F. Ceccarelli

Autoantibody Production in Anti‐TNF‐α‐Treated Patients

Abstract:u2002 Targeting tumor necrosis factor alpha (TNF‐α) has offered an additional therapeutic strategy against several rheumatic inflammatory disorders. The current use of TNF‐α inhibitors allows physicians who manage these diseases and patients themselves to testify to an extraordinary efficacy, even though caution for possible adverse events must be maintained. Among these, the occurrence of autoimmune phenomena, encompassing new autoantibody formation and triggering of clinical manifestations, continues to be noted in published reports. Here, we review the current knowledge regarding the autoimmune phenomena linked to anti‐TNF‐α therapy in patients with rheumatic inflammatory disorders.

AB0004 Polymorphisms in Genes in The IL-17 Pathway and B Cell Mediated Immune Response Modulate The Development of Specific Autoimmune Manifestations in Systemic Lupus Erythematosus

Background Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease in which a complex interaction between genetic, environmental and immunological factors determines the susceptibility and the phenotype. Dysregulations in IL-17 pathway as well as in B-cell mediated immune response have been observed in SLE. We have recently demonstrated that polymorphisms in TRAF3IP2 gene are associated with susceptibility for SLE and can predispose for the development of pericarditis (1). TRAF3IP2 encodes for Act1, a molecule that acts as a negative regulator of B cell by inhibiting CD40-mediated signaling and as a positive signaling adapter in IL-17-mediated cellular responses. TRAF6 is essential for the IL-17/Act1-mediated activation of NF-kB, while CD40 is the target for the Act1 mediated inhibition of B cell response. Objectives The primary goal of our study was to evaluate the association of polymorphisms in CD40, TRAF6 and TNFSF4 (OX40) genes with susceptibility to SLE. Secondary objectives were to assess the possible association of these polymorphisms with the clinical and laboratory features. Methods We recruited 315 Italian SLE patients and 278 healthy controls. Genotyping of rs4810485 in CD40, rs4755453 and rs5030437 in TRAF6, and rs2205960 and rs10489265 in TNFSF4 (OX40) SNPs was performed by allelic discrimination assay. A case/control association study and a genotype/phenotype correlation analysis were performed. Results Deviations from Hardy–Weinberg equilibrium for the three SNPs were not observed. None of the studies polymorphisms was associated with susceptibility for SLE. Only CD40 rs4810485 was associated at genotypic (P=0.034) but not at the allelic level. Nonetheless, these polymorphisms seem to contribute to define disease phenotype. TRAF6 was associated with the presence of anemia (P=0.019, OR=1.96), rs2205960 of TNFSF4 was associated with the pericarditis (P=0.013, OR=2.14), and rs4810485 of CD40 with the presence of anti-SSB/La (P=0.014, OR=2.26) and lupus nephritis (P=0.024, OR=1.8). Conclusions We were not able to confirm previous association of TRAF6 and CD40 polymorphisms with susceptibility for SLE. However, such SNPs seem to influence the disease phenotype. In particular, it is of interest the association of rs4810485 in CD40 with lupus nephritis and the presence of anti-SSB/La. Indeed, the modulation of CD40-mediated T cell-dependent antibody response has already been associated with the development of anti-SSB/La in association with SLE-like nephritis in a mouse model (2), probably due to an uncontrolled B cell activation signal. References Perricone C, Ciccacci C, Ceccarelli F, et al. TRAF3IP2 gene and systemic lupus erythematosus: association with disease susceptibility and pericarditis development. Immunogenetics. 2013 Oct;65(10):703–9 Qian Y, Giltiay N, Xiao J, et al. Deficiency of Act1, a critical modulator of B cell function, leads to development of Sjögrens syndrome. Eur J Immunol. 2008 Aug;38(8):2219–28. Disclosure of Interest None declared

Porphyromonas gingivalis in the tongue biofilm is associated with clinical outcome in rheumatoid arthritis patients

