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1.  General practitioners’ perspectives on campaigns to promote rapid help-seeking behaviour at the onset of rheumatoid arthritis 
Abstract
Objective. To explore general practitioners’ (GPs’) perspectives on public health campaigns to encourage people with the early symptoms of rheumatoid arthritis (RA) to seek medical help rapidly. Design. Nineteen GPs participated in four semi-structured focus groups. Focus groups were audio-recorded, transcribed verbatim, and analysed using thematic analysis. Results. GPs recognised the need for the early treatment of RA and identified that facilitating appropriate access to care was important. However, not all held the view that a delay in help seeking was a clinically significant issue. Furthermore, many were concerned that the early symptoms of RA were often non-specific, and that current knowledge about the nature of symptoms at disease onset was inadequate to inform the content of a help-seeking campaign. They argued that a campaign might not be able to specifically target those who need to present urgently. Poorly designed campaigns were suggested to have a negative impact on GPs’ workloads, and would “clog up” the referral pathway for genuine cases of RA. Conclusions. GPs were supportive of strategies to improve access to Rheumatological care and increase public awareness of RA symptoms. However, they have identified important issues that need to be considered in developing a public health campaign that forms part of an overall strategy to reduce time to treatment for patients with new onset RA. This study highlights the value of gaining GPs’ perspectives before launching health promotion campaigns.
doi:10.3109/02813432.2014.900239
PMCID: PMC4137901  PMID: 24635577
General practice; general practitioner; health promotion; primary care; qualitative; rheumatoid arthritis; seeking help; United Kingdom
2.  Identification of the Tyrosine-Protein Phosphatase Non-Receptor Type 2 as a Rheumatoid Arthritis Susceptibility Locus in Europeans 
PLoS ONE  2013;8(6):e66456.
Objectives
Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA). However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA.
Methods
A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data.
Results
Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10−9).
Conclusions
This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase) with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene.
doi:10.1371/journal.pone.0066456
PMCID: PMC3688762  PMID: 23840476
3.  Availability of 25-hydroxyvitamin D3 to antigen presenting cells controls the balance between regulatory and inflammatory T cell responses 
1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, exerts potent effects on several tissues including cells of the immune system, where it affects T cell activation, differentiation and migration. The circulating, inactive form of vitamin D, 25(OH)D3, is generally used as an indication of “vitamin D status”. However, utilization of this precursor depends on its uptake by cells and subsequent conversion by the enzyme 25(OH)D3-1α-hydroxylase (CYP27B1) into active 1,25(OH)2D3. Using human T cells, we now show that addition of inactive 25(OH)D3 is sufficient to alter T cell responses only when dendritic cells (DCs) are present. Mechanistically, CYP27B1 is induced in DCs upon maturation with LPS or upon T cell contact resulting in the generation and release of 1,25(OH)2D3 which subsequently affects T cell responses. In most tissues, vitamin D binding protein (DBP) acts as a carrier to enhance the utilization of vitamin D. However, we show that DBP modulates T cell responses by restricting the availability of inactive 25(OH)D3 to DC. These data indicate that the level of “free” 25(OH)D3 available to DCs determines the inflammatory/regulatory balance of ensuing T cell responses.
doi:10.4049/jimmunol.1200786
PMCID: PMC3504609  PMID: 23087405
4.  A mixed methods protocol to investigate medication adherence in patients with rheumatoid arthritis of White British and South Asian origin 
BMJ Open  2013;3(2):e001836.
Background
Low adherence to medicines is an important issue as up to 40% of patients with chronic diseases do not take their medications as prescribed. This leads to suboptimal clinical benefit. In the context of rheumatoid arthritis, there is a dearth of data on adherence to disease-modifying antirheumatic drugs among minority ethnic groups. This study aims to assess the relationship between adherence to medicines and biopsychosocial variables in patients with rheumatoid arthritis of South Asian and White British origin.
Methods/analysis
A mixed methods approach will be used, encompassing a cross-sectional survey of 176 patients collecting demographic and clinical data, including information on adherence behaviour collected using a series of questionnaires. This will be followed by indepth qualitative interviews.
Ethics and dissemination
This study has been approved by the South Birmingham (10/H1207/89) and Coventry and Warwickshire (12/WM/0041) Research Ethics Committees. The authors will disseminate the findings in peer-reviewed publications.
doi:10.1136/bmjopen-2012-001836
PMCID: PMC3586171  PMID: 23430593
5.  Characterisation of fibroblast-like synoviocytes from a murine model of joint inflammation 
Introduction
Fibroblast-like synoviocytes (FLS) play a central role in defining the stromal environment in inflammatory joint diseases. Despite a growing use of FLS isolated from murine inflammatory models, a detailed characterisation of these cells has not been performed.
