Inflammatory biomarkers do not distinguish between patients with sciatica and referred leg pain within a primary care population: results from a nested study within the ATLAS cohort.

BACKGROUND: There is increasing interest in the role of pro-inflammatory cytokines in the pathogenesis of sciatica and whether these could be potential targets for treatment. We sought to investigate serum biomarker levels in patients with low back-related leg pain, including sciatica. METHODS: Primary care consulters aged > 18 with low back-related leg pain were recruited to a cohort study (ATLAS). Participants underwent a standardised clinical assessment, lumbar spine MRI and a subsample (n = 119) had samples taken for biomarker analysis. Participants were classified having: a) clinically confirmed sciatica or referred leg pain, and then subdivided into those with (or without) MRI confirmed nerve root compression due to disc prolapse. Seventeen key cytokines, chemokines and matrix metalloproteinases (MMPs) implicated in sciatica pathogenesis including TNFα and IL-6, were assayed in duplicate using commercial multiplex detection kits and measured using a Luminex suspension array system. Median biomarker levels were compared between the groups using a Mann Whitney U test. Multivariate logistic regression analysis was used to investigate the association between clinical measures and biomarker levels adjusted for possible confounders such as age, sex, and symptom duration. RESULTS: No difference was found in the serum level of any of the 17 biomarkers tested in patients with (n = 93) or without (n = 26) clinically confirmed sciatica, nor between those with (n = 44) or without (n = 49) sciatica and MRI confirmed nerve root compression. CONCLUSION: In this cohort, no significant differences in serum levels of TNFα, IL-6 or any other biomarkers were seen between patients with sciatica and those with back pain with referred leg pain. These results suggest that in patients with low back-related leg pain, serum markers associated with inflammation do not discriminate between patients with or without clinically confirmed sciatica or between those with or without evidence of nerve root compression on MRI.

An initial investigation into endothelial CC chemokine expression in the human rheumatoid synovium.

Rheumatoid arthritis (RA) is a destructive and chronic autoimmune inflammatory disease. Synovial inflammation is a major feature of RA and is associated with leukocyte recruitment. Leukocytes cross the endothelial cells (ECs) into the synovial tissue and fluid and this migration is mediated via a range of chemokines and adhesion molecules on the ECs. As important mediators of leukocyte extravasation, a number of chemokines from each of the chemokine families have been established as expressed in the RA joint. However, as little information is available on which chemokines are expressed/presented by the ECs themselves, the purpose of the study was to ascertain which of the CC chemokines were localised in RA ECs. Immunofluoresence was used to assess the presence of the CC-family chemokines in RA synovial ECs using von-Willebrand factor (VWF) as a pan-endothelial marker and a range of human chemokine antibodies. The percentage of VWF positive vessels which were positive for the chemokines was determined. The presence of the four most highly expressed novel chemokines were further investigated in non-RA synovial ECs and the sera and synovial fluid (SF) from patients with RA and osteoarthritis (OA). Statistical analysis of immunofluorescence data was carried out by Student's t-test. For analysis of ELISA data, Kruskal-Wallis ANOVA followed by Dunn's multiple comparison test was utilised to analyse differences in sera and SF levels for each chemokine between RA and OA. Spearman rank correlations of sera and SF chemokine levels with a range of clinical variables were also performed. Chemokine detection varied, the least abundant being CCL27 which was present in 8.3% of RA blood vessels and the most abundant being CCL19 which was present in 80%. Of the 26 chemokines studied, 19 have not been previously observed in RA ECs. Four of these novel chemokines, namely CCL7, CCL14, CCL16 and CCL22 were present on ≥60% of vessels. CCL14 and CCL22 were shown to be increased in RA ECs compared to non-RA ECs, p=0.0041 and p=0.014 respectively. EC chemokines CCL7, CCL14, CCL16 and CCL22 also occurred in RA synovial fluid and sera as established by ELISA. CCL7 was shown to be significantly increased in sera and SF from RA patients compared to that from osteoarthritis (OA) patients (p<0.01), and to have a highly significant correlation with the level of anti-CCP (R=0.93, p=0.001). Less abundant chemokines shown to be present in RA ECs were CCL1-3, CCL5, CCL10-13, CCL15, CCL17, CCL18, CCL20, CCL21 and CCL23-28. In conclusion, this initial study is the first to show the presence of a number of CC chemokines in RA ECs. It provides evidence that further validation and investigation into the presence and functionality of these novel chemokines expressed at RA synovial ECs may be warranted.

