Josep M. Llobet

Functional consequences of platelet binding to T lymphocytes in inflammation

Expression of the scavenger receptor CD36 on lymphocytes is intriguing. We observed that a minor subpopulation of lymphocytes expressed CD36 on the cell surface. We investigated the source of CD36 and also the proliferation and cytokine production of these CD36+ CD4+ lymphocytes. Flow cytometry analysis and immunofluorescence microscopy showed that CD36+ platelets were responsible for CD36 detection on lymphocytes. CD36 was then used as a tool to characterize lymphocytes with bound platelets. Activation‐induced proliferation was lower in CD4+ lymphocytes with bound platelets than lymphocytes without bound platelets. IL‐17 and IFN‐γ production was also reduced in lymphocytes with bound platelets. We then studied the presence of CD36+ CD4+ lymphocytes in RA patients. We observed that the percentage of CD4+ lymphocytes with bound platelets was higher on RA patients than in healthy donors. RA patients with higher titers of anti‐CCP, RF levels, and cardiovascular risk index presented a lower percentage of CD4+ lymphocytes with bound platelets. These patients also had higher IL‐17 and IFN‐γ production. These results suggest that platelet‐binding modifies lymphocyte function. This binding could be a regulatory mechanism in RA that confers a less severe phenotype.

Predictive factors for induction of remission in patients with active rheumatoid arthritis treated with tocilizumab in clinical practice

OBJECTIVE To identify predictors of early response to tocilizumab (TCZ) in patients with active rheumatoid arthritis (RA) seen in daily routine clinical practice. METHODS A multicenter ambispective observational study of 126 RA patients treated with TCZ as a first- or second-line biological therapy. The variables associated to achieve the therapeutic goal (remission defined as a DAS28-ESR < 2.6) at 3 and 6 months were identified using regression analysis. RESULTS TCZ was administered as the first biologic in 26% of patients. Overall, 34% of patients received TCZ as monotherapy. EULAR response and remission were obtained in 82% and 31% of patients at 3 months and in 86% and 40% at 6 months. In the multivariate analysis, the predictive factors increasing the likelihood of clinical remission at 3 months were baseline ESR > 30 mm/h (OR = 19.07, 95% CI: 2.720-133.716), baseline CRP > 10 mg/L (OR = 4.95; 95% CI: 1.464-13.826), and the presence of extra-articular manifestations of the disease (OR = 15.45, 95% CI: 2.334-102.319). The factors that decreased it were higher concentrations of hemoglobin (OR = 0.53, 95% CI: 0.319-0.910), higher baseline DAS28-ESR (OR = 0.30, 95% CI: 0.145-0.635) and the number of previous DMARDs (OR = 0.41, 95% CI: 0.221-0.779), and biological therapies used (OR = 0.33, 95% CI: 0.155-0.734). The only factor that remained statistically significant at 6 months was higher baseline DAS28-ESR (OR = 0.55, 95% CI: 0.347-0.877). No relationship was found with the neutrophil count or with the RF or ACPA positivity. CONCLUSION In routine clinical practice, strong acute phase response, the presence of extra-articular manifestations, and the number of previous DMARDs and biological therapies used may help to identify patients who will have a rapid response to TCZ. However, it is likely that no parameter will predict response if taken separately.

Comparative effectiveness of tocilizumab with either methotrexate or leflunomide in the treatment of rheumatoid arthritis.

Objective In agreement with EULAR recommendations, a DMARD in combination with a biotherapy is the reference treatment because of the superior long-term clinical and radiographic outcomes. Methotrexate (MTX) is the cornerstone of combination therapy but is in some cases contra-indicated or poorly tolerated. This observational study aimed to compare the effectiveness and safety of TCZ in combination with either MTX or leflunomide (LEF) in the treatment of patients with active rheumatoid arthritis (RA) and an inadequate response to one or more DMARDs and/or biological agents in a real-world setting. Methods We performed an ambispective review of 91 patients with active RA who were routinely treated with TCZ plus MTX or LEF. A comparative study between the two combinations of treatment was performed at 6 months of follow-up considering 3 outcomes: improvement of RA disease activity, evolution of functional disability, and tolerability and side effect profile. Results Of the 91 patients, 62 received TCZ with MTX and 29 received TCZ with LEF. Eighty-one patients were followed for 6 months, and the remaining 10 patients discontinued treatment due to serious adverse events. At baseline, there were no significant differences between the groups in terms of the main clinical and laboratory data or in the number of previous DMARDs and biological agents used. At 6 months, there were no significant differences between the combinations in terms of disease activity and functional disability. Serious adverse events occurred in 11% and 10% of the patients treated in combination with MTX and LEF, respectively. Conclusion Our preliminary data support the argument that LEF is an effective and safe (equivalent) alternative to MTX for combination treatment with TCZ.