Methods
In this study, FLS were isolated from inflamed joints of mice expressing both the T cell receptor transgene KRN and the MHC class II molecule Ag7 (K/BxN mice) and their purity in culture determined by immunofluorescence and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Basal expression of proinflammatory genes was determined by real-time RT-PCR. Secreted interleukin 6 (IL-6) was measured by enzyme-linked immunosorbent assay (ELISA), and its regulation by tumor necrosis factor-alpha (TNF-α and corticosterone (the major glucocorticoid in rodents) measured relative to other mesenchymal cell populations.
Results
Purity of FLS culture was identified by positive expression of fibronectin, prolyl 4-hydroxylase, cluster of differentiation 90.2 (CD90.2) and 248 (CD248) in greater than 98% of the population. Cultured FLS were able to migrate and invade through matrigel, a process enhanced in the presence of TNF-α. FLS isolated from K/BxN mice possessed significantly greater basal expression of the inflammatory markers IL-6, chemokine ligand 2 (CCL-2) and vascular cell adhesion molecule 1 (VCAM-1) when compared to FLS isolated from non-inflamed tissue (IL-6, 3.6 fold; CCL-2, 11.2 fold; VCAM-1, 9 fold; P < 0.05). This elevated expression was abrogated in the presence of corticosterone at 100 nmol/l. TNF-α significantly increased expression of all inflammatory markers to a much greater degree in K/BxN FLS relative to other mesenchymal cell lines (K/BxN; IL-6, 40.8 fold; CCL-2, 1343.2 fold; VCAM-1, 17.8 fold; ICAM-1, 13.8 fold; P < 0.05), with secreted IL-6 mirroring these results (K/BxN; con, 169 ± 29.7 versus TNF-α, 923 ± 378.8 pg/ml/1 × 105 cells; P < 0.05). Dose response experiments confirmed effective concentrations between 10 and 100 nmol/l for corticosterone and 1 and 10 ng/ml for TNF-α, whilst inflammatory gene expression in FLS was shown to be stable between passages four and seven.
Conclusions
This study has established a well characterised set of key inflammatory genes for in vitro FLS culture, isolated from K/BxN mice and non-inflamed wild-type controls. Their response to both pro- and anti-inflammatory signalling has been assessed and shown to strongly resemble that which is seen in human FLS culture. Additionally, this study provides guidelines for the effective characterisation, duration and treatment of murine FLS culture.
doi:10.1186/ar4158
PMCID: PMC3672796  PMID: 23363614
6.  Synovial DKK1 expression is regulated by local glucocorticoid metabolism in inflammatory arthritis 
Arthritis Research & Therapy  2012;14(5):R226.
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.
doi:10.1186/ar4065
PMCID: PMC3580537  PMID: 23079210
7.  Predictive Value of Antibodies to Cyclic Citrullinated Peptide in Patients with Very Early Inflammatory Arthritis 
The Journal of rheumatology  2005;32(2):231-238.
Objective
To study the prognostic value of antibodies to cyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF), alone and in combination, in patients with very early synovitis.
Methods
A cross-sectional study was performed in patients with established inflammatory and noninflammatory disease to validate the assay in our unit and confirm previously reported sensitivities and specificities of anti-CCP antibodies. Subsequently, patients with synovitis of ≤ 3 months’ duration were followed for 72 weeks and the ability of anti-CCP antibodies and RF to predict the development of rheumatoid arthritis (RA) and persistent inflammatory arthritis was assessed.
Results
One hundred twenty-four patients were assessed in the initial cross-sectional study. Anti-CCP antibodies and RF were detected by ELISA in only 4% of patients with non-RA inflammatory disease and in no patient with noninflammatory disease. Ninety-six patients with very early synovitis were assessed longitudinally. In these patients with early arthritis, the combination of anti-CCP antibodies and RF had a specificity, positive predictive value (PPV), sensitivity, and negative predictive value (NPV) for a diagnosis of RA of 100%, 100%, 58%, and 88%, respectively. The specificity, PPV, sensitivity, and NPV of this antibody combination for the development of persistent disease-fulfilling classification criteria for RA were 97%, 86%, 63%, and 91%, respectively.
Conclusion
In patients with synovitis of ≤ 3 months’ duration, a combination of anti-CCP antibodies and RF has a high specificity and PPV for the development of persistent RA. This autoantibody combination can be used to identify patients with disease destined to develop RA who may be appropriate for very early intervention.
