Putative Association between Low Baseline Gene Expression in the Peripheral Blood and Clinical Remission in Rheumatoid Arthritis Patients Treated with Tofacitinib

We investigated the importance of the baseline expression of genes involved in energy generation, as prognostic biomarkers of the treatment response to tofacitinib in patients with rheumatoid arthritis (RA). Peripheral blood samples were obtained from 28 patients with RA who received 3 months of tofacitinib therapy from 26 healthy controls. Clinical response was evaluated based on the disease activity score, the erythrocyte sedimentation rate (DAS28-ESR), and the serum levels of ACPA, RF, CRP, and ESR. Clinical remission was assessed based on DAS28 score <2.6. Protein concentrations were measured using ELISA. Total RNA isolated from whole blood was used for gene expression analysis using quantitative RT-PCR. All patients were diagnosed with Steinbrocker’s radiographic stage II-III at baseline, and most showed erosive arthritis with ACPA and RF positivity. Tofacitinib treatment significantly decreased the disease activity. Upon study completion, seven patients showed remission. Before and after TOFA therapy, a significantly higher expression of succinate dehydrogenase and pyruvate kinase genes was observed in all the examined patients compared to healthy subjects. However, the pre-therapy expression of these genes and corresponding proteins was significantly (p ≤ 0.05) lower in patients who showed remission than in other patients with RA. Moreover, we observed that, during follow-up, patients who developed remission showed an increasing trend in the expression of the examined genes, whereas the others showed some decreases in gene expression, although this was not statistically significant. We concluded that, compared with RA patients maintaining persistent moderate or high disease activity, those with clinical remission following tofacitinib treatment showed a significantly lower baseline expression of genes involved in energy generation.

Assessment of the frequency of tarsal tunnel syndrome in rheumatoid arthritis

Objectives: In this study, we aimed to determine the frequency of tarsal tunnel syndrome (TTS) in rheumatoid arthritis (RA) patients. Patients and methods: Thirty RA patients (1 male, 29 females; mean age: 41.9±10.1 years; range, 26 to 65 years) who met the American College Rheumatology (ACR) classification criteria and 20 healthy volunteers (1 male, 19 females; mean age: 39.3±10.8 years; range, 26 to 60 years) without any complaints between August 2006 and October 2007 were included in the study. Demographic characteristics of the study group were assessed and neurological examinations were performed. The Tinel’s sign was checked to provoke the TTS symptoms. Disease severity was measured using Visual Analog Scale (VAS), Disease Activity Score-28 (DAS28), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). The health-related quality of life and disability status were determined using the Health Assessment Questionnaire (HAQ), Short Form 36 (SF-36), Foot Function Index (FFI), and VAS (0-100 mm). The positional relationship of the foot pain was questioned with VAS. The 100-m walking distance of the patient and control groups were calculated. Results: Bilateral TTS was detected in 10 of the patients (33.3%) with rheumatoid arthritis. No relationship with the TTS disease duration, seropositivity, rheumatoid nodule, joint deformities, corticosteroid use, and DAS28 score were found. In correlation with TTS, foot and ankle joint were the first involved joints at the beginning of RA disease (p<0.005). The Tinel’s sign was found to be 45% positive in patients with TTS. The 100-m walking time was significantly longer in RA patients compared to the control group (p<0.0001). Conclusion: Tarsal tunnel syndrome is commonly seen in RA and its incidence increases in patients with primary foot involvement. Therefore, caution should be taken against the entrapment neuropathies in these patients, and they should be supported by electrophysiological practices, when the diagnosis is necessary.

