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Background Thrombosis of arteries in the central nervous system (CNS) represents the most frequent CNS manifestation and the most frequent arterial event in antiphospholipid syndrome (APS). Previous in vivo studies demonstrated that beta2 glycoprotein I (β2GPI) - the main antigenic target for anti-phospholipid antibodies (aPL) - does not localize in brain in resting conditions. However, β2GPI is detectable on the brain vascular endothelium in association with IgG and complement elements in immunized mice treated with lipopolysaccharide. These data suggest that after an appropriate second hit, β2GPI can play a critical role of in APS CNS damage, likely interacting with the complement system. To date, the pathophysiological functions of β2GPI in brain ischemic injury have not been investigated. Objectives To investigate the presence and the distribution of the activated form of β2GPI in the ischemic brain at different time points. Methods Eleven-week old male mice underwent focal cerebral ischemia induced by 609 transient middlecerebral artery occlusion (tMCAo), or sham-operation. Mice were sacrified at 909, 6h, 24h, 48h, 4d or 7d after tMCAo to obtain brains and plasma. The presence and time course of β2GPI deposition within the ischemic territory and its spatial distribution relative to blood vessels and brain cells were defined the by post mortem immunofluorescence and confocal microscopy. The activated β2GPI was detected by anti-beta2 glycoprotein I minibody (MBB2) – a human monoclonal IgG antibody targeting β2GPI domain I. Results β2GPI was present in the ischemic brain tissue starting from 909 after ischemic onset and was still detectable at 7d. β2GPI localized within blood vessels and in brain parenchyma at all times. Notably, at 24h β2GPI was mainly extravascular and colocalized with neurons (NeuN positive cells). Sham-operated mice showed only a faint signal for β2GPI. Conclusions β2GPI is present in its activated conformation in the brain vessels and tissue after experimental cerebral ischemia. It localizes on neurons in brain ischemic areas. These data suggest that an ischemic/inflammatory hit may up-regulate β2GPI tissue presence and may expose the N-terminal domain I of the molecule which is thougth to be the main target for pathogenic aPL. Disclosure of Interest None declared

Background An early diagnosis of connective tissue disease (CTD) provides a window of opportunity for monitoring and treating patients. Objectives We examined the role of history, capillaroscopy and autoantibodies to differentiate between patients with primary Raynaud9s phenomenon (RP) and secondary RP due to a CTD at their first medical evaluation. Methods One hundred fifteen consecutive adults with RP were studied. Patient profiles based on history, physical examination, capillaroscopy and laboratory investigations were examined. Logistic regression models were used in the statistical analysis. Results RP was bilateral in 92.7% of patients. The middle finger was significantly more affected. A lack of association between fingers affected by RP and fingers with capillary abnormalities was observed OR=0.75 (0.34-1.66). Fingers affected by RP with the cyanotic phase had a higher risk to have hemorrhages at capillaroscopy OR=4.46 (1.50-13.30) and giant capillaries OR=24.85 (1.48-41.74). None of the variables can discriminate between primary and secondary RP. Among these, thumb involvement and the presence of three colour phases have an OR of 1.48 and 1.85 respectively. Patients with SSc had a greater value of VAS pain (p=0.011). The presence of anti-centromere antibodies was significantly associated with a higher risk of SSc (p Conclusions Markers of a potential development of a CTD in patients with RP include: severe RP symptoms (high VAS pain), positive autoantibodies and capillary abnormalities. The presence of three colour changes and the involvement of thumb may also be a warning for secondary forms. Disclosure of Interest None declared

Background Raynaud9s phenomenon (RP) may predate an overt Connective Tissue Disease (CTD) of the “Scleroderma spectrum” such as Systemic sclerosis (SSc). The predictive role of auto-antibodies (auto-Abs) such as ANA for the development of a CTD in patients with isolated RP (i.e. with no other signs or symptoms of CTD) is well known, whereas data regarding the prevalence of anti-ENA in such patients are lacking. Objectives Our aim was to evaluate the profile of anti-ENA in patients with isolated RP. Methods Sera were analyzed for anti-ENA by EUROLINE “Scleroderma profile” (antigens: Scl70, CENP A, CENP B, RP11, RP155, fibrillarin (Fib), NOR90, Th/To, PM-Scl100, PM-Scl75, Ku, PDGFR, Ro-52) and “Myosites profile” (antigens: Mi-2, Ku, PM-Scl100, PM-Scl75, Jo-1, SRP, PL-7, PL-12, EJ, OJ, Ro-52) [EUROIMMUN AG Leuback, Germany]. Results A total of 115 adults (105 females and 10 males) with isolated RP were enrolled in two Italian Rheumatology Centers (G. Pini Institute, Milan and Moriggia-Pelascini Hospital, Gravedona) from March 2011 to September 2013. Forty-four of 115 (38.0%) subjects were positive for at least one anti-ENA; 71 out of 110 (61.7%) were negative both for Scleroderma and Myosites profiles (see table 1 and 2). Conclusions Up to 38% of RP subjects were positive for at least one anti-ENA. The detection of anti-ENA since the first Rheumatologic evaluation may aid the specialist in achieving an earlier diagnosis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5955

