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Multi-ancestry genome-wide association analyses identify 124 risk loci for rheumatoid arthritis, of which 34 are novel. A polygenic risk score based on multi-ancestry data showed comparable performance between populations of European and East Asian ancestries.Rheumatoid arthritis (RA) is a highly heritable complex disease with unknown etiology. Multi-ancestry genetic research of RA promises to improve power to detect genetic signals, fine-mapping resolution and performances of polygenic risk scores (PRS). Here, we present a large-scale genome-wide association study (GWAS) of RA, which includes 276,020 samples from five ancestral groups. We conducted a multi-ancestry meta-analysis and identified 124 loci (P < 5 x 10(-8)), of which 34 are novel. Candidate genes at the novel loci suggest essential roles of the immune system (for example, TNIP2 and TNFRSF11A) and joint tissues (for example, WISP1) in RA etiology. Multi-ancestry fine-mapping identified putatively causal variants with biological insights (for example, LEF1). Moreover, PRS based on multi-ancestry GWAS outperformed PRS based on single-ancestry GWAS and had comparable performance between populations of European and East Asian ancestries. Our study provides several insights into the etiology of RA and improves the genetic predictability of RA.

The question of how the heart develops, and the genetic networks governing this process have become intense areas of research over the past several decades. This research is propelled by classical developmental studies and potential clinical applications to understand and treat congenital conditions in which cardiac development is disrupted. Discovery of the tinman gene in Drosophila, and examination of its vertebrate homolog Nkx2.5, along with other core cardiac transcription factors has revealed how cardiac progenitor differentiation and maturation drives heart development. Careful observation of cardiac morphogenesis along with lineage tracing approaches indicated that cardiac progenitors can be divided into two broad classes of cells, namely the first and second heart fields, that contribute to the heart in two distinct waves of differentiation. Ample evidence suggests that the fate of individual cardiac progenitors is restricted to distinct cardiac structures quite early in development, well before the expression of canonical cardiac progenitor markers like Nkx2.5. Here we review the initial specification of cardiac progenitors, discuss evidence for the early patterning of cardiac progenitors during gastrulation, and consider how early gene expression programs and epigenetic patterns can direct their development. A complete understanding of when and how the developmental potential of cardiac progenitors is determined, and their potential plasticity, is of great interest developmentally and also has important implications for both the study of congenital heart disease and therapeutic approaches based on cardiac stem cell programming.

Background/Aims Pain remains a common, life-changing symptom for patients with rheumatoid (RA) and psoriatic arthritis (PsA). Its current care focuses on prescribing analgesics, particularly opioids, despite minimal supportive trial evidence and potential associated harms. Intensive management with disease-modifying anti-rheumatic drugs (DMARDs) reduces disease activity and improves function in RA and PsA. Its impacts on pain are less well established. We examined the impact of varying intensities of DMARD treatment on pain in patients with RA/PsA in a secondary analysis of three trials. Methods The trials comprised MIPA in PsA (methotrexate vs. placebo), CARDERA in early RA (triple therapy vs. monotherapy), and TITRATE in established RA (monthly assessments with DMARD uptitration and psychosocial support vs. standard care). All measured 100mm pain intensity visual analogue scores. Regression models evaluated the relationship between active vs. control treatment and: (a) achieving “low” endpoint pain scores of ≤ 34, (b) changes in pain scores between baseline and endpoint, (c) achieving ≥30% reductions in pain scores between baseline and endpoint. Results We evaluated 765 patients (from 1,023 [75%]) with endpoint pain intensity scores available. In all trials more patients achieved low endpoint pain scores (≤34) with active than control treatment (MIPA 70% vs. 42%, P=0.003; CARDERA 71% vs. 56%, P=0.011; TITRATE 67% vs. 50%; P=0.008). Multivariable models showed the associations remained significant after treatment effects were adjusted for age, sex, and baseline pain: odds ratios (ORs) 2.8 (P=0.012), 1.9 (P=0.019) and 2.1 (P=0.006) in MIPA, CARDERA, and TITRATE. The trials differed in the impact of intensive treatment on changes in pain scores. In MIPA mean changes in pain intensity scores over the trial period were similar with active and control treatment. In CARDERA (P=0.019) and TITRATE (P=0.004) mean changes in pain scores were significantly greater with active than control treatment in multivariable models (estimated differences of 6.8 and 9.4 units). The trials also differed in the impact of treatment on the proportion of patients having large reductions in pain scores (≥30). In MIPA proportionally more patients achieved this with active vs. control treatment but this was not statistically significant. In CARDERA (P=0.009) and TITRATE (P=0.002) a significantly greater proportion of patients achieved large reductions in pain scores in multivariable models with active treatment (ORs 1.9 and 2.2). Conclusion In our secondary analysis all forms of intensive management provided beneficial effects on pain in active RA and PsA. More patients achieved “low” endpoint pain scores with active treatment in all trials. In the two trials employing the most intensive management strategies (CARDERA/TITRATE) significantly greater reductions in pain scores and a greater proportion achieving substantial pain improvements with active treatment were seen. Our results support the use of intensive management to improve pain in patients with active RA/PsA. Disclosure I.C. Scott: None. D.L. Scott: None. F. Ibrahim: None.

Background/Aims Analgesics are widely prescribed in patients with inflammatory arthritis (IA) despite limited evidence of efficacy and uncertainties about potential harms. The assessment of harms is complex as some drug-related adverse events (AE), including myocardial infarctions, are part of the IA comorbidity spectrum. As trials have limited follow-up periods and often exclude patients with comorbidities, safe prescribing requires an understanding of analgesic harms in “real-world” settings. We consequently undertook the first systematic review of observational studies reporting risks of adverse events with analgesics in adults with rheumatoid arthritis (RA) and spondyloarthritis (SpA). Methods A pre-specified protocol was PROSPERO-registered (CRD42019154277). Systematic searches were conducted in Web of Science/Embase/Medline without language/date restriction on the 18/1/22. Due to marked methodological and statistical heterogeneity, studies were combined in a structured narrative synthesis. Results Studies: Of 6,444 citations screened, thirty-five studies met eligibility criteria, comprising 21 cohort (200 to 143,433 patients); 12 case-control (41 to 7,102 cases); 1 self-controlled case series; 1 case-crossover. Two evaluated opioids in RA, 23 oral NSAIDs in RA, 9 oral NSAIDs in SpA, and 1 NSAIDs and opioids in RA. None evaluated paracetamol/gabapentinoids. Fourteen were rated “good”, 19 “fair” and 1 “poor” quality. Adverse Event Risks: All studies of opioids reported significantly increased risks of fractures/serious infection. For NSAIDs renal, upper gastrointestinal (UGI), and cardiovascular (CV) events were most often reported. There was consistent evidence that NSAIDs significantly increased renal (3/3 studies) and UGI (3/4 studies) AEs. Findings with CV AEs varied: 7/18 studies showed significantly increased risks; 3/18 showed significantly reduced risks, and 3/18 showed significantly increased risks with some NSAIDs and reduced risks with others. There was no evidence NSAIDs increased risks of lower/functional gastrointestinal AEs, or fractures. With regards to mortality, one study showed a significantly reduced risk of death with NSAID use in SpA; the other showed a significantly reduced risk of death with NSAID use in inflammatory polyarthritis (but not RA). Differences Between RA And SpA: More studies reported a significantly reduced risk of CV events/fractures/deaths with NSAIDs in SpA (6/10) than RA (4/24). Differences Between IA and Non-IA Populations: Two of four studies found significant differences in AE risks in RA/SpA compared to OA/controls. One reported a significantly higher risk of myocardial infarction in SpA than OA and another a significantly lower risk of composite CV events in RA than controls. Conclusion Observational study data on risks of analgesic harms in IA are limited. All studies of opioids showed increased risks of harms. Studies of NSAIDs gave variable results, reflecting heterogeneity and potential confounding. Overall, oral NSAIDs increased renal and UGI AEs, with some evidence they reduced fractures/mortality in SpA. Further high-quality observational studies are needed to better characterise analgesic harms in IA. Disclosure C.R. White: None. O. Babatunde: None. J. Higgs: None. S. Muller: None. C.D. Mallen: None. S.L. Hider: None. I.C. Scott: None.

