Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network.

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

Prescription Patterns of Daratumumab in Patients with Multiple Myeloma in Underprivileged Circumstances: A Multicenter Experience in Mexico.

BACKGROUND: Despite novel therapies, multiple myeloma (MM) remains an incurable malignancy, daratumumab (DARA) being a major game changer, may be a good option for treatment. AIMED OF THE STUDY: To assess the prescription patterns of DARA in patients with MM in Mexico. METHODS: 47 patients with MM were analyzed in 13 different hospitals in Mexico. RESULTS: Five (10.5%) of patients received DARA as first line therapy, 13 (27.5%) as second line, whereas 29 (62%) received its in ≥3rd line. In 32% DARA was used in combination with dexamethasone, 64% received DARA on a triple combination, and 4% as a 4 drug combination. Eighty three percent of patients had a response, including 32% in complete remission. Progression free survival (PFS) was higher in patients in ISS stage 1, and in patients achieving ≥PR. Overall survival (OS) was lower in patients not achieving ≥PR, in patients having increased LDH, and extramedullary disease. Grade 1-2 infusion related reactions were present in 34%, and grade 3-4 neutropenia and lymphopenia in 25 and 17% of the patients. CONCLUSIONS: In Mexico, 62% of patients with MM received DARA as a third or further line of treatment. DARA employed as a doublet or triplet combination is useful in relapsed/refractory patients with tolerable adverse events.

Influence of Mesenchymal Stem Cell Sources on Their Regenerative Capacities on Different Surfaces.

Current gold-standard strategies for bone regeneration do not achieve the optimal recovery of bone biomechanical properties. To bypass these limitations, tissue engineering techniques based on hybrid materials made up of osteoprogenitor cells-such as mesenchymal stem cells (MSCs)-and bioactive ceramic scaffolds-such as calcium phosphate-based (CaPs) bioceramics-seem promising. The biological properties of MSCs are influenced by the tissue source. This study aims to define the optimal MSC source and construct (i.e., the MSC-CaP combination) for clinical application in bone regeneration. A previous iTRAQ analysis generated the hypothesis that anatomical proximity to bone has a direct effect on MSC phenotype. MSCs were isolated from adipose tissue, bone marrow, and dental pulp, then cultured both on a plastic surface and on CaPs (hydroxyapatite and ß-tricalcium phosphate), to compare their biological features. On plastic, MSCs isolated from dental pulp (DPSCs) presented the highest proliferation capacity and the greatest osteogenic potential. On both CaPs, DPSCs demonstrated the greatest capacity to colonise the bioceramics. Furthermore, the results demonstrated a trend that DPSCs had the most robust increase in ALP activity. Regarding CaPs, ß-tricalcium phosphate obtained the best viability results, while hydroxyapatite had the highest ALP activity values. Therefore, we propose DPSCs as suitable MSCs for cell-based bone regeneration strategies.

Adverse effects of xenogenic scaffolding in the context of a randomized double-blind placebo-controlled study for repairing full-thickness rotator cuff tears.

PURPOSE: The purpose of the study was to compare the safety and efficacy of autologous mesenchymal stem cells (MSCs) embedded in a xenogenic scaffold for repairing the supraspinatus tendon. METHODS: This was a randomized, double-blind and placebo-controlled trial evaluating patients with full-thickness rotator cuff tears (Eudra-CT, 2007-007630-19). Effectiveness was evaluated using the Constant score and a visual analogue pain scale (VAS). Constant score has four domains including pain (15 possible points), activities of daily living (20 possible points), mobility (40 possible points), and strength (25 possible points). Scores range from 0 points (most disability) to 100 points (least disability). The structural integrity of the repaired tendon was assessed by magnetic resonance imaging (MRI) according to Patte and Thomazeau classification criteria. The primary study end point was an improvement in the Constant score by 20 points at one year compared to initial assessment. RESULTS: The trial was stopped due to adverse effects observed in both groups. Only thirteen patients were included and analyzed. The Constant questionnaire showed a significant improvement in the MSC treatment group compared with the preoperative data (p = 0.0073). Secondary outcome measures were similar in both groups. CONCLUSIONS: Our study showed preliminary inconclusive clinical outcomes in the patients treated with MSCs. Adverse events revealed the need for further approaches using scaffolds of a different nature or perhaps no scaffolds, in the context of small joints. TRIAL REGISTRATION: Eudra-CT, 2007-007630-19 . Registered on 30 January 2008. LEVEL OF EVIDENCE: A Level 1 of evidence treatment study.

