disease, The Second Clinical College, China Medical University, Shenyang 110003, Liaoning Province, China
| | - Gui-Zhen Zhao
- Department of Infectious Disease, The Second Clinical College, China Medical University, Shenyang 110003, Liaoning Province, China
| | - Li-Lan Shi
- Department of Infectious Disease, The Second Clinical College, China Medical University, Shenyang 110003, Liaoning Province, China
| | - Li Cao
- Department of Infectious Disease, The Second Clinical College, China Medical University, Shenyang 110003, Liaoning Province, China
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Liang XS, Lian JQ, Zhou YX, Nie QH, Hao CQ. A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo. World J Gastroenterol 2003; 9:1008-13. [PMID: 12717847 PMCID: PMC4611363 DOI: 10.3748/wjg.v9.i5.1008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA).
METHODS: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was tansfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccinas line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay.
RESULTS: Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus.
CONCLUSION: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication.
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Affiliation(s)
- Xue-Song Liang
- The center of diagnosis and treatment for infectious diseases of Tangdu Hospital of Military Medical University of PLA, Xi'an. 710038, Shaanxi Province, China
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Abstract
AIM: To study hepatic virus C (HCV) RNA and HCV protein expression in peripheral blood mononuclear cells (PBMCs) of patients with HCV infection, and explore the relationship between the HCV RNA in the PBMCs and response to interferon (IFN) therapy.
METHODS: Type-specific primers were designed and RT-nested PCR was used to detect the plus- and minus- strands of HCV RNA in PBMCs of 54 patients with HCV infection; Indirect immunofluorescence assay was applied to identify HCVNS5 protein expression in PBMCs; 6 month-, 3 MU-IFN regiment was administrated to observe the responses to IFN in 35 chronic hepatitis C patients with different HCV RNA status in PBMCs.
RESULTS: HCV plus strand RNA was found in 10 of 19 (52.6%) acute hepatitis C patients and 22 of 35 (62.9%) chronic hepatitis C patients. HCV minus strand RNA was detected in 14 of 35 (40.0%) chronic hepatitis C patients, but only one patient (5.3%) with acute HCV infection was found to be minus HCV RNA positive. Though no HCV NS5 protein expression was found in the examined 10 cases of acute HCV infection, it was positive in 17 of 20 (85.0%) chronic hepatitis C patients by indirect immunofluoresence assay. There are significant differences of positive rate of the minus-strand and HCVNS5 protein between acute and chronic hepatitis C groups (u = 2.07, P < 0.05 and u = 4.43, P < 0.01 respectively). The patients with minus-strand HCV RNA showed a significantly lower 6-month sustained response (SR-6) to IFN compared to those without minus-strand HCVRNA in PBMCs (biologically 14.3% vs 42.8%, χ2 = 4.12, P < 0.05 and virologically 7.1% vs 23.9%, χ2 = 4.24, P < 0.05).
CONCLUSION: HCV is capable of infecting and replicating in PBMCs, and HCVNS5 protein was expressed in PBMCs. The patients with minus strand HCV RNA in PBMCs showed a significantly lower 6-month sustained response to IFN, suggesting that minus-strand HCV RNA in PBMCs may be one of the factors influencing response to IFN therapy.
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Affiliation(s)
- Guo-Zhong Gong
- Center for Liver Diseases, Second Xiangya Hospital, Xiangya Medical School, Central South University, 86 Middle Renmin Street, Changsha 410011, Hunan Province, China.
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Liang XS, Lian JQ, Zhou YX, Nie QH, Hao CQ. Inhibitory effect of IRES specific inhibitor RNA on HCV IRES mediated protein translation. Shijie Huaren Xiaohua Zazhi 2003; 11:157-160. [DOI: 10.11569/wcjd.v11.i2.157] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the inhibitory effect of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA) on HCV IRES mediated protein translation in vivo.
METHODS: Human hepatic carcinoma cell line (HHCC) was transfected with the eukaryotic vectors of IRNA or mIRNA (pcRz-IRNA or pcRz-mIRNA), and then selected with G418 for 4 weeks. HHCC expressing IRNA or mIRNA was cotransfected with pCMVNCRluc containing HCV IRES. HHCC stably expressing pcHCVcluc was transfected with pcRz-IRNA, and pcRz-mIRNA, respectively, the luciferase activity was examined at desired time post-transfection.
RESULTS: The pCMVNCRluc was efficiently suppressed in HHCC expressing IRNA rather than the cell line expressing mIRNA. The IRES specific IRNA inhibited expression of HCV IRES mediated luc gene by 20% to 80% in pcHCVcluc expressing cell after transfection; However, no inhibitory effect of the mutant IRNA was observed.
CONCLUSION: pcHCVcluc could be expressed successfully in HHCC, and IRNA inhibited HCV IRES mediated gene expression in vivo.