Background/Aims Pain is a common symptom experienced by patients with inflammatory arthritis (IA). Emerging data indicate IA pain treatment focuses on prescribing analgesics despite an absence of evidence for long-term efficacy. Delivering optimal, equitable IA pain care in England requires understanding current practice. To address this, we evaluated analgesic prescribing in patients diagnosed with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and axial spondyloarthritis (SpA) contributing data to the Clinical Practice Research Datalink Aurum, a primary care electronic health record database covering 20% of England. Methods Cross-sectional analyses (2004-2020) evaluated the annual prevalence of analgesic prescriptions in patients with RA/PsA/axial SpA. These were reported stratified by age, gender, ethnicity, deprivation, and geographical region. Analgesic prescriptions were classified using Bedson et al’s hierarchical system with an additional gabapentinoid group. Joinpoint regression models evaluated time-points when a significant change in the trends of prescription annual prevalence occurred. Results Although declining over time, the annual prevalence of analgesic prescriptions remained high (84.2% [95% CI 83.9, 84.5] in 2004; 64.5% [95% CI 64.2, 64.8] in 2020). In 2004, oral NSAIDs were the commonest analgesic (prevalence 56.1% [95% CI 55.8, 56.5]). By 2020, this fell to 22.3% (95% CI 22.1, 22.6) with opioids being the commonest analgesic (prevalence 39.0% [95% CI 38.7, 39.2]). Most opioid prescriptions were “strong/very strong” and long-term. Annual prevalence of non-NSAID analgesic prescriptions was highest in RA and of NSAIDs was highest in axial SpA. The annual prevalence of all analgesic types except NSAIDs differed by age, sex, deprivation, ethnicity, and region. Annual prevalence of non-NSAID analgesic prescriptions increased with age and deprivation, were higher in females than males, and higher in Northern than Southern England. The opposite pattern was seen with age for oral NSAIDs. NSAIDs were more commonly prescribed in males than females. Very little difference was seen in NSAID prescribing by deprivation, ethnicity, and region. There was a significant change in the trend of opioid prescription annual prevalence in 2015 (year of Public Health England’s “Opioid Aware” release) when the rate of decline increased, and at 2013 and 2018 for gabapentinoids when the annual prevalence increase slowed then declined (years of NHS England’s release of information on gabapentinoid risks, and their reclassification as controlled substances). Conclusion Whilst overall national analgesic prescribing in IA has fallen over 17 years - with interventions to deliver safer opioid/gabapentinoid prescribing potentially contributing - it remains common. This is at variance with evidence of analgesic efficacy and risks. The differences in prescribing of some analgesic types by patient sociodemographic factors and regions suggest unwarranted variation. Interventions are needed to improve the delivery of effective, equitable IA pain treatment. Disclosure I.C. Scott: None. R. Whittle: None. J. Bailey: None. H. Twohig: None. S.L. Hider: None. C.D. Mallen: None. S. Muller: None. K.P. Jordan: None.

Abstract Objective To assess the longer term impact of the COVID-19 pandemic on the self-reported physical and mental health of people with inflammatory rheumatic diseases (IRDs). Methods Two thousand twenty-four patients with IRDs were randomly selected from electronic health records. Survey invitations were sent (August 2021 coinciding with relaxation of UK COVID-19 restrictions) using SMS and postal approaches. Self-reported data included demographics, shielding status and physical (MSK-HQ) and mental health (PHQ8 and GAD7). Results Six hundred thirty-nine people completed the survey (mean (SD) age 64.5 (13.1) years, 384 (60%) female). Moderate/severe impact of the pandemic on physical and mental health was reported by 250 (41%) and 241 (39%) respectively. One hundred seventy-two (29%) reported moderate/severe depression (PHQ8 ≥ 10) and 135 (22%) moderate/severe anxiety (GAD7 ≥ 10). Females reported greater impacts of the pandemic on physical health (44% vs 34%), mental health (44% vs 34%), arthritis symptoms (49% vs 36%) and lifestyle factors (weight gain and reduced exercise and physical activity) than males. The physical and mental impacts were less in people with RA compared with other IRDs. Physical health impacts did not differ between age groups, but younger patients reported greater impacts on mental health. Conclusion The COVID-19 pandemic has had a significant impact on the physical and mental health of people with IRDs. These effects were greatest in females. Recovery needs to address the negative impact of the pandemic on lifestyle factors to minimise the long-term impacts for people with IRDs. Key Points• The pandemic had a significant impact on long term physical and mental health in almost 40% of people with IRDs.• The impact of the pandemic was greater in women for physical health, mental health and arthritis symptoms.• Many people reported negative pandemic impacts on lifestyle factors including weight and physical activity.

Objective The aim was to evaluate the impact of the coronavirus disease 2019 (COVID-19) pandemic and stringent social isolation measures on patients with rheumatic disease (RD) from the beginning of the pandemic (April 2020). Methods In this UK-based single-centre, prospective, observational cohort study, all RD follow-up patients at our centre were invited by SMS text message in April 2020 to participate in the study. Participants completed questionnaires at four time points between April 2020 and December 2021. We collected demographics, clinically extremely vulnerable (CEV) status, short form 12 mental (MCS) and physical health component scores (PCS) for health-related quality of life, vaccination status, COVID-19 infection rates and incidence of long COVID. Results We enrolled 1605 patients (female, 69.0%; CEV, 46.5%); 906 of 1605 (56.4%) completed linked responses to our final questionnaire. MCS improved (+0.6, P Conclusions Mental and physical health in RD patients has changed throughout the pandemic; outcomes for both metrics of health were worse in CEV patients, although there were no differences in infection rates between the groups. These data might assist the understanding and planning of future health-care policy and social restrictions in RD patients. Trial registration ClinicalTrials.gov, www.clinicaltrials.gov, NCT04542031.

Contemporary data on rheumatoid arthritis (RA), psoriatic arthritis (PsA) and axial spondyloarthritits (SpA) epidemiology in England are lacking. This knowledge is crucial to planning healthcare services. We updated algorithms defining patients with diagnoses of RA, PsA, and axial SpA in primary care and applied them to describe their incidence and prevalence in the Clinical Practice Research Datalink Aurum, an electronic health record (EHR) database covering ∼20% of England.Algorithms for ascertaining patients with RA, axial SpA, and PsA diagnoses validated in primary care EHR databases using Read codes were updated (to account for the English NHS change to SNOMED CT diagnosis coding) and applied. Updated diagnosis and synthetic disease-modifying anti-rheumatic drug code lists were devised by rheumatologists and general practitioners. Annual incidence/point-prevalence of RA, PsA, and axial SpA diagnoses were calculated from 2004 to 2020 and stratified by age/sex.Point-prevalence of RA/PsA diagnoses increased annually, peaking in 2019 (RA 0·779% [95% confidence interval (CI) 0·773, 0·784]; PsA 0·287% [95% CI 0·284, 0·291]) then falling slightly. Point-prevalence of axial SpA diagnoses increased annually (except in 2018/2019), peaking in 2020 (0·113% [95% CI 0·111, 0·115]). RA diagnosis annual incidence was higher between 2013-2019 (after inclusion in the Quality and Outcomes Framework, range 49·1 [95% CI 47·7, 50·5] to 52·1 [95% CI 50·6, 53·6]/100,000 person-years) than 2004-2012 (range 34·5 [95% CI 33·2, 35·7] to 40·0 [95% CI 38·6, 41·4]/100,000 person-years). Increases in the annual incidence of PsA/axial SpA diagnosis occurred following new classification criteria publication. Annual incidence of RA, PsA and axial SpA diagnoses fell by 40·1%, 67·4%, and 38·1%, respectively between 2019 and 2020, likely reflecting the COVID-19 pandemic's impact on their diagnosis.Recorded RA, PsA, and axial SpA diagnoses are increasingly prevalent in England, underlining the importance of organising healthcare services to provide timely, treat-to-target care to optimise the health of1% of adults in England.National Institute for Health and Care Research (NIHR300826).

GATA4/5/6 transcription factors play essential, conserved roles in heart development. To understand how GATA4/5/6 modulates the mesoderm-to-cardiac fate transition, we labeled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at precardiac time points spanning zebrafish gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was substantially altered. In addition to the expected absence of cardiac mesoderm, we confirmed a concomitant expansion of cranial-pharyngeal mesoderm. Moreover, Gata5/6 loss led to extensive changes in chromatin accessibility near cardiac and pharyngeal genes. Functional analyses in zebrafish and the tunicate Ciona , which has a single GATA4/5/6 homolog, revealed that GATA4/5/6 acts upstream of tbx1 to exert essential and cell-autonomous roles in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, cardiac and pharyngeal mesoderm fate choices are achieved through an evolutionarily conserved GATA4/5/6 regulatory network.

Background Pain is the main concern of patients with rheumatoid arthritis (RA) while reducing disease activity dominates specialist management. Disease activity assessments like the disease activity score for 28 joints with the erythrocyte sedimentation rate (DAS28-ESR) omit pain creating an apparent paradox between patients’ concerns and specialists’ treatment goals. We evaluated the relationship of pain intensity and disease activity in RA with three aims: defining associations between pain intensity and disease activity and its components, evaluating discordance between pain intensity and disease activity, and assessing temporal changes in pain intensity and disease activity. Methods We undertook secondary analyses of five trials and one observational study of RA patients followed for 12 months. The patients had early and established active disease or sustained low disease activity or remission. Pain was measured using 100-mm visual analogue scales. Individual patient data was pooled across all studies and by types of patients (early active, established active and established remission). Associations of pain intensity and disease activity were evaluated by correlations (Spearman’s), linear regression methods and Bland-Altman plots. Discordance was assessed by Kappa statistics (for patients grouped into high and low pain intensity and disease activity). Temporal changes were assessed 6 monthly in different patient groups. Results A total of 1132 patients were studied: 490 had early active RA, 469 had established active RA and 173 were in remission/low disease activity. Our analyses showed, firstly, that pain intensity is associated with disease activity in general, and particularly with patient global assessments, across all patient groups. Patient global assessments were a reasonable proxy for pain intensity. Secondly, there was some discordance between pain intensity and disease activity across all disease activity levels, reflecting similar discrepancies in patient global assessments. Thirdly, there were strong temporal relationships between changes in disease activity and pain intensity. When mean disease activity fell, mean pain intensity scores also fell; when mean disease activity increased, there were comparable increases in pain intensity. Conclusions These findings show pain intensity is an integral part of disease activity, though it is not measured directly in DAS28-ESR. Reducing disease activity is crucial for reducing pain intensity in RA.

Centronuclear myopathy (CNM) is a congenital neuromuscular disorder caused by pathogenic variation in genes associated with membrane trafficking and excitation-contraction coupling (ECC). Bi-allelic autosomal recessive mutations in striated muscle enriched protein kinase (SPEG) account for a subset of CNM patients. Previous research has been limited by the perinatal lethality of Speg knockout mice. Thus, the precise biological role of SPEG in skeletal muscle remains unknown. To address this issue, we generated zebrafish spega, spegb, and spega/spegb (speg-DKO) mutant lines. We demonstrate that speg-DKO zebrafish faithfully recapitulate multiple phenotypes associated with human CNM, including disruption of the ECC protein machinery, dysregulation of calcium homeostasis during ECC, and impairment of muscle performance. Taking advantage of the availability of zebrafish models of multiple CNM genetic subtypes, we compared novel and known disease markers in speg-DKO with mtm1-KO and DNM2-S619L transgenic zebrafish. We observed desmin (DES) accumulation common to all CNM subtypes, and DNM2 upregulation in muscle of both speg-DKO and mtm1-KO zebrafish. In all, we establish a new model of SPEG-related CNM, and identify abnormalities in this model suitable for defining disease pathomechanisms and evaluating potential therapies.Summary StatementWe created a novel zebrafish speg mutant model of centronuclear myopathy that recapitulates key features of the human disorder and provides insight into pathomechanisms of the disease.

Centronuclear myopathy (CNM) is a congenital neuromuscular disorder caused by pathogenic variation in genes associated with membrane trafficking and excitation–contraction coupling (ECC). Bi-allelic autosomal-recessive mutations in striated muscle enriched protein kinase (SPEG) account for a subset of CNM patients. Previous research has been limited by the perinatal lethality of constitutive Speg knockout mice. Thus, the precise biological role of SPEG in developing skeletal muscle remains unknown. To address this issue, we generated zebrafish spega, spegb and spega;spegb (speg-DKO) mutant lines. We demonstrated that speg-DKO zebrafish faithfully recapitulate multiple phenotypes associated with CNM, including disruption of the ECC machinery, dysregulation of calcium homeostasis during ECC and impairment of muscle performance. Taking advantage of zebrafish models of multiple CNM genetic subtypes, we compared novel and known disease markers in speg-DKO with mtm1-KO and DNM2-S619L transgenic zebrafish. We observed Desmin accumulation common to all CNM subtypes, and Dnm2 upregulation in muscle of both speg-DKO and mtm1-KO zebrafish. In all, we establish a new model of SPEG-related CNM, and identify abnormalities in this model suitable for defining disease pathomechanisms and evaluating potential therapies. This article has an associated First Person interview with the joint first authors of the paper.

BackgroundRespiratory viral infections are closely associated with COPD exacerbations, hospitalisations, and significant morbidity and mortality. The consequences of the persisting inflammation and differentiation status in virus associated severe disease is not fully understood. The aim of this study was to evaluate barrier function, cellular architecture, the inflammatory response in severe COPD bronchial epithelium to human rhinovirus (HRV) induced pathological changes and innate immune responses.MethodsWell-differentiated primary bronchial epithelial cells (WD-PBECs) derived from severe COPD patients and age-matched healthy controls were cultured in the air-liquid interface (ALI) model. The differentiation phenotype, epithelial barrier integrity, pathological response and cytokine secreting profile of these cultures before and after HRV infection were investigated.ResultsWD-PBECs derived from severe COPD patients showed aberrant epithelium differentiation with a decreased proportion of ciliated cells but increased numbers of club cells and goblet cells compared with healthy controls. Tight junction integrity was compromised in both cultures following HRV infection, with heightened disruptions in COPD cultures. HRV induced increased epithelial cell sloughing, apoptosis and mucus hypersecretion in COPD cultures compared with healthy controls. A Th1/Th2 imbalance and a strong interferon and pro-inflammatory cytokine response was also observed in COPD cultures, characterized by increased levels of IFNγ, IFNβ, IP-10, IL-10 and decreased TSLP and IL-13 cytokine levels prior to HRV infection. Significantly enhanced basolateral secretion of eotaxin 3, IL-6, IL-8, GM-CSF were also observed in both mock and HRV infected COPD cultures compared with corresponding healthy controls. In response to HRV infection, all cultures displayed elevated levels of IFNλ1 (IL-29), IP-10 and TNFα compared with mock infected cultures. Interestingly, HRV infection dramatically reduced IFNλ levels in COPD cultures compared with healthy subjects.ConclusionAn altered differentiation phenotype and cytokine response as seen in severe COPD WD-PBECs may contribute to increased disease susceptibility and an enhanced inflammatory response to HRV infection.

Airway epithelium plays a pivotal role in maintaining immune homeostasis and orchestrates the innate and adaptive responses of the lung against inhaled insults. However, the aetiology of pathological changes on airway epithelium during COPD pathogenesis is not completely understood, in particular factors linked to the susceptibility to severe disease. To address this, we characterized well-differentiated primary bronchial epithelial cells (WD-PBECs) derived from severe COPD patients undergoing transplantation and age-matched healthy controls employing an air-liquid interface (ALI) 3D in vitro/ex-vivo culture model. WD-PBECs derived from severe COPD patients showed an indistinguishable morphology compared with controls. However, aberrant epithelium differentiation in terms of a decrease in the number of ciliated cells and an increase in the number of Club cells and goblet cells in was evidenced in COPD cultures compared with healthy controls. Tight junction integrity was also compromised in the former. Moreover, there was enhanced basolateral secretion of IL-6, IL-8, GM-CSF, IL-1alpha, IP-10, CCL1 as well as altered secretion of Th1 and Th2 cytokines by COPD cells. Our data demonstrate that primary bronchial epithelial cells from patients with severe COPD show alterations in individual cell types during differentiation and enhanced secretion of inflammatory mediators including an alteration in Th1 and Th2 cytokines. Such changes in COPD bronchial epithelium during severe disease may represent an underlying perturbation in cellular function in these cells which may contribute to inflammation and infection in the COPD airways.

Objective\ud Treat-to-target in rheumatoid arthritis (RA) recommends targeting remission, with low disease activity (LDA) being an alternative goal. When deciding to target remission or LDA, important considerations are the likelihood of attaining them, and their impacts on function and healthrelated quality of life (HRQoL). We have addressed this by studying: (a) the frequency of remission and LDA/remission; (b) DAS28-ESR trends after remission; (c) ability of remission vs. LDA to identify patients with normal function (HAQ ≤0.5) and HRQoL (EQ-5D ≥ the normal population).\ud \ud Methods\ud We studied 571 patients in two clinical trials, and 1,693 patients in a 10-year routine care cohort. We assessed the frequency and sustainability of remission and LDA/remission, variability in DAS28-ESR after remission, and sensitivity/specificity of remission and LDA/remission at identifying patients with low disability levels and normal HRQoL using Receiver Operator Characteristic (ROC) curves.\ud \ud Results\ud Point remission and remission/LDA were common (achieved by 35-58% and 49-74% of patients, respectively), but were rarely sustained (sustained remission and remission/LDA achieved by 5-9% and 9-16% of patients, respectively). Following attaining remission, DAS28-ESR levels varied substantially. Despite this, of those patients attaining point remission, the majority (53-61%) were in remission at study end-points. Whilst remission was highly specific at identifying patients with low disability (85-91%) it lacked sensitivity (51-57%); similar findings were seen for normal HRQoL (specificity 78-86%; sensitivity 52-59%). The optimal DAS28-cut-off to identify individuals with low disability and normal HRQoL was around the LDA threshold.\ud \ud Conclusions\ud Our findings support both the treat-to-target goals. Attaining remission is highly specific for attaining low disability and normal HRQoL, although many patients with more active disease also have good function and HRQoL. Attaining a DAS28-ESR ≤3.2 has a better balance of specificity and sensitivity for attaining these outcomes, with the benefit of being more readily achievable. Although sustaining these targets over time is rare, even attaining them on a oneoff basis leads to better function and HRQoL outcomes for patients.

Letter

GATA4/5/6 transcription factors play essential, conserved roles in heart development. How these factors mediate the transition from multipotent mesoderm progenitors to a committed cardiac fate is unclear. To understand how GATA4/5/6 modulate cell fate decisions we labelled, isolated, and performed single-cell gene expression analysis on cells that expressgata5at pre-cardiac time points spanning gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamicgata5/6expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was dramatically altered. In addition to the expected absence of cardiac mesoderm, we observed a concomitant expansion of cranial-pharyngeal mesoderm. Functional genetic analyses in zebrafish and the invertebrate chordateCiona, which possess a single GATA4/5/6 homolog, revealed an essential and cell-autonomous role for GATA4/5/6 in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, the maintenance and repression of GATA4/5/6 activity plays a critical, evolutionarily conserved role in early development.

Rana et al. demonstrate that white adipose tissue-resident multipotent stromal cells (WAT-MSC) support IL-33– and LFA-1/ICAM-1–mediated proliferation and type-2 cytokine expression by ILC2. Inversely, ILC2-derived IL-4 and IL-13 promote WAT-MSC eotaxin production and eosinophil recruitment, further maintaining type-2 immune homeostasis., Group-2 innate lymphoid cells (ILC2), type-2 cytokines, and eosinophils have all been implicated in sustaining adipose tissue homeostasis. However, the interplay between the stroma and adipose-resident immune cells is less well understood. We identify that white adipose tissue–resident multipotent stromal cells (WAT-MSCs) can act as a reservoir for IL-33, especially after cell stress, but also provide additional signals for sustaining ILC2. Indeed, we demonstrate that WAT-MSCs also support ICAM-1–mediated proliferation and activation of LFA-1–expressing ILC2s. Consequently, ILC2-derived IL-4 and IL-13 feed back to induce eotaxin secretion from WAT-MSCs, supporting eosinophil recruitment. Thus, MSCs provide a niche for multifaceted dialogue with ILC2 to sustain a type-2 immune environment in WAT., Graphical Abstract

Quebec Platelet Disorder (QPD) is an autosomal dominant bleeding disorder with a unique, platelet-dependent gain-of-function defect in fibrinolysis, without systemic fibrinolysis. The hallmark feature of QPD is a >100-fold overexpression of PLAU specifically in megakaryocytes. This overexpression leads to >100-fold increased platelet stores of urokinase plasminogen activator (PLAU/uPA), subsequent plasmin-mediated degradation of diverse a-granule proteins, and platelet-dependent, accelerated fibrinolysis. The causative mutation is a 78kb tandem duplication of PLAU. How this duplication causes megakaryocyte-specific PLAU overexpression is unknown. To investigate the mechanism that causes QPD, we used epigenomic profiling, comparative genomics, and chromatin conformation capture approaches to study PLAU regulation in cultured megakaryocytes from QPD participants and unaffected controls. We show that the QPD duplication leads to ectopic interactions between PLAU and a conserved megakaryocyte enhancer found within the same topologically associating domain (TAD). Our results support a unique disease mechanism whereby the reorganization of subTAD genome architecture results in a dramatic, cell-type specific blood disorder phenotype.

Background Obesity predisposes to a pro-inflammatory state. Studies have reported worse clinical outcomes in patients with inflammatory arthritis who are obese. The relationship between obesity and outcomes in patients with polymyalgia rheumatica (PMR), another common inflammatory rheumatic condition, has not been assessed. We examined this in a cohort study of primary care-recruited patients with PMR. Methods The PMR Cohort Study is an inception cohort of patients with incident PMR, recruited from 382 general practices. Self-completed questionnaires at 0, 12, and 24-months captured: (a) PMR-related pain (0-10 numeric rating scale [NRS]); (b) PMR-related stiffness (0-10 NRS); (c) anxiety (GAD7); (d) depression (PHQ8); (e) fatigue (FACIT-Fatigue); (f) function (mHAQ); (g) quality of life (EQ-5D-3L). Height was self-reported at baseline, and weight at baseline, 12 and 24 months. Patients were categorised as underweight (BMI Results 652 patients were included (62% female). At baseline, mean age was 72 years, median BMI 26.6 (IQR 24.0-30.2), 34% were normal/under-weight, 40% overweight, and 26% obese. Compared to normal/underweight patients, obese patients had the following significantly poorer outcomes (P Conclusion Obesity is associated with poorer outcomes in patients with PMR. Consideration should be given to providing weight management support to patients with PMR and obesity. Further research should examine the impacts of weight-based steroid dosing on outcomes. Disclosures I.C. Scott None. R. Bajpai None. S.L. Hider None. T. Helliwell None. S. Muller None. C.D. Mallen None.

