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OBJECTIVE: Clinical heterogeneity, a hallmark of systemic autoimmune diseases, impedes early diagnosis and effective treatment, issues that may be addressed if patients could be classified into groups defined by molecular pattern. This study was undertaken to identify molecular clusters for reclassifying systemic autoimmune diseases independently of clinical diagnosis. METHODS: Unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data on 955 patients with 7 systemic autoimmune diseases and 267 healthy controls was undertaken. In addition, an inception cohort was prospectively followed up for 6 or 14 months to validate the results and analyze whether or not cluster assignment changed over time. RESULTS: Four clusters were identified and validated. Three were pathologic, representing "inflammatory," "lymphoid," and "interferon" patterns. Each included all diagnoses and was defined by genetic, clinical, serologic, and cellular features. A fourth cluster with no specific molecular pattern was associated with low disease activity and included healthy controls. A longitudinal and independent inception cohort showed a relapse-remission pattern, where patients remained in their pathologic cluster, moving only to the healthy one, thus showing that the molecular clusters remained stable over time and that single pathogenic molecular signatures characterized each individual patient. CONCLUSION: Patients with systemic autoimmune diseases can be jointly stratified into 3 stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of nonresponse to therapy, marking a paradigm shift in our view of systemic autoimmune diseases. ispartof: ARTHRITIS & RHEUMATOLOGY vol:73 issue:6 pages:1073-1085 ispartof: location:United States status: published

Background/objectives The European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) 2019 classification criteria for systemic lupus erythematosus system showed high specificity, while attaining also high sensitivity. We hereby analysed the performance of the individual criteria items and their contribution to the overall performance of the criteria. Methods We combined the EULAR/ACR derivation and validation cohorts for a total of 1197 systemic lupus erythematosus (SLE) and n=1074 non-SLE patients with a variety of conditions mimicking SLE, such as other autoimmune diseases, and calculated the sensitivity and specificity for antinuclear antibodies (ANA) and the 23 specific criteria items. We also tested performance omitting the EULAR/ACR criteria attribution rule, which defines that items are only counted if not more likely explained by a cause other than SLE. Results Positive ANA, the new entry criterion, was 99.5% sensitive, but only 19.4% specific, against a non-SLE population that included other inflammatory rheumatic, infectious, malignant and metabolic diseases. The specific criteria items were highly variable in sensitivity (from 0.42% for delirium and 1.84% for psychosis to 75.6% for antibodies to double-stranded DNA), but their specificity was uniformly high, with low C3 or C4 (83.0%) and leucopenia Conclusions Changing the position of the highly sensitive, non-specific ANA to an entry criterion and the attribution rule resulted in a specificity of >80% for all items, explaining the higher overall specificity of the criteria set.

Funder: American College of Rheumatology Research and Education Foundation; FundRef: http://dx.doi.org/10.13039/100000960, Funder: National Institute of Arthritis and Musculoskeletal and Skin Diseases; FundRef: http://dx.doi.org/10.13039/100000069, Funder: European League Against Rheumatism; FundRef: http://dx.doi.org/10.13039/501100008741, OBJECTIVES: The European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) 2019 Classification Criteria for systemic lupus erythematosus (SLE) have been validated with high sensitivity and specificity. We evaluated the performance of the new criteria with regard to disease duration, sex and race/ethnicity, and compared its performance against the Systemic Lupus International Collaborating Clinics (SLICC) 2012 and ACR 1982/1997 criteria. METHODS: Twenty-one SLE centres from 16 countries submitted SLE cases and mimicking controls to form the validation cohort. The sensitivity and specificity of the EULAR/ACR 2019, SLICC 2012 and ACR 1982/1997 criteria were evaluated. RESULTS: The cohort consisted of female (n=1098), male (n=172), Asian (n=118), black (n=68), Hispanic (n=124) and white (n=941) patients; with an SLE duration of 1 to

Background Clinical heterogeneity, a hallmark of systemic autoimmune diseases (SADs) impedes early diagnosis and effective treatment, issues that may be addressed if patients could be grouped into a molecular defined stratification. Methods With the aim of reclassifying SADs independently of the clinical diagnoses, unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data of 918 patients with 7 SADs and 263 healthy controls was undertaken. An inception cohort prospectively followed for 6 and 14 months was studied to validate the results in early cases and analyze if cluster assignment was modified with time. Results Four clusters were identified Three aberrant clusters were ‘acute phase inflammatory’, ‘T cell immunity’, and ‘interferon’, each including all diagnoses, were defined by genetic, clinical, serological and cellular features. A fourth cluster showed no specific molecular pattern, to which 74% of healthy controls clustered with patients. The inception cohort showed that most patients were either assigned always to the same cluster or moved from the healthy-like cluster to a single aberrant cluster resembling the relapsing-remitting dynamic of these diseases, showing that single aberrant molecular signatures characterize each individual patient. Conclusions Patients with SADs share molecular signatures and can be therefore stratified into three disease clusters differentiating each patient into a specific molecular disease pathway. Such assignment is stable with time. These results have important implications for understanding disease progression and therapy design marking a paradigm shift in our view of SADs. Acknowledgment This work has been supported through a grant from the Innovative Medicines Initiative Joint Undertaking No. 115565 and in-kind and in-cash contributions from the EFPIA partners. G.B. is supported by the Instituto de Salud Carlos III (ISCIII, Spanish Health Ministry), through the Sara Borrell subprogram (CD18/00153). The authors would like to particularly express their gratitude to the patients, nurses and many others who helped directly or indirectly in the consecution of this study.

Background Supported by both the ACR and EULAR, the EULAR/ACR 2019 Classification Criteria for SLE employ positive ANA (ever) as an entry criterion and use a weighted scheme with values ranging from 2 to 10, for a classification cut-off of 10. Criteria items are attributed to SLE only if there is no more likely alternative diagnosis in the individual patients. Items are organized in domains, and only the highest ranking item within a domain is counted. These criteria have been validated in a cohort of 696 SLE patients and 574 non-SLE patients from a total of 21 centers, reaching an overall sensitivity of 96.1% and a specificity of 93.4%. To at least estimate the performance in groups underrepresented in the validation cohort of this transatlantic project, we analyzed this cohort for patient subsets with regard to sex, ethnicity, and disease duration. Methods The full EULAR/ACR 2019 classification criteria validation cohort was analyzed for female (n=1,098) and male (n=172) patients, Asian (n=118), Black (n=68), Hispanic (n=124) and White (n=941) patients, and patients with an SLE duration of less than 1 year (n=34), one to less than 3 years (n=196), 3 to less than 5 years (n=157), and 5 or more years (n=879). Sensitivity and specificity were calculated for the EULAR/ACR 2019 criteria, the SLICC 2012 criteria and the ACR 1997 criteria each. Results As shown in table 1, most of the point estimates for sensitivity and specificity in subsets lay within the 95% confidence intervals of the sensitivity and specificity of the EULAR/ACR 2019 criteria validation. In particular, sensitivity and specificity for all ethnic groups were within the confidence intervals or even higher. Formally, the sensitivity was slightly lower for male patients, corresponding to a higher specificity, but the male 95% confidence intervals (0.86–0.98 for sensitivity, 0.90–0.99 for specificity) overlapped. While sensitivity appeared independent of disease duration from year 1 on, sensitivity was only 89% in the first year of disease, identical to the SLICC criteria (89%) and numerically higher than the ACR criteria (56%), but all confidence intervals overlapped. Conclusion While not all subgroups of SLE patients in the validation cohort are of adequate size to fully explore the sensitivity and specificity of the EULAR/ACR 2019 SLE classification criteria in the respective subsets, the point estimates of sensitivity and specificity suggest that the new criteria perform at least reasonably well in all ethnic groups, in men and in early disease. Nevertheless, sensitivity and specificity should be independently validated in larger groups of Asian, Black and Hispanic patients, male patients and in early disease.

SUMMARYBackgroundClinical heterogeneity, a hallmark of systemic autoimmune diseases (SADs) impedes early diagnosis and effective treatment, issues that may be addressed if patients could be grouped into a molecular defined stratification.MethodsWith the aim of reclassifying SADs independently of the clinical diagnoses, unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data of 918 patients with 7 SADs and 263 healthy controls was undertaken. In addition, an inception cohort was prospectively followed for 6 and 14 months to validate the results and analyze if cluster assignment changed or not with time.ResultsFour clusters were identified. Three clusters were aberrant, representing ‘inflammatory’, ‘lymphoid’, and ‘interferon’ patterns each including all diagnoses and defined by genetic, clinical, serological and cellular features. A fourth cluster showed no specific molecular pattern and accumulated also healthy controls. An independent inception cohort showed that with time, the molecular clusters remain stable, showing that single aberrant molecular signatures characterize each individual patient.ConclusionsPatients with SADs can be jointly stratified into three stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of therapy non-responsiveness marking a paradigm shift in the view of SADs.

