Charis Pericleous

Antiphospholipid antibodies induce a pro-inflammatory response in first trimester trophoblast via the TLR4/MyD88 pathway

Problem  Women with antiphospholipid antibodies (aPL) are at risk for recurrent miscarriage, pre‐eclampsia, and pre‐term labor. aPL target the placenta directly by binding to beta2‐glycoprotein I (β2GPI) expressed on the surface of trophoblast cells. The objective of this study was to determine the effects of aPL on trophoblast function and the mechanisms involved.

Novel Assays of Thrombogenic Pathogenicity in the Antiphospholipid Syndrome Based on the Detection of Molecular Oxidative Modification of the Major Autoantigen β2-Glycoprotein I

Objective Beta-2-glycoprotein I (β2GPI) constitutes the major autoantigen in the antiphospholipid syndrome (APS), a common acquired cause of arterial and venous thrombosis. We recently described the novel observation that β2GPI may exist in healthy individuals in a free thiol (biochemically reduced) form. The present study was undertaken to quantify the levels of total, reduced, and posttranslationally modified oxidized β2GPI in APS patients compared to various control groups. Methods In a retrospective multicenter analysis, the proportion of β2GPI with free thiols in serum from healthy volunteers was quantified. Assays for measurement of reduced as well as total circulating β2GPI were developed and tested in the following groups: APS (with thrombosis) (n = 139), autoimmune disease with or without persistent antiphospholipid antibodies (aPL) but without APS (n = 188), vascular thrombosis without APS or aPL (n = 38), and healthy volunteers (n = 91). Results Total β2GPI was significantly elevated in patients with APS (median 216.2 μg/ml [interquartile range 173.3–263.8]) as compared to healthy subjects (median 178.4 μg/ml [interquartile range 149.4–227.5] [P < 0.0002]) or control patients with autoimmune disease or vascular thrombosis (both P < 0.0001). The proportion of total β2GPI in an oxidized form (i.e., lacking free thiols) was significantly greater in the APS group than in each of the 3 control groups (all P < 0.0001). Conclusion This large retrospective multicenter study shows that posttranslational modification of β2GPI via thiol-exchange reactions is a highly specific phenomenon in the setting of APS thrombosis. Quantification of posttranslational modifications of β2GPI in conjunction with standard laboratory tests for APS may offer the potential to more accurately predict the risk of occurrence of a thrombotic event in the setting of APS.

Modulation of trophoblast angiogenic factor secretion by antiphospholipid antibodies is not reversed by heparin.

Citation Carroll TY, Mulla MJ, Han CS, Brosens JJ, Chamley LW, Giles I, Pericleous C, Rahman A, Sfakianaki AK, Paidas MJ, Abrahams VM. Modulation of trophoblast angiogenic factor secretion by antiphospholipid antibodies is not reversed by heparin. Am J Reprod Immunol 2011; 66: 286–296

Effects of Polyclonal IgG Derived from Patients with Different Clinical Types of the Antiphospholipid Syndrome on Monocyte Signaling Pathways

A major mechanism of hypercoagulability in the antiphospholipid syndrome (APS) is antiphospholipid Ab-mediated upregulation of tissue factor (TF) on monocytes via activation of TLRs, p38 MAPK, and NF-κB pathways. We examined whether monocyte signaling pathways are differentially activated by IgG from patients with vascular thrombosis (VT) alone compared with IgG from patients with pregnancy morbidity (PM) alone. We purified IgG from 49 subjects. A human monocyte cell line and ex vivo healthy monocytes were treated with 100 μg/ml IgG for 6 h, and cell extracts were examined by immunoblot using Abs to p38 MAPK and NF-κB. To further investigate intracellular signaling pathways induced by these IgGs, specific inhibitors of p38 MAPK, NF-κB, TLR4, and TLR2 were used to determine their effect on TF activity. Only IgG from patients with VT but no PM (VT+/PM−) caused phosphorylation of NF-κBand p38 MAPK and upregulation of TF activity in monocytes. These effects were not seen with IgG from patients with PM alone (VT−/PM+), anti-phospholipid Ab-positive patients without APS, or healthy controls. TF upregulation caused by the VT+/PM− samples was reduced by inhibitors of p38 MAPK, NF-κB, and TLR4. The effects of VT+/PM− IgG on signaling and TF upregulation were concentrated in the fraction that bound β-2-glycoprotein I. Our findings demonstrate that IgGs from patients with diverse clinical manifestations of APS have differential effects upon phosphorylation of NF-κB and p38 MAPK and TF activity that may be mediated by differential activation of TLR4.

Measuring IgA Anti-β2-Glycoprotein I and IgG/IgA Anti-Domain I Antibodies Adds Value to Current Serological Assays for the Antiphospholipid Syndrome

Introduction Currently available clinical assays to detect antiphospholipid antibodies (aPL) test for IgG and IgM antibodies to cardiolipin (aCL) and β2-glycoprotein I (aβ2GPI). It has been suggested that testing for IgA aPL and for antibodies to Domain I (DI), which carries the key antigenic epitopes of β2GPI, could add value to these current tests. We performed an observational, multicenter cohort study to evaluate the utility of IgG, IgM and IgA assays to each of CL, β2GPI and DI in APS. Methods Serum from 230 patients with APS (n = 111), SLE but not APS (n = 119), and 200 healthy controls were tested for IgG, IgM and IgA aCL, aβ2GPI and aDI activity. Patients with APS were further classified into thrombotic or obstetric APS. Logistic regression and receiver operator characteristic analyses were employed to compare results from the nine different assays. Results All assays displayed good specificity for APS; IgG aCL and IgG aβ2GPI assays however, had the highest sensitivity. Testing positive for IgA aβ2GPI resulted in a higher hazard ratio for APS compared to IgM aβ2GPI. Positive IgG, IgM or IgA aDI were all associated with APS, and in subjects positive for aCL and/or aβ2GPI, the presence of aDI raised the hazard ratio for APS by 3–5 fold. IgG aCL, aβ2GPI, aDI and IgA aDI were associated with thrombotic but not obstetric complications in patients with APS. Conclusion Measuring IgG aDI and IgA aβ2GPI and aDI may be useful in the management of patients with APS, particularly thrombotic APS.