Background The Musculoskeletal Health Questionnaire (MSK-HQ) is a recently developed generic patient-reported outcome measure (PROM), evaluating impacts of musculoskeletal (MSK) conditions on patients’ health. Its scores range 0-56; higher scores indicate better health. Its performance (a) in patients with MSK-pain in primary care, and (b) compared to MSK pain-site reference PROMs, is uncertain. We addressed these uncertainties through secondary analysis of a previous primary-care based study, enrolling 524 patients with shoulder, neck, lower back, knee, or multi-site MSK pain managed by GPs, from 8 West Midlands practices. Methods The study captured the following self-report questionnaire data at 0 and 6 months: MSK-HQ, EQ-5D-5L, Roland-Morris Disability Questionnaire (back pain), Neck Disability Index (neck pain), Shoulder Pain and Disability Index (shoulder pain), Knee Injury and Outcome Score (knee pain), Short-Form-12 (multisite pain). At 6 months, patients self-rated their global change in MSK pain, from -5 (“very much worse”) to + 5 (“completely recovered”). Receiver operating characteristic curves evaluated the ability of 6-month changes in each PROM to discriminate between patients improving/not improving on global change scores. Minimal Clinically Important Differences (MCID) were calculated (cut-off optimising discriminatory sensitivity/specificity). Results Most (60%) reported pain improvements, with mean baseline MSK-HQ scores rising from 29.4 to 37.4 over 6-months. The MSK-HQ had a moderate ability to discriminate between patients improving vs. no change/worsening in MSK pain (area under the curve [AUC] 0.81; 95% CI 0.78-0.85). Its discriminative ability was better than the EQ-5D-3L (AUC 0.68; 95% CI 0.62-0.73) and at least as good as site-specific PROMs (Table). MCID for the 6-month change in MSK-HQ was 5.5 across all patients. Minor variation ( Conclusion In patients consulting in primary care with MSK pain, the MSK-HQ appears at least as good as existing pain-site specific PROMs at identifying patients self-reporting pain improvements, and superior to the EQ-5D-5L. Our results support the use of the MSK-HQ in this setting. Disclosures I.C. Scott None. G. McCray None. G. Lancaster None. N.E. Foster None. J.C. Hill None.

Background There is little evidence analgesics improve pain in inflammatory arthritis (IA), and substantial evidence they have significant harms. Excepting NSAIDs in ankylosing spondylitis (AS), their long-term use is not recommended in guidelines. Despite this, international data suggest pain management in IA involves the substantial and sustained use of opioids (∼40% of North American patients with rheumatoid arthritis [RA] are chronic opioid users). We evaluated analgesic prescribing practice in NHS-managed patients with IA. Methods This study was undertaken in the Consultations in Primary Care Archive (CiPCA), containing primary care consultation/prescription data from 9 general practices (∼200,000 patients) in North Staffordshire (UK) from 2000-2015. Repeated cross-sectional analyses evaluated annual prevalence of analgesic prescriptions in (a) patients with IA (RA; psoriatic arthritis [PsA]; AS), and (b) five age, sex, and practice-matched controls without inflammatory rheumatic conditions. Patients without full calendar-years of data were excluded. The numerator was the number of patients receiving oral analgesic prescriptions in a calendar-year; the denominator was the number of patients contributing data within that calendar-year. Analgesic prescriptions were classified into basic, opioids, gabapentinoids, and NSAIDs. Patients were grouped into users, or non-users of analgesics. Users were sub-classified into chronic (≥3 prescriptions across a calendar year), or intermittent (1-2 prescriptions) users. Results In all years, the majority (65.1-77.8%) of patients with IA received analgesics, compared with the minority (38.1-42.0%) of controls (Table). Opioid prescribing in IA fell slightly between 2000-2015, but remained common with 45.0% receiving an opioid, and 32.5% being chronic opioid users in 2015. The proportion of patients with IA prescribed gabapentinoids increased from 0% in 2000, to 9.4% in 2015. In contrast, the proportion prescribed NSAIDs fell from 53.3% in 2000, to 24.9% in 2015. In all years, analgesic use was commoner in patients with RA, compared to PsA and AS, and 2-4 times higher in IA than controls. Conclusion Analgesic prescribing - particularly opioids - in IA is commonplace, with most prescribed long-term. This is at variance with existing evidence, and guidelines. Interventions are needed to improve analgesic prescribing in this population. Disclosures I.C. Scott None. J. Bailey None. C. White None. C.D. Mallen None. S. Muller None.

Summary Splenic red pulp macrophages (RPMs) contribute to erythrocyte homeostasis and are required for iron recycling. Heme induces the expression of SPIC transcription factor in monocyte-derived macrophages and promotes their differentiation into RPM precursors, pre-RPMs. However, the requirements for differentiation into mature RPMs remain unknown. Here, we have demonstrated that interleukin (IL)-33 associated with erythrocytes and co-cooperated with heme to promote the generation of mature RPMs through activation of the MyD88 adaptor protein and ERK1/2 kinases downstream of the IL-33 receptor, IL1RL1. IL-33- and IL1RL1-deficient mice showed defective iron recycling and increased splenic iron deposition. Gene expression and chromatin accessibility studies revealed a role for GATA transcription factors downstream of IL-33 signaling during the development of pre-RPMs that retained full potential to differentiate into RPMs. Thus, IL-33 instructs the development of RPMs as a response to physiological erythrocyte damage with important implications to iron recycling and iron homeostasis., Graphical Abstract, Highlights • IL-33 signaling promotes development of monocyte-derived red pulp macrophages (RPMs) • Il33−/− and Il1rl1−/− mice have decreased RPMs and splenic erythrophagocytosis • ERK activation is required for RPM development and is potentiated by hemin and IL-33 • GATA2 instructs RPM development and is aberrant in Il1rl1−/− RPM precursors, Splenic red pulp macrophages (RPMs) are critical for iron recycling. Here, Lu et al. implicate a role for IL-33 signaling in the regulation of GATA2, which controls the transition of monocytes to produce pre-RPMs that are competent to terminally differentiate into mature RPMs.

Interleukin (IL)-1 family cytokines are important initiators of innate immunity and host defence; however, their uncontrolled activities can cause tissue-damaging inflammation. Consequently, IL-1 family cytokines have sophisticated regulatory mechanisms to control their activities including proteolytic processing for their activation and the deployment of soluble receptors and receptor antagonists to limit their activities. IL-33 is a promoter of type 2 immunity and allergic inflammation through its alarmin activity that can rapidly initiate local immune responses by stimulating innate immune cells following exposure to environmental insults, pathogens, or sterile injury. Recent publications have provided new insights into how the range and duration of IL-33 activity is regulated by direct sensing of host-derived and exogenous proteolytic activities as well as oxidative changes during tissue damage. Here, we discuss how this impacts our understanding of the roles of IL-33 in initiating immune responses and the evidence that these sensing mechanisms might regulate the activities of other IL-1 family cytokines and their biological functions. Finally, we discuss translational challenges these discoveries pose for the accurate detection of different forms of these cytokines.

As primary care clinicians are typically the first point of contact for patients with musculoskeletal problems, they are crucial to the early diagnosis and treatment of patients with an incident inflammatory arthritis, like rheumatoid arthritis. Current UK and international guidelines recognise this, recommending the prompt referral of patients with suspected persistent synovitis to secondary care. In England and Wales, this is advised to occur within 3 working days. However, recent audit data suggests this recommendation is infrequently met, with some patients waiting many months for referral. In this review article we will discuss the various challenges to achieving the early referral of patients with a new-onset inflammatory arthritis from primary to secondary care. We will also describe how these challenges could potentially be overcome, with the ultimate goal of ensuring that the right patients are referred to the right services, and at the right time.

