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Background There is no agreement on the prevalence of anti-phospholipid antibodies (aPLs) and the correlation with atherosclerosis and cardiovascular (CV) events in the general population. Methods We performed a cross-sectional study on 1712 randomly enrolled subjects from a Northern Italian city to investigate the presence of aPLs and the association with subclinical atherosclerosis (using the carotid artery intima media thickness measured as inter-adventitia common carotid artery diameters - ICCAD) and retrospectively collected CV factors and events (i.e. acute myocardial infarction, stroke, and peripheral obliterans arterial vasculopathy) using physician-assisted questionnaires. We tested serum IgG, IgM, and IgA anti-cardiolipin, anti-beta2glycoprotein I (aGPI), and anti-phosphatidylserine-prothrombin antibodies. Results Positive aPLs were found in 15.1% of the subjects, with no differences between sex but with higher rates in older subjects. Carotid subclinical atherosclerosis was more frequent in aPL positive subjects; more specifically, aGPI IgA were associated with higher ICCAD average (adjusted beta 0.51, 95% confidence interval (CI)0.17–0.84; p = 0.003). A positive history of CV events was also more frequent in aPL positive subjects (odds ratio (OR) 1.67, 95%CI 1.08–2.54; p = 0.012), particularly peripheral obliterans arterial vasculopathy ( OR 2.02 ; 95%CI 1.14–3.57; p = 0.015). Among subjects with a Framingham risk score >20, and/or diabetes, and/or body mass index >3 5 kg/m 2 , aPL positivity was associated to the highest risk of CV events (OR 2.52, 95%CI 1.24–5.11; p = 0.011). Conclusions APL prevalence in the general population is higher than previously reported. CV events and subclinical atherosclerosis are more frequent in the presence of aPL, particularly when a high CV risk coexists.

The identification of anti-NXP2 antibodies is considered a serological marker of dermatomyositis (DM), with calcinosis, severe myositis and, in some reports, with cancer. Historically, these associations with anti-NXP2 antibodies have been detected by immunoprecipitation (IP), but in the last few years commercial immunoblotting assays have been released. The aim of this collaborative project was to analyse the clinical features associated to anti-NXP2 antibodies, both with commercial line blot (LB) and IP. Myositis-specific and myositis-associated autoantibodies were detected in single centres by commercial line blot (LB); available sera were evaluated in a single centre by protein and RNA immunoprecipitation (IP), and IP-Western blot. Sixty patients anti-NXP2+ (NXP2+) positive by LB were compared with 211 patients anti-NXP2 negative with idiopathic inflammatory myositis (IIM). NXP2+ showed a younger age at IIM onset (p = 0.0014), more frequent diagnosis of dermatomyositis (p = 0.026) and inclusion-body myositis (p = 0.009), and lower rate of anti-synthetase syndrome (p

IntroductionAntimitochondrial antibodies (AMAs) are the hallmark of primary biliary cholangitis (PBC) but can be identified also in patients with connective tissue disease, namely, systemic sclerosis (SSc). Protein immunoprecipitation (IP) and IP-Western blot (WB) can be used to confirm AMA positivity directed at the pyruvate dehydrogenase complex (PDC) subunits E1α, E1β, E2/E3, and E3BP in patients showing a cytoplasmic reticular pattern at indirect immunofluorescence when performed in a screening setting before the onset of overt cholestasis in rheumatic patients.Patients and MethodsWe studied sera from 285 patients affected by connective tissue disease [SSc, n = 144; dermato/polymyositis (DM/PM), n = 56; and undifferentiated connective tissue disease (UCTD), n = 85] by indirect immunofluorescence (IIF), protein-IP, and IP-WB to identify specific PDC subunits recognized by AMA.ResultsTwenty percent (57/285) of sera from patients with connective tissue disease had a cytoplasmic reticular pattern at IIF, and in 77% (44/57, including 20 SSc, 12 PM/DM, and 12 UCTD) of these, we detected different titers of autoantibodies against the PDC subunits, specifically against PDC-E2. Among these sera, 4 (9%) tested positive for anti-E1α, 15 (34%) for anti-E1β, and 16 (36%) for anti-E3BP. Four of the 20 AMA-positive SSc cases (20%) had been already diagnosed with PBC, and all were positive for autoantibodies against the subunits PDC-E2, E3, and E3BP.ConclusionsUsing IIF and IP, we confirm that autoantibodies against the PDC components are detected in rheumatic patients with PBC or without liver dysfunction. In view of the strong predictive value of AMA for PBC, a strict follow-up of these latter patients is warranted for an early diagnosis of the disease.

PURPOSE OF REVIEW: The aim of the present review is to analyze the link between autoimmune diseases and environmental factors, in particular severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19) as it shares numerous features with the interstitial lung disease associated with connective tissue diseases positive for rare autoantibodies directed at highly specific autoantigens (i.e., MDA5 and RIG1) among the intracellular sensors of SARS-CoV-2 in the innate response against viruses. RECENT FINDINGS: As shown in recent publications and in our original data, specific autoantibodies may be functionally relevant to COVID-19 infection. We evaluated sera from 35 hospitalized patients with COVID-19 to identify antinuclear antibodies and autoantibodies directed against specific antigenic targets, and we identified anti-nuclear antibodies (ANA) in 20/35 of patients with COVID-19 (57%), in patients with need for supplemental oxygen (90% vs. 20% in ANA-negative cases; P 