ObjectiveNon-pharmacological interventions support patients with connective tissue diseases to better cope with and self-manage their diseases. This study aimed to map existing evidence on non-pharmacological interventions in patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and mixed connective tissue diseases regarding content, feasibility and potential suitability in an e-health setting.MethodsA literature search was performed in eight different databases in July 2020. The intervention’s content was extracted using the ‘Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide’. A Sankey diagram and descriptive statistics were used to analyse the data and illustrate the relationships between the interventions.ResultsOf 8198 identified records, 119 papers were eligible. One hundred and four of them (87.4%) were conducted between 2000 and 2020, mainly in the USA (SLE n=24 (21.2%), SSc n=16 (14.2%)), Brazil (SLE n=8 (7.1%), SSc n=5 (4.4%)) and Italy (SLE n=0 (0%), SSc n=12 (10.6%)). Fifty-two studies (SLE n=24 (21.2%), SSc n=28 (24.8%)) used multicomponent interventions. The single interventions were physical exercises (SLE n=16 (14.2%), SSc n=17 (15.0%)), coaching/counselling (SLE n=11 (18.0%), SSc n=0 (0%)) and education (SLE n=2 (1.8%), SSc n=3 (2.7%)). Primary outcomes focused on physical function (SLE n=1 (0.9%), SSc n=15 (13.3%)), mouth opening in SSc (n=4 (5.9%)) and physical capacity (SLE n=2 (1.8%), SSc n=1 (0.9%)). No interventions for mixed connective tissue disease were found.ConclusionThere was a great variety in the intervention’s content due to differences in body structure, activity limitations and participation restrictions in SLE and SSc. These results highlight the need for personalised, multicomponent, non-pharmacological interventions, which could be delivered as e-health interventions.

Summary Autoantibodies play an important role in the pathophysiology of renal involvement in systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), systemic vasculitis, and anti–glomerular basement membrane disease (or Goodpasture syndrome). Direct removal of autoantibodies therefore has been tried in various ways, first by plasma exchange. Today, immunoadsorption is the extracorporeal method that most effectively removes (pathogenic) immune complexes and antibodies. Although past data have shown efficacy and biocompatibility of immunoadsorption in (renal) SLE, it is still an experimental and expensive procedure, and evidence from randomized controlled trials is needed. Nevertheless, immunoadsorption is being used as a rescue therapy in life-threatening situations of SLE patients because of its fast mode of action and its acceptable safety profile. In granulomatosis with polyangiitis (GPA) (or Wegener’s granulomatosis), microscopic polyangiitis (MPA), and anti–glomerular basement membrane disease, the current standard is plasma exchange. Immunoadsorption, which probably would reduce the autoantibody burden more effectively, might be an even better more effective option, but sufficient evidence is lacking.

Background: Autoimmune Connective Tissue Disorders (CTDs), including Mixed Connective Tissue Disease (MCTD), systemic sclerosis (SSc) and systemic lupus erythematous (SLE) can lead to Raynaud phenomenon, involvement of internal organs, pain in joints, skin abnormalities and muscle weakness. Consequently, people with CTDs experience limitations in activities of daily life and restrictions in participation. Programmes promoting patient education, self-management, physical activity, and healthy life style, provided by health professionals are therefore essential to these patients. However, there is a substantial gap of knowledge about the content which should be included in non-pharmacological intervention programmes. Objectives: To identify, map and appraise the content of existent face-to-face, digital and/or distance-learning non-pharmacological intervention programmes for patients with MCTD, SSc and/or SLE to inform a EULAR funded project with the overall aim to develop a new distance learning programme realizable with digital technologies. Methods: A scoping literature review with a nested descriptive meta-analysis was performed in MEDLINE [Pubmed], EMBASE [OVID], CINAHL [EBSCO], PsycINFO [ProQUEST], the Cochrane Database of Systematic Reviews, OTSeeker, PEDro, and SciELO in September 2018. Furthermore, an international task-force identified additional grey literature and ongoing research projects. Eligibility checks of the articles based on pre-defined in- and exclusion criteria and data extractions were done independently by two researchers. The interventions contained in these programmes were extracted using the "Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide". A Sankey diagram and descriptive statistics were used to illustrate the relationships between the interventions and other characteristics of the studies. The protocol was pre-published on ResearchGate. Results: Of 6667 identified records, 94 papers were eligible. Sixty-one studies (65%) were conducted between 2000 and 2018, mainly in the USA (35 [37%]), Brazil (11 [12%]) and Italy (10 [11%]). Thirty-three studies (35%) were randomised controlled trials, 17 (18%) were one-group pre-post-test designs and 12 (16%) clinical trials without randomisation (Figure 1). Overall, 4274 patients participated; 2955 had SLE [69%] and 1319 had SSc [31%], thus no studies with MCTD where found. The main content of the intervention programmes included exercises (22 [23%]), self-management (9 [10%]) and education (7 [7%]), with treatment goals focusing mainly on physical function (27 [29%]), quality of life (19 [20%]) and fatigue (12 [16%]). The interventions (67 [72%]) included or were based on an initial face-to-face component. Conclusion: There was a variety of programmes content and interventions due to different consequences regarding activity limitations and participation restrictions in autoimmune CTDs. This highlights the need for personalized, multicomponent, non-pharmacological interventions. Disclosure of Interests: Valentin Ritschl: None declared, Ricardo Ferreira: None declared, Ruben Fernandes: None declared, Eduardo Santos: None declared, Kim Fligelstone: None declared, Helena Gaspar: None declared, Linda Schraven: None declared, Judy Ammerlaan: None declared, Georg Stummvoll: None declared, Maria Joao Salvador: None declared, Janet L Poole: None declared, Cornelia van den Ende: None declared, Carina Bostrom: None declared, Tanja Stamm Grant/research support from: TS has received grant support from AbbVie., Paid instructor for: TS has received speaker fees from AbbVie, Janssen, MSD, Novartis, and Roche.