PMCID: PMC3160476  PMID: 15693082
CYCLIC CITRULLINATED PEPTIDE; ANTI-CYCLIC CITRULLINATED PEPTIDE ANTIBODY; RHEUMATOID ARTHRITIS; DIAGNOSIS; EARLY ARTHRITIS; RHEUMATOID FACTOR
8.  Targeting Stromal Cells in Chronic Inflammation 
Discovery medicine  2007;7(37):20-26.
Summary
Why chronic inflammatory reactions persist in specific sites, such as rheumatoid arthritis in the joints, remains a mystery. Current models of inflammation have concentrated upon the responses of lymphocytes such as B and T cells to specific antigens, and have attempted, often unsuccessfully, to address the causative agent. However recent studies have shown that stromal cells such as macrophages, endothelial cells, and fibroblasts play important roles in the switch that turns a spontaneously resolving acute inflammatory response within a tissue into chronic and persistent disease. Therapeutic manipulation of the stromal microenvironment has been particularly effective in treating cancer and is likely to provide novel therapies to achieve improved control of chronic inflammatory disease.
PMCID: PMC3160478  PMID: 17343801
9.  Altered Expression of MicroRNA-203 in Rheumatoid Arthritis Synovial Fibroblasts and Its Role in Fibroblast Activation 
Arthritis and rheumatism  2011;63(2):373-381.
Objective
MicroRNA (miRNA) are recognized as important regulators of a variety of fundamental biologic processes. Previously, we described increased expression of miR-155 and miR-146a in rheumatoid arthritis (RA) and showed a repressive effect of miR-155 on matrix metalloproteinase (MMP) expression in RA synovial fibroblasts (RASFs). The present study was undertaken to examine alterations in expression of miR-203 in RASFs and analyze its role in fibroblast activation.
Methods
Differentially expressed miRNA in RASFs versus osteoarthritis synovial fibroblasts (OASFs) were identified by real-time polymerase chain reaction (PCR)–based screening of 260 individual miRNA. Transfection of miR-203 precursor was used to analyze the function of miR-203 in RASFs. Levels of interleukin-6 (IL-6) and MMPs were measured by real-time PCR and enzyme-linked immunosorbent assay. RASFs were stimulated with IL-1β, tumor necrosis factor α (TNFα), lipopolysaccharide (LPS), and 5-azacytidine (5-azaC). Activity of IκB kinase 2 was inhibited with SC-514.
Results
Expression of miR-203 was higher in RASFs than in OASFs or fibroblasts from healthy donors. Levels of miR-203 did not change upon stimulation with IL-1β, TNFα, or LPS; however, DNA demethylation with 5-azaC increased the expression of miR-203. Enforced expression of miR-203 led to significantly increased levels of MMP-1 and IL-6. Induction of IL-6 by miR-203 overexpression was inhibited by blocking of the NF-κB pathway. Basal expression levels of IL-6 correlated with basal expression levels of miR-203.
Conclusion
The current results demonstrate methylation-dependent regulation of miR-203 expression in RASFs. Importantly, they also show that elevated levels of miR-203 lead to increased secretion of MMP-1 and IL-6 via the NF-κB pathway and thereby contribute to the activated phenotype of synovial fibroblasts in RA.
doi:10.1002/art.30115
PMCID: PMC3116142  PMID: 21279994
10.  Treating very early rheumatoid arthritis 
Rheumatoid arthritis (RA) is common and leads to joint damage due to persistent synovitis. The persistence of inflammation is maintained by hyperplastic stromal tissue, which drives the accumulation of leukocytes in the synovium. Aggressive treatment after the first 3–4 months of symptoms, with either disease modifying anti-rheumatic drugs or anti-tumor necrosis factor (TNF)-α therapy, reduces the rate of disease progression. However, it rarely switches off disease such that remission can be maintained without the continued need for immunosuppressive therapy. There is increasing evidence that the first few months after symptom onset represent a pathologically distinct phase of disease. This very early phase may translate into a therapeutic window of opportunity during which it may be possible to permanently switch off the disease process. The rationale for, and approaches to, treatment within this very early window are discussed.