Summary Tofacitinib is an oral small molecule targeting the intracellular Janus kinase–signal transducer and activator of transcription (JAK-STAT) pathways approved for the treatment of active rheumatoid arthritis (RA). We investigated the effects of tofacitinib on the response of RA lymphocytes to B and T cell collagen epitopes in their native and post-translationally modified forms. In particular, peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy subjects were cultured with type II collagen peptides (T261-273, B359-369, carT261-273, citB359-369) or with phorbol myristate acetate (PMA)/ionomycin/CD40L in the presence or absence of 100 nM tofacitinib for 20 h and analyzed by fluorescence activated cell sorter (FACS). Cultures without brefeldin A were used for cytokine supernatant enzyme-linked immunosorbent assay (ELISA) analysis. Tofacitinib down-regulated inflammatory cytokines by stimulated B [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] and T [interferon (IFN)-γ, IL-17 or TNF-α] cells in the short term, while a significant reduction of IL-17 and IL-6 levels in peripheral blood mononuclear cell (PBMC) supernatant was also observed. IL-10 was significantly reduced in collagen-stimulated B cells from patients with RA and increased in controls, thus mirroring an altered response to collagen self-epitopes in RA. Tofacitinib partially prevented the IL-10 down-modulation in RA B cells stimulated with collagen epitopes. In conclusion, the use of tofacitinib exerts a rapid regulatory effect on B cells from patients with RA following stimulation with collagen epitopes while not reducing inflammatory cytokine production by lymphocytes.

Objectives Serum autoantibodies are pivotal for the early detection of systemic autoimmune rheumatic diseases such as Systemic Sclerosis (SSc) and Poly/Dermatomyositis (PM/DM), and in some cases are associated with organ complications such as interstitial lung disease (ILD). A paradigmatic example is provided by the autoantibody against the Eukaryotic Initiation Factor 2B (eIF2B) that has been recently detected in SSc. Methods Sera from 118 patients with SSc, 8 Poly/Dermatomyositis, 2 overlap SSc/Polymyositis, 4 undifferentiated connective tissue disease-UCTD and 3 healthy controls were tested first by indirect immunofluorescence for anti-nuclear antibodies-ANA pattern. Further, we employed prot ein-radioimmunoprecipitation (IP) and IP- Western Blot for the detection and confirmation of anti-eIF2B antibodies. Serum findings were further correlated with the clinical features of patients. Results We identified 3 SSc cases (2.5%) positive for anti-eIF2B antibodies while this autoantibody was not detected in control sera. Using protein-IP all three patients manifested the 38kD protein which is the antigenic target of anti-eIF2B antibodies, and this was associated with a cytoplasmic pattern at indirect immunofluorescence. The presence of anti-eIF2B was associated with ILD and a diffuse SSc variant, in one case in association with anti-Scl70/topoI. Conclusions Our data confirm that a small subgroup (2.5%) of patients with SSc have detectable anti-eIF2B with cytoplasmic-positive staining at immunofluorescence and this reactivity is associated with ILD., Highlights • A new and rare autoantibody called anti-eIF2B is detected in 2.5% of SSc sera. • Anti-eIF2B antibodies are associated to diffuse SSc and ILD. • Specific autoantibodies confer a diagnostic and prognostic value for ILD in SSc and PM/DM patients.

Background: Anti-mitochondrial antibodies (AMA) are found in >90% of primary biliary cholangitis patients. Anti-rods/rings antibodies (anti-RR) are most commonly associated with interferon-α and ribavirin treatment in hepatitis C patients. Clinical laboratories routinely screen for AMA and anti-RR using indirect immunofluorescence on HEp-2 cells (HEp-2-IFA). Therefore, we sought to establish reference materials for use in AMA and anti-RR testing. Methods: AMA-positive and anti-RR-positive human plasma samples (AMA-REF and RR-REF), identified as potential reference materials based on preliminary data, were further validated by multiple laboratories using HEp-2-IFA, immunoprecipitation (IP), western blotting, IP-western, line immunoassay (LIA), addressable laser bead immunoassay (ALBIA) and enzyme-linked immunosorbent assay (ELISA). Results: AMA-REF showed a strong positive cytoplasmic reticular/AMA staining pattern by HEp-2-IFA to ≥1:1280 dilution and positive signal on rodent kidney/stomach/liver tissue. AMA-REF reacted with E2/E3, E3BP, E1α and E1β subunits of the pyruvate dehydrogenase complex by IP and western blotting and was positive for AMA antigens by LIA, ALBIA and ELISA. RR-REF showed a strong positive rods and rings staining pattern by HEp-2-IFA to ≥1:1280 dilution. RR-REF reacted with inosine monophosphate dehydrogenase by IP, IP-western and ALBIA. RR-REF also produced a nuclear homogenous staining pattern by HEp-2-IFA, immunoprecipitated proteins associated with anti-U1RNP antibody and reacted weakly with histones, nucleosomes, Sm and nRNP/Sm by LIA. Conclusions: AMA-REF and RR-REF are useful reference materials for academic or commercial clinical laboratories to calibrate and establish internal reference standards for immunodiagnostic assays. AMA-REF and RR-REF are now available for free distribution to qualified laboratories through Plasma Services Group.

