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Originally published online as doi:10.1189/jlb.0106047 on May 30, 2006

Published online before print May 30, 2006
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(Journal of Leukocyte Biology. 2006;80:298-308.)
© 2006 by Society for Leukocyte Biology

Dynamics of CD81 expression on lymphocyte subsets during interferon-{alpha}-based antiviral treatment of patients with chronic hepatitis C

Bernd Kronenberger*,1, Eva Herrmann*, W. Peter Hofmann*, Heiner Wedemeyer{dagger}, Martina Sester{ddagger}, Ulrike Mihm*, Temam Ghaliai*, Stefan Zeuzem* and Christoph Sarrazin*,1

* Klinik für Innere Medizin II and
{ddagger} IV, Universitätsklinikum des Saarlandes, Homburg/Saar, Germany; and
{dagger} Medizinische Hochschule Hannover, Germany

1Correspondence: Klinik für Innere Medizin II, Universitätsklinikum des Saarlandes, Kirrberger Strasse, D-66421 Homburg/Saar, Germany. E-mails: bernd.kronenberger{at}email.de and christoph.sarrazin{at}uniklinik-saarland.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD81 is a hepatitis C virus (HCV) coreceptor with important functions in lymphocytes. During treatment, CD81 expression may be changed directly by the antiviral therapy or indirectly by reduction of the HCV serum level. The regulation of CD81 on lymphocyte subtypes has not been investigated so far and may be relevant for the control of viral infection and treatment response. CD81 was analyzed by flow cytometry in CD8(+), CD4(+), CD19(+), and CD56(+) lymphocyte subtypes from 20 patients with chronic hepatitis C before, during, and after antiviral treatment with pegylated interferon-{alpha} (IFN-{alpha}) and ribavirin. A sustained virologic response (SVR) was achieved in 11 patients. Dynamics of CD81 were investigated in correlation with HCV-RNA dynamics and the outcome of therapy. During treatment, the following typical patterns of CD81 regulation were observed: down-regulation on CD8(+) T cells (P=0.022) and most significantly, on CD56(+) natural killer cells (P<0.001), transient up-regulation on CD19(+) B cells (P=0.006), and weak and late down-regulation on CD4(+) T cells (P=0.028). During treatment, CD81 expression was not associated with the HCV-RNA serum level on all lymphocyte subtypes. After end of treatment, CD81 increased again in CD8(+) and CD56(+) cells (P=0.001, P=0.002). On CD8(+) T cells post-treatment, CD81 remained lower in patients who achieved a SVR compared with patients who failed to eliminate HCV after treatment (P=0.033). Lymphocyte subsets show different patterns of CD81 response before and during antiviral treatment, which are associated with administration of IFN-{alpha} and antiviral response.

Key Words: host-cell interaction • antiviral immune response


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis C virus (HCV) is an enveloped, positive-stranded RNA virus, which preferentially replicates in hepatocytes with continuous de novo infection and a high turnover of infected cells [1 , 2 ]. Patients with chronic hepatitis C show a characteristic dynamic equilibrium between virus production and clearance [3 , 4 ]. Chronic hepatitis C often causes necroinflammation and fibrosis in the liver and frequently progresses to liver cirrhosis and its sequelae [5 ]. Despite a large number of activated T cells in the liver of patients with chronic hepatitis C, only small amounts of HCV-specific T cells can be detected, which in addition, show an impairment of their functional properties [6 7 8 ].

HCV-RNA has been detected in peripheral blood lymphocytes (PBL), mainly CD8(+) and CD19(+) cells, suggesting that HCV is also a lymphotropic virus [9 10 11 ]. The lymphotropism of HCV may be responsible for the impaired, specific immune response and the development of autoimmune diseases in patients with chronic hepatitis C [12 13 14 15 16 17 ]. Recently, CD81 was detected as an essential but not sufficient HCV receptor for infection of hepatocytes [13 , 18 ]. Furthermore, binding of the HCV structural E2 protein to CD81 on B cells was demonstrated, providing a mechanism by which HCV may alter the activation status of lymphocytes [12 13 14 5 16 17 ]. Conclusive data about the interaction between HCV-E2 and CD81 in vitro, leading to a lowered activation threshold for B and T lymphocytes and inhibition of natural killer (NK) cells, have been presented [12 , 13 , 15 , 19 20 21 22 ].

In patients with chronic hepatitis C, an overexpression of CD81 on total PBL [23 ], CD19(+) [24 ], and CD5(+) B cells [25 ] was shown. The level of CD81 expression could be associated with infection of target cells and the alteration of their functional properties. During treatment with interferon-{alpha} (IFN-{alpha}), significant down-regulation of CD81 on total PBL was observed in patients with an initial virologic response [23 ]. In addition, a decline of CD81 expression on CD5(+) B cells after the end of treatment (EOT), with respect to pretreatment levels, was observed in patients with a sustained virologic response (SVR), and in nonresponders (NR) overexpression of CD81 was unchanged [25 ].

The expression profile of CD81 on the different major subtypes of lymphocytes in patients with chronic hepatitis C during IFN-{alpha}-based antiviral treatment is unknown. The aim of the present study was to compare the dynamics of CD81 expression on CD8(+), CD4(+), CD19(+), and CD56(+) lymphocyte subtypes during IFN-{alpha}-based antiviral treatment in vivo. During treatment, CD81 expression may be changed directly by IFN-{alpha}-based antiviral therapy or indirectly, by reduction of the viral load. Therefore, CD81 expression on lymphocyte subtypes was correlated with the corresponding HCV-RNA levels before and during treatment. Furthermore, CD81 on lymphocyte subtypes in correlation with treatment outcome was investigated. To analyze the association between CD81 on lymphocyte subtypes and virologic response, CD81 expression was correlated with the infected cell loss and the efficacy of antiviral treatment derived from mathematical viral kinetic analysis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
In the present study, 20 patients with chronic hepatitis C were investigated. Patients’ characteristics are shown in Table 1 . The diagnosis of chronic hepatitis C was based on the consistent detection of HCV-RNA (≥6 months), positive anti-HCV antibodies by third-generation enzyme immunoassay, and histologic examination, which was performed by an experienced, local pathologist. Grading and staging were semiquantitatively assessed, according to Knodell et al. [26 ]. All patients were negative for hepatitis B surface antigen and antibodies to the human immunodefiency virus types 1 and 2. Patients with liver disease of different origin, autoimmune disease, consumption of more than 20 g/day alcohol, and past and present intravenous drug abuse and patients with decompensated liver disease were excluded. All patients were treated with a combination of pegylated IFN-{alpha}-2a 180 µg once weekly and ribavirin (800–1200 mg, Hoffmann-La Roche, Basel, Switzerland) for 24–48 weeks in the outpatient clinic of the University of Frankfurt (Germany). Eight healthy, nonmatched volunteers, without consumption of drugs and alcohol and without liver diseases, served as controls (Table 1) . All patients gave written, informed consent, and the Ethics Committee of Medical Research in Frankfurt gave approval in accordance with the 1975 Declaration of Helsinki.