doi:10.1016/j.berh.2006.05.005
PMCID: PMC3145120  PMID: 16980210
rheumatoid arthritis; early arthritis; synovitis; therapy; remission; DMARD; anti-TNF-α therapy
11.  The role of chemokines in leucocyte-stromal interactions in rheumatoid arthritis 
New dimensions in our understanding of immune cell trafficking in health and disease have been opened by the discovery of chemokines and their receptors. This family of chemo-attractant cytokines performs essential roles in the recruitment and subsequent positioning of leucocyte subsets within tissue microenvironments. Investigation of chemokine networks offers a novel approach to understand the mechanisms by which inflammatory cells persist in diseases such as rheumatoid arthritis (RA), where evidence is mounting that the inappropriate temporal and spatial expression of chemokines and/or their receptors may impair the resolution of leucocyte infiltrates. The recognition that stromal cells such as fibroblasts, as active components of tissue specific microenvironments, are able to determine the type and persistence of inflammatory infiltrates has opened new vistas in research. Stromal cells are active contributors to cytokine and inflammatory chemokine networks which result in immune cell recruitment and activation. However an intriguing role of stromal cells has been demonstrated in the inappropriate expression of constitutive, housekeeping chemokines, which contribute to the persistence of inflammation by actively blocking its resolution.
PMCID: PMC3145134  PMID: 17981742
Chemokine; chemokine receptor; Stromal; Fibroblast; Rheumatoid arthritis; Inflammation; Microenvironment; Review
12.  Tumor Necrosis Factor α Activates Release of B Lymphocyte Stimulator by Neutrophils Infiltrating the Rheumatoid Joint 
Arthritis and rheumatism  2007;56(6):1776-1786.
Objective
The tumor necrosis factor (TNF) family member B lymphocyte stimulator (BLyS) is an important regulator of B cell–dependent autoimmunity. Similar to other TNF family members, it is generally expressed as a transmembrane protein and cleaved from the surface to release its active soluble form. This study was undertaken to investigate the expression of BLyS and regulation of BLyS release from the surface of neutrophils infiltrating the rheumatoid joint.
Methods
BLyS expression was studied in neutrophils from the synovial fluid and peripheral blood of patients with rheumatoid arthritis (RA) and healthy controls, by flow cytometry, Western blotting, and immunofluorescence analyses. Peripheral blood neutrophils cultured with 50% RA synovial fluid were study for membrane expression of BLyS. Neutrophils were exposed to a range of proinflammatory cytokines to study the mechanisms of surface loss of BLyS.
Results
Expression of BLyS was detected on the surface of peripheral blood neutrophils from both RA patients and healthy controls, whereas BLyS expression on synovial fluid neutrophils was very low. Constitutive expression of BLyS was observed in neutrophils, both on the cell membrane and in intracellular stores; however, BLyS release from each of these sites was found to be regulated independently. Of the various cytokine stimuli, only TNFα triggered release of BLyS from the neutrophil membrane. This process led to release of physiologically relevant quantities of soluble BLyS, which was dependent on the presence of the pro-protein convertase furin. In contrast, stimulation of neutrophils with granulocyte colony-stimulating factor induced BLyS release from the intracellular stores. Incubation of peripheral blood neutrophils with RA synovial fluid led to TNFα-dependent shedding of BLyS from the cell surface.
Conclusion
These findings indicate that as neutrophils enter the site of inflammation, they release surface-expressed BLyS in a TNFα-dependent manner, and thus may contribute to local stimulation of autoimmune B cell responses.
doi:10.1002/art.22697
PMCID: PMC3132453  PMID: 17530706
13.  Differential Survival of Leukocyte Subsets Mediated by Synovial, Bone Marrow, and Skin Fibroblasts 
Arthritis and rheumatism  2006;54(7):2096-2108.
Objective
Synovial fibroblasts share a number of phenotype markers with fibroblasts derived from bone marrow. In this study we investigated the role of matched fibroblasts obtained from 3 different sources (bone marrow, synovium, and skin) to test the hypothesis that synovial fibroblasts share similarities with bone marrow–derived fibroblasts in terms of their ability to support survival of T cells and neutrophils.
Methods
Matched synovial, bone marrow, and skin fibroblasts were established from 8 different patients with rheumatoid arthritis who were undergoing knee or hip surgery. Resting or activated fibroblasts were cocultured with either CD4 T cells or neutrophils, and the degree of leukocyte survival, apoptosis, and proliferation were measured.
Results
Fibroblasts derived from all 3 sites supported increased survival of CD4 T cells, mediated principally by interferon-β. However, synovial and bone marrow fibroblasts shared an enhanced site-specific ability to maintain CD4 T cell survival in the absence of proliferation, an effect that was independent of fibroblast activation or proliferation but required direct T cell–fibroblast cell contact. In contrast, fibroblast-mediated neutrophil survival was less efficient, being independent of the site of origin of the fibroblast but dependent on prior fibroblast activation, and mediated solely by soluble factors, principally granulocyte–macrophage colony-stimulating factor.
