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BackgroundElevated rheumatoid factor (RF) in patients with rheumatoid arthritis (RA) is associated with higher disease activity and increased risk for disease progression1.Recent publications indicate significantly lower efficacy of TNF inhibitors (TNFi) in RA patients with high RF levels compared with low/negative RF subgroup2,3. Fab therapy with Certolizumab pegol (CZP), a PEGylated, Fc-free monoclonal antibody (mAb), has shown comparable efficacy and consistent serum levels irrespective of baseline RF4.RF binds the Fc region of IgG1, the subtype used to engineer the majority of mAbs5. Formation of large immune complexes may likely explain the inceased clearance of mAbs in patients with high RF titers and reported reduced TNFi efficacy.ObjectivesWe aimed to evaluate in clinical practice whether RF levels in RA patients influence serum drug levels of 3 TNFi with different molecular structures.MethodsWe evaluated retrospectively a cohort of RA patients from La Paz University Hospital (RA-Paz Registry, 1999-2019) treated with Infliximab (IFX), Adalimumab (ADA) or CZP. Clinical and demographic data were collected at baseline (T0) and after 6 months (T6) of treatment. RF titers and serum drug levels were measured at T0 and T6 using nephelometry and ELISA respectively. Association between baseline RF titers and drug levels was assessed using non-parametric test (Mann-Whitney).Results168 patients were evaluated: 90 received IFX, 48 ADA and 32 CZP. Characteristics at T0 are shown in Table 1. All patients had active disease at baseline and 76% were RF positive: ADA subgroup had lower percentage of positive RF than IFX and CZP subgroups. Patients were stratified into quartiles based on baseline RF titers: low (20-57 IU/ml), medium (57-380 IU/ml), high (>380 IU/ml) and seronegative (Table 1.Baseline characteristics.CharacteristicsTotal (n=168)IFX (n=90)ADL (n=48)CZP (n=32)p valueAge, years*55.5(45.3-66)57(46-65)50(42-64)61(47-70)0.08Body mass index, Kg/m2*24.5(21.7-29)24.2(21.8-27.7)24.7(21.5-30.3)24.6(22.2-30.3)0.3Male, n(%)28(17%)14(15%)9(19%)5(17%)0.2Disease duration, years*8.7(4.5-14.3)8.4 (4.4-14.3)8.8 (3.9-16)9.7(5-12)0.06Smoking status, n(%)0.03Currently/ex-smoker66(39%)29(32%)22(48%)16(57%)Non-smoker96(57%)61(68%)24(52%)12(43%)RF, n(%)128(76%)75(83%)28(58%)25(81%)0.002ACPA, n(%)134(80%)73(81%)35(73%)27(84%)0.3DAS28**5.1(1.3)5.4(1.3)4.5(1.3)4.9(1.3)0.002CRP levels*7.8(3-21.8)10.3(3.2-25.2)5.1(1.4-10.1)7.8(2.3-18.2)0.1Prior bDMARDs, n(%)26(15%)10 (11%)10 (21%)6(20%)0.2Monotherapy, n(%)16(10%)8(9%)8(17%)00.2csDMARDS, n(%)152(90%)82(91%)82(91%)32(100%)Methotrexate, n(%)112(67%)64(78%)33(83%)17(53%)0.2Other csDMARDs, n(%)24(24%)18(22%)7(18%)15(50%)0.0008Prednisone, n(%)85(51%)49(54%)21(44%)16(50%)0.6*Median and interquartile range;**mean and standard deviationDrug levels of IFX and ADA at T6 were significantly lower in those patients who had higher RF titers at T0 compared to seronegative. In contrast, CZP levels remained stable irrespectively of baseline RF titers, without significant differences among quartiles (Figure 1).ConclusionHigher baseline RF titers are associated with lower IFX and ADA levels at T6 in a cohort of RA patients. A concentration-response association has been clearly established for TNFi, and baseline RF levels appear to influence drug levels.Reduced immune complexes formation with CZP may result in a limited impact of baseline RF titers on drug levels.References[1]Aletaha D. Arthritis Res Ther2015;17(1):229.[2]Bobbio-Pallavicini F. Ann Rheum Dis 2007;66(3):302–7.[3]Potter C. Ann Rheum Dis 2009;68(1):69–74.[4]Tanaka Y. APLAR 2020. Oral Communication.[5]Levy RA. Immunotherapy 2016;8(12):1427-1436.AcknowledgementsThis study was funded by an anrestricted reserch grant from UCB pharma.Disclosure of InterestsANA MARTÍNEZ-FEITO: None declared, Borja Hernández-Breijo: None declared, Marta Novella-Navarro Grant/research support from: UCB, Alejandro Villalba: None declared, Diana Peiteado: None declared, Pilar Nozal: None declared, DORA PASCUAL-SALCEDO Speakers bureau: Abbvie, Pfizer, Novartis, Takeda, Menarini and MSD., Grant/research support from: Abbvie, Pfizer, Novartis, Takeda, Menarini and MSD., Alejandro Balsa Speakers bureau: Pfizer, AbbVie, Galapagos, Lilly, Gilead, UCB, Nordic, Sandoz, Consultant of: Galapagos, Pfizer, AbbVie, Lilly, UCB, Nordic, Grant/research support from: Pfizer, Abbvie, UCB, Chamaida Plasencia Speakers bureau: Abbvie, Pfizer, UCB, Sandoz, Sanofi, Biogen, Lilly, Roche and Novartis, Grant/research support from: Abbvie, Pfizer, UCB, Sandoz, Sanofi, Biogen, Lilly, Roche and Novartis