Background:EULAR/ACR 2019 SLE Classification Criteria were validated in an international cohort.Objectives:To evaluate performance characteristics of SLE classification systems in sex, race/ethnicity, and disease duration subsets.Methods:Sensitivity and specificity of the EULAR/ACR 2019, SLICC 2012 and ACR 1982/1997 criteria were evaluated in the validation cohort.Results:The cohort consisted of female (n=1098), male (n=172), Asian (n=118), Black (n=68), Hispanic (n=124) and White (n=941) patients; and patients with an SLE duration of 1-3 years (n=196), 3-5 years (n=157), and ≥5 years (n=879). Among patients with 1-3 years disease duration, the EULAR/ACR criteria had better sensitivity than the ACR criteria (97% (95%CI 92-99%) vs 81% (95%CI 72-88%). The new criteria performed well in men (sensitivity 93%, specificity 96%) and women (sensitivity 97%, specificity 94%). The new criteria had better sensitivity than the ACR criteria in White (95% vs 83%), Hispanic (100% vs 86%) and Asian patients (97% vs 77%).Conclusion:The EULAR/ACR 2019 criteria perform well in patients with early disease, and across sexes and ethnicities.Disclosure of Interests:Sindhu Johnson Grant/research support from: Boehringer Ingelheim, Corbus Pharmaceuticals, GlaxoSmithKline, Roche, Merck, Bayer, Consultant of: Boehringer Ingelheim, Ikaria, Ralph Brinks: None declared, Karen Costenbader Grant/research support from: Merck, Consultant of: Astra-Zeneca, David Daikh: None declared, Marta Mosca: None declared, Rosalind Ramsey-Goldman: None declared, Josef S. Smolen Grant/research support from: AbbVie, Eli Lilly, Janssen, Merck Sharp & Dohme, Pfizer, Roche – grant/research support, Consultant of: AbbVie, Amgen Inc., AstraZeneca, Astro, Celgene Corporation, Celtrion, Eli Lilly, Glaxo, ILTOO, Janssen, Medimmune, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Samsung, Sanofi, UCB – consultant, Speakers bureau: AbbVie, Amgen Inc., AstraZeneca, Astro, Celgene Corporation, Celtrion, Eli Lilly, Glaxo, ILTOO, Janssen, Medimmune, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Samsung, Sanofi, UCB – speaker, David Wofsy: None declared, Dimitrios Boumpas Grant/research support from: Unrestricted grant support from various pharmaceutical companies, Diane L Kamen Consultant of: Consulted on SLE survey development for Lilly and consulted on SLE trial protocol development for EMD Serono in 2019, David Jayne Grant/research support from: ChemoCentryx, GSK, Roche/Genentech, Sanofi-Genzyme, Consultant of: Astra-Zeneca, ChemoCentryx, GSK, InflaRx, Takeda, Insmed, Chugai, Boehringer-Ingelheim, Ricard Cervera: None declared, Nathalie Costedoat-Chalumeau Grant/research support from: UCB to my institution, Betty Diamond: None declared, Dafna D Gladman Grant/research support from: AbbVie, Amgen Inc., BMS, Celgene Corporation, Janssen, Novartis, Pfizer, UCB – grant/research support, Consultant of: AbbVie, Amgen Inc., BMS, Celgene Corporation, Janssen, Novartis, Pfizer, UCB – consultant, Bevra H. Hahn Grant/research support from: Janssen Research & Development, LLC, Falk Hiepe: None declared, Soren Jacobsen: None declared, Dinesh Khanna Shareholder of: Eicos Sciences, Inc./Civi Biopharma, Inc., Grant/research support from: Dr Khanna was supported by NIH/NIAMS K24AR063120, Consultant of: Acceleron, Actelion, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Corbus Pharmaceuticals, Horizon Therapeutic, Galapagos, Roche/Genentech, GlaxoSmithKline, Mitsubishi Tanabe, Sanofi-Aventis/Genzyme, UCB, Kirsten Lerstrom: None declared, Elena Massarotti: None declared, William Joseph McCune: None declared, Guillermo Ruiz-Irastorza: None declared, Jorge Sanchez-Guerrero: None declared, Matthias Schneider: None declared, Murray B Urowitz: None declared, George Bertsias Grant/research support from: GSK, Consultant of: Novartis, Bimba F. Hoyer: None declared, Nicolai Leuchten: None declared, Chiara Tani: None declared, Sara Tedeschi: None declared, Zahi Touma: None declared, Gabriela Schmajuk Grant/research support from: Pfizer, Branimir Anic: None declared, Florence Assan: None declared, Tak Chan: None declared, Ann E Clarke: None declared, Mary K. Crow: None declared, László Czirják Consultant of: Actelion, BI, Roche-Genentech, Lilly, Medac, Novartis, Pfizer, Bayer AG, Andrea Doria Consultant of: GSK, Pfizer, Abbvie, Novartis, Ely Lilly, Speakers bureau: UCB pharma, GSK, Pfizer, Janssen, Abbvie, Novartis, Ely Lilly, BMS, Winfried Graninger: None declared, Bernadett Halda-Kiss: None declared, Sarfaraz Hasni: None declared, Peter Izmirly: None declared, Michelle Jung: None declared, Gabor Kumanovics Consultant of: Boehringer, Teva, Speakers bureau: Roche, Lilly, Novartis, Xavier Mariette: None declared, Ivan Padjen: None declared, Jose M Pego-Reigosa: None declared, Juanita Romero-Diaz Consultant of: Biogen, Iñigo Rua-Figueroa: None declared, Raphaèle Seror Consultant of: BMS, Medimmune, Novartis, Pfizer, GSK, Lilly, Georg Stummvoll: None declared, Yoshiya Tanaka Grant/research support from: Asahi-kasei, Astellas, Mitsubishi-Tanabe, Chugai, Takeda, Sanofi, Bristol-Myers, UCB, Daiichi-Sankyo, Eisai, Pfizer, and Ono, Consultant of: Abbvie, Astellas, Bristol-Myers Squibb, Eli Lilly, Pfizer, Speakers bureau: Daiichi-Sankyo, Astellas, Chugai, Eli Lilly, Pfizer, AbbVie, YL Biologics, Bristol-Myers, Takeda, Mitsubishi-Tanabe, Novartis, Eisai, Janssen, Sanofi, UCB, and Teijin, Maria Tektonidou Grant/research support from: AbbVie, MSD, Novartis and Pfizer, Consultant of: AbbVie, MSD, Novartis and Pfizer, Carlos Vasconcelos: None declared, Edward Vital Grant/research support from: AstraZeneca, Roche/Genentech, and Sandoz, Consultant of: AstraZeneca, GSK, Roche/Genentech, and Sandoz, Speakers bureau: Becton Dickinson and GSK, Daniel J Wallace: None declared, Sule Yavuz: None declared, Pier Luigi Meroni: None declared, Marvin Fritzler: None declared, Raymond Naden: None declared, Thomas Dörner Grant/research support from: Janssen, Novartis, Roche, UCB, Consultant of: Abbvie, Celgene, Eli Lilly, Roche, Janssen, EMD, Speakers bureau: Eli Lilly, Roche, Samsung, Janssen, Martin Aringer Consultant of: Boehringer Ingelheim, Roche, Speakers bureau: Boehringer Ingelheim, Roche

ObjectiveTo develop new classification criteria for systemic lupus erythematosus (SLE) jointly supported by the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR).MethodsThis international initiative had four phases. (1) Evaluation of antinuclear antibody (ANA) as an entry criterion through systematic review and meta-regression of the literature and criteria generation through an international Delphi exercise, an early patient cohort and a patient survey. (2) Criteria reduction by Delphi and nominal group technique exercises. (3) Criteria definition and weighting based on criterion performance and on results of a multi-criteria decision analysis. (4) Refinement of weights and threshold scores in a new derivation cohort of 1001 subjects and validation compared with previous criteria in a new validation cohort of 1270 subjects.ResultsThe 2019 EULAR/ACR classification criteria for SLE include positive ANA at least once as obligatory entry criterion; followed by additive weighted criteria grouped in seven clinical (constitutional, haematological, neuropsychiatric, mucocutaneous, serosal, musculoskeletal, renal) and three immunological (antiphospholipid antibodies, complement proteins, SLE-specific antibodies) domains, and weighted from 2 to 10. Patients accumulating ≥10 points are classified. In the validation cohort, the new criteria had a sensitivity of 96.1% and specificity of 93.4%, compared with 82.8% sensitivity and 93.4% specificity of the ACR 1997 and 96.7% sensitivity and 83.7% specificity of the Systemic Lupus International Collaborating Clinics 2012 criteria.ConclusionThese new classification criteria were developed using rigorous methodology with multidisciplinary and international input, and have excellent sensitivity and specificity. Use of ANA entry criterion, hierarchically clustered and weighted criteria reflect current thinking about SLE and provide an improved foundation for SLE research.

Background This 4 phase project1 jointly supported by EULAR and ACR has led to draft criteria.2 Objectives To simplify and validate the new criteria in a large international cohort. Methods 23 expert centres each contributed up to 100 patients with SLE and with non-SLE diagnoses. Diagnoses were verified by 3 independent reviewers for 1,193 SLE and 1059 non-SLE patients. 500 randomly selected SLE and non-SLE patients formed the derivation cohort and the remainder the validation cohort. Results The criteria were fine-tuned and simplified, using ANA of ≥1:80 as entry criterion and a classification threshold of 10. Sensitivity was close to the SLICC 2012 criteria, specificity maintained at the level of the ACR 1997 criteria. This performance was independently confirmed in the validation cohort. Conclusions The new criteria developed with EULAR/ACR support achieved sensitivity close to the SLICC criteria, while maintaining the specificity of the ACR criteria. References [1] Aringer, et al. Ann Rheum Dis2017;76(S2):4. [2] Tedeschi, et al. Ann Rheum Dis2017;76(S2):50. Disclosure of Interest None declared

Background Pristane induced lupus (PIL) is an established murine model of induced systemic lupus erythematosus (SLE). Mice develop specific autoantibodies and show symptoms of SLE including arthritis, glomerulonephritis and haemorrhagic pulmonary capillaritis. Objectives To investigate the therapeutic effects of in vitro-induced regulatory T cells (iTreg) in the murine model of PIL. Methods BALB/c mice were injected i.p. with 0.5 ml of pristane (PIL) or PBS (control). Naive CD4+ thymocytes were sorted and cultured and cell suspensions with >80% CD4+FoxP3+ cells (iTreg) were injected i.v. (i) once when PIL was induced (5 × 106 iTreg (iTreg-single)) or (ii) every 4 weeks (1 × 106 iTreg, iTreg-rep). Mice were monitored for paw swelling and grip strength. After 8 months, histological analysis quantified cartilage degradation, number of osteoclasts, extent of inflammation and bone erosion. Glomerulonephritis and pneumonitis were quantified using the kidney biopsy score and a histomorphometric image analysis system; inflammatory tissue was analysed by tissue cytometry. Serum levels of auto-antibodies were measured by ELISA. Frequencies of B cells, activated and regulatory CD4+ T cells and Th1, Th2, Th17 cells were measured by flow cytometry. FlowCytomix Pro was used to measure serum cytokines. RT-qPCR was used to measure expression levels of interferon (IFN)-signature and T-cell subset related as well as inflammation-associated genes. Results Monthly injections of 1 × 106 iTreg reduced the clinical and histological severity of PIL-arthritis. This was seen by a higher mean grip strength, less mean paw swelling, retardation of symptom onset and a lower summative arthritis severity score (figure A). There was significant reduction of arthritis severity in all histological parameters and in the percentage of affected mice with erosive arthritis (33% of iTreg-rep mice vs 62% of PIL-mice). A single boost of 5 × 106 iTreg could not prevent joint manifestation, however a slight retardation in ‘loss of grip strength’ and significantly less erosive area was seen. In regards to cellular composition of the inflammatory tissue in paws, a significantly increased relative amount of CD4 +Foxp3+cells was seen in the iTreg-rep group compared to PIL group. Repeatedly injected mice (iTreg-rep) had significantly less renal disease (glomerular activity score) (figure B) and pulmonary involvement (perivascular inflammatory area) (figure C) compared to PIL. iTreg-rep mice had significantly lower serum levels of disease-associated auto-antibodies (figure D). Upon restimulation, splenic CD4+ cells in iTreg-rep showed a less pronounced Th1, Th2 and Th17 response as well as lower percentages in B- and activated T-cells, whereas the percentage of Treg was higher than in all other groups (figure E). FlowCytomix analysis showed reduced cytokine production after repeated iTreg administration. Correspondingly, iTreg-rep showed decreased expressions of IFN-signature-, T-cell subset- and inflammatory cytokine related genes (figure F). Conclusions Repeated injections of iTreg ameliorate the clinical, histological, serological as well as the genetic severity of PIL-manifestations. A single boost of iTreg at time of disease induction does not prevent manifestations, but retards the onset of symptoms. Thus iTreg have significant positive effects on PIL, which may have consequences for future approaches in treating SLE. Disclosure of Interest None declared