Conclusion
These results suggest an important functional role for fibroblasts in the differential accumulation of leukocyte subsets in a variety of tissue microenvironments. The findings also provide a potential explanation for site-specific differences in the pattern of T cell and neutrophil accumulation observed in chronic inflammatory diseases.
doi:10.1002/art.21930
PMCID: PMC3119431  PMID: 16802344
14.  Endothelial cells, fibroblasts and vasculitis 
Rheumatology (Oxford, England)  2005;44(7):860-863.
One of the most important questions in vasculitis research is not why inflammation of blood vessels occurs but why it persists, often in a site-specific manner. In this review we illustrate how stromal cells, such as fibroblasts and pericytes, might play an important role in regulating the site at which vasculitis occurs. Smooth muscle cells and fibroblasts directly influence the behaviour of overlying vascular cells, amplifying the response of the endothelium to proinflammatory agents such as TNF-α and allowing enhanced and inappropriate leucocyte recruitment. An abnormal local vascular stromal environment can therefore influence local endothelial function and drive the persistence of local vascular inflammation. However, such local vascular inflammation can have distant effects on the systemic vascular system, leading to widespread endothelial cell dysfunction. Vascular endothelial dysfunction is common in a range of immune-mediated inflammatory diseases, is seen in multiple vascular beds, and is reversible following the induction of disease remission. The mechanisms that drive such systemic vascular endothelial dysfunction are unclear but factors such as TNF-α and CRP may play a role. Persistence of such widespread endothelial dysfunction in systemic vasculitis appears to have long-term consequences, leading to the acceleration of atherosclerosis and premature ischaemic heart disease. It may also underlie the accelerated atherosclerosis seen in other immune-mediated rheumatic diseases, such as rheumatoid arthritis.
doi:10.1093/rheumatology/keh542
PMCID: PMC3119433  PMID: 15644388
Vasculitis
15.  Galectin 3 Induces a Distinctive Pattern of Cytokine and Chemokine Production in Rheumatoid Synovial Fibroblasts via Selective Signaling Pathways 
Arthritis and rheumatism  2009;60(6):1604-1614.
Objective
High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts.
Methods
Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor α (TNFα), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-κB activation assay.
Results
Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte–macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3–induced secretion of TNFα, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFα blockade ruled out autocrine TNFα-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-κB activation was required for production of both IL-6 and CCL5.
Conclusion
Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell–recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.
doi:10.1002/art.24574
PMCID: PMC3116228  PMID: 19479862
16.  Utility of ultrasound joint counts in the prediction of rheumatoid arthritis in patients with very early synovitis 
Annals of the Rheumatic Diseases  2010;70(3):500-507.
Objectives
Early therapy improves outcomes in rheumatoid arthritis (RA). It is therefore important to improve predictive algorithms for RA in early disease. This study evaluated musculoskeletal ultrasound, a sensitive tool for the detection of synovitis and erosions, as a predictor of outcome in very early synovitis.
Methods
58 patients with clinically apparent synovitis of at least one joint and symptom duration of ≤3 months underwent clinical, laboratory, radiographic and 38 joint ultrasound assessments and were followed prospectively for 18 months, determining outcome by 1987 American College of Rheumatology (ACR) and 2010 ACR/European League Against Rheumatism criteria. Sensitivity and specificity for 1987 RA criteria were determined for ultrasound variables and logistic regression models were then fitted to evaluate predictive ability over and above the Leiden rule.
Results
16 patients resolved, 13 developed non-RA persistent disease and 29 developed RA by 1987 criteria. Ultrasound demonstrated subclinical wrist, elbow, knee, ankle and metatarsophalangeal joint involvement in patients developing RA. Large joint and proximal interphalangeal joint ultrasound variables had poor predictive ability, whereas ultrasound erosions lacked specificity. Regression analysis demonstrated that greyscale wrist and metacarpophalangeal joint involvement, and power Doppler involvement of metatarsophalangeal joints provided independently predictive data. Global ultrasound counts were inferior to minimal power Doppler counts, which significantly improved area under the curve values from 0.905 to 0.962 combined with the Leiden rule.
Conclusion
In a longitudinal study, extended ultrasound joint evaluation significantly increased detection of joint involvement in all regions and outcome groups. Greyscale and power Doppler scanning of metacarpophalangeal joints, wrists and metatarsophalangeal joints provides the optimum minimal ultrasound data to improve on clinical predictive models for RA.
doi:10.1136/ard.2010.131573
PMCID: PMC3033529  PMID: 21115552
17.  Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression, but little interleukin-22 and interleukin-23R expression 
Arthritis Research & Therapy  2010;12(5):R184.
Introduction
Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.
Methods
Flow cytometry was used to analyse the phenotype and cytokine production of mononuclear cells isolated from peripheral blood (PBMC) (n = 44), synovial fluid (SFMC) (n = 14) and synovium (SVMC) (n = 10) of RA patients and PBMC of healthy controls (n = 13).
Results
The frequency of IL-17-producing CD4 T cells was elevated in RA SFMC compared with RA PBMC (P = 0.04). However, the frequency of this population in RA SVMC was comparable to that in paired RA PBMC. The percentage of IL-17-producing CD4 T cells coexpressing tumor necrosis factor alpha (TNFα) was significantly increased in SFMC (P = 0.0068). The frequency of IFNγ-producing CD4 T cells was also significantly higher in SFMC than paired PBMC (P = 0.042). The majority of IL-17-producing CD4 T cells coexpressed IFNγ. IL-17-producing CD4 T cells in RA PBMC and SFMC exhibited very little IL-22 or IL-23R coexpression.
Conclusions
These findings demonstrate a modest enrichment of IL-17-producing CD4 T cells in RA SFMC compared to PBMC. Th17 cells in SFMC produce more TNFα than their PBMC counterparts, but are not a significant source of IL-22 and do not express IL-23R. However, the percentage of CD4 T cells which produce IL-17 in the rheumatoid joint is low, suggesting that other cells may be alternative sources of IL-17 within the joints of RA patients.
doi:10.1186/ar3152
PMCID: PMC2991017  PMID: 20929536
18.  The relationship between the presence of anti-cyclic citrullinated peptide antibodies and clinical phenotype in very early rheumatoid arthritis 
Background
Anti-cyclic citrullinated peptide (anti-CCP) antibodies are highly specific for RA, but are not detectable in all RA patients. The aim of this study was to establish whether the clinical phenotypes of anti-CCP positive and negative disease are distinct at the earliest clinically apparent phase of disease.
Methods
Patients were recruited from the Birmingham early inflammatory arthritis clinic. Participants were included in the current study if they presented within 3 months of symptom onset and fulfilled 1987 ACR criteria for RA at some point during an 18 month follow-up. Data were collected on demographic variables, joint symptoms and tender (n = 68) and swollen (n = 66) joint counts. CRP, ESR, rheumatoid factor and anti-CCP2 status were measured.
Results
92 patients were included (48 anti-CCP positive). The anti-CCP positive and negative groups were comparable in terms of demographic variables, inflammatory markers, joint counts and 1987 ACR classification criteria, except that more anti-CCP positive patients were rheumatoid factor positive (83.3% vs. 11.4%, p < 0.01). There was no significant difference in the pattern of joint involvement, except for an increased prevalence of knee joint swelling in anti-CCP positive patients (42.9% vs. 22.2%, p = 0.03).
Conclusions
Patients with and without anti-CCP antibodies present in a similar way, even within three months of clinically apparent disease that eventually develops into RA.
doi:10.1186/1471-2474-11-187
PMCID: PMC2936346  PMID: 20731815
19.  Investigation of potential non-HLA rheumatoid arthritis susceptibility loci in a European cohort increases the evidence for nine markers 
Annals of the Rheumatic Diseases  2010;69(8):1548-1553.
Background
Genetic factors have a substantial role in determining development of rheumatoid arthritis (RA), and are likely to account for 50–60% of disease susceptibility. Genome-wide association studies have identified non-human leucocyte antigen RA susceptibility loci which associate with RA with low-to-moderate risk.
Objectives
To investigate recently identified RA susceptibility markers using cohorts from six European countries, and perform a meta-analysis including previously published results.
Methods
3311 DNA samples were collected from patients from six countries (UK, Germany, France, Greece, Sweden and Denmark). Genotype data or DNA samples for 3709 controls were collected from four countries (not Sweden or Denmark). Eighteen single nucleotide polymorphisms (SNPs) were genotyped using Sequenom MassArray technology. Samples with a >95% success rate and only those SNPs with a genotype success rate of >95% were included in the analysis. Scandinavian patient data were pooled and previously published Swedish control data were accessed as a comparison group. Meta-analysis was used to combine results from this study with all previously published data.
