Biologics such as TNF antagonists are a new class of drugs that have greatly improved Rheumatoid Arthritis (RA) treatment. However, for unknown reasons, individual patients with RA respond to one of these drugs but not to others even those targeting the same molecule. Methods to predict response are sorely needed because these drugs are currently selected by trial and error, what is very inefficient and prejudicial for the patient and the healthcare system. Here, we have explored the discovery of protein biomarkers in serum from patients treated with infliximab, one of the major anti-TNF drugs. The study was based in a quantitative proteomics approach using 8-plex iTRAQ labeling. It combined depletion of the most abundant serum proteins, two-dimensional LC fractionation, protein identification and relative quantification with a hybrid Orbitrap mass spectrometer. This approach allowed the identification of 315 proteins of which 237 were confidently quantified with two or more peptides. The detection range covered up to 6 orders of magnitude including multiple proteins at the ng/mL level. A new set of putative biomarkers was identified comprising 14 proteins significantly more abundant in the non-responder patients. The differential proteins were enriched in apolipoproteins, components of the complement system and acute phase reactants. These results show the feasibility of this approach and provide a set of candidates for validation as biomarkers for the classification of RA patients before the beginning of treatment, so that anticipated non-responders could be treated with an alternative drug. Rheumatoid Arthritis (RA) treatment. However, for unknown reasons, individual 3 patients with RA respond to one of these drugs but not to others even those targeting the 4 same molecule. Methods to predict response are sorely needed because these drugs are 5 currently selected by trial and error, what is very inefficient and prejudicial for the 6 patient and the healthcare system. Here, we have explored the discovery of protein 7 biomarkers in serum from patients treated with infliximab, one of the major anti-TNF 8 drugs. The study was based in a quantitative proteomics approach using 8-plex iTRAQ 9 labeling. It combined depletion of the most abundant serum proteins, two-dimensional 10 LC fractionation, protein identification and relative quantification with a hybrid 11Orbitrap mass spectrometer. This approach allowed the identification of 315 proteins of 12 which 237 were confidently quantified with two or more peptides. The detection range 13 covered up to 6 orders of magnitude including multiple proteins at the ng/mL level. A 14 new set of putative biomarkers was identified comprising 14 proteins significantly more 15 abundant in the non-responder patients. The differential proteins were enriched in 16 apolipoproteins, components of the complement system and acute phase reactants. 17These results show the feasibility of this approach and provide a set of candidates for 18 validation as biomarkers for the class...

Response to treatment of rheumatoid arthritis shows large inter-individual variability. This heterogeneity is observed with all the anti-rheumatic drugs, including the commonly used TNF inhibitors. It seems that drug-specific and target-specific factors lead individual patients to respond or not to a given drug, although this point has been challenged. The search of biomarkers distinguishing responders from non-responders has included shotgun proteomics of serum, as a previous study of response to infliximab, an anti-TNF antibody. Here, we have used the same study design and technology to search biomarkers of response to a different anti-TNF antibody, adalimumab, and we have compared the results obtained for the two anti-TNF drugs. Search of biomarkers of response to adalimumab included depletion of the most abundant serum proteins, 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) labeling, two-dimensional liquid chromatography fractionation and relative quantification with a hybrid Orbitrap mass spectrometer. With this approach, 264 proteins were identified in all the samples with at least 2 peptides and 95% confidence. Nine proteins showed differences between non-responders and responders (P < 0.05), representing putative biomarkers of response to adalimumab. These results were compared with the previous study of infliximab. Surprisingly, the non-responder/responder differences in the two studies were not correlated (rs = 0.07; P = 0.40). This overall independence with all the proteins showed two identifiable components. On one side, the putative biomarkers of response to either adalimumab or infliximab, which were not shared and showed an inverse correlation (rs = -0.69; P = 0.0023). On the other, eight proteins showing significant non-responder/responder differences in the analysis combining data of response to the two drugs. These results identify new putative biomarkers of response to treatment of rheumatoid arthritis and indicate that they are notably drug-specific.

