PEGylated Domain I of Beta-2-Glycoprotein I Inhibits Thrombosis in a Chronic Mouse Model of the Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is an autoimmune disorder in which autoantibodies cause clinical effects of vascular thrombosis and pregnancy morbidity. The only evidence-based treatments are anticoagulant medications such as warfarin and heparin. These medications have a number of disadvantages, notably risk of haemorrhage. Therefore, there is a pressing need to develop new, more focused treatments that target the actual pathogenic disease process in APS. The pathogenic antibodies exert their effects by interacting with phospholipid-binding proteins, of which the most important is beta-2-glycoprotein I. This protein has five domains, of which the N-terminal Domain I (DI) is the main site for binding of pathogenic autoantibodies. We previously demonstrated bacterial expression of human DI and showed that this product could inhibit the ability of IgG from patients with APS (APS-IgG) to promote thrombosis in a mouse model. Since DI is a small 7kDa protein, its serum half-life would be too short to be therapeutically useful. We therefore used site-specific chemical addition of polyethylene glycol (PEG) to produce a larger variant of DI (PEG-DI) and showed that PEG-DI was equally effective as the non-PEGylated DI in inhibiting thrombosis caused by passive transfer of APS-IgG in mice. In this paper, we have used a mouse model that reflects human APS much more closely than the passive transfer of APS-IgG. In this model, the mice are immunized with human beta-2-glycoprotein I and develop endogenous anti-beta-2-glycoprotein I antibodies. When submitted to a pinch stimulus at the femoral vein, these mice develop clots. Our results show that PEG-DI inhibits production of thromboses in this model and also reduces expression of tissue factor in the aortas of the mice. No toxicity was seen in mice that received PEG-DI. Therefore, these results provide further evidence supporting possible efficacy of PEG-DI as a potential treatment for APS

Antiphospholipid antibody positivity in early systemic lupus erythematosus is associated with subsequent vascular events

OBJECTIVE: Antiphospholipid antibodies (aPL) are found in the blood of 20-30% of patients with systemic lupus erythematosus (SLE). Although aPL cause vascular thrombosis in the antiphospholipid syndrome it is not clear whether positive aPL levels in early SLE increase risk of subsequent vascular events (VE). In a previous analysis of 276 patients with SLE, we found that early positivity for ≥2 of IgG anti-cardiolipin (anti-CL), IgG anti-beta2glycoprotein I (anti-β2GPI) and anti-Domain I of beta2glycoprotein I (anti-DI) showed a possible association with VE. Here we have extended that analysis. METHODS: Serum samples taken from 501 patients with SLE early in their disease had been tested for IgG anti-CL, anti-β2GPI and anti-DI by ELISA. Complete VE history was available for 423 patients of whom 23 were excluded because VE occurred before the diagnosis of SLE. For the remaining 400 patients we carried out Kaplan-Meier survival analysis to define groups at higher risk of VE. RESULTS: Of 400 patients, 154 (38.5%) were positive for one or more aPL; 27 (6.8%) were double/triple-positive and 127 (31.8%) were single-positive. There were 91 VE in 77 patients, of whom 42 were aPL-positive in early disease. VE were significantly increased in aPL-positive versus aPL-negative patients (p= 0.041) and in double/triple-positive versus single-positive versus aPL-negative patients (p= 0.0057).Omission of the IgG anti-DI assay would have missed 14 double/triple-positive patients of whom 6 had VE. CONCLUSION: Double/triple-positivity for IgG anti-CL, anti- anti-β2GPI and anti-DI in early SLE identifies a population at higher risk of subsequent VE

Serum nitrated nucleosome levels in patients with systemic lupus erythematosus: a retrospective longitudinal cohort study

