OP0249 SERUM PROTEOMIC BIOMARKERS DEFINE PATIENTS WITH SYSTEMIC SCLEROSIS WITH INTERSTITIAL LUNG DISEASE

Background:Systemic sclerosis (SSc) is a systemic condition affecting multiple organs and thus being burdened by high morbidity and mortality; disease management is based largely on the early detection of organ involvement, particularly in the case of interstitial lung disease (ILD), ideally through noninvasive biomarkers. Beside serum autoantibodies associated with diffuse SSc, there is currently no reliable serum marker to predict the onset of SSc organ involvement, monitor its progression, and foresee the response to treatments. Proteomic analysis based on aptamer technology is a powerful method with the potential to address this unmet need in SSc.Objectives:To identify serum biomarkers associated with ILD in SSc.Methods:Serum samples from 6 women with SSc (3 with ILD at high-resolution pulmonary CT scan) and 7 age-matched female healthy controls (HC) were analyzed using the SOMAscan platform (SomaLogic, Inc., Boulder, CO, USA) to test more than 1300 proteins even at femtomolar concentration. Subsequent validation of candidate proteins was performed using ELISA in an independent cohort of 88 patients with SSc and 48 HC. Statistical analysis included Student’s t-test and was assessed using the SomaSuite software (SomaLogic, Boulder, CO, USA).Results:The proteomic analysis identified 33 proteins with significantly different serum levels in SSc cases compared to HC and 9 proteins differentiating SSc patients according to ILD (Table 1). Compared to HC, SSc sera manifested an altered expression of proteins involved in extracellular matrix formation and cell-cell adhesion (with higher Calpain, EphA5, IDS, MATN2, MMP-12, TNR4, and lower desmoglein-1, SNP25), angiogenesis (with higher anti-angiogenetic factors as angiopoietin-2 and kininogen high molecular weight) lymphocyte recruitment, activation, and signaling (with higher CXCL-1, LAG3 and lower SH21A) with an overall inhibition of neutrophil function (with lower G-CSF-R, CD177, calgranulin B).Table 1.Significantly altered proteins at serum proteomic analysis of systemic sclerosis (SSc) with or without interstitial lung disease (ILD) and healthy controls (HC)SSc versus healthy controlsSSc with ILD versus SSc without ILD and healthy controlsIncreasedReducedIncreasedReducedAldolase AAngiopoietin-2*C1QR1CalpainCOLEC12 EotaxinEphA5Fractalkine/CXCL-1GranulinsIDS Kininogen, HMVLAG-3Lamin-B1LRP1bMATN2MMP-12STAT1 TMR4AdrenomedullinASGR1C1sC5Calgranulin BCD177Desmoglein-1Flt-3 ligandG-CFS-RIL-1RaLeptinLypd3SH21ASNP25TPBS2FCRL3IL-22BP**MCP-3PDE11PGP9.5sICAM-5StratifinBAFFDERM*significantly increased also at ELISA** significantly increased at ELISA only in SSc with ILD versus HCThe majority of proteins with higher levels in SSc with ILD compared to SSc without ILD were involved in intracellular signaling and cell cycle (FCRL3, PDE11, Stratifin), along with higher MCP-3, a monocyte chemoattractant, and sICAM-5, ligand for the leukocyte adhesion protein LFA-1. Of note, we found that increased IL-22BP, antagonist of IL-22, and decreased BAFF levels characterized SSc with ILD.Conclusion:Aptamer proteomic analysis allowed to define serum profiles differentiating SSc patients from healthy controls and SSc with ILD from SSc without ILD; the proteins identified are involved in SSc pathogenic pathways and after further investigation on larger cohorts they can be used as reliable biomarkers.Characters from table content including title and footnotes: 631Disclosure of Interests:None declared

Altered Phosphorylation of Cytoskeleton Proteins in Peripheral Blood Mononuclear Cells Characterizes Chronic Antibody-Mediated Rejection in Kidney Transplantation

Chronic antibody-mediated rejection (CAMR) is the major cause of kidney transplant failure. The molecular mechanisms underlying this event are still poorly defined and this lack of knowledge deeply influences the potential therapeutic strategies. The aim of our study was to analyze the phosphoproteome of peripheral blood mononuclear cells (PBMCs), to identify cellular signaling networks differentially activated in CAMR. Phosphoproteins isolated from PBMCs of biopsy proven CAMR, kidney transplant recipients with normal graft function and histology and healthy immunocompetent individuals, have been investigated by proteomic analysis. Phosphoproteomic results were confirmed by Western blot and PBMCs’ confocal microscopy analyses. Overall, 38 PBMCs samples were analyzed. A differential analysis of PBMCs’ phosphoproteomes revealed an increase of lactotransferrin, actin-related protein 2 (ARPC2) and calgranulin-B in antibody-mediated rejection patients, compared to controls. Increased expression of phosphorylated ARPC2 and its correlation to F-actin filaments were confirmed in CAMR patients. Our results are the first evidence of altered cytoskeleton organization in circulating immune cells of CAMR patients. The increased expression of phosphorylated ARPC2 found in the PBMCs of our patients, and its association with derangement of F-actin filaments, might suggest that proteins regulating actin dynamics in immune cells could be involved in the mechanism of CAMR of kidney grafts.