Antiphospholipid antibodies represent a heterogeneous group of autoantibodies directed against anionic phospholipids (PLs) usually linked to protein cofactors. Their presence during the antiphospholipid syndrome is associated with risks of thrombosis and fetal losses. Among 5 randomly selected monoclonal antiphospholipid antibodies, all originating from a single patient suffering from this autoimmune disease, only 1 induced fetal losses when passively injected into pregnant mice. Its antiphospholipid activity was dependent on annexin A5, and its variable regions contained mainly 3 replacement mutations. To clarify the role of these mutations in the pathogenicity of the antibody, they were in vitro reverted to the germ line configuration. The resulting "germ line" antibody reacted with multiple self-antigens and only partially lost its reactivity against PLs, but it was no more dependent on annexin A5 and, more importantly, was no more pathogenic. This study illustrates that the in vivo antigen-driven maturation process of natural autoreactive B cells can be responsible for pathogenicity. (Blood.

Globoside (Gb4Cer), Ku80 autoantigen, and ␣5␤1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptormediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections.

The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) IntroductionAntiphospholipid autoantibodies (aPLs) 1 are associated with thrombosis, recurrent fetal loss, and thrombocytopenia in patients with antiphospholipid syndrome (APS). [2][3][4] The mechanism of the characteristic prothrombotic state related to aPLs during APS is still under debate. 5 Much of it is related to the definition of the precise nature of aPL specificity, which remains unclear because of the number of possible antigenic targets discovered over the last few years. It is now accepted that plasma proteins play an important role in aPL reactivity. 6,7 The most common protein cofactors are ␤2GP1 8,9 and prothrombin. [10][11][12] Other phospholipid (PL)-binding proteins such as protein C9, protein S9, and annexin V 13 have also been found to have cofactor properties. On one hand, studies have shown that the binding of aPLs to cardiolipin (CL) is enhanced by the addition of one cofactor (␤2GP1, prothombin, protein C, protein S), which suggests that aPLs react to a complex compounded of anionic PLs and a cofactor 8,10,11 or to an epitope that becomes exposed when a protein is bound to a PL. [14][15][16] On the other hand, other studies have shown that aPLs are able to bind directly to immobilized ␤2GP1 [17][18][19] or PT 20,21 in the absence of PL. In addition, some studies have shown that anti-␤2GP1 antibodies (Abs) are primarily associated with thrombosis in APS 22-24 although other reports have involved others Abs as anti-PT 25 or lupus anticoagulant. 26 It appears that most of these difficulties are related to the use of polyclonal Abs (serum or purified immunoglobulin [Ig]) 7,8,10,11 or a few monoclonal IgM 15,27-29 or IgG 30-33 aPLs originating from different patients. We therefore considered it necessary to concentrate our attention on aPL reactivity of each Ab making up the polyclonal aPL reactivity, breaking down this reactivity at the clonal level in a single patient.The major methods currently in use for the production of human monoclonal antibodies (mAbs) are Epstein-Barr virus transformation, 34 mouse-human hybridomas, 35 or human-human hybridomas 36 and fusion of Epstein-Barr virus-transformed B lymphocytes with a malignant cell line. 37 These methods have their dr...