BackgroundRegular physical activity is recommended for all patients in the ASAS/EULAR recommendations for the management of axial spondyloarthritis (axSpA). However, there is a lack of outcome measures that assess the amount and type of physical activity in patients with axSpA. For this matter, the modified Short QUestionnaire to Assess Health enhancing physical activity (mSQUASH) was developed and validated, originally in Dutch1.ObjectivesTo translate and cross-culturally adapt the mSQUASH into Spanish and to test the equivalence of the translated version in patients with axSpA.MethodsThe mSQUASH was translated into Spanish and then back-translated into Dutch, following forward-backward procedure as described by Beaton2 (Figure 1). Two bi-lingual translators (native speakers for European Spanish) produced independent forward translations of the item content, response options, and instructions of the mSQUASH into Spanish. Both versions were harmonized in a meeting among the Spanish translators, a methodologist and a rheumatologist into a consensual version. Another translator (native speaker for Dutch), blinded for the original version, back translated the synthesized version into Dutch. An expert committee, including all translators, one methodologist and a rheumatologist, reached consensus on discrepancies, ensuring equivalence between the Dutch and Spanish versions, and developed a pre-final version of the Spanish mSQUASH. The field test with cognitive debriefing involved a sample of 10 patients with axSpA covering the full spectrum of the disease -radiographic axSpA (r-axSpA) and non-radiographic axSpA (nr-axSpA)- with different gender, age, disease duration, and educational background. Each patient was interviewed to check understandability, interpretation and cultural relevance of the translation.Figure 1.Cross-cultural adaptation of the mSQUASHResultsThe translation process of the mSQUASH was completed without major complications following the forward-backward procedure. The first translation needed several iterations due to small discrepancies in the wording. Back-translation was performed without difficulties, and the expert committee agreed upon a final version of the questionnaire. A total of 10 patients with axSpA participated in the field test (Table 1). Seven were male, mean age (SD) was 38.9 (14.4) years; 6 patients had r-axSpA, 9 were HLA-B27+. Cognitive debriefing showed the Spanish questionnaire to be, relevant, understandable and comprehensive. The preliminary version was accepted with minor modifications. As a result of the interviews, minor spelling errors were corrected, and the wording of the response categories was homogenized (“despacio/ligero”). Besides, the term “colegio”- translated literally from the Dutch “school”- was found not comprehensive enough to reflect possibilities on education (i.e. it does not include university), so it was adapted to “el lugar de estudio”.Table 1.Patients’ characteristics#GenderAgeWorking statusEducationaxSpA subtypeDisease durationHLA-B27DrugBASDAI1Male63WorkingUniversityr-axSpA35 y+NSAIDs2.32Male24StudentSecondaryr-axSpA6 y+NSAIDs03Male37WorkingUniversityr-axSpA5 y+ADA2.54Male66RetiredUniversityr-axSpA23 y+IFN3.15Male29WorkingUniversityr-axSpA11 y+ADA06Female26WorkingUniversitynr-axSpA2 y+NSAIDs-7Male24StudentUniversitynr-axSpA1 y+ETA4.58Male35WorkingUniversityr-axSpA12 y+GOL09Female40WorkingSecondarynr-axSpA4 y+NSAIDs-10Female45UnemployedPrimarynr-axSpA9 y-GOL8.2ConclusionThe resulting Spanish version of the mSQUASH showed good linguistic and face validity according to the field test, revealing potential for use in both clinical practice and research settings. In order to conclude the cross-cultural adaptation of the mSQUASH into Spanish, the next step is the assessment of psychometric properties of the Spanish version.References[1]Beaton DE, et al. Spine. 2000; 25:3186-91[2]Carbo et al. Semin Arthritis Rheum. 2021; 51:719-27Disclosure of InterestsDiego Benavent Speakers bureau: Jannsen, Roche, Grant/research support from: Novartis, Andrea Jochems: None declared, DORA PASCUAL-SALCEDO Speakers bureau: Pfizer, Menarini, Takeda, Abvvie., Grant/research support from: Pfizer, Menarini, Takeda, Abvvie., Gijs Jochems: None declared, Chamaida Plasencia Speakers bureau: Pfizer, Abbvie, Lilly, Sandoz, Sanofi, Biogen, Roche and Novartis, Grant/research support from: Pfizer and Abbvie, Sofia Ramiro Speakers bureau: Eli Lilly, MSD, Novartis, UCB, Consultant of: AbbVie, Eli Lilly, MSD, Novartis, Pfizer, UCB, Sanofi, Grant/research support from: AbbVie, Galapagos, Novartis, Pfizer, UCB, Suzanne Arends: None declared, Anneke Spoorenberg Consultant of: AbbVie, Novartis, Pfizer; UCB, Lilly, Grant/research support from: AbbVie, Pfizer, Alejandro Balsa Speakers bureau: Pfizer, Abbvie, Lilly, Galapagos, BMS, Sandoz, Nordic Pharma, Gebro, Roche, Sanofi, UCB, Consultant of: Pfizer, Abbvie, Lilly, Galapagos, BMS, Nordic Pharma, Sanofi, UCB, Grant/research support from: Pfizer, Abbvie, BMS, Nordic Pharma, Gebro, Roche, UCB, Victoria Navarro-Compán Speakers bureau: AbbVie, Eli Lilly, Janssen, MSD, Novartis, Pfizer, UCB Pharma, Consultant of: AbbVie, Eli Lilly, MSD, Novartis, Pfizer, UCB Pharma, Grant/research support from: AbbVie and Novartis

BackgroundRheumatic diseases substantially affect the lives of patients, with complex associations between disease severity and self-perceived health status. In this regard, the Coping with Rheumatic Stressors (CORS) questionnaire was developed to measure how patients with rheumatoid arthritis cope with stressors such as pain or dependence. There is no validated instrument to measure coping in axial spondyloarthritis (axSpA) and therefore the adaptation of the CORS would be of great value.ObjectivesTo cross-culturally adapt the CORS into Spanish and to test the conceptual equivalence of the translated version in patients with axSpA.MethodsA translation of the CORS into Spanish was performed, followed by a back-translation into Dutch, following forward-backward procedure as described by Beaton1(Figure 1). Two bi-lingual translators (native speakers for Spanish), one of them informed of the content of the questionnaire and the other not informed, produced independent forward translations of the item content, response options, and instructions of the CORS into Spanish. Both versions were harmonized in a consensual version. Another translator (native speaker for Dutch), not informed of the concepts used in the questionnaire, back translated the synthesized version into Dutch. An expert committee including all translators, one methodologist and a rheumatologist, held a meeting and reached consensus on discrepancies to develop a pre-final version of the Spanish CORS. The field test with cognitive debriefing involved a sample of 10 patients with axSpA covering the full spectrum of the disease and with different sociodemographic backgrounds.Figure 1.Cross-cultural adaptation of the CORSResultsThe translation process of the CORS was completed following the forward-backward procedure, after discussion of the discrepancies throughout the process. The first translation was done without major complications. However, several discrepancies appeared in the back-translation, in which there were minor modifications in the wording in one response option (“muchas veces” to “muy a menudo”) and 15 questionnaire items. As an example, “Ik ga de deur uit”, literally meaning “I go out by the door”, was initially translated as such (“salgo por la puerta”); however, it conceptually represents “I go away”, and it was adapted like this (“me voy a la calle”). Thus, a pre-final consensus version of the CORS was agreed by the expert committee. This pre-final version was field tested in 10 patients with axSpA: mean age (SD) was 38.9 (14.4) years, 7 patients were male, 6 had radiographic axSpA, and 9 were HLA-B27+. The Spanish questionnaire appeared clear and understandable to all patients. However, some minor modifications were proposed in some items (Table 1). As a result of the cognitive debriefing, two changes were implemented (one instruction and one item), whereas two other suggestions did not lead to any change due to minor wording discrepancies with similar conceptual equivalence. The final version of the Spanish CORS is shown at shorturl.at/cimC6.Table 1.Cognitive debriefing queries and decisions from the expert committeeOriginal Dutch itemSpanish translation pre-final# Patient queriesQueriesFinal version(….) aan te geven hoe vaak u het beschreven gedrag uitvoert.(…) indique cuán a menudo usted ha llevado a cabo dicho comportamiento.1Literal discrepancies(…) indique la frecuencia con que usted ha tenido dicho comportamiento.Ik rust op tijd uitMe voy a tiempo a descansar1Literal discrepanciesNo changesIk probeer er het beste van te makenIntento aprovechar al máximo1Literal discrepanciesNo changesIk houd rekening met anderenTengo en cuenta a los demás2Meaning doubtsTengo en consideración a los que me ayudan/cuidanConclusionThe Spanish version of the CORS showed good cross-cultural validity and good face validity in patients with axSpA according to the field test. Before the Spanish CORS is implemented, further validation is in progress to test the psychometric properties of the instrument.References[1]Beaton DE, et al. Spine. 2000; 25:3186-91Disclosure of InterestsDiego Benavent Speakers bureau: Jannsen, Roche, Grant/research support from: Novartis, Andrea Jochems: None declared, DORA PASCUAL-SALCEDO Speakers bureau: Pfizer, Menarini, Takeda, Abvvie., Grant/research support from: Pfizer, Menarini, Takeda, Abvvie., Gijs Jochems: None declared, Chamaida Plasencia Speakers bureau: Pfizer, Abvvie, Lilly, Sandoz, Sanofi, Biogen, Roche, Novartis., Grant/research support from: Pfizer, Abvvie., Sofia Ramiro Speakers bureau: Eli Lilly, MSD, Novartis, UC, Consultant of: AbbVie, Eli Lilly, MSD, Novartis, Pfizer, UCB, Sanofi, Grant/research support from: AbbVie, Galapagos, Novartis, Pfizer, UCB, Wim van Lankveld: None declared, Alejandro Balsa Speakers bureau: Pfizer, Abbvie, Lilly, Galapagos, BMS, Sandoz, Nordic Pharma, Gebro, Roche, Sanofi, UCB, Consultant of: Pfizer, Abbvie, Lilly, Galapagos, BMS, Nordic Pharma, Sanofi, UCB, Grant/research support from: Pfizer, Abbvie, BMS, Nordic Pharma, Gebro, Roche, UCB, Victoria Navarro-Compán Speakers bureau: AbbVie, Eli Lilly, Janssen, MSD, Novartis, Pfizer, UCB Pharma, Consultant of: AbbVie, Eli Lilly, MSD, Novartis, Pfizer, UCB Pharma, Grant/research support from: AbbVie and Novartis

