Hepatitis C Virus Replicates in the Same Immune Cell Subsets in Chronic Hepatitis C and Occult Infection
2008; Elsevier BV; Volume: 134; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.12.011
ISSN1528-0012
AutoresTram N. Q. Pham, Dawn King, Sonya A. MacParland, Jerry McGrath, S. Bharati Reddy, Ford Bursey, Tomasz I. Michalak,
Tópico(s)Diabetes and associated disorders
ResumoBackground & Aims: Infection of the lymphatic system by hepatitis C virus (HCV) appears to be an intrinsic characteristic of chronic hepatitis C (CHC) and low-level (occult) HCV infection, but the subsets of immune cells involved were not defined. The aim of this study was to characterize HCV replication status and to assess virus compartmentalization in CD4+ and CD8+ T lymphocytes, B cells, and monocytes in CHC, and silent infection persisting after resolution of hepatitis C. Methods: Immune cell subtypes isolated from 7 patients with CHC and 7 individuals with occult infection were analyzed for HCV-RNA–positive and –negative strands and, in selected cases, nonstructural protein 5A display and HCV variants. Results: All subtypes of immune cells investigated support HCV replication in both forms of infection, although significant differences were found between patients, and virus loads in the cells were greater in CHC than in occult infection. Although HCV RNA occurred at a comparable frequency in all cell subtypes in CHC, monocytes contained the greatest loads. In contrast, B cells tended to carry the highest virus quantities in occult infection, whereas monocytes appeared to be the least frequently infected. Detection of HCV nonstructural protein 5A and HCV variants that were not found in plasma confirmed virus replication in different immune cell types. Conclusions: This work documents that the immune system supports HCV replication regardless of clinical appearance of infection and identifies immune cells that are reservoirs of HCV in symptomatic and occult infections. Background & Aims: Infection of the lymphatic system by hepatitis C virus (HCV) appears to be an intrinsic characteristic of chronic hepatitis C (CHC) and low-level (occult) HCV infection, but the subsets of immune cells involved were not defined. The aim of this study was to characterize HCV replication status and to assess virus compartmentalization in CD4+ and CD8+ T lymphocytes, B cells, and monocytes in CHC, and silent infection persisting after resolution of hepatitis C. Methods: Immune cell subtypes isolated from 7 patients with CHC and 7 individuals with occult infection were analyzed for HCV-RNA–positive and –negative strands and, in selected cases, nonstructural protein 5A display and HCV variants. Results: All subtypes of immune cells investigated support HCV replication in both forms of infection, although significant differences were found between patients, and virus loads in the cells were greater in CHC than in occult infection. Although HCV RNA occurred at a comparable frequency in all cell subtypes in CHC, monocytes contained the greatest loads. In contrast, B cells tended to carry the highest virus quantities in occult infection, whereas monocytes appeared to be the least frequently infected. Detection of HCV nonstructural protein 5A and HCV variants that were not found in plasma confirmed virus replication in different immune cell types. Conclusions: This work documents that the immune system supports HCV replication regardless of clinical appearance of infection and identifies immune cells that are reservoirs of HCV in symptomatic and occult infections. Hepatitis C virus (HCV) infects more than 170 million people worldwide and causes chronic hepatitis C (CHC) in up to 85% of patients. The induced CHC often advances to cirrhosis and hepatocellular carcinoma, and is accompanied at a higher frequency by disorders of the lymphatic system, including type II mixed cryoglobulinemia and non-Hodgkin's lymphoma.1Blackard J.T. Kemmer N. Sherman K.E. Extrahepatic replication of HCV: insights into clinical manifestations and biological consequences.Hepatology. 