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Introduction: Cryopyrin associated periodic syndromes (CAPS) are rare autoinflammatory disorders associated with dominantly gain-offunction mutations in the NLRP3 gene that result in overactivation of the inflammasome, increased secretion of interleukin (IL)-1beta and IL-18, and systemic inflammation. It has been reported that oligomeric particles of the adaptor ASC (apoptosis-associated Speck-like protein with a caspase-recruitment domain) are released together with IL-1beta and active caspase-1 subunits after activation of the inflammosome complex and that patients with CAPS show an increased serum concentration of ASC+ particles. Objectives: The diagnosis of CAPS is a critical factor due to both the lack of specific laboratory results and the sharing of similar clinical manifestations with other autoinflammatory diseases, our aim is to develop a simple assay to evaluate the levels of ASC particles in the serum of CAPS patients to provide novel biomarkers facilitating early disease diagnosis and able to monitor treatment responses. Methods: We developed an ELISA for the quantification of ASC particles in serum and plasma of normal and pathological subjects. We analysed samples from CAPS patients and from patients with autoimmune disorders (Multiple Sclerosis (MS), Type 1 Diabetes (T1D) and juvenile idiopathic arthritis), to confirm that ASC presence in the serum is not due to other chronic inflammatory processes characterizing autoimmunity. In addition, we also evaluated the concentration of ASC in the sera of TNF receptor–associated periodic syndrome (TRAPS) patients to reinforce the concept of specificity of this biomarker in CAPS patients and not in individuals suffering from others inflammatory disorders. Results: We observed that untreated CAPS patients are characterized by the presence of a significant higher amount of ASC particles when compared with healthy controls (HS) and with patients suffering from MS and T1D. This tendency was also evident in patients with arthritis and TRAPS even if the difference was not statistically significant due to the small number of samples. In addition there is a tendency through a reduction of ASC levels in CAPS patients after pharmacological treatment, which require future investigations. Conclusion: These data suggest that ELISA quantitation of ASC protein could represent a novel and additional strategy for the diagnosis and monitoring of CAPS.

Objectives Microcrystal-induced arthritis is still an unresolved paradigm for medicine. Overt inflammation may be absent even when crystals occur in SF. Recently, the production of neutrophil extracellular traps (NETs) embedding MSU crystals has been proposed as a possible mechanism of the auto-resolution of the inflammatory phase during gout. We aimed to verify and quantify the release of NETs in SFs during gout and pseudogout attacks and to compare any differences with respect to crystals and neutrophils number, and to analyse activation of necroptosis pathway in SF from crystal-induced arthritis. Methods SF samples were obtained by arthrocentesis from 22 patients presenting acute crystal-induced arthritis, gout or pseudogout (n = 11 each group), and from 10 patients with acute non-crystal arthritis as controls. NETosis was quantified in SF by nucleic acid stain and by quantification of human neutrophil elastase. Activation of phosphorylated MLKL was assessed by western blot. Results We observed that SF neutrophils encountering MSU and CPPD crystals during episodes of gout and pseudogout release NETs in relation to the number of crystals in SF and irrespective of neutrophil density and type of crystal. This release was accompanied by necroptosis through the activation of the MLKL pathway. Conclusions Our findings suggest that a role of NETs in crystal-induced arthritis is to ‘trap extracellular particles’, including microcrystals. Embedding crystals in aggregates of NETs may be the basis of tophi and CPPD deposition, and may have implications for disease evolution rather than for spontaneous resolution of the acute attack.

The study aims are to describe the activity of our Unit on the diagnostics of monogenic autoinflammatory diseases (AIDs), and to apply the clinical classification criteria for periodic fevers from the Eurofever Registry to our cohort of patients, thus evaluating their usefulness in the real life. We retrospectively analyzed data from patients referring to our Center for recurrent fever attacks, and undergoing genetic analysis between April 2014 and July 2016, and we applied the classification criteria to both genetically positive and -negative patients. We visited 195 patients (101 females, 94 males); 126 (64.6%) were adults and 192 (98.5%) Caucasians; 12.3% carried mutations and 12.7% of adults were genetically positive. No statistically significant differences were identified in the frequency of genetic diagnosis between adults and children (p = 0.82) as well as in the frequency of genetic diagnosis, based on the number of genes evaluated (p = 0.57). When we applied the Eurofever criteria, 126/195 (64.6%) patients were classified for at least one among the four main monogenic AIDs; 22 (11.3%) patients fulfilled criteria for 2 diseases and 4 (2.1%) for 3 diseases. Among patients carrying mutations, 12/24 (50%) correctly fulfilled the score, 3/24 (12.5%) fulfilled criteria differently from their genetic diagnosis; 9/22 (40.9%) recieved no classification. An expanded genetic testing does not seem useful, while a correct interpretation of patients' clinical picture may allow performing specific genetic testing. The classification criteria from the Eurofever Registry have shown to be a beneficial tool in the evaluation of patients with a suspected monogenic AID.