Background The term spondyloarthritis (SpA) encompass a group of crhonic inflammatory disease with predominant axial involvement. Anti-TNF therapy (AT) has stirred up the management of these diseases. Nevertheles, according to nationwide registries of the drug continuation rate in several countries, the rate of treatment failure is considerable. Nowdays there are insufficient data to evaluate the differences between responders (Resp-AT) and non-responders (Nonresp-AT) to anti-TNF therapy. Objectives To compare the main sociodemographic, clinical and analytical features at baseline, after 6 months of treatment and at the end of the first anti-TNF therapy of responders to a first anti-TNF therapy and non-responders to several anti-TNF therapies form our cohort of patients diagnosed with SpA. We also analized the association between drug levels (DL) and antidrug antibody (ADA) in non-responders to several anti-TNF. Methods 181 patients were included, 155 (85%) kept receiving their first AT therapy (responders) and 26 (15%) had discontinued at least two different AT therapies. The main demographic features, disease activity (BASDAI, ASDAS, CRP and ESR) were measured at baseline, after 6 months (v-6) and when the first AT was discontinued. Serum drug levels and/or ADA were measured at drug discontinuation of anti-TNF treatment and last visit in non-responders and responders respectively. Results The mean age was 55.3±15 and 50±10.8 in the resp-AT and nonresp-AT groups, respectively. The demographic and clinical characteristics of both groups are shown in Table 1. At baseline, a lower BMI was observed in the resp-AT group (25.6±5 resp-AT, 28.7±5 nonresp-AT, p=0.013); 59% and 35% were in concomitant treatment with DMARDS at baseline in the resp-AT and nonresp-AT groups respectively (p=0.032); 33% of the resp-AT were in monotherapy in the last visit compared to 62% of the nonresp-AT when discontinued the 1st AT (p=0.008). There were no differences in BASDAI (5.6±2 resp-AT, 6.1±1.9 nonresp-AT, p=0.2) and ASDAS (3.3±1.1 resp-AT, 3.4±1.1 nonresp-AT, p=0.8) at baseline. In v-6, the resp-AT group had a better clinical response in terms of BASDAI (3.2±2.3 in resp-AT, 4.7±2.4 in nonresp-AT, p=0.004) and ASDAS (1.7±0.9 in resp-AT, 2.7±1.2 in nonresp-AT, p=0.00), as well as in the delta-ASDAS (ΔASDAS) (1.56±1.2 resp -AT, 0.7±0.9 nonresp-AT, p=0.04). However, delta-BASDAI (ΔBASDAI) showed no difference between the two groups (2.3±2.3 resp-AT, 1.5±1.6 nonresp-AT, p=0.1). 24% of the nonresp-AT were ADA + at drug discontinuation, while only 0.7% of the AT-resp (AT) were ADA + (p=0.00) at last visit. Conclusions In our cohort of patients with axial SpA, a significant improvement in BASDAI, ASDAS and ΔASDAS after 6 months of treatment is associated with a lower frequency of drop-out of the first AT. Moreover a lower BMI, DMARDS at baseline and absence of ADA determine a better response to AT treatment. Disclosure of Interest None declared

Orthopedic surgery is described as an event with a high risk of thromboembolic diseases. This is probably a consequence of a synergistic combination of different risk factors in the patients subjected to this type of surgery, including age, immobilization, anesthesia and different hypercoagulable states. After surgery patients develop an acute-phase response that leads to changes in several plasma proteins. One of these proteins is the complement regulator C4b-binding protein (C4BP). We have recently shown that in some acute-phase patients C4BP is incorrectly controlled (with elevation of the C4BPΒ-containing isoforms), leading to a potential hypercoagulable state by decreasing the plasma levels of free (active) protein S. Here we have studied whether patients subjected to orthopedic surgery have an appropriate modulation of the C4BP isoforms during their postoperative acute-phase responses. We have analyzed the evolution of the C4BP isoforms in serial samples from 11 patients who have undergone knee (or hip) prosthesis surgery (mean age 70 years), or scoliosis surgery (mean age 18 years). Our data suggest a similar evolution of C4BP isoforms in all these patients, with an almost exclusive increase of C4BP isoforms lacking C4BPΒ polypeptides and steady levels of free protein S.

Immunogenicity of anti-Tumor Necrosis Factor agents is one of the mechanisms behind treatment failure.

Rheumatoid arthritis (RA) is a chronic polygenic inflammatory disease associated with accelerated atherosclerosis and high risk of cardiovascular disease (CVD). In this study, we evaluated the potential association of 9p21.3 single-nucleotide polymorphisms (SNPs) - previously linked to coronary artery disease - and CVD risk in 2001 Spanish RA patients genotyped for 9p21.3 SNPs using TaqMan™ assays. Carotid intima media thickness (cIMT) and presence of carotid plaques were also analyzed. Cox regression model did not disclose significant differences between patients who experienced CVD and those who did not. Neither association was found between cIMT or carotid plaques and SNPs allele distribution. In conclusion, results do not support a role of rs10116277 or rs1537375 SNPs in CVD risk in Spanish RA patients.

Endothelial cells (EC) play a key role in the inflammatory response, both by the production of proinflammatory cytokines and by their interaction with leukocytes. Molecular genetic analysis has demonstrated that functional NF-kappa B sites are involved in the transcription of interleukin-6 (IL-6), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in response to inflammatory mediators. Thus, we have explored the effect of two inhibitors of the NF-kappa B activation, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), on the production of these cytokines by EC. Both PDTC and NAC inhibited, in a dose-dependent manner, the synthesis of IL-6, IL-8, and GM-CSF induced by tumor necrosis factor (TNF)-alpha or bacterial lipopolysaccharides (LPS) in human umbilical vein endothelial cells (HUVEC). PDTC appeared to prevent IL-6, IL-8, and GM-CSF gene transcription, as it blocked the induction of specific mRNA by TNF-alpha or LPS. The TNF-alpha mediated transcriptional activation of a chloramphenicol acetyltransferase (CAT) plasmid containing three copies of the -72 kappa B binding site from the IL-6 promoter was abrogated by PDTC. According to transfection experiments, electrophoretic mobility shift assays (EMSA) demonstrated that the antioxidant prevented the induction of NF-kappa B DNA-binding activity by TNF-alpha. Under the same conditions, PDTC by itself or in combination with TNF-alpha, enhanced the DNA-binding activity of AP-1, as well as c-fos and c-jun mRNA levels. Altogether, these results indicate that the antioxidant PDTC specifically inhibits the transcription of IL-6, IL-8, and GM-CSF genes through the inhibition of the NF-kappa B activation, while increasing the expression of AP-1. Our data make evident the antiinflammatory and immunoregulatory potential of the pharmacological inhibition of the NF-kappa B activation. In addition, PDTC and related molecules may be a useful tool to explore the expression of genes involved in the inflammatory response.

