The BILAG-2004 index is associated with development of new damage in SLE.

OBJECTIVE: To determine whether BILAG-2004 index is associated with the development of damage in a cohort of SLE patients. Mortality and development of damage were examined. METHODS: This was a multicentre longitudinal study. Patients were recruited within 12 months of achieving fourth ACR classification criterion for SLE. Data were collected on disease activity, damage, SLE-specific drug exposure, cardiovascular risk factors, antiphospholipid syndrome status and death at every visit. This study ran from 1 January 2005 to 31 December 2017. Descriptive statistics were used to analyse mortality and development of new damage. Poisson regression was used to examine potential explanatory variables for development of new damage. RESULTS: A total of 273 SLE patients were recruited with total follow-up of 1767 patient-years (median 73.4 months). There were 6348 assessments with disease activity scores available for analysis. During follow-up, 13 deaths and 114 new damage items (in 83 patients) occurred. The incidence rate for development of damage was higher in the first 3 years before stabilizing at a lower rate. Overall rate for damage accrual was 61.1 per 1000 person-years (95% CI: 50.6, 73.8). Analysis showed that active disease scores according to BILAG-2004 index (systems scores of A or B, counts of systems with A and BILAG-2004 numerical score) were associated with development of new damage. Low disease activity (LDA) states [BILAG-2004 LDA and BILAG Systems Tally (BST) persistent LDA] were inversely associated with development of damage. CONCLUSIONS: BILAG-2004 index is associated with new damage. BILAG-2004 LDA and BST persistent LDA can be considered as treatment targets.

Flares in patients with systemic lupus erythematosus.

OBJECTIVE: SLE is characterized by relapses and remissions. We aimed to describe the frequency, type and time to flare in a cohort of SLE patients. METHODS: SLE patients with one or more 'A' or 'B' BILAG-2004 systems meeting flare criteria ('new' or 'worse' items) and requiring an increase in immunosuppression were recruited from nine UK centres and assessed at baseline and monthly for 9 months. Subsequent flares were defined as: severe (any 'A' irrespective of number of 'B' flares), moderate (two or more 'B' without any 'A' flares) and mild (one 'B'). RESULTS: Of the 100 patients, 94% were female, 61% White Caucasians, mean age (s.d.) was 40.7 years (12.7) and mean disease duration (s.d.) was 9.3 years (8.1). A total of 195 flares re-occurred in 76 patients over 781 monthly assessments (flare rate of 0.25/patient-month). There were 37 severe flares, 32 moderate flares and 126 mild flares. By 1 month, 22% had a mild/moderate/severe flare and 22% had a severe flare by 7 months. The median time to any 'A' or 'B' flare was 4 months. Severe/moderate flares tended to be in the system(s) affected at baseline, whereas mild flares could affect any system. CONCLUSION: . In a population with active SLE we observed an ongoing rate of flares from early in the follow-up period with moderate-severe flares being due to an inability to fully control the disease. This real-world population study demonstrates the limitations of current treatments and provides a useful reference population from which to inform future clinical trial design.

Patterns and predictors of skin score change in early diffuse systemic sclerosis from the European Scleroderma Observational Study.

OBJECTIVES: Our aim was to use the opportunity provided by the European Scleroderma Observational Study to (1) identify and describe those patients with early diffuse cutaneous systemic sclerosis (dcSSc) with progressive skin thickness, and (2) derive prediction models for progression over 12 months, to inform future randomised controlled trials (RCTs). METHODS: The modified Rodnan skin score (mRSS) was recorded every 3 months in 326 patients. 'Progressors' were defined as those experiencing a 5-unit and 25% increase in mRSS score over 12 months (±3 months). Logistic models were fitted to predict progression and, using receiver operating characteristic (ROC) curves, were compared on the basis of the area under curve (AUC), accuracy and positive predictive value (PPV). RESULTS: 66 patients (22.5%) progressed, 227 (77.5%) did not (33 could not have their status assessed due to insufficient data). Progressors had shorter disease duration (median 8.1 vs 12.6 months, P=0.001) and lower mRSS (median 19 vs 21 units, P=0.030) than non-progressors. Skin score was highest, and peaked earliest, in the anti-RNA polymerase III (Pol3+) subgroup (n=50). A first predictive model (including mRSS, duration of skin thickening and their interaction) had an accuracy of 60.9%, AUC of 0.666 and PPV of 33.8%. By adding a variable for Pol3 positivity, the model reached an accuracy of 71%, AUC of 0.711 and PPV of 41%. CONCLUSIONS: Two prediction models for progressive skin thickening were derived, for use both in clinical practice and for cohort enrichment in RCTs. These models will inform recruitment into the many clinical trials of dcSSc projected for the coming years. TRIAL REGISTRATION NUMBER: NCT02339441.