Objective We herein examine the role of endogenous miR155 in the development of systemic manifestations in pristane induced lupus. Materials and methods Systemic lupus in miR155-deficient and wild type mice was induced upon injection of pristane and analyzed after 8 months, PBS-injected mice served as controls. Glomerulonephritis and pneumonitis were quantified using the kidney biopsy score and a newly adapted histomorphometric image analysis system; lung tissue was further analyzed by tissue cytometry. Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Frequencies of B cells, activated and regulatory CD4+ T cells as well as Th1, Th2, Th17 cells were measured by flow cytometry. RT-qPCR was used to measure expression levels of interferon-signature and T-cell subset related as well as miR155-associated genes. Results After induction of lupus, miR155-deficient mice had significant less pulmonary involvement (perivascular inflammatory area in mm2/mm2 lung area 0.00092±0.00015 vs. 0.0027±0.00075, p = 0.0347) and renal disease (glomerular activity score 1.95±0.19 vs 3±0.26, p = 0.0029) compared to wild types. MiR155-deficient mice had significantly lower serum levels of disease-associated auto-antibodies and decreased frequencies of activated CD4+CD25+ (Foxp3-) cells. Upon restimulation, CD4+ cells showed a less pronounced Th2 and Th17 and a slightly decreased Th1 response in mir155-deficient mice. Pristane-treated wild types showed significantly up-regulated expression of genes related to the INF-signature (MX1, IP10, IRF7, ISG15). Conclusions MiR155-deficient mice had less severe organ involvement, lower serum auto-antibody levels, a less prominent T cell response and lower expressions of genes jointly responsible for disease development. Thus, antagonizing miR155 might be a future approach in treating SLE.

Summary Systemic lupus erythematosus (SLE) is a heterogeneous disease with a vast variety of clinical manifestations. Timely diagnosis is important for gaining access to specific therapy and care. In this survey, we asked SLE patients with an established diagnosis to report about their personal history and their daily life with SLE in order to gain knowledge about diagnostics, treatment pathways and potential problems in daily living and functioning. In most cases, the diagnosis of SLE was made by a specialist in rheumatology or dermatology. Of the patients 41.5% were diagnosed within the first year after onset of disease symptoms, while 37.3% of the patients waited for 3 or more years for the final diagnosis of SLE. Interestingly, we found no differences with respect to patients living in urban or rural areas. Specific therapy worked well in many but not in all patients: the majority of patients reported problems with paid work, social life and leisure activities including traveling. Patients reported a need for better information for the general public about SLE. In addition, they wanted be better informed themselves. Despite all successful efforts in recent years, there is still room for improvement with respect to early diagnosis, early start of specific therapy and for better information of the public on the mysterious disorder named SLE.

Background. Systemic lupus erythematosus (SLE) is characterized by pathogenic autoantibodies, which can be removed by extracorporeal procedures. While previous studies have shown short-term efficacy of immunoadsorption (IAS) in SLE, no information on long-term benefit and safety is available. Methods. IAS was offered to patients with highly active renal disease when conventional therapy had failed. Eleven patients entered the prolonged IAS programme and were followed for up to 10 years (mean 6.4 6 3.5). Efficacy of IAS was determined by reduction in proteinuria (primary outcome), global disease activity [SLE Disease Activity Index (SLEDAI)] and anti-double-stranded DNA (antidsDNA) levels (secondary outcomes). Full/partial remission was defined as � 0.5/� 1.0 g/day for proteinuria, � 5/ � 8 for SLEDAI and � 25/� 50 IU/mL for anti-dsDNA levels. We further assessed flares, infections, malignancies and procedure-related adverse events. Results. Short-term IAS (� 1 year) resulted in a significant reduction of proteinuria (9.2 6 3.7 to 2.3 6 2.4, P ¼ 0.0001), disease activity (SLEDAI 19 6 8t o 46 2, P ¼ 0.0004) and dsDNA levels (168 6 205 to 45 6 34, P ¼ 0.001). In patients without remission after 1 year (n ¼ 5), prolonged IAS decreased proteinuria from 4.3 6 2.4 to 0.5 6 0.4 g/day, P ¼ 0.02. At the end of observation, complete remission in proteinuria was achieved in seven patients (64%) and partial remission in two (18%) additional patients. One patient flared and was discontinued; in all other patients, disease activity and anti-dsDNA stabilized at remission levels. Flares (0.28 6 0.30) and infections (0.66 6 0.70 per patient/year) were relatively uncommon; no malignancies, anaphylactic or orthostatic adverse events were observed. Conclusion. IAS is effective in short-term use but prolonged IAS can provide additional therapeutic benefit while showing an acceptable safety profile. The vast majority of initially therapy-refractory patients met the remission criteria at the end of observation.

ObjectiveTo describe the experiences of people with systemic sclerosis (SSc) in different European countries of functioning and health and to link these experiences to the WHO International Classification of Functioning, Disability and Health (ICF) to develop a common understanding from a bio-psycho-social perspective.MethodA qualitative multicentre study with focus-group interviews was performed in four European countries: Austria, Romania, Sweden and Switzerland. The qualitative data analysis followed a modified form of ‘meaning condensation’ and the concepts that emerged in the analysis were linked to the ICF.Results63 people with SSc participated in 13 focus groups. In total, 86 concepts were identified. 32 (37%) of these were linked to the ICF component body functions and structures, 21 (24%) to activities and participation, 26 (30%) to environmental factors, 6 (7%) to personal factors and 1 (1%) to the health condition itself. 19 concepts (22%) were identified in all four countries and included impaired hand function, household activities, paid work, drugs, climate and coldness, support from others and experiences with healthcare institutions, non-pharmacological treatment, social security and benefits.ConclusionConcepts identified in all four countries could be used for guiding clinical assessment, as well as interdisciplinary team care and rheumatological rehabilitation for patients with SSc. For a full understanding of the aspects of the disease that were most relevant to people with SSc, people with SSc from multiple countries needed to be involved.

CD4+ T cells from the TCR transgenic TxA23 mouse recognize a peptide from the H/K-ATPase α-chain. When TxA23 CD4+ thymocytes are differentiated into Th1, Th2, and Th17 lines, all three subpopulations induced autoimmune gastritis (AIG) upon transfer into nu/nu recipients. The induction of AIG by naive T cells or by Th1 or Th2 cell lines could be prevented by the cotransfer of polyclonal Foxp3+ T regulatory cells (nTreg), whereas Th17-induced AIG was resistant to suppression. We compared the capacity of different types of Treg to suppress Th17-mediated AIG. Cotransfer of either nTreg or polyclonal TGFβ-induced Treg (iTreg) did not prevent AIG, while cotransfer of TGFβ-induced Ag-specific TxA23 iTreg completely prevented the development of disease. Ag-specific iTreg were able to suppress Th17-mediated disease when injected 6 days after the Th17 effectors. The implications of these results for the use of Treg for the cellular biotherapy of autoimmune disease are discussed.

Th cells can be subdivided into IFN-γ-secreting Th1, IL-4/IL-5-secreting Th2, and IL-17-secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H+K+-ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced disease only at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in humans.

Objective:To address key aspects of anti-histone autoimmunity in systemic lupus erythaematosus (SLE), we performed a detailed characterisation of cellular and humoral autoreactivity to histone H1 and the four core histones H2A, H2B, H3, H4 in patients with SLE and healthy controls.Methods:Peripheral blood mononuclear cells of 41 patients with SLE and 28 healthy controls were exposed to individual histones and proliferation was measured by [3H]-thymidine incorporation. H1-reactive T cell clones were obtained by limiting dilution. Cytokines and total IgG in culture supernatants was measured by ELISA, and autoantibodies to histones were determined by ELISA and immunoblotting.Results:Proliferative responses to H1 were more frequent and more pronounced in cell cultures from patients with SLE (pConclusions:Histone H1 constitutes a major B cell and T cell autoantigen in SLE, triggering a proinflammatory Th1 response and driving autoantibody production. This suggests that histone H1 may be of considerable relevance for the pathogenesis of SLE.