Several studies have suggested a link between human microbiome and rheumatoid arthritis (RA) development. Porphyromonas gingivalis seems involved in RA initiation and progression, as supported by the high occurrence of periodontitis. In this case–control study, we analysed tongue P. gingivalis presence and quantification in a large healthy and RA cohort. We enrolled 143 RA patients [male/female (M/F) 32/111, mean ± standard deviation (s.d.), age 57·5 ± 19·8 years, mean ± s.d. disease duration 155·9 ± 114·7 months); 36 periodontitis patients (M/F 11/25, mean ± s.d., age 56 ± 9·9 years, mean ± s.d. disease duration 25·5 ± 20·9 months); and 57 patients (M/F 12/45, mean ± s.d., age 61·4 ± 10·9 years, mean ± s.d. disease duration 62·3 ± 66·9 months) with knee osteoarthritis or fibromyalgia. All subjects underwent a standard cytological swab to identify the rate of P. gingivalis/total bacteria by using quantitative real‐time polymerase chain reaction. The prevalence of P. gingivalis resulted similarly in RA and periodontitis patients (48·9 versus 52·7%, P = not significant). Moreover, the prevalence of this pathogen was significantly higher in RA and periodontitis patients in comparison with control subjects (P = 0·01 and P = 0·003, respectively). We found a significant correlation between P. gingivalis rate in total bacteria genomes and disease activity score in 28 joints (DAS28) (erythrocyte sedimentation rate) (r = 0·4, P = 0·01). RA patients in remission showed a significantly lower prevalence of P. gingivalis in comparison with non‐remission (P = 0·02). We demonstrated a significant association between the percentage of P. gingivalis on the total tongue biofilm and RA disease activity (DAS28), suggesting that the oral cavity microbiological status could play a role in the pathogenic mechanisms of inflammation, leading to more active disease.

Efficacy of Spa Therapy, Mud-Pack Therapy, Balneotherapy, and Mud-Bath Therapy in the Management of Knee Osteoarthritis. A Systematic Review

Background Osteoarthritis (OA) is the most common musculoskeletal disease in the world. OA is the result of an inflammatory and degenerative process affecting the entire joint. Osteoarthritis, especially involving the knee, has a relevant socioeconomic impact in terms of drugs, hospital admissions, work absences, and temporary or permanent invalidity. Therapy of knee osteoarthritis is based on pharmacological and nonpharmacological measures. Methods We conducted a systematic review of the studies published between 2002 and 2017 on spa therapy, mud-pack therapy, balneotherapy, and mud-bath therapy in the treatment of knee osteoarthritis in order to investigate the evidence of the efficacy of such treatment on pain, functional limitation, drug use, and quality of life. Overall, 35 studies were examined among which 12 were selected and included in the review if they are trial comparative. We have been able to illustrate the main results obtained in the individual studies and to elaborate these results in order to allow as much a unitary presentation as possible and hence an overall judgment. Results Because the studies we reviewed differed markedly from one another in terms of the methods used, we were unable to conduct a quantitative analysis (meta-analysis) of pooled data from the 12 studies. For the purposes of the present review, we reevaluated the results of the different studies using the same statistical method, Students t-test, which is used to compare the means of two frequency distributions. Among all the studies, the most relevant indexes used to measure effectiveness of spa therapy were improved including VAS, Lequesne, and WOMAC Score. Conclusions The mud-pack therapy, balneotherapy, mud-bath therapy, and spa therapy have proved to be effective in the treatment and in the secondary prevention of knee osteoarthritis, by reducing pain, nonsteroidal anti-inflammatory drug consumption, and functional limitation and improving quality of life of affected patients.

AB0163 Increased eryptosis levels in erythrocytes treated with antibodies from aps patients: eryptosis as new actor in aps pathogenesis