2006; 44: 15-22Google Scholar HCV is a single-stranded RNA virus that replicates via synthesis of a negative strand. Within the genome, the 5′-untranslated region (5′-UTR) is most conserved and serves as the internal ribosomal entry site (IRES) essential to viral RNA translation.2Bartenschlager R. Lohmann V. Replication of hepatitis C virus.J Gen Virol. 2000; 81: 1631-1648Crossref Scopus (589) Google Scholar The presence of HCV quasispecies is a direct consequence of rapid replication driven by an error-prone polymerase.2Bartenschlager R. Lohmann V. Replication of hepatitis C virus.J Gen Virol. 2000; 81: 1631-1648Crossref Scopus (589) Google Scholar Limited evidence suggesting that HCV variants in cells of the immune system in patients with CHC are distinct from those in the plasma or liver 3Laporte J. Bain C. Maurel P. et al.Differential distribution and internal translation efficiency of hepatitis C virus quasispecies present in dendritic and liver cells.Blood. 2003; 101: 52-57Google Scholar, 4Roque-Afonso A.M. Ducoulombier D. Di Liberto G. et al.Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells.J Virol. 2005; 79: 6349-6357Google Scholar, 5Navas S. Martin J. Quiroga J.A. et al.Genetic diversity and tissue compartmentalization of the hepatitis C virus genome in blood mononuclear cells, liver and serum from chronic hepatitis C patients.J Virol. 1998; 72: 1640-1646Crossref Google Scholar implies that cellular milieu may promote propagation of tissue-specific virus variants.4Roque-Afonso A.M. Ducoulombier D. Di Liberto G. et al.Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells.J Virol. 2005; 79: 6349-6357Google Scholar, 6Ducoulombier D. Roque-Afonso A.-M. Di Liberto G. et al.Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes.Hepatology. 2004; 39: 817-825Google Scholar The pathogenic relevance of this compartmentalization remains unknown. The notion that HCV is a lymphotropic virus is supported by findings of replicating HCV found in B cells,7Pal S. Sullivan D.G. Kim S. et al.Productive replication of hepatitis C virus in perihepatic lymph nodes in vivo: implications of HCV lymphotropism.Gastroenterology. 2006; 130: 1107-1116Abstract Full Text Full Text PDF Scopus (137) Google Scholar T cells,8MacParland S.A. Pham T.N.Q. Gujar S.A. et al.Infection and propagation of wild-type hepatitis C virus in human T lymphocytes in vitro.J Gen Virol. 2006; 87: 3577-3586Google Scholar and peripheral blood mononuclear cells (PBMCs) from patients with CHC.1Blackard J.T. Kemmer N. Sherman K.E. Extrahepatic replication of HCV: insights into clinical manifestations and biological consequences.Hepatology. 2006; 44: 15-22Google Scholar, 4Roque-Afonso A.M. Ducoulombier D. Di Liberto G. et al.Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells.J Virol. 2005; 79: 6349-6357Google Scholar, 9Kao J.H. Chen P.J. Lai M.Y. et al.Positive and negative strand of hepatitis C virus RNA sequences in peripheral blood mononuclear cells in patients with chronic hepatitis C: no correlation with genotypes 1b, 2a and 2b.J Med Virol. 1997; 52: 270-274Google Scholar, 10Pham T.N.Q. Mulrooney-Cousins P.M. Mercer S.E. et al.Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection.J Viral Hepat. 2007; 14: 537-548Google Scholar Recently, occult HCV infection, defined as the presence of low levels of HCV genomes in serum, PBMCs, and/or liver in the absence of clinically evident liver disease, was identified in patients years after apparent complete resolution of hepatitis C.11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar, 12Radkowski M. Gallegos-Orozco J.F. Jablonska J. et al.Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C.Hepatology. 2005; 41: 106-114Google Scholar, 13Castillo I. Rodriguez-Inigo E. Lopez-Alcorocho J.M. et al.Hepatitis C virus replicates in the liver of patients who have a sustained response to antiviral treatment.Clin Infect Dis. 2006; 43: 1277-1283Google Scholar, 14Di Liberto G.D. Roque-Afonso A.M. Kara R. et al.Clinical and therapeutic implications of hepatitis C virus compartmentalization.Gastroenterology. 