Mohammed Akil

Methylation status of a single CpG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis

OBJECTIVE Genetic variation in the gene for interleukin-6 (IL-6) contributes to the pathogenesis of inflammatory arthritis, but the role, if any, of epigenetic variability has not been reported. The aims of this study were to compare the DNA methylation status of the IL6 promoter in rheumatoid arthritis (RA) patients and control subjects and to study the effects on gene expression. METHODS Genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) obtained from RA patients and healthy controls. Macrophages from healthy controls were isolated and stimulated with lipopolysaccharide (LPS). Methylation status was determined using bisulfite genomic sequencing and IL6 messenger RNA (mRNA) levels by quantitative polymerase chain reaction. Gel shift assays were performed with methylated or unmethylated probes and HeLa cell nuclear extract. RESULTS The proximal CpG motifs (-666 to +27) were predominantly unmethylated and the upstream motifs (-1099 to -1001) were highly methylated in PBMCs from patients and controls. Methylation of individual CpG motifs was similar, except at -1099C, which was less methylated in the patients than in the controls (58% versus 98%; P = 1 x 10(-6)). To test whether this observation might relate to gene regulation, LPS-stimulated macrophages were grouped according to their IL6 mRNA stimulation index (SI). The level of methylation at -1099C was significantly lower in the group with high (SI >75) compared with the group with low (SI <10) induced mRNA levels (71% versus 93%; P = 0.007). Gel shift assays revealed decreased protein binding to the -1099C unmethylated probe. CONCLUSION These data suggest that methylation of a single CpG in the IL6 promoter region may affect IL6 gene regulation and may play a role in the pathogenesis of RA.

Aspire Registry: assessing the spectrum of pulmonary hypertension identified at a referral centre

Pulmonary hypertension (PH) is a heterogeneous condition. To date, no registry data exists reflecting the spectrum of disease across the five diagnostic groups encountered in a specialist referral centre. Data was retrieved for consecutive, treatment-naïve cases diagnosed between 2001 and 2010 using a catheter-based approach. 1,344 patients were enrolled, with a mean follow-up of 2.9 yrs. The 3-yr survival was 68% for pulmonary arterial hypertension (PAH), 73% for PH associated with left heart disease, 44% for PH associated with lung disease (PH-lung), 71% for chronic thromboembolic PH (CTEPH) and 59% for miscellaneous PH. Compared with PAH, survival was inferior in PH-lung and superior in CTEPH (p<0.05). Multivariate analysis demonstrated that diagnostic group independently predicted survival. Within PAH, Eisenmenger’s survival was superior to idiopathic PAH, which was superior to PAH associated with systemic sclerosis (p<0.005). Within PH-lung, 3-yr survival in sleep disorders/alveolar hypoventilation (90%) was superior to PH-lung with chronic obstructive pulmonary disease (41%) and interstitial lung disease (16%) (p<0.05). In CTEPH, long-term survival was best in patients with surgically accessible disease undergoing pulmonary endarterectomy. In this large registry of consecutive, treatment-naïve patients identified at a specialist PH centre, outcomes and characteristics differed between and within PH groups. The current system of classification of PH has prognostic value even when adjusted for age and disease severity, emphasising the importance of systematic evaluation and precise classification.

Potential Novel Biomarkers of Disease Activity in Rheumatoid Arthritis Patients : CXCL13, CCL23, Transforming Growth Factor α, Tumor Necrosis Factor Receptor Superfamily Member 9, and Macrophage Colony-Stimulating Factor

OBJECTIVE To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients. METHODS Plasma proteins (n = 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 age- and sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial least-squares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti-tumor necrosis factor alpha (anti-TNFalpha). RESULTS The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate < or = 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colony-stimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor alpha, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity (kappa = 0.64). In anti-TNFalpha-treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score. CONCLUSION This exploratory study for biomarker discovery led to the identification of several proteins predictive of RA disease activity that may be useful in the definition of disease subphenotypes and in the measurement of response to therapy in clinical studies.

The use of Systemic Lupus Erythematosus Disease Activity Index-2000 to define active disease and minimal clinically meaningful change based on data from a large cohort of systemic lupus erythematosus patients