RNA sequencing of mesenchymal stem cells reveals a blocking of differentiation and immunomodulatory activities under inflammatory conditions in rheumatoid arthritis patients.

INTRODUCTION: Mesenchymal stem cells (MSCs) have the ability to differentiate into different types of cells of the mesenchymal lineage, such as osteocytes, chondrocytes, and adipocytes. It is also known that under inflammatory stimuli or in the appropriate experimental conditions, they can also act as regulators of inflammation. Thus, in addition to their regenerating potential, their interest has been extended to their possible use in cell therapy strategies for treatment of immune disorders. OBJECTIVE: To analyze, by RNA-seq analysis, the transcriptome profiling of allogenic MSCs under RA lymphocyte activation. METHODS: We identified the differentially expressed genes in bone marrow mesenchymal stem cells after exposure to an inflammatory environment. The transcriptome profiling was evaluated by means of the precise measurement of transcripts provided by the RNA-Seq technology. RESULTS: Our results evidenced the existence of blocking of both regenerative (differentiation) and immunomodulatory phenotypes under inflammatory conditions characterized by an upregulation of genes involved in immune processes and a simultaneous downregulation of genes mainly involved in regenerative or cell differentiation functions. CONCLUSIONS: We conclude that the two main functions of MSCs (immunomodulation and differentiation) are blocked, at least while the inflammation is being resolved. Inflammation, at least partially mediated by gamma-interferon, drives MSCs to a cellular distress adopting a defensive state. This knowledge could be of particular interest in cases where the damage to be repaired has an important immune-mediated component.

Functional implications of single nucleotide polymorphisms rs662 and rs854860 on the antioxidative activity of paraoxonase1 (PON1) in patients with rheumatoid arthritis.

BACKGROUND: Atherosclerosis leading to cardiovascular disease (CVD) is the main cause of mortality and morbidity in patients with rheumatoid arthritis (RA). Paraoxonase1 (PON1) is the best understood member of plasma paraoxonases with anti-atherogenic properties. PATIENTS AND METHODS: Spanish RA (n = 549) consecutively recruited from 1 single center and 477 ethnically matched healthy controls were included in a case-control study. The concentration of PON1 was evaluated by means of an enzyme-linked immunosorbent sssay (ELISA). An arylesterase/paraoxonase assay kit was used to evaluate PON1 activity. Sample genotyping was performed by using TaqMan assays-on-demand. All results were expressed as medians ± interquartile range. One-way ANOVA comparisons were done using a nonparametric Kruskall-Wallis test. P values under 0.05 were considered to be significant. RESULTS: The concentration of PON1 in the RA group was higher than in control group (p = 0.0003), although the differences were not significant when PON1 activities were compared between both groups. No significant differences were found related to distributions of rs662 genotypes in RA patients compared to healthy controls. Among rs854860 polymorphisms, overall genotype was widely distributed between RA patients and controls. Overall PON1 concentration in plasma was not significantly different between individuals carrying any of rs662 (p = 0.8501) or rs854860 (p = 0.2741) polymorphisms. Although PON1 levels were not associated with any of the SNPs in the study, differences appear when enzyme activities are compared for each SNP separately. CVD in RA patients correlate with increased PON1 levels and lower PON1 activity. CONCLUSIONS: Although protective role of PON1 against oxidative damage in vivo could be related to other activities, in our study arylesterase activity was useful to identify phenotypic differences with emphasis placed on two SNPs coding for nonconservative amino acid changes in the functional protein.