2006; 131: 76-84Abstract Full Text Full Text PDF Scopus (82) Google Scholar However, it was undefined as to which immune cells support HCV replication and whether distinct HCV variants may occur in these cells in CHC and occult infection. Previously, we showed that ex vivo stimulation of lymphoid cells from individuals with occult HCV infection profoundly augmented HCV replication, allowing for efficient virus detection,10Pham T.N.Q. Mulrooney-Cousins P.M. Mercer S.E. et al.Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection.J Viral Hepat. 2007; 14: 537-548Google Scholar, 11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar, 15Pham T.N.Q. MacParland S.A. Coffin C.S. et al.Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells.J Gen Virol. 2005; 86: 657-666Google Scholar although such an increase was not universal.10Pham T.N.Q. Mulrooney-Cousins P.M. Mercer S.E. et al.Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection.J Viral Hepat. 2007; 14: 537-548Google Scholar In fact, when HCV load was about 103–104 virus genome equivalents (vge)/107 cells, as it typically is seen in CHC, mitogen stimulation caused a swift decrease or even elimination of HCV expression.10Pham T.N.Q. Mulrooney-Cousins P.M. Mercer S.E. et al.Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection.J Viral Hepat. 2007; 14: 537-548Google Scholar This finding was associated consistently with heightened endogenous transcription of interferon-alfa (IFN-alfa) and myxovirus (influenza) resistance A (MxA).10Pham T.N.Q. Mulrooney-Cousins P.M. Mercer S.E. et al.Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection.J Viral Hepat. 2007; 14: 537-548Google Scholar In the current study, HCV replication status was assessed in individual immune cell subsets isolated from patients with CHC or occult infection continuing after resolution of hepatitis C. We found that monocytes, B cells, and CD4+ and CD8+ T lymphocytes can support HCV propagation in both forms of infection, although different subsets were infected to varying degrees in different patients. Furthermore, sequence analysis of the HCV IRES from the immune cells in CHC and occult infection revealed variants that were distinct from those in sera, suggesting that IRES polymorphism may have a role in virus persistence in the lymphatic system. Overall, our study provides definitive proof that the immune system supports active HCV replication regardless of the clinical appearance of infection and identifies immune cell subsets that serve as reservoirs of replicating HCV in symptomatic and occult infections. Fourteen randomly selected patients were investigated (Table 1). Seven patients had CHC as diagnosed by virologic, biochemical, and histologic criteria. Of the 7 individuals who terminated hepatitis, 2 spontaneously recovered and 5 resolved CHC after achieving a sustained virologic response (SVR) after pegylated IFN-alfa and ribavirin therapy. At the time of study, these 7 individuals had been followed up for 5–31 months (Table 1). In all patients, serum HCV RNA was repeatedly negative by Roche Amplicor HCV v2.0 assay (sensitivity, 500 IU/mL or 1000 vge/mL; Roche Molecular Diagnostics, Pleasanton, CA) and liver function tests were normal.Table 1Clinical and Virologic Characteristics of Patients With CHC or Occult HCV InfectionCase/sampleAge, y/sexHCV genotypeType/duration of antiviral treatment, moFollow-up period after SVR to treatment, moHCV RNA in plasma,aHCV RNA measured by real-time RT-PCR (sensitivity, ∼100 vge/mL) or by nested RT-PCR/NAH (sensitivity, <10 vge/mL) using RNA extracted from 250 μL plasma, unless otherwise stated.vge/mLCHC 155/F3aNoneNA1.7 × 105 252/FNANoneNA5.2 × 105 3abTwo samples were available for analysis.50/F1bNoneNA8.3 × 105 3bbTwo samples were available for analysis.1bAs aboveNA5 × 105 450/F2bNoneNA5.3 × 107 549/M3aNoneNA1.6 × 107 641/M3aNoneNA2.0 × 104 724/M1aNoneNA5 × 102 HCV-RNA positivity/total tested (%)7/7 (100)Resolved hepatitis C 823/F1bNANA<40cRNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. 