Background Tocilizumab (TCZ), a humanized anti-IL-6 receptor antibody, represents a new treatment strategy for RA patients.Several studies have demonstrated the association between high serum levels of TNF-inhibitors and a good clinical response in patients with RA. Little evidence exists on this relationship for other biological therapies. Objectives To evaluate the association between TCZ serum through levels and disease activity in a cohort of RA patients after one year of treatment with TCZ. Methods 34 RA patients treated with Tocilizumab were included. Clinical activity was assessed using the Disease Activity Score 28 (DAS28-ESR) and the Clinical and Simplified Activity Index (CDAI and SDAI), serological improvement by C-Reactive Protein- CRP- and Erythrocyte Sedimentation Rate-ESR, clinical improvement by the delta-DAS 28 and response to treatment using EULAR criteria at baseline and after one year of treatment. We stratified all patients into quartiles according to the tocilizumab levels (μg/ml) as follows: IQR1: 18.5. A last observation carried forward analysis (LOCF) was performed at the first year of treatment, so patients that dropped out of the therapy before this period were included. Blood samples were collected just before intravenous (i.v) infusion. Serum drug levels were determined by a capture enzymelinked immunosorbent assay (ELISA). Results The baseline demographic and clinical characteristics are shown in Table 1. When patients were categorised into quartiles, IQR1 comprised 8 (24,2%) patients; IQR2, 7 (21,2%); IQR3, 8 (24,2%) and IQR4, 10 (30,3%). Clinical activity (DAS28, CDAI and SDAI) was statistically significant higher in patients with lower serum through levels at the first year [DAS28 (IQR1: 4,46 ±1,5, IQR2: 2,58 ± 0,87, IQR 3: 3,6± 0,79; IQR4: 2,17 ± 0,74; p=0,001), CDAI (IQR1: 23±13,9, IQR2: 7,72 ± 4,44, QIR 3: 13,38± 4,35, IQR4: 6,33 ± 4,62; p=0,003) and SDAI (IQR1: 19,46 ±15,5, IQR2: 9,51± 7,4, IQR3: 12,59 ± 6,31, IQR4: 4,55 ± 3,73; p=0,005)] and also was the number of swollen joints (IQR1: 7,5 ± 6,6, IQR2: 2,29 ±2,06, IQR3: 5,63 ± 4,3, IQR4: 1,1 ± 1,6, p=0,013) and tender joints (IQR1: 5,5 ± 5,7, IQR2: 1,71 ± 2,06, IQR3: 3,13 ± 2,8, IQR4: 0,8 ± 0,9, p=0,014). In addition, the EULAR reponse was worse in those patients with lower serum drug levels [non-responders: 4 (66,7%) in IQR1 vs 1 (16,7%) in IQR2 vs 1 (16,7%) in IQ3 vs 0 (0%) in IQR4, moderate responders: 3 (37,5%) in IQR1 vs 0 (0%) in IQR2 vs 4 (50%) in IQR3 vs 1 (12,5%) in IQR4 and good responders: 1 (5,3%) in IQR1 vs 6 (31,6%) in IQR2 vs 3 (37,5%) in IQR3 vs 9 (47,4%) in IQR4, p=0,006]. In terms of acute-phase reactants, the mean ESR tended to be higher in patients with lower TCZ levels (IQR1: 22,6±14,8 vs IQR2: 5,6±1,6 vs IQR 3: 9,4 ±3,5 vs IQR4: 6,4 ± 3,5, p=0,005), and also CRP levels (RIQ1: 10,2±17,4 vs RIQ2: 2,3 ± 3,4 vs RIQ3 0,56 ± 0,4 vs RIQ 4: 0,42 ± 0,66, p=NS)]. Conclusions The presence of low serum Tocilizumab levels correlates with a worse clinical disease activity. Consequently, measurement of Tocilizumab levels is a valuable tool that might help in the clinical management of patients with Rheumatoid Arthritis. Disclosure of Interest C. Tornero Marin Grant/research support from: Funded by an unrestricted medical grant from Pfizer., C. Plasencia: None declared, D. Pascual Salcedo: None declared, T. Jurado: None declared, M. B. Paredes: None declared, I. Monjo: None declared, E. Moral: None declared, A. Pieren: None declared, G. Bonilla Hernan: None declared, D. Peiteado: None declared, P. Bogas: None declared, L. Nuno: None declared, A. Villalba Yllan: None declared, E. Martin Mola: None declared, A. Balsa Criado: None declared

Background The first infliximab (IFX) biosimilar (CT-P13) in Europe is marketed as two brand names, Inflectra (IFT) and Remsima (RMS), that contain the same IFX molecule produced with the same manufacturing process. One of the main questions is whether patients treated with Remicade (RMC) can be effectively and safely switched to CT-P13. Objectives To determine if antibodies to infliximab (ATI) in RMC-treated patients cross-react with CT-P13. Methods Consecutive patients (250) with active rheumatoid arthritis (RA) and spondyloarthritis (SpA) under RMC treatment and never exposed to CT-P13, were retrospectively selected according to clinical information and anti-drug antibody (ADA) status. 37 IFX-naive rheumatic patients, 19 IFX-naive rheumatic patients with anti-adalimumab antibodies (AAA), and 21 healthy individuals were assayed as controls. ATI trough levels were measured in parallel with three different ELISA assays: #1 Promonitor-ANTI-IFX (Progenika, Spain) which uses RMC to crosslink patient ADA; #2 the same assay but using RMS, and #3 the same assay but using IFT. Results In total, 126 samples out of 250 Remicade-treated patient samples were tested positive with Promonitor-ANTI-IFX (50.4%, 131 patients). All were ATI-positive when either IFT or RMC bridging assays were used (Fig1). Positive and Negative Percent Agreements were 100%/100% for any comparison: RMC vs. IFT or RMC vs. RMS or IFT vs. RMS, respectively. Spearman9s coefficients were determined to be 0.995, 0.996 and 0.998, for RMC vs. IFT, RMC vs. RMS, and IFT vs. RMS, respectively (p Conclusions ATI of RMC-treated patients cross-react with either IFT or RMS. Results suggest that the epitopes raising the immune response to the innovator drug are responsible for the same degree of reactivity when sera are confronted to the biosimilar molecule, although it must be noted that the biosimilar might present new epitopes due to different glycosylation pattern or impurities, and even conformational epitopes due to potential aggregations. ATI-positive patients treated with RMC should not be switched to a biosimilar treatment, since preexisting ATI will interact with the new drug, enhance clearance and potentially lead to loss of response and infusion-related reactions. Further studies with biosimilar-treated patients are warranted to increase our knowledge about potentially different immunogenic responses. Results also demonstrate that Promonitor-ANTI-IFX test can be used to monitor antibodies to CT-P13 in biosimilar-treated patients. This finding supports the utility for therapeutic drug monitoring before a switching strategy is considered. References Ben-Horin S et al Cross-immunogenicity: antibodies to infliximab in Remicade-treated patients with IBD similarly recognise the biosimilar Remsima. Gut 2015 [Epub ahead of print] Disclosure of Interest B. Ruiz-Arguello Employee of: Employee of Progenika Grifols, A. Maguregui Employee of: Employee of Progenika Grifols, A. Ruiz del Agua Employee of: Employee of Progenika Grifols, D. Pascual-Salcedo: None declared, A. Martinez: None declared, T. Jurado: None declared, C. Plasencia: None declared, A. Balsa: None declared, J. Rosas: None declared, F. Llinares-Tello: None declared, N. Torres Employee of: Employee of Progenika Grifols, A. Martinez Employee of: Employee of Progenika Grifols, D. Nagore Employee of: Employee of Progenika Grifols

Background The majority of patients with axial spondyloarthritis (SpA) response to anti-TNF therapy. However, discontinuation of this therapy due to different reasons is still a relevant problem. Currently, there is not enough data to know exactly which is the prevalence and causes of interruption of anti-TNF therapies in clinical practice. Objectives First, to evaluate the frequency and causes of discontinuating adalimumab or infliximab as the first anti-TNF in patients with axial SpA in clinical practice. Second, to investigate the influence of anti-drug antibodies (ADA) on these causes. Methods A total of 326 patients with axial SpA who had received adalimumab (34%) or infliximab (66%) as a first anti-TNF therapy were included in this retrospective, observational study performed in a tertiary hospital. Disease activity (BASDAI, ASDAS, CRP and ESR) was measured before starting anti-TNF therapy, after 6 months and when interrupting the therapy to assess properly whether the reason for discontinuation was primary or secondary failure. Serum drug levels and/or ADA were measured at 6 months visit and at the end of anti-TNF treatment. Results A total of 99 (30.4%) patients discontinued treatment. Mean (SD) under anti-TNF therapy until discontinuation was 2.5 (2.9) years. Characteristics of these patients when initiating anti-TNF therapy are shown in Table 1. The reason to interrupt treatment was: primary failure in 22.2%, secondary failure in 36.4%, side effects 32.3%), and other reasons in 9.1%. Serum drug levels and ADA were available in 83 patients. In most patients with ADA positive (14/17), the reason for discontinuation was secondary failure. Out of those patients who discontinued due to secondary failure, 38.9% had ADA positive. Conclusions In our cohort of patients with axial SpA treated with adalimumab/infliximab, 30% of patients discontinued anti-TNF therapy. The main reason to discontinue treatment was secondary failure, which was related to the presence of ADA in almost 40% of patients. Disclosure of Interest E. Moral Grant/research support from: Funded by an unrestricted medical grant from Pfizer, C. Plasencia: None declared, V. Navarro-Compan: None declared, D. Pascual Salcedo: None declared, T. Jurado: None declared, C. Tornero: None declared, A. Pierens: None declared, M. B. Paredes: None declared, P. Bogas: None declared, I. Monjo: None declared, E. Martin Mola: None declared, A. Balsa: None declared