Binding of Platelets to Lymphocytes: A Potential Anti-Inflammatory Therapy in Rheumatoid Arthritis

Soluble factors released from platelets can modulate the immune response of leukocytes. We and others have recently found that T lymphocytes with bound platelets have reduced proliferation and IFN-γ and IL-17 production. Thus, we speculate that if we induce the binding of platelets to lymphocytes, we will be able to regulate the inflammatory response. When we cocultured platelets with lymphocytes at different ratios, we were able to increase the percentage of lymphocytes with bound platelets. The coculture of platelets with lymphocytes in the presence of stimulation decreased the production of IFN-γ and TNF-α, T cell proliferation, and the expression of CD25, PD-L1, and SLAM. However, this coculture increased CD39 expression. All of these effects were dependent on the dose of platelets and operated indistinctly with platelets from different healthy donors. When platelets were cocultured in the same compartment with lymphocytes, we observed less IFN-γ and TNF-α production and T lymphocyte proliferation than in cultures with platelets separated from lymphocytes by a 0.4-μm pore size filter. The binding of platelets to lymphocytes was blocked with anti–P-selectin Abs, and when this occurred we observed higher IFN-γ and TNF-α production than in nonblocked conditions. The cocultures of platelets with synovial fluid cells from rheumatoid arthritis patients reduced inflammatory cytokine production and increased IL-10 production. These results suggest that platelet binding to lymphocytes effectively regulates T lymphocyte function. This mechanism could be easily applied to reduce inflammatory responses.

Adalimumab regulates intracellular TNFα production in patients with rheumatoid arthritis.

IntroductionAdalimumab is a fully human anti–tumor necrosis factor α (anti-TNFα) monoclonal antibody that specifically blocks the interaction of TNFα with its receptors. It binds both soluble and transmembrane TNFα. We hypothesized that blocking these TNFα signals regulates the altered TNFα production in rheumatoid arthritis (RA) patients.MethodsWe compared, by flow cytometry, Toll-like receptor induction levels of membrane and intracellular TNFα in monocytes (iTNFα + CD14+ cells) from 12 patients before and after adalimumab treatment with those from 5 healthy donors.ResultsBefore starting the treatment, the percentage of iTNFα+ CD14+ cells in the RA patients was significantly lower than that in healthy donors (mean ± SEM = 33.16 ± 4.82% vs 66.51 ± 2.4%, P < 0.001). When we added in vitro TNFα to healthy donor culture cells, levels of iTNFα+ CD14+ cells decreased, suggesting that the TNFα signal was responsible for the iTNFα+ CD14+ cell downregulation observed in the RA patients. After 2, 6 and 12 adalimumab injections, we observed significant blocking of membrane and soluble TNFα and a progressive increase in iTNFα+ CD14+ cells in ten patients with a good to moderate response as defined by the European League Against Rheumatism (EULAR) criteria. Levels of iTNFα+ CD14+ cells after 12 injections in these 10 patients were comparable to levels in healthy donors. In two patients, iTNFα+ CD14+ cell upregulation was not observed, and their EULAR-defined responses had not improved. The first patient developed antiadalimumab antibodies, explaining why adalimumab was not able to block membrane and soluble TNFα. In the second patient, adalimumab was discontinued because of adverse effects, which led to a decrease in iTNFα+ CD14+ cells to levels measured before treatment.ConclusionsOur findings suggest that adalimumab treatment in RA patients can return iTNFα levels to those of healthy donors. This effect was not observed in the presence of neutralizing antiadalimumab antibodies.

SAT0606 Impact of Musculoskeletal Ultrasound in Treatment Decision in Routine Daily Care of Rheumatoid Arthritis (Impulsar Study): Table 1

