Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates with Alanine Aminotransferase Serum Activity
ABSTRACT Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity.
Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry.
Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027).
CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis.
- SourceAvailable from: Zuquan Weng[Show abstract] [Hide abstract]
ABSTRACT: Like most cell types, hepatocytes constantly produce extracellular vesicles (EVs) such as exosomes and microvesicles that are released into the circulation to transport signaling molecules and cellular waste. Circulating EVs are being vigorously explored as biomarkers of diseases and toxicities, including drug induced liver injury (DILI). Emerging data suggest that a) blood-borne EVs contain liver-specific mRNAs and microRNAs (miRNAs), b) the levels can be remarkably elevated in response to DILI, and c) the increases correlate well with classical measures of liver damage. The expression profile of mRNAs in EVs and the compartmentalization of miRNAs within EVs or other blood fractions were found to be indicative of the offending drug involved in DILI, thus providing more informative assessment of liver injury than using alanine aminotransferase (ALT) alone. EVs in the urine and cell culture medium were also found to contain proteins or mRNAs that were indicative of DILI. However, major improvements in EV isolation methods are needed for the discovery, evaluation and quantification of possible DILI biomarkers in circulating EVs.Toxicology Letters 01/2014; · 3.36 Impact Factor
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ABSTRACT: MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.PLoS ONE 09/2013; 8(9):e75184. · 3.53 Impact Factor
- Hepatitis Monthly 05/2014; 14(5):e13514. · 1.80 Impact Factor
Soluble Serum CD81 Is Elevated in Patients with Chronic
Hepatitis C and Correlates with Alanine
Aminotransferase Serum Activity
Martin-Walter Welker1, David Reichert1, Simone Susser1, Christoph Sarrazin1, Yolanda Martinez1, Eva
Herrmann2, Stefan Zeuzem1, Albrecht Piiper1, Bernd Kronenberger1*
1Medizinische Klinik 1, Klinikum der Johann Wolfgang Goethe-Universita ¨t, Theodor-Stern-Kai 7, Frankfurt am Main, Germany, 2Fachbereich Medizin, Institut fu ¨r
Biostatistik und mathematische Modellierung, Johann Wolfang Goethe-Universita ¨t, Frankfurt am Main, Germany
Aim: Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81
in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the
exosomal serum fraction is associated with HCV replication and inflammatory activity.
Patients and Methods: Four cohorts were investigated, patients with chronic hepatitis C (n=37), patients with chronic HCV
infection and persistently normal ALT levels (n=24), patients with long term sustained virologic response (SVR, n=7), and
healthy volunteers (n=23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation
ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry.
Results: Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p=0.03) and cured
patients (p=0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR
had similar soluble CD81 levels as healthy controls (p.0.2). Overall, soluble CD81 levels were associated with ALT levels
(r=0.334, p=0.016) and severe liver fibrosis (p=0.027).
Conclusion: CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be
associated with inflammatory activity and severity of fibrosis.
Citation: Welker M-W, Reichert D, Susser S, Sarrazin C, Martinez Y, et al. (2012) Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates
with Alanine Aminotransferase Serum Activity. PLoS ONE 7(2): e30796. doi:10.1371/journal.pone.0030796
Editor: Johan K. Sandberg, Karolinska Institutet, Sweden
Received October 17, 2011; Accepted December 29, 2011; Published February 15, 2012
Copyright: ? 2012 Welker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the clinical research unit KFO 129, funded by the German Research Foundation (DFG), and by the Scolari Stiftung. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Bernd.Kronenberger@kgu.de
Chronic hepatitis C virus (HCV) infection is a major cause of
liver cirrhosis, and hepatocellular carcinoma worldwide [1,2].
Recently the protease inhibitors boceprevir and telaprevir, which
are associated with considerably enhanced sustained virologic
response (SVR) rates in HCV genotype 1 infected patients in
combination with pegylated interferon-alfa and ribavirin, have
been approved by the European Medicines Agency and the Food
and Drug Administration [3–5]. Nevertheless, approximately one
quarter of patients with HCV genotype 1 infection are assumed
not to achieve SVR even with triple therapy including either
boceprevir or telaprevir .