Differential Expression of HOX Genes in Mesenchymal Stem Cells from Osteoarthritic Patients Is Independent of Their Promoter Methylation.

Skeletogenesis, remodeling, and maintenance in adult tissues are regulated by sequential activation of genes coding for specific transcription factors. The conserved Homeobox genes (HOX, in humans) are involved in several skeletal pathologies. Osteoarthritis (OA) is characterized by homeostatic alterations of cartilage and bone synthesis, resulting in cartilage destruction and increased bone formation. We postulate that alterations in HOX expression in Mesenchymal Stem cells (MSCs) are likely one of the causes explaining the homeostatic alterations in OA and that this altered expression could be the result of epigenetic regulation. The expression of HOX genes in osteoarthritic-derived MSCs was screened using PCR arrays. Epigenetic regulation of HOX was analyzed measuring the degree of DNA methylation in their promoters. We demonstrate the downregulated expression of HOXA9 and HOXC8 in OA-MSCs. However, their expression does not correlate with promoter methylation status, suggesting that other epigenetic mechanisms could be implicated in the regulation of HOX expression. Studies on the role of these genes under active differentiation conditions need to be addressed for a better knowledge of the mechanisms regulating the expression of HOX, to allow a better understanding of OA pathology and to define possible biomarkers for therapeutic treatment.

Long-term continuation of methotrexate therapy in giant cell arteritis patients in clinical practice.

OBJECTIVES: To assess the long-term continuation of methotrexate (MTX) in a cohort of patients with giant cell arteritis (GCA) in daily clinical practice. Factors associated with its discontinuation rate were also investigated. METHODS: A longitudinal study from 1991-2014, was performed. GCA patients with MTX and followed-up in a rheumatology outpatient clinic of Madrid during the study period were included. PRIMARY OUTCOME: discontinuation of MTX due to: adverse drug reactions (ADR moderate and severe); inefficacy; sustained clinical response; patient decision. Covariables: sociodemographic, clinical and therapy. Incidence rates (IR) of MTX discontinuation per 100 patient-years with their 95% confidence interval (CI) were estimated using survival techniques. Factors associated to specific discontinuation causes were analysed using Cox models. RESULTS: We included 108 patients (244 patient-years). The IR was 37.2 [30.3-45.7]. The IR due to ADR, severe ADR, sustained clinical response and inefficacy was 20.8 [15.8-27.4]; 5.7 [3.3-9.6]; 8.2 [5.3-12.7] and 2.8 [1.3-6.0], respectively. Regarding multivariate analysis, younger patients, baseline cardiovascular disease, taking more glucocorticoids and lower initial doses of MTX were associated to a higher discontinuation rate due to inefficacy. Factors influencing the suspension due to ADRs were: older age, baseline. Chronic obstructive pulmonary disease, higher baseline erythrocyte sedimentation rate, several specific clinical patterns at diagnosis, and higher maximum dose of MTX during the follow up. In the final model for sustained clinical response older patients and more recurrences were independently associated to less discontinuation rate. CONCLUSIONS: We provide further data of the potential safety of long-term MTX in the management of GCA. We have also found several factors influencing the continuation of MTX.

Treatment in rheumatoid arthritis and mortality risk in clinical practice: the role of biologic agents.