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  		Table 1. Patients’ Characteristics

 
Detection and quantification of HCV-RNA
The HCV-RNA serum level before and during treatment was determined using a quantitative RT-PCR chain-reaction assay (Cobas Amplicor HCV MonitorTM 2.0, Roche Diagnostic Systems, Branchburg, NJ) with a lower detection limit of 600 IU/mL. HCV-RNA detection after treatment was performed with a qualitative assay and a lower detection limit of 50 IU/ml (Cobas AmplicorTM HCV, Roche Diagnostic Systems). Patients who had undetectable HCV-RNA for at least 6 months post-treatment were classified as SVR, and those who had detectable HCV-RNA post-treatment, as NR. HCV genotyping was performed by reverse hybridization assay (Inno LiPA HCV II, Innogenetics, Gent, Belgium).

Quantification of CD81 expression on lymphocyte subsets
PBL were isolated at baseline before treatment (pre), 4 weeks and 12 weeks after beginning of treatment (TW4, TW12, respectively), at the EOT, and during follow-up between 12 and 24 weeks after EOT (post). For isolation, blood from patients with chronic hepatitis C was collected in sterile collection tubes containing heparin. Blood was diluted in phosphate-buffered saline (PBS), and lymphocytes were isolated by Ficoll gradient and centrifugation at 1000 g. The cell suspension was washed three times in PBS and stored in liquid nitrogen until use. For staining, cells were incubated with 20 µl R-phycoerythrin (PE)-conjugated anti-CD81 antibodies [Clone JS-81, mouse anti-human immunoglobulin G (IgG), BD PharMingen, San Diego, CA]. For labeling of lymphocyte subsets, peripheral blood mononuclear cells (PBMC) were costained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (Clone RPA-T4, mouse anti-human IgG, BD PharMingen), anti-CD8 (Clone HIT8a, mouse anti-human IgG, BD PharMingen), anti-CD19 (Clone 4G7, mouse anti-human, BD PharMingen), and anti-CD56 antibodies (Clone NCAM 16.2, mouse anti-human IgG, BD PharMingen). For characterization of lymphocyte subsets according to CD3 expression, cells were stained with a combination of allophycocyanin (APC)-conjugated anti-CD3 (Clone SK7, mouse anti-human, BD PharMingen), PE-conjugated anti-CD81 (Clone JS-81), FITC-conjugated anti-CD56 (NCAM 16.2), and peridinin chlorophyll protein (PerCP)-conjugated anti-CD8 antibodies (Clone SK1, mouse anti-human, BD PhaMingen). Staining of lymphocytes with FITC-conjugated anti-CD8 Clone HIT8a and PerCP-conjugated anti-CD8 Clone SK1 resulted in a similar labeling of CD8(+) lymphocytes. For analysis of CD5-expressing B cells, PBMC were stained with a combination of PE-conjugated anti-CD5 (Clone L17F12, mouse anti-human, BD PharMingen), FITC-conjugated anti-CD81 (Clone JS-81), and PerCP-conjugated CD19 antibodies (Clone SJ25C1, mouse anti-human IgG, BD PharMingen). Staining of lymphocytes with FITC-conjugated anti-CD19 Clone 4G7 and PerCP-conjugated anti-CD19 Clone SJ25C1 resulted in a similar labeling of CD19(+) lymphocytes. PBMC were incubated with the respective antibodies for 30 min at 4°C and subsequently washed with PBS twice. For each time-point, the same amount of antibody was used. Mean fluorescence intensity (MFI) of CD81 was analyzed with a fluorescence-activated cell sorter (FACSCalibur, Becton Dickinson, Heidelberg, Germany). FITC-, PE-, PerCP-, and APC-conjugated mouse IgG antibodies (BD PharMingen) were used as isotype controls for the quantification of background fluorescence. All time-points of lymphocyte preparations relating to one patient were stained and quantified on the same day.

Mathematical model and statistics
Viral kinetics were calculated according to the model described by Herrmann et al. [27 ]. In brief, compartments were used for the free viral load, productively infected and uninfected hepatocytes. Estimates were derived for the efficacy of antiviral therapy to block virus production {epsilon} and the infected cell loss {delta}, using a nonlinear least squares approach of the logarithmic viral load.

Correlations were calculated using the Spearman test. Individual parameters from two different groups were compared using the Mann-Whitney U-test. Paired parameters were analyzed with the matched Wilcoxon-test. P values ≤0.05 were considered to be significant. Data are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD81 expression on CD8(+) lymphocytes
CD81 expression on CD8(+) lymphocytes from untreated patients with chronic hepatitis C was 482.3 ± 43.6 MFI. During antiviral treatment, a reduction of mean CD81 expression on CD8(+) lymphocytes was observed at TW4 and EOT (Fig. 1A and 1B ). Mean CD81 expression on CD8(+) lymphocytes increased after withdrawal of antiviral therapy (Fig. 1B) .