HCV is a plus-strand RNA virus without known ability to
integrate its genome into the host genome. Chronic hepatitis C is
characterized by high turnover of infected cells and persistence of
HCV requires continuous de novo infection [7–10]. Therefore,
inhibition of cell entry is a promising approach in antiviral therapy
against HCV. Moreover, cell entry inhibitors may be useful in
prevention of otherwise inevitable graft infection after orthotopic
liver transplantation in HCV associated liver cirrhosis and
hepatocellular carcinoma. Proof-of-concept studies suggest that
the new class of HCV entry inhibitors has a substantial capability
to widen preventive and therapeutic options in HCV infection
Four essential HCV hepatocyte entry receptors, CD81, the
scavenger receptor B1, claudin-1, and occludin have been
identified, yet [12–15]. CD81 is of particular interest because
CD81 is not only expressed on hepatocytes but also on peripheral
blood mononuclear cells (PBMC). Interaction of HCV with CD81
expressed on PBMC was suggested to be involved in an attenuated
innate and adaptive immune response against HCV as well as in
development of extrahepatic HCV manifestations [16–20].
However, these results were challenged by a recent study showing
that HCV envelope 2 proteins have no modulatory effect on
natural killer cell functions when expressed as part of cell culture
infectious viral particles . The different observations could be
explained by differences in the configuration of viral envelope
proteins in protein expression systems compared to systems using
compete HCV particles [20,21].
PLoS ONE | www.plosone.org1 February 2012 | Volume 7 | Issue 2 | e30796
Yet well known as a cell surface protein, CD81 is also a typical
component of exosomes. Exosomes are membrane vesicles se-
creted by different eukaryotic cells, e.g. hepatocytes and
lymphocytes [22–24]. In two studies, exosomes were enriched by
differential centrifugation from human plasma from patients with
chronic hepatitis C [23,25]. Exosomal CD81 was reliably
detectable in the pellet of the fifth fraction of the centrifugation
process. Moreover, CD81 exosomes were found to be associated
with HCV RNA in plasma obtained from patients chronically
infected with HCV . However, neither CD81 levels nor HCV
RNA were quantified in enriched plasma or serum, which makes it
difficult to assess clinical importance of these findings. Neverthe-
less, exosomal CD81 may be of relevance in chronic HCV
infection as HCV particles bound to exosomal CD81 could
represent an additional HCV compartment with putative impact
on the HCV infection rate or viral persistence, respectively .
Aim of the present study was to perform a comprehensive
analysis if soluble CD81 is increased in patients with chronic
hepatitis C compared to healthy controls, patients with chronic
HCV infection but persistently normal alanine transaminase
(ALT) levels, and patients with cured hepatitis C. Therefore, a
method to quantify soluble CD81 in the exosomal serum fraction
was established, and the association of soluble CD81 in the
exosomal serum fraction with clinical and virological parameters
within the different cohorts was investigated.
The present study is a case control study with 23 healthy
controls and 37 patients with chronic hepatitis C and elevated
alanine aminotransferase (ALT) level measured at least at 2 time
points 14 days or more apart within 6 months before study entry.
Furthermore, 24 patients with chronic HCV infection and
persistently normal ALT serum levels as documented by three
occasional measurements within the last 6 months before study
entry and a minimum interval of 4 weeks between measurements,
and 7 patients with long-term sustained virologic response 5 years
after antiviral therapy with pegylated interferon-alfa 2a and
ribavirin were included. All cohorts were independently recruited.
Patients with decompensated liver cirrhosis or co-infection with
hepatitis B virus or human immunodeficiency virus were excluded.
Diagnosis of chronic HCV infection (.6 months) was confirmed
by positive anti-HCV antibodies by third-generation enzyme
immunoassay and detectable HCV RNA in serum. Grading and
staging of chronic hepatitis C was done by a local, experienced
pathologist according to the histological activity index described
by Knodell et al. . Patient cohorts with persistently normal
ALT levels and patients with elevated ALT levels were derived
from clinical trials performed at the university hospitals Frankfurt/
Main, Germany and Homburg/Saar, Germany. Study protocols
were conform to the ethical guidelines of the 1975 Declaration of
Helsinki and were approved by the ethic committees of the
university hospitals Frankfurt/M, Germany and Homburg/Saar,
Germany. Written, informed consent was obtained from every
human subject when the samples were collected under the terms of
Isolation of CD81 from serum and plasma
Serum and plasma for ALT and AST levels, CD81 assessment,
and HCV-RNA quantification were collected at the same time
point and stored at 280uC until use. Enrichment of exosomes was
performed according to Masciopinto et al. . In this former
study, the CD81 enriched exosomal fraction was identified by
immunoblotting in the pellets from the fourth (p4) and - mainly -
the fifth (p5) centrifugation fraction from plasma . In the
current study, differential centrifugation was performed at 200 g
for 10 min (p1), at 500 g (p2), at 2000 g for 15 min (p3), at
10,000 g for 30 min (p4), and finally 100,000 g for 30 min (p5).
