Patricia Moya

Polymorphisms in genes involved in the mechanism of action of methotrexate: are they associated with outcome in rheumatoid arthritis patients?

BACKGROUND Methotrexate (MTX) is the first-line treatment option for newly diagnosed rheumatoid arthritis (RA) patients. However, 50-70% of the patients respond to treatment and 30% suffer toxicity. AIM To identify pharmacogenetic markers of outcome in RA patients treated with MTX. PATIENTS & METHODS We analyzed 27 genetic variants in DHFR, TYMS, MTHFR, ATIC and CCND1 genes. RESULTS We included 124 RA patients treated with MTX monotherapy. In multivariate analyses two variants in the MTHFR gene were associated with response, rs17421511 (p = 0.024) and rs1476413 (p = 0.0086), as well as one in the DHFR gene, rs1643650 (p = 0.026). The ATIC rs16853826 variant was associated with toxicity (p = 0.039). CONCLUSION MTHFR, DHFR and ATIC genetic variants can be considered as pharmacogenetic markers of outcome in RA patients under MTX monotherapy.

IL-6 blockade reverses the abnormal STAT activation of peripheral blood leukocytes from rheumatoid arthritis patients.

Considering the interplay of multiple STATs in response to cytokines, we investigated how IL-6 and its blocking affect STAT signaling in rheumatoid arthritis (RA). Leukocytes obtained from RA patients before and after tocilizumab treatment and healthy donors (HDs) were cytokine-stimulated and STAT phosphorylation was analyzed by cytometry. RA patients had significantly fewer pSTAT1+, pSTAT3+, and pSTAT6+ monocytes and pSTAT5+ lymphocytes than HDs. After 24weeks of treatment, percentages of IFNγ-induced pSTAT1+ and IL-10-induced pSTAT3+ monocytes in RA patients increased, reaching levels comparable to HDs. pSTAT1+ and pSTAT3+ cells correlated inversely with RA disease activity index and levels of pSTAT+ cells at baseline were higher in patients with good EULAR response to tocilizumab. IFNγ-induced pSTAT1+ cells correlated inversely with memory T cells and anti-CCP levels. IL-10-induced pSTAT3+ cells correlated with Treg/Teff ratio. Our findings suggest that IL-6 blocking reduces the inflammatory mechanisms through the correction of STAT1 and STAT3 activation status.

Association Between Nailfold Capillaroscopy Findings and Pulmonary Function Tests in Patients with Systemic Sclerosis

Objective. To determine whether there is an association between different capillaroscopic findings and pulmonary function tests in systemic sclerosis (SSc). Methods. We did a retrospective observational study in a cohort of patients with SSc and early SSc. Patients with at least 1 nailfold videocapillaroscopy (NVC) magnified 120× were included. Pathological findings were giant capillaries, angiogenesis, and density loss. Findings were compared with lung function values: percent expected value of forced vital capacity (FVC), DLCO, and FVC/DLCO ratio. Other variables collected were sex and SSc type, and the presence of digital ulcers (DU), interstitial lung disease (ILD), scleroderma renal crisis, and/or pulmonary hypertension (PH). Results. Of 136 patients with SSc, 85 had undergone an NVC. The frequency of ILD, DU, and PH was 24.1%, 28.7%, and 17.2%, respectively. Data analysis showed that patients with density loss had worse FVC% (86.91 ± 19.42 vs 101.13 ± 16.06, p < 0.01) and DLCO% (71.43 ± 21.19 vs 85.9 ± 19.81, p < 0.01) compared to those without. Conclusion. Patients with loss of density present worse FVC and DLCO values. Prospective studies are warranted to determine whether NVC is useful for studying pulmonary function in SSc.

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.

The combination of IL-6 and its soluble receptor is associated with the response of rheumatoid arthritis patients to tocilizumab

BACKGROUND IL-6 contributes significantly to the chronic inflammatory process of rheumatoid arthritis (RA). Tocilizumab, a humanized anti-human IL-6 receptor antibody that blocks the signaling originated by the IL-6/IL-6R complex, is an effective treatment. However, predictors of the response to tocilizumab are still required. We aimed to combine IL-6 and soluble IL-6R (sIL-6R) levels to identify groups of responses. METHODS Heparinized blood and clinical data from 63 RA patients were collected before treatment and after 3 and 6 months. Two-step clustering (SPSS v.18) was used to establish the relationship between IL-6 and sIL-6R. Then, we compared European League Against Rheumatism (EULAR) response criteria with remission achievement in the groups of patients. RESULTS Three statistical significant clusters of RA patients (i.e., g1, g2, and g3) were defined by serum concentrations of IL-6 and sIL-6R at baseline. All groups of RA patients had higher IL-6 and sIL-6R levels than healthy donors. The levels of IL-6 expressed as median (IQR) in g1 patients were 124(90-183)pg/ml, in g2 12.3(4.4-24)pg/ml, and in g3 60.1(30-146)pg/ml (p < 0.001). The levels of sIL-6R expressed as mean ± sd in g1 patients were 250.5 ± 72ng/ml, in g2 269.1 ± 125ng/ml, and in g3 732.7 ± 243ng/ml (p < 0.001). Disease activity score (DAS)28, C-reactive protein, and erythrocyte sedimentation rate were comparable in the three groups at baseline. Disease duration in g3 was the longest (median(IQR) years: g1 = 11(5-15), g2 = 12(8-20), and g3 23(16-26); p = 0.006), with years of disease evolution being correlated with sIL-6R levels (R = 0.417, p < 0.001). Simple and Clinical Disease Activity Index (SDAI and CDAI) decreased significantly in the three groups. However, EULAR response criteria and remission achievement at 6m was different in the three groups (p = 0.03 and 0.04, respectively). In all. 17 out of the 18 patients in g1 had a good or moderate response to tocilizumab. Conversely, the percentage of patients with no response to tocilizumab was higher in g3 than in g1 and g2. We also observed different changing patterns of IL-6 and sIL-6R levels among the three groups. CONCLUSIONS RA patients could be easily stratified prior to therapeutic intervention with two molecules related to the pathway blocked by tocilizumab. G1 patients, who had the best response to tocilizumab, had the highest level of IL-6 and the lowest level of sIL-6R.