Results
After quality control, 3209 patients and 3692 controls were included in the study. Eight markers (ie, rs1160542 (AFF3), rs1678542 (KIF5A), rs2476601 (PTPN22), rs3087243 (CTLA4), rs4810485 (CD40), rs5029937 (6q23), rs10760130 (TRAF1/C5) and rs7574865 (STAT4)) were significantly associated with RA by meta-analysis. All 18 markers were associated with RA when previously published studies were incorporated in the analysis. Data from this study increased the significance for association with RA and nine markers.
Conclusions
In a large European RA cohort further evidence for the association of 18 markers with RA development has been obtained.
doi:10.1136/ard.2009.121020
PMCID: PMC2938898  PMID: 20498205
20.  1,25-dihydroxyvitamin D3 and interleukin-2 combine to inhibit T cell production of inflammatory cytokines and promote development of regulatory T cells expressing CTLA-4 and FoxP3 
The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunomodulatory properties that have promoted its potential use in the prevention and treatment of infectious disease and autoimmune conditions. A variety of immune cells, including macrophages, dendritic cells and activated T cells express the intracellular vitamin D receptor (VDR) and are responsive to 1,25(OH)2D3. Despite this, how 1,25(OH)2D3 regulates adaptive immunity remains unclear, and may involve both direct and indirect effects on the proliferation and function of T cells. To further clarify this issue we have assessed the effects of 1,25(OH)2D3 on human CD4+ CD25− T cells. We observed that stimulation of CD4+ CD25− T cells in the presence of 1,25(OH)2D3 inhibited production of pro-inflammatory cytokines including IFN- γ, IL-17 and IL-21 but did not substantially affect T cell division. In contrast to its inhibitory effects on inflammatory cytokines, 1,25(OH)2D3 stimulated expression of high levels of CTLA-4 as well as FoxP3, the latter requiring the presence of IL-2. T cells treated with 1,25(OH)2D3 could suppress proliferation of normally responsive T cells indicating that they possessed characteristics of adaptive Tregs. Our results suggest that 1,25(OH)2D3 and IL-2 have direct synergistic effects on activated T cells, acting as potent anti-inflammatory agents and physiologic inducers of adaptive Tregs.
doi:10.4049/jimmunol.0803217
PMCID: PMC2810518  PMID: 19843932
Human; T cells; Cell differentiation; Tolerance
21.  Differential regulation of nuclear and mitochondrial Bcl-2 in T cell apoptosis 
Apoptosis  2007;13(1):109-117.
Activated T cells require anti-apoptotic cytokines for their survival. The anti-apoptotic effects of these factors are mediated by their influence on the balance of expression and localisation of pro- and anti-apoptotic members of the Bcl-2 family. Among the anti-apoptotic Bcl-2 family members, the expression level of Bcl-2 itself and its interaction with the pro-apoptotic protein Bim are now regarded as crucial for the regulation of survival in activated T cells. We studied the changes in Bcl-2 levels and its subcellular distribution in relation to mitochondrial depolarisation and caspase activation in survival factor deprived T cells. Intriguingly, the total Bcl-2 level appeared to remain stable, even after caspase 3 activation indicated entry into the execution phase of apoptosis. However, cell fractionation experiments showed that while the dominant nuclear pool of Bcl-2 remained stable during apoptosis, the level of the smaller mitochondrial pool was rapidly downregulated. Signals induced by anti-apoptotic cytokines continuously replenish the mitochondrial pool, but nuclear Bcl-2 is independent of such signals. Mitochondrial Bcl-2 is lost rapidly by a caspase independent mechanism in the absence of survival factors, in contrast only a small proportion of the nuclear pool of Bcl-2 is lost during the execution phase and this loss is a caspase dependent process. We conclude that these two intracellular pools of Bcl-2 are regulated through different mechanisms and only the cytokine-mediated regulation of the mitochondrial pool is relevant to the control of the initiation of apoptosis.
doi:10.1007/s10495-007-0143-z
PMCID: PMC2668593  PMID: 17957472
Bcl-2; Cytokines; Survival; Lymphocytes; Programmed cell death
22.  Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha 
Introduction
A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (TH17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect TH17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.
Methods
IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.
Results
TH17-expressing CD4+ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASFIL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASFIL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASFIL-17/TNF-mediated neutrophil rescue.