OBJECTIVES: To assess the mortality rate (MR) and the mortality risk of a rheumatoid arthritis (RA) inception cohort, with and without biologic agents (BAs). Other factors associated to mortality were also investigated. METHODS: Retrospective longitudinal study of RA patients, attending the rheumatology outpatient clinic of a tertiary Hospital (Madrid), collected over 5 years (2000-2004), and followed from the diagnosis of RA up to the patients' death, lost to follow-up or September 2013. The dependent variable was death and the independent variable was exposure to BAs. Covariables: sociodemographic, clinical and therapy variables. MR was expressed per 1,000 patient-years with the 95% confidence interval [CI]. BA influence on MR was analysed by multivariable Cox models. Clinical and therapy variables were used in a time-dependent manner. The results are expressed in hazard ratio (HR) and [CI]. RESULTS: We included 576 patients and 711 courses of therapy. 19.6% were taking BA, 86% disease-modifying anti-rheumatic drugs (DMARDs) (70% on methotrexate - MTX), and 12% were untreated. There were 133 deaths during 4,981.64 patient-years at risk. The MR for BA was 12.6 [6-26], for DMARDs was 22.3 [18.4-27.1], and for those without treatment was 89.1 [61.9-128.2]. The adjusted HR for mortality in those exposed to BA versus those not exposed was 0.75 [0.32-1.71]). Other variables independently associated with mortality were: age, rheumatoid factor, hospital admissions, Health Assessment Questionnaire (HAQ), and MTX use (HR: 0.44 [0.29-0.66]). CONCLUSIONS: BAs and standard DMARDs are more effective in decreasing mortality compared to no therapy. Patients exposed to Bas were not associated with a significant increase or decrease in mortality when compared to patients with non-biological DMARDs. The use of MTX remains the only drug that has independently shown a beneficial effect on mortality.

Dose down-titration of biological DMARDs in patients with rheumatoid arthritis over time and in daily clinical practice.

OBJECTIVES: To describe and compare dosing optimisation in biological DMARDs (bDMARDs) and relapses after that, in a cohort of rheumatoid arthritis (RA) during clinical practice. METHODS: Observational retrospective longitudinal study of RA patients taking bDMARDs from December 1999 to November 2013. Optimisation was defined as a 15% decrease in dose either reducing single dose or separating dose interval administration, for at least 4 times the recommended period between dosages. Relapse was defined as suspension or starting again with the recommended dose after optimisation. Incidence rates (IR) per 100 patient-years were estimated using survival techniques. Cox multivariate models were conducted to compare bDMARDs expressed in hazard ratios (HR) and confidence intervals [95%CI]. RESULTS: 443 patients and 752 different courses of bDMARDs treatments were included. We observed 146 optimisations with an IR of 8.1. The HR of optimisation in: a) adalimumab, etanercept and rituximab compared to infliximab was 1.56 [1.01-2.4], 1.5 [0.9-2.4] and 0.6 [0.3-1.4], respectively; b) adalimumab, etanercept compared to rituximab were 2.3 [1.2-4.5] and 2.2 [1.2-4.3]. There were no statistically significant differences between adalimumab and etanercept. Following optimisation, 36% relapsed (78% due to disease activity). The IR related to disease activity was 6.3, and was lower for adalimumab and etanercept compared to infliximab (HR: 0.42; [0.19-0.94]; HR: 0.34; [0.13-0.89], respectively). There were no statistically significant differences between etanercept and adalimumab. No patients on rituximab relapsed. CONCLUSIONS: Optimisation was similar between adalimumab and etanercept, and was lower for infliximab and rituximab. After optimisation, rituximab did not relapse, but infliximab did with the highest hazard.

Altered Expression of Wnt Signaling Pathway Components in Osteogenesis of Mesenchymal Stem Cells in Osteoarthritis Patients.

INTRODUCTION: Osteoarthritis (OA) is characterized by altered homeostasis of joint cartilage and bone, whose functional properties rely on chondrocytes and osteoblasts, belonging to mesenchymal stem cells (MSCs). WNT signaling acts as a hub integrating and crosstalking with other signaling pathways leading to the regulation of MSC functions. The aim of this study was to evaluate the existence of a differential signaling between Healthy and OA-MSCs during osteogenesis. METHODS: MSCs of seven OA patients and six healthy controls were isolated, characterised and expanded. During in vitro osteogenesis, cells were recovered at days 1, 10 and 21. RNA and protein content was obtained. Expression of WNT pathway genes was evaluated using RT-qPCR. Functional studies were also performed to study the MSC osteogenic commitment and functional and post-traslational status of ß-catenin and several receptor tyrosine kinases. RESULTS: Several genes were downregulated in OA-MSCs during osteogenesis in vitro. These included soluble Wnts, inhibitors, receptors, co-receptors, several kinases and transcription factors. Basal levels of ß-catenin were higher in OA-MSCs, but calcium deposition and expression of osteogenic genes was similar between Healthy and OA-MSCs. Interestingly an increased phosphorylation of p44/42 MAPK (ERK1/2) signaling node was present in OA-MSCs. CONCLUSION: Our results point to the existence in OA-MSCs of alterations in expression of Wnt pathway components during in vitro osteogenesis that are partially compensated by post-translational mechanisms modulating the function of other pathways. We also point the relevance of other signaling pathways in OA pathophysiology suggesting their role in the maintenance of joint homeostasis through modulation of MSC osteogenic potential.