9NANANANA0cRNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. 10abTwo samples were available for analysis.61/F1aP-IFN/R (12)24150 10bbTwo samples were available for analysis.1aAs above310cRNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. 1151/M2bP-IFN/R (6)12<40cRNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. 12abTwo samples were available for analysis.38/M2bP-IFN/R (6)6<40cRNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. 12bbTwo samples were available for analysis.2bAs above1580cRNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. 13abTwo samples were available for analysis.40/M3aP-IFN/R (6)6<40cRNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. 13bbTwo samples were available for analysis.3aAs above150cRNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. 1446/M1aP-IFN/R (12)580 HCV-RNA positivity/total tested (%)7/10 (70)NA, not applicable; P-IFN, pegylated IFN-alfa; R, ribavirin.a HCV RNA measured by real-time RT-PCR (sensitivity, ∼100 vge/mL) or by nested RT-PCR/NAH (sensitivity, <10 vge/mL) using RNA extracted from 250 μL plasma, unless otherwise stated.b Two samples were available for analysis.c RNA extracted from a pellet recovered after ultracentrifugation of 4 mL plasma. Open table in a new tab NA, not applicable; P-IFN, pegylated IFN-alfa; R, ribavirin. Plasma and PBMCs were obtained from the same blood sample for all patients. In addition, a second pair of plasma and PBMCs (referred to as sample b in Table 1) was acquired 7–9 months after the first from 1 patient with CHC (case 3) and 3 with SVR (cases 10, 12, and 13). The study was approved by the local Human Investigation Committee and samples were collected after informed consent had been obtained. PBMCs from patients and healthy volunteers (n = 5) were isolated by Ficoll-HyPaque (Pharmacia Biotech, Quebec, Canada) gradient centrifugation.11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar Approximately 1 × 107 PBMCs were cryopreserved for HCV-RNA analyses. CD8+, CD4+ T cells and monocytes were isolated successively from fresh PBMCs by positive selection on microbeads coated with anti-human CD8 monoclonal antibody (mAb), anti-CD4 mAb, and anti-CD14 mAb, respectively (Miltenyi Biotec Inc., Auburn, CA). The leftover fraction contained more than 95% B cells, as determined by flow cytometry with anti-CD19 mAb (data not shown). CD4+ and CD8+ T cells and monocytes were more than 98% pure (data not shown). PBMCs, CD4+ T cells, and, when feasible, CD8+ T cells were cultured for 72 hours with T-cell activating mitogen phytohemagglutinin (PHA; Sigma, Mississauga, Canada) and interleukin-2 (IL-2; Roche).11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar Because of insufficient cell numbers, B cells and monocytes were not stimulated before examination. RNA was extracted from PBMCs or immune cell subsets using Trizol (Invitrogen Life Technologies, Burlington, Canada) and from plasma using Trizol LS (Invitrogen).11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar When HCV RNA was not detectable in 250 μL plasma, 4 mL of plasma was ultracentrifuged for 20 hours at 40,000 rpm using the SW-55T1 rotor and Sorvall Discovery 100SE ultracentrifuge (Mandel Scientific Company Inc., Guelph, Canada). RNA was extracted from the pellet recovered. RNA from lymphoid cells (1–2 μg RNA; equivalent of 1–2 × 106 cells) or plasma (equivalent of 250 μL or 4 mL) was reversely transcribed, and complementary DNA (cDNA) was subjected to polymerase chain reaction (PCR)/nucleic acid hybridization (NAH) using 5′ UTR-specific primers and conditions described elsewhere.11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar Ten-fold serial dilutions of HCV synthetic RNA (sRNA) were used as quantitative standards. The sensitivity of this assay was less than 