Background The options for treatment of rheumatic diseases is constantly growing. Tocilizumab (TCZ) is an anti-interleukin 6 receptor monoclonal antibody indicated for the treatment of severe active and progressive rheumatoid arthritis (RA) and active systemic juvenile idiopathic arthritis (sJIA). Monitoring of drug levels (DL) and anti-drug antibodies (ADA) in clinical practice is highly advisable in order to establish normal therapeutic ranges, complement clinical assessment and optimise patient treatment. As a consequence several tests and technologies have been developed. However, a common objection that limits implementation of biological drug monitoring in medical practice is the claim that the magnitude of measurements varies among assays and that assay standardisation is lacking. Objectives The purpose of this study was to evaluate the correlation and agreement between different commercially available tests to measure TCZ and antibodies to TCZ (ATT) in patients with rheumatic diseases. Methods Trough levels of TCZ and ATT were analyzed in a retrospective cohort of 26 rheumatic patients (42 serum samples, 88% RA and 12% sJIA) under maintenance therapy with TCZ for up to two years. Samples were assayed using Promonitor-TCZ and Promonitor-ANTI-TCZ ELISA kits (PR, Progenika, Spain), LISA-TRACKER Duo Tocilizumab ELISA kit (LT, Theradiag, France), and by Sanquin Blood Supply testing service (SQ, Amsterdam, The Netherlands) that uses ELISA and RIA technologies to measure TCZ and ATT levels, respectively. One-way analysis of variance (ANOVA) and Spearman9s test was used to study correlation between the DL tests; the difference (bias) in values obtained with DL assays was also assessed with Bland-Altman analysis. Percentage of positive and negative agreement was used to study the association between the ADA assays. Results TCZ trough level in patients are much higher than other biological treatments like anti-TNF infliximab and adalimumab. ANOVA analysis showed that the differences in the median values among the three DL assays are not statistically different (median 4.4 vs 4.7 vs 5.4 μg/mL, IQR 0 – 8.7 μg/mL vs 0 – 7.8 μg/mL vs 0 – 9.4 μg/mL for Promonitor-TCZ, LISA-TRACKER and Sanquin ELISA tests, respectively, p =0.693). Spearman9s coefficient showed a very high correlation between the each pair of assays ( r =0.951, 0.935 and 0.944 for PR vs LT, PR vs SQ and LT vs SQ, respectively, p 0.05). Antibodies to TCZ were not detected with any of the assays in the patient cohort confirming the low immunogenicity of TCZ. Conclusions Despite different reagents, assay formats and technologies, all tests studied here provide exactly the same results and measure the same magnitude of TCZ in rheumatic patients. Assays can be safely exchanged in a clinical setting even in the absence of a gold standard test, and should lead to similar drug therapeutic ranges and actions. Acknowledgement This work was supported by the Basque Country Government (Dep. Education, Universities and Research, Ref. IT687–13) and ZabaldUZ 2012, UPV/EHU. Disclosure of Interest S. Martin Grant/research support from: PhD student research support from Progenika, A. Ruiz del Agua Employee of: Employee of Progenika Grifols, N. Torres Employee of: Employee of Progenika Grifols, D. Pascual-Salcedo: None declared, C. Plasencia: None declared, T. Jurado: None declared, A. Martinez: None declared, A. Balsa: None declared, B. Ruiz-Arguello Employee of: Employee of Progenika Grifols, A. Martinez Employee of: Employee of Progenika Grifols, R. Navarro: None declared, D. Nagore Employee of: Employee of Progenika Grifols

Objective. To analyse the relationship between the presence of auto-antibodies [rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP)], HLA-DRB1 alleles and PTPN22 1858 C/T polymorphism and test the value of their combination as susceptibility markers for rheumatoid arthritis (RA). Methods. Patients with early arthritis were included. At entry in the cohort or during follow-up, 191 patients fulfilled the criteria for RA and 184 individuals suffered from other arthropathies. RF was measured by nephelometry and anti-CCP antibody by enzyme-linked immunosorbent assay. HLA class II alleles were determined by polymerase chain reaction. Samples were genotyped for PTPN22 1858C/T variants using a TaqMan 5 0 -allele discrimination assay. Results. The presence of shared epitope (SE) alleles was strongly associated with anti-CCP and RF-positive RA [P ¼ 7.05 � 10 � 10 , odds ratio (OR) 4.57, 95% confidence interval (CI) 2.76–7.57 and P ¼ 1.68 � 10 � 6 , OR 2.99, 95% CI 1.89–4.74, respectively). The combination of the PTPN22 1858T variant and anti-CCP antibodies gave a high specificity for the disease, and was significantly associated with RA (P ¼ 8.86 � 10 � 5 , OR 10.05, 95% CI 1.88–53.73). Conclusion. The combination of the T variant of the 1858 polymorphism of the PTPN22 gene in combination with the presence of anti-CCP antibodies, preferentially in a SE-positive individual, is associated with the development of RA.

Study of the role of a functional polymorphism of MHC2TA in rheumatoid arthritis in three ethnically different populations.

To study the serum levels of IL-15 in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), seronegative spondyloarthropathies (SSd) and healthy donors.The IL-15 serum levels were measured by ELISA in sera from 50 RA patients, 30 patients with SLE, 30 patients with SSd and 30 healthy donors. In RA patients, several clinical and demographic parameters were also obtained at the time of sample collection. IL-15 levels were compared in different RA subpopulations (positive or negative rheumatoid factor [RF], long term or recent onset disease, high or low disease activity). In addition, the possible association with other demographic and clinical parameters (gender, age, disease duration, etc) was also analysed.RA patients had significantly higher serum levels of IL-15 (102.4 +/- 150 pg/ml; p = 0.0001) than SLE patients (9.8 +/- 15.3 pg/ml), SSd patients (7.9 +/- 14.6 pg/ml) and healthy donors' (5.2 +/- 11.6 pg/ml). RA patients with a disease evolution less than 2 years showed lower IL-15 levels (33.7 +/- 62.2 pg/ml) than those with long-term disease (152.4 +/- 64.6 pg/ml; p = 0.004). In addition, a significant correlation between IL15 in serum and the number of disease-modifying antirheumatic drugs (DMARDs) prescribed was detected in RA patients (r = 0.42; p = 0.002). No association between IL-15 levels and age, gender, RF or disease activity was observed in this group.IL-15 is elevated in RA patients, specially in those with long term disease, compared to other rheumatic disorders. This finding supports that IL-15 is involved in the perpetuation of RA synovitis.

To investigate the association of the poly(ADP-ribose) polymerase 1 (PARP-1) gene promoter polymorphism with rheumatoid arthritis (RA) predisposition.An association study with 213 Spanish RA patients and 242 healthy subjects was carried out to investigate the association of all known PARP-1 gene promoter polymorphisms, i.e., a CA microsatellite repeat, a poly(A)(n), and 3 single point mutations (C410T, C1362T, and G1672A), with disease susceptibility. Additionally, we analyzed the distribution of PARP-1 polymorphisms in 58 Spanish families with 1 or more affected members.Upon complete genotyping of the panel of 455 samples, strong linkage disequilibrium was observed among the 5 PARP-1 polymorphisms. Only 2 PARP-1 haplotypes were detected: haplotype A (410T-[A](10)-[CA](10-12)-1362C, which includes short PARP-1 CA alleles) and haplotype B (410C-[A](11)-[CA](13-20)-1362T, always paired with long PARP-1 CA variants). Regarding the G1672A variation, although linkage disequilibrium was detected, it did not seem to be part of the conserved haplotypes described. Haplotype B was statistically overrepresented in the RA patient group compared with the healthy subjects (odds ratio 1.42, 95% confidence interval 1.06-1.91, P = 0.019). In addition, a significant dose effect of PARP-1 haplotype carriage on disease predisposition was observed. Of note, within haplotype B, the PARP-1 CA 97-bp allele was found to be the RA-predisposing marker (odds ratio 2.17, 95% confidence interval 1.27-3.72, P = 0.003, corrected P0.05).Our results demonstrate the existence of 2 unique PARP-1 haplotypes in the Spanish population and provide the first evidence that PARP-1 haplotypes play a role in susceptibility to RA.