Figure 1
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  		Figure 1. CD81 expression on CD8(+) lymphocytes. (A) Pretreatment of CD81 expression on CD8(+) lymphocytes from a 49-year-old female SVR after gating for lymphocytes (dot-blot on the left). The lower-left quadrant indicates the range of the isotype control for PE- and FITC-conjugated antibodies, respectively. The box indicates the gate for CD8(+) lymphocytes. The percentage of CD81-positive and CD81-negative CD8(+) lymphocytes is indicated in the upper-right and lower-right quadrants, respectively. The histogram (right) shows CD81 expression on gated CD8(+) lymphocytes from the same patient before treatment (pre, solid curve) and at the EOT 48 weeks after initiation of therapy (EOT, bold line, open curve). The thin line indicates PE fluorescence of the isotype control. (B) Dynamics of CD81 expression on CD8(+) lymphocytes from 20 patients with chronic hepatitis C. Bars indicate MFI of CD81 expression ± SEM before treatment (pre), 4 and 12 weeks after initiation of treatment (TW4, TW12, respecitvely), at the EOT, and after treatment (post). Furthermore, CD81 expression on CD8(+) lymphocytes is shown for healthy individuals (n=8). CD81 was labeled with PE-conjugated anti-CD81 antibodies, and CD8 with FITC-conjugated anti-CD8 antibodies. *, A significantly different CD81 expression in patients with chronic hepatitis C compared with healthy individuals (two-sided P≤0.05). (C) Correlation between the HCV-RNA serum level and CD81 expression on CD8(+) lymphocytes from the patients with chronic hepatitis C before treatment, 4 and 12 weeks after initiation of treatment, and at the EOT. The viral load is shown on the left vertical axis. Short, horizontal bars indicate the lower detection limit of the HCV-RNA serum level (600 IU/ml=2.8 log10 IU/ml). CD81-MFI (PE-conjugated anti-CD81 antibodies) on CD8(+) lymphocytes (FITC-conjugated anti-CD8 antibodies) is shown on the right vertical axis. Correlation coefficients are shown for each time-point, and two-sided P values ≤0.05 are shown where appropriate. (D) Comparison of CD81 expression before treatment (pre) and after treatment (post) between patients who achieved a SVR (n=11) and those who remained infected after treatment (NR, n=9). Furthermore, CD81 expression on CD8(+) lymphocytes from healthy individuals is shown (n=8). Bars indicate MFI of CD81 expression ± SEM (PE-conjugated anti-CD81 antibodies) on CD8(+) cells (FITC-conjugated anti-CD8 antibodies). Two-sided P values ≤0.05 were considered significant. *, A significantly different CD81 expression in patients with chronic hepatitis C compared with healthy individuals (two-sided P≤0.05). (E) CD81 expression in CD3-negative and CD3-positive CD8(+) lymphocytes from the same patient as in A before treatment (pre), at Treatment Week 12 (TW12), at EOT, and after treatment (post). All dot-blots show CD81 expression (vertical axis), according to CD3 expression (horizontal axis) in CD8 (PerCP)-gated lymphocytes. The lower-left quadrant indicates the range of the isotype control for PE- and APC-conjugated antibodies. The percentage of cells in the quadrants and MFI of CD81 expression is shown in the respective quadrants. (F) Dynamics of CD81 expression in CD3-positive and CD3-negative CD8(+) lymphocytes. Dots indicate the change of mean CD81 expression as percentage of baseline CD81 expression on CD3(+) and CD3(–) CD8 (PerCP)-gated lymphocytes from three SVR (24 years, 48 years, 49 years, as in A, all infected with genotype 1) with a typical decline of CD81 on CD8(+) and CD56(+) cells during treatment and increase after treatment. *, Significant decline of CD81 expression compared with baseline (P≤0.05).

 
CD81 expression on CD8(+) lymphocytes from patients with chronic hepatitis C was compared with CD81 expression on CD8(+) lymphocytes from healthy individuals. Patients with chronic hepatitis C had a significantly higher CD81 expression on CD8(+) cells than healthy individuals before treatment and 4 weeks and 12 weeks after initiation of antiviral therapy (Fig. 1B) . At EOT, CD81 expression in patients with chronic hepatitis C became similar to CD81 expression in healthy individuals (Fig. 1B) . After treatment, CD81 expression in patients with chronic hepatitis C was higher again than CD81 expression in healthy individuals (Fig. 1B) .

CD81 expression may be associated with the HCV-RNA serum level. Therefore, the correlation between CD81 and the HCV-RNA serum level was assessed. The baseline HCV-RNA level was correlated inversely with the baseline CD81 expression on CD8(+) lymphocytes (Fig. 1C) . However, during treatment, HCV-RNA levels did not correlate with the respective CD81 expression levels on CD8(+) lymphocytes (Fig. 1C) .

To investigate a potential HCV effect on CD81 expression, the level of CD81 was compared between patients who achieved a SVR and those patients who remained infected after treatment (NR). SVR patients showed a significantly lower CD81 expression than NR patients at TW12 (332.9±23.2 MFI vs. 483.3±50.6 MFI, P=0.019) and at the EOT (271.2±20.3 MFI vs. 388.6±39.1 MFI; P=0.008). After treatment, CD81 expression in SVR patients remained lower than CD81 expression in NR patients, was lower compared with the respective pretreatment level, and was similar to CD81 expression on CD8(+) lymphocytes from healthy individuals (Fig. 1D) .

The correlation between CD81 and viral kinetic parameters was assessed to get insight into a potential mechanism of CD81 regulation. The efficacy of antiviral therapy {epsilon} correlated inversely with CD81 expression on CD8(+) lymphocytes at TW4 (r=–0.745, P=0.008).

CD81 expression may be associated with the percentage of CD8(+) lymphocytes. The percentage of CD8(+) lymphocytes did not change during treatment (36.2±3.3% at TW4, 34.5±2.4% at TW12, 33.1±2.4% at EOT, 32.3±2.4% after treatment vs. 34.3±2.6% pretreatment; P>0.2 for all time-points compared with pretreatment). The percentage of CD8(+) lymphocytes did not correlate with CD81 expression on CD8(+) lymphocytes (P>0.2 for all time-points).

CD8 may be expressed by more than one cell type. CD3 is a marker of T lymphocytes and was used for analysis of CD81 on CD8(+) T lymphocyte subtypes. The majority of CD8(+) lymphocytes was CD3(+) T lymphocytes (Fig. 1E) . As CD81 expression may be different in CD8(+) lymphocyte subpopulations, CD81 expression was compared between CD3(+) and CD3(–) CD8(+) lymphocytes in three SVR with a decline of CD81 on CD8(+) cells during treatment and an increase of CD81 on CD8(+) cells after treatment. The dynamics of CD81 expression during treatment were similar in CD3-positive and CD3-negative CD8(+) lymphocytes (Fig. 1E and 1F) . In all patients, CD3-positive and CD3-negative CD8(+) lymphocyte subsets showed a decline of CD81 at TW12 and at EOT. Post-treatment levels of CD81 were similar to pretreatment levels (P>0.2).

CD81 expression on CD56(+) cells
The pretreatment level of CD81 expression on CD56(+) cells was 427.9 ± 25.6 MFI. IFN-based antiviral therapy was associated with a strong and rapid decline of CD81 expression on CD56(+) cells (Fig. 2A and 2B ). After treatment, CD81 expression on CD56(+) cells increased significantly compared with the EOT level (Fig. 2B) .