10 vge/mL (≤2 IU/mL). HCV-RNA–negative strand was determined by strand-specific reverse-transcription (RT)-PCR/NAH using recombinant Thermus thermophilus (Promega Corporation, Madison, WI) DNA polymerase.11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar Ten-fold serial dilutions of HCV-sRNA–negative strand were used as semiquantitative standards, whereas those of HCV-sRNA–positive strand served as specificity control. This assay detects approximately 100 vge of the correct (negative) strand, while nonspecifically detecting greater than 106 vge of the positive strand.11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar In all reactions, a sample containing water instead of test cDNA and a mock were included as contamination controls, and cDNA from PBMCs of a healthy donor served as a negative control. The specificity of PCR amplicons and the validity of the controls were confirmed by NAH (Southern blot hybridization) using 32Melon S. Galarraga M.C. Villar M. et al.Hepatitis C virus reactivation in anti-hepatitis C virus positive renal transplant recipients.Transplant Proc. 2005; 37: 2083-2085Google ScholarP-labeled recombinant HCV UTR-E2 as a probe.11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar HCV RNA in plasma and PBMCs was quantified, when feasible, by real-time RT-PCR.11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar However, when HCV quantification was not possible by direct real-time RT-PCR, cDNA was subjected to nested real-time RT-PCR.12Radkowski M. Gallegos-Orozco J.F. Jablonska J. et al.Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C.Hepatology. 2005; 41: 106-114Google Scholar Briefly, test cDNA along with 10-fold serial dilutions of recombinant HCV UTR-E2 plasmid were amplified for 20 cycles by conventional PCR using primers and cycling conditions reported before.11Pham T.N.Q. MacParland S.A. Mulrooney P.M. et al.Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Google Scholar Then, 2 μL of the product was used as template for real-time PCR. Although this method is more sensitive than the direct real-time RT-PCR, allowing for accurate measurement of HCV loads between 10 and 100 vge, its sensitivity does not supersede that of our nested RT-PCR/NAH (ie, <10 vge/mL). Freshly isolated and, in certain cases, mitogen-treated PBMCs or affinity-purified immune cells from patients with CHC or occult infection and Huh7 cells as controls (naive or carrying HCV AB12-A2 replicon; kindly provided by Drs. Joyce Wilson and Christopher Richardson, Ontario Cancer Institute, Canada) were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 10% normal goat serum, and double-stained with rat antitubulin (Chemicon International, Temecula, CA) and with either mouse anti-HCV nonstructural protein 5A (NS5A) mAb (Chemicon) or mouse isotype control (BD Biosciences, Mississauga, Canada). Subsequently, cells were incubated with Cy2-labeled donkey anti-mouse and Cy5-conjugated donkey anti-rat antibodies (both from Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and examined under an Olympus BX50WI microscope with a FlouView FV300 confocal system (both from Olympus America Inc., Melville, NY). 5′-UTR amplicons were cloned using the TOPO-TA cloning system (Invitrogen). The highly conserved 5′-UTR was chosen because it would allow for reliable identification of lasting compartment-specific variants. Nine to 10 clones from each PCR product were sequenced bidirectionally using universal forward and reverse M13 primers, and samples were read on an Applied Biosystems 3730 XL sequencer (Applied Biosystems Canada, Streetsville, Canada). Sequence analysis was performed using Sequencher software version 4.7 (Gene Codes Corp., Ann Arbor, MI) and Biology Workbench version 3.2 (Department of Bioengineering, University of California at San Diego, San Diego, CA, available at: http://workbench.sdsc.edu). Data were analyzed using GraphPad Prism 4 (San Diego, CA) software. Statistical significance was evaluated by 2-tailed Mann–Whitney