Disability, fatigue, pain and their associates in early diffuse cutaneous systemic sclerosis: the European Scleroderma Observational Study.

Objectives: Our aim was to describe the burden of early dcSSc in terms of disability, fatigue and pain in the European Scleroderma Observational Study cohort, and to explore associated clinical features. Methods: Patients completed questionnaires at study entry, 12 and 24 months, including the HAQ disability index (HAQ-DI), the Cochin Hand Function Scale (CHFS), the Functional Assessment of Chronic Illness Therapy-fatigue and the Short Form 36 (SF36). Associates examined included the modified Rodnan skin score (mRSS), current digital ulcers and internal organ involvement. Correlations between 12-month changes were also examined. Results: The 326 patients recruited (median disease duration 11.9 months) displayed high levels of disability [mean (s.d.) HAQ-DI 1.1 (0.83)], with 'grip' and 'activity' being most affected. Of the 18 activities assessed in the CHFS, those involving fine finger movements were most affected. High HAQ-DI and CHFS scores were both associated with high mRSS (ρ = 0.34, P < 0.0001 and ρ = 0.35, P < 0.0001, respectively). HAQ-DI was higher in patients with digital ulcers (P = 0.004), pulmonary fibrosis (P = 0.005), cardiac (P = 0.005) and muscle involvement (P = 0.002). As anticipated, HAQ-DI, CHFS, the Functional Assessment of Chronic Illness Therapy and SF36 scores were all highly correlated, in particular the HAQ-DI with the CHFS (ρ = 0.84, P < 0.0001). Worsening HAQ-DI over 12 months was strongly associated with increasing mRSS (ρ = 0.40, P < 0.0001), decreasing hand function (ρ = 0.57, P < 0.0001) and increasing fatigue (ρ = -0.53, P < 0.0001). Conclusion: The European Scleroderma Observational Study highlights the burden of disability in early dcSSc, with high levels of disability and fatigue, associating with the degree of skin thickening (mRSS). Impaired hand function is a major contributor to overall disability.

HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.

Treatment outcome in early diffuse cutaneous systemic sclerosis: the European Scleroderma Observational Study (ESOS).

OBJECTIVES: The rarity of early diffuse cutaneous systemic sclerosis (dcSSc) makes randomised controlled trials very difficult. We aimed to use an observational approach to compare effectiveness of currently used treatment approaches. METHODS: This was a prospective, observational cohort study of early dcSSc (within three years of onset of skin thickening). Clinicians selected one of four protocols for each patient: methotrexate, mycophenolate mofetil (MMF), cyclophosphamide or 'no immunosuppressant'. Patients were assessed three-monthly for up to 24 months. The primary outcome was the change in modified Rodnan skin score (mRSS). Confounding by indication at baseline was accounted for using inverse probability of treatment (IPT) weights. As a secondary outcome, an IPT-weighted Cox model was used to test for differences in survival. RESULTS: Of 326 patients recruited from 50 centres, 65 were prescribed methotrexate, 118 MMF, 87 cyclophosphamide and 56 no immunosuppressant. 276 (84.7%) patients completed 12 and 234 (71.7%) 24 months follow-up (or reached last visit date). There were statistically significant reductions in mRSS at 12 months in all groups: -4.0 (-5.2 to -2.7) units for methotrexate, -4.1 (-5.3 to -2.9) for MMF, -3.3 (-4.9 to -1.7) for cyclophosphamide and -2.2 (-4.0 to -0.3) for no immunosuppressant (p value for between-group differences=0.346). There were no statistically significant differences in survival between protocols before (p=0.389) or after weighting (p=0.440), but survival was poorest in the no immunosuppressant group (84.0%) at 24 months. CONCLUSIONS: These findings may support using immunosuppressants for early dcSSc but suggest that overall benefit is modest over 12 months and that better treatments are needed. TRIAL REGISTRATION NUMBER: NCT02339441.

Tight control of disease activity fails to improve body composition or physical function in rheumatoid arthritis patients.