Background Treatment with biological drugs in the last fifteen years has changed the therapy of most inflammatory diseases, mainly in the field of rheumatology. The use of these drugs has supposed a revolution in the treatment of disease, making this type of medication the fastest growing class of therapeutics today. Monoclonal antibodies targeting tumor necrosis factor (TNF) were among the first biologicals to appear on the market, and they are the most often used to date. They have proven to be powerful drugs applied to such inflammatory rheumatic diseases as rheumatoid arthritis, ankylosing spondylitis. All patients may benefit from routine measurement of serum trough levels of biologicals. Immunogenicity. Drug and antibody levels Several methods have been developed to measure the serum concentration of these drugs, mainly by capture or sandwich ELISA, or a reported gene-based assay based on a bioluminescent reaction produced by cells growing in the presence of TNF. Many publications have associated the good clinical response of a treatment with biologics with the circulating amount of the drug. However, biologics are recognized by the immune system as foreign and they provoke an immune response. Antibodies to the drugs (ADA) recognize strange sequences in the antigen-binding site of the monoclonal antibodies, or in the fusion part of the proteinconstructs. Besides the nature of the biological, antibody responses may also be influenced by patient-related factors such as genetic background, underlying disease, immunomodulating therapy, and dosing schedule. If a patient develops ADA, circulating levels of the biological will be less than expected or even absent in the serum. ADA can form a complex with the drug, thereby inhibiting drug activity, which can result in treatment failure. The production of ADA is associated with treatment failure, increased risk of infusion-related reactions and early drop-out of the treatment. Two are the main methods used in the clinical routine to measure the appearance of ADA: bridging ELISA and Antigen Binding Test (ABT)). ELISA takes advantage of the fact that IgG1,2,3 isotypes can bridge the same molecule (the biological) by both arms. It is easy to perform and suitable to implement in any lab. Drawbacks of this system are the potentially false positive results given by rheumatoid factor and the difficulty to detect IgG4-ADA, because of its monovalency. ABT is based on the detection of the radiolabel drug to the serum antibody bound to protein A Sepharose beads. ABT detects IgG4-ADA as well as the other isotypes. However, with both kind of assays only excess of the antibody present in serum is detected, due to the drug interference. Complexes of ADA-drug are not detected in the assays to measure drug neither in the ADA assays. Although it is admitted that the therapeutic effect of a drug is associated with the amount of free drug available to bind the antigen (TNF), efforts are being made to develop assays that can detect the earliest production of antibodies, and to investigate its clinical relevance. These assays are based on the acid-dissociation of the complexes together with neutralization of the free drug before re-naturalization. However, if we realize that methods to measure ADA are still not well standardized, the most easy way to detect whether a patient is making ADA is to monitor the amount of the circulating drug levels at any moment of the treatment. Analysis of the drug and antibody levels The most useful application of the measurement of drug and ADA in serum is the therapeutic drug monitoring (TDM) as a tool of personalized medicine to improve patient care by adjusting the drug dosage at the individual patient levels. By performing this monitoring a range of the optimal amount of circulating drug – in relation with the clinical response – can be drawn, per patient and per kind of biological. This procedure will allow: A) To avoid overtreatment or under treatment, both expensive procedures because of a waste of resources B) To help the performance of switching in patients who are non-responders to the therapy. Patients not responding to therapy associated to ADA formation, may switch to a biological with a similar target. In contrast, non-responders with adequate drug levels who do not develop ADA may benefit from biologics with another mode of action. C) To perform a dose tapering of the administered amount of drug in patients that enter in remission or maintain low clinical activity, D) To help in the decision to increase the amount of drug when there are no other therapeutic options. Thus all these options allow for a therapy optimization of individual patients. The personalized medicine approach benefits the patient and reduces costs associated to a more efficient therapy implementation. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.6203

To describe clinical characteristics and the homogeneity of disease expression between involved members in multicase Spanish rheumatoid arthritis (RA) families.73 families with two or more siblings with RA were found, with a total of 149 patients distributed in 79 pairs (70 sib pairs, and 3 sib trios). Demographic, clinical and radiological characteristics were recorded in a standard questionnaire.Clinical characteristics were similar to those of sporadic RA with a high frequency of women (78%), positive rheumatoid factor (RF) (86%), erosions (89%) and a 25% of the patients having extraarticular disease. The most important variable in disease severity was disease duration. The concordance between family members of the same age and calendar year of disease onset, and the pattern of disease expression, was not higher than expected, showing that the disease is heterogenic.Environmental factors seem to be more important in RA susceptibility. Clinical characteristics of familiar RA in Spain do not seem to be different from sporadic RA, although differences were found in disease expression within families that may be due to variation of genetic or environmental factors, responsible for the susceptibility and disease duration.

It has been proposed that noninherited maternal antigens (NIMA) (HLA-DR antigens) might play a role in susceptibility to rheumatoid arthritis (RA), especially in patients who are not genetically predisposed, such as those who are HLA-DR4 and/or shared epitope (SE) negative. The present study was undertaken to test the NIMA hypothesis in a large cohort of European RA patients assembled by the European Consortium on RA Families (ECRAF).HLA-DRB1 oligotyping was performed in families of European RA patients for whom both parents were alive. These families were consecutively recruited by the ECRAF between 1996 and 1998, for association studies. The frequencies of HLA-DR NIMA were compared with those of the noninherited paternal antigens (NIPA) after stratification for HLA-DR*04, *0401 and/or *0404, and SE status. NIMA or NIPA that coincided with inherited HLA-DR antigens were considered redundant and excluded from analysis. Calculations concerning the whole group and restricted to patients lacking parental RA were performed.One hundred seventy families from France (n = 81), Belgium (n = 23), Spain (n = 24), Italy (n = 19), Portugal (n = 14), and The Netherlands (n = 9) were oligotyped. The group of probands was predominantly female (88%), positive for rheumatoid factor, DR*04, and SE (71%, 58%, and 75%, respectively), and had erosive disease (75%). Parental RA was reported in 21 families. Using the NIPA as control, the frequency of HLA-DRB1*04, *0401 and/or *0404-, or SE-positive NIMA was not found to be increased in patients lacking these susceptibility alleles. The same was true when the 21 probands with parental RA were excluded from analysis. In DRB1*04-positive patients, we found no evidence of a relevant effect of HLA-DR3 or DR6 in the NIMA.Our results do not support the notion that noninherited maternal antigens have a role in susceptibility to RA in the offspring.

The integrin VLA-4 mediates attachment of myeloma cells to multiple myeloma (MM) bone marrow stroma. The alternatively-spliced CS-1 region of fibronectin (FN) and VCAM-1 are main ligands for VLA-4 and are both expressed on MM stroma. The H1 region is present in all FN isoforms and represents an additional binding site for VLA-4. We employed FN fragments FN-H89 and FN-H0, that contain either the CS-1 and H1, or only the H1 sites, respectively, as well as soluble VCAM-1 (sVCAM-1), to characterize VLA-4-mediated adhesion pathways used by myeloma cells to attach to MM stroma. CD38highCD45RA- cells from MM bone marrow, and the myeloma-derived cell lines NCI-H929, IM-9 and RPMI 8226, specifically adhered, by different degrees, to FN-H89, FN-H0 and sVCAM-1, and their VLA-4-dependent adhesion was substantially up-regulated by the anti-beta1 antibody TS2/16, which increases the affinity of VLA-beta1 integrins. Furthermore, VLA-4 function on NCI-H929 cells was enhanced by TS2/16 during adhesion to MM stroma. The alpha4beta7 integrin mediated a small portion of myeloma cell line adhesion to FN-H89, mainly upon integrin activation with Mn2+. These results indicate that myeloma cells use VLA-4 to interact with CS-1/FN, H1/FN and VCAM-1 on MM stroma, and that its function can be potentially up-regulated, enabling higher degrees of cell adhesion to these VLA-4 ligands, which might influence myeloma cell localization in the bone marrow.