Background Remission or low disease activity is the therapeutic target in the ACR-EULAR recommendation for the management of rheumatoid arthritis (RA). In multiple studies, musculoskeletal ultrasound (US) has shown to be more reliable and sensitive than physical examination in both the diagnosis and the assessment of RA activity. However, the real impact of this technique on routine daily care of RA and treatment decisions has not been studied. Objectives To assess the proportion of therapeutic decisions that are modified by the results of the US examination in patients with RA. To determine which group of patients would get more benefit from a musculoskeletal US to optimize treatment. Methods Seventy eight consecutive patients diagnosed with RA by ACR 1987 criteria and visited between July and November 2014, were included. All patients were initially visited by their usual rheumatologist and a therapeutic decision was made according to physical examination and clinical and laboratory findings. Subsequently, a musculoskeletal US was performed by an expert sonographer of our center and the usual rheumatologist was asked to reassess his therapeutic decision in light of the US findings. We classified the change in the therapeutic attitude as: negative (maintenance of therapeutic attitude) and positive (increase or reduction in treatment, compared to the initial decision). Demographic, clinical and laboratory data were collected from the clinical history and activity scores were calculated. Results Clinical, demographic, laboratory, treatment and activity score data are shown in table below. In 29 patients [37.2% (95% CI 26.5 to 48.9)] the findings in the US examination conditioned a change in the therapeutic decision of the usual rheumatologist. In 18 patients (62.07%) the change was towards an intensification of treatment, while in 11 patients (37.93%) a decrease was possible. Change of treatment was more frequent in patients with intermediate disease activity (mild and moderate activity) than in those with extreme activities (remission and high activity), 41.4% vs 25%, although this difference was not statistically significant. A higher frequency of change was found in men (53.8% vs 33.8%) and erosive RA forms (43.6% vs 21.7%), but the results did not reach statistical significance.Table 1 Total (n=78) Age (years ± SD) 63,27 (±12,94) Disease duration (years ± SD) 15,49 (±10,66) Female (%) 65 (83,33%) Erosive form (%) 55 (70,51%) RF positive (%) 49 (62,82%) Anti-CCP positive (%) 59 (75,64%) DMARDs  Methotrexate 33 (42,31%)  Leflunomide 5 (6,41%)  Salazopyrin 7 (8,97%)  Hydroxychloroquine 2 (2,56%) Biologic therapies  Anti-TNF 14 (17,95%)  Others 11 (14,10%) DAS28-ESR (mean ± SD) 3,19 (±1,09) DAS28 activity  high 1 (1,28%)  moderate 36 (46,15%)  mild 22 (28,20%)  remission 19 (24,36%) Conclusions Musculoskeletal ultrasound, when added to routine rheumatologic investigation, is an important tool for treatment decisions in the routine daily care of rheumatoid arthritis. Patients with intermediate activities of the disease might get more benefit from the use of an US examination. Disclosure of Interest None declared

AB0692 Radiographic Damage in A Group of Patients with Axial Spondyloarthritis

Background Recent studies in the field of axial spondyloarthropathies (SpA-ax) have shown a group of patients with chronic lower back pain without evidence of radiographic damage (non-radiographic axial spondyloarthritis). Currently, data is lacking that would confirm whether it is an early stage of the disease or a group of SpA-ax that is different, in which time of progression and radiographic damage show no relationship. Objectives Describe our cohort of patients diagnosed with radiographic axial spondyloarthritis. Assess whether those with both sacroiliac and spinal radiographic involvement have a longer duration of the disease and the relationship between this most-affected group and the different items that constitute the ASAS criteria for SpA-ax. Methods All patients diagnosed with SpA-ax by ASAS or NY criteria, who were seen in a monographic consultation of spondyloarthritis of our center between January 2012 and December 2013, were reviewed. Only those with sacroiliitis of grade II or higher were included; patients who did not have spinal radiographs were excluded. The following clinical and laboratory variables were collected from clinical history: age, sex, duration of disease, inflammatory back pain, arthritis, enthesitis, uveitis, dactylitis, psoriasis, inflammatory bowel disease, positive response to NSAIDs, family history of SpA, HLA-B27 and elevated CRP. The presence of 3 or more syndesmophytes in the dorsal and/or lumbar region was considered as spinal involvement. For statistical analysis, students t-test, chi-square test or Fishers exact test were performed where applicable. Results Of the 72 patients diagnosed with SpA-ax, 58 met the inclusion and exclusion criteria. The mean age of the study patients was 53.69 years and 69% were men. The mean disease duration was 19.48 years and 36 patients had spinal involvement. Only the age of patients, the sex and the duration of the disease showed significant results regarding the structural damage (see table). Table 1 Total Spinal X-ray − Spinal X-ray + p (n=58) (n=22) (n=36) Age, years 53.69±13.41 47.36±13.05 57.56±13.45 *p=0.006 Disease duration, years 19.48±12.06 14.86±9.57 22.31±12.67 *p=0.021 % male 40 (69) 11 (50) 29 (80.5) *p=0.02 Inflammatory back pain (%) 48 (82.75) 20 (90.9) 28 (77.8) p=0.29 Arthritis (%) 31 (53.45) 11 (50) 20 (55.5) p=0.79 Enthesitis (%) 25 (43.1) 9 (40.9) 16 (44.4) p=1.00 Uveitis (%) 12 (20.7) 6 (27.3) 6 (16.7) p=0.50 Dactylitis (%) 6 (10.3) 3 (13.6) 3 (8.3) p=0.70 Psoriasis (%) 20 (34.5) 10 (45.4) 10 (27.8) p=0.25 Inflammatory bowel disease (%) 2 (3.45) 1 (4.5) 1 (2.8) p=1.00 Good response to NSAIDs (%) 47 (81) 20 (90.9) 27 (75) p=0.29 Family history of SpA (%) 13 (22.4) 4 (18.2) 9 (25) p=0.75 HLA-B27 positive (%) 33 (56.9) 12 (54.5) 21 (58.3) p=0.78 CRP >5mg/l (%) 45 (77.6) 15 (68.2) 30 (83.3) p=0.21 * p<0.05. Conclusions In our cohort, patients with more severe radiographic involvement were those of a longer disease duration and an older age, with a predominance of men. No significant differences for the remaining assessed variables were found, including signs of inflammation and HLA-B27, which were similar in both groups of patients. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4483