INTRODUCTION: Circulating nucleosomes released from apoptotic cells are important in the pathogenesis of systemic lupus erythematosus (SLE). Both nucleosomes and anti-nucleosome antibodies are deposited in inflamed tissues in patients with SLE. Active inflammation promotes nitration of tyrosine residues on serum proteins. Our hypothesis was that levels of nitrated nucleosomes would be elevated in patients with SLE and could be associated with disease activity. We therefore carried out a retrospective longitudinal study to investigate factors affecting levels of nitrated nucleosomes (NN) in patients with SLE. METHODS: A novel serum ELISA was developed to measure serum NN and modified to measure serum nitrated albumin (NA). Levels of both NN and NA were measured in 397 samples from 49 patients with SLE followed through periods of disease flare and remission for a mean of 89 months. Anti-nucleosome antibody (anti-nuc) levels were measured in the same samples. The effects of 24 different clinical, demographic and serological variables on NN, NA and anti-nuc levels were assessed by univariable and multivariable analysis. RESULTS: Patients with SLE had higher mean NN than healthy controls or patients with other autoimmune rheumatic diseases (P =0.01). Serum samples from 18 out of 49 (36.7%) of SLE patients were never positive for NN. This group of 18 patients was characterized by lower anti-double stranded DNA antibodies (anti-dsDNA), disease activity and use of immunosuppressants. In the remaining 63.3%, NN levels were variable. High NN was significantly associated with anti-Sm antibodies, vasculitis, immunosuppressants, hydroxychloroquine and age at diagnosis. NN levels were raised in neuropsychiatric flares. NN levels did not completely parallel NA results, thus providing additional information over measuring nitration status alone. NN levels were not associated with anti-nuc levels. CONCLUSIONS: NN are raised in a subset of patients with SLE, particularly those who are anti-Sm positive. Elevated NN may be a marker of vascular activation and neuropsychiatric flares in these patients

Activation and contraction of human ‘vascular’ smooth muscle cells grown from circulating blood progenitors

Blood outgrowth smooth muscle cells offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering and personalised medicine. However, little is known about these cells which has meant their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of blood outgrowth smooth muscle cells and vessel derived smooth muscle cells to sense the thromboxane mimetic U46619 by measuring intracellular calcium elevation and contraction. U46619 (10 26 -6 M) increased cytosolic calcium in blood outgrowth smooth muscle cells fibroblasts. Increased calcium signal peaked between 10-20 seconds after U46619 in both smooth muscle cell types. Importantly, U46619 (10-9 to 10-6 M) induced concentration-dependent contractions of both blood outgrowth smooth muscle cells and vascular smooth muscle cells but not in fibroblasts. In summary, we show that functional responses of blood outgrowth smooth muscle cells are in line with vascular smooth muscle cells providing critical evidence of their application in biomedical research

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 (beta(2)GPI) 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 beta(2)GPI 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 beta(2)GPI in APS patients compared to various control groups.Methods. In a retrospective multicenter analysis, the proportion of beta(2)GPI with free thiols in serum from healthy volunteers was quantified. Assays for measurement of reduced as well as total circulating beta(2)GPI 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 beta(2)GPI was significantly elevated in patients with APS (median 216.2 mu g/ml [interquartile range 173.3-263.8]) as compared to healthy subjects (median 178.4 mu g/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 beta(2)GPI 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 beta(2)GPI via thiol-exchange reactions is a highly specific phenomenon in the setting of APS thrombosis. Quantification of posttranslational modifications of beta(2)GPI 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

PEGylated Domain I of Beta-2-Glycoprotein I Inhibits the Binding, Coagulopathic, and Thrombogenic Properties of IgG From Patients With the Antiphospholipid Syndrome

APS is an autoimmune disease in which antiphospholipid antibodies (aPL) cause vascular thrombosis and pregnancy morbidity. In patients with APS, aPL exert pathogenic actions by binding serum beta-2-glycoprotein I (β2GPI) via its N-terminal domain I (DI). We previously showed that bacterially-expressed recombinant DI inhibits biological actions of IgG derived from serum of patients with APS (APS-IgG). DI is too small (7 kDa) to be a viable therapeutic agent. Addition of polyethylene glycol (PEGylation) to small molecules enhances the serum half-life, reduces proteolytic targeting and can decrease immunogenicity. It is a common method of tailoring pharmacokinetic parameters and has been used in the production of many therapies in the clinic. However, PEGylation of molecules may reduce their biological activity, and the size of the PEG group can alter the balance between activity and half-life extension. Here we achieve production of site-specific PEGylation of recombinant DI (PEG-DI) and describe the activities in vitro and in vivo of three variants with different size PEG groups. All variants were able to inhibit APS-IgG from: binding to whole β2GPI in ELISA, altering the clotting properties of human plasma and promoting thrombosis and tissue factor expression in mice. These findings provide an important step on the path to developing DI into a first-in-class therapeutic in APS

Oral abstracts 3: RA Treatment and outcomesO13. Validation of jadas in all subtypes of juvenile idiopathic arthritis in a clinical setting