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.

Background: Dominantly gain-of-function mutations in the NLRP3 gene lead to Cryopyrin associated periodic syndromes (CAPS) characterized by constitutive activation of the inflammasome, increased secretion of interleukin (IL)-1beta and IL-18, and systemic inflammation. IL-1beta and active caspase-1 subunits are released in the serum together with the oligomeric particles of the adaptor ASC (apoptosis-associated Speck-like protein with a caspase-recruitment domain) after activation of the inflammosome complex and, as a consequence, patients with CAPS show an increased serum concentration of ASC+ particles. Objectives: Patients suffering from CAPS are characterized by clinical manifestation similar to other autoinflammatory diseases. This phenomenon together with the lack of specific laboratory tests makes difficult the diagnosis of CAPS. In this context the development of a test for the evaluation of serum ASC levels could provide novel biomarkers facilitating early disease diagnosis and able to monitor treatment responses. Methods: We analysed, with a novel ELISA assay, the levels of ASC particles in serum and plasma of normal subjects, CAPS patients and patients with autoimmune disorders (Multiple Sclerosis (MS), Type 1 Diabetes (T1D) and juvenile idiopathic arthritis), to confirm that ASC presence in the serum is not due to other chronic inflammatory processes characterizing autoimmunity. To evaluate the specificity of this biomarker in CAPS patients and not in individuals suffering from others inflammatory disorders, we also analysed sera from TNF receptor–associated periodic syndrome (TRAPS) patients. Results: ASC particles were higher in untreated CAPS patients with respect to healthy controls and patients suffering from MS and T1D. This tendency was also evident in patients with arthritis and TRAPS even if the difference was not statistically significant due to the small number of samples. In addition after pharmacological treatment there is a tendency to be confirmed through a reduction of ASC levels in CAPS patients. Conclusion: These data suggest that ELISA quantitation of ASC protein could represent a novel and additional strategy for the diagnosis and monitoring of CAPS. Disclosure of Interests:: Fortunata Carbone: None declared, Teresa Micillo: None declared, Luca Cantarini: None declared, Maria Alessio: None declared, Alma Nunzia Olivieri: None declared, Maria Francesca Gicchino: None declared, Antonella Insalaco: None declared, Maria Cristina Maggio: None declared, Orso Maria Lucherini: None declared, Roberto Scarpioni: None declared, Matteo Piga: None declared, Maria Maddalena Angioni: None declared, Laura Obici: None declared, Antonella Simpatico: None declared, Pietro Leccese: None declared, Rita Consolini: None declared, Raffaele Manna: None declared, Paolo Sfriso: None declared, Sara Bindoli: None declared, Paola Galozzi: None declared, Ida Orlando: None declared, Sabrina Chiesa: None declared, Marco Gattorno Grant/research support from: MG has received unrestricted grants from Sobi and Novartis, Giuseppe Matarese Grant/research support from: Matarese reports research grants from Merck-Serono, Biogen Idec, Novartis and IBSA.

Adipocytokines, including adiponectin, resistin, and visfatin may play an important role in the pathophysiology of osteoarthritis (OA). Spa therapy is one of the most commonly used non-pharmacological approaches for OA, but its mechanisms of action are not completely known. The aim of the present study was to assess whether a cycle of mud-bath therapy (MBT) influences the serum levels of adiponectin, resistin, and visfatin in patients with knee OA. As part of a prospective randomized, single blind-controlled trial evaluating the efficacy of MBT in knee OA, we included in this study 95 outpatients. One group (n = 49) received a cycle of MBT at the spa center of Chianciano Terme (Italy) in addition to the usual treatment, and one group (control group; n = 46) continued their regular care routine alone. Patients were assessed at basal time and at the end of the study (15 days) for clinical and biochemical parameters. Clinical assessments included spontaneous pain on a visual analog scale (VAS) score and the Western Ontario and McMaster Universities index (WOMAC) subscores for knee OA evaluated as total pain score (W-TPS), total stiffness score (W-TSS), and total physical function score (W-TPFS). Adiponectin, resistin and visfatin serum levels were assessed by enzyme immunoassay methods. At the end of the mud-bath therapy, serum adiponectin levels showed a significant decrease (p