Two different Janus kinase (JAK) inhibitors-baricitinib and tofacitinib-are effective and licensed in active rheumatoid arthritis (RA). There have been recent concerns about potential thromboembolic risks with these drugs. Concerns about baricitinib focus on clinical trial findings. Using all publically available data, we estimate thromboembolic risks are approximately five events per 1000 patient years with 4 mg baricitinib daily. Concerns about tofacitinib have been raised by analyses of the Federal Drug Administration Adverse Event Reporting System (FAERs). These show some evidence of increased risks of pulmonary thrombosis, though not pulmonary embolism or venous thrombosis. Observational studies suggest in the general population and non-RA controls there are one to four thromboembolic events per 1000 patient years. In RA, thromboembolic risks increase to three to seven per 1000 patient years. The impact of biologics and disease-modifying anti-rheumatic drugs (DMARDs) on disease risk appears minimal, and the number of thromboembolic events is between four and eight per 1000 patient years. In the short term, full details of thromboembolic events in trials of JAK inhibitors need to be published. As the numbers of thromboembolic events will be small and patients enrolled in trials are not representative of all RA patients who may receive JAK inhibitors, this information is unlikely to provide definitive answers. Consequently, in the longer term, large observational studies are needed to accurately quantify thromboembolic risks attributable to JAK inhibitors and other drugs used to treat RA, and differentiate these from risks attributable to RA itself and its comorbidities.

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy1,2,3. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations2. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes1,3. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1)1,3,4. Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat., This work was supported by an Alex's Lemonade Stand Young Investigator Award (S.C.M.), The CIHR Banting Fellowship (S.C.M.), The Cancer Prevention Research Institute of Texas (S.C.M., RR170023), Sibylle Assmus Award for Neurooncology (K.W.P.), the DKFZ-MOST (Ministry of Science, Technology & Space, Israel) program in cancer research (H.W.), James S. McDonnell Foundation (J.N.R.) and NIH grants: CA154130 (J.N.R.), R01 CA169117 (J.N.R.), R01 CA171652 (J.N.R.), R01 NS087913 (J.N.R.) and R01 NS089272 (J.N.R.). R.C.G. is supported by NIH grants T32GM00725 and F30CA217065. M.D.T. is supported by The Garron Family Chair in Childhood Cancer Research, and grants from the Pediatric Brain Tumour Foundation, Grand Challenge Award from CureSearch for Children’s Cancer, the National Institutes of Health (R01CA148699, R01CA159859), The Terry Fox Research Institute and Brainchild. M.D.T. is also supported by a Stand Up To Cancer St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113).

BACKGROUND In this study, we reveal a previously undescribed role of the HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) tumor suppressor protein in normal vertebrate heart development using the zebrafish (Danio rerio) model. We examined the link between the cardiac phenotypes associated with hace1 loss of function to the expression of the Rho small family GTPase, rac1, which is a known target of HACE1 and promotes ROS production via its interaction with NADPH oxidase holoenzymes. RESULTS We demonstrate that loss of hace1 in zebrafish via morpholino knockdown results in cardiac deformities, specifically a looping defect, where the heart is either tubular or "inverted". Whole-mount in situ hybridization of cardiac markers shows distinct abnormalities in ventricular morphology and atrioventricular valve formation in the hearts of these morphants, as well as increased expression of rac1. Importantly, this phenotype appears to be directly related to Nox enzyme-dependent ROS production, as both genetic inhibition by nox1 and nox2 morpholinos or pharmacologic rescue using ROS scavenging agents restores normal cardiac structure. CONCLUSIONS Our study demonstrates that HACE1 is critical in the normal development and proper function of the vertebrate heart via a ROS-dependent mechanism. Developmental Dynamics 247:289-303, 2018. © 2017 Wiley Periodicals, Inc.

Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects learning and memory in adult fish. Here, we develop a contextual fear conditioning paradigm using an electric shock as the aversive stimulus. We find that contextual fear conditioning is modulated by shock intensity, prevented by inhibition of (N-methyl-D-aspartate) NMDA receptors, lasts at least 14 days, and exhibits extinction. Furthermore, fish of various background strains (AB, Tu, and TL) are able to acquire fear conditioning, but differ in fear extinction rates. Taken together, we find that contextual fear conditioning in zebrafish shares many similarities with the widely used contextual fear conditioning paradigm in rodents. Combined with the amenability of genetic manipulation in zebrafish, we anticipate that our paradigm will prove to be a useful complementary system in which to examine the molecular basis of vertebrate learning and memory.

Chondrogenesis in the developing skeleton requires transformation of chondrocytes from a simple mesenchymal condensation to cells with a highly enriched extracellular matrix (ECM). This transition is in part accomplished by alterations in the chondrocyte protein transport machinery to cope with both the increased amount and large size of ECM components. In a zebrafish mutagenesis screen to identify genes essential for cartilage development, we uncovered a mutant that disrupts the gene encoding Sec1 family domain containing 1 (scfd1). Homozygous scfd1 mutant embryos exhibit a profound craniofacial abnormality caused by a failure of chondrogenesis. Loss of scfd1 was found to hinder ER to Golgi transport of ECM proteins and is accompanied with activation of the unfolded protein response in chondrocytes. We further demonstrate a conserved role for Scfd1 in differentiation of mammalian chondrocytes, in which loss of either SCFD1 or STX18, a SLY1 interacting t-SNARE, severely impair transport of type II collagen. These results show that the existence of a specific export pathway, mediated by a complex containing SCFD1 and STX18 that plays an essential role in secretion of large ECM proteins during chondrogenesis.

Primary care is key to the early diagnosis and ongoing care of people with inflammatory rheumatic diseases. Primary care is usually the first point of call for health problems and is the setting wh...

Introduction: Interleukin (IL)-33 is released from the airway epithelium and can sense the proteolytic and oxidative environment (Cohen, E.S. et al. Nat Comms 2015; 6:8327; Scott, I.C. et al. Sci Rep 2018; 8:3363). IL-33 amplifies inflammation via its receptor ST2 and is associated with type 2 immunity. IL-33 exists in reduced and oxidized forms of IL-33 but can be sequestered by soluble ST2 (sST2), however, there are no assays that distinguish forms. Inconsistencies with IL-33 detection with commercial assays might be due to the differential detection of these antigenically distinct forms. Here we describe new assays for IL-33 forms and measurements in patient samples. Methods: anti-IL-33 mAb were paired in S-plex assays (MSD) with lower limit of detection (LLOD): reduced IL-33 (~16 fg/ml), oxidized (ox) (~100 fg/ml) or IL-33/sST2 complexes (~100 fg/ml). Assays were evaluated in serum from healthy (n=20), asthmatics (n=14) and COPD patients (n=16) or nasal mucosal lining fluid (MLF) from atopics (n=6) following allergen challenge (NAC, 0-24h). Results: We detected comparable levels of IL-33/sST2 complex (~1-5 pg/ml) in serum from healthy individuals and patients. Total sST2 in serum was similar between patients but at high levels (~10-100 ng/ml). Reduced and oxIL-33 were undetected in serum. In contrast, we detected reduced and oxIL-33 in MLF from atopics with levels increased post-NAC that peaked after 1 h (20-1000 pg/ml). In comparison, PGD2 (1-3 ng/ml) peaked after 15 min and IL-5 (~25-300 pg/ml) after 3-10 h. IL-33/sST2 complexes were low in MLF. Conclusions: We have generated novel assays for detection of IL-33 forms that will provide insights into mechanisms regulating IL-33 activity.