Figure 2
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  		Figure 2. CD81 expression on CD56(+) cells. (A) Pretreatment of CD81 expression on CD56(+) cells from the same patient as in Figure 1A after gating for lymphocytes (dot-blot on the left). The lower-left quadrant indicates the range of the isotype control for PE- and FITC-conjugated antibodies. The box indicates the gate for CD56(+) lymphocytes. The percentage of CD81-positive and CD81-negative CD56(+) lymphocytes is indicated in the upper-right and lower-right quadrants, respectively. The histogram (right) shows CD81 expression of gated CD56(+) lymphocytes from the same patient before treatment (pre, solid curve), and CD81 expression at the EOT 48 weeks after initiation of therapy (EOT, bold line, open curve). The thin line indicates fluorescence of the isotype control. (B) Dynamics of CD81 expression on CD56(+) cells from the 20 patients with chronic hepatitis C. Bars indicate MFI of CD81 expression ± SEM on CD56(+) lymphocytes before treatment (pre), 4 and 12 weeks after initiation of treatment (TW4, TW12, respectively), at the EOT, and after treatment (post). Furthermore, CD81 expression on CD56(+) cells is shown for healthy individuals (n=8). CD81 was labeled with PE-conjugated anti-CD81 antibodies, and CD56 with FITC-conjugated anti-CD56 antibodies. *, A significantly different CD81 expression in patients with chronic hepatitis C compared with healthy individuals (two-sided P≤0.05). (C) Correlation between the HCV-RNA serum level and CD81 expression on CD56(+) lymphocytes from the patients with chronic hepatitis C before treatment, 4 and 12 weeks after initiation of treatment, and at the EOT. The viral load is shown on the left vertical axis. Short, horizontal bars indicate the lower detection limit of the HCV-RNA serum level (600 IU/ml=2.8 log10 IU/ml). The MFI of CD81 expression (PE) on CD56(+) cells (FITC) is shown on the right vertical axis. Correlation coefficients are shown for each time-point, and two-sided P values ≤0.05 are shown where appropriate. (D) Comparison of CD81 expression before treatment (pre) and after treatment (post) between patients who achieved a SVR (n=11) and those who remained infected after treatment (NR, n=9). Furthermore, CD81 expression on CD56(+) lymphocytes from healthy individuals is shown (n=8). Bars indicate MFI of CD81 expression (PE) ± SEM on CD56(+) cells (FITC). Two-sided P values ≤0.05 were considered significant. *, A significantly different CD81 expression in patients with chronic hepatitis C compared with healthy individuals (two-sided P≤0.05). (E) CD81 expression in CD3-negative and CD3-positive CD56(+) lymphocytes from the same patient as in A before treatment (pre), at Treatment Week 12 (TW12), at EOT, and after treatment (post). All dot-blots show CD81 expression (vertical axis) according to CD3 expression (horizontal axis) in CD56 (FITC)-gated lymphocytes. The lower-left quadrant indicates the range of the isotype control for PE- and APC-conjugated antibodies. The percentage of cells in the quadrants and MFI of CD81 expression is shown in the respective quadrants. (F) Dynamics of CD81 expression in CD3-positive and CD3-negative CD56(+) lymphocytes. Dots indicate the change of mean CD81 expression as percentage of baseline CD81 expression on CD3(+) and CD3(–) CD56 (FITC)-gated lymphocytes from the same patients as in Figure 1F . *, Significant decline of CD81 expression compared with baseline (P≤0.05).

 
The relative decline from baseline of CD81 in CD56(+) cells was significantly higher than the relative decline from baseline of CD81 in CD8(+) T cells (P=0.03 for TW4, P<0.001 for TW12, and P=0.031 for EOT).

The pretreatment level of CD81 on CD56(+) cells from patients with chronic hepatitis C was significantly higher than CD81 expression on CD56(+) cells from healthy individuals (Fig. 2B) . During treatment, CD81 expression on CD56(+) cells from patients with chronic hepatitis C became similar to or lower than CD81 expression on CD56(+) cells from healthy individuals (Fig. 2B) . After treatment, CD81 expression on CD56(+) lymphocytes from patients with chronic hepatitis C was similar to CD81 expression on CD56(+) lymphocytes from healthy individuals (Fig. 2B) .

CD81 expression on CD56(+) cells was not associated with the respective HCV-RNA serum level (Fig. 2C) . Furthermore, no association with viral kinetic parameters {epsilon} and {delta} was observed.

The post-treatment level of CD81 expression on CD56(+) cells was not different between SVR and NR patients (Fig. 2D) . In SVR and NR patients, the post-treatment level of CD81 on CD56(+) cells was lower compared with the respective pretreatment level and was similar to CD81 expression on CD56(+) lymphocytes from healthy individuals (Fig. 2D) .

The percentage of CD56(+) cells increased during treatment at TW12 and EOT compared with the percentage of CD56(+) cells before treatment (16.7±1.7% at TW12 and 22.6±2.5% at EOT vs. 11.7±1.9% pretreatment, P=0.04 and P=0.001, respectively). After treatment, the percentage of CD56(+) cells was similar to the percentage of CD56(+) before treatment (11.0±1.3% vs. 11.7±1.9%, P>0.2). The percentage of CD56(+) cells was not correlated with CD81 expression (P>0.2).

CD56 may be expressed by more than one cell type. Therefore, CD81 was investigated on CD56(+) cells according to CD3 expression in the three patients with a typical decline of CD81 on CD8(+) cells. All three patients also showed a decline of CD81 on CD56(+) cells. The majority of CD56(+) cells was CD3-negative NK cells (Fig. 2E) . The decline of CD81 expression during treatment was similar in CD3-negative CD56(+) NK cells and CD3-positive CD56(+) lymphocytes (Fig. 2E and 2F) .

CD81 expression on CD4(+) lymphocytes
CD4(+) lymphocytes showed a baseline CD81 expression level of 418.7 ± 23.3 MFI. In contrast to CD8(+) lymphocytes and CD56(+) lymphocytes, antiviral treatment induced only weak changes of CD81 expression on CD4(+) lymphocytes (Fig. 3A and 3B ). A weak but significant, late reduction of mean CD81 expression compared with baseline was observed at EOT (Fig. 3B) .


