Despite inducing a strong host cellular and humoral immune response, the helminth Echinococcus granulosus is a highly successful parasite that develops, progresses, and ultimately causes chronic disease. Although surgery remains the preferred therapeutic option, pharmacological research now envisages antihelminthic strategies. To understand the mechanisms that E. granulosus uses to escape host immunosurveillance and promote chronic infection, we investigated how two hydatid cyst components, purified antigen B (AgB) and sheep hydatid fluid (SHF), act on host dendritic cell (DC) differentiation from monocyte precursors and how they influence maturation of DC that have already differentiated. We evaluated the immunomodulatory potential of these antigens by performing immunochemical and cytofluorimetric analyses of monocyte-derived DCs from healthy human donors. During monocyte differentiation, AgB and SHF downmodulated CD1a expression and upregulated CD86 expression. Compared with immature DCs differentiated in medium alone (iDCs), AgB-and SHF-differentiated cells stimulated with lipopolysaccharide included a significantly lower percentage of CD83؉ cells (P < 10 ؊4 ) and had weaker costimulatory molecule expression. When stimulated with AgB and SHF, iDCs matured and primed lymphocytes towards the Th2 response typical of E. granulosus infection. SHF and particularly AgB reduced the production of interleukin-12p70 (IL-12p70) and tumor necrosis factor alpha in lipopolysaccharide-stimulated iDCs. Anti-IL-10 antibodies increased the levels of IL-12p70 secretion in AgB-and SHF-matured DCs. AgB and SHF induced interleukin-1 receptor-associated kinase phosphorylation and activated nuclear factor-B, suggesting that Toll-like receptors could participate in E. granulosus-stimulated DC maturation. These results suggest that E. granulosus escapes host immunosurveillance in two ways: by interfering with monocyte differentiation and by modulating DC maturation.

). AgB skewed the Th1/Th2 cytokine ratios towards a preferentially immunopathology-associated Th2 polarization, predominantly in patients with progressive disease. AgB-stimulated patients' PBMC also proliferated less than SHF-stimulated PBMC (P ‫؍‬ 9 ؋ 10 ؊3 ). In vitro Th2 cytokine production was reflected in vivo by elevated specific immunoglobulin E (IgE) and IgG4 antibodies binding to AgB. These findings confirm that AgB plays a role in the escape from early immunity by inhibiting PMN chemotaxis. They also add new information on the host-parasite relationship, suggesting that AgB exploits the activation of T helper cells by eliciting a nonprotective Th2 cell response.Cystic echinococcosis (CE) is a widely endemic helminthic disease caused by infection with metacestodes (larval stage) of the tapeworm Echinococcus granulosus. It affects humans and a wide range of livestock species (28). The disease is characterized by the growth in the host internal organs, mostly liver and lungs, of steadily growing fluid-filled, unilocular cysts surrounded by a two-layered hydatid cyst wall. The main feature of the host-parasite relationship is the coexistence of the chronic infection with detectable humoral and cellular responses against the parasite. Parasite survival in vivo depends on efficient evasion mechanisms starting to operate as the parasite develops toward a hydatid cyst. A fibrotic host capsule of variable thickness usually develops, thus forming together with the parasite-derived acellular laminated layer a formidable cystic structure. As a consequence, the actively dividing germinal layer within the cyst along with its associated brood capsule and enclosed protoscoleces are effectively sequestered from the host immune responses. In addition to this physical barrier, the hydatid cyst has probably evolved other strategies for immune evasion. Although older models suggested a more passive role for parasites in immune evasion-for example sequestration, antigenic masking by host proteins, and global immunosuppression-later studies suggest in human parasitoses the active deployment of strategies that manipulate and exploit the host response (12, 30).

The human plasma protein ␤ 2 -glycoprotein I (␤ 2 -GPI) is the most common target for antiphospholipid antibodies associated with thrombotic events in chronic disorders related to endothelial cell dysfunction. Crucial information is needed to clarify why this self-abundant protein is targeted by autoimmune responses. In this study, we investigated whether oxidative modification of ␤ 2 -GPI, either spontaneous in culture wells or induced by treatment with H 2 O 2 , renders this selfprotein able to activate immature monocyte-derived dendritic cells (DCs) from healthy human donors. Oxidized ␤ 2 -GPI caused DCs to mature so that CD83 appeared and CD80, CD86, human leukocyte antigen-D region related (HLA-DR), and CD40 increased. The interaction between oxidized ␤ 2 -GPI and DCs specifically stimulated these cells to secrete interleukin 12 (IL-12), IL-1␤, IL-6, IL-8, tumor necrosis factor ␣ (TNF-␣), and IL-10. Oxidized ␤ 2 -GPI-stimulated DCs had increased allostimulatory ability and primed naive T lymphocytes, thus inducing T helper 1 (Th1) polarization. The interaction between oxidized ␤ 2 -GPI and DCs involved interleukin-1 receptor associated kinase (IRAK) phosphorylation and nuclear factor B (NFB) activation. Pretreatment of ␤ 2 -GPI with the antioxidant ␣-tocopherol prevented DC maturation. These findings show that human oxidized ␤ 2 -GPI, probably by interacting with a member of the Toll-like receptor (TLR) family, causes DCs to mature. Because this key ␤ 2 -GPI function requires oxidative modification, in several chronic disorders related to endothelial cell dysfunction oxidative stress might trigger the "autoimmune spiral. Introduction␤ 2 -glycoprotein I (␤ 2 -GPI), a human plasma protein that binds to negatively charged phospholipids, is the most common target for antiphospholipid antibodies (aPLs). These autoantibodies are associated with thrombotic events in systemic lupus erythematosus 1 and primary antiphospholipid antibody syndrome 2,3 and are proatherogenic. 4 ␤ 2 -GPI also activates lipoprotein lipase, 5 lowers the triglyceride level, 6 and binds to oxidized low-density lipoprotein (LDL) 7 and to nonself particles or apoptotic bodies to allow their clearance. [8][9][10] Another property of ␤ 2 -GPI is its ability to bind at the monocyte surface, thus promoting tissue factor and thereby increasing the risk of thrombotic events. 11 ␤ 2 -GPI can also be expressed on the endothelial cell membrane 12 and on macrophages. 13 Among the several candidate ␤ 2 -GPI cell receptors, annexin II, megaline, and apolipoprotein E receptor 2Ј (apoER2Ј) are involved in activating endothelial cells and platelets. [14][15][16] Recent findings demonstrate that anti-␤ 2 -GPI antibodies react with their antigen probably in association with a member of the Toll-like receptor (TLR)/interleukin 1 (IL-1) receptor family on the endothelial cell surface and directly induce activation. 17,18 Once bound to endothelial cells, ␤ 2 -GPI offers suitable epitopes targeting circulating anti-␤ 2 -GPI antibodies that affect cell functions a...