Signature of microRNA expression during osteogenic differentiation of bone marrow MSCs reveals a putative role of miR-335-5p in osteoarthritis.

BACKGROUND: The aim of this study was to evaluate, the existence of a signature of differentially expressed microRNAs (miRNAs) during osteogenic differentiation of bone marrow MSCs from OA and healthy donors and to describe their possible implication in joint regeneration through modulation of molecular mechanisms involved in homeostatic control in OA pathophysiology. METHODS: Following phenotypic assessment of BM-MSCs obtained from OA diagnosed patients (n = 10) and non-OA (n = 10), total small RNA was isolated after osteogenic induction for 1, 10 and 21 days, miRNA profiles were generated using a commercial expression array of 754 well-characterized miRNAs. MiRNAs, with consistent differential expression were selected for further validation by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. RESULTS: A total of 246 miRNAs were differentially expressed (fold change ≥ ± 2, P ≤0.05) between OA and non-OA BM-MSC samples; these miRNAs showed variable interactions depending on the cell and differentiation status. Two miRNAs, hsa-miR-210 and hsa-miR-335-5p out of 21 used for validation showed a significant downregulated expression during induced osteogenesis. In particular hsa-miR-335-5p, a critical regulator in bone homeostasis, was further studied. hsa-miR-335-5p downregulation in OA-MSCs, as well as their host coding gene, MEST, were also assessed. CONCLUSIONS: To our knowledge, this study represents the most comprehensive assessment to date of miRNA expression profiling in BM-MSCs from OA patients and their role during osteogenic differentiation. We describe the existence of a correlation between miR-335-5p expression and OA indicating the putative role of this miRNA in OA features. These findings, may contribute to our understanding of the molecular mechanisms involved in MSCs mediated homeostatic control in OA pathophysiology that could be applicable in future therapeutic approaches.

Selected reaction monitoring assays in mesenchymal stem cells from osteoarthritis patients.

Osteoarthritis (OA) is considered the most prevalent form of arthritis. The aim of this study was to verify potential protein OA biomarkers by applying Selected Reaction Monitoring (SRM) assays to protein extracts obtained from Bone Marrow-Mesenchymal Stem Cells (BM-MSCs) isolated from OA patients. BM aspirates were obtained from the femoral channel of OA patients at the time of surgery and from the femoral channel of hip fracture subjects without OA during hip joint replacement surgery for the treatment of subcapital fracture. SRM results verified the differential expression of several protein biomarkers in BM-MSCs from OA patients.

Orthopedic surgery in rheumatoid arthritis in the era of biologic therapy.

OBJECTIVE: To analyze sociodemographic and clinic-related factors associated with the use of orthopedic surgical procedures in rheumatoid arthritis (RA), focusing on the potential role of new biologic therapies. METHODS: A retrospective medical record review was performed in a probability sample of 1272 patients with RA from 47 units distributed in 19 Spanish regions. Sociodemographic and clinical features, use of drugs, and arthritis-related joint surgeries were recorded following a standardized protocol. RESULTS: A total of 94 patients (7.4%) underwent any orthopedic surgery during their disease course, with a total of 114 surgeries; 47 (41.2%) of these surgeries were total joint replacement (TJR). The median time to first orthopedic procedure was 7.9 years from the onset of RA symptoms, and the rate of orthopedic surgery (excluding TJR) was 4.5 procedures per 100 person-years from the beginning of RA, while the rate of TJR was 2.25 interventions per 100 person-years. A higher risk of undergoing an orthopedic surgical procedure was associated with taking nonsteroidal antiinflammatory drugs (NSAID) in the previous 2 years, female sex, longterm disease, and the presence of extraarticular complications. The risk factors for undergoing a TJR were being old, having a longterm disease, and taking biologic therapies. CONCLUSION: In the era of biologics, our national audit found a low percentage of patients who underwent orthopedic surgery, probably reflecting a thorough management of the RA. Sociodemographic factors, longterm RA, extraarticular complications, and NSAID were associated with orthopedic surgery.

