We monitored the number of intravascular platelet-leukocyte aggregates (PLAs) and thrombotic occlusions (TOs) by intravascular microscopy in the mesentery of rats receiving antiphospholipid (aPL) immunoglobulin G (IgG) purified from the sera of patients with antiphospholipid syndrome. aPL IgG had no procoagulant effect, but it caused rapid endothelial deposition of fibrinogen, followed by PLA and TO in rats receiving an intraperitoneal injection of bacterial lipopolysaccharide 3 hours before IgG infusion. Anti-␤2-glycoprotein I-depleted aPL IgG failed to induce PLAs and TOs. C3 and C9 colocalized with aPL IgG on the mesenteric vessels. The number of PLAs and TOs was markedly reduced in C6-deficient rats and in animals treated with anti-C5 miniantibody, suggesting the contribution of the terminal complement (C) complex to the aPL antibody-mediated intravascular thrombosis. In conclusion, our data indicate that antibodies to ␤2-glycoprotein I trigger coagulation subsequent to a priming proinflammatory factor and that the terminal C complex is the main mediator of the coagulation process. (Blood. 2005; 106:2340-2346)

Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa−/−) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa−/− mice. Bone marrow (BM) chimeras between C1qa−/− and WT mice identify non-BM-derived cells as the main local source of C1q that can promote cancer cell adhesion, migration and proliferation. Together these findings support a role for locally synthesized C1q in promoting tumour growth.

In vitro studies have documented ␤2 glycoprotein I (␤2GPI) binding to endothelial cells (ECs) and trophoblast using antiphospholipid antibodies. The in vivo binding of ␤2GPI to these cells and the conditions that favor their interaction have not been investigated. We analyzed the in vivo distribution of cyanine 5.5-labeled ␤2GPI in mice and evaluated the effect of pregnancy and circulating antibodies on its tissue localization. The signal was detected in the liver by whole body scan and ex vivo analysis. The ␤2GPI failed to bind to the vascular endothelium and reacted only with the ECs of uterine vessels. In pregnant mice the protein was localized on ECs and trophoblast at the embryo implantation sites. Immunized mice showed a similar ␤2GPI biodistribution to naive mice but the immunized pregnant animals exhibited a significant increase in fetal loss associated with C3 and C9 deposition at the implantation sites. Treatment of mice with LPS after ␤2GPI-Cy5.5 injection promoted protein localization on gut and brain ECs associated with IgG, C1q, and C9 deposition in immunized mice. These findings indicate that ␤2GPI binding to EC requires priming with pro-inflammatory factors which is not needed for uterine and placental localization probably dependent on hormonal changes. (Blood. 2011;118(15): 4231-4238) IntroductionAntiphospholipid syndrome (APS) is characterized by vascular thrombosis and adverse pregnancy outcome associated with circulating antiphospholipid antibodies (aPL) which are believed to play an important pathogenic role in the development of the clinical manifestations of the syndrome. 1,2 Human ␤2-glycoprotein I (␤2GPI) has been recognized as the major antigenic target for antiphospholipid antibodies and in vivo models have shown that antibodies directed against this molecule are able to mediate thrombus formation. 3,4 Beta2GPI is a heavily glycosylated glycoprotein that circulates in blood at a concentration of 150-300 g/mL 5 and is synthesized mainly in the liver, although expression of ␤2GPI mRNA has also been detected in endothelial cells (ECs), central nervous system, astrocytes, and placenta. The physiologic function of this protein is still unclear, but the apparently healthy life of humans and mice deficient in ␤2GPI suggests that its role is not all that critical. [6][7][8][9] Most of the information now available on ␤2GPI has been collected following the observation that this protein is the main target of antiphospholipid (aPL) antibodies. 10,11 Patients with circulating antibodies to ␤2GPI are at increased risk of venous and arterial thrombosis as well as of pregnancy complications including miscarriage, preeclampsia and retarded fetal growth. 1,12 For this reason antibodies with this specificity have been included among the criteria for the diagnosis of aPL syndrome (APS). 13,14 The induction of fetal loss and promotion of thrombosis in animal models as a result of immunization with ␤2GPI or passive transfer of antibodies further support the pathogenic role of these antibodies. [15][16][17...