The corresponding pellets (p1, p2, etc.) were each resuspended in
50 ml lysis buffer (100 mL 0.5 M TrisHCl pH 7.4, 1.5 mM NaCl,
2.5% desoxycholic acid, 10% NP-40, 10 mM ethylenediamine-
tetraacetic acid, diluted 1:10 in aqua bidest., Merck, Darmstadt,
Germany, and Sigma-Aldrich, Taufkirchen, Germany) and stored
at 220uC until further analysis. As usage of serum may have some
advantages over plasma, e.g. better reflection of biological
conditions and easier sample processing, we further optimized
detection of CD81 in enriched serum. Therefore, we first
compared isolation of not cell-bound CD81 from plasma and
serum samples, and then tested different volumes of serum
(30 mL, 15 mL, 7 mL, and 1 mL) to evaluate a possible minimum
amount for reliable detection of CD81. In contrast to plasma,
serum samples were allowed to clot for 10 min before centrifu-
gation. After that, serum specimen of the healthy controls and
distinct patient groups were processed using the developed
optimized serum sample processing protocol.
Immunoblotting of CD81
Serum derived proteins from different centrifugation steps were
separated by dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE). As thiol compounds may have a negative impact on
anti-CD81 antibody binding , a thiol free SDS sample buffer
was used. In detail, after dilution of specimens in loading buffer
(5 mL glycerin, 2 mL 0.625 M TrisHCl, pH 6,8, 0.2 g SDS,
0.1 mL bromphenoleblue as 1% ethanol dilution, 2.4 mL aqua
bidest.), the samples were separated by SDS-page (50 mA, 90 min)
and electrophoretic transferred (110 mA, 90 min) to nitrocellulose
membranes Whatman Schleicher & Schell, Dassel, Germany).
Nonspecific binding sites were blocked by incubation in 5% skim
milk in buffer TBST (150 mM NaCl; 30 mM Tris, pH 7.4; 0.05%
Tween20) for 12 hours at +4uC. Primary anti-CD81 antibody
(mouse-anti-human antibody, clone JS-81, Pharmingen, Heidel-
berg, Germany) were allowed to bind for a minimum of 2 hours.
Detection of CD81/antibody immune complexes was performed
using horse radish peroxidase-conjugated antibodies (Bio-Rad
Laboratories, Hercules CA, U.S.A.) and enhanced chemilumines-
cence (Millipore GmbH, Schwalbach, Germany). By staining with
monoclonal anti-CD81 antibodies and consecutive polyclonal
horse raddish peroxidase-conjugated antibodies or staining with
polyclonal horse raddish peroxidase-conjugated antibodies only,
specific detection of CD81 was confirmed (data not shown).
Further semi-quantitative analysis was performed by automated
Quantification of CD81
Huh7 cells are characterized by high CD81 expression .
Serial dilutions of a cell lysate of 104Huh7 were used as standard
for semi-quantitative assessment of serum CD81. Aliquots of the
lysate were stored at 280uC until use, and applied as the same
standard for all experiments. CD81 in Huh7 cell lysates was
detected by immunoblotting and densitometric analysis by use of
an analysis program (Gelscan 5, BioSciTec GmbH, Frankfurt,
Germany). Concentration of serum derived CD81 was assessed
semi-quantitatively in relation to the standard curve obtained from
the Huh7 cell lysate dilutions by densitometry and expressed as
relative units (RU).
Exosomal CD81 in HCV
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Measurement of HCV RNA from native serum and HCV
Quantitative assessment of HCV RNA in serum was performed
by a quantitative reverse transcription polymerase chain reaction
assay (Cobas Amplicor HCV MonitorTM 2.0, Roche Diagnostic
Systems, Branchburg, NJ; lower detection limit, 600 IU/mL).