Role of the human endogenous retrovirus HERV-K18 in autoimmune disease susceptibility: study in the Spanish population and meta-analysis.

BACKGROUND: Human endogenous retroviruses (HERVs) are genomic sequences that resulted from ancestral germ-line infections by exogenous retroviruses and therefore are transmitted in a Mendelian fashion. Increased HERV expression and antibodies to HERV antigens have been found in various autoimmune diseases. HERV-K18 in chromosome 1 was previously associated with type one diabetes and multiple sclerosis (MS). The etiology of these complex conditions has not been completely elucidated even after the powerful genome wide association studies (GWAS) performed. Nonetheless, this approach does not scrutinize the repetitive sequences within the genome, and part of the missing heritability could lie behind these sequences. We aimed at evaluating the role of HERV-K18 in chromosome 1 on autoimmune disease susceptibility. METHODS: Two HERV-K18 SNPs (97Y/C and 154W/Stop substitutions) conforming three haplotypes were genotyped in Spanish cohorts of multiple sclerosis (n = 942), rheumatoid arthritis (n = 462) and ethnically matched controls (n = 601). Our findings were pooled in a meta-analysis including 5312 autoimmune patients and 4032 controls. RESULTS: Significant associations of both HERV-K18 polymorphisms in chromosome 1 with MS patients stratified by HLA-DRB1*15:01 were observed [97Y/C p = 0.02; OR (95% CI) = 1.5 (1.04-2.17) and 154W/Stop: p = 0.001; OR (95% CI) = 1.6 (1.19-2.16)]. Combined meta-analysis of the previously published association studies of HERV-K18 with different autoimmune diseases, together with data derived from Spanish cohorts, yielded a significant association of the HERV-K18.3 haplotype [97Y-154W: p(M-H) = 0.0008; OR(M-H) (95% CI) = 1.22 (1.09-1.38)]. CONCLUSION: Association of the HERV-K18.3 haplotype in chromosome 1 with autoimmune-disease susceptibility was confirmed through meta-analysis.

Alternative splicing and proteolytic rupture contribute to the generation of soluble IL-6 receptors (sIL-6R) in rheumatoid arthritis.

OBJECTIVE: To describe the relationship between the two mechanisms involved in sIL6R generation in rheumatoid arthritis (RA). METHOD: RA patients were selected from a group of subjects genotyped for the rs8192284 SNP, located at the proteolytic cleavage site of IL-6R. sIL6R and protease levels (ADAM17) were measured and the contribution of alternative splicing in the generation of sIL-6R was evaluated through qRT-PCR. RESULT: Increased sIL-6R plasma levels and expression of spliced isoform generating sIL-6R are genotype dependent. ADAM17 concentrations were independent of the genotype studied. CONCLUSION: Alternative splicing and proteolytic cleavage participate in sIL-6R generation in RA. The rs8192284 polymorphism determines the sIL-6R plasma level through differential proteolytic rupture controlled by ADAM17.

NPSR1 gene is associated with reduced risk of rheumatoid arthritis.