Background Erythrocytes (RBCs) are highly sensitive cells constantly exposed to several stress stimuli including inflammatory mediators. Despite the absence of nuclei and the lack of crucial elements in the machinery of apoptosis, they have developed a rapid self-destruction process called eryptosis. During this process the externalization of phosphatidylserine (PS) activates the correct elimination of erythrocytes by phagocytes preventing inflammation and intravascular hemolysis. It has been recently demonstrated that PS-exposing erythrocytes are able to adhere to endothelial cells causing an impairment of circulation,1 suggesting a possible involvement of eryptosis in the increased risk of thrombotic episodes typical of antiphospholipid syndrome (APS). Objectives Enhanced eryptosis is known to contribute to several pathological conditions but the role of this process in APS has not been investigated yet. For this reason, we evaluated the effect of antibodies (Abs) purified from APS patients and healthy subjects positive for antiphospolipid antibodies without clinical manifestations (aPL carriers) on eryptosis activation. Moreover, spontaneous eryptosis levels in APS, aPL carriers, autoimmune haemolytic anaemia (AIHA) and healthy donors (HD) were analyzed. Methods 30 patients with primary APS (M/F 7/23, mean age 50.5±8.2 years), 17 aPL carriers (M/F 4/13, mean age 48.6±8.3 years) were recruited after written informed consent. Moreover 13 AIHA patients and 17 HD were also enrolled as positive and negative control group respectively. Ammonium sulfate precipitation is used to purify Abs from sera of APS and aPL carriers subjects. RBCs, isolated from whole blood by centrifugation, were incubated with APS and aPL carriers Abs at concentration of 20ug/mL and after 4 hours the percentage of annexin V-positive cells (PS-exposing cells) was analyzed by flow cytometry. The same technique was used to estimate spontaneous eryptosis levels in all cohorts studied. Results In vitro Abs from APS induced eryptosis in RBCs isolated from HD after 4 hours of culture compared to untreated and RBCs stimulated with Intravenous Immunoglobulins (IVIG), both p= 0.02. On the contrary, Abs from aPL carriers had no effect on the percentage of PS-exposing RBCs (figure 1). Ex vivo, APS patients showed higher levels of spontaneous eryptosis compared to HD (p=0.001). As expected, eryptosis was upregulated in AIHA patients compared to all populations studied (p<0.0001). Interestingly, the percentage of annexin V- positive RBCs was lower in aPL carriers respect to APS patients (p=0.001). No significant correlation between eryptosis and clinical parameters was found.Abstract AB0163 – Figure 1 Conclusions In this study we demonstrated a new aspect of APS pathogenesis based on the capacity of Abs isolated from APS patients, and not those from aPL carriers, to stimulate eryptosis suggesting a possible contribution of this process in APS clinical manifestations. Reference [1] Borst O, Abed M, Alesutan I, et al. Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX. Am J Physiol Cell Physiol 2012;302:C644–51. Disclosure of Interest None declared

THU0013 Towards the definition of a risk model profile of pericarditis in systemic lupus erythematosus: a genetic study

Background We have recently demonstrated that polymorphisms (SNPs) in TRAF3IP2 gene are associated with susceptibility for systemic lupus erythematosus (SLE) and can predispose to the development of pericarditis (1). Moreover, we found that other genes previously associated with susceptibility to SLE (MIR1279, STAT4, PTPN2) seem to be involved in the development of pericarditis (2). At this purpose, we built a genetic risk model for the development of pericarditis in SLE (3). Objectives We aimed to expand the knowledge on the genetic risk of pericarditis in SLE by studying the role of rs2205960 (TNFSF4) and rs2233945 (ATG16L1) SNPs, previously associated with SLE susceptibility, improving our genetic risk model. Methods We recruited SLE patients (diagnosed according to 1997 revised ACR criteria) and healthy subjects served as controls. Study protocol included complete physical examination and blood drawing. The clinical and laboratory data were collected in a standardized, computerized, electronically-filled form (3). Clinical and laboratory features were assessed with a dichotomous score (present=1; absent=0). SNPs genotyping was performed by allelic discrimination assay. A case/control association study and a genotype/phenotype correlation analysis were performed and a risk profile model for pericarditis in SLE was built. Results Three-hundred fifteen SLE patients [285 F (90.5%), 30 M (9.5%), mean age 43.11±11.28 years, mean age at onset 32.19±11.84 years] and 278 healthy controls were enrolled. Pericarditis was present in 56 (17.8%) SLE patients. Deviations from Hardy–Weinberg equilibrium for the studied SNPs were not observed. The variant alleles of the rs2205960 (TNFSF4, P=0.013, OR=2.14) and of the rs2233945 (ATG16L1 P=0.009, OR=2.32) were significantly associated with susceptibility to pericarditis. A risk profile model for pericarditis considering the risk alleles of TRAF3IP2, MIR1279, STAT4, PTPN2, TNFSF4 and ATG16L1 showed that patients with more than 5 risk alleles have a significantly higher risk to develop pericarditis (P<0.001, OR=8.01). Anti-Sm antibodies were the only laboratory parameter associated with the development of pericarditis. Thus, a multivariate analysis by binary regression analysis, considering as dependent variable the presence or absence of pericarditis and as independent variables all the studied SNPs associated with pericarditis, was performed. In a stepwise approach including anti-Sm, TRAF3IP2, MIR1279, STAT4, PTPN2, TNFSF4 and ATG16L1 SNPs, the model explains about 25% (R2 Cox &Snell) of the variability involved in the susceptibility to pericarditis. Conclusions We describe for the first time the contribution of TNFSF4 and ATG16L1 SNPs in pericarditis development in SLE patients. We improved our genetic risk profile model to better and earlier identify SLE patients more susceptible to develop this complication. References Perricone C, Ciccacci C, et al. Immunogenetics. 2013 Oct;65(10):703–9. Ciccacci C, Perricone C, et al. PLoS One. 2014 Nov 4;9(11):e111991. Ciccacci C, Perricone C, et al. Lupus. 2016. doi: 10.1177/0961203316679528. Disclosure of Interest None declared