Figure 3
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  		Figure 3. CD81 expression on CD4(+) lymphocytes. (A) Pretreatment of CD81 expression on CD4(+) lymphocytes from the same patient as in Figure 1A after gating for lymphocytes (dot-blot on the left). The lower-left quadrant indicates the range of the isotype control for PE- and FITC-conjugated antibodies. The box indicates the gate for CD4(+) lymphocytes. The percentage of CD81-positive and CD81-negative CD4(+) lymphocytes is indicated in the upper-right and lower-right quadrants, respectively. The histogram (right) shows CD81 expression of gated CD4(+) lymphocytes before treatment (pre, solid curve) and CD81 expression at the EOT 48 weeks after initiation of therapy (EOT, bold line, open curve). The thin line indicates PE fluorescence of the isotype control. (B) Dynamics of CD81 expression on CD4(+) lymphocytes from the 20 patients with chronic hepatitis C. Bars indicate MFI of CD81 expression ± SEM pretreatment (pre), 4 and 12 weeks after initiation of treatment (TW4, TW12, respectively), at the EOT, and post-treatment (post) on CD4-gated lymphocytes. Furthermore, CD81 expression on CD4(+) cells is shown for healthy individuals (n=8). CD81 was labeled with PE-conjugated anti-CD81 antibodies, and CD4 with FITC-conjugated anti-CD4 antibodies. *, A significantly different CD81 expression in patients with chronic hepatitis C compared with healthy individuals (two-sided P≤0.05). (C) Correlation between the HCV-RNA serum level and CD81 expression on CD4(+) lymphocytes from the patients with chronic hepatitis C before treatment, 4 and 12 weeks after initiation of treatment, and at the EOT. The viral load is shown on the left vertical axis. Short, horizontal bars indicate the lower detection limit of the HCV-RNA serum level (600 IU/ml=2.8 log10 IU/ml). The MFI of CD81 expression (PE) on CD4(+) cells (FITC) is shown on the right vertical axis. Correlation coefficients are shown for each time-point, and two-sided Pvalues ≤0.05 are shown where appropriate. (D) Comparison of CD81 expression before treatment (pre) and after treatment (post) between patients who achieved a SVR (n=11) and those who remained infected after treatment (NR, n=9). Furthermore, CD81 expression on CD4(+) lymphocytes from healthy individuals is shown (n=8). Bars indicate MFI of CD81 expression ± SEM. Two-sided Pvalues ≤0.05 were considered significant. *, A significantly different CD81 expression in patients with chronic hepatitis C compared with healthy individuals (two-sided P≤0.05). (E) Pretreatment of CD81 expression in CD3-negative and CD3-positive CD4(+) lymphocytes from the same patient as in A. The dot-blot shows CD81 expression (vertical axis) according to CD3 expression (horizontal axis) on CD4 (FITC)-gated lymphocytes. The lower-left quadrant indicates the range of the isotype control for PE- and APC-labeled antibodies. The percentage of cells in the quadrants and MFI of CD81 expression is shown.

 
CD81 expression on CD4(+) lymphocytes from patients with chronic hepatitis C was higher than CD81 expression on CD4(+) lymphocytes from healthy individuals before treatment and remained higher during and after treatment (Fig. 3B) .

Pretreatment CD81 on CD4(+) lymphocytes correlated inversely with the HCV-RNA concentration at baseline (Fig. 3C) and positively with CD81 on CD8(+) cells (r=0.747, P=0.001). Treatment of CD81 on CD4(+) lymphocytes, however, was not correlated with the respective HCV-RNA level (Fig. 3C) , virologic response, and viral kinetic parameters.

The post-treatment level of CD81 expression on CD4(+) cells was not different between SVR and NR patients and in both groups higher than CD81 expression on CD4(+) lymphocytes from healthy individuals (Fig. 3D) .

Before treatment, 31.8 ± 2.8% of lymphocytes was CD4(+). Almost all CD4(+) lymphocytes were CD3-positive before treatment (Fig. 3E) . The percentage of CD4(+) cells did not change during treatment and after treatment compared with baseline (29.1±3.7% at TW4, 29.9±2.7% at TW12, 30.1±3.6% at EOT, 33.7±3.7% post-treatment vs. 31.8±2.8% pretreatment, P>0.2, for all time-points compared with pretreatment) and was not associated with the respective CD81 expression.

CD81 expression on CD19(+) B lymphocytes
Pretreatment of CD81 expression on CD19(+) B lymphocytes was 528.0 ± 50.5 MFI. One patient showed a two- to threefold overexpression of CD81 on CD19(+) B lymphocytes of >1250 MFI at all time-points and therefore, was excluded from further analysis.

The EOT level and the post-treatment level of CD81 on CD19(+) B lymphocytes were not different compared with the pretreatment level (Fig. 4A and 4B ). In contrast to CD8(+), CD56(+), and CD4(+) cells, antiviral treatment induced a transient increase of the mean CD81 expression level on CD19(+) B lymphocytes with a maximum expression level at TW12 (Fig. 4B) . Before, during, and after treatment, CD81 expression was higher on CD19(+) B lymphocytes from patients with chronic HCV than on CD19(+) B lymphocytes from healthy individuals (Fig. 4B) .


Figure 4
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  		Figure 4. CD81 expression on CD19(+) lymphocytes. (A) Pretreatment of CD81 expression on CD19(+) lymphocytes from the same patient as in Figure 1A after gating for lymphocytes (dot-blot on the left). The lower-left quadrant indicates the range of the isotype control for PE- and FITC-conjugated antibodies. The box indicates the gate for CD19(+) lymphocytes. The percentage of CD81-positive and CD81-negative CD19(+) lymphocytes is indicated in the upper-right and lower-right quadrants, respectively. The histogram (right) shows CD81 expression of gated CD19(+) lymphocytes from the same patient before treatment (pre, solid curve) and CD81 expression at the EOT 48 weeks after initiation of therapy (EOT, bold line, open curve). The thin line indicates PE fluorescence of the isotype control. (B) Dynamics of CD81 expression on CD19(+) B cells from the 20 patients with chronic hepatitis C. Bars indicate MFI of CD81 expression ± SEM pretreatment (pre), 4 and 12 weeks after initiation of treatment (TW4, TW12, respectively), at the EOT, and post-treatment (post). Furthermore, CD81 expression on CD19(+) B cells is shown for healthy individuals (n=8). CD81 was labeled with PE-conjugated anti-CD81 antibodies, and CD19 with FITC-conjugated anti-CD19 antibodies. *, A significantly different CD81 expression in patients with chronic hepatitis C compared with healthy individuals (two-sided P≤0.05). (C) Correlation between the HCV-RNA serum level and CD81 expression on CD19(+) lymphocytes from the patients with chronic hepatitis C before treatment, 4 and 12 weeks after initiation of treatment, and at the EOT. The viral load is shown on the left vertical axis. Short, horizontal bars indicate the lower detection limit of the HCV-RNA serum level (600 IU/ml=2.8 log10 IU/ml). The MFI of CD81 expression (PE) on CD19(+) cells (FITC) is shown on the right vertical axis. Correlation coefficients are shown for each time-point, and two-sided P values ≤0.05 are shown where appropriate. (D) Comparison of CD81 expression before treatment (pre) and after treatment (post) between patients who achieved a SVR (n=11) and those who remained infected after treatment (NR, n=9). Furthermore, CD81 expression on CD19(+) cells from healthy individuals is shown (n=8). Bars indicate MFI of CD81 expression ± SEM. Two-sided Pvalues ≤0.05 were considered significant. *, A significantly different CD81 expression in patients with chronic hepatitis C compared with healthy individuals (two-sided P≤0.05). (E) CD81 expression in CD5-negative and CD5-positive CD19(+) lymphocytes from the same patient as in A before treatment (pre), at Treatment Week 12 (TW12), at EOT, and after treatment (post). All dot-blots show CD81 expression (vertical axis) according to CD5 expression (horizontal axis) in CD19 (PerCP)-gated lymphocytes. The lower-left quadrant indicates the range of the isotype control for FITC- and PE-conjugated antibodies. The percentage of cells in the quadrants and CD81 MFI is shown. (F) Dynamics of CD81 expression in CD5-positive and CD5-negative CD19(+) lymphocytes. Dots indicate the change of mean CD81 expression as percentage of baseline CD81 expression on CD5(+) and CD5(–) CD19 (PerCP)-gated lymphocytes from the same three patients as in Figure 1F . *, Significant decline of CD81 expression compared with baseline (P≤0.05).