FRI0224 Predictive factors of response to tocilizumab in patients with active rheumatoid arthritis

Objectives To evaluatethe efficacy and identify predictors of response to tocilizumab (TCZ) in patients with rheumatoid arthritis. Methods A multi-center ambispective observational study in 126 patients with active rheumatoid arthritis treated with TCZ. The variables associated to achieve the therapeutic goal (remission of the disease defined as a DAS28 <2.6) at 3 and 6 months, were identified by a logistic regression model by using the backward approach. SAS 9.3 was used for the management and statistical analysis. Results Patients’ demographic and clinical characteristics are shown in table 1, as well as response rates at 3 and 6 months of treatment. Of the 126 patients included, TCZ was administered as first biologic in 33 cases (26%), while in the remaining 93 patients was used after inadequate response (inefficacy or intolerance) to one or more biological therapies (the average of previous biological drugs tested per patient was 2.18 ±1.1, range 1-6). Only 43 (35%) patients received TCZ as monotherapy. Of all the variables analyzed by the multivariate analysis, the predictive factors that increase the likelihood of response DAS28-ESR 2.6 to 3 months were ESR> 30 mm / h (OR=19.07, 95% CI: 2.7, 133.7) and the presence of extra-articular manifestations of the disease (OR = 15.5, 95% CI 2.3, 102.3). Factors that decreased it were hemoglobin (OR = 0.53, 95% CI: 0.32, 0.91), the baseline DAS28-ESR (OR = 0.30, 95% CI: 0.15, 0.64) and the number of previous DMARDs used (OR = 0.42, 95% CI: 0.22, 0.78). The only significant factor that reduced the possibility of DAS28-ESR response <2.6 at 6 months was the baseline DAS28-ESR (OR = 0.55, 95% CI: 0.35, 0.88). No relationship was found in the number of previous biological therapies, nor in the decrease of the neutrophils count nor in the positivity RF/anti-PCC. Conclusions Some clinical and laboratory variables can help in the selection of the best candidates for treatment with tocilizumab. Disclosure of Interest: None Declared

THU0184 TNF Production is Regulated by Adalimumab Treatment in Rheumatoid Arthritis Patients

Background Adalimumab is a neutralizing anti-TNFα monoclonal antibody that blocks specifically TNFα action. It has been shown to be effective in clinical and quality of life improvement in rheumatoid arthritis (RA) patients. However, the influence of adalimumab on TNFα production and secretion remains unknown. Methods Twelve rheumatoid arthritis patients with no previous biological therapy were treated with adalimumab according to clinical practice. We collected demographic (age and gender) clinical (disease duration and DAS28) and laboratory data (RF, a-CCP, ESR and CRP) before and 4, 12 and 24 weeks after the initiation of adalimumab. In whole blood stimulated with LPS, we evaluated: a) free secreted TNFα levels in supernatant (not neutralized by adalimumab) by ELISA; b) TNFα expression on monocyte surface (not neutralized by adalimumab) by flow cytometry; c) Intracellular TNFα production by flow cytometry. Results Ten RA patients completed all time of treatment; two RA patients withdraw treatment due to adverse effects. Baseline characteristics of RA patients were: mean age 56.6 ± 12.6 years, 90 % were female, mean duration of disease was 13.7 ± 11.2 years, 70 % of RA patients were RF or anti-CCP positive, mean DAS28 5.05 ± 0.5, mean CRP was 15.6 ± 17.8 mg/L and mean ESR 40.1 ± 23.6 mm/h. Intracellular TNFa production was different in RA patients compared to controls (healthy donors) (33.15 ± 16.71 vs 71.28 ± 1.04% of CD14+TNFα+ cells respectively, p<0.05). After treatment with adalimumab, intracellular TNFα levels increased. Twenty-four weeks after initiating treatment, the intracellular TNFa levels in patients were comparable to controls. Levels of TNFα on monocyte surface were significantly reduced at four weeks and these levels remained low thereafter (see table). Significant changes in intracellular TNFα, surface TNFα and TNFα secretion were detected in RA patients with a good/moderate EULAR response to adalimumab. However, none of these changes were observed in RA patients with bad EULAR response. Conclusions Active RA patients showed a defective regulation of TNFα production. Blocking TNFα with adalimumab corrected this defect. After receiving adalimumab, intracellular TNFa levels in RA patients and controls were comparable. Disclosure of Interest None Declared