AB0424 Il-6 receptor blockade induced a different immune response in rheumatoid arthritis patients with and without remission

Background Tocilizumab (TCZ) is a humanised antibody that blocks IL-6 receptor. Despite its effectiveness in rheumatoid arthritis (RA), there are patients that do not respond to the IL-6R blockade. The immune characteristics that would explain this lack of response are not known. Objectives Our aim was to determine the tocilizumab-induced changes in CD4 +T cells of patients that achieve, or not, remission at 12 m. Methods Prospective, multicenter study in 47 RA patients treated with TCZ during one year following standard clinical practice. Demographic, disease and treatment characteristics were collected at each visit. Ultrasound (US) grey scale and power doppler were assessed for joints and tendons using a semiquantitative scale from 0–3 points. Phenotyping of T lymphocytes was determined by flow cytometry and the plasma cytokine concentration was quantified by ELISA. Results Forty seven patients were treated with a mean age of 54±11 y and 85% were women. Years of disease were 13±8. We segregated patients according to the DAS28-remission. 44% achieved remission at month 12. We observed that absolute counts of neutrophils and CD4 +T lymphocytes decreased significantly in the remission group but not in the other one. Both memory and naïve CD4 +T cells decreased in the remission group. The analysis of T cells classified according to chemokine receptors showed that memory (29.1±4.0 vs 22.7±2.7 × 104 cells/mL; p=0.06) and naïve (22.6±4.1 vs 17.2±2.8; p=0.04) CD4 +with CXCR3 +and with CCR4 +were the subsets that decreased significantly in the remission group but not in the non-remission group. Since the expression of chemokine receptors defines the different Th subpopulations, we analysed them in the two groups of patients. Th1 tended to decreased in the remission group (3.5±0.7 vs 2.5±0.4; p=0.06) and Th9 decreased significantly in both groups (R: 5.0±0.8 vs 2.5±0.3; p=0.006 and Non R 5.5±0.8 vs 3.1±0.4; p=0.001). In regard to the cytokines produced by CD4 +T lymphocytes, IL-17 (2.1±1.1 vs 1.2±0.5 ng/ml; p=0.04) and VEGF (0.5±0.2 vs 0.3±0.1 ng/ml; p=0.05) but not IL-6 and IL-22 changed significantly in the remission group. Interestingly, IL-17 and VEGF correlated with US findings before the initiation of the treatment (grey scale R=0.378, p=0.01 and R=0.322, p=0.03; power Doppler R=0.415, p=0.004 and R=0.320, p=0.03 respectively). Conclusions Tocilizumab induced changes in specific subsets of CD4 +T cells and their inflammatory associated cytokines in the remission group. Disclosure of Interest None declared

SAT0666 Inflamation beyond clinical remission: ultrasound as a tool to guide us to remission

Background Among patients with RA in remission, subclinical synovitis (SS) has a prevalence of 45% and is associated with an increased risk of clinical relapse and progression to structural damage. US is a sensitive and accessible tool for evaluating SS1,2,3. Objectives To analyse US as a tool for evaluating SS in RA patients treated with tocilizumab (TCZ), in order to assess remission, and from there on propose therapeutic tapering. Methods Multicenter, 1 year follow up study in 45 patients with RA treated with TCZ. The project was aproved by Ethics Committees and all the patients gave their informed consent. At each visit: DAS28, SDAI, CDAI, mHAQ, US grey scale (GS) and Power Doppler (PD) parameters for 32 joints (J) and 28 tendons (T), with a semiquantitative scale from 0–3 points. A quantitative index was obtained for J and T in GS and PD and overall (EG +PD) for each patient/visit. SS was considered as the presence of synovitis with PD(+)≥2. Our intra and interobserver kappa index was 0.8. Results A significant reduction of all clinical indexes and US variables was observed in all patients. Patients were divided into two groups: remission (R) and no remission (NR) according to whether they achieved DAS28 ≤2.6 at 12 months. Group R achieved DAS28 ≤2.6 after mo 3, whereas US showed SS (GS+, PD >2) until mo 12. The final overall PD value in the R group was 0.6 (±0.9). Conclusions A significant number of patients achieve clinical remission within a few months of starting TCZ. The R group, achieved a good EULAR response since mo 3; a progressive improvement in PD, persisted until mo 12. We consider that these findings refer to SS. From our data we consider that dose tapering should not be initiated until at least 9 months from the start of remission. References [1] doi 10.1002/art.22190 [2] PMID 27050636 [3] doi 10.1093/rheumatology/kex084 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