Background:Psoriatic disease is a chronic inflammatory disorder spanning from skin disease (psoriasis) to psoriatic arthritis (PsA). The genetic background is insufficient to explain disease onset as illustrated by not very informative Genome Wide Association Studies and monozygotic (MZ) twin studies recently performed. It is strongly assumed that epigenetics may contribute to disease susceptibility modulating gene expression. DNA methylation has been found involved in several autoimmune inflammatory rheumatic diseases. Here we have analysed the DNA methylation profile of a selected cohort of MZ twins discordant for psoriasis/PsA.Objectives:To identify the methylome associated with psoriasis and PsA in the peripheral blood of MZ twins discordant for these conditions.Methods:Peripheral blood from 7 couples of MZ twins discordant for psoriatic disease was collected and DNA extracted for a genome-wide evaluation of the DNA methylation profile, with the Infinium MethylationEPIC BeadChip. Minfi and the packages of the Bioconductor were used to analyse the data obtained. Quality control and exclusion criteria were applied to the raw data having a final number of 762.451 probes, which accounts for 88% of the total.Results:The approach first identified 2564 differentially methylated positions (DMPs; *pConclusion:We report the first DNA methylation approach in MZ twins discordant for psoriatic disease. We believe that the observed changes in SMAD3 and SMARCA/BRG1 genes may suggest an epigenetic imbalance of chromatin remodelling factors involved in inflammation pathways with a potential role in PsA/psoriasis immunopathogenesis.Disclosure of Interests:None declared

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

Summary Nuclear interferon-inducible protein 16 (IFI16) and anti-IFI16 antibodies have been detected in subjects with several rheumatic diseases, often correlating with disease severity, and in this study we investigated their prevalence and clinical associations in psoriatic arthritis (PsA) compared to psoriasis (Pso). We tested sera and synovial fluids of patients with PsA for IFI16 protein levels by capture enzyme-linked immunosorbent assay (ELISA) and for anti-IFI16 immunoglobulin (Ig)G and IgA by ELISA, protein radio-immunoprecipitation and immunoprecipitation-Western blot of IgG. Sera from patients with Pso and healthy subjects were used as controls, and in a subgroup of patients with PsA we also studied sera after treatment with etanercept. IFI16 was detectable in the sera of 66% of patients with Pso, 46% with PsA and 19% of controls. Among PsA cases, 51% of IFI16-positive cases had elevated levels of C-reactive protein (CRP) compared to 31% of patients with undetectable IFI16. Anti-IFI16 of both IgG and IgA isoforms were detected with significantly higher frequency in PsA and Pso compared to healthy controls, with higher IgG titres in patients with elevated C-reactive protein (CRP) (P = 0·015). Immunoprecipitation confirmed the presence of anti-IFI16 IgG antibodies and these preferentially recognized epitopes outside the N-terminus of the protein. Lastly, IFI16 was detected in one of seven and anti-IFI16 in three of seven synovial fluids from patients with PsA. Therefore, IFI16 and anti-IFI16 are detectable in serum and synovial fluid of PsA patients, especially in cases of elevated CRP.

Serum rheumatoid factors are autoantibodies of different isotypes directed against the Fc fraction of immunoglobulin G (IgG) and represent paradigmatic autoantibodies that have been largely used in clinical practice for decades. Traditionally IgG has been associated with rheumatoid arthritis and more recently included also in the classification criteria for Sjӧgren's syndrome. Researchers have established that rheumatoid factors are positive in a variety of infectious, autoimmune, and neoplastic disorders, thus requiring a comprehensive evaluation of seropositive patients. Of note, hepatitis B and C viruses represent a crossroad that includes the high rheumatoid factor seroprevalence and chronic inflammatory disease, as well as progression to non-Hodgkin's lymphomas. Chronic antigen stimulation is the likely common ground of these processes and rheumatoid factors may represent mere bystanders or drivers of pathology. Mixed cryoglobulinemia and lymphoproliferative disease are prime examples of the deleterious effects of rheumatoid factor-B cell activity, possibly associated with hepatitis B and C. More importantly, they show a clear association in a physiological host response to infection, chronic inflammation, and the slide toward autoimmunity and malignancy. The association between hepatitis B and C infections and the appearance of serum rheumatoid factors is further supported by prevalence data, which support a coexistence of these markers in a significant proportion of cases, with viral infections being frequent causes of rheumatoid factors in patients without a rheumatic condition. We provide a comprehensive overview of the known connections between hepatitis B and C infections and rheumatoid factors.