Clinical efficacy of oral alendronate in ankylosing spondylitis: a randomised placebo-controlled trial.

OBJECTIVES: A prospective, double blind, randomised, placebo controlled trial over 2 years was performed to test the efficacy of alendronate, an oral aminobisphosphonate, in improving symptoms and arrest disease progression in patients with mild to severe ankylosing spondylitis (AS). METHODS: 180 patients with AS were randomised to receive weekly alendronate 70 mg or placebo (1:1 randomisation). BAS-G was the primary outcome measure with Bath indices as secondary outcomes. Vertebral x-rays were performed at 0 and 24 months. Biomarkers (including CRP, IL-1beta, IL6, VEGF, MMP-1, and MMP-3) were collected during the first 12 months. RESULTS: There was no significant difference between the placebo and treatment groups in any of the recorded outcomes over the 2 years including clinical indices, biomarkers, and radiology. The change in BAS-G, the primary outcome measure, was -0.21 for the treatment group and -0.42 for the placebo group p=0.57. Change in all other clinical outcome measures were also non-significant; BASDAI p=0.86, BASFI p=0.37, BASMI p=0.021. Sub-group analysis of those subjects with a baseline BASDAI >4 were also non-significant. CONCLUSIONS: This prospective study demonstrates that alendronate 70mg weekly for 2 years was no more efficacious than placebo in improving clinical or laboratory measures of disease activity or measures of physical impact in subjects with mild to severe active AS. TRIAL REGISTRATION: ID SRCTN12308164, registered on 15.12.2015.

The role of parvovirus B19 and the immune response in the pathogenesis of acute leukemia.

In this article, we review the evidence suggesting a possible role for B19 virus in the pathogenesis of a subset of cases of acute leukemia. Human parvovirus B19 infection may complicate the clinical course of patients with acute leukemia and may also precede the development of acute leukemia by up to 180 days. Parvovirus B19 targets erythroblasts in the bone marrow and may cause aplastic crisis in patients with shortened-red cell survival. Aplastic crisis represents a prodrome of acute lymphoblastic leukemia in 2% patients. There is a significant overlap between those HLA classes I and II alleles that are associated with a vigorous immune response and development of symptoms during B19 infection and those HLA alleles that predispose to development of acute leukemia. Acute symptomatic B19 infection is associated with low circulating IL-10 consistent with a vigorous immune response; deficient IL-10 production at birth was recently found to be associated with subsequent development of acute leukemia. Anti-B19 IgG has been associated with a particular profile of methylation of human cancer genes in patients with acute leukemia, suggesting an additional hit and run mechanism. The proposed role for parvovirus B19 in the pathogenesis of acute leukemia fits well with the delayed infection hypothesis and with the two-step mutation model, which describes carriage of the first mutation prior to birth, followed by suppression of hematopoiesis, which allows rapid proliferation of cells harboring the first mutation, acquisition of a second activating mutation, and expansion of cells carrying both mutations, resulting in acute leukemia.

Polymorphism in the vascular endothelial growth factor A (VEGFA) gene is associated with serum VEGF-A level and disease activity in rheumatoid arthritis: differential effect of cigarette smoking.

OBJECTIVE: To assess the impact of common genetic variants in the vascular endothelial growth factor A (VEGFA) gene on circulating VEGF-A levels and disease activity in patients with rheumatoid arthritis (RA). METHODS: A cohort (n=419) of consecutively recruited RA patients of Caucasian origin was studied. Disease activity (DAS28) was recorded on a regular basis (0, 12 and 24 months). Smoking history (never, past and current) was obtained. PCR-RFLP assays were used to determine the genotypes of VEGFA single-nucleotide polymorphisms (SNP) including VEGFA-2578 (rs699947), -460 (rs833061), +405 (rs2010963) and +936 (rs3025039). Circulating levels of VEGF-A were measured in serum samples using a fluorescent bead-based assay system (Luminex®). Associations were analyzed using univariate and multivariate statistics. RESULTS: VEGFA-2578 AA genotype was associated with lower serum VEGF-A levels, as was the most frequent haplotype (A(-2578)-C(-460)-G(+405), 48.1%) within the 5'-flanking region of the gene. The same genotype and haplotype were also associated with decreased disease activity in RA. This was seen only in patients who had never smoked. In multivariate multiple regression models, the VEGFA-2578 SNP was shown to be associated with disease activity at presentation (p=0.029) and over time (p=0.016) in patients who never smoked, independent of serum VEGF-A levels and other confounding factors. CONCLUSION: Genetic variation in the VEGFA gene is associated with serum VEGF-A levels in RA, and shows an association with disease activity in RA patients who have never smoked, independent of serum VEGF-A levels.

