The tetraspanin CD81 is an essential HCV hepatocyte cell entry receptor 
. Interplay between HCV and CD81 expressed on PBMC has been related to HCV persistence as well as modulation of extrahepatic manifestations of chronic HCV infection 
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 
. 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 
. Here, CD81 is an exosomal marker protein, and the CD81 content in the exosomal fraction can be considered to be mainly of exosomal origin 
. 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 histological liver lesions and increased ALT levels over time in these patients 
, indicating that this specific cohort may develop 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 
. 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.