OBJECTIVE: RA typically features rheumatoid cachexia [loss of muscle mass (MM) and excessive total fat mass (TFM), especially trunk FM], which contributes to physical disability. Since rheumatoid cachexia is driven by inflammation, it would be anticipated that the success of tight control of disease activity, such as treat-to-target (T2T), in attenuating inflammation would benefit body composition and physical function. This aim of this cross-sectional study was to assess the impact of T2T on body composition and objectively assessed function in RA patients. METHODS: A total of 82 RA patients exclusively treated by T2T, were compared with 85 matched sedentary healthy controls (HCs). Body composition was estimated by DXA, with appendicular lean mass the surrogate measure of total MM. Physical function was assessed by knee extensor strength, handgrip strength, 30 s sit-to-stands, 8' up and go, and 50' walk (tests which reflect the ability to perform activities of daily living). RESULTS: Although generally well treated (mean DAS28 = 2.8, with 49% in remission), RA patients had ∼10% proportionally less appendicular lean mass and were considerably fatter (by ∼27%), particularly in the trunk (∼32%), than HCs. All measures of function were 24-34% poorer in the RA patients relative to HC. CONCLUSIONS: Despite marked improvements in disease control (most patients achieving or approaching remission), the relative loss of MM and increased adiposity in RA patients compared with matched HCs was similar to that observed pre-T2T. Additionally, performance of objective function tests was unchanged from that reported by our group for pre-T2T RA patients. Thus T2T, even in responsive RA patients, did not attenuate rheumatoid cachexia or improve objectively assessed function.

Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.

Global subcellular characterization of protein degradation using quantitative proteomics.

Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ~5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.

Rheumatoid arthritis-related interstitial lung disease: associations, prognostic factors and physiological and radiological characteristics--a large multicentre UK study.

OBJECTIVES: The prevalence of interstitial lung disease (ILD) in RA is ∼5%. Previous work identified increasing age, active articular disease and articular damage as risk factors for RA-associated ILD (RA-ILD). The roles of high-resolution CT (HRCT) and lung function testing in defining the nature and extent of pulmonary involvement have recently been explored. This study is the first to examine predictive and prognostic factors for the development of RA-ILD and to report on the physiological and radiological characteristics of the condition from a large multicentre UK network. METHODS: We collected data from centres across the UK on patients with both RA and ILD (proved on HRCT) diagnosed over a 25-year period from 1987 to 2012 using a standard pro forma. Potential predictors of RA-ILD were analysed. Baseline lung function data were recorded and related to HRCT findings. We analysed HRCT for subtype and extent of lung involved and examined the relationship between these and both all-cause and pulmonary mortality. We compared our results with case controls matched for age and gender using computer-generated selection from the RA population from one contributing centre. RESULTS: A total of 230 patients were identified from across the UK with proven RA-ILD diagnosed over 25 years. Median age at diagnosis was 64 years and the male:female ratio was 1:1.09. Univariate analysis showed anti-CCP antibody titres to be the single most strongly associated predictor of RA-ILD. Male gender, age at onset, smoking and RF were all independently associated with RA-ILD on multivariate analysis. Vital capacity (VC) was preserved in limited disease but reduced in extensive disease, while gas transfer was reduced in both. Usual interstitial pneumonia (UIP) was the most common subtype on HRCT and both this and extensive disease were associated with increased all-cause mortality. CONCLUSION: This is the largest study of RA-ILD in the UK. Anti-CCP antibodies were strongly associated with RA-ILD in both sexes. Smoking was strongly associated with ILD in males, which may explain the higher frequency of RA-ILD in men. The predominant HRCT pattern was UIP and most patients had limited disease at presentation. The presence of UIP and extensive disease are associated with increased mortality. Baseline gas transfer is a useful screening tool for ILD, while the preservation of VC at baseline might predict limited disease on HRCT.

Systematic analysis of protein pools, isoforms, and modifications affecting turnover and subcellular localization.

In higher eukaryotes many genes encode protein isoforms whose properties and biological roles are often poorly characterized. Here we describe systematic approaches for detection of either distinct isoforms, or separate pools of the same isoform, with differential biological properties. Using information from ion intensities we have estimated protein abundance levels and using rates of change in stable isotope labeling with amino acids in cell culture isotope ratios we measured turnover rates and subcellular distribution for the HeLa cell proteome. Protein isoforms were detected using three data analysis strategies that evaluate differences between stable isotope labeling with amino acids in cell culture isotope ratios for specific groups of peptides within the total set of peptides assigned to a protein. The candidate approach compares stable isotope labeling with amino acids in cell culture isotope ratios for predicted isoform-specific peptides, with ratio values for peptides shared by all the isoforms. The rule of thirds approach compares the mean isotope ratio values for all peptides in each of three equal segments along the linear length of the protein, assessing differences between segment values. The three in a row approach compares mean isotope ratio values for each sequential group of three adjacent peptides, assessing differences with the mean value for all peptides assigned to the protein. Protein isoforms were also detected and their properties evaluated by fractionating cell extracts on one-dimensional SDS-PAGE prior to trypsin digestion and MS analysis and independently evaluating isotope ratio values for the same peptides isolated from different gel slices. The effect of protein phosphorylation on turnover rates was analyzed by comparing mean turnover values calculated for all peptides assigned to a protein, either including, or excluding, values for cognate phosphopeptides. Collectively, these experimental and analytical approaches provide a framework for expanding the functional annotation of the genome.