Hypoalbuminaemia, systemic albumin leak and endothelial dysfunction in peritoneal dialysis patients.

BACKGROUND: Inflammation, hypoalbuminaemia and peritoneal protein clearance are important predictors of survival in patients treated with peritoneal dialysis (PD). We hypothesized that the common link is abnormal endothelial barrier function. To test this, we explored associations between hypoalbuminaemia, systemic albumin leak and soluble markers of systemic inflammation and endothelial injury. METHODS: This was a cross-sectional study of 41 prevalent PD patients. Endothelial barrier function was measured as transcapillary escape rate of (125)I albumin [transcapillary escape rate of albumin (TER(alb))]. Seventeen plasma biomarkers including pro-inflammatory cytokines, endothelial biomarkers and metalloproteinases were measured. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) were used to explore the hypothesis. RESULTS: The mean TER(alb) was 13.7 ± 8.9 (%/h), higher than in non-uraemic subjects 8.22 ± 5.8 (%/h). Three patient clusters were defined from HCA according to their biomarker patterns. Cluster 1 was characterized by inflammation, hypoalbuminaemia, overhydration and intermediate TER(alb). Cluster 2 was non-inflamed, preserved muscle mass and more normal TER(alb). Cluster 3 had highest TER(alb), platelet activation, preserved plasma albumin and intermediate high-sensitivity C-reactive protein levels. Two principal components (PCs) were identified from the biomarker matrix, PC1, indicating platelet activation and PC2, pro-inflammatory. TER(alb) was positively related to PC1 but not PC2. Diabetes and ischaemic heart disease were associated with PC1 and PC2, respectively. CONCLUSIONS: This exploratory analysis indicates that endothelial barrier function is decreased in PD patients and is associated with diabetic status and markers of platelet activation more than inflammation. In contrast, hypoalbuminaemia is associated more with inflammation and atherosclerotic disease indicating a more complex relationship between systemic endothelial barrier function, inflammation and hypoalbuminaemia which requires further validation.

Age at onset of rheumatoid arthritis: association with polymorphisms in the vascular endothelial growth factor A(VEGFA) gene and an intergenic locus between matrix metalloproteinase (MMP) 1 and 3 genes.

OBJECTIVES: The present paper aims to investigate whether polymorphisms in the vascular endothelial growth factor A (VEGFA) gene and the loci of matrix metalloproteinase (MMP) 1 and 3 genes are associated with age at onset of RA. METHODS: A sample of 413 hospital-based RA patients of Caucasian origin was studied. Common single-nucleotide polymorphisms (SNP) with likely importance were typed, including rs699947, rs833061, rs2010963 and rs3025039 in VEGFA, and rs1799750 in the MMP1 gene, rs3025058, rs679620 in the MMP3 gene and rs495366 located within the region between the MMP1 and MMP3 genes. Age at onset of RA was obtained on each patient. Demographic variables, smoking information, and a core set of clinical characteristics measured at recruitment were recorded. Hazard ratios (HR) that measured the effect size of genetic risk on age at RA onset were computed using Cox regression models. RESULTS: The T allele at rs3025039 was associated with an increased risk of early onset (HR=1.25 [95% CI 1.0-1.58] for the risk over time; HR=1.84 [95% CI 1.20-2.83] for the risk of onset <40 years old). The AA genotype at rs495366 was also associated with an increased risk (HR=1.92 [95% CI 1.27-2.89] over time; HR=2.54 [95% CI 1.30-4.95] for onset <40 years old). These associations were independent of other risk factors such as sex, smoking and anti-CCP status. CONCLUSIONS: Polymorphisms in the VEGFA gene and the MMP1-3 intergenic locus may influence age at onset of RA.

Depression in RA patients treated with anti-TNF is common and under-recognized in the rheumatology clinic.

