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26.  Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl-glutaryl-CoA reductase autoantibodies 
Introduction
Necrotizing autoimmune myopathies (NAM) have recently been defined as a distinct group of severe acquired myopathies, characterized by prominent myofiber necrosis without significant muscle inflammation. Because of the lack of appropriate biomarkers, these diseases have been long misdiagnosed as atypical forms of myositis. NAM may be associated to autoantibodies directed against signal recognition particle (SRP) or 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). The objective of this work was to quantify anti-HMGCR autoantibodies in patients with suspicion of NAM through the development of a new addressable laser bead immunoassay (ALBIA).
Methods
Recombinant HMGCR C-domain was bound to fluorescent beads. After incubation with serum, autoantibodies were revealed using class- or subclass-specific anti-human immunoglobulin G (IgG) antibodies. Anti-HMGCR levels were assayed in 150 patients with suspicion of NAM, 142 controls with different inflammatory/autoimmune diseases and 100 healthy donors. Inhibition with free recombinant HMGCR and immunoprecipitation experiments confirmed test specificity. Reproducibility and repeatability were determined from sera with various levels of anti-HMGCR autoantibodies. A multiplex assay (ALBIA-NAM) was also developed to permit the simultaneous quantification of anti-HMGCR and anti-signal recognition particle autoantibodies.
Results
No controls scored positive. Of 150 patients with suspicion of NAM, 24% were positive for anti-HMGCR autoantibodies with levels ranging from 24 to 2,656 AU/mL. Anti-HMGCR positivity could be associated to a cytoplasmic pattern in immunofluorescence assay on HEp-2 cells. Anti-HMGCR-positive patients had high creatine kinase (CK) levels (mean 6,630 IU/L) and only 40% of them had been exposed to statins. Multiplex ALBIA-NAM was equally as effective as monoplex anti-HMGCR and anti-SRP ALBIA.
Conclusions
Both monoplex ALBIA-HMGCR and multiplex ALBIA-NAM reliably detect and quantify anti-HMGCR autoantibodies. A positive result allows ascribing patients with a necrotizing myopathy to an autoimmune form. Anti-HMGCR autoantibodies may be found in patients who have not taken statins.
doi:10.1186/ar4468
PMCID: PMC3979083  PMID: 24484965
27.  Expression and function of visfatin (Nampt), an adipokine-enzyme involved in inflammatory pathways of osteoarthritis 
Introduction
Visfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA). With a homodimeric conformation, it exerts nicotinamide phosphoribosyltransferase (Nampt) enzymatic activity, essential for nicotinamide adenine dinucleotide biosynthesis. We examined the tissular origin and conformation of visfatin/Nampt in human OA joints and investigated the role of visfatin/Nampt in chondrocytes and osteoblasts by studying Nampt enzymatic activity.
Methods
Synovium, cartilage and subchondral bone from human OA joints were used for protein extraction or incubated for 24 hours in serum-free media (conditioned media), and synovial fluid was obtained from OA patients. Visfatin/Nampt expression in tissular extracts and conditioned media was evaluated by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Nampt activity was assessed in OA synovium by colorimetric assay. Primary cultures of murine chondrocytes and osteoblasts were stimulated with visfatin/Nampt and pretreated or not with APO866, a pharmacologic inhibitor of Nampt activity. The effect on cytokines, chemokines, growth factors and hypertrophic markers expression was examined by quantitative reverse transcriptase polymerase chain reaction and/or ELISA.
Results
In tissular explants, conditioned media and synovial fluid, visfatin/Nampt was found as a homodimer, corresponding to the enzymatically active conformation. All human OA joint tissues released visfatin/Nampt (synovium: 628 ± 106 ng/g tissue; subchondral bone: 195 ± 26 ng/g tissue; cartilage: 152 ± 46 ng/g tissue), with significantly higher level for synovium (P <0.0005). Nampt activity was identified ex vivo in synovium. In vitro, visfatin/Nampt significantly induced the expression of interleukin 6, keratinocyte chemoattractant and monocyte chemoattractant protein 1 in chondrocytes and osteoblasts. APO866 decreased the mRNA and protein levels of these pro-inflammatory cytokines in the two cell types (up to 94% and 63% inhibition, respectively). Levels of growth factors (vascular endothelial growth factor, transforming growth factor β) and hypertrophic genes were unchanged with treatment.