Background Serum anti-mitochondrial antibodies (AMA) are the hallmark of primary biliary cholangitis (PBC) and are identified also in systemic sclerosis (SSc) patients with limited variant and serum anti-centromere antibodies (ACA). Indirect immunofluorescence (IIF) shows the cytoplasmic pattern that characterizes AMA positivity, but it is usually performed when a suspect of PBC is already present. Protein-immunoprecipitation (IP) and IP-Western Blot (WB) have the advantage of 1) identify AMA in an early screening phase, before the onset of PBC and liver function abnormalities; 2) detect the proteins that constitute the pyruvate dehydrogenase complex (PDC) complex (the subunits E1α, E1β, E2/E3, and E3BP) which seem to be associated to different risk of PBC onset. Objectives To determine the prevalence and clinical significance of the PDC complex subunits in AMA positive SSc patients with and without PBC. Methods Sera from 279 consecutive patients with rheumatic disease (146 with SSc, 77 with undifferentiated connective tissue disease –UCTD-, and 56 with polymyositis/dermatomyositis -PM/DM) were tested by protein-IP for the screening of serum autoantibodies. In 58/279 (21%) sera we report a complete or partial protein-IP pattern of 75-50-40-34kD bands corresponding to the 4 proteins of the PDC complex recognized by AMA. In these sera we performed IIF and IP-WB to confirm the identity of these bands, by using established protocols. Clinical charts were used to analyze clinical and laboratory data for possible statistical correlation. Results In 15/58 (26%) patients with protein-IP AMA pattern we confirm a diagnosis of PBC, and all of them had a diagnosis of SSc without additional rheumatic disease. Their expression of the PDC subunits is variable by IP-WB (Figure panel A), but this variability is not associated with specific clinical and laboratory features. In the 42/58 (72%) cases with AMA protein-IP pattern but without clinical and histological evidence of PBC, the E2 component of the PDC was detected in all except one patient, while the expression of the other proteins of the PDC was significantly less represented, i.e. E3BP in 14/42 (33%), E1β in 13/42 (31%), E3 in 6/42 (14%), and E1α in 4/42 (10%) patients. The IIF analysis on Hep2 cells of AMA positive samples also shows different patterns of expression, which spans from cytoplasmic positive staining with a reticular pattern in AMA positive sera for all the PDC proteins recognized by AMA, to cytoplasmic speckled pattern in cases with incomplete expression of PDC antigens (Figure panel B). So far, no specific clinical correlation was identified in these patients, as they did not have a diagnosis of PBC or other autoimmune liver disease or manifested elevated liver function tests. Conclusion The expression of the PDC antigenic components is variable both in SSc patients with AMA positive PBC and in SSc patients AMA positive without PBC or altered liver function tests, but we could not identify a clinical significance of this variability. It may be necessary to maintain a strict follow-up of these patients and to perform longitudinal studies to determine the prognostic value of this variable expression of PDC components in the onset of PBC. References [1] Fujimoto M, et al. Arthritis Rheum. 1995Jul; 38(7): 985-9 [2] Ceribelli A, et al. J Immunol Methods. 2018Jan; 452: 1-5 Disclosure of Interests Angela Ceribelli: None declared, Natasa Isailovic: None declared, Carolina Gorlino: None declared, Elena Generali: None declared, Maria De Santis: None declared, Giacomo Maria Guidelli: None declared, Marta Caprioli: None declared, Piercarlo Sarzi-Puttini: None declared, Minoru Satoh: None declared, Carlo Selmi Grant/research support from: AbbVie, Janssen, MSD, Novartis, Pfizer, Consultant for: AbbVie, Alfa-Sigma, Biogen, Bristol-Myrs Squibb, Celgene, Eli-Lilly, Janssen, Merck Sharp and Dohme, Novartis, Pfizer, Roche, Sanofi-Genzyme, UCB, Speakers bureau: AbbVie, Alfa-Sigma, Biogen, Bristol-Myrs Squibb, Celgene, Eli-Lilly, Janssen, Merck Sharp and Dohme, Novartis, Pfizer, Roche, Sanofi-Genzyme, UCB

Background: In this work, we aimed to establish a clinical target in the management of knee osteoarthritis (KOA) and to propose good clinical practice (GCP) statements for carrying out a treat-to-target strategy. Methods: A steering committee of seven experts had formulated a provisional set of recommendations that were exposed for discussion and modification to a technical expert panel (TEP) of 25 multidisciplinary experts from Europe, North America, South America and Asia. The level of evidence and strength of each recommendation was discussed. The TEP formulated overarching principles and GCP statements based on the level of agreement for each item with a vote using a 10-point numerical scale. Results: Two overarching principles and 10 GCP statements were formulated by the TEP. These GCP statements suggest: treatment should achieve clinical improvement bringing the patient to the Patient Acceptable Symptom State (PASS); pharmacological and nonpharmacological treatment should begin as early as possible, with an early diagnosis of symptomatic KOA; the patient should be evaluated every 3–6 months; risk factors of KOA progression should be identified and managed with patients at the beginning of the treatment and monitored regularly; treatment should be adapted according to patient phenotype and disease severity; healthy lifestyle must be promoted and monitored. The level of agreement average ranged from 8.7 to 9.6 on scale. Conclusions: The proposed overarching principles and GCP statements have the aim of involving patients, general practitioners and multidisciplinary specialists in sharing a therapeutic treat-to-target strategy for KOA management based on the best evidence and expert opinions.

The identification of serum autoantibodies is central in the diagnosis of systemic autoimmune rheumatic disease (SARD), and an increasing number of specificities have been detected in the past years. This allows an early diagnosis in the active phases of diseases, with the identification of specific disease subsets that may ultimately improve the disease outcomes. Thanks to the use of old and new laboratory techniques that are becoming increasingly available worldwide, the number of rheumatic patients with a specific autoantibody is increasing and this is improving also our knowledge of disease trigger mechanisms. The paradigmatic example is the plethora of serum autoantibodies described in polymyositis and dermatomyositis, coined myositis-specific antibodies (MSA) which include antibodies directed against tRNA synthetases, anti-SRP, anti-Mi-2, and anti-TIF-1γ and can discriminate disease subtypes, particularly when associated with the risk of cancer. As a further example, anti-HMGCR antibodies have been reported in several studies in association with necrotizing autoimmune myositis that may follow statin use. To clarify the current knowledge on these rare specificities, we performed a systematic literature review. We focused on the main features associated to specific autoantibodies that are rarely identified in rheumatic disease, to increase the awareness and scientific knowledge on these autoantibodies in different ethnic groups worldwide. Fil: Ceribelli, Angela. Humanitas Research Hospital; Italia. Università degli Studi di Milano; Italia Fil: Isailovic, Natasa. Humanitas Research Hospital; Italia Fil: De Santis, Maria. Humanitas Research Hospital; Italia Fil: Generali, Elena. Humanitas Research Hospital; Italia Fil: Gorlino, Carolina Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Humanitas Research Hospital; Italia Fil: Palermo, Bianca. Humanitas Research Hospital; Italia Fil: Selmi, Carlo. Università degli Studi di Milano; Italia. Humanitas Research Hospital; Italia