Objective Increasing evidence indicates systemic inflammation as a new potential cause of acquired long QT syndrome (LQTS), via cytokine-mediated changes in cardiomyocyte ion channels. Torsade de pointes (TdP) is a life-threatening polymorphic ventricular tachycardia occurring in patients with LQTS, usually when multiple QT-prolonging factors are simultaneously present. Since classical risk factors cannot fully explain TdP events in a number of patients, we hypothesised that systemic inflammation may represent a currently overlooked risk factor contributing to TdP development in the general population. Methods Forty consecutive patients who experienced TdP (TdP cohort) were consecutively enrolled and circulating levels of C-reactive protein (CRP) and proinflammatory cytokines (interleukin-6 (IL-6), tumour necrosis factor alpha (TNFα), interleukin-1 (IL-1)) were compared with patients with active rheumatoid arthritis (RA), comorbidity or healthy controls. An additional 46 patients with different inflammatory conditions (acute infections, n=31; immune-mediated diseases, n=12; others, n=3) and elevated CRP (inflammatory cohort) were prospectively enrolled, and corrected QT (QTc) and cytokine levels were measured during active disease and after a CRP decrease of >75% subsequent to therapy. Results In the TdP cohort, 80% of patients showed elevated CRP levels (median: ~3 mg/dL), with a definite inflammatory disease identifiable in 18/40 cases (acute infections, n=12; immune-mediated diseases, n=5; others, n=1). In these subjects, IL-6, but not TNFα and IL-1, was ~15–20 times higher than in controls, and comparable to RA patients. In the inflammatory cohort, where QTc prolongation was common (mean values: 456.6±30.9 ms), CRP reduction was associated with IL-6 level decrease and significant QTc shortening (−22.3 ms). Conclusion The data are first to show that systemic inflammation via elevated IL-6 levels may represent a novel QT-prolonging risk factor contributing to TdP occurrence in the presence of other classical risk factors. If confirmed, this could open new avenues in antiarrhythmic therapy.

Mounting evidence indicates that in chronic inflammatory arthritis (CIA), QTc prolongation is frequent and correlates with systemic inflammatory activation. Notably, basic studies demonstrated that inflammatory cytokines induce profound changes in potassium and calcium channels resulting in a prolonging effect on cardiomyocyte action potential duration, thus on the QT interval on the electrocardiogram. Moreover, it has been demonstrated that in rheumatoid arthritis (RA) patients, the risk of sudden cardiac death is significantly increased when compared to non-RA subjects. Conversely, to date no data are available about torsades de pointes (TdP) prevalence in CIA, and the few cases reported considered CIA only an incidental concomitant disease, not contributing factor to TdP development. We report three patients with active CIA developing marked QTc prolongation, in two cases complicated with TdP degenerating to cardiac arrest. In these patients, a blood sample was obtained within 24 h from TdP/marked QTc prolongation occurrence, and levels of IL-6, TNFα, and IL-1 were evaluated. In all three cases, IL-6 was markedly elevated, ~10 to 100 times more than reference values. Moreover, one patient also showed high circulating levels of TNFα and IL-1. In conclusion, active CIA may represent a currently overlooked QT-prolonging risk factor, potentially contributing in the presence of other “classical” risk factors to TdP occurrence. In particular, a relevant role may be played by elevated circulating IL-6 levels via direct electrophysiological effects on the heart. This fact should be carefully kept in mind, particularly when recognizable risk factors are already present and/or the addition of QT-prolonging drugs is required.

Purpose The aim of our study was to determine the prevalence, clinical significance of antibodies to individual histone components and to evaluate their correlation with other autoantibody specificities in a cohort of Italian SSc patients. Some authors, demonstrated high prevalence of anti-histone antibodies in Italian SSc patients, associated with cardiac and renal involvement, suggesting a prognostic value of these autoantibodies; however, these data need to be confirmed. Methods Serum from 112 adult SSc patients, classified as diffuse (dc) and limited cutaneous (lc) SSc subsets were analyzed for autoantibodies by indirect immunofluorescence, fluoroenzyme immunoassay and enzyme immunoassay. Results AHA were found in 13 patients (11.6%), nine with lcSSc and four with dcSSc. Among them, five patients were anti-Scl70+ and four were anti-CENP B+. The presence of AHA was not associated with multi-organ involvement or with diffuse subset, as already described. Anti-Scl70 was detected in 43% of patients, anti-CENP B in 32% and anti-RNA polymerase III in 7.1%. We confirmed the association between anti-Scl70 antibodies and pulmonary fibrosis (OR 15.75, p