Background Beta2 glycoprotein I has been suggested as LPS scavenger protein and the complex b2GPI-LPS is uptaken by monocytes. In vivo/in vitro indirect data suggest the role of Toll Like Receptor (TLR) in aPL-mediated endothelial cell (EC) activation. Furthermore TLR4 has recently been suggested as a component of a multi-protein complex on the EC surface involved in beta2GPI/anti- beta2GPI antibody-induced signalling pathway. However, what is/are the molecule(s) eventually involved in this interaction is still an open question. Objectives To investigate the role of endothelial TLR4 in beta2GPI/anti- beta2GPI antibody interaction. Methods i) direct binding and internalization of beta2GPI was evaluated by confocal microscopy in living CHO/TLR4 cells; ii) anti- beta2GPI antibody binding and activation (as adhesion molecule up-regulation) were investigated in TLR4- and AnnexinA2-silenced HUVEC. Receptor expression was evaluated both at mRNA and protein level by real time RT-PCR and western blotting, respectively. Results incubation of living CHO/TLR4 cells, but not TLR4-negative CHO Mock cells, with beta2GPI-FITC resulted in a strong membrane signal shortly after the addition of the protein. After about 15 min the fluorescent signal was detected within the cytoplasm as granules, progressively more abundant in the following 20-30 min of observation, when membrane signal faded. Immunofluorescence studies on fixed cells showed the interaction between beta2GPI and TLR4 both on the cell membrane and in intracytoplasmic granules. beta2GPI/LPS complex induced EC activation evaluated as ELAM upregulation, while LPS alone or in combination with BSA had no effect. TLR4 and AnnexinA2 silencing significantly down-regulated mRNA and protein expression in HUVEC. Both tlr4 and annexinA2 siRNA transfection strongly inhibited anti-beta2GPI antibody binding to HUVEC. On the other hand aPL-induced ELAM/ICAM-1 up-regulation was significantly decreased in tlr4 but not annexinA2 silenced cells. This observation is in line with the fact that AnnexinA2 lacks an intracytoplasmic tail and needs co-receptors to induce cell signalling. No synergic effects were observed in tlr4 and annexinA2 simultaneously silenced HUVEC. Conclusions Our findings strongly support the binding of beta2GPI to TLR4 on EC offering the rationale for the TLR4 involvement in EC signalling and activation by aPL. At the same time, the interaction of beta2GPI with LPS and in turn with TLR4 may represent the molecular basis to explain the “two hit” theory in APS. Disclosure of Interest None Declared