THU0696 Anti-carbamylated proteins antibodies in sle patients with joint involvement: a possible new biomarker for erosive damage

Background The traditional concept of non-erosive arthritis in Systemic Lupus Erythematosus (SLE) patients changed during the last years, thanks to the use of more sensitive imaging techniques, such as ultrasonography (US). The predictive role of Rheumatoid Arthritis (RA)-specific autoantibodies for bone loss in SLE patients has been investigated. In particular, anti-citrullinated peptide antibodies (ACPA) have been identified in about 50% of SLE patients with x-Ray detected erosive arthritis. More recently, anti-carbamylated proteins (anti-CarP) antibodies have been demonstrated in seronegative RA patients, with a prevalence of about 16% and a significant association with erosive damage. Objectives In the present cross-sectional study, we aimed at assessing the association between anti-CarP antibodies and erosive damage in a large cohort of SLE patients with joint involvement. Methods For this purpose, we evaluated 152 SLE patients (1997 ACR criteria; M/F 11/141, mean±SD age 46.4±11.3 years, mean±SD disease duration 144.9±110.5 months) with joint involvement (arthralgia/arthritis). The clinical and laboratory data were collected in a standardized computerized electronically filled form. All patients underwent blood draws to detect Rheumatoid Factor (RF) and ACPA, by using commercial ELISA kits according to the manufacturers instructions, and anti-CarP antibodies by home-made ELISA (results were expressed in arbitrary units (AU)/ml and values above 340 IU/ml were considered positive). US was performed to assess the bone surfaces of metacarpophalangeal and proximal interphalangeal. At each joint, according with OMERACT definition, the presence of erosions was registered with a dichotomous value (0/1), allowing the possibility to obtain a total score, ranging from 0 to 20. Results The prevalence of anti-CarP antibodies was 28.3%, similar to RF (27.6%) and significantly higher to ACPA (11.2%, p=0.003). The mean±SD titer of anti-CarP antibodies was 890.5±794.9 IU/ml. Thirty-nine patients (25.6%) showed erosive arthritis: all the patients referred the occurrence of at least one episode of clinical synovitis. Erosive arthritis was significantly associated with anti-CarP antibodies (p=0.004) and ACPA (p=0.0008). Moreover, a significant correlation between anti-CarP antibody titer and US-erosive score was observed (r=0.2, p=0.01). Of note, anti-CarP antibodies were identified in 24.5% of double negative (ACPA-/RF-) patients, with erosive damage in 25% of them. Interestingly, anti-CarP antibodies resulted significantly associated with the presence of anti-dsDNA (P=0.01). Conclusions In the present study, for the first time, we identified a significant association between anti-CarP antibodies and erosive damage in SLE-related arthritis, in terms of frequency and severity. We found these antibodies in about 25% of ACPA-/RF- SLE patients, a prevalence higher than that described in seronegative RA patients. Taken together, our results suggest that anti-CarP antibodies could be considered as a candidate biomarker of severity in SLE patients with joint involvement. Disclosure of Interest None declared