OBJECTIVE: Neuropeptide S receptor 1 (NPSR1) is a G protein-coupled receptor involved in immune response and is associated with several inflammatory diseases. We investigated the possible contribution of several polymorphisms in the intronic region of NPSR1 to rheumatoid arthritis (RA). METHODS: Genotyping of 7 single-nucleotide polymorphisms (SNP) was performed in a total of 1232 patients with RA and 983 healthy controls of Spanish white origin by real-time polymerase chain reaction technology, using the TaqMan 5'-allele discrimination assay. RESULTS: One out of the 7 SNP analyzed (rs740347) was associated with RA [p after Bonferroni correction (p(BNF)) = 1.2 × 10(-3), OR 0.73]. An association was also observed with rheumatoid factor-positive and shared epitope-positive RA (p(BNF) = 0.011, OR 0.73; p(BNF) = 0.037, OR 0.75, respectively). CONCLUSION: Our results show that variations in the NPSR1 intronic region are associated with low risk in patients with RA, supporting other evidence that this locus represents a common genetic factor in inflammatory diseases.

Combined influence of genetic and environmental factors in age of rheumatoid arthritis onset.

To study the combined effect of both genetic and environmental factors in the age of rheumatoid arthritis onset. Patients (n = 507). Shared epitope characterization was performed using Lifecodes HLA-SSO. Genotyping of protein tyrosine phosphatase non-receptor 22 (PTPN22) rs2476601 and signal transducers and activators of transcription 4 (STAT4) rs7574865 polymorphism was performed using fast real-time PCR System. Shared epitope, antibodies directed against cyclic citrulinated peptide (anti-CCP) antibodies and a higher level of education were associated with a younger age at disease onset (P = 0.033, P = 0.004 and P < 0.0001, respectively). Neither carriers of the minor allele of PTPN22 rs2476601 nor STAT4 rs7574 polymorphisms showed a significant association with a younger age at disease onset (P = 0.355, P = 0.065, respectively). We found an additive effect of the three genetic markers in the age at onset: subjects with three markers were associated with a disease onset 9.56, 8.61, and 6.41 years before than those with none, one, or two genetic markers (P = 0.004, P = 0.006 and P = 0.043, respectively). We also described the additive effect of shared epitope, anti-CCP antibodies, educational level, PTPN22, and STAT4 polymorphisms in age at onset. Patients with two, three, four, or five variables were associated with a significant younger age of disease onset (4.72 [0.05-9.38] years (P = 0.048), 9.56 [4.72-14.40] years (P < 0.0001), 12.74 [6.84-18.64] years (P < 0.0001), and 20.87 [10.40-37.17] years (P < 0.0001)). Risk factors for the development of rheumatoid arthritis are also associated, with an additive effect, with a younger age at disease onset.

Plasma soluble IL-6 receptor concentration in rheumatoid arthritis: associations with the rs8192284 IL6R polymorphism and with disease activity.

Soluble interleukin-6 receptor α subunit (sIL-6R) is primarily generated by shedding of the membrane-bound form. This process is influenced by the single nucleotide polymorphism rs8192284 (A > C) resulting in an aspartic acid to alanine substitution (D358A) at the proteolytic cleavage site. The aim of this study was to determine whether plasma levels of sIL6R are influenced by the rs8192284 polymorphism in patients with rheumatoid arthritis and to assess the association between plasma sIL-6R levels and disease activity as reflected by anti-CCP status. Thirty-nine patients were randomly selected from a cohort of patients with RA of Spanish descent. Plasma sIL-6R concentrations were measured using sandwich ELISA. Genotyping of the rs8192284 (A > C) polymorphism was done using a Fast Real-Time PCR System. DAS 28 scores were used to assess disease activity. Plasma sIL-6R levels were positively associated with the number of C alleles (AA: 35.27 (3.50) ng/ml, AC: 45.50 (4.58) ng/ml, CC: 52.55 (3.18) ng/ml, P = 0.0001). DAS28 and plasma sIL-6R levels were positively associated in the anti-CCP-positive subgroup (r (2) = 0.45, P = 0.0336) and negatively associated in the anti-CCP-negative subgroup (r (2) = -0.45, P = 0.0825). No association between anti-CCP status and sIL-6R level was found. Our findings show that the rs8192284 polymorphism is operative in patients with RA. The presence of anti-CCP antibodies determines the relationship between sIL-6R concentration and disease activity.