Objectives. To examine SLEDAI-2000 cut-off scores for definition of active SLE and to determine the sensitivity to change of SLEDAI-2000 for the assessment of SLE disease activity and minimal clinically meaningful changes in score. Methods. Data from two multi-centre studies were used in the analysis: in a cross-sectional and a longitudinal fashion. At every assessment, data were collected on SLEDAI-2000 and treatment. The cross-sectional analysis with receiver operating characteristic (ROC) curves was used to examine the appropriate SLEDAI-2000 score to define active disease and increase in therapy was the reference standard. In the longitudinal analysis, sensitivity to change of SLEDAI-2000 was assessed with multinomial logistic regression. ROC curves analysis was used to examine possible cut-points in score changes associated with change in therapy, and mean changes were estimated. Results. In the cross-sectional analysis, the most appropriate cut-off scores for active disease were 3 or 4. In the longitudinal analysis, the best model for predicting treatment increase was with the change in SLEDAI-2000 score and the score from the previous visit as continuous variables. The use of cut-points was less predictive of treatment change than the use of continuous score. The mean difference in the change in SLEDAI-2000 scores, adjusted for prior score, between patients with treatment increase and those without was 2.64 (95% CI 2.16, 3.14). Conclusions. An appropriate SLEDAI-2000 score to define active disease is 3 or 4. SLEDAI-2000 index is sensitive to change. The use of SLEDAI-2000 as a continuous outcome is recommended for comparative purposes.

British Isles Lupus Assessment Group 2004 index is valid for assessment of disease activity in systemic lupus erythematosus

Objective To determine the construct and criterion validity of the British Isles Lupus Assessment Group 2004 (BILAG-2004) index for assessing disease activity in systemic lupus erythematosus (SLE). Methods Patients with SLE were recruited into a multicenter cross-sectional study. Data on SLE disease activity (scores on the BILAG-2004 index, Classic BILAG index, and Systemic Lupus Erythematosus Disease Activity Index 2000 [SLEDAI-2K]), investigations, and therapy were collected. Overall BILAG-2004 and overall Classic BILAG scores were determined by the highest score achieved in any of the individual systems in the respective index. Erythrocyte sedimentation rates (ESRs), C3 levels, C4 levels, anti–double-stranded DNA (anti-dsDNA) levels, and SLEDAI-2K scores were used in the analysis of construct validity, and increase in therapy was used as the criterion for active disease in the analysis of criterion validity. Statistical analyses were performed using ordinal logistic regression for construct validity and logistic regression for criterion validity. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. Results Of the 369 patients with SLE, 92.7% were women, 59.9% were white, 18.4% were Afro-Caribbean and 18.4% were South Asian. Their mean ± SD age was 41.6 ± 13.2 years and mean disease duration was 8.8 ± 7.7 years. More than 1 assessment was obtained on 88.6% of the patients, and a total of 1,510 assessments were obtained. Increasing overall scores on the BILAG-2004 index were associated with increasing ESRs, decreasing C3 levels, decreasing C4 levels, elevated anti-dsDNA levels, and increasing SLEDAI-2K scores (all P < 0.01). Increase in therapy was observed more frequently in patients with overall BILAG-2004 scores reflecting higher disease activity. Scores indicating active disease (overall BILAG-2004 scores of A and B) were significantly associated with increase in therapy (odds ratio [OR] 19.3, P < 0.01). The BILAG-2004 and Classic BILAG indices had comparable sensitivity, specificity, PPV, and NPV. Conclusion These findings show that the BILAG-2004 index has construct and criterion validity.

Numerical scoring for the BILAG-2004 index

Objective. To develop an additive numerical scoring scheme for the BILAG-2004 index. Methods. SLE patients were recruited into this multi-centre cross-sectional study. At every assessment, data were collected on disease activity and therapy. Logistic regression was used to model an increase in therapy, as an indicator of active disease, by the BILAG-2004 index score in the nine systems. As both indicate inactivity, scores of D and E were set to 0 and used as the baseline in the fitted model. The models were used to determine the numerical values for Grades A–C. Different scoring schemes were compared. Results. There were 1510 assessments from 369 SLE patients. The coding schemes suggested for the Classic BILAG index (A = 12, B = 5, C = 1, D/E = 0 and A = 9, B = 3, C = 1, D/E = 0) did not fit the data well. A coding scheme (A = 12, B = 8, C = 1 and D/E = 0) was recommended, based on analysis results and consistency with the numerical coding scheme of the Classic BILAG index. Conclusion. A reasonable additive numerical scoring scheme based on treatment decision for the BILAG-2004 index is A = 12, B = 8, C = 1, D = 0 and E = 0.

The BILAG-2004 index is sensitive to change for assessment of SLE disease activity

Objective. To determine if the BILAG-2004 index is sensitive to change for assessment of SLE disease activity. Methods. This was a prospective multi-centre longitudinal study of SLE patients. At every assessment, data were collected on disease activity (BILAG-2004 index) and treatment. Analyses were performed using overall BILAG-2004 index score (as determined by the highest score achieved by any of the individual systems) and all the systems scores. Sensitivity to change was assessed by determining the relationship between change in disease activity and change in therapy between two consecutive visits. Statistical analyses were performed using multinomial logistic regression. Results. There were 1761 assessments from 347 SLE patients that contributed 1414 observations for analysis. An increase in therapy between visits occurred in 22.7% observations, while 37.3% had a decrease in therapy and in 40.0% therapy was unchanged. Increase in overall BILAG-2004 index score was associated with increase in therapy and inversely associated with decrease in therapy. Decrease in overall BILAG-2004 index score was associated with decrease in therapy and was inversely associated with increase in therapy. Changes in overall BILAG-2004 index score were differentially related to change in therapy, with greater change in score having greater predictive power. Increase in the scores of most systems was independently associated with an increase in treatment and there was no significant association between decreases in the score of any system with an increase in therapy. Conclusions. The BILAG-2004 index is sensitive to change and is suitable for use in longitudinal studies of SLE.