OBJECTIVES: Depression is common in RA and may be influenced by both disease activity and severity. The aims of this study were to investigate the prevalence of depression in RA patients starting anti-TNF therapy, to investigate how mood alters after exposure to anti-TNF and to determine whether depression is recognized and appropriately managed in the clinic. METHODS: Patients starting anti-TNF therapy were assessed for depression using the Hospital Anxiety and Depression Scale (HADS-D), and classified as depressed with an HADS-D of > or =8. Change in mood was assessed at 6 weeks, 4 months and 12 months. Disease activity data were recorded at baseline, 3 and 12 months. Patients who remained persistently depressed at 4 months had their clinical case notes reviewed to determine whether their low mood had been recognized or treated. RESULTS: Depression was common in this cohort. Depressed patients had higher disease activity scores (DAS28) at all time points, and patients with persistent depression had smaller reductions in DAS28 [median (interquartile range or IQR) change DAS28 1.71 (0-2.6) vs 2.2 (1.5-3.2); P = 0.005]. Only 57% (13/23) patients with persistent depression at 4 months had their depression recognized or managed within the rheumatology clinic. CONCLUSIONS: Depression is common but under-recognized in RA patients starting on anti-TNF therapy. Patients with persistent depression tended to respond less well to anti-TNF, with smaller reductions in DAS28. Given that a significant reduction in DAS28 is a requirement for continuing therapy, recognition and appropriate management of depression may improve TNF effectiveness.

Soluble syndecan-3 binds chemokines, reduces leukocyte migration in vitro and ameliorates disease severity in models of rheumatoid arthritis.

BACKGROUND: Syndecans are heparan sulfate proteoglycans that occur in membrane-bound or soluble forms. Syndecan-3, the least well-characterised of the syndecan family, is highly expressed on synovial endothelial cells in rheumatoid arthritis patients. Here, it binds pro-inflammatory chemokines with evidence for a role in chemokine presentation and leukocyte trafficking into the joint, promoting the inflammatory response. In this study, we explored the role of soluble syndecan-3 as a binder of chemokines and as an anti-inflammatory and therapeutic molecule. METHODS: A human monocytic cell line and CD14+ PBMCs were utilised in both Boyden chamber and trans-endothelial migration assays. Soluble syndecan-3 was tested in antigen-induced and collagen-induced in vivo arthritis models in mice. ELISA and isothermal fluorescence titration assays assessed the binding affinities. Syndecan-3 expression was identified by flow cytometry and PCR, and levels of shedding by ELISA. RESULTS: Using in vitro and in vivo models, soluble syndecan-3 inhibited leukocyte migration in vitro in response to CCL7 and its administration in murine models of rheumatoid arthritis reduced histological disease severity. Using isothermal fluorescence titration, the binding affinity of soluble syndecan-3 to inflammatory chemokines CCL2, CCL7 and CXCL8 was determined, revealing little difference, with Kds in the low nM range. TNFα increased cell surface expression and shedding of syndecan-3 from cultured human endothelial cells. Furthermore, soluble syndecan-3 occurred naturally in the sera of patients with rheumatoid arthritis and periodontitis, and its levels correlated with syndecan-1. CONCLUSIONS: This study shows that the addition of soluble syndecan-3 may represent an alternative therapeutic approach in inflammatory disease.

Preexisting psychological stress predicts acute and chronic fatigue and arthritis following symptomatic parvovirus B19 infection.

BACKGROUND: Psychological stress is thought to be an important factor in the pathogenesis of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME). Therefore, we sought to examine this relationship in the context of parvovirus B19 infection. METHODS: Thirty-nine patients with laboratory-documented acute parvovirus B19 infection were asked to complete questionnaires on negative life events, perceived stress, and negative affect relevant to the time of onset of parvovirus infection and during the preceding 12 months. These scores were combined into an overall stress index, which was then examined for associations with particular parvovirus-associated symptoms at acute infection and during the ensuing 1-3 years. Additional characteristics monitored included presence of parvovirus antibodies and nucleic acid, cortisol level, dehydroepiandrosterone level, autoantibodies, levels of a range of serum cytokines, and human leukocyte antigen class I and II alleles. RESULTS: Stress index was significantly associated with development of fatigue during the acute phase of parvovirus B19 infection and also with chronic fatigue and arthritis occurring 1-3 years following acute parvovirus B19 infection. Logistic regression that included all clinical variables indicated that a high stress index at the time of onset of infection was the primary predictor of CFS/ME 1-3 years following acute parvovirus B19 infection (odds ratio, 25.7; 95% confidence interval, 1.7-121.9; P=.005). CONCLUSIONS: We report a highly significant association between psychological stress and development of acute and chronic fatigue and arthritis several years following laboratory-documented acute parvovirus B19 infection.

Anti-citrullinated protein antibody positive rheumatoid arthritis is primarily determined by rheumatoid factor titre and the shared epitope rather than smoking per se.

OBJECTIVE: To analyse the relationship between rheumatoid factor (RF) titre, smoking and HLA-DRB1 alleles coding a "shared epitope" (SE) in relation to anti-citrullinated protein antibody (ACPA) positivity in rheumatoid arthritis (RA). METHODS: RA patients (n = 658) attending rheumatology clinics in Cornwall, UK (cohort 1) were stratified according to RF and ACPA titre, and smoking pack years at diagnosis. A further 409 RA patients from North Staffordshire, UK (cohort 2) were studied to confirm the relationship between RF levels, smoking and ACPA positivity in relation to SE status. RESULTS: In cohort 1 there was a trend (p<0.01) of increasing ACPA positivity rates with increasing levels of RF without statistically significant differences between patients who had never smoked and smokers (never smoked: 15/71 (21%) RF -ve, vs. 43/64 (67%) RF weak +ve, vs 88/100 (88%) RF strong +ve, ever smoked: 18/70 (26%) RF -ve vs. 66/83 (80%) RF weak +ve vs. 196/210 (93%) RF strong +ve). No significant gender difference was observed. No significant difference between smoking and ACPA positivity was seen in RF negative patients. Smoking >20 pack years conferred an increased risk of anti-CCP positive RA (158/200 (79%)), compared to having never smoked (146/235 (62%), p = <0.01), but this increased risk correlated with smokers' RF positivity as the principal determinant on subsequent regression analysis of cohort 2. In cohort 2, ACPA positivity rates significantly increased with RF positivity and carriage of 1 or 2 SE alleles (p<0.01). Little or no relationship was observed in patients lacking SE. CONCLUSIONS: ACPA positivity in RA strongly associates with increasing RF titre independent of smoking. This relationship is dependent on carriage of SE alleles. There is no relationship between ACPA and smoking in RF negative patients.

DNA methylation at diagnosis is associated with response to disease-modifying drugs in early rheumatoid arthritis.

AIM: A proof-of-concept study to explore whether DNA methylation at first diagnosis is associated with response to disease-modifying antirheumatic drugs (DMARDs) in patients with early rheumatoid arthritis (RA). PATIENTS & METHODS: DNA methylation was quantified in T-lymphocytes from 46 treatment-naive patients using HumanMethylation450 BeadChips. Treatment response was determined in 6 months using the European League Against Rheumatism (EULAR) response criteria. RESULTS: Initial filtering identified 21 cytosine-phosphate-guanines (CpGs) that were differentially methylated between responders and nonresponders. After conservative adjustment for multiple testing, six sites remained statistically significant, of which four showed high sensitivity and/or specificity (≥75%) for response to treatment. Moreover, methylation at two sites in combination was the strongest factor associated with response (80.0% sensitivity, 90.9% specificity, AUC 0.85). CONCLUSION: DNA methylation at diagnosis is associated with disease-modifying antirheumatic drug treatment response in early RA.

Genome-wide profiling in treatment-naive early rheumatoid arthritis reveals DNA methylome changes in T and B lymphocytes.

AIM: Although aberrant DNA methylation has been described in rheumatoid arthritis (RA), no studies have interrogated this epigenetic modification in early disease. Following recent investigations of T and B lymphocytes in established disease, we now characterize in these cell populations genome-wide DNA methylation in treatment-naive patients with early RA. PATIENTS & METHODS: HumanMethylation450 BeadChips were used to examine genome-wide DNA methylation in lymphocyte populations from 23 early RA patients and 11 healthy individuals. RESULTS: Approximately 2000 CpGs in each cell type were differentially methylated in early RA. Clustering analysis identified a novel methylation signature in each cell type (150 sites in T lymphocytes, 113 sites in B lymphocytes) that clustered all patients separately from controls. A subset of sites differentially methylated in early RA displayed similar changes in established disease. CONCLUSION: Treatment-naive early RA patients display novel disease-specific DNA methylation aberrations, supporting a potential role for these changes in the development of RA.

DNA methylation profiling of synovial fluid FLS in rheumatoid arthritis reveals changes common with tissue-derived FLS.

AIM: Alterations in DNA methylation contribute to the abnormal phenotype of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). We profiled genome-wide DNA methylation in these cells from synovial fluid, a more readily accessible source of disease-associated cells. PATIENTS & METHODS: Genome-wide DNA methylation was interrogated in fluid-derived FLS from five RA and six osteoarthritis patients using Human Methylation 450 Bead Chip and bisulfite pyrosequencing. RESULTS: Array analysis identified 328 CpGs, representing 195 genes, that were differentially methylated between RA and osteoarthritis fluid-derived FLS. Comparison with the genes identified in two independent studies of tissue-derived FLS revealed 73 genes in common (~40%), of which 22 shared identity with both studies. Pyrosequencing confirmed altered methylation of these genes. CONCLUSION: Synovial fluid-derived RA FLS show methylation changes common with tissue-derived FLS, supporting the use of fluid-derived FLS for future investigations.

HLA-DRB1 status affects endothelial function in treated patients with rheumatoid arthritis.

PURPOSE: To examine endothelial function in rheumatoid arthritis patients and to assess whether clinical or genetic factors affect the development of endothelial dysfunction. METHODS: Fifty-five patients fulfilling the 1987 American College of Rheumatology classification criteria for rheumatoid arthritis were recruited from Hospital Xeral-Calde, Lugo, Spain. Patients were required to have been treated for at least 5 years, including current treatment with one or more disease-modifying antirheumatic drugs. Patients with diabetes mellitus, renal insufficiency, or cardiovascular disease were excluded. Thirty-one age-, sex-, and ethnically matched controls were also studied. Endothelium-dependent (postischemia) and -independent (postnitroglycerin) vasodilatation were measured by brachial ultrasonography. Patients were genotyped for human leukocyte antigen (HLA)-DRB1. RESULTS: Patients had decreased endothelium-dependent vasodilatation (mean [+/- SD], 3.8% +/- 4.9%) compared with controls (8.0% +/- 4.5%; P <0.001). There were no differences in endothelium-independent vasodilatation. Clinical features were not associated with endothelial dysfunction. Endothelium-dependent vasodilatation was lower in the 30 rheumatoid arthritis patients with the HLA-DRB1*04 shared epitope alleles (2.4% +/- 4.1%) than in the remaining patients (5.5% +/- 5.3%; P = 0.01). Similar results were seen for patients with the HLA-DRB1*0404 shared epitope allele (-0.4% +/- 2.5%) compared with other patients (4.4% +/- 4.9%; P = 0.01). CONCLUSION: Patients with chronically treated rheumatoid arthritis had evidence of endothelial dysfunction, especially those with certain HLA-DRB1 genotypes. If confirmed, our results suggest that HLA-DRB1 status may be a predictor of cardiovascular risk in these patients.

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations.

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.

Influence of adult height on rheumatoid arthritis: association with disease activity, impairment of joint function and overall disability.

OBJECTIVES: To investigate whether normal variation of adult height is associated with clinical characteristics in rheumatoid arthritis (RA), including disease activity (DAS28), impairment of joint function (mechanical joint score, MJS) and overall disability (health assessment questionnaire, HAQ). METHODS: A cohort (134 males, 287 females) of consecutively recruited RA patients of Northern European origin was studied. Height, weight and demographic information were obtained. A core set of disease measurements, including DAS28, MJS and HAQ, were recorded at baseline, 12 and 24 months. Other clinical variables (e.g. disease duration, IgM rheumatoid factor, antibodies to cyclic citrullinated peptide, C-reactive protein, erythrocyte sedimentation rate) were recorded at baseline. Socioeconomic status, smoking status, comorbid condition, other autoimmune conditions and drug therapy were also recorded. Associations were analyzed using univariate statistics and multivariate linear regression models. Mediation tests were also carried out for evaluating the relationship between gender, height and disease measures. RESULTS: In males, height was inversely associated with DAS28, MJS and HAQ (at baseline and over 24 months) independent of other factors (e.g. weight, body mass index, age, disease duration, osteoporosis, autoantibodies, erosive disease, joint replacement, steroid use, smoking status, socioeconomic status and comorbid disease). In females, a similar trend was seen but the relationships were non significant. In the whole population, the association of female gender with more active disease and poor function disappeared after adjustment for height. Mediation analysis indicated that height served as a full mediator in the relationship of gender with disease activity and overall disability. Confirmation of these findings was demonstrated in a second RA population (n = 288). CONCLUSION: Adult height is inversely associated with disease activity, impairment of joint function and overall disability in RA, particularly in males. The association of female sex with more severe disease activity and disability appears to be mediated by smaller stature.