Proteomic Approaches to Defining Remission and the Risk of Relapse in Rheumatoid Arthritis

ObjectivesPatients with Rheumatoid Arthritis (RA) are increasingly achieving stable disease remission, yet the mechanisms that govern ongoing clinical disease and subsequent risk of future flare are not well understood. We sought to identify serum proteomic alterations that dictate clinically important features of stable RA, and couple broad-based proteomics with machine learning to predict future flare.MethodsWe studied baseline serum samples from a cohort of stable RA patients (RETRO, n = 130) in clinical remission (DAS28&lt;2.6) and quantified 1307 serum proteins using the SOMAscan platform. Unsupervised hierarchical clustering and supervised classification were applied to identify proteomic-driven clusters and model biomarkers that were associated with future disease flare after 12 months of follow-up and RA medication withdrawal. Network analysis was used to define pathways that were enriched in proteomic datasets.ResultsWe defined 4 proteomic clusters, with one cluster (Cluster 4) displaying a lower mean DAS28 score (p = 0.03), with DAS28 associating with humoral immune responses and complement activation. Clustering did not clearly predict future risk of flare, however an XGboost machine learning algorithm classified patients who relapsed with an AUC (area under the receiver operating characteristic curve) of 0.80 using only baseline serum proteomics.ConclusionsThe serum proteome provides a rich dataset to understand stable RA and its clinical heterogeneity. Combining proteomics and machine learning may enable prediction of future RA disease flare in patients with RA who aim to withdrawal therapy.

Platelet indices parameters in the new disease activity score of rheumatoid arthritis with ankle involvement: A comparative analytic study

Background: Platelet indices (PIs) are platelet parameters that are correlated with platelet activity. Despite being widely available, inexpensive, and feasible; their use in clinical settings is limited. Recently, we developed a new score (EgyDAS), which relies on PIs and assesses disease activity in rheumatoid arthritis (RA). Objectives: This study explored the practicability and validity of EgyDAS in RA with ankle involvement, considering that ankle is neglected in the commonly used DAS28 score. Methods: This comparative case-control study included 2-groups of RA patients, group1 (control): without and group 2: with ankle involvement. Results: Ankle involvement in RA showed no gender or age differences, however, it was associated with higher platelet count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), platelet distribution width (PDW), visual analogue scale (VAS), tender joint count (TJC), and lower hemoglobin (Hb) and mean platelet volume (MPV). DAS28 categorized a higher proportion of patients to have high disease activity compared with EgyDAS; moreover, it did not detect those in remission in group 2 patients. Highly significant differences in the 2-scores were observed between the two groups. Further analyses revealed superiority of EgyDAS in assessing disease activity in group 2 patients. Finally, both scores were found correlated together in the study groups. Conclusions: Over or underestimation of RA disease activity could occur when using DAS28. PIs were found correlated with ankle involvement in RA. PIs and EgyDAS are the best tools to assess disease activity in RA patients with ankle involvement. However, the study recommended the use of both scores together.

Impact of social support on severity of depressive symptoms by remission status in patients with rheumatoid arthritis

ABSTRACT Objectives We aimed to examine the psychosocial characteristics of patients with rheumatoid arthritis (RA) by remission status and determine the impacts of social support on severity of depressive symptoms. Methods We enrolled RA patients aged 40–79 years who visited university hospitals’ outpatient clinics. Severity of depressive symptoms (Beck Depression Inventory-II), physical disability (Health Assessment Questionnaire), and support were evaluated. Furthermore, RA disease activity was evaluated by 28-point Disease Activity Score (DAS28) calculation. The independent impacts of instrumental and emotional social support on depressive symptoms by remission status defined as DAS28 score &lt; 2.6 were estimated by multivariable regression analysis. Results This study included 360 RA patients. In the remission group, emotional support showed a statistically significant negative impact on depressive symptoms, whereas instrumental support had an extremely limited contribution to severity of depressive symptoms. In the non-remission group, instrumental support showed a negative tendency of impact on severity of depressive symptoms, whereas emotional support had a wide range of influence. Conclusions Favourable association between emotional support and depressive symptoms is confirmed only among RA patients in remission status. The influence of emotional support in non-remission patients and that of instrumental support regardless of remission status are inconclusive.