Recombinant osteoprotegerin (OPG) promoted the adhesion of both primary polymorphonuclear neutrophils (PMNs) and leukemic HL60 cells to endothelial cells. Leukocyte/endothelial cell adhesion was promoted by short (peak at 1 hour) preincubation of either endothelial cells or PMNs with OPG, and the peak of proadhesive activity was observed in the same range of OPG concentrations detected in the sera of patients affected by cardiovascular diseases. Although the cognate high-affinity ligands for OPG, membrane receptor activator of nuclear factor-B ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were detected at significant levels on both PMNs and HL60 cells, they were not expressed on the surface of endothelial cells. However, preincubation of OPG with heparin abrogated its proadhesive activity, whereas pretreatment of endothelial cells with chondroitinase plus heparinases significantly decreased the proadhesive activity of OPG. Taken together, these findings suggest the involvement of both the ligand binding and the N-terminal heparin-binding domains of OPG in mediating its pro-adhesive activity. The relevance of these in vitro findings was underscored by in vivo experiments, in which the topical administration of recombinant OPG increased leukocyte rolling and adhesion to rat mesenteric postcapillary venules. Our data suggest that a pathological increase of OPG serum levels might play an important role in promoting leukocyte/endothelial cell adhesion. IntroductionOsteoprotegerin (OPG), a soluble member of the tumor necrosis factor (TNF) receptor superfamily, was originally characterized for its ability to suppress osteoclast formation. 1 OPG inhibits osteoclastogenesis by binding to receptor activator of nuclear factor-B (NF-B) ligand (RANKL), a member of the TNF superfamily of cytokines, and preventing the interaction of RANKL with its high-affinity transmembrane receptor (receptor activator of NF-B [RANK]). 2 It has been shown that in the low nanomolar range, the binding interaction of OPG and RANKL is 1:1 3 and that dimerization of OPG results from noncovalent interactions mediated by the death domains and, to a lesser extent, by a C-terminal heparinbinding region. OPG dimer formation is required for the mechanism of inhibition of the RANKL/RANK receptor interaction. 3 OPG can also interact with another member of the TNF superfamily, TNF-related apoptosis inducing ligand (TRAIL), 4 which can kill a variety of cancer cell types both in vivo and in vitro. 5 A role for OPG as a neutralizing receptor for TRAIL under physiological conditions has been questioned in early studies, but mounting evidence now suggests that the OPG/TRAIL interaction is biologically important, at least in in vitro culture systems. 6 OPG has been shown to act in a paracrine or autocrine manner by binding TRAIL and promoting the survival of prostate cancer cells, 7 breast cancer cells, 8 and multiple myeloma cells. 9 Moreover, when a rationally designed small molecule mimic of OPG was examined for associa...

Key Points• A recombinant antibody recognizing the D1 domain of b2 glycoprotein I induces fetal loss and clot formation in animal models.• The CH2-deleted antibody fails to activate complement and prevents the procoagulant and proabortive effects of patient antibodies.A single-chain fragment variable (scFv) recognizing b2-glycoprotein 1 (b2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against b2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-b2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-b2GPI antibodies from APS patients and displaced b2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy. (Blood. 2014;123(22):3478-3487)

Recurrent thrombosis and miscarriages are the main clinical manifestations of antiphospholipid syndrome (APS). Although most patients display both clinical signs, some patients can have isolated vascular or obstetric variants. Emerging data raise the question of whether obstetric and vascular APS are the same or different diseases. An important difference between the two conditions is that a thrombophilic state is a common feature in vascular APS, whereas clot occlusions of the decidual spiral arteries are seldom observed in obstetric APS, and infarctions are found in only one-third of APS placentae. Conversely, inflammation, which is undetectable in vascular APS, is frequently observed in the placentae of patients with obstetric APS and has been documented in the placentae of pregnant mice with fetal loss mediated by antiphospholipid antibodies. Attempts to identify different antibodies or epitopes responsible for the two clinical manifestations of APS have so far been unsuccessful. Possible mechanisms exist that might explain the development of the two clinical presentations, including the tissue distribution and expression level of the main target antigen of antiphospholipid antibodies, β2 glycoprotein I (β2GPI). The identification of the factors that promote the onset of either obstetric or vascular APS has important diagnostic and therapeutic implications.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of β2 integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection.