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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 and aim: Effective and safe behavioral health interventions in primary care are critical during pandemic and other disaster situations. California shelter-in-place orders necessitated rapid transition of an effective mindfulness-based medical group visit (MGV) program from in-person to videoconferenced sessions (VCSs). Aim: to Describe procedures, acceptability, and feasibility of converting from in-person to VCS. Patients and methods: Methods: qualitative. Dataset: primary care. Intervention: a six-session 2-h MGV program with educational and mindfulness components was converted. Four in-person sessions and two VCSs were held. General Anxiety Disorder and Patient Health Questionnaire-9 were administered at first and last sessions. A semi-structured focus group was conducted after session six. Population studied: six primary care patients (42 ± 11 years) with stress, anxiety, or depression participated. Results: Procedural changes included remote material distribution, scheduling, hosting, and facilitation functions using the Zoom platform. The focus group revealed that patients preferred in-person sessions during initial visits, but appreciated transitioning to VCS, which provided continued support during a challenging time. Instruction on technical (e.g. logging on) and social (e.g. signaling next speaker) aspects of VCS was suggested. Building relationships through conversations was an important part before and after in-person sessions missing from VCS. Patients suggested combining in-person and VCS to allow relationship building while also improving access. Conclusion: While many procedural changes were needed to facilitate conversion to VCS, primary care patients seeking stress, anxiety, and depression interventions found VCS acceptable during COVID-19. Future iterations of this program are proposed which incorporate procedural changes and facilitate relationship building between patients in VCS.

Mutations in two genes can result in activated PI3Kδ syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3Kδ inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase 1b study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15–25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 n = 3; APDS2 n = 4) received seletalisib; five completed the phase 1b study. For the extension study, four patients entered, one withdrew consent (week 24), three completed ≥84 wk of treatment. In the phase 1b study, patients had improved peripheral lymphadenopathy (n = 2), lung function (n = 1), thrombocyte counts (n = 1), and chronic enteropathy (n = 1). Overall, effects were maintained in the extension. In the phase 1b study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase 1b study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase 1b study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk–benefit profile was maintained for ≤96 wk.

Objectives This phase 2 proof-of-concept study (NCT02610543) assessed efficacy, safety and effects on salivary gland inflammation of seletalisib, a potent and selective PI3Kδ inhibitor, in patients with moderate-to-severe primary Sjögren’s syndrome (PSS). Methods Adults with PSS were randomized 1:1 to seletalisib 45 mg/day or placebo, in addition to current PSS therapy. Primary end points were safety and tolerability and change from baseline in EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI) score at week 12. Secondary end points included change from baseline at week 12 in EULAR Sjögren’s Syndrome Patient Reported Index (ESSPRI) score and histological features in salivary gland biopsies. Results Twenty-seven patients were randomized (seletalisib n = 13, placebo n = 14); 20 completed the study. Enrolment challenges led to early study termination with loss of statistical power (36% vs 80% planned). Nonetheless, a trend for improvement in ESSDAI and ESSPRI [difference vs placebo: –2.59 (95% CI: –7.30, 2.11; P=0.266) and –1.55 (95% CI: –3.39, 0.28), respectively] was observed at week 12. No significant changes were seen in saliva and tear flow. Serious adverse events (AEs) were reported in 3/13 of patients receiving seletalisib vs 1/14 for placebo and 5/13 vs 1/14 discontinued due to AEs, respectively. Serum IgM and IgG concentrations decreased in the seletalisib group vs placebo. Seletalisib demonstrated efficacy in reducing size and organisation of salivary gland inflammatory foci and in target engagement, thus reducing PI3K-mTOR signalling compared with placebo. Conclusion Despite enrolment challenges, seletalisib demonstrated a trend towards clinical improvement in patients with PSS. Histological analyses demonstrated encouraging effects of seletalisib on salivary gland inflammation and organisation. Trial registration https://clinicaltrials.gov, NCT02610543.

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.

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.

Sjogren’s syndrome (SS) is a chronic autoimmune disease characterised by B-cell hyper-activation and exocrine gland infiltration that results in loss of glandular function, systemic manifestations and autoantibody production. The phosphatidylinositol 3–kinase delta isoform (PI3Kδ) belongs to a family of intracellular lipid kinases that regulate metabolism, survival, proliferation, apoptosis, growth, and cell migration and has been successfully targeted in B-cell malignancies. Given the central role of the B-cells in the pathogenesis of SS we investigated evidence for the engagement of the PI3Kδ pathway in SS and the functional consequences of blocking PI3Kδ in an animal model of SS. PI3K pathway activation was investigated in salivary gland (SG) biopsies from patients with SS or non-specific chronic sialoadenitis (NSCS). Samples were stained for phosphorylated ribosomal protein S6 (pS6), a downstream effector in the PI3Kδ pathway. UCB5857, a small molecule inhibitor of PI3Kδ, was used either prophylactically or therapeutically in vivo in an inducible model of ectopic lymphoneogenesis in murine SGs that mimics SS. Flow cytometry, immunofluorescence and quantitative PCR was used on the murine isolated SG to evaluate the samples at peak of inflammation which is day 15 post-cannulation. Histological staining for pS6 showed significant expression of pS6 in SS samples relative to NSCS control, confirming engagement of the PI3K pathway. pS6 was detected within lymphoid aggregates in both T- and B-cell areas and on the periphery of the lymphoid foci in SG biopsies. pS6 staining was predominantly found on CD138 + plasma cells in SG biopsies. Mice treated either prophylactically or therapeutically with UCB5857 displayed decreased lymphocyte infiltration in cannulated salivary glands relative to vehicle treated mice. Additionally, a gene expression profile associated with ectopic lymphoneogenesis (CXCL13, CCL19, CCL21) was significantly impaired in mice treated with UCB5857. Lymphoid aggregates in these mice were characterised by decrease in focus score, smaller size and reduced T/B-cell follicular organisation. Preliminary data implicates activation of PI3Kδ pathway in several cells within the SGs of SS patients that may contribute towards pathology. Accordingly, prophylactic and therapeutic blocking of PI3Kδ results in disaggregation of the inflammatory foci and resolution of SG inflammation in an animal model of SS.

Background: Seletalisib is a potent, selective oral inhibitor of phosphoinositide-3 kinase delta (PI3Kδ). Preclinical data have shown that the PI3Kδ pathway is upregulated within salivary glands of patients with PSS and contributes to disease pathogenesis.1 Objectives: To assess the efficacy and safety of seletalisib in patients with PSS. Methods: In this Phase II, double-blind, proof of concept study (NCT02610543), patients with PSS having an EULAR Sjogren’s Syndrome Disease Activity Index (ESSDAI) score ≥5 were randomised 1:1 to seletalisib once daily or placebo (PBO) in addition to current PSS therapy for 12 weeks. The primary endpoint was change from baseline in ESSDAI at Week 12. The study was designed to have 80% power to detect a difference of 3.8 points in change from baseline in ESSDAI between seletalisib and PBO at Week 12 and required 58 patients to complete treatment. Other endpoints included EULAR Sjogren’s Syndrome Patient Reported Index (ESSPRI), salivary gland biopsy changes, Schirmer’s I test, immunoglobulin concentrations and incidence of treatment-emergent adverse events (TEAEs). Results: The study was terminated early due to slow recruitment, which led to study power decreasing to 36%. Twenty of 27 patients randomised (seletalisib n=13, PBO n=14) completed treatment. Demographic characteristics were generally similar between groups. Mean (SE) change from baseline in ESSDAI at Week 12 was seletalisib –5.4 (1.7) vs PBO –2.8 (1.5); treatment difference vs PBO (95% CI) was –2.59 (–7.30, 2.11; p=0.266). The percentages of patients achieving a ≥3 point reduction in ESSDAI were seletalisib 66.7% vs PBO 54.5%. Post-hoc Bayesian analyses of treatment difference showed an 86.5% probability of being superior to PBO and a 48.8% probability of a >3 point difference from PBO. Clinically notable improvements in some secondary endpoints were also observed in the seletalisib group (Table 1). Minor salivary gland biopsies had broadly similar histological features across groups at baseline. At Week 12, seletalisib treatment led to a reduction in the size and cellular organisation of mononuclear inflammatory cell foci vs PBO (Table 2). TEAEs were reported by 13/13 (100.0%) seletalisib and 13/14 (92.9%) PBO patients; most frequently reported: diarrhoea (5/13 [38.5%] vs 0/14 [0%]) and headache (3/13 [23.1%] vs 2/14 [14.3%]). Serious TEAEs were reported by 3/13 (23.1%) vs 1/14 (7.1%), and discontinuations due to TEAEs by 5/13 (38.5%) vs 1/14 (7.1%) seletalisib and PBO patients, respectively. Conclusion: Although this Phase II PSS study was terminated early due to slow recruitment, seletalisib demonstrated a trend to clinical improvement in patients with PSS and acceptable safety and tolerability. Histological analyses demonstrated encouraging effects of seletalisib on the organisation and extent of salivary gland lymphocytic infiltration in patients with PSS. References [1] Nayar S, et al. Ann Rheum Dis. 2019;78:249–260 Acknowledgement: Funded by UCB Pharma Disclosure of Interests: Maria Juarez Shareholder of: UCB Pharma, Employee of: UCB Pharma, Nieves Diaz Shareholder of: UCB Pharma, Employee of: UCB Pharma, Geoffrey I. Johnston Shareholder of: UCB Pharma, Pfizer, AstraZeneca, Employee of: UCB Pharma (and AstraZeneca + Pfizer previously), Saba Nayar: None declared, Andrew Payne Employee of: UCB Pharma, Eric Helmer Employee of: Galapagos, UCB Pharma, Takeda, Johnson & Johnson, Dionne Cain Employee of: UCB Pharma, Paulette Williams Shareholder of: UCB Pharma, Employee of: UCB Pharma, Wan Fai Ng: None declared, Benjamin Fisher Consultant for: Novartis, Roche, MedImmune, Bristol-Myers Squibb, Jacques-Eric Gottenberg Grant/research support from: Bristol-Myers Squibb, Grant/research support from: Bristol-Myers Squibb, Consultant for: Bristol-Myers Squibb, Lilly, Pfizer, Sanofi-Genzyme, UCB Pharma, Consultant for: Bristol-Myers Squibb, Eli Lilly, UCB, Sanofi-Genzyme, Pfizer, Giuliana Guggino Grant/research support from: Laborest, Pfizer, Consultant for: Novartis, Abbvie, Speakers bureau: Sandoz, Xavier Mariette Grant/research support from: Servier, Consultant for: AstraZeneca, Bristol-Myers Squibb, GlaxoSmithKline, Janssen, Pfizer, UCB Pharma, Marika Kvarnstrom: None declared, Valerie Devauchelle-Pensec Grant/research support from: Roche-Chugai, Speakers bureau: MSD, BMS, UCB, Roche, Jose Rosas Consultant for: Abbvie, Amgen, Bristol, Janssen, Lilly, Merck Sharp & Dohme, Pfizer, UCB Pharma, Speakers bureau: Abbvie, Amgen, Bristol, Janssen, Lilly, Merck Sharp & Dohme, Pfizer, UCB Pharma, Juan Sanchez-Burson Speakers bureau: Lilly, Janssen, Pfizer, Roberto Giacomelli Grant/research support from: Pfizer, Actelion, Speakers bureau: Actelion, Bristol-Myers Squibb, Merck Sharp & Dohme, Abbvie, Pfizer, Sobi, Roche, Francesca Barone Grant/research support from: GlaxoSmithKline, Roche, UCB Pharma, Actelion, ONO Pharmaceutical, Consultant for: GlaxoSmithKline, Roche, Actelion, ONO Pharmaceutical, Simon J. Bowman Grant/research support from: Previously UCB Pharma (to University of Birmingham) and Roche, Consultant for: 2016-7: Novartis, Mitsubishi Tanabe Pharma 2017-8: AstraZeneca, MedImmune, GFK, Xtlbio, ONO Pharmaceutical 2018-9: Novartis, AstraZeneca, UCB Pharma

The delta isoform of phosphoinositide 3-kinase (PI3Kδ) regulates various lymphocyte functions. Considering the key pro-inflammatory role of IL-17A and IL-17F cytokines in psoriasis and spondyloarthritis (SpA), we investigated the potential of PI3Kδ blockade to suppress IL-17A, IL-17F and associated pro-inflammatory cytokines that could synergize with IL-17A and IL-17F. Using in vitro studies with primary human cells and ex vivo studies with inflamed target tissues, we assessed if seletalisib, a selective PI3Kδ inhibitor, suppresses cytokine production by T cells and innate-like lymphocytes, and if seletalisib modulates the inflammatory responses in stromal cell populations in psoriasis (human dermal fibroblasts (HDF)) and SpA (fibroblast-like synoviocytes (FLS)). In vitro, seletalisib inhibited the production of pro-inflammatory cytokines, including IL-17A and IL-17F, from peripheral blood mononuclear cells (PBMCs), T helper 17 (Th17) cells as well as γδ-T cells and mucosal-associated invariant T cells. This inhibition resulted in decreased inflammatory activation of HDF in co-culture systems. Seletalisib was also efficacious in inhibiting SpA PBMCs and synovial fluid mononuclear cells (SFMCs) from producing pro-inflammatory cytokines. Furthermore, supernatant derived from cultured seletalisib-treated Th17 cells showed reduced potency for activating inflammatory responses from cultured SpA FLS and decreased their osteogenic differentiation capacity. Finally, analysis of inflamed SpA synovial tissue biopsies revealed activation of the PI3K-Akt-mTOR pathway. We observed that ex vivo seletalisib treatment of inflamed synovial tissue reduced IL-17A and IL-17F expression. Collectively, inhibition of PI3Kδ reduces the production of pro-inflammatory cytokines from IL-17-producing adaptive and innate-like lymphocytes and thereby inhibits downstream inflammatory and tissue remodeling responses. PI3Kδ-targeting may therefore represent a novel therapeutic avenue for the treatment of IL-17-mediated chronic inflammatory diseases such as psoriasis and SpA.

Objectives The PRECISESADS project aims at using OMICs, and bioinformatics to identify new classifications for systemic autoimmune diseases (SADs) known to share common pathophysiological mechanisms in view of personalised treatments. Multi OMICs parameters collected in addition to routine clinical data in a cross-sectional study involving patients suffering from systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjogren’s syndrome (Sjs), rheumatoid arthritis (RA), primary antiphospholipid syndrome (PAPs), mixed connective tissue disease (MCTD), undifferentiated connective tissue disease (UCTD) and healthy controls (HC) will be analysed to identify clinically relevant clusters. Methods A European multi centre, non-randomised, cross-sectional clinical study was conducted in 18 sites and 9 countries. Collection of OMIC data including genetic, epigenomic, transcriptomic (from peripheral blood and from isolated cells), flow cytometry, metabolomics and proteomic in plasma and urine, exosome analysis and classical serology (antibodies and autoantibodies) was organised. Novel and innovative methodologies including fine flow cytometry were conducted. Quality procedures were established to ensure standardisation of samples collection, processing, transportation and storage. Techniques were validated to ensure reproducibility of analyses. Unsupervised bioinformatics and biostatistics approaches will be applied. Results Recruitment started in December 2014 and ended in October 2017. A total of 2656 participants were recruited: 377 RA, 470 SLE, 402 SSc, 385 SjS, 99 MTCD, 106 PAPs, 166 UCTD patients and 651 HCs. Median age was between 46 and 59 years and was consistent with each disease onset peak. 97% of the population was Caucasian. Most of the patients were treated with standard of care therapies and less than 10% were on biologics. OMICs and bioinformatics analyses are on-going. Conclusions We have established one of the largest collaborative multi-OMICs studies from patients with SADs. The most important challenge is now the integration of all these novel data to support hypothesis-free, machine learning-led analytical protocols. It is expected that the integration of data from affected patients, in comparison with well-matched controls, will provide new biomarker-led descriptions of clusters of potentially etiologically distinct disease entities. Acknowledgements This work has received support from the EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS grant n° 1 15 565 Disclosure of Interest None declared

Background Autoantibodies directed against antigens with post-translational modifications (PTMs), such as citrullination (ACPA), are a hallmark of rheumatoid arthritis (RA)1. ACPA titres increase prior to disease onset, but are thought to be relatively stable after symptomatic inflammation is established2. The temporal profile of antibodies against acetylated (AAPA) and carbamylated (ACarPA) peptides has not been so comprehensively characterised following the onset of joint swelling3. Objectives We aimed to track serum levels of anti-PTM antibodies over 18 months in patients with newly-presenting RA in our prospective observational cohort. Methods Patients with treatment-naive inflammatory arthritis donated serum at baseline, 6 and 18 months. 103 patients satisfying ACR/EULAR 2010 criteria for RA underwent testing for IgG and IgA antibodies against peptides with citrulline (ACPA), carbamylated lysine (ACarPA), and acetylated lysine (AAPA) PTMs using ELISA as previously described4. Results 57% of participants were female, and 48% and 50% patients were anti-CCP2 or rheumatoid factor positive respectively. Mean age was 56 years (s.d. 15.2), symptom duration 55 days (s.d 22.4), and DAS28CRP 4.4 (s.d. 1.3) at enrolment. Comparing baseline and 18 month median antibody levels measured by optical density, a decrease was observed over time for IgG (0.26 vs 0.17, p Conclusions Median AAPA and, to a lesser extent, ACPA levels fell over time, regardless of therapy. This was most marked for the IgA isotype. Differential isotype effects may represent maturation of the autoantibody repertoire from mucosal IgA antibodies involved in the breach of tolerance. Lability of AAPA levels may reflect the relative reversibility of acetylation of a lysine amino acid residue by comparison with citrullination and carbamylation modifications. By contrast, and as observed previously3, IgG ACarPA levels increased over time, perhaps reflecting the tendency of the human proteome to accrue carbamylation modifications due to ageing, and metabolic or inflammatory stress. References [1] Trouw LA, et al. Nat Rev Rheumatol2017;13: 331–9. [2] Modi S, et al. Clin Exp Immunol2013;173(1):8–17. [3] Spinelli FR, et al. J Rheum Dis Treat2015;1:13. [4] Juarez M, et al. Ann Rheum Dis2016;75:1099–1107. Acknowledgements This work was funded via an EU FP7 grant (EuroTeam). KR is funded by the Birmingham NIHR Biomedical Research Centre. Disclosure of Interest None declared

Background PI3Kδ is predominantly expressed in lymphocytes; the role it plays in immune disease has encouraged the development of inhibitors targeting its kinase activity. Inhibitors of PI3Kδ have been approved or are in clinical development for the treatment of B cell malignancies; their therapeutic potential in inflammatory and autoimmune disease is being explored. Seletalisib is a selective and potent PI3Kδ inhibitor that has been profiled in preclinical and early clinical studies. Objectives To assess the therapeutic potential of seletalisib in inflammatory and autoimmune disease. Methods In vitro cell-based studies were performed on blood samples taken from healthy volunteers (HV) or from patients with primary Sjogren9s Syndrome (pSS) and/or psoriasis. In addition, samples were analysed from a single ascending dose study in HV (NCT02207595) and a multiple ascending dose study in HV and psoriasis patients (NCT02303509). T and B cell responses in the presence and absence of seletalisib were assessed by flow cytometry or a Meso-Scale discovery assay (Meso-Scale Diagnostics, MD, USA), following activation of the T cell- or B cell-receptor by receptor cross-linking or via stimulation with specific antigens. The functional selectivity of seletalisib was assessed in the BioMap system (DiscoverX, CA, USA). Translational studies used flow cytometry and/or immunofluorescence to demonstrate the presence of active PI3K signalling in diseased tissue, through expression of phosphorylated-AKT (pAKT) or -ribosomal protein S6 (pS6). The effect of seletalisib on PI3K signalling was determined by phospho-flow cytometry. Target engagement was determined through measurement of basophil degranulation in healthy subjects, and assessment of lesion severity score in skin biopsies from patients with psoriasis that were treated with seletalisib. Results Preclinical studies showed seletalisib potently inhibited T cell differentiation and function (IC 50 range: 2–31 nM). Further, it blocked activation and proliferation of B cells (IC 50 range: 16–49 nM). When profiled in a wide range of primary cell assay systems, including fibroblasts, epithelial, endothelial and vascular smooth muscle cells, seletalisib showed significant activity only in those systems containing lymphocytes, demonstrating its functional selectivity towards PI3Kδ-expressing cells. Expression of the PI3K pathway in lymphocytes was shown at the site of disease in clinical samples both from patients with psoriasis and pSS. Seletalisib inhibited PI3K signalling, measured by a reduction in pAKT and pS6 expression, in T cells derived from patients with psoriasis. In first-in-man studies, mean seletalisib plasma concentration-time profiles increased with increasing dose after single and multiple dosing, with no major deviations from dose proportionality. There was no unexpected accumulation or loss of exposure after multiple dosing (time-independent pharmacokinetic (PK) profile) and apparent t 1/2 values (approx. 20h) were supportive of once-daily dosing. Inhibition of basophil degranulation in healthy subjects and effects on the cellular composition in lesional skin biopsies from patients with psoriasis, provided indications of target engagement, following treatment with seletalisib. Conclusions Seletalisib is a potent, selective PI3Kδ inhibitor with an attractive preclinical and human PK profile; clinical studies are ongoing. Disclosure of Interest A. Payne Shareholder of: UCB Pharma, Employee of: UCB Pharma, M. Juarez Shareholder of: UCB Pharma, Employee of: UCB Pharma, G. Johnston Shareholder of: UCB Pharma, Employee of: UCB Pharma, E. Helmer Shareholder of: UCB Pharma, Employee of: UCB Pharma, M. Thomson Shareholder of: UCB Pharma, Employee of: UCB Pharma

Although carbon (C) additions to soil have been used in restoration to combat invasive species through changes in soil nitrogen (N) availability, carbon amendments to soil derived from plant material can impact soil N availability in a species-specific manner. As such, amendment-driven feedbacks on N may impact invasive species success and woody encroachment. Soil amendments like biochar, which is often added to soil to increase C storage in grassland systems, may unintentionally encourage woody encroachment into these grasslands by changing soil N dynamics. Few studies have examined biochar impacts on non-agricultural species, particularly invasive species. Woody encroachment of Eastern Red Cedar (Juniperus virginiana) into grasslands provides an ideal context for examining the impact of biochar in grasslands. In the greenhouse, we examined the effect of biochar or leaf litter derived from native and exotic grasses on J. virginiana seedling growth. Juniperus virginiana seedlings grew 40% bigger in biochar amended soil as compared to seedlings grown in litter amended soil. Additionally, we found a more than 2 order of magnitude increase in available NH4+ in the biochar treatments compared to the litter amended soils. Furthermore we found that biochar feedstock type did not have an impact on the effect of biochar, as both native and exotic grass biochar had similar impacts on soil N levels and J. virginiana growth. Our work suggests that once grassland litter is converted to biochar, species impacts on soil N may disappear. In conclusion, our data suggests soil amendments of biochar may encourage woody encroachment into grasslands.

Mexico is one of the most important contributors of multidrug resistance tuberculosis (MDR-TB) in Latin-America, however little is known about the molecular characteristics of these strains. For this reason, the objective of this work was to determine the genotype and characterize polymorphisms in genes associated with resistance to rifampicin, isoniazid, and second-line drugs in isolates from two regions of Mexico with high prevalence of drug resistant tuberculosis. Clinical isolates from individuals with confirmed MDR-TB were genotyped using MIRU-VNTR 12 loci. To characterize the polymorphisms in genes associated with resistance to rifampicin, isoniazid and second-line drugs; rpoB, katG, inhA, rrs, eis, gyrA, gyrB and tlyA were sequenced. 22 (41%) of the 54 MDR-TB isolates recovered were from the state of Baja California, while 32 (59%) were from Veracruz. The results show the katGS315T mutation was observed in 20% (11/54) of the isolates, while rpoBS315L was present in 33% (18/54). rrs had three polymorphisms (T1239C, ntA1401C and ntA1401G), gyrB presented no modifications, whereas gyrA showed five (S95T, F60Y, A90V, S91P and P124A), eis two (G-10A and A431G) and tlyA one (insertion at codon 67). Only 20% (11/54) of isolates were confirmed as MDR-TB by sequencing, and no mutations at any of the genes sequenced were observed in 43% (23/54) of the strains. Two isolates were recognized with the proper set of mutations like pre-XDR and one was XDR-TB. Eighteen isolates were classified as orphans and the remaining thirty-six were distributed in fourteen lineages, the most frequent were S (11%), Haarlem (9%), Ghana (9%) and LAM (7%). Out of the fourteen clusters identified, seven included unknown genotypes and nine had lineages. This is one of the most detailed analyses of genotypic characteristics and mutations associated with drug resistance to first and second-line drugs in MDR-TB isolates from Mexico. An important genetic variability and significant discrepancy between phenotypic tests and polymorphisms was observed. Our results set the need to screen additional loci as well as implement a molecular epidemiological surveillance system of MDR-TB in the country.

In an urban jail population, 3 months of isoniazid and rifapentine (3HP) was associated with an 85% latent tuberculosis infection treatment completion rate compared with 18% in a standard 9-month isoniazid treatment group. Among the 91 patients who started 3HP therapy, there were 2 treatment discontinuations from adverse drug reactions.

Background Synovial fibroblasts play a key role in joint destruction and regulation of the inflammatory infiltrate in established rheumatoid arthritis (RA). The mechanisms by which this occurs in the earliest stages of RA are largely unknown. We investigated the role of Dickkopf-related protein 1 (DKK1) produced by synovial fibroblasts of patients with very early rheumatoid arthritis (VeRA). Methods Fibroblasts were isolated from the disease-modifying anti-rheumatic drug–naive Birmingham early arthritis cohort of patients with new onset of clinically apparent arthritis and inflammatory symptoms of ≤12 weeks’ duration, who at follow-up had either resolving arthritis or RA. Endothelial fibroblast co-cultures were formed using porous filters, and lymphocyte adhesion to co-cultures was assessed using phase-contrast microscopy. DKK1 gene expression and secretion were quantified by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results Synovial fibroblasts from patients with VeRA expressed significantly higher levels of DKK1 messenger RNA than those from patients with resolving arthritis. A similar trend was observed for DKK1 protein secretion. In co-culture constructs, more DKK1 tended to be secreted in co-cultures incorporating fibroblasts from VeRA than in co-cultures from non-inflamed joints and resolving arthritis. DKK1 secretion during co-culture positively correlated with lymphocyte adhesion. Conclusions Alterations in DKK1 could be involved in the pathogenesis and perpetuation of the inflammatory response in the earliest clinically apparent stages of RA.

Ethambutol inhibits arabinogalactan and lipoarabinomannan biosynthesis in mycobacteria. The occurrence of mutations in embB codon 306 in ethambutol-susceptible isolates and their absence in resistant isolates has raised questions regarding the utility of this codon as a potential marker for resistance against ethambutol. The characterization of mutations on embB 306 will contribute to a better understanding of the mechanisms of resistance to this drug; therefore, the purpose of this study was to investigate the association between embB 306 mutations and first-line drug resistance profiles in tuberculosis isolates. We sequenced the region surrounding the embB 306 codon in 175 tuberculosis clinical isolates, divided according to drug sensitivity, in three groups: 110 were resistant to at least one first-line drug, of which 61 were resistant to ethambutol (EMB r ), 49 were sensitive to ethambutol (EMB s ) but were resistant to another drug, and 65 were pansensitive isolates (P s ). The associations between embB 306 mutations and phenotypic resistance to all first-line drugs were determined, and their validity and safety as a diagnostic marker were assessed. One of the P s isolates (1/65), one of the EMB s isolates (1/49), and 20 of the EMB r isolates (20/61) presented with an embB 306 mutation. Four different single-nucleotide polymorphisms (SNPs) at embB 306 were associated with simultaneous resistance to ethambutol, isoniazid, and rifampin (odds ratio [OR], 17.7; confidence interval [CI], 5.6 to 56.1) and showed a positive predictive value of 82%, with a specificity of 97% for diagnosing multidrug resistance associated with ethambutol, indicating its potential as a molecular marker for several drugs.

This article explores the influence of demographic, sociocultural, and risk proneness indicators on age at first intercourse, number of lifetime sexual partners, and number of pregnancies in a sample of 670 Latina adolescents age 14-19 recruited from two publicly funded familyplanning clinics. Acculturation accounted for a small but statisticallysignificant amount of the variation explained for all three reproductive behaviors. Being less acculturated was associated with older age at initiation of sexual intercourse, fewer lifetime sexual partners, and fewer pregnancies. Substance experimentation was associated with earlier sexual debut and more sexual partners but with fewer pregnancies. Gender role orientation was significant onlyfor age at first intercourse. More traditional young women were older at sexual debut. These results suggest that gender role orientation is important for delaying first intercourse, but once sexually active, acculturation and substance experimentation become more important influences on sexual risk taking among Latina adolescents.

ObjectiveTo investigate serum antibody reactivity against a panel of post-translationally modified vimentin peptides (PTMPs) in patients with early inflammatory arthritis.MethodsA panel of PTMPs was developed. Microtitre plates were coated with peptides derived from vimentin that were identical in length and composition except at one amino acid that was changed to introduce one of three post-translational modifications (PTMs)—either a citrullinated, carbamylated or acetylated residue. Sera of 268 treatment-naive patients with early inflammatory arthritis and symptoms ≤3 months' duration were tested. Patients were assigned to one of three outcome categories at 18-month follow-up (rheumatoid arthritis (RA), persistent non-RA arthritis and resolving arthritis).ResultsAntibodies against citrullinated, carbamylated and acetylated vimentin peptides were detected in the sera of patients with early inflammatory arthritis. The proportion of patients seropositive for all antibody types was significantly higher in the RA group than in the other groups. Anti cyclic citrullinated peptide (CCP)-positive patients with RA had higher numbers of peptides recognised and higher levels of antibodies against those peptides, representing a distinct profile compared with the other groups.ConclusionsWe show for the first time that antibodies against acetylated vimentin are present in the sera of patients with early RA and confirm and extend previous observations regarding anticitrullinated and anticarbamylated antibodies.

Para dar respuesta a la pregunta ¿Cómo es la historia natural de la silicosis en una población de trabajadores mineros de Perú?, se aplicó el análisis de supervivencia de Kaplan – Meier a la data de 4056 mineros peruanos que laboraron entre 1955 y 2006, y que buscaron voluntariamente asistencia médica entre los años 2003 y 2006. Se encontró casos de Silicosis aguda, acelerada y crónica, que progresa rápidamente una vez instalada -Toma 4.7 años pasar de Silicosis 0 a Silicosis III - disminuyendo la esperanza de vida en todos los casos. El periodo de latencia desde el inicio de la exposición laboral hasta el diagnostico de Silicosis 0 fue de 20.11 años, pero para trabajadores mineros de subsuelo este periodo de latencia fue de 13.45 años, y para trabajadores de minería de superficie fue de 21.07 años. La probabilidad de sobrevivir sin diagnostico de silicosis los primeros 5 años de vida laboral fue de 0.9871 +/- 0.004. La mediana de supervivencia global al diagnostico de silicosis fue de 25.7 años. La probabilidad de sobrevivir sin diagnostico de silicosis luego de 50 años de exposición laboral, aun cuando la exposición haya cesado fue de cero. Se demostró que en la historia natural de la silicosis influyen factores como edad, número de empresas en que laboró el huésped, área de superficie corporal, peso, índice de masa corporal, presión arterial, tipo de minería donde laboró el huésped (superficie, subsuelo). Tesis

In order to understand and explore the function and roles of fibroblasts, it is necessary to understand their lineage relationships to other mesenchymal cells. Fibroblasts are ubiquitous non-epithelial, non-endothelial, and non-haematopoietic adherent cells that have the capacity to produce and remodel extracellular matrix. In addition to their well-known ’landscaping’ function which determines the unique structure and function of different organs, they play an important role in wound healing, immune tolerance, and disease. In cancer, epithelial-stromal interactions have been implicated in disease initiation and progression. In rheumatoid arthritis, synovial fibroblasts at diseased sites become persistently activated and behave abnormally, orchestrating joint inflammation and contributing to joint destruction. Recent evidence suggests that the activated phenotype of fibroblasts in pathology may result from epigenetic programming, which is becoming a major focus for development of new therapeutics.

Introduction: Inflammatory arthritis is associated with increased bone resorption and suppressed bone formation. The Wnt antagonist dickkopf-1 (DKK1) is secreted by synovial fibroblasts in response to inflammation and this protein has been proposed to be a master regulator of bone remodelling in inflammatory arthritis. Local glucocorticoid production is also significantly increased during joint inflammation. Therefore, we investigated how locally derived glucocorticoids and inflammatory cytokines regulate DKK1 synthesis in synovial fibroblasts during inflammatory arthritis.Methods: We examined expression and regulation of DKK1 in primary cultures of human synovial fibroblasts isolated from patients with inflammatory arthritis. The effect of TNFα, IL-1β and glucocorticoids on DKK1 mRNA and protein expression was examined by real-time PCR and ELISA. The ability of inflammatory cytokine-induced expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to sensitise fibroblasts to endogenous glucocorticoids was explored. Global expression of Wnt signalling and target genes in response to TNFα and glucocorticoids was assessed using a custom array.Results: DKK1 expression in human synovial fibroblasts was directly regulated by glucocorticoids but not proinflammatory cytokines. Glucocorticoids, but not TNFα, regulated expression of multiple Wnt agonists and antagonists in favour of inhibition of Wnt signalling. However, TNFα and IL-1β indirectly stimulated DKK1 production through increased expression of 11β-HSD1.Conclusions: These results demonstrate that in rheumatoid arthritis synovial fibroblasts, DKK1 expression is directly regulated by glucocorticoids rather than TNFα. Consequently, the links between synovial inflammation, altered Wnt signalling and bone remodelling are not direct but are dependent on local activation of endogenous glucocorticoids.

An increase in tissue levels of glucocorticoids (GCs) is an important component of the inflammatory response (1). Impairment of these counterregulatory responses (e.g., by impaired GC synthesis or GC receptor blockage) is associated with high mortality in inflammatory states in humans and animals (2, 3). The antiinflammatory actions of GCs are mediated through inhibition of proinflammatory signaling pathways such as NF-κB, activator protein 1 (AP-1), and MAPKs. At the tissue level, the action of GCs is regulated by activity of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) (4, 5), which interconverts inactive GCs such as cortisone and dehydrocorticosterone with their active counterparts cortisol and corticosterone. Expression of 11β-HSD1 appears to be a common feature in all cell types that have a mesodermal origin (6). Although 11β-HSD1 activity can be bidirectional, in these cells the activity is primarily in the reductase direction (converting inactive GCs to their active form). In osteoblasts, synovial fibroblasts, adipocytes, and myocytes, 11β-HSD1 expression, and consequent GC activation, has been postulated to play a role in the development of inflammation-associated osteoporosis, arthritis, obesity, and myopathy, respectively (7–11). We have previously reported that proinflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) increase the expression and activity of 11β-HSD1 in these mesenchymal stromal cell types and tissues (7, 10, 12, 13). In contrast, proinflammatory cytokines have no effect on 11β-HSD1 expression in hepatocytes, monocytes, or lymphocytes (10, 14, 15). Furthermore, combined treatment with GCs and proinflammatory cytokines synergistically increases expression and activity of 11β-HSD1 in osteoblasts, synovial fibroblasts, and myocytes (13). This ability of GCs to further stimulate, rather than inhibit, inflammation-associated 11β-HSD1 expression in mesenchymal stromal cells may be a feedforward mechanism to selectively increase local GC action in these cells during inflammation (16). The molecular mechanisms involved in regulating expression of 11β-HSD1 in cells such as hepatocytes, monocytes, and lymphocytes have been explored previously (14, 15, 17). The best-characterized of these mechanisms is the increase in 11β-HSD1 expression in hepatocytes in response to GCs; this is mediated by members of the CCAAT/enhancer binding protein family and requires new protein synthesis (17). However, to date none of these studies have characterized signaling systems involved in mediating the effects of proinflammatory cytokines and GCs in mesenchymal stromal cells. This raises the possibility that novel regulatory pathways regulate these effects. Furthermore, the presence of distinct regulatory mechanisms in musculoskeletal cells might enable tissue-specific regulation of 11β-HSD1 activity. In this study we examined the mechanisms underlying the regulation of 11β-HSD1 expression and activity in osteoblasts, synovial fibroblasts, and myoblasts.

Rheumatoid arthritis is a common chronic inflammatory disease that causes progressive synovial inflammation resulting in irreversible joint destruction, chronic disability and premature mortality. Although it is recognised that in rheumatoid arthritis, inflammation and its persistence result from complex interactions between haematopoietic and stromal cells, research into the pathogenesis of the disease has traditionally concentrated on cells and cytokines of the immune system, neglecting the role of stromal cells. As a consequence, new biologic treatments have been developed, which have led to a step-change in the management of the disease. Nevertheless, these treatments do not reverse tissue damage or lead to disease cure and are not effective for all patients. Furthermore, at best they induce a significant clinical response (ACR70) in less than 60% of patients, most of whom will relapse on treatment withdrawal, suggesting that additional therapeutic targets, responsible for complete resolution of inflammation, remain to be discovered. An increasing body of evidence implicates rheumatoid arthritis synovial fibroblasts in driving the persistent, destructive characteristics of the disease. In this paper, we discuss the evidence implicating synovial fibroblasts in the pathogenesis of rheumatoid arthritis and explore their role as therapeutic targets.

A partir d'une enquete de terrain realisee a Tulum dans les annees 1990, l'A. examine les relations entre mondialisation, tendances economiques, et modeles generationnels de mariage durant le 20 eme siecle chez les femmes mayas de Quintana Roo (Mexique), Mayas de la Guerre des Castes du Yucatan. Elle decrit d'abord les pratiques matrimoniales du debut du siecle. Elle soutient que les mariages arranges de la cette generation avaient pour fonction de structurer le travail et reproduire les familles et l'organisation sociale dans le cadre d'une economie mixte commerciale et de subsistance. Cependant, elle montre aussi que les notions d'amour et d'attraction sexuelle n'etaient pas etrangeres au choix du conjoint. Elle decrit ensuite les changements dans les pratiques et significations matrimoniales pour les trois generations suivantes. Elle montre comment les conceptions de l'amour, du mariage, de la famille, et de la sexualite changent dans le contexte d'une economie de plus en plus capitaliste et industrielle. Elle suggere que les mariages d'amour des plus jeunes peuvent contribuer a l'autonomie de la femme, mais egalement generer d'autres formes d'oppression.

Fuente TESIUNAM

To assess factors affecting follow-up care among low-income women after identification of a breast abnormality.Women with a newly discovered breast abnormality were selected from one public hospital and two comprehensive health centers (CHCs) in Los Angeles county. Twelve months later, a telephone survey and medical chart review were conducted.Of the 535 respondents, 8.6% received no follow-up care. Among those with follow-up care, 29.4% received inadequate care (did not initiate follow-up in a timely manner or did not complete all recommended procedures). Factors affecting receipt of any follow-up care included having the index visit at a CHC vs. a hospital (OR 2.79, CI 1.20-6.50), patient uncertainty about where to receive care (OR 0.24, CI 0.07-0.77), and recommendation of a clinical breast examination (CBE) (OR 0.12, CI 0.04-0.40) or 6-month mammogram (OR 0.11, CI 0.04-0.31) vs. a diagnostic mammogram as a first follow-up procedure. Factors affecting receipt of adequate follow-up care included index visit at a CHC vs. a hospital (OR 1.90, CI 1.13-3.20), being white/Asian Pacific Islander/other vs. Latina (OR 5.33, CI 1.71-16.68), recommendation of a 6-month mammogram vs. a diagnostic mammogram (OR 0.06, CI 0.02-0.14), and a family history of breast cancer (OR 0.44, CI 0.22-0.89).To maximize return for follow-up among low-income women with a breast abnormality, clear information should be provided about where to obtain care, particularly to patients in hospital settings. The importance of complete and timely follow-up care should be emphasized, especially with referrals for clinical breast examinations or 6-month mammograms.

Objective To investigate family physicians' and pediatricians' practice of and perceived barriers to smoking cessation counseling among patients 18 years and younger. Design Cross-sectional mail survey conducted between November 1, 1997, and January 31, 1998. Participants A stratified random sample selected from the 1997 American Medical Association Physician Masterfile of 1000 family physicians and pediatricians who practice in urban California, work at least 10% of the time in ambulatory care, and have at least 10% of patients 18 years and younger. Main Outcome Measures Physicians' adherence to 5 components of the National Cancer Institute's smoking cessation counseling recommendations (anticipate, ask, advise, assist, and arrange) and their perceived barriers to smoking cessation counseling. Results A total of 429 physicians participated in the study. Physicians of both specialties were more likely to anticipate, ask, and advise patients about smoking than to assist with and arrange cessation activities. Family physicians were more likely than pediatricians to assist and arrange, including scheduling follow-up visits to discuss quitting (25.1% vs 11.7%; odds ratio [OR], 3.07; 95% confidence interval [CI], 1.22-7.73) and directing nursing staff to counsel patients (17.1% vs 10.9%; OR, 3.70; 95% CI, 1.30-10.60). The most common perceived barrier to counseling was the belief that children would provide inaccurate responses due to either the presence of parents (86.4%) or the fear that parents would be notified of their answers (74.0%). Pediatricians reported lack of counseling skills as a barrier to providing smoking interventions in greater proportion than did family physicians (24.9% vs 54.8%; OR, 0.29; 95% CI, 0.14-0.63;P Conclusion Improvement in smoking cessation counseling skills and practices is needed among physicians treating children and adolescents.

Background Cathepsin K is a protease expressed by osteoclasts that plays an important role in osteoclastic mediated bone resorption. It degrades organic bone matrix, primarily type 1 collagen. Postmenopausal osteoporosis is characterised by an increase in bone resorption that manifests as elevation of bone remodelling makers. This phenomenon starts in the peri-menopausal period. The potential role of cathepsin K levels as markers of bone remodelling in postmenopausal osteoporosis is being evaluated, but there is paucity of data. There is increasing interest in identifying markers able to differentiate the number and activity of osteoclasts to clarify the mechanism of action of current anti-osteoporotic therapies. It is also suggested that such markers might help in the development of more specific novel treatments. Objectives To evaluate the role of cathepsin K as bone remodelling marker in females with postmenopausal osteoporosis. Methods Cross sectional study. Two groups were evaluated: postmenopausal females and healthy controls (this group was subdivided into pre and postmenopausal females). Cathepsin K levels in sera were determined by polyclonal antibody ELISA (BI-20432). Data were analysed with STATA10. Differences in cathepsin K levels between groups were determined. A quantile regression model was built to assess cathepsin K leves for the median, first and third quantile according to the variables of interest. A p value of

Background Human synovial fibroblasts are implicated in rheumatoid arthritis (RA) pathogenesis at two levels: they promote cartilage and bone destruction and contribute to persistent joint inflammation. Significant damage to RA joints occurs within the first year of disease and thus early aggressive treatment is advocated. Special attention has been given to the “early window of opportunity” in the first 3 months of disease. Despite this, whereas much is known about synovial fibroblasts taken from very late disease at joint replacement, little is known about fibroblast biology in early RA. We hypothesised that by studying synovial fibroblasts from patients in this early window we would identify novel stromal biomarkers and therapeutic targets in very early disease. Objectives To describe the gene expression pattern of human synovial fibroblasts from patients with early RA of up to three months symptom duration and compare it to that of synovial fibroblasts from patients with longstanding RA. Methods Synovial tissue was obtained from patients with longstanding RA undergoing joint replacement (n=8, mean disease duration 20 years) or patients with synovitis of at least one joint and symptom duration of up to 3 months fulfilling 1987 criteria for RA by 18 months follow-up (n=14) via ultrasound guided synovial biopsy. Normal (n=8) and resolving arthritis (n=16) fibroblasts were used as controls. RNA was extracted and treated with the RNA clean and concentrator kit. Samples were hybridised onto Agilent Sure Print G3 8x60k v2 arrays. Scanned images were analysed with Agilent Feature Extraction Software. Partek Genomics Suite was used to analyse gene expression using a 1-way fixed model ANOVA to create pairwise contrasts with a corrected step-up p-value (False Discovery Rate by Benjamini) and a fold change for differences in gene expression. Age, gender and joint of origin were found to contribute to variance and thus included in the ANOVA model. Antibody status did not contribute to variance of the datasets and was thus excluded. Results 73 out of a total of 50,599 probes, detected differentially expressed transcripts between early and late RA (FDR To ascertain those genes that were uniquely differentially expressed between early and late RA, normal and resolving samples were compared to late RA samples. In total 13 probes were shared between this analysis and that of early and late RA, leaving 60 probes exclusively differentiating early RA. Of these, 27 were upregulated and 33 downregulated in early RA compared to late RA. Gene ontology revealed that 38.2% of the differentially expressed genes were involved in transcription regulation and 23.5% in RNA metabolic processes. Detailed analysis and validation of differentially expressed targets is ongoing. Conclusions We have identified a distinct gene expression pattern in synovial fibroblasts of patients with early RA. By analysing normal and resolving arthritis samples, we have been able to ensure that this pattern is unique to early RA fibroblasts and not due to non-specific joint inflammation. Ongoing in depth analysis and validation of targets will allow unravelling of modifiable disease mechanisms in early RA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3703

Background and Objectives The importance of B cells in the pathogenesis of rheumatoid arthritis (RA) is highlighted by the clinical effectiveness of rituximab. Potential mechanisms for B cells in driving disease processes include autoantibody production, antigen presentation and cytokine secretion. Our recent findings suggest a pro-inflammatory and destructive role for B cells via production of RANKL, TNF-α and IL-6. A memory B cell subset in tonsils which expresses Fc receptor-like 4 (FcRL4) has previously been found to produce RANKL. We sought to determine whether the RANKL-expressing B cells present in the RA synovium belong to this subset. Methods: Synovial fluid and peripheral blood cells from RA patients were analysed for B cell differentiation markers by flow cytometry. Synovial tissue samples from RA patients were assessed using immunofluorescence and immunohistochemistry. FcRL4 mRNA expression was determined in synovial samples from RA patients and uninflamed control subjects by real-time PCR. FcRL4+ and FcRL4- CD19 + B cells were sorted from synovial fluid using a MoFlo cell sorter. mRNA expression of 48 cytokines was determined using Taqman microfluidic cards. Results FcRL4 expressing B cells were detected in the synovial fluid (median 14% of B cells) at a significantly higher frequency compared to peripheral blood (median 0.04%; p = 0.004). RANKL was significantly enriched in the FcRL4+ B cell population compared to the FcRL4- population. FcRL4+ B cells were predominantly switched memory B cells, expressing significantly higher levels of CD80, CD86, CD95, CD20 and CD11c than FcRL4- cells. Sorted FcRL4+ B cells expressed significantly higher mRNA levels of RANKL and TNF-α than FcRL4- B cells. FcRL4+ B cells expressing RANKL were present in synovial tissue, and were distributed under the synovial lining layer and in perivascular cuffs. FcRL4 mRNA was detected in synovium from RA patients but not uninflamed controls, and FcRL4 expression in RA synovium correlated positively with the degree of leukocyte infiltration. Conclusions We have identified a novel pro-inflammatory subset of B cells in the RA synovium which expresses RANKL and TNF-α and shows considerable expression of co-stimulatory molecules indicating a potential pathogenic role in RA. As these cells express high levels of CD20 it is conceivable that their removal contributes to the anti-inflammatory clinical effect of rituximab.