tests and P values less than .05 were considered significant. HCV-RNA load was presented as mean ± SD vge/μg total RNA, unless otherwise stated. To determine which immune cell subsets constitute reservoirs of replicating HCV in CHC, CD4+ and CD8+ T cells, B lymphocytes, and monocytes were isolated to high purity and examined for HCV-RNA–positive and replicative strands. PBMCs, CD4+ T cells, and, when possible, CD8+ T cells were analyzed fresh and after treatment with PHA/IL-2, whereas B cells and monocytes only were analyzed fresh. Nonstimulated PBMCs from all patients were reactive for positive and negative strands with average loads of 2.2 × 103 ± 3.4 × 103 and 75 ± 32 vge/μg RNA, respectively (Table 2). As in our recent study of lymphoid cells with high HCV loads,10Pham T.N.Q. Mulrooney-Cousins P.M. Mercer S.E. et al.Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection.J Viral Hepat. 2007; 14: 537-548Google Scholar HCV-RNA expression in treated PBMCs was decreased profoundly (up to 94%) in 2 cases and completely inhibited in 4 others (Figure 1), leading to overall detection of positive and negative strands in 43% (3 of 7) and 50% (1 of 2) of samples tested, respectively (Table 2). Among the cases studied, case 3 was unique in that although expression of HCV-RNA–positive strand in the PBMC sample was eliminated after mitogen treatment (Figure 1A), that in sample b collected 9 months later was augmented by approximately 100-fold after stimulation (Table 2).Table 2Detection of HCV-RNA–Positive and HCV-RNA–Negative Strands in Individual Immune Cell Subsets From Patients With CHC and Occult HCV InfectionCategory of infection/samplePBMCsCD4+ T cellsCD8+ T cellsB cellsMonocytesCase overall HCV positivityaDefined by the presence of HCV RNA in serum, PBMCs, and/or any immune cell subtype, after cells were treated or not with PHA/IL-2.UTTUTTUTTUTUTHCV-RNA–positive strand/HCV-RNA–negative strandbHCV-RNA–positive strand was analyzed by direct and, if necessary, nested real-time RT-PCR or RT-PCR/NAH, whereas HCV-RNA–negative strand was analyzed by RT-PCR/NAH as described in the Materials and Methods section. HCV load is expressed in vge/μg total RNA.CHC 1500/10030/010/NA0/NT20/NA0/NT500/100cPurification of B cells and monocytes was not feasible and the total non–T-cell fraction was analyzed.+ 2104/250/NT0/NT0/NT0/NT0/NT10/0105/104+ 3a4 × 103/1000/NT104/1000/NT3 × 103/25NA103/25104/500+ 3b103/100105/102103/50NA104/102NA125/50100/0 4103/500/NT104/1000/NT100/0NA750/100750/25+ 5100/100NA10/00/NT20/NANA50/NAcPurification of B cells and monocytes was not feasible and the total non–T-cell fraction was analyzed.+ 6500/NA100/NA100/NA20/NA250/Na20/NA103/NAcPurification of B cells and monocytes was not feasible and the total non–T-cell fraction was analyzed.+ 7200/500/NT0/NT0/NT0/NTNA750/NA0/NT+ % positivity of samples tested100/10043/5075/7514/NA75/7533/Na100/75dPercentage of HCV-RNA positivity was determined taking under consideration the number of only cases in which purified B cells and monocytes were analyzed.80/75dPercentage of HCV-RNA positivity was determined taking under consideration the number of only cases in which purified B cells and monocytes were analyzed.100Resolved hepatitis C 80/NT50/00/NT0/NT0/NT10/0500/250/NT+ 90/NT0/NT0/NA50/250/NTNA10/00/NT+ 10a0/NT50/00/NT250/250/NTNA0/NT0/NT+ 10b0/NT0/NT0/NT50/250/NT50/250/NT0/NT 110/NT0/NT0/NT0/NT0/NT0/NT0/NT0/NT+ 12a0/NT75/250/NT0/NT100/0NA200/500/NT+ 12b0/NT0/NT0/NT10/050/25NA100/250/NT 13a0/NT0/NT0/NT100/250/NTNA0/NT105/25+ 13b100/250/NT0/NT0/NT10/0NA0/NT10/0 140/NT100/250/NT200/250/NT150/50100/10100/10+ % positivity of samples tested10/10040/500/NA60/8330/3375/7550/8030/67100UT, untreated; T, treated with PHA/IL-2; NA, not available; NT, not tested or not applicable.a Defined by the presence of HCV RNA in serum, PBMCs, and/or any immune cell subtype, after cells were treated or not with PHA/IL-2.b HCV-RNA–positive strand was analyzed by direct and, if necessary, nested real-time RT-PCR