Objective T cell intracytoplasmic antigen 1 (TIA-1) and TIA-1–related protein (TIAR) are involved in posttranscriptional regulation of the expression of tumor necrosis factor α (TNFα) and other proteins. Given the pivotal role of TNFα in chronic inflammatory diseases, this study was undertaken to analyze sera from patients with systemic autoimmune diseases for the presence of autoantibodies to TIA proteins and to investigate the expression of these proteins in inflamed tissue. Methods The presence of autoantibodies to TIA proteins in sera from 385 patients with rheumatic diseases and healthy controls was determined by immunoblotting using recombinant antigens. Expression of TIA proteins in skin and kidney tissue was analyzed by immunohistochemistry. Serum levels of TNFα were measured by enzyme-linked immunosorbent assay. Results Autoantibodies to TIA-1 and/or TIAR were detected in 61% of patients with systemic lupus erythematosus (SLE), 42% of patients with systemic sclerosis (SSc), 15–31% of patients with other rheumatic diseases, and 6% of healthy controls. Compared with patients negative for anti-TIA antibody, anti-TIA antibody–positive SLE patients had higher disease activity (P = 0.01), elevated antibodies to double-stranded DNA (P = 0.0003), and increased serum TNFα levels (P = 0.018). In SLE patients, anti-TIAR antibodies were associated with lupus nephritis (P = 0.02), while in patients with SSc, anti–TIA-1 was associated with lung involvement (P = 0.02). Immunohistochemical analysis of skin and kidney tissue revealed aberrant expression of TIA proteins in skin lesions from SLE and SSc patients, as well as in glomerular cells from SLE patients. Conclusion Aberrant expression of TIA proteins in inflammatory tissue may lead to systemic autoantibody responses, particularly in SLE and SSc. Increased occurrence of anti-TIA autoantibodies in patients with severe organ involvement may point to a possible pathogenetic role.

The LE cell has been one of the first immunological signs of active systemic lupus erythematosus, included into the ACR criteria. LE cells consist of a phagocyte engulfing material of disputed origin, which was interpreted as either cellular remnants from necrotic cells or as early apoptotic cells. It is well established that LE cell formation is dependent on autoantibodies against the linker histone H1. In view of this fact, we investigated whether anti-histone H1 antibodies and LE cell positive sera bound to cells where apoptosis had been induced by gliotoxin or actinomycin D or which were necrotic after heating. Necrotic cell remnants, but not (early) apoptotic cells were bound by anti-histone H1 antibodies and LE cell positive sera, establishing that the process of LE cell formation, which is dependent on anti-H1 binding, leads to engulfment of necrotic (or late apoptotic) material, but not of early apoptotic cells.

To test whether the active metabolite of leflunomide (LEF-M), in addition to blocking the proliferation of activated lymphocytes by inhibiting dihydro-orotate dehydrogenase (DHODH), influences the transendothelial migration (TEM) of peripheral blood mononuclear cells (PBMC).In an in vitro model of PBMC transmigration through an endothelial cell (EC) barrier, PBMC were re-collected in three groups: cells not adherent to the EC, cells bound to, and cells which had migrated through, the EC layer. Experiments in which cells were pretreated with LEF-M (in the absence or in the presence of uridine) were compared with parallel experiments in the presence of medium alone.Preincubation of EC with LEF-M led to a 36 (SEM 16)% reduction in PBMC TEM (p0.05). Likewise, preincubation of PBMC induced a reduction in their TEM of 39 (9)% (p0.005). Incubation of both PBMC and EC with LEF-M had an additive effect (mean reduction of 48 (6)%, p0.005). Incubation of PBMC with LEF-M also decreased monocytic CD44 expression (p0.005) and PBMC-hyaluronan binding (p0.05). Incubation of cells with LEF-M and uridine in addition to LEF-M reversed the inhibition of migration, suggesting that the observed effects were due to DHODH inhibition. Fluorocytometric analysis of PBMC subsets within the migrated population showed a decrease of monocytes, but not of B or T cells, after LEF-M treatment.LEF-M reduces monocytic adhesion molecule expression and TEM and may thus interfere with monocyte and EC activities in RA. Thus, the clinical effects of leflunomide may, at least in part, be due to blocking cell traffic into the inflamed synovia.

OBJECTIVE: In systemic lupus erythematosus (SLE), extracorporeal procedures aiming at reduction of immunoglobulin (Ig) and immune complexes (IC) are used as a rescue therapy. Plasma exchange (PE) has not been proven overall effective in SLE, and long-term treatment in particular has been associated with severe bacterial and viral infections. Immunoadsorption (IAS), in contrast, selectively removes Ig and IC and may thus be safer. We therefore investigated the rate of infections in SLE patients who were undergoing long-term IAS. METHODS: 16 SLE patients were treated with ≥10 courses of IAS, and nine patients with highly active disease received pulse cyclophosphamide (IVCP) therapy in parallel. We retrospectively analysed the records of all these patients for the occurrence of infections. Patients receiving IAS therapy plus IVCP were compared with 25 patients with similarly active disease treated with standard IVCP therapy within the same observation period. Patients receiving IAS without additional IVCP were compared with patients with similarly moderate disease activity receiving neither IAS nor IVCP. RESULTS: No potentially life-threatening viral infection occurred in IAS-treated patients and episodes of herpes zoster were equally distributed. No severe infection was observed during IAS without concomitant cyclophosphamide. As expected, more patients with highly active disease receiving IVCP experienced infections than those with less active disease (16 of 34 [47%] vs. 2 of 22 [9%], p < 0.04). On comparing the two groups with highly active disease, infections were similar (IAS plus IVCP, 3 of 9 patients [33%]; IVCP only, 5 of 25 [20%]), but one patient receiving IAS plus IVCP died of septicaemia. Disease activity significantly decreased in both groups treated with IAS. CONCLUSION: IAS has an acceptable safety profile with regard to severe infections and appears safe with regard to severe viral disease. Highly active disease and IVCP therapy increase the risk of severe infections in SLE.

Objectives In Primary Sjogren’s Syndrome (PSS), there is an apparent lack of data concerning the perspectives of patients, their needs, preferences and difficulties of daily life. This qualitative study was conducted to explore perspectives and needs of patients with PSS that influence health related quality of life (HRQL). Methods We recruited 20 PSS patients fulfilling the American-European consensus classification criteria out of the PSS cohort of the Medical University Graz, Austria. In total, 6 focus group sessions (with three to four patients per group) were performed. A modified meaning condensation procedure was used to analyse the data. Results The interview analysis resulted in 484 meaning units, 254 subconcepts and 86 concepts. The identified concepts were grouped into three dimensions: physical dimension, psychological & emotional challenges and social life & daily living. A dependency between the three categories was identified. The concepts most commonly reported by patients were related to the physical dimension: pain and dryness as well as complaints associated with/provoked by these symptoms. Patients also reported shortness of breath, fatigue und constipation. Conclusions This qualitative study underpins that HRQL in PSS patients is affected by several factors. The problems are not limited to dryness, pain and fatigue while the complaints secondary to these symptoms are important to patients with PSS significantly affecting physical, psychological and social life components of HRQL. A disease-specific patient related outcome measures for clinical practice and trials should be developed considering the different aspects of HRQL in PSS.

Introduction: Scurfy mice are deficient in regulatory T cells (Tregs), develop a severe, generalized autoimmune disorder that can affect almost every organ and die at an early age. Some of these manifestations resemble those found in systemic lupus erythematosus (SLE). In addition, active SLE is associated with low Treg numbers and reduced Treg function, but direct evidence for a central role of Treg malfunction in the pathophysiology of lupus-like manifestations is still missing. In the present study, we characterize the multiorgan pathology, autoantibody profile and blood count abnormalities in scurfy mice and show their close resemblances to lupus-like disease. Methods: Scurfy mice have dysfunctional Tregs due to a genetic defect in the transcription factor Forkhead box protein 3 (Foxp3). We analyzed skin, joints, lung and kidneys of scurfy mice and wild-type (WT) controls by conventional histology and immunofluorescence (IF) performed hematological workups and tested for autoantibodies by IF, immunoblotting and enzyme-linked immunosorbent assay. We also analyzed the intestines, liver, spleen and heart, but did not analyze all organs known to be affected in scurfy mice (such as the testicle, the accessory reproductive structures, the pancreas or the eyes). We transferred CD4+ T cells of scurfy or WT mice into T cell-deficient B6/nude mice. Results: We confirm previous reports that scurfy mice spontaneously develop severe pneumonitis and hematological abnormalities similar to those in SLE. We show that scurfy mice (but not controls) exhibited additional features of SLE: severe interface dermatitis, arthritis, mesangioproliferative glomerulonephritis and high titers of anti-nuclear antibodies, anti-double-stranded DNA antibodies, anti-histone antibodies and anti-Smith antibodies. Transfer of scurfy CD4+ T cells (but not of WT cells) induced autoantibodies and inflammation of lung, skin and kidneys in T cell-deficient B6/nude mice. Conclusion: Our observations support the hypothesis that lupus-like autoimmune features develop in the absence of functional Tregs.