 
CD81 on CD19(+) B lymphocytes, before and during antiviral therapy, was not associated with the HCV-RNA serum level and virologic response (Fig. 4C) .

The post-treatment level of CD81 on CD19(+) B lymphocytes was not different between SVR and NR patients (Fig. 4D) . SVR and NR patients showed a higher CD81 expression on CD19(+) lymphocytes before and after treatment compared with CD81 expression on CD19(+) cells from healthy individuals (Fig. 4D) .

The CD5-positive subpopulation of B lymphocytes has been implicated in the development of autoimmune manifestations of chronic hepatitis C [17 ]. CD81 expression may be different between CD5-positive and CD5-negative B lymphocytes. Therefore, CD81 expression was compared between CD5-positive and CD5-negative CD19(+) B lymphocytes in the three SVR, with down-regulation of CD81 on CD8(+) and CD56(+) cells. In contrast to CD81 expression on CD8(+) and CD56(+) cells, CD81 expression was not down-regulated on CD5-positive CD19(+) B lymphocytes (Fig. 4E and 4F) .

The percentage of CD19(+) lymphocytes did not change during and after treatment (8.3±1.6% at TW4, 7.7±2.0% at TW12, 8.1±1.6% at EOT, 8.6±1.4% post-treatment vs. 10.1±1.6% pretreatment; P>0.1 for all time-points compared with pretreatment). The percentage of CD5-positive CD19(+) cells also did not change after treatment compared with baseline (22.3±5.3% vs. 19.3±7.1%; P>0.2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD81 expression is down-regulated by IFN-{alpha} in PBL from patients with chronic hepatitis C and in cultured CD8(+), CD4(+), and CD19(+) cells from healthy individuals [23 ]. In the present study, we investigated CD81 dynamics during antiviral treatment on CD8(+), CD4(+), CD19(+), and CD56(+) lymphocyte subtypes from patients with chronic hepatitis C. The major finding of the present study was that lymphocyte subtypes from patients with chronic hepatitis C show different CD81 patterns during treatment. We observed that the overall decline of CD81 on PBL was mainly caused by a down-regulation of CD81 on CD8(+) and CD56(+) lymphocytes. In contrast, we observed weak and late changes of CD81 expression on CD4(+) T cells and a transient increase of CD81 on CD19(+) B cells. These results suggest that the regulation of CD81 in response to IFN-{alpha}-based treatment underlies different mechanisms in the respective lymphocyte subtypes.

CD8 and CD56 may be expressed by more than one cell type. The T cell marker CD3 allows a better distinction of CD3-positive CD8(+) T cells and CD3-negative CD56(+) NK cells. We observed that down-regulation of CD81 expression during treatment was similar in CD3-positive and CD3-negative CD8(+) lymphocytes and CD3-negative and CD3-positive CD56(+) lymphocytes, respectively.

To obtain insights into potential HCV-associated mechanisms leading to alteration of CD81 expression, we investigated the correlation between the HCV-RNA serum level and CD81 expression on the lymphocyte subtypes at baseline. We observed that the HCV-RNA serum level correlated inversely with the CD81 expression on CD8(+) and CD4(+) lymphocytes. This inverse correlation may be explained by direct interaction between the virus and the cell, e.g., binding of HCV particles to the CD81 receptor. A similar dose-dependent decrease of CD81 on B cells by recombinant HCV-E2 proteins has been reported in the study by Pileri et al. [13 ] where the presence of bound HCV-E2 to the cell surface of B cells leads to a subsequent inhibition of binding of anti-CD81 antibodies.

During treatment, changes of CD81 may be caused by changes of the HCV concentration or by a direct IFN-{alpha} effect on CD81 expression. We therefore investigated CD81 expression during treatment in correlation with the HCV-RNA level. Our results indicate that CD81 expression during treatment is not associated with the HCV-RNA level. Therefore, changes of CD81 during treatment seem to be caused mainly by an effect of IFN-{alpha}.

Especially, the reduction of CD81 expression on CD56(+) cells in the present study seems to be a mere IFN-{alpha} effect. Reduction of CD81 on CD56(+) cells was stronger compared with changes of CD81 on other lymphocyte subtypes. After treatment, CD81 levels on CD56(+) cells increased in SVR and NR patients. The mechanisms by which IFN-{alpha} alters CD81 expression are unclear. Potentially involved mechanisms are reduced transcription of CD81 mRNA, reduced translation of CD81 mRNA, and enhanced degradation of CD81 protein. Our previous study indicated that CD81 reduction in vitro occurs at the post-translational/transcriptional level, as CD81 protein down-regulation is associated with an increase of CD81 mRNA [23 ].

CD81 is an essential coreceptor for HCV [1 , 18 , 28 ]. It has been reported that HCV alters the expression of CD81 to higher levels [17 , 23 , 24 ]. In the present study, we observed a higher expression of CD81 on CD8(+), CD56(+), CD4(+), and CD19(+) lymphocytes from patients with chronic hepatitis C compared with CD81 expression on the respective lymphocyte subtypes from healthy individuals. If HCV alters CD81 expression, a difference in post-treatment CD81 expression between patients who are cured by antiviral therapy and patients who remain infected can be expected. In support of this, we observed in the present study a significantly lower CD81 expression after treatment in CD8(+) T cells in patients who were cured by antiviral therapy (SVR) compared with patients who remained infected with HCV after treatment (NR). Furthermore, in SVR patients, post-treatment CD81 expression in CD8(+) cells but not in CD4(+) and CD19(+) B cells was similar to CD81 expression in the respective cells from healthy individuals.