Introduction Antiphospholipid antibodies (aPL) have been associated with organ damage and certain features in systemic lupus erythematosus(SLE) patients. Our aim was to investigate the differences between SLE patients according to the presence of aPL and/or clinical antiphospholipid syndrome (APS). Materials and methods Patients from the RELESSER-T registry were included. RELESSER-T is a Spanish multicenter, hospital-based, retrospective, SLE registry. Results We included 2398 SLE patients, 1372 of whom were positive for aPL. Overall 1026 patients were classified as SLE, 555 as SLE-APS and817 as SLE-aPL. Regarding cardiovascular risk factors, SLE-APS patients had higher rates of hypertension, dyslipidemia and diabetes than those with SLE-aPL and SLE ( p < 0.001). SLE-APS patients showed higher rates of neuropsychiatric, cardiac, pulmonary, renal and ophthalmological manifestations than the other groups ( p < 0.001). SLE-APS patients presented greater damage accrual with higher SLICC values (1.9 ± 2.2 in SLE-APS, 0.9 ± 1.4 in SLE-aPL and 1.1 ± 1.6 in SLE, p < 0.001) and more severe disease as defined by the Katz index (3 ± 1.8 in SLE-APS, 2.7 ± 1.7 in SLE-aPL and 2.6 ± 1.6 in SLE, p  < 0.001). SLE-APS patients showed higher mortality rates ( p < 0.001). Conclusions SLE-APS patients exhibited more severe clinical profiles with higher frequencies of major organ involvement, greater damage accrual and higher mortality than SLE-aPL and SLE patients.

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Background: Systemic Lupus Erythematosus (SLE) is an autoimmune disorder of unknown origin with a high mortality pattern due to the development of a premature cardiovascular disease. The presence in these patients of a dysfunctional endothelium together with a hypercoagulable milieu may contribute to an increased incidence of thrombotic events. Increased thrombin generation, elevated levels of circulating microparticles and plasmatic levels of PAI-1 may contribute to the prothrombotic phenotype of the disease but data are scarce. Objectives: 1-To characterize the prothrombotic state in SLE by rotational thromboelastometry (ROTEM) and thrombin generation linked to tissue factor bearing microparticles. 2- To evaluate endothelial damage in patients with SLE and its relationship with the prothrombotic state of the disease. 3- To evaluate the influence of PAI-1 in the prothrombotic state of SLE. Material and methods: 39 patients with SLE and 25 sex and age matched healthy subjects were included. Whole blood was drawn in standard BD sodium citrate tubes (3.2%). ROTEM was performed in naTEM condition. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]), the time that clot takes to increase from 2mm above baseline to 20mm above baseline (CFT) and A5, amplitude at 5 min, were recorded. To evaluate the presence of tissue factor bearing microparticles, thrombin generation was determined by Calibrated Automated Thrombogram (CAT) in presence of 4 mM phospholipid (MP-Reagent, Diagnostica Stago, Spain). The endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak were evaluated. Antigenic levels of E-selectin and PAI-1 were determined by ELISA (R&D Systems, MN, USA and Affymetrix eBioscience, Vienna, Austria) respectively. Results: ROTEM parameters showed a hypercoagulable profile in LES patients. CT and CFT were shorter (p<0.001 in both cases), and MCF, alpha angle and A5 were higher (p<0.001, p<0.02 and p<0.001 respectively) when compared to healthy controls. On the other hand, CAT parameters did not show differences between both groups. Nevertheless, in LES patients but not in controls, CAT parameters significantly correlated with ROTEM ones (Table 1). Table 1.Correlations in the LES patients group between CAT and ROTEM parameters. Analyses were performed with Spearman test. N: number of determinations; p* denotes significance.CTCFTMCFMCF-talphaA5A10ETP(MP)r-,301-,375*,369*-,368*,378*,364*,393*p,0840,028*0,031*0,032*0,027*0,034*0,021*N34343434343434Peak(MP)r-,353*-,396*,372*-,345*,396*,393*,402*p0,040*0,020*0,030*0,045*0,020*0,021*0,018*N34343434343434ttPeak(MP)r,240,222-,242,217-,223-,209-,210p,171,207,168,217,205,236,232N34343434343434 In order to evaluate endothelial damage, plasma E-selectin and PAI-1 were determined. No differences were found in E-selectin level whereas increased PAI-1 levels were seen in LES group (p<0.006). PAI-1 did not correlate to ROTEM parameters. Conclusions: ROTEM can detect a hypercoagulable state in patients with SLE. The hypercoagulable state may be linked to increased tissue factor bearing microparticles and increased PAI-1 plasma levels. Disclosures No relevant conflicts of interest to declare.