POS0460 ASSOCIATION OF ANTI-CITRULLINATED PROTEIN ANTIBODIES OF IgA SUBCLASSES WITH SUSTAINED REMISSION AND FLARE IN RHEUMATOID ARTHRITIS

Background:Understanding key mechanisms of flare development and sustained remission is one of the acute goals in modern rheumatology. Anti-citrullinated protein antibodies (ACPA) are the most abundant and specific autoantibodies in rheumatoid arthritis (RA) patients. However, the impact of ACPA of IgA isotype is poorly defined. IgA ACPA were previously shown to have a higher percentage of IgA2 in comparison to total IgA; and a correlation between IgA2% ACPA with the DAS28 score was observed in a previous study [1]. Of note, IgA1 and IgA2 were shown to exhibit different effector functions, with IgA2 being pro-inflammatory, which might be the background for its role in RA [1].Objectives:We aimed to investigate, whether IgA ACPA could be used as a predictive factor for flare development in RA; and to look further into the changes in IgA ACPA levels in patients remaining in stable remission versus patients developing flare.Methods:We analysed serum of 111 patients from a multicentre randomized controlled trial ‘RETRO’. The study observational period was 12 months. Patients in the trial had to be in stable remission (DAS28-ESR<2.6) for a minimum of 6 months and were randomized into 3 different treatment arms: continuation of treatment, tapering by 50% or a gradual tapering until discontinuation [2]. IgA ACPA concentrations were measured with an enzyme-linked immunosorbent assay on CCP2-pre-coated plates.Results:60% of patients had IgG-ACPA. IgA ACPA levels were higher among the IgG-ACPA-positive patients (median 4.7 versus 2.24 µg/ml, p<0.0001). Baseline IgA1 and 2 ACPA levels were not different between patients who had a flare later on in the study period and those remaining in remission, showing no predictive value for flare development. However, the percentage of IgA2 in ACPA was correlating with the first registered DAS28 after flare (r=0.36, p=0.046). After the 12 months study period, IgA2 ACPA as well as IgA2% ACPA decreased significantly in patients who remained in stable remission by 17.5% (median, p<0.0001) and 13.6% (p=0.0006), respectively. By contrast, there was no significant change in IgA2 ACPA levels over time in patients who developed a flare. IgA1 ACPA levels remained stable over time. Disease management strategies did not seem to influence IgA ACPA levels in a specific way, as baseline levels were similar between patients on biological and conventional DMARDs and changes in levels after 12 months did not depend on the assignment to either of the study arms.Conclusion:Neither IgA1 nor IgA2 ACPA levels were predictive of flare development or associated with treatment strategies (though rituximab, JAK-inhibitors and abatacept were not amongst treatment options). However, in patients remaining in sustained remission after 1 year a decrease in IgA2 and IgA2% ACPA was observed and IgA2% ACPA was associated with DAS28 score registered after flare. This could be an indication towards ACPA of IgA2 isotype contributing to the severity of flare, alongside other factors, and its reduction being associated with a prolonged state of remission.References:[1]Steffen U, Koeleman CA, Sokolova MV, et al. IgA subclasses have different effector functions associated with distinct glycosylation profiles. Nat Commun 11, 120 (2020).[2]Haschka J, Englbrecht M, Hueber AJ, et al. Relapse rates in patients with rheumatoid arthritis in stable remission tapering or stopping antirheumatic therapy: interim results from the prospective randomised controlled RETRO study. Ann Rheum Dis. 75:45-51 (2016).Disclosure of Interests:None declared

OP0185 RADIOGRAPHIC PROGRESSION DESPITE PERSISTENT LDA OR REMISSION IS INFLUENCED BY CURRENT SMOKING RATHER THAN THE RESPECTIVE DAS 28 LEVEL, RESULTS OF THE SWISS RHEUMATOID ARTHRITIS REGISTER (SCQM)