Objective Arthritis is frequently seen in human lupus, but rarely in lupus models. Pristane-induced lupus (PIL) can be induced in various mouse strains such as BALB/c and C57BL/6. We herein characterize clinical and histological features of arthritis in the context of systemic lupus and provide a prudent comparison with models of rheumatoid arthritis (RA). Methods A total of 57 BALB/c mice received pristane (PIL group) and were analyzed for serum autoantibodies (anti-chromatin-, -histone, -Sm, -dsDNA), as well as for clinical features and histopathology of joints, lungs and kidneys. Joint pathology was quantified by image analysis and tissue cytometry. Ten C57BL/6 mice (Bl/6-PIL) and historical groups of two different RA models were analyzed accordingly. Results In BALB/c PIL, clinical arthritis started at three months, occurred finally in 79% of PIL (but not in controls, p 0.7, p Conclusion PIL in BALB/c mice is characterized by severe organ involvement, typical autoabs and by a mild-erosive arthritis with similarities to, but also with distinct differences from, RA. PIL may help to study arthritis along with other key features of systemic lupus erythematosus after therapeutic interventions or in knock-out models based on a BALB/c but not on a C57BL/6 background.

Background Spondyloarthritis (SpA) is a common debilitating inflammatory disorder. Establishing the diagnosis is often difficult, since abnormalities in conventional X-ray develop with a latency of several years and only HLA-B27 is used as a laboratory marker. The goal of our study was to identify new autoantibodies as diagnostic markers of SpA. Methods Protein array technology was used to screen for new autoantigens in ankylosing spondylitis. Then, the results were confirmed by ELISA using Class II-associated invariant chain peptide domain of CD74 as antigen. Sera for the ELISA were obtained from 216 patients with axial (n=156) and peripheral (n=60) SpA. Sera of patients with psoriatic arthritis without axial involvement as another subtype of peripheral SpA, rheumatoid arthritis, systemic lupus erythematosus, HIV infection and blood donors served as controls. All donors provided informed consent for the study which was approved by the local ethics committee (project number 4928). Results Using protein arrays, we detected IgG antibodies against CD74 in SpA sera. Using ELISA technology on sera that had previously been frozen for several years, IgG autoantibodies against CD74 were found in 67% of the SpA patients and were even more frequent in patients with a short disease duration. In the controls, the prevalence of the new autoantibodies was 18/40 (45%) in psoriatic arthritis without axial involvement, 9/80 (11%) in rheumatoid arthritis, 6/40 (15%) in systemic lupus erythematosus, 1/40 (2.5%) in HIV and 1/125 (0.8%) in blood donors. Conclusions Antibodies against CD74 could provide an important additional tool for diagnosis of SpA.

Background Deregulation of endogenous miR155 was observed in many autoimmune conditions, including systemic lupus erythematosus (SLE). Objectives We herein examined the role of miR155 in the development of systemic manifestations in mice with pristane-induced lupus (PIL). Methods MiR155-deficient and C57/Bl6 mice were injected with pristane or PBS as control and analyzed after 8 months. Glomerulonephritis and pneumonitis were quantified by using the composite kidney biopsy score (KBS) and by analyzing the numbers of affected lung-vessels and the area of the inflammatory lung-infiltrate, respectively. Specimens were stained with B220 (B), CD3 (T), Neu7/4 (neutrophils) and F4/80 (macrophages) and analyzed by cell-identification algorithms for nuclear segmentation (HistoQuest). Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Frequencies of B cells, activated and regulatory CD4+ T cells as well as Th1, Th2, Th17 cells were measured by flow cytometry. Quantitative real-time polymerase chain reaction was used to measure expression levels of interferon-signature and T-cell subset related as well as miR155-associated genes. Results All pristane-injected mice showed signs of pneumonitis, while controls did not. Compared to wild types treated with pristane, similarly treated knockouts showed significantly decreased perivascular inflammatory area with B cells being the most prominent inflammatory cell. Wildtypes had a more severe renal involvement in the KBS than knockouts. Corresponding with reduced organ involvement, miR155 deficient mice had significantly lower serum levels of anti-dsDNA antibodies, anti-chromatin and anti-histone antibodies and decreased frequencies of activated CD4 + CD25 + (Foxp3 – ) cells. Interestingly, also frequencies of CD4 + CD25 + Foxp3 + regulatory T cells were lower in these mice. Upon restimulation, CD4 + cells showed a more pronounced Th2 response in wild types, but no significant differences in Th1 and Th17 phenotype. Regarding INF-signature and T-cell subset activation, pristane-treated wild types showed significantly up-regulated gene-expression patterns whereas equivalently treated mutants showed the same levels as PBS-treated controls. Conclusions MiR155 deficiency in PIL mice did not prevent disease, but was associated with less severe organ involvement, lower serum auto-abs levels, lower frequencies of Th2 cells, whereas lower expressions of genes jointly responsible for disease development may be one key mechanism. Thus, antagonisation of miR155 might be a future approach in treating SLE. Disclosure of Interest None declared

Background Even though joint involvement is a major burden in patients suffering from systemic lupus erythematosus (SLE), it is not focus of scientific research and treatment strategies continue to be limited. Studying lupus arthritis and possible treatment options can be done with the murine model of Pristane-induced lupus (PIL). Objectives We herein investigate if in vitro induced regulatory T-cells ( i Treg) are capable of ameliorating PIL arthritis and help us evaluate possible new treatment options. Methods BALB/c mice were injected i.p. with either 0.5ml of pristane (PIL-group) or PBS (controls). Naive CD4 + thymocytes were sorted and cultured and cell suspensions with >80% of CD4 + FoxP3 + cells ( i Treg) were injected intravenously (i) once when PIL was induced (5x10 6 i Treg, i Treg-boost), or (ii) every 4 weeks (1x10 6 i Treg, i Treg-rep). Animals were monitored for paw swelling and grip strength. After 8 months histological analysis evaluated for cartilage degradation, number of osteoclasts and the extent of inflammation and bone erosion. In addition, the cellular composition of the inflammatory tissue was determined by a cell-identification algorithm for nuclear segmentation (HistoQuest). Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Results Monthly injections of 1x10 6 i Treg reduced the clinical as well as the histological severity of PIL-arthritis, seen by a higher mean grip strength (2.964±0.024 vs. 2.732± 0.063, p i Treg-rep mice had erosive arthritis. There was a significant reduction of arthritis severity in all histological parameters (inflammatory area 0.188±0.0574 vs. 0.688±0.113; erosive area 0.011 ±0.009 vs. 0.069±0.017; number of osteoclasts 2.000± 1.125 vs. 9.143±1.999; cartilage degradation 0.059±0.004 vs. 0.187±0.033). The single boost of 5x10 6 i Treg could not prevent joint manifestation. However, a slight retardation in “loss of grip strength” and a significantly less erosive area was seen. In regards to the cellular composition of the inflammatory tissue, a significantly increased relative amount of Foxp3 cells was seen in the i Treg-rep group compared to the PIL group (5.2±2.3 vs. 0.6±0.2). Corresponding to the reduced severity in joint involvement, the i Treg-group had significantly lower serum levels of antibodies. Conclusions Repeated injections of i Treg ameliorate the clinical and histological severity of PIL- arthritis. A single boost of i Treg at the time of disease induction does not prevent joint manifestation, but retards the onset of symptoms and progression of erosive bone degradation. Thus, i Treg have significant positive effects on PIL arthritis, which may have consequence for future therapeutic considerations. Disclosure of Interest None declared

Immunoadsorption (IAS) is used as a rescue therapy in severely ill SLE patients who are refractory to conventional therapies. This extracorporeal method aims at the rapid and extensive removal of pathogenic immunocomplexes (ICs) and (auto-)antibodies (Abs). Although past data have shown short- to mid-term efficacy and biocompatibility of IAS in (renal) SLE, it is still an experimental and rather expensive procedure – and evidence from randomized controlled trials (RCTs) is lacking. Nevertheless, IAS is successfully used in life-threatening situations because of its fast mode of action and its acceptable safety profile.

