Infection with hepatitis C virus (HCV), a leading cause of chronic liver diseases, can associate with B lymphocyte proliferative disorders, such as mixed cryoglobulinemia and non-Hodgkin lymphoma. The major envelope protein of HCV (HCV-E2) binds, with high affinity CD81, a tetraspanin expressed on several cell types. Here, we show that engagement of CD81 on human B cells by a combination of HCV-E2 and an anti-CD81 mAb triggers the JNK pathway and leads to the preferential proliferation of the naïve (CD27 ؊ ) B cell subset. In parallel, we have found that B lymphocytes from the great majority of chronic hepatitis C patients are activated and that naïve cells display a higher level of activation markers than memory (CD27 ؉ ) B lymphocytes. Moreover, eradication of HCV infection by IFN therapy is associated with normalization of the activation-markers expression. We propose that CD81-mediated activation of B cells in vitro recapitulates the effects of HCV binding to B cell CD81 in vivo and that polyclonal proliferation of naïve B lymphocytes is a key initiating factor for the development of the HCV-associated B lymphocyte disorders.monoclonal antibody ͉ multimeric engagement ͉ B cell antigen receptor ͉ cryoglobulinemia H epatitis C virus (HCV) is a positive-stranded RNA virus of the Flaviviridae family (1). The HCV genome is 9.6 kb in length, with one large translational ORF encoding a single polyprotein, which is processed by host and viral proteases into at least three structural and seven nonstructural proteins with various enzymatic activities (1). Two heavily N-glycosylated proteins E1 and E2 are virion-envelope proteins and form heterodimers in vitro (2). An estimated 170 million individuals are infected with HCV worldwide (3). HCV infection is associated with the development of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (4). B cell abnormalities, including cryoglobulinemia (5) and an increased risk of B cell non-Hodgkin lymphoma (6, 7), have been reported in a minority of HCV infections.Until very recently, it was not possible to grow HCV in cell culture, therefore studies of virus interaction with human cells have been surrogated by the assessment of binding and entry of HCV recombinant glycoproteins (8) or virus pseudotypes (9).We have previously reported that HCV-E2 protein binds with high affinity to the large extracellular loop of human CD81 (CD81-LEL) and that ''bona fide'' HCV particles bind human CD81 (10). Recently, it has been demonstrated that CD81 is required for entry and infection of human cells by in vitro-generated infectious HCV (11). CD81 is a widely distributed cell-surface tetraspanin that participates in different molecular complexes on various cell types, including B, T, and natural killer (NK) cells (12). On human B cells, CD81 is known to form a costimulatory complex with CD19 and CD21 (13,14) and that coligation of the B cell antigen receptor (BCR) with any of the components of this costimulatory complex lowers the threshold required for BCR-mediated B cell proliferat...

Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomicproteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.Chlamydia pneumoniae is an obligate intracellular bacterium and a common human pathogen (48). It is a significant cause of pneumonia in both hospital and outpatient settings, accounting for approximately 7 to 10% of cases of community-acquired pneumonia among adults. C. pneumoniae has also been associated with atherosclerotic and cardiovascular disease, as suggested by results of seroepidemiologic studies, detection of the organism in atherosclerotic plaque specimens, experimental in vitro cell culture studies, animal model studies, and two small secondary prevention antibiotic treatment trials (12,13,15,19,20,28,45).Like all obligate intracellular parasites, for its survival and propagation C. pneumoniae must accomplish several essential tasks which include adhering to and entering host cells, creating an intracellular niche for replication, exiting host cells for subsequent invasion of neighboring cells, and also avoiding host defense mechanisms. To carry out all these functions, C. pneumoniae has developed a unique biphasic life cycle involving two developmental forms, a spore-like infectious form (elementary bodies [EBs]) and an intracelluar replicative form (reticulate bodies [RBs]). Adhesion, host cell colonization capabilities, and the ability to cope with host defense mechanisms when outside the cell presumably rely in large part on EB surface organization.Because of the intrinsic difficulty in working with C. pneumoniae and the lack of adequate methods for its genetic ma...

Chronic viral hepatitis is characterized by a dramatic lymphocyte infiltrate in the liver. Although it is one of the most common chronic inflammatory diseases in humans, little information is available on the functional state of these intra-hepatic lymphocytes (IHL). To address this issue, we have optimized cytofluorimetric techniques to assess directly ex vivo the functions, dynamics and repertoires of IHL isolated from biopsies of patients with chronic hepatitis C. We estimate that 1% of the total body lymphocytes infiltrate the inflamed liver and find that, at variance with peripheral blood lymphocytes (PBL) isolated from the same patients, most IHL display an activated phenotype and produce Th1 type lymphokines when stimulated in vitro. Virtually all IHL are found in the G0/G1 state of the cell cycle, while a sizeable percentage of them is undergoing programmed cell death in vivo, as detected by the TUNEL assay performed on freshly isolated cells. In contrast again to PBL from the same patients, IHL show a preferential compartmentalization of NK and TCRgamma/delta+ cells, and a remarkable (up to 20-fold) enrichment for Valpha24+ T cells. Together our data suggest that in a liver injured by chronic hepatitis C, most IHL are pro-inflammatory activated cells which are highly enriched for effectors of innate resistance. These IHL do not undergo clonal expansion in the liver but rather display effector function and die in situ at a high rate, suggesting that maintenance of the IHL pool is dependent on continuous migration from extra-hepatic sites.