Samples with HCV RNA concentration above the upper detection
limit above 800.000 IU/mL were diluted appropriately. HCV
genotyping was performed by reverse hybridization assay (Inno
LiPA HCV II, Innogenetics, Gent, Belgium). All contamination
prevention measures suggested by Kwok and Higuchi were strictly
Measurement of HCV RNA in enriched serum
For comparison with serum HCV RNA and correlation to
CD81 concentration within enriched serum fraction p5, HCV
RNA was assessed in enriched serum, also. Therefore, differential
centrifugation was performed in accordance to the CD81
enrichment protocol. Each pellet from a given centrifugation step
was resuspended in 0.25 mL PBS, and HCV RNA was extracted
fully automatic (COBAS-Ampliprep, Roche, Mannheim, Ger-
many). Quantification of HCV RNA was performed standardized
by real-time polymerase chain reaction (COBAS-Taqman 48
Analyzer Roche, Mannheim, Germany).
Unless indicated otherwise, all tests were two tailed and p
values,0.05 were considered significant. Clinical and biochemical
characteristics of patients were expressed as mean 6 standard
deviation (SD) or median and range as appropriate. Correlations
between two variables were calculated by the spearman test.
Furthermore, Kruskal-Wallis test, Friedman test, Wilcoxon-paired
sample test and Wilcoxon Mann-Whitney U test were applied as
appropriate. A Bonferroni correction was applied, if multiple
comparisons were performed. A receiver operating characteristic
analysis was performed to compare CD81-p5 and severity of liver
Detection of CD81 in the exosomal serum fraction
Detection of exosomal CD81 in plasma from patients with
chronic hepatitis C has been previously described by Masciopinto
et al. . In the present study, we investigated whether exosomal
CD81 is also detectable in human serum. For this purpose,
differential centrifugation for enrichment of exosomal CD81 was
performed with human plasma and serum as previously described
for plasma, followed by immunoblotting with CD81 specific
monoclonal antibodies . Lysates from Huh7 cells character-
ized by high CD81 expression were used as positive control.
Consistently, CD81 was absent in fractions p2 and p3, weakly
detectable in fraction p4 and reliably detectable in the exosomal
fraction p5 of both plasma and serum samples (Figure 1 A, B). The
only difference in detection of CD81 in serum vs. plasma samples
was found in the p1 fraction corresponding to the cellular fraction.
Here, CD81 was detectable in plasma samples only. Fraction p5
containing exosomal CD81 was used for subsequent analyses.
Enrichment of CD81 from different volumes of serum ranging
from 1 mL to 30 mL showed that robust detection of CD81 was
feasible with the smallest tested volume of 1 mL serum. This
volume was used in all further analyses.
Quantification of serum CD81
We next investigated whether CD81 concentration in the p5
fraction can be quantified. For this purpose, different dilutions of
Huh7cell lysates were detected by Western blot analysis and
quantified by densitometry. As shown in Figure 1 C and D, this
approach allowed semiquantitative assessment of CD81 in the p5
fraction (CD81-p5). In the subsequent experiments, different
dilutions of Huh7 lysates covering the range of serum CD81-p5
were used as reference standard. Stock solutions from the same
lysate were used for all analyses (Figure 2A).
Association of serum CD81-p5 with chronic hepatitis C
Aim of this study was to investigate whether soluble CD81 levels
are increased in patients with chronic hepatitis C. Therefore, the
concentration of soluble CD81 in the exosomal fraction p5 was
compared between a cohort of 23 healthy controls and 37 patients
with chronic hepatitis C. As shown in Figure 2, the serum CD81
concentration in fraction p5 was significantly higher in patients
with chronic hepatitis C compared with healthy controls (Figure 2
A, B). In patients with chronic hepatitis C, CD81-p5 was neither
associated with patients characteristics such as age and gender
(Table 1). Apparently, there was no association between CD81-p5
and HCV genotype, however, reliable conclusions on the
association between HCV genotype and soluble CD81 cannot
be drawn due to imbalanced genotype distribution in the different
cohorts. To investigate whether HCV infection is associated with
an increase of serum obtained CD81, CD81-p5 was analyzed in a
cohort of patients with long term cure of HCV after pegylated
interferon-alfa based antiviral therapy and compared with CD81-
p5 in healthy controls and in patients with chronic hepatitis C. In
patients with cured hepatitis C the CD81-p5 level was similar to
healthy controls and significantly lower than in patients with
chronic hepatitis C (Figure 2 B).