Background: Juvenile Arthritis Disease Activity Score (JADAS) is a 4 variable composite disease activity (DA) score for JIA (including active 10, 27 or 71 joint count (AJC), physician global (PGA), parent/child global (PGE) and ESR). The validity of JADAS for all ILAR subtypes in the routine clinical setting is unknown. We investigated the construct validity of JADAS in the clinical setting in all subtypes of JIA through application to a prospective inception cohort of UK children presenting with new onset inflammatory arthritis. Methods: JADAS 10, 27 and 71 were determined for all children in the Childhood Arthritis Prospective Study (CAPS) with complete data available at baseline. Correlation of JADAS 10, 27 and 71 with single DA markers was determined for all subtypes. All correlations were calculated using Spearman's rank statistic. Results: 262/1238 visits had sufficient data for calculation of JADAS (1028 (83%) AJC, 744 (60%) PGA, 843 (68%) PGE and 459 (37%) ESR). Median age at disease onset was 6.0 years (IQR 2.6-10.4) and 64% were female. Correlation between JADAS 10, 27 and 71 approached 1 for all subtypes. Median JADAS 71 was 5.3 (IQR 2.2-10.1) with a significant difference between median JADAS scores between subtypes (p < 0.01). Correlation of JADAS 71 with each single marker of DA was moderate to high in the total cohort (see Table 1). Overall, correlation with AJC, PGA and PGE was moderate to high and correlation with ESR, limited JC, parental pain and CHAQ was low to moderate in the individual subtypes. Correlation coefficients in the extended oligoarticular, rheumatoid factor negative and enthesitis related subtypes were interpreted with caution in view of low numbers. Conclusions: This study adds to the body of evidence supporting the construct validity of JADAS. JADAS correlates with other measures of DA in all ILAR subtypes in the routine clinical setting. Given the high frequency of missing ESR data, it would be useful to assess the validity of JADAS without inclusion of the ESR. Disclosure statement: All authors have declared no conflicts of interest. Table 1Spearman's correlation between JADAS 71 and single markers DA by ILAR subtype ILAR Subtype Systemic onset JIA Persistent oligo JIA Extended oligo JIA Rheumatoid factor neg JIA Rheumatoid factor pos JIA Enthesitis related JIA Psoriatic JIA Undifferentiated JIA Unknown subtype Total cohort Number of children 23 111 12 57 7 9 19 7 17 262 AJC 0.54 0.67 0.53 0.75 0.53 0.34 0.59 0.81 0.37 0.59 PGA 0.63 0.69 0.25 0.73 0.14 0.05 0.50 0.83 0.56 0.64 PGE 0.51 0.68 0.83 0.61 0.41 0.69 0.71 0.9 0.48 0.61 ESR 0.28 0.31 0.35 0.4 0.6 0.85 0.43 0.7 0.5 0.53 Limited 71 JC 0.29 0.51 0.23 0.37 0.14 -0.12 0.4 0.81 0.45 0.41 Parental pain 0.23 0.62 0.03 0.57 0.41 0.69 0.7 0.79 0.42 0.53 Childhood health assessment questionnaire 0.25 0.57 -0.07 0.36 -0.47 0.84 0.37 0.8 0.66 0.4

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Hydroxychloroquine as an Immunomodulatory and Antithrombotic Treatment in Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is an acquired highly prothrombotic disorder in which thrombo-inflammatory antiphospholipid antibodies (aPL) cause thrombosis via multiple mechanisms, including endothelial damage and activation. Obstetric complications in APS are caused by placental thrombosis, inflammation and complement activation. Anticoagulation is poorly effective in some patients especially those with triple positive aPL who are at ~30% risk of thrombosis recurrence within 10 years. Increasing therapeutic anticoagulation intensity may be beneficial but leads to excess bleeding with serious complications, such as intracerebral haemorrhage. Nonetheless, anticoagulation is still the mainstay of treatment despite the autoimmune nature of APS. The antimalarial immunomodulatory drug hydroxychloroquine (HCQ) has been used for many years for the treatment of inflammatory rheumatic diseases. HCQ has complex pleiotropic mechanisms of action upon multiple cell types. The proposed biological processes that HCQ regulates support the hypothesis that it may be a successful adjunctive treatment in the prevention of recurrent thrombosis and pregnancy complications