Background Systemic sclerosis (SSc) is an autoimmune fibrotic disease characterised by variable clinical manifestations based on the predominant organ involvement. With the exceptions of acute fulminant myocarditis with pericardial effusion and heart failure, myocardial involvement in SSc is clinically silent until arrhythmias appear, while representing the major cause of sudden death or atrio-ventricular (AV) blocks in SSc. There are no reliable screening algorithm to discriminate myocardial involvement nor established treatments. Objectives To identify early/clinically silent myocardial involvement in SSc and define a possible treatment. Methods We used Holter electrocardiography (ECG) to investigate our cohort of patients with SSc (n=221, all fulfilling the ACR classification criteria) for ventricular ectopic beats (VEB)/AV blocks/unexplained tachycardia as sensitive signs of myocardial involvement. In 24 patients (women 23%–95.8%, anti-centromere-ACA 16%–66.6%, anti-topoisomerase-aScl70 3%–12.5%, anti-RNA polimerase III 3%–12.5%, anti-Ku 2%–8.3%, limited skin disease-lSSc 12%–50%, diffuse skin disease 5%–20.8%, sine scleroderma 7%–29.2%, median age 66.5 years, IQR 57.8–72.5), never treated with anti-fibrotic agents, we performed heart magnetic resonance (hMR) searching for myocardial oedema (on T2 STIR sequences, using the signal ratio between myocardium and skeletal muscle with a cut-off value of ≥1.9 for oedema) or fibrosis (presence of any late gadolinium enhancement (LGE) with intramyocardial or subepicardial pattern). Patients with myocardial oedema at hMR were treated with mycophenolate mofetil (MFM) 2 g/day or with azathioprine (AZA) 100 mg/day and underwent a control hMR after six months. Results In 10/24 (42%) of the SSc cases with Holter ECG alterations (8 VEB, 1 tachycardia, 1 type II AV block) we observed SSc myocardial involvement at hMR. In more detail, 6 patients had myocardial oedema at T2 STIR sequences (including cases as follows: 1 aScl70-positive with lSSc and lung fibrosis, 2 ACA-positive with lSSc, 3 ACA-positive sine scleroderma), and 4 only fibrosis (1 Scl70-positive with lSSc and lung fibrosis, 1 ACA-positive with lSSc, 1 ACA-positive sine scleroderma, 1 anti-Ku-positive sine scleroderma). At 6 months of medical treatment, myocardial oedema disappeared in 3 patients treated with Aza and in 1 treated with MFM, the additional 2 patients are still receiving MFM treatment at the time of analysis. Conclusions We observed that 42% of patients with SSc and minor arrhythmias on 24 hour Holter ECG have clinically silent myocardial involvement at hMR; those with potential reversible disease (oedema rather than fibrosis) favourably respond to immunosuppressants. Detectable serum ACA and the absence of skin involvement are over-represented in this subgroup of patients. Disclosure of Interest None declared

Background Increased TNF alpha (TNFα) levels have been associated to pregnancy complications such as intra-uterine growth retardation and fetal loss. Even if pregnancy has classically been considered as having a positive impact on RA, disease flares potentially leading to poor obstetrical outcomes are not uncommon, raising the challenge of RA management in the early stages of pregnancy. Among TNFα inhibitors indicated for rheumatoid arthritis (RA), the use of certolizumab pegol (CTZ) has been reported as safe during pregnancy. As such, CTZ is allowed during pregnancy as provided by the factory-issued product’s sheet. Objectives To assess the CTZ use safety during RA pregnancies, including maternal-fetal outcomes. Methods We retrospectively evaluated twelve women with RA, fulfilling the 2010 ACR/EULAR criteria, who had been exposed to CTZ throughout pregnancy. All women had signed an informed consent prior to treatment initiation. Results All cases were free from any potentially teratogenic drug, stopped at least 6 months before conceiving. Due to the underlying disease activity, and thanks to the approval for CTZ to be continued throughout the whole pregnancy, we carried on the treatment. Mean maternal age at conception was 31.6±40 months; mean disease duration dated of 51±26 months. All patients were on low-dose daily oral prednisone ranging from 2.5 to 5 mg; four subjects were on sulfasalazine 500 mg TID and four others on hydroxychloroquine 200 mg TID. We observed 12 singleton pregnancies; 8/12 mothers were primiparous. Mean gestational duration was 37±3 weeks and mean birth weight 3.037±0.58 grams. No stillbirths or fetal deaths were recorded. Five patients experienced elective caesarean section, while the others had vaginal delivery (four labour were induced by intravenous oxytocin; no dystocic births were reported). Mean APGAR scores after 1, 5 and 10 min from delivery were, respectively, 8±2; 8±1 and 9±1. No obstetric, perinatal or neonatal complications were observed. Eight/12 newborns were breastfed. After a 12 months observational gap, all babies were healthy and the development index above the 75th percentile. All children underwent the Italian scheduled vaccine program, without complications. Their antibody vaccine response is nowadays being investigated by our Research team. Conclusions The data from our case series confirm the safety of CTZ administration during pregnancy in women with RA, regarding both the mothers and the offspring. No infection rate increase, neither major maternal-fetal issue were reported. The higher caesarean section rate in our patients, in comparison to the average of northern Italy (20%), could have a iatrogenic explanation, as suggested by the fact that all had been scheduled ahead of time. Similarly, the relative low rate of breastfeeding could be justified by the lack of information given to mothers. Further large data collection and perspective, controlled studies are needed to confirm this statement. References [1] Mariette X, et al. Ann Rheum Dis. 2018;77(2):228–233. [2] Forger F, et al. Joint Bone Spine. 2016;83(3):341–3. [3] Clowse ME, et al. J Rheumatol. 2015;42(12):2270–8. Disclosure of Interest None declared