Background Refractory disease is defined as not achieving a low disease activity target despite DMARDs. This definition does not account for patients with well controlled inflammation who experience persistent symptoms, or who have a high perceived disease impact. Furthermore, variations in professionals understanding of refractory disease and related concepts exists, with potential discordance when compared to patients understanding. Objectives To qualitatively explore patients and professionals understanding and experiences of refractory disease and persistent symptoms. Methods Semi-structured interviews were conducted with 13 RA and 3 Polyarticular JIA patients (on bDMARD not responding with DAS28>3.2) attending four UK Rheumatology clinics. Thirty-two healthcare professionals (working in Rheumatology >1year) were interviewed across 11 UK hospitals. Inductive thematic analysis was conducted, with descriptive statistics reported for quantitative data. Results Patients had not responded to on average 3 csDMARDs and 3.5 bDMARDs, with mean MSK-HQ=24.6, mean DAS28=4.60, and mean Patient Global=55.4. Healthcare professionals interviewed were predominantly adult trained (25/32), with mean 11.7 years’ experience, comprising rheumatologists, nurses, physiotherapists, occupational therapists, psychologists, pharmacist, podiatrist, and social worker. Key themes for both patients and professionals’ experiences of refractory disease and persistent symptoms explored were: 1) Frustrations with the disease, non-response to medication and side effects, 2) Importance of areas not captured by the DAS28, 3) Patient-centred targets/care, 4) Role of other specialties, 5) Role of comorbidities, infections and/or joint damage, and 6) Patient acceptance, adjustment and resilience. Although patients expressed hopeful expectations despite poor experiences, this was in discordance to professionals’ accounts of loss of trust/hope, disengagement and limited treatment options, with hope mainly for stratified medicines. Patient specific emergent themes identified: 1) Disease not controlled fully but manageable, 2) Drugs do not work: at all versus over time, 3) Persistent pain, fatigue and restricted mobility are most problematic symptoms, 4) Life-limiting impact: practically, psychologically/emotionally and socially, and 5) Good support from rheumatology team but holistic approach needed. Professionals grouped refractory disease into four key areas: 1) Refractory Inflammation vs Refractory Symptomology, 2) Biological processes, 3) Drug inefficacy/tolerability, and 4) Patient health beliefs/behaviours. Those with JIA had a greater variety in their experiences compared to those with RA. In addition to above themes they all highlighted the challenges of having a long-term illness so young, e.g. not knowing a life without pain or disease and lack of understanding in both themselves and others. Paediatric/Adolescent professionals noted differences in treating JIA compared to RA, e.g. joints affected and treatment guidelines/availability not based on DAS equivalent scores. Conclusion These results highlight the potential need for a broader definition for refractory disease, and the need for holistic approaches to treatment that better support both patients, and professionals, in managing their condition. Disclosure of Interests Hema Chaplin: None declared, Ibone Verhey: None declared, Nora Ng: None declared, James Galloway Consultant for: Pfizer Inc, Ian Scott: None declared, Debajit Sen: None declared, Rachel Tattersall: None declared, Heidi Lempp: None declared, Sam Norton: None declared

Objective To establish if gravidity and parity associate with the development of rheumatoid arthritis (RA), and to establish if this effect is influenced by the time elapsed since pregnancy/childbirth, the number of pregnancies/childbirths, and serological status, through systematically reviewing the literature and undertaking a meta-analysis. Methods We searched Medline/EMBASE (from 1946 to 2018) using the terms “rheumatoid arthritis.mp” or “arthritis, rheumatoid/” and “pregnancy.mp” or “pregnancy/” or “parity.mp” or “parity/” or “gravidity.mp” or “gravidity/” (observational study filter applied). Case-control/cohort studies that examined the relationship between parity/gravidity and the risk of RA in women were included. Studies reporting effect size data for RA in ever vs. never parous/gravid women as ORs/RRs with 95% confidence intervals were included in a meta-analysis. Other relationships (i.e. risk by pregnancy/childbirth numbers) were analysed descriptively. Results Twenty studies (from 626 articles) met our inclusion criteria, comprising 14 case-control (4799 cases; 11,941 controls) and 6 cohort studies (8575 cases; 2,368,439 individuals). No significant association was observed in the meta-analysis of studies reporting the risk of RA in ever vs. never parous women (OR 0.91; 95% CI 0.80–1.04) and ever vs. never gravid women (OR 0.86; 95% CI 0.46–1.62). No consistent evidence of a relationship between the number of pregnancies/childbirths and RA risk was seen. No significant association was observed between being pregnant, or in the immediate post-partum period, and the risk of developing RA. Conclusion Our systematic review does not support the concept that gravidity and parity are associated with the risk of RA development.

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes, and other structures in cell culture, to date there have been only a few studies on living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 h of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches., Graphical Abstract, Highlights • Implementation of a proximity-dependent biotinylation in zebrafish embryos. • Use of miniTurbo and TurboID for proximal interaction detection in zebrafish embryos. • Optimization of mRNA injection and transgenics-based enzyme delivery. • Benchmarking conditions and approaches on the nuclear lamina., In Brief Proximity-dependent biotinylation approaches such as APEX and BioID are used to illuminate cellular organization in cell culture studies but are not often applied to whole organisms. Here, Rosenthal et al. report the development and optimization of a toolkit for proximity-dependent biotinylation with miniTurbo and TurboID in zebrafish embryos. They describe, using the lamin A component of the lamina, protocols and benchmarks for a rapid injection-based strategy that is appropriate for early development and a flexible heat shock inducible transgenic system.

Background:Evaluating the long-term impacts of healthcare policies on patient’s health and treatment costs for people with axial spondyloarthritis (axSpA) is challenging due to its chronic nature, and the variation in individual patient journeys post-diagnosis. Agent-based simulations are a novel approach to interrogating this complexity, and allow the consequences of different policy scenarios on outcomes to be explored.Objectives:Develop and validate an agent-based simulation of the UK axial spondyloarthritis healthcare system, using real-world data.Interrogate the effects of earlier biologic treatment on costs and patient outcomes.Methods:Anonymised data were obtained from the UK National Early Inflammatory Arthritis Audit, and BSR Biologics Register (BSRBR-AS). This provided data on 162 units, and 702 patients with 1,631 patient-years of follow-up. An agent-based model was designed and programmed on the Netlogo platform to simulate patients and units individually over time. New patients were created based on national disease prevalence statistics. Patients’ disease journeys were modelled with a Bath AS Disease Activity Index (BASDAI) score. The model included hospital outpatient attendances, treatment histories, drug costs, and key patient demographics. The baseline simulation was run for two simulated years, repeated 10 times, and assessed against the BSRBR-AS dataset for validation. The model was subsequently used to explore five experimental scenarios in which the time between the date of diagnosis, to first introduction to biologics (d-b) was varied by increasing the number of appointments. The experiment was run 10 times for each parameter setting.Results:In the baseline model in a typical two year run, 13,631 new patients attended 5,167 baseline, and 6,966 follow-up appointments. Of these, 6,324 and 623 were prescribed ≥1NSAID, and biologics, respectively. The validation comparison tests showed a high-level of similarity between simulated output and target datasets. In the target data, d-b was 250 days. In the experimental scenarios, as might be expected, earlier biologic access improved outcomes but at higher-costs (Figure 1; Table 1). Reducing d-b to 150 days doubled the number of patients on biologics at 2 years from 623 to 1,286. It also led to 8% more patients achieving a BASDAI of 0 to 2.5 at 2 years, with 5%, 1%, and 2% less patients achieving 2.5 - 5, 5 to 7.5 and 7.5 to 10 BASDAI, respectively. Reducing d-b to 150 days increased drug costs from £3.2 million to £8.8 million. However, the total number of appointments (a proxy for staff costs) increased proportionality less from 16,000 to 20,000.Table 1.Influence of varying the time between diagnosis to biologic treatment (d-b) on drug-use and staffing costsDiagnosis to Biologic (d-b)Drug Costs Unit (£k)Total AppointmentsNo. patients prescribed NSAIDsNo. patients prescribed Biologics1508,79620,3847,1541,2832205,70218,5926,7969712503,25916,2146,3246212602,05414,7005,9683822651,29713,4115,562233Figure 1.Influence of varying the time between diagnosis to biologic treatment (d-b) on 2 year BASDAI outcomeConclusion:We have successfully developed, and validated an agent-based approach to model the effect of key policy changes on the whole healthcare system, providing output estimates of cost and patient outcomes, based on integrated real-world data. To our knowledge this is the first attempt to explore the patient journey in people with axSpA in this way. The model provides a useful tool for exploring the effects of changing the way healthcare is delivered to patients with this disease. Our experimental analysis lends support to the case for increasing staffing and drug expenditure to achieve current NICE standards of care in AS.Acknowledgments:Financial support National Axial Spondyloarthritis Society (NASS), data access BSR.Disclosure of Interests:Alan Roach Grant/research support from: I was awarded an I-CRP grant from Pfizer for a similar simulation in RA, this was for about £50k and ran from 1/9/15 28/2/17., Ian Scott: None declared, Gary Macfarlane: None declared, Gareth T. Jones Grant/research support from: Pfizer, AbbVie, UCB, Celgene and GSK., Alex MacGregor: None declared