Background:The therapeutic aim for rheumatoid arthritis (RA) is to control disease activity and prevent radiographic progression. Various clinical scores are utilized to describe disease activity in RA patients. The DAS28 score can define states of low disease activity (LDA) and remission. Despite achieving LDA or remission, radiographic progression may nevertheless occur. However, the rates and frequency of this occurrence have not been analyzed in detail.Objectives:To describe the frequency and rate of radiographic progression in patients with persistent LDA or remission.Methods:Analysis of RA patients from the SCQM cohort. Persistent LDA or remission were defined as DAS 28 ≤3.2 or <2.6 respectively, at two subsequent follow up time points in the database. We included patients with at least two sets of radiographs within these intervals of LDA and/or remission. Radiographic progression was measured with the Ratingen-score (range 0-190), which describes joint erosions numerically. Repair was defined as an improvement in the Ratingen score >5 points/year and progression as >2 or >5 points change in the Ratingen score within one year.Results:Among 10’141 RA patients, 4’342 episodes of remission occurred in 3’927 patients with 1’776 sets of X rays available within these episodes. Similarly, 8’136 episodes of LDA in 6’765 patients and 2’358 sets of X rays were present within these intervals. For patients in LDA or remission, rates of repair were 5.5% and 4.8%, respectively, while for radiographic progression >5 points in the Ratingen score/year were 10.3% in both groups and for >2 points change of Ratingen score/year were 27.7 and 25.4%, respectively).No differences for demographic factors or measures of disease activity, rheumatoid factor or ACPA were found comparing patients with radiographic progression or non-progression despite LDA or remission at the beginning of the episode of LDA and/or remission.Interestingly, 42.9% of patients in LDA with progression of >5 points in the Ratingen score/year were current smokers vs 29.4% among the non-progressors (X2 = 6.55, p = 0.01). This significant difference vanished when the cut-off for radiographic progression was set at >2 points yearly change in Ratingen score or in patients in remission.Conclusion:Radiographic progression despite LDA or remission are more frequent than expected. No differences in radiographic progression were found comparing LDA and remission suggesting that the goal of LDA is appropriate. Smoking seems to be an independent risk factor for radiographic progression despite LDA. Why the effect of smoking could was not demonstrated in patients in remission, remains unclear.Disclosure of Interests:Lena Brandt: None declared, Hendrik Schulze-Koops: None declared, Thomas Hügle Consultant of: GSK, Abbvie, Pfizer, Jansen, Novartis, Eli Lilly., Michael J. Nissen Consultant of: Abbvie, Celgene, Eli-Lilly, Janssen, Novartis and Pfizer, Hasler paul Consultant of: Abbvie, Lilly, Rudiger Muller Consultant of: AbbVie, Novartis, Grant/research support from: Gebro

POS0389 NEUTROPHIL AND MONOCYTE EXTRACELLULAR TRAPS IN RHEUMATOID ARTHRITIS: POTENTIAL SOURCE OF DIAGNOSTIC BIOMARKERS

Background:Up-to date quite high rheumatoid arthritis (RA) prevalence has also a trend towards steady increase for several decades. Earliest possible diagnosis and personalized treatment of RA are commonly believed to be principal way to prevent or diminish joint destruction. Emerging biomarkers retrieval has therefore critical importance for improvement of RA treatment outcomes, especially for its biological treatment. One of upcoming sources of new biomarkers is newly discovered phenomenon of neutrophil extracellular traps (NET) and monocyte extracellular traps (MET) formation, specifically in RA.Objectives:Evaluation of peripheral blood neutrophils and monocytes ability to generate NET and MET spontaneously and after induction in vitro in RA.Methods:The research was carried out in agreement with the WMA Declaration of Helsinki principles. 30 patients with verified RA according to the ACR/EULAR 2010 criteria were included in the study. 30 healthy volunteers were enrolled as a reference group. RA disease activity was assessed using DAS28 score did not exceeded 2.6 in every patient at the inclusion timepoint. Neutrophils and monocytes were isolated with one-step centrifugation procedure using three-layer ficoll-amidotrizoate density gradient with density of upper, intermediate and lower layers 1068 kg/m3, 1080 kg/m3, and 1090 kg/m3, respectively. The cell types in the resulting fractions were identified histochemically, and the extent of cell activation was assessed using common nitro-blue tetrazolium test. Generation of NETs was stimulated by phorbol-12-myristate-13-acetate (PMA). Generation of METs was stimulated using bacterial LPS. The shape and size of NETs and ETs were assessed using fluorescence microscopy with SYBR green.Results:Mean contamination fraction of neutrophils and monocytes in the reference group did not exceeded 3% and 2%, respectively. Mean purity of neutrophil fraction in RA group was 93.1±6.1%, and cell viability in every sample was above 95.4±5.1%. Mean purity of monocyte fraction in RA group was 98.4±6.4%, cell viability in every sample was above 93.4±4.8%. Spontaneous NET and MET formation was observed in neutrophils and monocytes isolated from both RA patients and, significantly less, in healthy controls. Neutrophils from ACPA-positive RA patients were found to reveal increased spontaneous and induced NETs formation compared to ACPA- negative RA patients. Monocytes did not demonstrate any difference between these subgroups.Conclusion:NETs could probably be considered as a candidate source of citrulline autoantigen participating in autoantibody production, whereas METs may play less important role in this phenomenon. NETs and ETs can be considered as potential diagnostic biomarkers of RA. Further studies of NETosis and ETosis in RA patients can promote emerging researches for targeted therapy of RA.Disclosure of Interests:None declared