CD81-p5 concentration and inflammatory activity
Exosomal CD81 may also be associated with inflammatory
activity. To investigate a potential association between the CD81
concentration in the p5 fraction and liver inflammation, CD81-p5
was analyzed in a cohort of patients with chronic hepatitis C and
persistently normal ALT levels. In the present study, patients with
chronic hepatitis C and persistently normal ALT levels showed
lower CD81-p5 levels than patients with chronic hepatitis C and
elevated ALT levels and comparable levels of CD81-p5 with
healthy controls and patients with cured HCV.
Overall, the CD81-p5 level was associated with serum ALT
activity in patients with chronic hepatitis C with or without ALT
elevation (r=0.372, p=0.003, Figure 2C). Furthermore, we
observed a significant association between CD81-p5 and aspartate
aminotransferase serum levels (r=0.306, p=0.017; Figure 2D).
Both associations were significant for the complete cohort of
patient with chronic HCV infection only, but not for subgroups
with normal or elevated ALT levels, respectively (p.0.2 for both).
Gamma-glutamyl transferase levels were significantly associated
with ALT and AST serum levels but not with CD81-p5 (r=0.589;
p,0.001; r=0.576, p,0.001; r=0.228, p=0.08, respectively).
We also analyzed the association between CD81-p5 and
histological liver damage. CD81-p5 was significantly associated
with severe vs. non severe fibrosis (area under curve 0.699,
p=0.027, Figure 3). However, no correlation was noticed between
CD81-p5 and histological necroinflammatory activity (p.0.2).
Exosomal CD81 in HCV
PLoS ONE | www.plosone.org3February 2012 | Volume 7 | Issue 2 | e30796
Serum ALT and AST levels also did not show significant
correlations with necroinflammatory activity (p.0.2 for both).
CD81-p5 and HCV RNA
As HCV binds to CD81, the CD81 concentration in fraction p5
may be associated with the HCV RNA level. The HCV RNA
concentration was quantified in unfractionated serum and in the
enriched serum fraction p5 in all patients with chronic HCV
infection (Figure 4 A, B). The HCV RNA concentration in
unfractionated serum was significantly correlated with the HCV
RNA concentration in fraction p5 (p,0.001; r=0.696, Figure 4C).
However, the CD81 concentration in fraction p5 neither
correlated with the HCV RNA concentration in unfractionated
serum (p=0.373; r=0.135, Figure 4A) nor with the HCV RNA
concentration in fraction p5 (p=0.39; r=0.15, Figure 4D).
Accordant analyses of the subgroups of patients with persistently
normal or elevated ALT serum levels showed no correlation
between HCV RNA concentration in serum or HCV RNA
concentration in fraction p5 with CD81-p5, respectively (p.0.2
for both subgroups).
The tetraspanin CD81 is an essential HCV hepatocyte cell
entry receptor [13,14]. Interplay between HCV and CD81
expressed on PBMC has been related to HCV persistence as well
as modulation of extrahepatic manifestations of chronic HCV
CD81 containing exosomes have not been studied in larger
cohorts of patients with chronic hepatitis C, so far. Exosomes are
small vesicles secreted by vital cells only, amongst others
considered to modify immune response in viral and malignant
diseases [23,30–33]. For instance, exosome mediated infection of
cells has been described in human immunodeficiency virus
infection . Moreover, exosome mediated intercellular transfer
of CD81 and enhancement of CD81 concentration on cell surfaces
by mergence has been observed . Therefore, exosomal CD81
taken up by target cells, e.g. hepatocytes, could increase HCV
receptor density and facilitate HCV entry, and HCV particles
bound to exosomal CD81 could moreover represent an additional
HCV compartment with putative relevance for the HCV infection
rate. Aim of the present study was to quantify and compare CD81
in the exosomal serum fraction in patients with chronic hepatitis C
compared to healthy controls and patients with cured hepatitis,
and furthermore to investigate whether soluble CD81 in the
exosomal serum fraction is associated with inflammatory activity
in chronic hepatitis C.