The aim of our study was to evaluate the clinical and HLA-class II allele associations of some anti-cofactor antibodies in a homogeneous group of European patients with SLE. One hundred thirty-six patients with SLE, fulfilling four or more of the ACR 1997 revised criteria for the classification of the disease, coming from 7 European countries, were enrolled consecutively. Anti-prothrombin (anti-PT), anti-annexin V (anti-AnnV), anti-protein C (anti-Cprot) and anti-protein S (anti-Sprot) were determined by using commercial ELISA kits. Molecular typing of HLA-DRB1, DRB3, DRB4, DRB5, DQA1, DQB1 and DPB1 loci was performed by using PCR-SSOP method, carried out using digoxygenin (DIG) labeled probes. The prevalence of anti-AnnV, anti-PT, anti-Cprot and anti-Sprot was 19%, 10.4%, 4.4% and 8.1%, respectively. Twenty-seven % of anti-AnnV positive patients reported migraine vs 5.5% of anti-AnnV negatives (p = 0.003, but p not significant, odds ratio (OR) = 6.4, 95% confidence interval (CI) = 2-21). Anti-PT, anti-AnnV and anti-Sprot were positively associated with some HLA alleles, but pc was not significant. In this study we have shown that some HLA alleles carry the risk to produce antibodies against phospholipid-binding proteins, but these association need confirmation in other studies, because they have never been reported and appear to be weak associations.

The aim of this study was to evaluate serum biomarkers, used in clinical routine, to predict the American College of Rheumatology (ACR) response to long-term anti-TNF alpha treatment (adalimumab). Sera from 29 consecutive rheumatoid arthritis patients were analysed for anti-cyclic citrullinated peptide (anti-CCP), cartilage oligomeric matrix protein (COMP) and IgM and IgA RFs (class-specific rheumatoid factors) at the start of treatment with adalimumab and after 3, 6 and 12 months. The response to the therapy was evaluated by ACR 20, 50, 70 and by DAS 28 scores. The mean serum COMP level of the population did not change after treatment. However, patients with low serum COMP levels (10 U/l) at baseline showed a significant (p0.02) higher ACR70 response (50%) within 3 months, and also at 6 months, than patients with higher COMP values (ACR7020%). This was also reflected by significantly higher decrease in DAS score at 3 (p0.02) and 6 months (p0.01) treatments. The IgM RF titre decreased significantly (p=0.02) after the therapy, but the percentage of serum positivity for anti-CCP and IgA/IgM RF did not change. No significant correlation was shown between serum COMP levels and C-reactive protein/erythrocyte sedimentation rate during the follow-up. Neither were any correlations shown between ACR/DAS 28 scores and anti-CCP, Ig M/IgA RFs. Our data indicate that low (10 U/l) serum COMP before starting anti-TNF alpha treatment predicts a rapid (within 3 months) and high ACR70 response compared to RA patients with higher COMP values. This might reflect different mechanisms in the cartilage process in the RA disease at that time of treatment with different therapeutic sensitivity to anti-TNF alpha treatment.

Serum amyloid-A (SAA) is an acute phase protein, synthesized by the liver and previously investigated as a marker of disease activity in many rheumatologic disorders. Its significance in Behcet’s disease (BD), a chronic inflammatory disorder at the crossroad between autoimmune and autoinflammatory syndromes, is still unraveled. Our aim was to assess the role of SAA levels as a potential marker of disease activity in patients with BD. According to our findings, the occurrence of oral aphthosis, neurological impairment, and ocular disease is significantly associated with SAA serum levels higher than 30, 50, and 150 mg/L, respectively. We also suggest that increased SAA levels might identify a thrombotic risk in BD with previous or concurrent vascular involvement.