FRI0705 Progression of chronic damage in systemic lupus erythematosus patients after 5-year follow-up: data from a large monocentric cohort

Background The prevention of chronic damage represents one of the most important target in the management of patients affected by Systemic Lupus Erythematosus (SLE). About 50% of patients develop chronic damage, especially in the early disease phase, due to disease activity, treatment adverse events and comorbidities. Longitudinal studies demonstrated a progressive increase of damage, evaluated by using SLICC Damage Index (SDI). Objectives Moving from these evidences, we aimed at evaluating the progression of chronic damage in a monocentric SLE cohort with at least 5-year follow-up and identifying the factors associated with damage progression. Methods We analyzed 658 SLE patients diagnosed according to the ACR 1997 revised criteria, referring to a dedicated outpatient clinic. For the present analysis, we evaluated only patients with a minimum follow-up of 5 years and at least one visit per year. Clinical and laboratory data were collected in a standardized, computerized and electronically-filled form, including demographics, past medical history, comorbidities and concomitant treatments. In all patients, chronic damage was determined by using SDI, with the evaluation of 12 organ systems. Disease activity was evaluated by SLEDAI-2K. Furthermore, we calculated the number of flares during the follow-up, defined as an increase in SLEDAI-2K score ≥4 from the previous visit, with a minimum interval of 2 months between visits. Results According with the inclusion criteria, we analyzed data deriving from 198 SLE patients (17 M/181 F, mean±SD age 46.8±12.1 years, mean±SD disease duration 131±99.7 months). At the first visit 60 patients (30.3%) showed SDI>0; in particular SDI=1 was identified in 65%, SDI=2 in 18.3%, SDI=3 in 10%, SDI=4 in 6.7%). At baseline, the presence of chronic damage was significantly associated with age (P<0.0001) and disease duration (P=0.001). After 5 years, we registered the progression of chronic damage in 69 patients (34.8%), with a significant increase of SDI values (baseline: median SDI 0.0, IQR 0–1; follow-up SDI: median 0 IQR 0–2, P=0.009). SDI progression resulted significantly more frequent in subjects with damage at baseline (55.0%) in comparison with free-damage patients (26.1%, P=0.0001, Fisher exact test). The progression of chronic damage was significantly associated with neuropsychiatric involvement (P=0.01), disease activity in terms of number of flares during the 5-year follow-up (P=0.001), concomitant anti-phospholipid syndrome (P=0.02) and arterial hypertension (P=0.001). Conclusions In our study, we observed a progression of chronic damage in almost 30% of SLE patients after 5 years. In particular, the presence of previous chronic damage seem to be a risk factor for new damage development. Moreover, the lack of disease control and the presence of comorbidities allows damage progression. Taken together, the results of this study underline the need of a better management of SLE patients in order to prevent damage accrual. Disclosure of Interest None declared

AB1125 Prediction of chronic damage in systemic lupus erythematosus by using machine-learning models