Inflammatory neutrophils retain susceptibility to apoptosis mediated via the Fas death receptor

Apoptosis and clearance of neutrophils is essential for successful resolution of inflammation. Altered signaling via the Fas receptor could explain the observed prolongation of neutrophil lifespan and associated tissue injury at inflammatory sites. We therefore compared inflammatory neutrophils extracted from joints of rheumatoid arthritis patients, with peripheral blood neutrophils. Inflammatory neutrophils underwent constitutive apoptosis in culture more rapidly than peripheral blood neutrophils; this was not explained by changes in surface expression of Fas or by induction of Fas ligand. Inflammatory neutrophils remained sensitive to Fas‐induced death, at levels comparable to those seen in peripheral blood neutrophils. Similarly, granulocyte‐macrophage colony‐stimulating factor reduced apoptosis but did not abolish signaling via Fas. These data provide evidence for the rate of apoptosis in inflammatory neutrophils being continually modulated by death and survival signals in the inflammatory milieu. This allows for rapid resolution of inflammation as levels of survival factors fall, and suggests new strategies for inducing resolution of inflammation. J. Leukoc. Biol. 67: 662–668; 2000.

Ventricular mass index correlates with pulmonary artery pressure and predicts survival in suspected systemic sclerosis-associated pulmonary arterial hypertension

OBJECTIVE The ventricular mass index (VMI) has been proposed as a diagnostic tool for the assessment of patients with suspected pulmonary hypertension (PH). We hypothesized that in patients with SSc it may predict the presence or absence of PH. METHODS Details of all consecutive SSc patients undergoing MRI and right heart catheterization were collected prospectively. Subsequently, the VMI for all patients was calculated, and further baseline data were collected. RESULTS Data for 40 patients, 28 of whom were diagnosed with PH at rest (PH(REST)), were analysed. VMI correlated strongly with mean pulmonary artery pressure (mPAP; r = 0.79). Using a VMI threshold of 0.56, positive predictive value (PPV) for PH(REST) was 88% and negative predictive value (NPV) was 100%. Using a threshold of 0.7, PPV was found to be 100% and NPV 53%. Echocardiographically obtained tricuspid gradient (TG) also demonstrated a strong correlation with mPAP. Two-year survival in patients with VMI <0.7 and > or =0.7 was 91 and 43%, respectively (P < 0.001). CONCLUSION VMI correlates well with mPAP in patients with SSc and may have a role in non-invasively excluding clinically significant PH in breathless SSc patients in whom echocardiographic screening has failed. Further study in larger groups of patients is justified.

The BILAG multi-centre open randomized controlled trial comparing ciclosporin vs azathioprine in patients with severe SLE

OBJECTIVE To determine whether low-dose ciclosporin was a more effective corticosteroid-sparing agent than AZA in patients with SLE. METHODS Patients with SLE requiring a change or initiation of a corticosteroid-sparing agent and who were taking > or =15 mg of prednisolone/day were randomized to receive either ciclosporin or AZA during this 12-month open-label multi-centre trial. There were strict guidelines for the reduction of prednisolone. The primary outcome was the absolute mean change in prednisolone. RESULTS Eighty-nine patients were randomized. Using an intention-to-treat analysis, the absolute mean change in prednisolone dose between baseline and 12 months, adjusted for baseline prednisolone dose, was 9.0 mg for ciclosporin (95% CI 7.2, 10.8) and 10.7 mg for AZA (95% CI 8.8, 12.7). The difference in the change between treatment groups was -1.7 mg (95% CI -4.4, 0.9; P = 0.2). No significant differences were detected for the secondary outcomes: change in disease activity [classic British Isles Lupus Assessment Group (BILAG) index], number of flares, development of new damage or change in quality of life. A similar number of patients in each arm stopped the study drugs due to adverse events and ineffectiveness. No patient developed severe hypertension or a persistent rise in creatinine. One patient in the ciclosporin arm developed a significant increase in proteinuria due to disease activity. CONCLUSIONS Both drugs were effective corticosteroid-sparing agents. Ciclosporin was not a more effective corticosteroid-sparing agent. Ciclosporin may be considered in patients who are unable to tolerate AZA. Patients on ciclosporin require close monitoring of blood pressure and creatinine. TRIAL REGISTRATION Current Controlled Trials, http://www.controlled-trials.com/, ISRCTN35919612.