A high CD81 expression level could be relevant for infection of target cells. HCV-RNA has been detected primarily in CD8(+) T cells and CD19(+) B cells, indicating a potential infection of these cell types [29 ]. In a previous study, we observed that CD81 during treatment was higher in PBMC positive for HCV-RNA compared with PBMC negative for HCV-RNA before treatment [23 ]. If a high CD81 expression level supports infection of target cells, it can be anticipated that lower levels of CD81 during treatment are associated with reduced infection of target cells. In potentially infected lymphocyte subtypes, e.g., CD8(+) T lymphocytes or subclasses of B lymphocytes, down-regulation of CD81 during treatment could reduce the amount of extrahepatic HCV replication. The reduction of HCV-infected extrahepatic cells could be another important antiviral mechanism of IFN-{alpha} in addition to blocking of HCV production [2 ].

CD81 overexpression has also been reported for the CD5(+) B cell subpopulation, potentially involved in the development of autoimmune manifestations in chronic hepatitis C [16 , 17 , 25 ]. Zuckerman et al [25 ]. reported that the achievement of a SVR after antiviral treatment is associated with reduction of CD81 expression on CD5(+) B cells and the improvement of autoimmune markers. The present study did not include patients with autoimmune manifestations of hepatitis C. It is interesting that in the present study, the three analyzed SVR patients showed neither a reduction of CD81 expression in the major CD19(+) B cell population nor in CD5(+) B cells although they showed a decline of CD81 expression in CD8(+) and CD56(+) cells. Furthermore, the reduction of post-treatment CD81 expression in CD8(+) and CD56(+) cells, in the present study, appeared weaker than the reduction of CD81 expression on CD5(+) cells reported by Zuckerman et al. [25 ] (90.2±11.6% and 78.1±0.2% of pretreatment CD81 expression vs. 66.8±19.8% of pretreatment CD81 expression).

CD81 has important functions in the activation process of lymphocytes [30 31 32 ]. Binding of the HCV-E2 protein to CD81 has been reported to counter the host’s immune response against HCV [12 , 15 , 33 , 34 ]. Down-regulation of CD81 during treatment, as observed in CD8(+) and CD56(+) cells in the present study, may reduce the interaction between HCV-E2 and target cells and thereby may enable a stronger antiviral immune response during treatment. Furthermore, CD81 down-regulation may reduce autoimmune phenomena during treatment.

The early prediction of SVR in patients with chronic hepatitis C during antiviral therapy is highly important. In the present study, we observed that 12 weeks after initiation of antiviral treatment, SVR developed significantly lower CD81 expression levels than patients who did not fully eradicate HCV after treatment. Thus, CD81 expression on CD8(+) cells might be useful for the prediction of the outcome of antiviral therapy. The potential use of CD81 expression to predict SVR should be tested in larger, prospective trials.

In summary, we observed different patterns of CD81 expression during antiviral treatment, which were associated with administration of IFN-{alpha} and antiviral response. The regulation of CD81 on lymphocyte subtypes during treatment could be relevant for the control of viral infection and for treatment response.


   ACKNOWLEDGEMENTS
 
This study was supported by a German Educational Ministry of Education and Research (BMBF) grant to B. K., C. S., and H. W. (German Kompetenznetz Hepatitis, HepNet TP15.1), by Homfor (2004), and by the Clinical Research Unit (KFO129), funded by the Deutsche Forschungsgemeinschaft (DFG).