During the phylotypic period, embryos from different genera show similar gene expression patterns, implying common regulatory mechanisms. Here we set out to identify enhancers involved in the initial events of cardiogenesis, which occurs during the phylotypic period. We isolate early cardiac progenitor cells from zebrafish embryos and characterize 3838 open chromatin regions specific to this cell population. Of these regions, 162 overlap with conserved non-coding elements (CNEs) that also map to open chromatin regions in human. Most of the zebrafish conserved open chromatin elements tested drive gene expression in the developing heart. Despite modest sequence identity, human orthologous open chromatin regions recapitulate the spatial temporal expression patterns of the zebrafish sequence, potentially providing a basis for phylotypic gene expression patterns. Genome-wide, we discover 5598 zebrafish-human conserved open chromatin regions, suggesting that a diverse repertoire of ancient enhancers is established prior to organogenesis and the phylotypic period., During early embryogenesis, critical cardiac specification events occur. Here the authors isolate cardiac progenitor cells from early zebrafish embryos and characterize accessible chromatin regions specific to this cell population, finding that many of these regions overlap with conserved non-coding elements that are ortholgous to accessible chromatin regions in human.

A key event in heart development is the timely addition of cardiac progenitor cells, defects in which can lead to congenital heart defects. However, how the balance and proportion of progenitor proliferation versus addition to the heart is regulated remains poorly understood. Here we demonstrate that Hey2 functions to regulate the dynamics of cardiac progenitor addition to the zebrafish heart. We found that the previously noted increase in myocardial cell number found in the absence of Hey2 function was due to a pronounced expansion in the size of the cardiac progenitor pool. Expression analysis and lineage tracing of hey2-expressing cells showed that hey2 is active in cardiac progenitors. Hey2 acted to limit proliferation of cardiac progenitors, prior to heart tube formation. Use of a transplantation approach demonstrated a likely cell autonomous (in cardiac progenitors) function for Hey2. Taken together, our data suggests a previously unappreciated role for Hey2 in controlling the proliferative capacity of cardiac progenitors, affecting the subsequent contribution of late-differentiating cardiac progenitors to the developing vertebrate heart.

During the phylotypic period embryos from different genera show similar gene expression patterns, implying common regulatory mechanisms. To identify enhancers involved in the initial events of cardiogenesis, which occurs during the phylotypic period, we isolated early cardiac progenitor cells from zebrafish embryos and characterized 3838 open chromatin regions specific to this cell population. Of these regions, 162 overlapped with conserved non-coding elements (CNEs) that also mapped to open chromatin regions in human. Most of the zebrafish conserved open chromatin elements tested drove gene expression in the developing heart. Despite modest sequence identity, human orthologous open chromatin regions could recapitulate the spatial temporal expression patterns of the zebrafish sequence, potentially providing a basis for phylotypic gene expression patterns. Genome-wide, we discovered 5598 zebrafish-human conserved open chromatin regions, suggesting that a diverse repertoire of ancient enhancers is established prior to organogenesis and the phylotypic period.

Interleukin (IL)-33 is an IL-1 family alarmin released from damaged epithelial and endothelial barriers to elicit immune responses and allergic inflammation via its receptor ST2. Serine proteases released from neutrophils, mast cells and cytotoxic lymphocytes have been proposed to process the N-terminus of IL-33 to enhance its activity. Here we report that processing of full length IL-33 can occur in mice deficient in these immune cell protease activities. We sought alternative mechanisms for the proteolytic activation of IL-33 and discovered that exogenous allergen proteases and endogenous calpains, from damaged airway epithelial cells, can process full length IL-33 and increase its alarmin activity up to ~60-fold. Processed forms of IL-33 of apparent molecular weights ~18, 20, 22 and 23 kDa, were detected in human lungs consistent with some, but not all, proposed processing sites. Furthermore, allergen proteases degraded processed forms of IL-33 after cysteine residue oxidation. We suggest that IL-33 can sense the proteolytic and oxidative microenvironment during tissue injury that facilitate its rapid activation and inactivation to regulate the duration of its alarmin function.

Failure to properly repair damaged due to myocardial infarction is a major cause of heart failure. In contrast with adult mammals, zebrafish hearts show remarkable regenerative capabilities after substantial damage. To characterize protein dynamics during heart regeneration, we employed an HPLC-ESI-MS/MS (mass spectrometry) approach. Myocardium tissues were taken from sham-operated fish and ventricle-resected sample at three different time points (2, 7, and 14 days); dynamics of protein expression were analyzed by an ion-current-based quantitative platform. More than 2000 protein groups were quantified in all 16 experiments. Two hundred and nine heart-regeneration-related protein groups were quantified and clustered into six time-course patterns. Functional analysis indicated that multiple molecular function and metabolic pathways were involved in heart regeneration. Interestingly, Ingenuity Pathway Analysis revealed that P53 signaling was inhibited during the heart regeneration, which was further verified by real-time quantitative polymerase chain reaction (Q-PCR). In summary, we applied systematic proteomics analysis on regenerating zebrafish heart, uncovered the dynamics of regenerative genes expression and regulatory pathways, and provided invaluable insight into design regenerative-based strategies in human hearts.

RNA helicases from the DEAD-box family are found in almost all organisms and have important roles in RNA metabolism, including RNA synthesis, processing and degradation. The function and mechanism of action of most of these helicases in animal development and human disease remain largely unexplored. In a zebrafish mutagenesis screen to identify genes essential for heart development we identified a mutant that disrupts the gene encoding the RNA helicase DEAD-box 39ab ( ddx39ab ). Homozygous ddx39ab mutant embryos exhibit profound cardiac and trunk muscle dystrophy, along with lens abnormalities, caused by abrupt terminal differentiation of cardiomyocyte, myoblast and lens fiber cells. Loss of ddx39ab hindered splicing of mRNAs encoding epigenetic regulatory factors, including members of the KMT2 gene family, leading to misregulation of structural gene expression in cardiomyocyte, myoblast and lens fiber cells. Taken together, these results show that Ddx39ab plays an essential role in establishment of the proper epigenetic status during differentiation of multiple cell lineages.

Methotrexate is the dominant initial drug in the management of rheumatoid arthritis (RA). Despite its widespread use, methotrexate is associated with a number of adverse effects. Tapering its dose to the minimal amount required to maintain RA remission is, therefore, an important clinical goal. While the complete withdrawal of disease-modifying anti-rheumatic drugs is associated with a definite risk of a disease flare, it is unclear as to what the risk is specific to methotrexate withdrawal and whether this can be minimized by gradual dose reduction (termed 'tapering'). This review examines studies of methotrexate tapering and withdrawal on RA outcomes. It covers three scenarios: tapering/withdrawing methotrexate monotherapy; tapering/withdrawing methotrexate as part of a 'step-down' combination disease-modifying anti-rheumatic drug regimen; and tapering/withdrawing methotrexate when it is being co-prescribed with biologic agents.

A key event in vertebrate heart development is the timely addition of second heart field (SHF) progenitor cells to the poles of the heart tube. This accretion process must occur to the proper extent to prevent a spectrum of congenital heart defects (CHDs). However, the factors that regulate this critical process are poorly understood. Here we demonstrate that Hey2, a bHLH transcriptional repressor, restricts SHF progenitor accretion to the zebrafish heart.hey2expression demarcated a distinct domain within the cardiac progenitor population. In the absence of Hey2 function an increase in myocardial cell number and SHF progenitors was observed. We found that Hey2 limited proliferation of SHF-derived cardiomyocytes in a cell-autonomous manner, prior to heart tube formation, and further restricted the developmental window over which SHF progenitors were deployed to the heart. Taken together, our data suggests a role for Hey2 in controlling the proliferative capacity and cardiac contribution of late-differentiating cardiac progenitors.