Background One of the hallmarks of overt Systemic Sclerosis (SSc) is the interstitial and perivascular fibrosis resulting in functional impairment of affected organs. During the fibrotic process, the increased collagen synthesis has been correlated to the increase of some biomarkers of collagen metabolism, but it is not know how early these biomarkers can be detected during the disease course. Objectives To evaluate the ongoing fibrotic process based on collagen biomarkers in a cohort of patients in different phases of the disease: from isolated Raynaud’s phenomenon (RP) to overt SSc. For this purpose tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), hyaluronic acid (HA), aminoterminal propeptide of type III procollagen (PIIINP) and Enhanced Liver Fibrosis (ELF) test were chosen. Methods 140 consecutive adult subjects referring in approximately 6 months time were enrolled. They were classified as: 1) primary RP (pRP) [1]; 2) RP suspected secondary to SSc; 3) very early SSc [2]; 4) limited cutaneous (lc)-SSc; and 5) diffuse cutaneous (dc)-SSc. Patients with any other fibrosing disorder or treated with interferon were excluded. TIMP-1, HA, PIIINP and ELF score (derived from an algorithm of the above mentioned biomarkers) were blindly determined by an independent commercial service (iQur, UK). Statistical analysis was performed by regression modelling and ROC curve analysis adjusting for age. Results The study population consisted in 50 pRP (47 women, median age 44.5 yrs), 43 RP suspected secondary to SSc (43 women, median age 43 yrs), 16 very early SSc (16 women, median age 57.5 yrs),15 lc-SSc (14 women, median age 67 yrs), and 15 dc-SSc (13 women, median age 65 yrs). 1 patient was excluded for a recent treatment with interferon. The ELF test and HA were significantly higher in patients with very early SSc compared to subjects with RP suspected secondary to SSc (ELF test: median 8,46 vs 7,84 ng/ml, p= 0.0286; HA: median 30,62 vs 13,6 ng/ml, p= 0.03). PIIINP was significantly higher in patients with dc-SSc compared to very early SSc (median 7,23 vs 5,42 ng/ml; p= 0.006). These differences were not confirmed when these results were weighted for age. When patients with overt SSc were studied an increase of these biomarkers was observed as a trend, but only PIIINP reached a statistical significance. This last result was also confirmed once being weighed for age. Conclusions In our study, the selected biomarkers were not able to distinguish among subjects between pRP, suspected secondary RP, and patients with very early SSc. Nevertheless, these fibrosis biomarkers showed an increasing trend in different subgroups and with age, with no relation to RP duration. These data support the hypothesis that the immune perturbations and vascular injury precede the development of fibrosis, which, in turn, further exacerbates vascular and immune damage. References LeRoy EC, Medsger TA, Jr. Clin Exp Rheumatol. 1992;10:485-8. Avouac J, Fransen J, Walker UA, Riccieri V, Smith V, Muller C, et al. Ann Rheum Dis. 2011;70:476-81. Disclosure of Interest None Declared

Background and Objectives antibodies anti-phospholipids (aPL) react to proteins bound to PL, mainly β2glycoprotein I (β2GPI). Antibodies against β2GPI (aβ2GPI) exert a pathogenic role and represent a risk-factor for clinical manifestations of anti-phospholipid syndrome (APS). However, some aβ2GPI-positive subjects never develop APS-related clinical manifestations. This observation may be explained by the heterogeneity of aβ2GPI population, with autoantibody subgroups targeting different β2GPI epitopes. In particular, antibodies anti-domain I (aDI) but not domains IV and V (aDIV/V) of β2GPI have been associated with thrombotic events. Therefore, the aim of this study was to assess the prevalence of aDI and aDIV/V IgG in a cohort of aPL-positive patients. Material and Methods 58 patients with a diagnosis of primary APS (PAPS) according to the 2006 Sydney criteria have been included in this study. 38 PAPS patients (65.5%) presented with venous and/or arterious thrombothic events while 20 subjects (34.5%) had obstetric manifestations only. 15 aPL asymptomatic carriers were also recruited. All samples had been tested for LA and for aCL and aβ2GPI with home-made assays according to international guidelines. In the thrombotic PAPS group, 35/38 subjects (92.1%) were aβ2GPI IgG positive; aβ2GPI IgG positivity rate was 85% in the obstetric PAPS group (17/20 women); 80% of the asymptomatic aPL carriers displayed aβ2GPI IgG. IgG specificities against whole β2GPI, DI and DIV/V have been evaluated with a novel solid-phase chemiluminiscent assay (BioFlash and ELISA, INOVA Diagnostics). Results Out of the 73 aPL positive patients: 21% were positive for aβ2GPI, aDI and aDIV/V; 41% were positive for aβ2GPI and aDI but negative for aDIV/V; 4% were positive for aβ2GPI and aDIV/V but negative for aDI; 21% were aβ2GPI positive only; 4% were positive for aDIV/V; 9% were negative for antibodies against the whole molecule and the studied domains. The prevalence of aDI was 74% among patients with thrombotic pAPS and 60% among women with obstetric manifestations. 40% of aPL asymptomatic carriers were positive for aDI. We observed a strong correlation between aβ2GPI and aDI (p Conclusions Most of the aβ2GPI positive sera displayed reactivity against DI, while aDIV/DV were detected in a low rate of patients. Our data suggest that DI is the immunodominant β2GPI epitope and that aDI are the main antibody population in APS patients. Future studies are warranted to better define the diagnostic and prognostic role of anti-DI in APS.