Background Skin psoriasis affects 3% of the general population and as much as 20%–40% of patients will develop psoriatic arthritis (PsA), with the two conditions representing a clinical and immunological continuum within a prototype for chronic inflammation. Different from most rheumatic diseases, no serum autoantibody is associated with PsA, and there are currently no biomarkers for an early diagnosis and prediction of PsA onset in psoriasis, frequently causing a delayed diagnosis. Monozygotic twins discordant for psoriasis/PsA represent a unique setting to investigate the influence of environmental and stochastic factors on disease phenotype. Through a high-throughput proteomic analysis (SomaLogic) we have previously identified a set of 13 proteins differentially expressed in the serum of monozygotic twins discordant for psoriatic disease. Objectives To validate serum proteomic biomarkers of psoriatic disease using commercially available ELISA in monozygotic twins and in unrelated patients with psoriasis or PsA. Methods Our study included sera from our cohort of monozygotic twins previously described and from 70 unrelated patients with psoriatic disease (psoriasis without PsA 34%–49%, PsA with/without psoriasis 36%–51%; women 46%, median age 52 years, interquartile range 41–59) followed at Humanitas Research Hospital, and 25 healthy subjects (52% women, median age 52 years, IQR 44–62). Candidate serum proteomic biomarkers obtained by SomaLogic analysis were validated using commercially available ELISA kits and proteins are herein anonymized due to a pending patent request. Results We found a significant correlation between SomaLogic results in monozygotic twins and ELISA results in unrelated psoriatic cases in the serum levels of 2 proteins (figure 1), which are involved in inflammatory and immune response, and one has been previously reported in psoriatic plaques. Four proteins showed a significantly different expression between psoriasis and PsA versus controls, in particular two proteins have a potential role in disease pathogenesis, as protein #1 acts as cell-surface receptor and regulates differentiation, proliferation and survival of dendritic cells, while protein #2 is involved in regulation of UV radiation-induced apoptosis and protein folding. Conclusions Two serum proteomic biomarkers, previously identified in a cohort of monozygotic twins discordant for psoriatic disease, can discriminate psoriatic disease, thus representing potential biomarkers of disease and possibly playing a pathogenetic role in disease. Disclosure of Interest None declared

Objective To describe a new immunoprecipitation pattern identified in Italian patients affected by systemic sclerosis (SSc), corresponding to the pyruvate dehydrogenase antigen complex recognized by anti-mitochondrial antibodies (AMA) in primary biliary cholangitis (PBC). Methods Autoantibodies in sera from 85 patients with SSc were tested by protein- and RNA-immunoprecipitation. Immunoprecipitation-Western blot was used to determine the identified proteins, and medical records re-evaluated for liver function tests and PBC. Results In 13/85 (15%) SSc sera, a unique set of 75-50-40-34 kD proteins that had not been previously reported, was noted. The four proteins were identified as the proteins X/E3BP, E1α, E1β, and E2/E3 of the pyruvate dehydrogenase antigen complex by immunoprecipitation-Western blot. From clinical record evaluation, 9/13 (69%) SSc patients with this new pattern were positive for AMA by routine indirect immunofluorescence, and 7/13 (54%) had a diagnosis of PBC, while 4/13 (31%) manifested no biochemical signs of cholestasis. Twelve of 13 patients with SSc and AMA by immunoprecipitation have a limited cutaneous form of SSc and anti-centromere antibodies. Conclusions We describe a pattern of 4 proteins in 15% of SSc patients, identified for the first time by protein-immunoprecipitation. This pattern corresponds to serum AMA against the pyruvate dehydrogenase antigen complex and it must be considered in the interpretation of protein-immunoprecipitation results.

The sequencing of the human genome is now well recognized as the starting point of personalized medicine. Nonetheless, everyone is unique and can develop different phenotypes of the same disease, despite identical genotypes, as well illustrated by discordant monozygotic twins. To recognize these differences, one of the easiest and most familiar examples of biomarkers capable of identifying and predicting the outcome of patients is represented by serum autoantibodies. In this review, we will describe the concept of personalized medicine and discuss the predictive, prognostic and preventive role of antinuclear antibodies (ANA), anti-citrullinated peptide antibodies (ACPA), rare autoantibodies and anti-drug antibodies (ADA), to evaluate how these can help to identify different disease immune phenotypes and to choose the best option for treating and monitoring rheumatic patients in everyday practice. The importance of ANA resides in the prediction of clinical manifestations in systemic sclerosis and systemic lupus erythematosus and their association with malignancies. ACPA have a predictive role in rheumatoid arthritis, they are associated with the development of a more aggressive disease, extra-articular manifestations and premature mortality in RA patients; moreover, they are capable of predicting therapeutic response. Rare autoantibodies are associated with different disease manifestations and also with a greater incidence of cancer. The determination of ADA levels may be useful in patients where the clinical efficacy of TNF-α inhibitor has dropped, for the assessment of a right management. The resulting scenario supports serum autoantibodies as the cornerstone of personalized medicine in autoimmune diseases.