A quantitative spatial proteomics analysis of proteome turnover in human cells.

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.

Evaluation of the efficacy of hydro-alcoholic extract of Evolvulus alsinoides L. for the treatment of primary hypertension - A randomized controlled trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Evolvulus alsinoides L. (Sankhaholi) has been traditionally used in Unani (Greco-Arabic) medicine to treat diverse cardiovascular disorders. Notably, preclinical and clinical investigations have substantiated its remarkable potential as an antihypertensive agent. AIM OF THE STUDY: The aim of this study was to compare the efficacy of hydroalcoholic extract of Evolvulus alsinoides L. and ramipril in treating hypertension using a higher dose of the test drug within the recommended limit. MATERIALS AND METHODS: In this open-label randomized controlled trial, 57 participants (29 in the test group, 28 in the control group) completed the 42-day study. The test group received 630 mg of dried hydro-alcoholic extract of Evolvulus alsinoides L. in capsule form orally once daily, while the control group received 5 mg of Ramipril orally once daily. Participants in both groups were advised to adhere to the Dietary Approaches to Stop Hypertension (DASH) eating plan in terms of diet and lifestyle adjustments recommended by JNC-8. The primary outcome measures were changes in systolic and diastolic blood pressure as well as changes in plasma levels of hsCRP and IL6. Secondary outcome measures included changes in symptoms such as palpitations, giddiness, headaches, fatigue and shortness of breath. Headaches, palpitations, and giddiness were assessed using a customized Visual Analog Scale (VAS) graded as "none," "mild," "moderate," and "severe". Fatigue was assessed on a binary scale as either absent or present, and dyspnea was assessed using the modified Medical Research Council (mMRC) scale for breathlessness. Both primary and secondary outcomes were assessed at baseline and each follow-up visit (2nd week, 4th week, and 6th week) until the completion of the trial. RESULTS: At the end of the trial, the mean differences for the primary outcomes were as follows:SBP:-1.8895%CI:-4.82,1.05,p=0.203,d=0.33, DBP: -2.8395%CI:-4.67,-0.10,p=0.003,d=0.8, hsCRP: -1.4095%CI:-2.80,-0.003,p=0.49,d=0.53, and IL6: -88.6795%CI:-148.90,-28.43,p=0.005,d=0.78. No statistically significant differences were observed between the two groups for any of the secondary outcomes. CONCLUSIONS: Based on the preliminary results, it can be inferred that the hydro-alcoholic extract of Evolvulus alsinoides L. exhibits significant antihypertensive potential, comparable to that of ramipril. Furthermore, it appears that Evolvulus alsinoides L. may be more effective than ramipril in reducing the biochemical markers of inflammation associated with primary hypertension. However, additional research is required to validate these findings.

The BILAG-2004 systems tally--a novel way of representing the BILAG-2004 index scores longitudinally.

OBJECTIVE: This was an exploratory analysis to develop a new way of representing BILAG-2004 system scores longitudinally that would be clinically meaningful and easier to analyse in comparison with multiple categorical variables. METHODS: Data from a multicentre longitudinal study of SLE patients (the BILAG-2004 index and therapy collected at every visit) were used. External responsiveness analysis of the index suggested the possibility of using counts of systems with specified transitions in scores as a basis to analyse the system scores. Exploratory analyses with multinomial logistic regression were used to examine the appropriateness of this new method of analysing BILAG-2004 system scores. Receiver operating characteristic (ROC) curve analysis was used to assess the performance of this approach. RESULTS: There were 1414 observations from 347 patients. A novel method was devised based on counts of systems with defined transitions in score (BILAG-2004 systems tally, BST). It has six components (systems with major deterioration, systems with minor deterioration, systems with persistent significant activity, systems with major improvement, systems with minor improvement and systems with persistent minimal or no activity). This was further simplified (simplified BST, sBST) into three components (systems with active/worsening disease, systems with improving disease and systems with persistent minimal or no activity). Both versions had expected associations with change in therapy. ROC curve analyses demonstrated that both versions had similar good performance characteristics (areas under the curve >0.80) in predicting increase in therapy. CONCLUSION: The BST and sBST provide alternative approaches to representing BILAG-2004 disease activity longitudinally. Further validation of their use is required.

Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners.

The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static "bead proteomes." The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.

Comparison of Responsiveness of British Isles Lupus Assessment Group 2004 Index, Systemic Lupus Erythematosus Disease Activity Index 2000, and British Isles Lupus Assessment Group 2004 Systems Tally.

OBJECTIVE: To compare the responsiveness of the British Isles Lupus Assessment Group 2004 index (BILAG-2004) and the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) disease activity indices and to determine whether there was any added value in combining BILAG-2004, BILAG-2004 system tally (BST), or simplified BST (sBST) with SLEDAI-2K. METHODS: This was a multicenter longitudinal study of SLE patients. Data were collected on BILAG-2004, SLEDAI-2K, and therapy on consecutive assessments in routine practice. The external responsiveness of the indices was assessed by determining the relationship between change in disease activity and change in therapy between 2 consecutive visits. Comparison of indices and their derivatives was performed by assessing the main effects of the indices using logistic regression. Receiver operating characteristic curves analysis was used to describe the performance of these indices individually and in various combinations, and comparisons of area under the curve were performed. RESULTS: There were 1,414 observations from 347 patients. Both BILAG-2004 and SLEDAI-2K maintained an independent relationship with change in therapy when compared. There was some improvement in responsiveness when continuous SLEDAI-2K variables (change in score and score of previous visit) were combined with BILAG-2004 system scores. Dichotomization of BILAG-2004 or SLEDAI-2K resulted in poorer performance. BST and sBST had similar responsiveness as the combination of SLEDAI-2K variables and BILAG-2004 system scores. There was little benefit in combining SLEDAI-2K with BST or sBST. CONCLUSION: The BILAG-2004 index had comparable responsiveness to SLEDAI-2K. There was some benefit in combining both indices. Dichotomization of BILAG-2004 and SLEDAI-2K leads to suboptimal performance. BST and sBST performed well on their own; sBST is recommended for its simplicity and clinical meaningfulness.

Mass spectrometry-based immuno-precipitation proteomics - the user's guide.

Immuno-precipitation (IP) experiments using MS provide a sensitive and accurate way of characterising protein complexes and their response to regulatory mechanisms. Differences in stoichiometry can be determined as well as the reliable identification of specific binding partners. The quality control of IP and protein interaction studies has its basis in the biology that is being observed. Is that unusual protein identification a genuine novelty, or an experimental irregularity? Antibodies and the solid matrices used in these techniques isolate not only the target protein and its specific interaction partners but also many non-specific 'contaminants' requiring a structured analysis strategy. These methodological developments and the speed and accuracy of MS machines, which has been increasing consistently in the last 5 years, have expanded the number of proteins identified and complexity of analysis. The European Science Foundation's Frontiers in Functional Genomics programme 'Quality Control in Proteomics' Workshop provided a forum for disseminating knowledge and experience on this subject. Our aim in this technical brief is to outline clearly, for the scientists wanting to carry out this kind of experiment, and recommend what, in our experience, are the best potential ways to design an IP experiment, to help identify possible pitfalls, discuss important controls and outline how to manage and analyse the large amount of data generated. Detailed experimental methodologies have been referenced but not described in the form of protocols.

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.

NOPdb: Nucleolar Proteome Database--2008 update.

An experimental data handling system has been created as an update to the previous Nucleolar Proteome Database (NOPdb3.0: http://www.lamondlab.com/NOPdb3.0/). This updated system is able to manage large data sets identified by multiple mass spectrometry and has been used to analyse highly purified preparations of human nucleoli from different cell lines. The newly created application includes a dynamic relational database, which is kept up to date by laboratory staff. The data are further annotated with information from specific external sources on the web, including the IPI and Gene Ontology databases. In addition, an Application Programming Interface provides external users with a portal to link into the nucleolar proteome database and hence, gain access to continually updated results. From the initial approximately 700 human proteins identified in the previous iteration of the NOPdb, we have now identified over 50 000 peptides contained in over 4500 human proteins from purified nucleoli, providing enhanced coverage of the nucleolar proteome.

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.