Conclusion
The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. TH17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.
doi:10.1186/ar2406
PMCID: PMC2453767  PMID: 18433499
23.  Hepatotoxicity associated with sulfasalazine in inflammatory arthritis: A case series from a local surveillance of serious adverse events 
Background
Spontaneous reporting systems for adverse drug reactions (ADRs) are handicapped by under-reporting and limited detail on individual cases. We report an investigation from a local surveillance for serious adverse drug reactions associated with disease modifying anti-rheumatic drugs that was triggered by the occurrence of liver failure in two of our patients.
Methods
Serious ADR reports have been solicited from local clinicians by regular postcards over the past seven years. Patients', who had hepatotoxicity on sulfasalazine and met a definition of a serious ADR, were identified. Two clinicians reviewed structured case reports and assessed causality by consensus and by using a causality assessment instrument. The likely frequency of hepatotoxicity with sulfasalazine was estimated by making a series of conservative assumptions.
Results
Ten cases were identified: eight occurred during surveillance. Eight patients were hospitalised, two in hepatic failure – one died after a liver transplant. All but one event occurred within 6 weeks of treatment. Seven patients had a skin rash, three eosinophilia and one interstitial nephritis. Five patients were of Black British of African or Caribbean descent. Liver enzymes showed a hepatocellular pattern in four cases and a mixed pattern in six. Drug-related hepatotoxicity was judged probable or highly probable in 8 patients. The likely frequency of serious hepatotoxicity with sulfasalazine was estimated at 0.4% of treated patients.
Conclusion
Serious hepatotoxicity associated with sulfasalazine appears to be under-appreciated and intensive monitoring and vigilance in the first 6 weeks of treatment is especially important.
doi:10.1186/1471-2474-9-48
PMCID: PMC2329632  PMID: 18405372
24.  Annexin-1 modulates T-cell activation and differentiation 
Blood  2006;109(3):1095-1102.
Annexin-1 is an anti-inflammatory protein that plays an important homeostatic role in innate immunity; however, its potential actions in the modulation of adaptive immunity have never been explored. Although inactive by itself, addition of annexin-1 to stimulated T cells augmented anti-CD3/CD28-mediated CD25 and CD69 expression and cell proliferation. This effect was paralleled by increased nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFATs), and activator protein-1 (AP-1) activation and preceded by a rapid T-cell receptor (TCR)–induced externalization of the annexin-1 receptor. Interestingly, differentiation of naive T cells in the presence of annexin-1 increased skewing in Th1 cells; in the collagen-induced arthritis model, treatment of mice with annexin-1 during the immunization phase exacerbated signs and symptoms at disease onset. Consistent with these findings, blood CD4+ cells from patients with rheumatoid arthritis showed a marked up-regulation of annexin-1 expression. Together these results demonstrate that annexin-1 is a molecular “tuner” of TCR signaling and suggest this protein might represent a new target for the development of drugs directed to pathologies where an unbalanced Th1/Th2 response or an aberrant activation of T cells is the major etiologic factor.
doi:10.1182/blood-2006-05-022798
PMCID: PMC1855438  PMID: 17008549
25.  Synovial fluid leukocyte apoptosis is inhibited in patients with very early rheumatoid arthritis 
Synovial leukocyte apoptosis is inhibited in established rheumatoid arthritis (RA). In contrast, high levels of leukocyte apoptosis are seen in self-limiting crystal arthritis. The phase in the development of RA at which the inhibition of leukocyte apoptosis is first apparent, and the relationship between leukocyte apoptosis in early RA and other early arthritides, has not been defined. We measured synovial fluid leukocyte apoptosis in very early arthritis and related this to clinical outcome. Synovial fluid was obtained at presentation from 81 patients with synovitis of ≤ 3 months duration. The percentages of apoptotic neutrophils and lymphocytes were assessed on cytospin preparations. Patients were assigned to diagnostic groups after 18 months follow-up. The relationship between leukocyte apoptosis and patient outcome was assessed. Patients with early RA had significantly lower levels of neutrophil apoptosis than patients who developed non-RA persistent arthritis and those with a resolving disease course. Similarly, lymphocyte apoptosis was absent in patients with early RA whereas it was seen in patients with other early arthritides. The inhibition of synovial fluid leukocyte apoptosis in the earliest clinically apparent phase of RA distinguishes this from other early arthritides. The mechanisms for this inhibition may relate to the high levels of anti-apoptotic cytokines found in the early rheumatoid joint (e.g. IL-2, IL-4, IL-15 GMCSF, GCSF). It is likely that this process contributes to an accumulation of leukocytes in the early rheumatoid lesion and is involved in the development of the microenvironment required for persistent RA.
doi:10.1186/ar2009
PMCID: PMC1779404  PMID: 16859518

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