Antibodies to domain I of β-2-glycoprotein I and IgA antiphospholipid antibodies in patients with ‘seronegative’ antiphospholipid syndrome

The standard serological tests included in the classification criteria1 for antiphospholipid syndrome (APS) are those to detect immunoglobulin G (IgG) and IgM antibodies to cardiolipin (aCL) or β-2-glycoprotein I (anti-β2GPI) and the lupus anticoagulant. It is increasingly recognised, however, that some patients have typical thrombotic and non-thrombotic features of APS but test repeatedly negative in these routinely used assays. It has been suggested that these patients have the so-called seronegative APS (SN-APS).2 In a retrospective study, there were no significant differences in clinical manifestations between 87 patients with seropositive APS and 67 with SN-APS.3 Several authors have suggested that in these ‘seronegative’ patients, clinically relevant antibodies can be detected by looking for different isotypes, particularly IgA2 and/or different antigen specificity4 or by using different techniques4 ,5 than those of the routine assays. In a recent …

Thrombin binding predicts the effects of sequence changes in a human monoclonal antiphospholipid antibody on its in vivo biologic actions.

The mechanisms by which antiphospholipid Abs (aPL) cause thrombosis are not fully understood. It is clear that binding to a number of phospholipid-associated Ags is important but it is difficult to identify which Ag-binding properties are most closely linked to the ability to cause biologic effects such as promotion of thrombosis and activation of endothelial cells. We have previously used an in vitro expression system to produce a panel of human monoclonal IgG molecules between which we engineered small differences in sequence leading to significant well-defined changes in binding properties. In this study, we assess the properties of five of these IgG molecules in assays of biologic function in vitro and in vivo. The i.p. injection of these IgG into mice subjected to a femoral vein pinch stimulus showed that only those IgG that showed strong binding to thrombin promoted in vivo venous thrombosis and leukocyte adherence. However, this finding did not hold true for the effects of these IgG on activation of cultured endothelial cells in vitro, where there was a less clear relationship between binding properties and biologic effects.

Gene expression studies on bio-electrosprayed primary cardiac myocytes

Cardiovascular pathology accounts for the greatest number of mortalities in the western world and thus the development of ex vivo cardiac tissue has vast potential in cardiac therapy. Bio‐electrosprays (BES), a recently discovered direct cell engineering protocol, has demonstrated tremendous applicability for regenerative and therapeutic medicine. For bio‐electrospraying to be carried forward as a novel method of cardiac tissue engineering, it is important that the process does not adversely affect cellular physiology. Our previous work has shown that bio‐electrospraying does not induce cell death, activate intracellular stress pathways or induce DNA damage in primary cardiac myocytes. Here we show for the first time using genome‐wide microarray analysis, that bio‐electrospraying has no negative effects on global gene expression in cardiac myocytes. Moreover, we show that bio‐electrospraying does not lead to endothelial cell activation. These data suggest that BES has minimal effect upon the physiology of cardiac myocytes and endothelial cells and thus paves the way for the development of BES in cardiac tissue engineering.

Evaluating the conformation of recombinant domain I of β2-glycoprotein I and its interaction with human monoclonal antibodies

Highlights ► Bacterial expressed human recombinant DI has a structure consistent with that of DI in the published β2GPI crystal structure. ► Mutating residues D8/D9 and R39 do not alter the overall DI protein fold but cause local changes in surface contour. ► Monoclonal aPL-derived antibodies and DI of β2GPI interactions are influenced by specific arginine residues in aPL and particular epitopes in DI.

New therapeutic targets for the antiphospholipid syndrome

Importance of the field: The antiphospholipid syndrome (APS) is an autoimmune condition whereby pathogenic antiphospholipid antibodies (aPL) cause vascular thrombosis and/or recurrent miscarriage, and carries a high burden of morbidity and mortality. Currently the only proven treatment is long-term anticoagulation, which is not effective in all patients and carries risk of haemorrhage. Areas covered in this review: Novel therapeutic targets that are currently being explored for APS in order to address the unmet needs of better, safer and ideally targeted therapy. These include B cell depletion, new-generation anticoagulants, interfering with aPL cell-mediated activation of endothelial cells and platelets both at the cell surface level and intracellularly, targeting components of the complement system and the novel concept of using decoy peptides to target only the pathogenic sub-population of aPL. What the reader will gain: An overview of the potential targets and rationale underpinning them. Take home message: Though current options remain limited for the treatment of APS, the future holds much promise with the identification of multiple targets, many of which are currently being explored. The challenge will be to undertake carefully designed prospective multi-centre trials to generate the evidence necessary to support integration of such candidates into clinical practice.