Received January 23, 2006; revised March 14, 2006; accepted April 3, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wakita, T., Pietschmann, T., Kato, T., Date, T., Miyamoto, M., Zhao, Z., Murthy, K., Habermann, A., Krausslich, H. G., Mizokami, M., Bartenschlager, R., Liang, T. J. (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome Nat. Med. 11,791-796[CrossRef][Medline]
  2. Perelson, A. S., Herrmann, E., Micol, F., Zeuzem, S. (2005) New kinetic models for the hepatitis C virus Hepatology 42,749-754[CrossRef][Medline]
  3. Zeuzem, S., Schmidt, J. M., Lee, J. H., Ruster, B., Roth, W. K. (1996) Effect of interferon {alpha} on the dynamics of hepatitis C virus turnover in vivo Hepatology 23,366-371[Medline]
  4. Zeuzem, S., Schmidt, J. M., Lee, J. H., von Wagner, M., Teuber, G., Roth, W. K. (1998) Hepatitis C virus dynamics in vivo: effect of ribavirin and interferon {alpha} on viral turnover Hepatology 28,245-252[CrossRef][Medline]
  5. Poynard, T., Mathurin, P., Lai, C. L., Guyader, D., Poupon, R., Tainturier, M. H., Myers, R. P., Muntenau, M., Ratziu, V., Manns, M., Vogel, A., Capron, F., Chedid, A., Bedossa, P. (2003) A comparison of fibrosis progression in chronic liver diseases J. Hepatol. 38,257-265[CrossRef][Medline]
  6. Nelson, D. R., Marousis, C. G., Davis, G. L., Rice, C. M., Wong, J., Houghton, M., Lau, J. Y. (1997) The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic hepatitis C J. Immunol. 158,1473-1481[Abstract]
  7. Wedemeyer, H., He, X. S., Nascimbeni, M., Davis, A. R., Greenberg, H. B., Hoofnagle, J. H., Liang, T. J., Alter, H., Rehermann, B. (2002) Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection J. Immunol. 169,3447-3458[Abstract/Free Full Text]
  8. Gruener, N. H., Lechner, F., Jung, M. C., Diepolder, H., Gerlach, T., Lauer, G., Walker, B., Sullivan, J., Phillips, R., Pape, G. R., Klenerman, P. (2001) Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus J. Virol. 75,5550-5558[Abstract/Free Full Text]
  9. Ducoulombier, D., Roque-Afonso, A. M., Di Liberto, G., Penin, F., Kara, R., Richard, Y., Dussaix, E., Feray, C. (2004) Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes Hepatology 39,817-825[CrossRef][Medline]
  10. Roque-Afonso, A. M., Ducoulombier, D., Di Liberto, G., Kara, R., Gigou, M., Dussaix, E., Samuel, D., Feray, C. (2005) Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells J. Virol. 79,6349-6357[Abstract/Free Full Text]
  11. Lerat, H., Rumin, S., Habersetzer, F., Berby, F., Trabaud, M. A., Trepo, C., Inchauspe, G. (1998) In vivo tropism of hepatitis C virus genomic sequences in hematopoietic cells: influence of viral load, viral genotype, and cell phenotype Blood 91,3841-3849[Abstract/Free Full Text]
  12. Crotta, S., Stilla, A., Wack, A., D’Andrea, A., Nuti, S., D’Oro, U., Mosca, M., Filliponi, F., Brunetto, R. M., Bonino, F., Abrignani, S., Valiante, N. M. (2002) Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein J. Exp. Med. 195,35-41[Medline]
  13. Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D., Grandi, G., Abrignani, S. (1998) Binding of hepatitis C virus to CD81 Science 282,938-941[Abstract/Free Full Text]
  14. Soldaini, E., Wack, A., D’Oro, U., Nuti, S., Ulivieri, C., Baldari, C. T., Abrignani, S. (2003) T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck Eur. J. Immunol. 33,455-464[CrossRef][Medline]
  15. Wack, A., Soldaini, E., Tseng, C., Nuti, S., Klimpel, G., Abrignani, S. (2001) Binding of the hepatitis C virus envelope protein E2 to CD81 provides a co-stimulatory signal for human T cells Eur. J. Immunol. 31,166-175[CrossRef][Medline]
  16. Zuckerman, E. (2003) Expansion of CD5+ B-cell overexpressing CD81 in HCV infection: towards better understanding the link between HCV infection, B-cell activation and lymphoproliferation J. Hepatol. 38,674-676[CrossRef][Medline]
  17. Zuckerman, E., Slobodin, G., Kessel, A., Sabo, E., Yeshurun, D., Halas, K., Toubi, E. (2002) Peripheral B-cell CD5 expansion and CD81 overexpression and their association with disease severity and autoimmune markers in chronic hepatitis C virus infection Clin. Exp. Immunol. 128,353-358[CrossRef][Medline]
  18. Cormier, E. G., Tsamis, F., Kajumo, F., Durso, R. J., Gardner, J. P., Dragic, T. (2004) CD81 is an entry coreceptor for hepatitis C virus Proc. Natl. Acad. Sci. USA 101,7270-7274[Abstract/Free Full Text]
  19. Bartosch, B., Dubuisson, J., Cosset, F. L. (2003) Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes J. Exp. Med. 197,633-642[Abstract/Free Full Text]
  20. McKeating, J. A., Zhang, L. Q., Logvinoff, C., Flint, M., Zhang, J., Yu, J., Butera, D., Ho, D. D., Dustin, L. B., Rice, C. M., Balfe, P. (2004) Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner J. Virol. 78,8496-8505[Abstract/Free Full Text]
  21. Zhang, J., Randall, G., Higginbottom, A., Monk, P., Rice, C. M., McKeating, J. A. (2004) CD81 is required for hepatitis C virus glycoprotein-mediated viral infection J. Virol. 78,1448-1455[Abstract/Free Full Text]
  22. Hsu, M., Zhang, J., Flint, M., Logvinoff, C., Cheng-Mayer, C., Rice, C. M., McKeating, J. A. (2003) Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles Proc. Natl. Acad. Sci. USA 100,7271-7276[Abstract/Free Full Text]
  23. Kronenberger, B., Ruster, B., Elez, R., Weber, S., Piiper, A., Lee, J. H., Roth, W. K., Zeuzem, S. (2001) Interferon {alpha} down-regulates CD81 in patients with chronic hepatitis C Hepatology 33,1518-1526[CrossRef][Medline]
  24. Hofmann, W. P., Herrmann, E., Kronenberger, B., Merkwirth, C., Welsch, C., Lengauer, T., Zeuzem, S., Sarrazin, C. (2004) Association of HCV-related mixed cryoglobulinemia with specific mutational pattern of the HCV E2 protein and CD81 expression on peripheral B lymphocytes Blood 104,1228-1229[Medline]
  25. Zuckerman, E., Kessel, A., Slobodin, G., Sabo, E., Yeshurun, D., Toubi, E. (2003) Antiviral treatment down-regulates peripheral B-cell CD81 expression and CD5 expansion in chronic hepatitis C virus infection J. Virol. 77,10432-10436[Abstract/Free Full Text]
  26. Knodell, R. G., Ishak, K. G., Black, W. C., Chen, T. S., Craig, R., Kaplowitz, N., Kiernan, T. W., Wollman, J. (1981) Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis Hepatology 1,431-435[Medline]
  27. Herrmann, E., Lee, J. H., Marinos, G., Modi, M., Zeuzem, S. (2003) Effect of ribavirin on hepatitis C viral kinetics in patients treated with pegylated interferon Hepatology 37,1351-1358[CrossRef][Medline]
  28. Lindenbach, B. D., Evans, M. J., Syder, A. J., Wolk, B., Tellinghuisen, T. L., Liu, C. C., Maruyama, T., Hynes, R. O., Burton, D. R., McKeating, J. A., Rice, C. M. (2005) Complete replication of hepatitis C virus in cell culture Science 309,623-626[Abstract/Free Full Text]
  29. Ducoulombier, D., Roque-Afonso, A. M., Di Liberto, G., Penin, F., Kara, R., Richard, Y., Dussaix, E., Feray, C. (2004) Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes Hepatology 39,817-825[CrossRef][Medline]
  30. Fritzsching, B., Schwer, B., Kartenbeck, J., Pedal, A., Horejsi, V., Ott, M. (2002) Release and intercellular transfer of cell surface CD81 via microparticles J. Immunol. 169,5531-5537[Abstract/Free Full Text]
  31. Levy, S., Todd, S. C., Maecker, H. T. (1998) CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system Annu. Rev. Immunol. 16,89-109[CrossRef][Medline]
  32. Witherden, D. A., Boismenu, R., Havran, W. L. (2000) CD81 and CD28 costimulate T cells through distinct pathways J. Immunol. 165,1902-1909[Abstract/Free Full Text]
  33. Tseng, C. T., Klimpel, G. R. (2002) Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions J. Exp. Med. 195,43-49[Medline]
  34. Rosa, D., Saletti, G., De Gregorio, E., Zorat, F., Comar, C., D’Oro, U., Nuti, S., Houghton, M., Barnaba, V., Pozzato, G., Abrignani, S. (2005) Activation of naive B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders Proc. Natl. Acad. Sci. USA 102,18544-18549[Abstract/Free Full Text]




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