Background Systemic sclerosis (SSc) is characterized in virtually all patients by anti-nuclear antibodies (ANA) with anti-centromere (ACA), anti-topoisomerase I (ant-Scl70), and anti-RNA polymerase III antibodies identifying patient subgroups. However, there are no reliable biomarkers predicting SSc susceptibility and internal organ involvement. Objectives We aimed to identify serum protein biomarkers associated with SSc and interstitial lung disease (ILD), using a highly multiplexed proteomic technology. Methods We analyzed serum samples of 3 patients with SSc and ILD (2 limited SSc: one ANA positive and one anti-Scl70 positive, and one diffuse SSc anti-Scl70 positive), 3 patients with SSc and no ILD (ACA positive limited SSc), and 4 healthy controls. All subjects were women and age matched. Serum proteomics profiling was performed using the SOMAscan platform (SomaLogic, Inc., Boulder, CO, USA). Statistical analysis included Student9s t-test and were performed using the SomaSuite software (SomaLogic, Boulder, CO, USA). Results Proteomic analysis identified 33 proteins which differentiate SSc from HC and 9 proteins which differentiate SSc patients with and without ILD. Compared to healthy controls, SSc cases showed an altered expression of proteins involved in extracellular matrix formation and cell-cell adhesion, angiogenesis, and lymphocyte recruitment, activation, and signaling, while an overall inhibition of neutrophil function was noted. An interferon and IL-1 signatures were also found. Patients with SSc and ILD manifested increased protein levels related to intracellular signaling and cell cycle, along with an increase of monocyte chemoattractants and ligands for the leukocyte adhesion compared to SSc without ILD. We further observed a decrease in B cell stimulating factor and IL-22 signaling in SSc with ILD. Conclusions Serum proteomic profiles can differentiate SSc from healthy controls and SSc patients with and without ILD; moreover, our results identify biomarkers with a putative pathogenetic significance. Disclosure of Interest None declared

This study aims to characterize myositis-specific antibodies in a well-defined cohort of patients with idiopathic inflammatory myopathy and to determine their association with cancer. Sera from 40 patients with polymyositis, dermatomyositis, and controls were tested by protein and RNA immunoprecipitation to detect autoantibodies, and immunoprecipitation-Western blot was used for anti-MJ/NXP-2, anti-MDA5, and anti-TIF1γ/α identification. Medical records were re-evaluated with specific focus on cancer. Anti-MJ/NXP-2 and anti-TIF1γ/α were the most common antibodies in dermatomyositis. In six dermatomyositis cases, we found five solid forms of cancer and one Hodgkin's lymphoma in long-term remission. Among patients with cancer-associated dermatomyositis, three were positive for anti-TIF1γ/α, two for anti-Mi-2, and one for anti-MJ/NXP-2. The strongest positivity of anti-TIF1γ was seen in two active forms of cancer, and this antibody was either negative or positive at low titers in the absence of cancer or in the 7-year remission Hodgkin's lymphoma. Four out of twenty (20 %) patients with polymyositis had solid cancer, but no specific association with autoantibodies was identified; further, none of the four cases of antisynthetase syndrome had a history of cancer. No serum myositis-associated autoantibody was observed in control sera, resulting in positive predictive value 75 %, negative predictive value 78.5 %, sensitivity 50 %, specificity 92 %, and area under the ROC curve 0.7083 for the risk of paraneoplastic DM in anti-TIF1γ/α (+) patients. Myositis-specific autoantibodies can be identified thanks to the use of immunoprecipitation, and their association with cancer is particularly clear for anti-TIF1γ/α in dermatomyositis. This association should be evaluated in a prospective study by immunoprecipitation in clinical practice.

Summary The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell- and B cell-epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4-related (antigen DR4− HLA-DR4)+ woman with early RA, her healthy monozygotic twin and an unrelated HLA-DR3+ woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7-aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)−17/IL-4/IL-10/IL-6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261-273), the K264 carbamylated T cell epitope (carT261–273), the native B cell epitope (B359–369) or the R360 citrullinated B cell epitope (citB359–369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4+ twins, but not in the DR3+ RA. The collagen-specific activation of CD4+ T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL-17 production in the RA twin, but a greater IL-4 and IL-10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL-6/IL-10 ratio in the RA twin compared to a greater IL-10 production in the healthy twin. Our data suggest that circulating collagen-specific T and B cells are found in HLA-DR4+ subjects, but only RA activated cells express co-stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.

Purpose Nailfold videocapillaroscopy (NVC) identifies the microvascular hallmarks of systemic sclerosis (SSc) and vascular endothelial growth factor (VEGF) and may play a pivotal role in the associated vasculopathy. The aim of the present study was to compare NVC alterations with clinical subsets, internal organ involvement, and serum VEGF levels in a cohort of selected SSc cases. Methods We studied 44 patients with SSc who were evaluated within 3 months from enrollment by NVC, skin score, severity index, pulmonary function tests, carbon monoxide diffusing capacity (DLCO), echocardiography, pulmonary high-resolution computed tomography (HRCT), gastroesophageal (GE) endoscopy or manometry or X-ray, and serum autoantibodies. Serum VEGF-A levels were determined by ELISA in 72 SSc patients and 31 healthy controls. Results Giant capillaries were inversely correlated with age (p = 0.034, r = −0.34) and to the extent of reticular pattern at HRCT (p = 0.04, r = −0.5). Avascular areas were directly correlated with capillaroscopy skin ulcer risk index (CSURI) (p = 0.006, r = +0.4) and severity index (p = 0.004, r = +0.5). The mean capillary density was directly correlated to the ulcer number (p = 0.02, r = +0.4) and to DLCO/alveolar volume (p = 0.02, r = +0.4) and inversely correlated with severity index (p = 0.01, r = −0.4) and skin score (p = 0.02, r = −0.4). Serum VEGF levels were higher in the SSc population vs controls (p = 0.03) and inversely correlated with DLCO (p = 0.01, r =−0.4) and directly with ground-glass and reticular pattern at HRCT (p = 0.04, r = +0.4 for both). Conclusions Our data suggest the importance of NVC not only for the diagnosis, but also for the global evaluation of SSc patients. Of note, serum VEGF levels may act as a biomarker of interstitial lung involvement.