The aim of this study was to determine if the measurement of anti-β2-glycoprotein I antibodies (aβ2-GPI) in serum levels contributes to the better characterization of the clinical situation of patients with antiphospholipid syndrome (APS). For this purpose aβ2-GPI of both isotypes was measured in 42 patients with APS and 32 SLE patients without APS. Clinical records of all patients were thoroughly reviewed. The presence of aβ2-GPI was correlated with the clinical manifestations of APS and compared with the presence of anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) activity. There was a positive correlation between levels of aCL and aβ2-GPI for both IgG and IgM isotypes (r of Spearman = 0.82 and 0.64 respectively, P = 0.0001). Both antibodies presented significantly higher titres in LA positive patients (P 0.05). The specificity for APS was 91% for IgG aβ2-GPI vs 75% for IgG aCL and 87% for IgM aβ2-GPI vs 81% for IgM aCL. 68% of patients with thrombosis of 100% of patients with thrombocytopenia showed positive tests for all three markers (aCL, LA, aβ2-GPI). Simultaneous presence of circulating LA and high titres of both aCL and aβ2-GPI identify a subset of patients with primary APS (PAPS) who have a more severe clinical course of the disease. Although the specificity of aβ2-GPI IgG is higher than that of aCL IgG, when all three tests are performed aβ2-GPI testing provides only additional information to that of aCL and LA. Therefore, we concluded that the aβ2-GPI test should not be considered as a substitute for conventional LA or aCL assays. However, performance of aβ2-GPI seems to be important in PAPS with high aCL titres, to alert the physician about the risk for the worst course of the illness.

We studied interleukin 6 (IL-6) and soluble IL-6 receptor (sIL-6R) in serum and synovial fluid (SF) to investigate their role in different arthropathies.IL-6 was measured by ELISA and bioassay and sIL-6R by ELISA, in 210 sera and 73 SF samples from 49 patients with rheumatoid arthritis (RA), 20 crystal deposition disease, 17 osteoarthritis (OA), 24 with other inflammatory arthropathies, and 100 controls. In all patients, disease activity was assessed by erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP); in patients with RA and other arthropathies pain, tender and swollen joints, and Ritchie index were also evaluated. Total leukocyte count in SF was determined.There was good correlation between IL-6 ELISA and bioassay levels both in serum (r = 0.62, p = 0.0001) and in SF (r = 0.72, p = 0.0001). Serum IL-6 was detected only in patients with inflammatory arthritis and SF IL-6 was detected in all patient groups. Serum IL-6 correlated with swollen joints (r = 0.35, p = 0.05), ESR (r = 0.46, p = 0.001), and CRP (r = 0.46, p = 0.001) in RA; and with CRP (r = 0.89, p = 0.0001) in crystal deposition disease. SF IL-6 correlated with ESR (r = 0.54, p = 0.007) and CRP (r = 0.42, p = 0.04) in RA; with SF total leukocyte count (r = 0.61, p = 0.004) in crystal deposition disease; and with SF total leukocyte count (r = 0.61, p = 0.009) in OA. No correlations were found in the group with other inflammatory diseases. No correlations were found between sIL-6R and IL-6 or between sIL-6R and disease activity variables in any group of patients.Unlike IL-6, sIL-6R is not produced at the site of inflammation and is not related to clinical or biological disease activity variables. Only in RA are both IL-6 and sIL-6R levels increased, suggesting that sIL-6R may reinforce the systemic effects of IL-6.

Background Spondyloarthropathies (SpA) include a heterogeneous group of rheumatic diseases that mainly affect the axial skeleton and entheses. Despite the anti-TNF therapy has been demonstrated effective in SpA patients, about 30% of them develop primary or secondary inefficacy. In rheumatoid arthritis (RA) has been shown that the development of antibodies (Ab) against the first anti-TNF determines the clinical response to a second anti-TNF. Objectives To assess if the effectiveness of second anti-TNF therapy is associated with the development of Ab against the first anti-TNF in SpA patients switching to a second anti-TNF. Methods We studied 33 SpA patients treated with a second anti-TNF after having an inadequate response to the first anti-TNF therapy. The diagnoses were: 23 (69.7%) Ankylosing Spondylitis (AS), 6 (18.2%) undifferentiated SpA, 2 (6.1%) psoriatic SpA, 1 (3%) SpA associated with inflammatory bowel disease and 1 (3%) reactive SpA. Clinical activity was measured by ASDAS-CRP at baseline to the 1st and 2nd anti-TNF treatment and after 6 months of the switching. Clinical improvement was assessed by the Delta-ASDAS (clinically important improvement ≥1.1). Drug through levels and anti-drug Ab were measured by ELISA at the end of the fist anti-TNF treatment. Statistical analysis was performed using SPSS 11.0. Results Our cohort included 33 patients, 18 (54.5%) male, mean age of 50.09±10 years and 21 (63.6%) HLA B27 positive. All of them were initially treated with an anti-TNF: 14 (42.4%) with infliximab (Ifx), 3 (9.1%) with adalimumab (Ada), 16 (48.5%) with etanercept (Eta). Nine out of 33 (27.3%) developed anti-drug Ab [8 anti-Ifx Ab (ATI) and 1 anti-Ada Ab (AAA)]. All patients switched to a 2nd anti-TNF, due to inefficacy: 7 (21.2%) to Ifx, 16 (48.5%) to Ada, 5 (15.2%) to Eta, 5 (15.2%) to golimumab. Clinical activity (ASDAS-CRP) was not different at baseline between first and second anti-TNF therapy (3.45±0.99 vs 3.22±0.96, p=0.24). No differences were observed between patients who developed or not anti-drug Ab at baseline to the first (3.34±0.87 with Ab vs 3.50±1.04 without Ab, p=0.93) and second (2.99±0.95 with Ab vs 3.31±0.96 without Ab, p=0.37) anti-TNF treatment. After 6 months of switching to a second anti-TNF, patients who had developed anti-drug Ab had lower clinical activity (ASDAS-CRP) than patients without anti-drug Ab (1.76±0.98 with Ab vs 2.79±1.10 without Ab, p=0.021). Moreover clinical improvement was higher in patients with anti-drug Ab (1.23±1.22 with Ab vs 0.52±1.08 without Ab, p=0.063). Most patients who had clinically important improvement had anti-drug Ab before switching [60% (6/10) vs 40% (4/10), p=0.010]. Conclusions Same as in RA, in SpA, the failure to a first anti-TNF therapy associated with the development of anti-drug Ab predicts a better clinical response to a second anti-TNF. The study of the immunogenicity in the biological treatment failure helps predict the response to a second biological treatment in SpA. Disclosure of Interest None Declared

The production of cytokines is a key event of inflammation. In this report we demonstrate that normal human T cells are capable to produce IL-1 alpha, IL-6, and TNF-alpha, cytokines formerly considered to be monokines. This production was optimal after stimulation with a combination of anti-CD2, PMA, and anti-CD28. All three cytokines were produced in a bioactive form. Both naive (CD4+CD45RA+) and memory (CD4+CD45RO+) subsets of T cells were shown to produce similar amounts of IL-6 and TNF-alpha. In contrast the production of IL-1 alpha was found to be completely restricted to the CD4+CD45RO+ subset. The finding that T cells are such potent producers of these important mediators of the inflammatory response might be a key observation in the appreciation of the role of T cells in chronic inflammation.