Conclusion
Visfatin/Nampt is released by all human OA tissues in a dimeric enzymatically active conformation and mostly by the synovium, which displays Nampt activity. The Nampt activity of visfatin is involved in chondrocyte and osteoblast activation, so targeting this enzymatic activity to disrupt joint tissue interactions may be novel in OA therapy.
doi:10.1186/ar4467
PMCID: PMC3978827  PMID: 24479481
28.  Low-density lipoprotein receptor–related protein 5 governs Wnt-mediated osteoarthritic cartilage destruction 
Introduction
Wnt ligands bind to low-density lipoprotein receptor–related protein (LRP) 5 or 6, triggering a cascade of downstream events that include β-catenin signaling. Here we explored the roles of LRP5 in interleukin 1β (IL-1β)- or Wnt-mediated osteoarthritic (OA) cartilage destruction in mice.
Methods
The expression levels of LRP5, type II collagen, and catabolic factors were determined in mouse articular chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild-type, Lrp5 total knockout (Lrp5-/-) and chondrocyte-specific knockout (Lrp5fl/fl;Col2a1-cre) mice was caused by aging, destabilization of the medial meniscus (DMM), or intra-articular injection of collagenase. The role of LRP5 was confirmed in vitro by small interfering RNA–mediated knockdown of Lrp5 or in Lrp5-/- cells treated with IL-1β or Wnt proteins.
Results
IL-1β treatment increased the expression of LRP5 (but not LRP6) via JNK and NF-κB signaling. LRP5 was upregulated in human and mouse OA cartilage, and Lrp5 deficiency in mice inhibited cartilage destruction. Treatment with IL-1β or Wnt decreased the level of Col2a1 and increased those of Mmp3 or Mmp13, whereas Lrp5 knockdown ameliorated these effects. In addition, we found that the functions of LRP5 in arthritic cartilage were subject to transcriptional activation by β-catenin. Moreover, Lrp5-/- and Lrp5fl/fl;Col2a1-cre mice exhibited decreased cartilage destruction (and related changes in gene expression) in response to experimental OA.
Conclusions
Our findings indicate that LRP5 (but not LRP6) plays an essential role in Wnt/β-catenin-signaling-mediated OA cartilage destruction in part by regulating the expression levels of type II collagen, MMP3, and MMP13.
doi:10.1186/ar4466
PMCID: PMC3978879  PMID: 24479426
29.  Patients’ preferences for osteoporosis drug treatment: a discrete-choice experiment 
Introduction
The patient’s perspective is becoming increasingly important in clinical and policy decisions. In this study, we aimed to evaluate the preferences of patients with, or at risk of, osteoporosis for medication attributes, and to establish how patients trade between these attributes.
Methods
A discrete choice experiment survey was designed and patients were asked to choose between two hypothetical unlabelled drug treatments (and an opt-out option) that vary in five attributes: efficacy in reducing the risk of fracture, type of potential common side-effects, mode and frequency of administration and out-of-pocket costs. An efficient experimental design was used to construct the treatment option choice sets and a mixed logit panel data model was used to estimate patients’ preferences and trade-offs between attributes.
Results
A total of 257 patients with, or at risk of, osteoporosis completed the experiment. As expected, patients preferred treatment with higher effectiveness and lower cost. They also preferred either an oral monthly tablet or 6-month subcutaneous injection above weekly oral tablets, 3-month subcutaneous, 3-month intravenous or yearly intravenous injections. Patients disliked being at risk of gastro-intestinal disorders more than being at risk of skin reactions and flu-like symptoms. There was significant variation in preferences across the sample for all attributes except subcutaneous injection.
Conclusions
This study revealed that osteoporotic patients preferred 6-month subcutaneous injection and oral monthly tablet, and disliked gastro-intestinal disorders. Moreover, patients were willing to pay a personal contribution or to trade treatment efficacy for better levels of other attributes. Preferences for treatment attributes varied across patients and this highlights the importance of clinical decision-making taking individual preferences into account to improve osteoporosis care.
doi:10.1186/ar4465
PMCID: PMC3979104  PMID: 24479410
30.  Good correlation between changes in objective and subjective signs of inflammation in patients with short- but not long duration of axial spondyloarthritis treated with tumor necrosis factor-blockers 
Introduction
The aim of this study was to investigate the influence of symptom duration on treatment response and on the correlation between improvements in patient reported outcomes (PRO) and objective inflammation in patients with axial spondylarthritis (SpA) treated with etanercept (ETA) or adalimumab (ADA).
Methods
Data from 112 patients with axial SpA originally enrolled in two randomized controlled clinical trials were pooled and analyzed after one year of treatment with ETA (n = 66) or ADA (n = 46). Patients with <4 years and ≥4 years of disease were compared for improvement in Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Ankylosing Spondylitis Disease Activity Score (ASDAS), C-reactive protein (CRP) and magnetic resonance imaging (MRI) score for sacroiliac joints (SIJ).