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Li G, Shu X, Ma HH, Chen W, Chen WS, Chen Q, Jiang YS, Yao JL. Detection of HBV, HCV and HBV YMDD mutants by DNA microarray. Shijie Huaren Xiaohua Zazhi 2003; 11:178-181. [DOI: 10.11569/wcjd.v11.i2.178] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effect of DNA microarray in detection of hepatitis B virus (HBV), hepatitis C virus (HCV) and HBV YMDD mutants.
METHODS: HBV and HCV in 40 serum samples were detected by mixed microarray and quantitative determination method as well; 20 serum samples from patients with hepatitis B treated with lamivudine were detected by microarray loaded HBV YMDD mutants gene, and were simultaneously tested with mismatched PCR and DNA sequencing for comparison.
RESULTS: The coincident rate of mixed microarray and quantitative determination of HBV DNA was 85% (34/40). The detectable rate of HBV by mixed microarray was 83% (19/23); 2 of 17 samples showed false positive reaction. The coincident rate of mixed microarray and HCV RNA quantitative determination was 85% (34/40). The detectable rate of HCV by mixed microarray was 58% (7/12). One of 28 samples showed false positive reaction. The coincident rate of HBV YMDD mutants microarray and mismatched PCR was 70% (14/20). Mixed infection of wild and mutant HBV or different mutants were detected by microarray.
CONCLUSION: Mixed microarray has high sensitivity and low non-specificity in detection of HBV, but has lower sensitivity and higher specificity in detection of HCV. Detection of HBV YMDD mutants and mixed infection with microarray had higher sensitivity and specificity.
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Zhong YW, Cheng J, Wang G, Shi SS, Li L, Zhang LX, Chen JM. Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry. World J Gastroenterol 2002; 8:863-7. [PMID: 12378631 PMCID: PMC4656576 DOI: 10.3748/wjg.v8.i5.863] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry.
METHODS: The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate. After five rounds of biopanning, 56 phage clones were identified specific to HCV E2 antigen. The selected scFv clones were digested by Sfi I/Not I and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E for expression as E-tagged soluble scFv. The liver tissue sections from normal person and patients with chronic hepatitis B and chronic hepatitis C were immunostained with HCV E2 scFv antibody.
RESULTS: The data of scFv-E2 DNA digestion and DNA sequencing showed that the scFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C E2 antigen has a specific binding character with hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with liver tissues from healthy persons or patients with chronic hepatitis B.
CONCLUSION: We have successfully screened and identified HCV E2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitis C.
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Affiliation(s)
- Yan-Wei Zhong
- Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of PLA, 26 Fengtai Road, Beijing 100039, China.
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Abstract
AIM: To investigate the dynamics of hepatitis C virus (HCV) variability through putative envelope genes during primary infection and the mechanism of viral genetic evolution in infected hosts.
METHODS: Serial serum samples prospectively collected for 12 to 34 mo from a cohort of acutely HCV-infected individuals were obtained, and a 1-kb fragment spanning E1 and the 5’ half of E2, including Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined a single-stranded conformational polymorphism (SSCP) and heteroduplex analysis (HDA) method to determine the number of clonotypes hypervariable region, was amplified by reverse transcriptase PCR and cloned. Nonsynonymous mutations per nonsynonymous site (dn), synonymous mutations per synonymous site (ds), dn/ds ratio and genetic distances within each sample were evaluated for intrahost evolutionary analysis.
RESULTS: Quasispecies complexity and sequence diversity were lower in early samples and a further increase after seroconversion, although ds value in the envelope genes was higher than dn value during primary infection. The trend, pronounced in most of samples, toward lower ds values in the E1 than in the 5' portion of E2. Quasispecies complexity was higher and E2 dn/ds ratio was a trend toward higher value in later samples during persistent viremia. We also found individual features of HCV genetic evolution in different subjects who were infected with different HCV genotypes.
CONCLUSION: Mutations of actively replicating virus arise stochastically with certain functional constaints. A complexity quasispecies exerted by a combination of either neutral evolution or selective forces shows clear differences in individuals, and associated with HCV persistence.
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Affiliation(s)
- Song Chen
- Department of Infectious Diseases, Southwest Hospital, Third Military Medical University,30 Gaotanyan Zhengjie, Shapingba District,Chongqing 400038, China.
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Zhou HC, Xu DZ, Wang XP, Zhang JX, Huang Y, Yan YP, Zhu Y, Jin BQ. Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes. World J Gastroenterol 2001; 7:583-6. [PMID: 11819836 PMCID: PMC4688680 DOI: 10.3748/wjg.v7.i4.583] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2001] [Revised: 03/05/2001] [Accepted: 03/12/2001] [Indexed: 02/06/2023] Open
Abstract
AIM To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.
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Affiliation(s)
- H C Zhou
- Department of Immunology, the Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China.
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