Evaluation of biomarkers related to zinc nutritional status, antioxidant activity and oxidative stress in rheumatoid arthritis patients

Background: Oxidative stress (OS) is an important process related to the pathophysiology of rheumatoid arthritis and can be increased by the low intake of antioxidants. Zinc (Zn) is an important antioxidant trace-element for human health and the assessment of the nutritional status of this micronutrient in these patients is of relevance. Aim: This study aimed to evaluate Zn nutritional status in rheumatoid arthritis patients and its relation to OS. Methods: A case–control study was carried out with 51 patients diagnosed with rheumatoid arthritis (RA group) recruited in Hospital São Paulo (São Paulo, Brazil) and 55 healthy women (CO group) from the campus of the University of São Paulo. Blood and 24-hour urine collection were used for biochemical parameters related to Zn status and OS. The assessment of dietary Zn was performed by three 24-hour dietary recalls. Results: The RA group presented significative low Zn intake ( p < 0.001) and plasma concentration ( p = 0.040) of this mineral compared to the CO group. However, both groups were Zn deficient and the disease activity (DAS28 score) for RA patients did not influence Zn biomarkers. In addition, the antioxidant enzymes (superoxide dismutase and glutathione peroxidase) activity and the urinary 8-isoprostanes were reduced in RA patients. Conclusion: The evaluation of dietary intake and biochemical biomarkers indicates that rheumatoid arthritis patients are zinc deficient and have increased OS.

Interactions between rheumatoid arthritis antibodies are associated with the response to anti-tumor necrosis factor therapy

Background Blocking of the Tumor Necrosis Factor (TNF) activity is a successful therapeutic approach for 50–60% of rheumatoid arthritis (RA) patients. However, there are yet no biomarkers to stratify patients for anti-TNF therapy. Rheumatoid factor (RF) and anti-cyclic-citrullinated antibodies (anti-CCP) have been evaluated as biomarkers of response but the results have shown limited consistency. Anti-carbamylated protein (anti-CarP) and anti-peptidylarginine deiminase type 4 (anti-PAD4) antibodies have been much less studied. Despite being linked to common immune processes, the interaction between these markers has not been evaluated yet. Our aim was to analyze the interaction between these four antibodies in relation to the response to anti-TNF therapy. Methods For this objective, a prospective cohort of n = 80 RA patients starting anti-TNF therapy was recruited. Serum determinations at baseline were performed for RF, anti-CCP, anti-CarP and anti-PAD4 antibodies using enzyme-linked immunosorbent assays (ELISA). The clinical response to anti-TNF therapy was determined at week 12 using the change in DAS28 score. Association was performed using multivariate linear regression adjusting for baseline DAS28, sex and age. Results The interaction between pairs of antibodies was tested by the addition of an interaction term. We found two highly significant antibody interactions associated with treatment response: anti-CarP with anti-PAD4 (p = 0.0062), and anti-CCP with RF (p = 0.00068). The latter antibody interaction was replicated in an independent retrospective cohort of RA patients (n = 199, p = 0.04). Conclusions The results of this study suggest that antibody interaction effects are important factors in the response to anti-TNF therapy in RA.