The pathogenesis of idiopathic inflammatory myositis (IIMs, including polymyositis and dermatomyositis) remains largely enigmatic, despite advances in the study of the role played by innate immunity, adaptive immunity, genetic predisposition, and environmental factors in an orchestrated response. Several factors are involved in the inflammatory state that characterizes the different forms of IIMs which share features and mechanisms but are clearly different with respect to the involved sites and characteristics of the inflammation. Cellular and non-cellular mechanisms of both the immune and non-immune systems have been identified as key regulators of inflammation in polymyositis/dermatomyositis, particularly at different stages of disease, leading to the fibrotic state that characterizes the end stage. Among these, a special role is played by an interferon signature and complement cascade with different mechanisms in polymyositis and dermatomyositis; these differences can be identified also histologically in muscle biopsies. Numerous cellular components of the adaptive and innate immune response are present in the site of tissue inflammation, and the complexity of idiopathic inflammatory myositis is further supported by the involvement of non-immune mechanisms such as hypoxia and autophagy. The aim of this comprehensive review is to describe the major pathogenic mechanisms involved in the onset of idiopathic inflammatory myositis and to report on the major working hypothesis with therapeutic implications.

Background Psoriatic arthritis (PsA) coexists with skin psoriasis (PsO) in 10-30% of patients. No serum autoantibody is consistently found in PsA, thus supporting the view of a chronic inflammatory rather than autoimmune pathogenesis. Further, the lack of serum biomarkers is responsible for delayed PsA diagnosis particularly in PsO. Objectives To apply protein- and RNA-immunoprecipitation (IP) for the identification of new serologic markers of PsA. Methods Sera from 29 adult consecutive patients with PsA were collected in the outpatient clinic of the Rheumatology Unit at the Humanitas Research Hospital. CASPAR criteria for PsA were used to establish the diagnosis in all cases. Sera were analyzed by immunoprecipitation (IP) of K562 (human erythroleukemia) cell extract radiolabeled with 35S-methionin. RNA in the immunoprecipitate was extracted with phenol/chloroform/isoamyl alcohol and analyzed by silver staining. Clinical information was obtained from clinical correlations. Results PsA cases are divided in polyarticular (n=23), mono/oligoarticular (n=4), and axial (n=2) phenotypes according to the pattern of joint and spine involvement. The female:male ratio is 2.2 and the mean age of PsA patients is 55 years (range 22-78 years). The mean duration of PsO is 10.3 years, while PsA mean duration is 6.8 years, with PsO preceding PsA in most individuals. One patient with PsO has polyarthritis and also positive Rheumatoid Factor and anti-CCP antibodies and is thus excluded from the series, while the other 29 PsA patients are consistently negative for both autoantibodies. Serum anti-nuclear antibodies are positive in 7/29 (24%) PsA and are speckled in 5 cases, 1 homogeneous and 1 nucleolar. IP analysis allowed the identification of specific bands corresponding to anti-Ago2/Su in 2 cases, anti-Ki/Sl in 2 cases and one weak anti-PM/Scl. None of these patients had clinical features suggestive for connective tissue disease in association with PsA. In 6/29 (20%) cases we were able to immunoprecipitate bands of variable molecular weight (48-180kD), but these do not correspond to known autoantibodies and no common band was detected in our subsets of PsA. RNA-IP did not show any specific autoantibody in the 29 PsA patients. One PsA patient later developed myositis, Raynaud9s phenomenon and sclerodactily but no specific autoantibody was identified by protein- and RNA-IP. Conclusions We report for the first time results of an extensive screening for new autoantibodies in PsA and identify specific bands and autoantibodies by protein-IP in our PsA patients, thus suggesting a possible autoimmune coexistance. The use of IP in a screening and follow-up setting may be useful to characterize new biomarkers for the early diagnosis of PsA in healthy subject and/or PsO patients. Disclosure of Interest None declared

The study aimed to determine the prevalence of rheumatoid factor (RF) and anti-citrullinated peptides (ACPA), to estimate the association with hepatitis B (HBV) or C (HCV) virus infections and the 15-year risk of developing RA in a large cohort from a Northern Italian region.In 1998, 15,907 subjects between the ages of 18 and 75 were randomly selected 1:4 for HBV and HCV testing; more recently, we tested a subgroup of sera for RF (n=2196) and ACPA (n=2525). Administrative databases were searched after 15 years for incident RA diagnoses occurring between 1998 and 2013.RF was positive in 8.1% of cases with 10% of RF-positive subjects having HBsAg (p=0.004) and 9% anti-HCV. ACPA were detected in 4.8% of subjects with 5% of the ACPA-positive subjects having HBsAg and 5.9% anti-HCV. Older subjects had higher positivity rates for both RF and ACPA. HBsAg and anti-HCV were detected in 5.5% and 4.3% of sera, respectively. Over 15 years, 10 RA cases were recorded (9 women, median age at diagnosis 52 years) with RF previously positive in 2/10 and ACPA in 5/10 cases. RF and ACPA were associated with relative risks for developing RA of 5.7 (adjusted for HBsAg status; 95% CI 1.2-26.3) and 13.2 (95% CI 3.8-46.3), respectively.Our data in a large cohort from an unselected general population confirm a higher risk of RA development associated with ACPA compared to RF. HBV exposure correlates with RF but not with ACPA positivity.