Background Neutrophil extracellular traps (NETs) are web-like structures, composed of nucleic acids, histones and granular enzymes, including myeloperoxidase and elastase, that have a physiological crucial role in innate immunity response by trapping microbes. In addition, NETs have been identified as regulators of many inflammatory and autoimmune disorders, including crystal-induced arthritis.[1,2] In particular, a key role in the pathogenesis of gout flares has been postulated for NETs. During gout attacks, NET aggregates should package MSU crystals with a concomitant degradation of proinflammatory cytokines, leading to the rapid resolution of the inflammatory phase.[3] Objectives To evaluate NET expression in synovial fluid from acute CPPD arthritis and to compare NET release between acute MSU and CPPD arthritis. Methods Synovial fluid was collected from patients with active gout or pseudogout attack (n=5 for each group) and immediately centrifuged (805 rad) to separate cells from supernatant. Polymorphonuclear leukocytes were counted and immediately processed for morphological evaluation under transmission electron microscopy (Zeiss EM 109; 4000x). NET expression was evaluated by fluorescence microscopy using SYTOX Green (Life Technologies) and neutrophil elastase specific antibody (ABCAM). Fluorometric quantification of NET components, DNA and neutrophil elastase, was performed in supernatant (Cary Eclipse Varian; exc/em 503/526). Results were normalized to cells/mm 3 . Peripheral blood neutrophils from healthy donors stimulated with PMA were used as positive controls whereas PMA-untreated neutrophils were used as negative ones. All the experiments were performed according with Helsinki guidelines. Results Extracellular material morphologically compatible with NETs was observed by electron microscopy in samples from both acute MSU and CPPD arthritis. NET structures were confirmed by the presence of extracellular DNA and neutrophil elastase at immunofluorescence. Fluorometric quantification of extracellular DNA and neutrophil elastase showed an increased NET release in both gout and pseudogout samples compared with negative controls (p Conclusions Beyond confirming NETs in acute MSU arthritis, we have evidence of NET release in synovial fluid from acute CPPD arthritis. Our preliminary data suggest that the release of NETs could be a common physiophatological pathway to both crystral induced arthropaties. Disclosure of Interest None declared

Objective Recent evidence suggests that anti-Ro/SSA antibodies, strongly associated with the development of congenital heart block, may also be arrhythmogenic for the adult heart. In fact, anti-Ro/SSA–positive patients with connective tissue disease (CTD) frequently display corrected QT (QTc) prolongation associated with an increase in ventricular arrhythmias. However, QTc prolongation prevalence markedly differs throughout the studies (10–60%), but the reason why is not yet clear. The aim of this study was to evaluate whether anti-Ro/SSA–associated QTc prolongation in adult patients with CTD is related to antibody level and specificity. Methods Forty-nine adult patients with CTD underwent a resting 12-lead electrocardiogram recording to measure QTc interval, and a venous withdrawal to determine anti-Ro/SSA antibody level and specificity (anti–Ro/SSA 52 kd and anti–Ro/SSA 60 kd) by immunoenzymatic methods and Western blotting. Results In our population, a direct correlation was demonstrated between anti–Ro/SSA 52-kd level and QTc duration (r = 0.38, P = 0.007), patients with a prolonged QTc had higher levels of anti–Ro/SSA 52 kd with respect to those with a normal QTc (P = 0.003), and patients with a moderate to high level (≥50 units/ml) of anti–Ro/SSA 52 kd showed a longer QTc interval (P = 0.008) and a higher QTc prolongation prevalence (P = 0.008) than those with a low positive/negative level (

Systemic sclerosis (SSc) is a connective tissue disease characterized by vascular and fibrotic changes in the skin and in internal organs. Endothelin-1 (ET-1) is a peptide that has a role in promoting both vascular injury and the fibrotic process in SSc; indeed, patients with systemic sclerosis have higher levels of ET-1 compared with healthy subjects. Moreover, ET-1 enhances expression of pro-inflammatory cytokines in animal model. Bosentan is a dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension and digital ulcers in scleroderma patients. In animal models and in vitro models, after treatment with Bosentan, a significant reduction of cytokine (TNFα, IFNγ, IL-8, IL-4) levels was observed. The aim of the study is to verify whether Bosentan treatment in SSc patients can reduce circulating cytokines levels. We enrolled 10 patients affected by SSc with digital ulcers and/or pulmonary hypertension, treated with Bosentan 125 mg twice daily. Patients were tested for cytokines and ET-1 level before treatment and after 12 months. The cytokines tested were IL-10, IL-2, IL-4, IL-5, IL-6, IL-8, GM-CSF, IFN-γ and TNF. Levels of ET-1, IL-10, IL-4, IL-5, GM-CSF and TNFα did not show consistent modification during treatment with Bosentan in respect to baseline, while IL-2, IL-6, IL-8 and IFN-γ were significantly decreased. Bosentan significantly reduced IL-2, IL-6, IL-8 and IFN-γ levels in SSc patients, probably slowing progression to fibrosis and vascular damage. This is the first report showing a decrease of profibrotic and proinflammatory cytokines levels in humans during treatment with Bosentan.