Introduction Pathogenic autoantibodies (Abs) are a hallmark of SLE and their rapid removal is beneficial in active SLE. Immunoadsorption (IAS) is effective in removing serum levels of all classes of immunoglobulin (Ig), immune complexes (IC) and anti-dsDNA Abs and appears superior to plasmapheresis with respect to side effects. IAS can be performed with different columns, which use different ligands to bind their target. In particular, high affinity columns are in the focus of interest. Their ligands are either sheep IgG directed against human Ig (Ig-column, Ig-Therasorb(®)), or staphylococcal Protein A (ProtA-column, Immunosorba(®)), or the synthetic peptide Gam146 (GAM-column, Globaffin(®)). In our experience Ig-columns have been effective in treating active renal SLE. However, no analysis has so far been published on which column type should be preferred in treating SLE patients. Patients and methods Among our SLE patients maintained on prolonged IAS therapy, we identified those with stable renal SLE and low to moderate disease activity who were successfully treated by using Ig-columns. Six of these patients were switched to ProtA-columns, keeping the rest of the protocol and the medication constant. In addition, two patients were switched from Ig- to GAM-columns. Results All types of columns significantly lowered the serum levels of IgG, IgM, and anti-dsDNA Abs. Disease activity was constantly low before and after the switch, as were parameters of renal function. In addition, patients with highly active disease were effectively treated when ProtA- (n=6) or GAM-columns (n=1) were used as first-line extracorporeal treatment. Conclusion Our data demonstrate that all columns are adequately effective in controlling key parameters of SLE. Thus, it is not the type of the ligand, but only the outcome, i.e. the successful removal of Ig, IC, and (auto-) Abs that is required for controlling SLE activity.

Immunoadsorption (IAS) with various methods is used as a rescue therapy in severely ill SLE patients who are refractory to conventional therapeutic procedures. The method aims at the rapid and extensive removal of pathogenic immunocomplexes (IC) and (auto-) antibodies (Abs). Long-term observational studies suggested efficacy and have not seen an increase in the risk of infections (as were seen in other extracorporeal procedures). Unfortunately, prospective, randomized controlled trials (RCT) are lacking. Recently, biologicals aiming at TNF-blockade or B-cell depletion have been used to treat severe SLE: They are easier to apply since they do not necessitate additional (expensive) hardware or specially trained staff. While there is emerging evidence for efficacy from uncontrolled observations, no RCT could so far demonstrate benefit in SLE. Under these circumstances, IAS still has a role in treating severe SLE, when other therapies are not effective enough or are contraindicated (as in pregnancy). These data are reviewed and illustrated in the case of a pregnant lupus patient with nephrotic syndrome.

Naive antigen-specific CD4(+) T cells (TxA23) induce autoimmune gastritis when transferred into BALB/c nu/nu mice. Transfer of in vitro pre-differentiated Th1 or Th17 TxA23 effector T cells into BALB/c nu/nu recipients induces distinct histological patterns of disease. We have previously shown that co-transfer of polyclonal naturally occurring Treg (nTreg) suppressed development of Th1-, but not Th17-mediated disease. Therefore, we analysed the suppressive capacity of different types of Treg to suppress Th1- and Th17-mediated autoimmune gastritis. We compared nTreg with polyclonal TGFbeta-induced WT Treg (iTreg) or TGFbeta-induced antigen-specific TxA23 iTreg in co-transfer experiments with Th1 or Th17 TxA23 effector T cells. 6 weeks after transfer in vitro pre-differentiated TxA23 Th1 and Th17 effector cells induced destructive gastritis. Th1-mediated disease was prevented by co-transfer of nTreg and also antigen-specific iTreg, whereas WT iTreg did not show an effect. However, Th17-mediated disease was only suppressed by antigen-specific iTreg. Pre-activation of nTreg in vitro prior to transfer did not increase their suppressive activity in Th17-mediated gastritis. Thus, antigen-specific iTreg are potent suppressors of autoimmune gastritis induced by both, fully differentiated Th1 and Th17 effector cells.

Objective: To analyse the effects of rigorous immunoglobulin removal by immunoadsorption (IAS) on proteinuria (primary outcome variable), disease activity (SIS, SLEDAI, ECLAM), and autoantibodies to double stranded DNA (anti-dsDNA) in active systemic lupus erythematosus (SLE). Methods: 16 patients with severe SLE and renal disease, in whom cyclophosphamide was contraindicated or failed to halt disease progression, were treated with IAS for 3 months. Patients achieving at least 20% improvement in two or more of the outcome measures were considered responders and offered a 9 months' extension period. Results: Within 3 months, 14 patients responded and 11 opted for an extension. Proteinuria decreased from 6.7 (4.6) g/day (mean (SD)) at baseline to 4.3 (3.5) g/day at 3 months and 2.9 (2.4) g/day at 12 months (p

CD4+ T lymphocytes play an important part in the pathogenesis of scleroderma (systemic sclerosis, SSc) and predominate in perivascular SSc skin lesions. Both soluble and membrane bound adhesion molecules are overexpressed in SSc, possibly influencing lymphocyte/endothelial cell (EC) contact.To assess the transendothelial migration capacity of peripheral lymphocytes in vitro.Collagen was covered with human umbilical vein endothelial cells (HUVEC), and peripheral blood mononuclear cells (PBMC) of patients and matched healthy controls (HC) were added in parallel experiments. Before and after fractionated harvest of non-adherent, bound, and migrated lymphocytes, the CD4/CD8 ratio and the lymphocytic expression of activation markers and adhesion molecules were analysed by fluorocytometry.13 (SD 12)% of the SSc PBMC migrated compared with only 5 (5)% HC PBMC (p0.0002); this increase was primarily due to the migration of CD3+ T lymphocytes and mainly to a larger proportion of CD4+ cells within this CD3+ fraction (71 (SD 14)% for SSc v 56 (14)% for HC, p0.03), leading to an increased CD4/CD8 ratio among migrated SSc lymphocytes in comparison with controls (3.3 (1.5) v 1.62 (0.93), p0.006). Among migrated SSc CD4+ T lymphocytes, the frequency of HLA-DR+ cells was increased; migrated lymphocytes highly expressed the adhesion molecules CD11a, CD49d, CD29, and CD44.Transendothelial migration of CD4+ T lymphocytes is enhanced in SSc, and migrating cells exhibit an activated phenotype. The data suggest that activated CD3+CD4+ lymphocytes as found in SSc peripheral blood are prone to transvascular migration, thus contributing to the formation of typical perivascular lymphocytic infiltrates.

In systemic lupus erythematosus (SLE) serum TNF is increased and correlates with its soluble receptors and with disease activity. We therefore investigated (i) whether the TNF in SLE serum is bioactive, (ii) whether SLE cells react to TNF and (iii) whether there are associations with cell death, which is regarded as pathogenic in SLE. Sera from active SLE patients induced an increase in fibroblast CD54, which was abolished by blocking antibodies against TNF, suggesting TNF bioactivity. SLE lymphocytes had a similar surface expression of TNF-R I as healthy lymphocytes, their expression of TNF-R II was slightly increased. Recombinant TNF induced cell death in PBMC of SLE patients, suggesting functional receptors. Serum levels of sTNF-RII (as a surrogate marker for TNF activity) correlated with sTNF-RI and disease activity, as expected, and also correlated with the percentage of dying lymphocytes and with lymphocytic CD95. SLE sera contain increased amounts of biologically active TNF. Peripheral blood lymphocytes of SLE patients express functional TNF receptors. Finally, associations with cell death and CD95 receptors suggest that TNF may be pathogenic in SLE.

Background MicroRNAs (miRs) recently came into view as an important class of regulators of gene expression that are associated with a variety of biological functions. Deregulation of endogenous miR155 was observed in many autoimmune conditions, including systemic lupus erythematosus (SLE). Objectives We herein examine the role of miR155 in the development of systemic manifestations in murine pristane induced lupus (PIL) by evaluating the severity of organ involvement and assessing serum antibody-levels and T helper cell homeostasis. Methods MiR155-deficient (miR155-PIL group) and C57/Bl6 (WT-PIL group) mice were injected i.p. with 0.5ml of pristane or PBS as control (WT-PBS group). PIL is an induced model of SLE where typical auto-abs profile and organ involvement develop over a considerable time period ranging from 6 to 10 months. In order to observe the effects of miR155 deficiency in fully developed SLE, we analyzed the mice 8 months after induction. Animals were monitored for clinical signs of glomerulonephritis (GN) by using urine assay strips. In order to analyze and compare disease severity, a blinded specialist appraised histological features of GN using the composite kidney biopsy score (KBS), as described before. Histological features of pneumonitis were quantified by an image analysis system (Osteomeasure®): Lungs were scored for the severity of perivascular inflammation by analyzing the numbers of affected vessels and the area of the inflammatory infiltrate. In order to assess the composition of these infiltrates, specimens were stained with B220 (B), CD3 (T), Neu7/4 (neutrophils) and F4/80 (macrophages) and analyzed by cell-identification algorithms for nuclear segmentation (HistoQuest®). Lymphocytes were isolated from spleens and analyzed separately for each mouse by standard FACS procedures. For analysis of the Th1, 2 or 17 subsets, respectively, cells were re-stimulated in vivo with anti-CD3 and anti-CD28abs. Results Lungs were affected in both pristane-treated groups (WT-PIL and miR155-PIL), but not in controls. MiR155 deficient mice (miR155-PIL) had reduced lupus severity as indicated by significantly decreased perivascular inflammatory area with B cells being the most prominent inflammatory cell type in the HistoQuest analysis (Fig. 1A). Without showing clinical abnormalities WT-PIL had a more severe renal involvement in the kidney biopsy score than miR-PIL (Fig. 1B). Corresponding with reduced severity in organ involvement, miR155-PIL had lower serum levels of anti-dsDNA-abs (Fig. 1C), decreased frequencies of CD4 + cells (14.24±0.7587 vs. 18.04±1.075, p=0.01) and slightly lower frequencies of activated CD4 + CD25 + Foxp3 – cells (1.539±0.1279 vs. 1.838±0.2259, p=ns.). Interestingly, also frequencies of CD4 + CD25 + Foxp3 + regulatory T cells were lower in Mir155-PIL (1.689±0.1388 vs. 2.375±0.2320, p=0.03). Upon restimulation, CD4 + cells showed a more pronounced Th2 and Th17 response in WT-PIL, but no significant differences in Th1 phenotype (Fig. 1D). Conclusions MiR155 deficiency in PIL mice did not prevent the development of disease, but was associated with less severe lung and kidney involvement, lower serum auto-abs levels and lower Th17 and Th2 frequencies when analyzed in fully established PIL after 8 months. Thus, antagonisation of miR155 might be a beneficial future approach in treating SLE. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5340