To optimize growth and Ig production of in vitro-cultured Epstein-Barr virus (EBV)-transformed B cells, a panel of six monoclonal EBV B-cell lines was analyzed for autocrine growth factor production and responsiveness to various cytokines. Three cell lines produced Il-I and four produced Il-6, although differences concerning the amount of lymphokines produced were observed. Interestingly a considerable tumor necrosis factor beta (lymphotoxin) activity was found in supernatants of all cell lines. One IgM-producing cell line that did not secrete either Il-1 or Il-6 was exceptional in its ability to respond to the addition of rIl-6 with a 5- to 10-times elevated IgM production. In contrast, cell lines in the panel capable of Il-6 production showed only a minimal elevation of Ig production on addition of exogenous Il-6. Ig production was slightly less in some cell lines when Il-6 was neutralized. Antibodies against lymphotoxin or Il-6 did not influence growth rate of the cell lines significantly, implying that neither Il-6 nor lymphotoxin had an autostimulatory effect on the analyzed cell lines. This study demonstrates a heterogeneity regarding the amount and type of lymphokines produced by long-term monoclonal EBV cell lines, which may account for the diverse responses exhibited by these cells towards exogenously added lymphokines.

Studying the production of IL-6 (interleukin-6) by monocytes, endothelial cells and smooth muscle cells we observed that cytokine inducers like IL-1, TNF alpha (tumor necrosis factor alpha), LPS (lipopolysaccharide), SAC (Staphylococcus Aureus Cowan 1) and PMA could be divided roughly into two categories. Bacterial products such as LPS or SAC have a potent IL-6 inducing effect on monocytes and minor or no effect on endothelial- and smooth muscle cells. The other category comprising IL-1, TNF alpha and PMA induces IL-6 production in endothelial- and smooth muscle cells. Only IL-1 induces IL-6 production in monocytes as well as in endothelial cells and smooth muscle cells. In addition to IL-6, also IL-1 and TNF alpha are produced by monocytes however with different kinetics. None of the stimuli had any inhibitory effect on IL-6 production with the exception of PMA. Whereas PMA induced IL-6 production in endothelial cells and it potentiated the induction of IL-6 by IL-1 in these cells, it inhibited LPS-stimulated IL-6 production in monocytes. In line with the effects of PMA, staurosporin induced IL-6 production in monocytes and it inhibited IL-1 driven IL-6 production by endothelial cells.

Studying the production of IL-6 by monocytes, endothelial cells and smooth muscle cells we observed that cytokine inducers like IL-1, TNF alpha, LPS, SAC and PMA could be divided roughly into two categories. One, that consisted of LPS and SAC, which had a potent IL-6 inducing effect on monocytes and minor or no effect on endothelial- and smooth muscle cells. The other category, consisting of IL-1, TNF alpha and PMA, induced IL-6 production in endothelial- and smooth muscle cells and of which IL-1 also induced IL-6 in monocytes. Only IL-1 induced IL-6 production in monocytes as well as in endothelial cells and smooth muscle cells. Besides IL-6, also IL-1 and TNF alpha were produced by monocytes however with different kinetics. None of the stimuli had any inhibitory effect on IL-6 production with the exception of PMA. Whereas PMA induced IL-6 production in endothelial cells and it potentiated the induction of IL-6 by IL-1 in these cells, it inhibited LPS-stimulated IL-6 production in monocytes. In line with the effects of PMA, staurosporin induced IL-6 production in monocytes and it inhibited IL-1 driven IL-6 production by endothelial cells.

Objective It has been proposed that noninherited maternal antigens (NIMA) (HLA–DR antigens) might play a role in susceptibility to rheumatoid arthritis (RA), especially in patients who are not genetically predisposed, such as those who are HLA–DR4 and/or shared epitope (SE) negative. The present study was undertaken to test the NIMA hypothesis in a large cohort of European RA patients assembled by the European Consortium on RA Families (ECRAF). Methods HLA–DRB1 oligotyping was performed in families of European RA patients for whom both parents were alive. These families were consecutively recruited by the ECRAF between 1996 and 1998, for association studies. The frequencies of HLA–DR NIMA were compared with those of the noninherited paternal antigens (NIPA) after stratification for HLA–DR*04, *0401 and/or *0404, and SE status. NIMA or NIPA that coincided with inherited HLA–DR antigens were considered redundant and excluded from analysis. Calculations concerning the whole group and restricted to patients lacking parental RA were performed. Results One hundred seventy families from France (n = 81), Belgium (n = 23), Spain (n = 24), Italy (n = 19), Portugal (n = 14), and The Netherlands (n = 9) were oligotyped. The group of probands was predominantly female (88%), positive for rheumatoid factor, DR*04, and SE (71%, 58%, and 75%, respectively), and had erosive disease (75%). Parental RA was reported in 21 families. Using the NIPA as control, the frequency of HLA–DRB1*04, *0401 and/or *0404–, or SE-positive NIMA was not found to be increased in patients lacking these susceptibility alleles. The same was true when the 21 probands with parental RA were excluded from analysis. In DRB1*04-positive patients, we found no evidence of a relevant effect of HLA–DR3 or DR6 in the NIMA. Conclusion Our results do not support the notion that noninherited maternal antigens have a role in susceptibility to RA in the offspring.

The case of a 23 year-old female with a diagnosis of systemic lupus erythematosus associated to selective IgA deficiency is reported. The patient persistently had serum IgA levels below 0.05 mg/dl, while the remaining serum immunoglobulins were normal. No salivary IgA was detected, and the karyotype disclosed no abnormalities of the chromosome 18. In the in vitro immunological study a normal number of B and T lymphocytes was found, and decreased production of all surface immunoglobulins was observed.

In vitro lymphocyte function of 13 patients with selective IgA deficiency was studied. IgG, IgA and IgM secretion by pokeweed mitogen-stimulated peripheral blood lymphocytes from normal donors and IgA deficient patients was measured by an enzyme-linked immunosorbent assay. Addition of concanavalin A or hydrocortisone and co-cultures of B and T cell enriched populations from patients and normal donors, allowed us to investigate B cell Ig production and T cell regulatory abnormalities. IgA production by these patients' B cells was either absent or very low, as compared to their total Ig production, even in the presence of optimal T cell help. Several T cell immunoregulatory abnormalities were seen in different patients. A moderate increase in suppressor activity selective for IgA production was observed in some, but does not appear to play a major role in the pathogenesis of the IgA deficiency. In others, with associated autoimmune phenomena, a decrease in suppressor T cell function was found.

Six patients with primary hypogammaglobulinaemia and hyper IgM were studied. All showed very low serum IgG and IgA concentrations. The in vitro pokeweed-mitogen (PWM)-induced immunoglobulin (Ig) production, including IgM, by their peripheral blood lymphocytes was low. Even when patients' B cells were cocultured with normal T cells, IgM production did not reach normal levels. These results and studies of Ig class on the surface of B lymphocytes point to a maturation arrest of these cells. T cells from all but one patient helped very little Ig production by patients' or normal B cells. Similar numbers of these T cells did not suppress Ig production by normal T plus B cells. Therefore a defect in T cell help for IgM, IgG and IgA was seen in most patients, in addition to the B cell abnormality.

Three patients with the Wiskott-Aldrich syndrome were studied. One of them had no past history of relevant infections. The other two presented different degrees of humoral and cellular immunodeficiency and their T cells in vitro showed a defect in regulatory activity of Ig production in PWM stimulated cultures. This defect was not observed in the third patient. All three had normal numbers of B cells, producing normal amounts of Ig in vitro when co-cultured with normal T cells. It is suggested that the immunoregulatory T cell abnormality might play an important role in the pathogenesis of the humoral immunodeficiency.

We describe a 2 year old girl with nucleoside phosphorylase (PNP) deficiency, who had low blood T cell numbers and T lymphocyte blastogenic response to mitogens, hypergammaglobulinaemia, high titres of antibodies to many common antigens, various autoantibodies, a monoclonal IgM-kappa protein, an increased frequency of mature Ig containing blood B cells and a high production of Ig in vitro in unstimulated cultures. E rosetting cells showed faint or no immunofluorescence staining with monoclonal antibodies directed against T cell membrane antigens. In vitro Ig production in response to pokeweed mitogen was defective, and no T cell helper or suppressor activity was observed. It is suggested that the immunoregulatory deficiency might have caused the B cell hyperactivity.