Enrichment of exosomes from plasma or serum, as performed
within the current study, can be performed by differential
centrifugation [23,25]. Here, CD81 is an exosomal marker
Figure 1. Enrichment and quantification of soluble CD81. (A, B) Immunoblotting of soluble CD81 is shown after enrichment by differential
centrifugation of frozen (A) plasma and (B) serum samples. Huh7 cells served as positive control, and HepG2 cells as negative control. While only a
marginal fraction was detectable in fraction p4, the main portion was detectable in fraction p5, comparable between plasma and serum samples. (C,
D). Soluble CD81 concentration was assessed semi-quantitatively by use of dilutions from a Huh7 cell lysate by densitometric analysis. The lysate
dilution immunoblot analysis and the corresponding obtained standard curve are shown in (C) and (D), respectively.
Exosomal CD81 in HCV
PLoS ONE | www.plosone.org 4February 2012 | Volume 7 | Issue 2 | e30796
protein, and the CD81 content in the exosomal fraction can be
considered to be mainly of exosomal origin [23,25]. Therefore, we
measured the concentration of soluble CD81 after differential
centrifugation of a standardized volume of 1 mL serum.
In the present study, soluble CD81 levels were increased in the
exosomal serum fraction p5 in patients with chronic hepatitis C
compared to healthy volunteers. Moreover, in patients cured from
hepatitis C, CD81 levels normalized to levels found in healthy
subjects. Overall these results suggest that infection with the
hepatitis C virus is associated with increase of soluble CD81.
Soluble CD81 could be associated with HCV replication.
Therefore, we assessed whether soluble CD81 correlates with the
HCV RNA level. In the present study, however, HCV RNA
serum levels were not correlated with the CD81 concentration in
the exosomal serum fraction p5. HCV RNA may also bind to
exosomal CD81 which is suggested by data from Masciopinto et
al., who have shown an association of HCV envelope proteins and
HCV RNA with exosomal CD81 in vitro . In the present study,
HCV RNA was enriched in the exosomal serum fraction;
however, the concentration of HCV RNA in the exosomal serum
fraction was not correlated with the soluble CD81 concentration.
Therefore, it may be concluded that exosomal CD81 is not
directly involved in HCV replication.
Recently, proinflammatory exosomes have been described to
contribute to inflammation e.g. in sarcoidosis . To investigate
whether increased soluble CD81 levels are associated with disease
activity in chronic HCV infection, we compared soluble CD81
serum levels in patients with chronic hepatitis C to a cohort of
patients with HCV infection but persistently normal ALT levels as
ALT is a surrogate marker for apoptosis and necroinflammation in
chronic hepatitis C. It is estimated that approximately 30% of
patients with HCV show normal ALT levels . The reason
therefore is unknown, but it was suggested that an arbitrary high
upper limit of normal range may contribute to this phenomenon
. The long term clinical outcome is not completely known.
Patients with HCV infection and persistently normal ALT levels
may show a more benign course than patients with elevated ALT
levels. Nevertheless, several studies described significant histolog-
ical liver lesions and increased ALT levels over time in these
patients [36–40], indicating that this specific cohort may develop
Figure 2. Quantification of soluble CD81 serum levels in different populations and correlation to serum ALT and AST levels. (A)
Immunoblot analysis from representative patient samples (P1–P5) and healthy controls (H1–H5) as well as Huh7 dilution series (D1–D3). Enrichment
of soluble CD81 had been standardized before sample processing, and a standardized volume of 1 mL serum was used for CD81 enrichment and
quantification in all study samples analyzed. (B) Enriched CD81 serum concentrations given in healthy volunteers (n=23), patients with chronic HCV
infection and persistently normal ALT levels (n=24), patients with chronic hepatitis C and elevated ALT levels (n=37), and patients with long-term
SVR (n=7). The CD81 concentration is given in relative units (RU)/mL as box-and-whisker plot showing median, 25 and 75 quartiles as well as the total
range. Differences between two groups were calculated by the Mann-Whitney U test. (C) Correlation of CD81 concentration in fraction p5 given in
RU/mL and serum ALT level given in U/mL in all patients with chronic HCV infection, including patients with elevated (n) and persistently normal (#)
ALT levels (n=61). Correlations between two variables were assessed by the Spearman’s rank correlation test. (D) Correlation of CD81 concentration
in fraction p5 given in RU/mL and serum AST level given in U/mL in all patients with chronic HCV infection, including patients with elevated (n) and
persistently normal (#) ALT levels (n=61). Correlations between two variables were assessed by the Spearman’s rank correlation test.