Background CD4 + T cells and the Th1 and Th17 subsets in particular play a pivotal role in SLE. Regulatory T cells (Treg) are essential for maintaining peripheral tolerance, but their number and function in SLE is decreased. Objectives We herein characterize CD4+T cell and Treg homeostasis and severity of organ involvement in a murine model of SLE and analyze the effects of in vitro –induced iTreg. Methods Mice were injected i.p. with 0.5ml of pristane or PBS as control and killed after 8 months. Induction of Treg: Naive CD4 + thymocytes were cultured under specific conditions (including TGFβ, IL-2) and tested for CD4 + Foxp3 + expression by FACS (induced Treg, iTreg). Cell suspensions with >80% purity for CD4 + FoxP3 + iTreg were injected intravenously either once at start of experiments (5x10e6, iTreg-boost) or monthly (1x10e6, iTreg-repeated). Animals were monitored for clinical signs of arthritis and in order to analyze and compare disease severity, histological features of arthritis and pneumonitis were quantified by an image analysis system (Osteomeasure®). Lungs were scored for the severity of perivascular inflammation by analyzing the numbers of affected vessels and the area of the inflammatory infiltrate. Lymphocytes were isolated from granulomas (ectopic lymphoid tissue within the peritoneum), lymph nodes (LN) and spleens and were analyzed separately for each mouse by FACS. For analyzes of the Th1, 2, 17 subsets, respectively, cells were restimulated in vivo plate bound with anti-CD3 and anti-CD28abs. Results PIL mice presented with involvement of inner organs, most frequently affected were the lungs (100% pneumonitis); 52% of PIL developed arthritis, both clinically and histologically. Monthly iTreg-injection significantly decreased clinical signs of arthritis and histological lung and joint parameters. Out of 6 mice treated, 4 (66%) did not show any signs of arthritis at all. (Fig. 1A-F) The iTreg-boost did not prevent joint manifestations or pneumonitis, but appeared to have a retarding effect (Fig.1A-F) indicated by a delayed onset of clinical symptoms and by a significantly decreased erosive area at the end of observation (Fig.1B). Intraperitoneal granuloma typical for PIL appeared to be the hotspots of inflammation showing a significantly elevated Teff/Treg ratio of 1.3. Upon re-stimulation, CD4+ cells showed a pronounced Th1 response (27% IFNγ producers) compared to cells from LN and spleens from both PIL and HC (with Th1 percentages ranging from 9-16%). In addition, frequencies of Th2 and Th17 cells were elevated in PIL, again with the highest yield in granuloma. The repeated application of iTreg reduced the Teff/Treg ratio in PIL granuloma to 0.7. Conclusions Repeated injections of exogenously induced iTregs reduce severity of pneumonitis and arthritis as well as the T eff /T reg ratio. A single injection of iTregs (even when applied before the clinical onset) is not effective, but appears to retard onset of symptoms and progression of arthritis and pneumonitis. Thus, iTreg have significant effects on lupus symptoms when applied repeatedly, which may have consequence for future therapeutic considerations. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5386

Background The disease modifying drug leflunomide is widely used in the therapy of rheumatoid arthritis (RA). Its active metabolite, A771726, leads to inhibition of dihydroorotate dehydrogenase, an enzyme necessary for pyrimidine de-novo synthesis. Since activated lymphocytes expand their pyrimidine pool, A771726 decreases their proliferation. A771726 also suppresses TNF mediated nuclear factor kappaB activation. We were therefore interested if A771726 also would be capable to influence transendothelial migration (TEM) of peripheral mononuclear cells (PBMC). Objectives Methods We investigated the TEM of PBMC, which migrated through endothelial cell monolayers in an in-vitro model. Human umbilical vein endothelial cells (EC) were cultured to confluence on collagen gels and then incubated with human PBMC of healthy blood donors. PBMC were recollected in three groups: 1) cells that did not adhere to the endothelium, 2) cells that bound to the endothelium, 3) cells that had migrated through the endothelium, and then counted by microscope. Experiments in which EC and PBMC as well as only EC were treated with A771726 (in the absence or presence of uridine) were compared to simultaneously performed control experiments. No increased toxicity on the PBMC treated with the doses of A771726 used in our experiments, was observed. Paired T Test was used for statistical evaluations. Results 24 h incubation of PBMC and EC with A771726 induced a significant decrease in TEM (8 ± 6% vs. 16 ± 12% migrated cells, p = 0.004). Furthermore, 24 h preincubation of solely the EC with A771726 inhibited the TEM of untreated PBMC (11 ± 9% vs. 15 ± 8% migrated cells, p = 0.043), indicating direct effects of A771726 on EC. Uridine significantly reversed the decrease in TEM when incubating PBMC and EC with both uridine and A771726 (15 ± 10% vs 9 ± 7% migrated cells, p = 0.023). Conclusion Our results demonstrate that leflunomide may have direct anti-inflammatory effects by inhibiting the extravasation of PBMC, possibly via downregulation of adhesion molecules on EC and mononuclear cells. These data suggest, that leflunomide, besides its influences on lymphocyte proliferation, also reduces recruitement of mononuclear cells to inflammatory sites.

Background Active SLE patients undergoing plasmapheresis and cyclophosphamid therapy are at high risk of severe bacterial and viral infections. Immune adsorption (IAS) is a more novel approach to autoantibody removal with an unclear risk of infections in SLE. Objectives We therefore decided to investigate the risk of infections in patients undergoing longterm IAS. Methods We analysed the records of ten consecutive active SLE patients treated with long term IAS (mean 38 months) for the occurrence of severe infectious disease. We compared the IAS patients to historical control groups1 of 9 patients under plasmapheresis and cyclophosphamid (PP) and 12 patients treated with cyclophosphamid (CX) alone for similar time periods. Results One IAS patient died from gram-negative septicemia. Three localised bacterial infections (two gram-positive, one gram-negative) were treated with antibiotics. All infections occurred in the subgroup of five highly active patients (SIS >15, mean 17.7 ± 0.9) who all underwent additional CX therapy, showing characteristics similar to the CX and PP control groups (SIS 18.7 ± 2.4 and 20.3 ± 3.7, respectively). Bacterial infections were more frequent than in the CX group (4/5 vs 1/12, p Conclusion IAS did not promote severe viral infections, but was associated with an increased rate of potentially life-threatening bacterial infections in SLE patients with very active disease. Reference Aringer M, Smolen JS, Graninger WB. Severe infections in plasmapheresis-treated systemic lupus erythematosus. Arthritis Rheum. 1998;41(3):414–20

Recent onset arthritis reminiscent of rheumatoid arthritis (RA) may be an early manifestation of an occult malignancy. In this report, we present two patients with cancer-associated polyarthritis. Both suffered from symmetric polyarthritis when initially visiting their physicians and did not achieve relief when treated with non-steroidal anti-rheumatic drugs (NSAIDs). In both patients, subsequent work-up led to the diagnosis of an underlying malignancy. One patient suffered from small cell lung cancer (SCLC), while the other was diagnosed with adenocarcinoma of the colon. In both, the arthritis spontaneously disappeared after successful treatment of the malignancy, i.e. chemotherapy and tumor resection, respectively. We discuss these cases in view of the existing literature, since awareness of the entity of cancer polyarthritis is necessary for its timely treatment and may potentially be life-saving.