Background The increased survival in Systemic Lupus Erythematosus (SLE) patients implies the development of chronic damage, occurring in up to 50% of cases after a follow-up of 10 years. Its prevention is a major goal in the SLE management. During the last years, it has been suggested that Artificial Neural Networks (ANNs) could be a useful prediction tool in medical scenarios, by using patients data as inputs and the specific outcomes as outputs. The International Conference on Advanced Computing and Communication Systems in 2015 underlined the possible application of sophisticated data analysis tools, such as machine learning methods, in SLE patients, in the light of their potential application to diagnostic and prediction purposes. Objectives In the present study, we aimed at predicting chronic damage in a large monocentric SLE cohort by using neural networks. Methods For the present analysis, we used data from 413 SLE patients (1997 ACR criteria; M/F 30/383; mean age ± SD 46.3±11.9 years; mean disease duration ± SD 174.6±112.1 months, mean follow-up period ±SD 63.9±30.7 months). At each visit, the patients underwent a complete physical examination and clinical and laboratory data were collected in a standardized, computerized, and electronically filled form. All the patients were evaluated at least twice per year. Autoantibodies and complement serum levels were also registered. Chronic damage was assessed by the SLICC/ACR Damage Index (SDI). We applied Recurrent Neural Networks (RNNs) as a machine-learning model to predict the risk of chronic damage. The clinical data sequences registered for each patient during the follow-up were used for building and testing the RNNs. We used 27 clinical and laboratory items for the mathematical model. Results At the first visit, 35.8% of patients had an SDI≥1, with a mean±SD value of 1.7±1.1. For the RNN model, two groups of patients were analyzed: patients with SDI=0 at the baseline, developing damage during the follow-up (N=38), and patients without damage (SDI=0). In particular, in the first group, we used all the visits before the development of damage, and in the second group, we considered patients with at least 5 visits and a follow-up of 2 years. We created a mathematical model with an AUC value of 0.77, able to predict damage development. A threshold value of 0.35 (sensitivity 0.74, specificity 0.76) seems able to identify patients at risk to develop damage. Conclusions We applied RNNs to identify a prediction model for SLE chronic damage. By using longitudinal data, including laboratory and clinical items, we created a mathematical model able to identify patients at higher risk to develop chronic damage. Disclosure of Interest None declared

AB0135 Plasmatic and urinary endothelial microparticles are increased in patients with lupus nephritis

Background Systemic lupus erythematosus (SLE) is a chronic autoimmune disease presenting with a wide array of clinical manifestations and incompletely understood pathogenesis. SLE is characterized by alterations in both the innate and adaptive immune system ultimately leading to the loss of immunologic tolerance and occurrence of autoantibodies against nuclear material. Lupus nephritis is one of the most severe features of SLE determining an increase in morbidity e mortality rates. Renal biopsy still represent a fundamental diagnostic and prognostic tool for LN. Therefore, non-invasive surrogate biomarkers of active LN are urgently needed. Circulating, heterogeneous subcellular microparticles (MPs) are released from cells and platelets constitutively and upon cellular activation or apoptosis. Such MPs may reflect the state of their parental cells and tissues, and could serve as markers of pathology. Particularly in SLE, MPs are potential biomarkers and triggers of autoimmunity. Recent studies have demonstrated increased levels of plasmatic EMPs in patients with SLE active disease and their reduction after treatment. Objectives The aim of this study was to investigate plasmatic and urinary levels of endothelial microparticles in a cohort of SLE patients with and without renal involvement compared to healthy controls. Methods Consecutive SLE patients and sex- and age-matched HC were included in the study. MPs were isolated from plasma and urine and characterized by flow cytometry using AnxV (a probe that binds to the exposed phosphatidilserine - PS) and antibodies against surface markers endothelial cells (CD31+CD41-). Mann-Whitney and Spearman correlation tests were used. A p value <0.05 was considered statistically significant. Results Sixty SLE patients (55F:5M, age 41,7±9.6 Y disease duration 149±112 months) and 29 healthy controls were studied. Twenty-eight patients had renal involvement.The total number of plasmatic MPs was lower in SLE patients than HC (p=0.001). In contrast there was no significant difference in levels of EMPs between the two groups. When the patients were divided according to renal involvement, the patients with active lupus nephritis (A-LN) showed lower plasmatic level of EMPs in comparison to inactive LN (I-LN) (p=0.01), while the patients with I-LN had higher levels of EMPs than HC (p=0.002). There was no significant difference of total urinary level of MPs between SLE patients and HC. Urinary levels of EMPs were higher in SLE and in lupus nephritis patients than HC. Conclusions The results of the present study show increased urinary and plasmatic levels of EMPs in patients with lupus nephritis in remission. Circulating EMPs have been considered as a potential biomarker of endothelial activation and damage in several autoimmune disorders, and higher EMP levels have been detected in patients with vasculitis and associated with disease activation. According to our results, plasmatic EMPs levels are higher in inactive LN patients than in healthy donors. These results may suggest a potential role of EMP as a biomarker of lupus nephritis Disclosure of Interest None declared