or RT-PCR/NAH, whereas HCV-RNA–negative strand was analyzed by RT-PCR/NAH as described in the Materials and Methods section. HCV load is expressed in vge/μg total RNA.c Purification of B cells and monocytes was not feasible and the total non–T-cell fraction was analyzed.d Percentage of HCV-RNA positivity was determined taking under consideration the number of only cases in which purified B cells and monocytes were analyzed. Open table in a new tab UT, untreated; T, treated with PHA/IL-2; NA, not available; NT, not tested or not applicable. In regard to CD4+ T cells, although HCV-RNA–positive strand was identified before stimulation in 75% (6 of 8) of samples with an average load of 2.6 × 103 ± 4.5 × 103 vge/μg RNA, only 14% (1 of 7) of them remained positive after mitogen treatment. Similarly, HCV-RNA–negative strand in untreated CD4+ T cells was found in 75% (3 of 4; 63 ± 48 vge/μg RNA) of samples tested (Table 2). In CD8+ T cells, of 75% (6 of 8) of samples carrying HCV-positive strand at 1.7 × 103 ± 3.5 × 103 vge/μg RNA, 75% (2 of 3) of those tested were reactive for the negative strand. As in PBMCs and CD4+ T cells, PHA/IL-2 stimulation down-regulated HCV-RNA expression in CD8+ T lymphocytes. When B cells and monocytes from patients with CHC were evaluated for HCV RNA, positive strand was detected in 100% (5 of 5; 5.3 × 102 ± 4.3 × 102 vge/μg RNA) and 80% (4 of 5; 2.2 × 104 ± 4.4 × 104 vge/μg RNA) of the samples, respectively. Further, the replicative intermediates were found in 75% (3 of 4) of samples of both B cells (44 ± 43 vge/μg RNA) and monocytes (2.6 × 103 ± 4.9 × 103 vge/μg RNA) that were positive-strand reactive. In cases in which only the non–T fraction was available for analysis, all samples tested were positive for HCV (Table 2). The data also showed that different immune cells support HCV to a varying extent in different patients with CHC. For example, although HCV-RNA–positive strand was found only in B cells and monocytes from case 2, it was readily detectable in all 4 cell subtypes from case 3a (Figure 1A). Importantly, except for B cells from case 2, monocytes from case 3b and fresh CD8+ T lymphocytes from case 4, the presence of HCV-RNA–positive strand always was accompanied by that of the negative strand (Figure 1B and Table 2). Furthermore, HCV-RNA expression was either reduced drastically or abolished in PBMCs, CD4+ T cells, or, when tested, CD8+ T cells after mitogen stimulation in all samples, with the exception of PBMCs from case 3b (Figure 1A and Table 2). In contrast to mitogen-induced inhibition of HCV-RNA expression in CHC, PHA/IL-2 treatment of lymphoid cells enhanced virus detection in occult infection (Table 2 and Figure 2). As shown (Table 2 and Figure 2A), although fresh PBMCs and CD4+ T cells from cases 8 to 14 (except case 13b) were seemingly HCV negative, the virus-positive strand was readily detectable after stimulation. The estimated average load in fresh and stimulated PBMCs and CD4+ T cells was 37.5 ± 43 and 66 ± 91 vge/μg RNA, respectively. Notably, the positive strand detection was accompanied almost universally by that of the negative strand, confirming enhanced HCV replication (Figure 2B and Table 2). In CD8+ T cells and B cells, HCV-positive strand was detected at levels of 37 ± 52 and 92 ± 16 × 102 vge/μg RNA, respectively. The negative strand was detected more frequently in naive B cells (4 of 5; 22 ± 19 vge/μg RNA) than in CD8+ T cells (3 of 6 of fresh and stimulated samples tested; 17 ± 20 vge/μg RNA). In addition, although HCV RNA was detected frequently and at high levels in monocytes in CHC, it was present in only 3 of 10 samples (22 ± 43 vge/μg RNA) in individuals with occult infection (Table 2). In some instances, mitogen-treated CD4+ T cells (cases 10b, 12b, and 13a), naive B cells (case 12b), monocytes (case 13a), and CD8+ T cells (case 10b), but not PBMCs, mitogen-treated or not, were HCV-RNA positive, suggesting possible
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