Exosomal CD81 in HCV
PLoS ONE | www.plosone.org5February 2012 | Volume 7 | Issue 2 | e30796
liver damage in the absence of surrogate cell death markers. In the
current study, soluble CD81 levels were significantly higher in
patients with hepatitis C compared to patients with HCV infection
and normal ALT levels. This may indicate that increase of soluble
CD81 in patients with chronic hepatitis C is mostly related with
HCV associated necroinflammation.
To further investigate the observed correlation between soluble
CD81 levels and necroinflammatory activity, we compared CD81
levels to ALT and AST levels. Here, a significant correlation
between serum ALT as well as AST levels and soluble CD81 was
observed. This may indicate an association between hepatocyte
cell death and higher CD81 serum levels. The association between
soluble CD81 and surrogate markers of cell death could be due to
release of CD81 containing exosomes or cell detritus from
hepatocytes during the necroinflammatory process. However,
the correlation coefficients between soluble CD81 levels and
biochemical markers of liver damage, even though statistically
significant, were weak. A final conclusion that soluble CD81 levels
are mostly derived from inflammatory processes in patients with
hepatitis C cannot be drawn from the results of the current study.
Another explanation for the increase of soluble CD81 levels in
patients with chronic hepatitis C is secretion of CD81 containing
exosomes by lymphocytes. Interaction of HCV particles with
CD81 expressed on PBMC as well as alteration of CD81
expression on PBMC during HCV infection is well known and
of clinical importance [18,19,41]. Potential interaction of PBMC
released CD81 exosomes with HCV particles may occur and
influence hepatic inflammation in chronic hepatitis C. Here,
further studies defining the source of soluble CD81 in HCV
infection are necessary.
In summary, the results of the present study show that chronic
HCV infection is associated with increase of soluble CD81 in the
exosomal serum fraction. CD81 in the exosomal fraction in
patients with chronic hepatitis C appears to be associated with
inflammatory activity. This is a new finding with potential
implications in the understanding of HCV persistence and
HCV-associated necroinflammation. In concordance, soluble
CD81 levels were associated with higher stage liver fibrosis.
Table 1. Baseline characteristics of patients and healthy controls.
Patients with chronic HCV infection Patients with SVR Healthy volunteers
(n=24)(n=37) (n=7) (n=23)
Age (mean 6 SD)50.8611.8 53.3610.263.6611.829.465.7
Gender (male/female) 10/1415/224/3 12/11
ALT (U/L; mean 6 SD)41.4612.8145.36108.3 28.7610.826.564.9
AST (U/L; mean 6 SD)39.9616.9106.7661.838.422.214.171.124
GGT (U/L; mean 6 SD) 49.3648.199.8671.324.5610.6 30.066.8
Histological activity index
I(median [min., max.])1 (1, 1) 1 (1, 3)n.d. n.a.
II(median [min., max.])1 (1, 1) 1 (1, 1)n.d.n.a.
III (median [min., max.]) 3 (1, 3)3 (1,3)n.d.n.a.
IV(median [min., max.]) 1 (1, 3) 3 (1,3) n.d. n.a.
Genotype distribution (1/2/3) 17/4/332/1/4n.d. n.a.
HCV RNA (log IU/mL, mean 6 SD)5.860.6 6.160.6n.d.n.a.
ALT, alanine transaminase; AST, aspartate transaminase; GGT, gamma-glutamyl transferase (the upper limit of normal was 50 U/L, 50 U/L, 39 U/L for ALT, AST and GGT);
max., maximum; min; minimum; n.a., not applicable; n.d., not done NR, nonresponse SD, standard deviation; SVR, sustained virologic response.
Figure 3. Receiver operating characteristic showing the
association between soluble CD81 serum concentration and
fibrosis. Higher CD81-p5 levels were associated with sever fibroses
(area under curve, AUC, 0.699; p=0.027).
Exosomal CD81 in HCV
PLoS ONE | www.plosone.org6February 2012 | Volume 7 | Issue 2 | e30796
Conceived and designed the experiments: MWW CS EH SZ AP BK.
Performed the experiments: DR SS YM. Analyzed the data: MWW DR
CS EH SZ AP BK. Contributed reagents/materials/analysis tools: CS EH
SZ AP BK. Wrote the paper: MWW CS SZ AP BK.
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