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To evaluate the diagnostic sensitivity and specificity of immunoelectrophoresis (IEP), indirect haemagglutination (IHA), enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB), we compared their ability in detecting IgG antibodies to a hydatid fluid fraction (HFF) and to native and recombinant antigen B of Echinococcus granulosus. We tested sera from patients who had cystic echinococcosis (CE) grouped according to their type of cysts (n = 204), from patients with other parasitic diseases (n = 21), lung or liver carcinomas (n = 6) or serous cysts (n = 26) and from healthy controls (n = 90). HFF-IB gave the highest sensitivity (80%) followed by ELISA (72%), IHA (54%) and IEP (31%), respectively. The diagnostic sensitivity significantly (P < 0.01) decreased as cysts matured from type I-II to type VII. Recombinant and native antigen B-IB yielded similar sensitivity (74%). A large number of clinically or surgically confirmed CE patients (20%) resulted negative. In these patients' sera, IB to assess the usefulness of the recombinant E. granulosus elongation factor-1 beta/delta in detecting IgE antibodies yielded 33% of positivity. Our findings underline the need to standardize techniques and antigenic preparations and to improve the performance of immunodiagnosis by characterizing new antigens and detecting distinct immunoglobulin classes.

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To investigate the role of T lymphocytes in the immune response to Echinococcus granulosus, using sheep hydatid fluid (SHF) and antigen B (AgB), we generated T-cell lines from patients with active, transitional and inactive hydatid cysts. We established 16 T-cell lines, eight specific to SHF and eight specific to AgB. At surface phenotyping 88-98% of cells displayed the helper/inducer CD4 antigen. In all patients, at all clinical stages of hydatid cyst disease, T-cell stimulation with SHF and AgB invariably amplified a large number of almost identical Vbeta subfamily fragments. Irrespective of antigen-specificity, the two cell lines from the patient with an inactive cyst had a Th1 profile, because they exclusively expressed and produced IFN-gamma. Conversely, the T-cell lines derived from the seven patients with active and transitional hydatid cysts had mixed Th1/Th2 and Th0 clones. The functional characteristics of the 16 T-cell lines differed markedly in the various clinical stages of cystic echinococcosis, thus providing new in vitro evidence that Th1 lymphocytes contribute decisively to the inactive stage of hydatid disease, Th2 lymphocytes in the active and transitional stages. The parasite-specific T-cell lines, especially the two Th1 lines from the patient with an inactive cyst, may help identify Th1 protective epitopes on SHF and AgB.

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4؉ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60 -65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.A major requirement for the susceptibility to CD95 (APO-1/ Fas)-mediated apoptosis in CD4ϩ T cells is the CD95 polarization on cell uropods, as a result of CD95 linkage to the actin cytoskeleton through ezrin (1). Ezrin, radixin, and moesin (ERM) 1 are closely related proteins involved in cellular polarization and in various cellular functions (2-4). ERM are found in microvilli, filopodia, membrane ruffles, and cell-to-cell contact sites, where they co-localize and associate with F-actin (5-10). ERM are members of the erythrocyte protein 4.1 superfamily, characterized by a ϳ300-residue globular N-terminal domain highly conserved in the ERM family (FERM domain) (3,(11)(12)(13)(14). ERM interact directly with various membrane proteins, such as CD43, CD44, and intercellular adhesion molecule-1, -2, and 3, through their FERM amino-terminal domain (reviewed in Ref.3). These proteins can also indirectly bind to H ϩ /Na ϩ exchanger 3 via the cytoplasmic protein EBP50 (15, 16). ERM are present both in an inactive/closed and in an active/opened form (9, 14), and a direct consequence of ERM activation is their recruitment to the plasma membrane, allowing the ERM linkage to the membrane molecules (17, 18). The ERM/membrane protein interaction is stabilized at the plasma membrane by the ERM association to phosphatidylinositol 4, 5-bisphosphate (19, 20) and regulated by tyrosine phosphorylation in Tyr 145 and Tyr 353 (21-26). However, whereas the ERM domains involved in actin binding are known (27-30), the specific sites accounting for the binding to membrane proteins are much less defined. Particularly, the specific a...