Results
Patients with <4 years of disease showed a significantly better improvement than longer diseased patients in BASDAI (3.2 (95% confidence interval (CI): 2.7 to 3.7) vs. 1.7 (1.1 to 2.2)), BASFI, BASMI and ASDAS (1.6 (1.4 to 1.8) vs. 0.9 (0.7 to 1.1)). The change in BASDAI showed a significant correlation with the change in SIJ score (Spearman’s rank correlation coefficient (rho) = 0.37, P = 0.01) and the change in CRP (rho = 0.45, P = 0.001) in patients with <4 years of disease. For long diseased patients this correlation was poor and did not achieve statistical significance (rho = 0.13, P = 0.46; rho = 0.22, P = 0.13 respectively).
Conclusion
The low correlation between change of PROs and change of objective signs of inflammation seen in axial SpA patients with longer symptom duration treated with tumor necrosis factor-blocker seems to indicate that inflammation is not the only cause of the patients’ symptoms, while inflammation seems to be the major cause in short diseased patients.
Trial registration
Clinical Trials.gov NCT00844142 (Trial 1); NCT00235105 (Trial 2)
doi:10.1186/ar4464
PMCID: PMC3978619  PMID: 24476416
31.  Influence of the ABCG2 gout risk 141 K allele on urate metabolism during a fructose challenge 
Introduction
Both genetic variation in ATP-binding cassette sub-family G member 2 (ABCG2) and intake of fructose-containing beverages are major risk factors for hyperuricemia and gout. This study aimed to test the hypothesis that the ABCG2 gout risk allele 141 K promotes the hyperuricaemic response to fructose loading.
Methods
Healthy volunteers (n = 74) provided serum and urine samples immediately before and 30, 60, 120 and 180 minutes after ingesting a 64 g fructose solution. Data were analyzed based on the presence or absence of the ABCG2 141 K gout risk allele.
Results
The 141 K risk allele was present in 23 participants (31%). Overall, serum urate (SU) concentrations during the fructose load were similar in those with and without the 141 K allele (PSNP = 0.15). However, the 141 K allele was associated with a smaller increase in SU following fructose intake (PSNP <0.0001). Those with the 141 K allele also had a smaller increase in serum glucose following the fructose load (PSNP = 0.002). Higher fractional excretion of uric acid (FEUA) at baseline and throughout the fructose load was observed in those with the 141 K risk allele (PSNP <0.0001). However, the change in FEUA in response to fructose was not different in those with and without the 141 K risk allele (PSNP = 0.39). The 141 K allele effects on serum urate and glucose were more pronounced in Polynesian participants and in those with a body mass index ≥25 kg/m2.
Conclusions
In contrast to the predicted responses for a hyperuricemia/gout risk allele, the 141 K allele is associated with smaller increases in SU and higher FEUA following a fructose load. The results suggest that ABCG2 interacts with extra-renal metabolic pathways in a complex manner to regulate SU and gout risk.
Clinical Trials Registration
The study was registered by the Australian Clinical Trials Registry (ACTRN12610001036000).
doi:10.1186/ar4463
PMCID: PMC3978630  PMID: 24476385
32.  MRI osteitis predicts cartilage damage at the wrist in RA: a three-year prospective 3T MRI study examining cartilage damage 
Introduction
Cartilage damage impacts on patient disability in rheumatoid arthritis (RA). The aims of this magnetic resonance imaging (MRI) study were to investigate cartilage damage over three years and determine predictive factors.
Methods
A total of 38 RA patients and 22 controls were enrolled at t = 0 (2009). After 3 years, clinical and MRI data were available in 28 patients and 15 controls. 3T MRI scans were scored for cartilage damage, bone erosion, synovitis and osteitis. A model was developed to predict cartilage damage from baseline parameters.
Results
Inter-reader reliability for the Auckland MRI cartilage score (AMRICS) was high for status scores; intraclass correlation coefficient (ICC), 0.90 (0.81 to 0.95) and moderate for change scores (ICC 0.58 (0.24 to 0.77)). AMRICS scores correlated with the Outcome MEasures in Rheumatoid Arthritis Clinical Trials (OMERACT) MRI joint space narrowing (jsn) and X-Ray (XR) jsn scores (r =0.96, P < 0.0001 and 0.80, P < 0.0001, respectively). AMRICS change scores were greater for RA patients than controls (P = 0.06 and P = 0.04 for the two readers). Using linear regression, baseline MRI cartilage, synovitis and osteitis scores predicted the three-year AMRICS (R2 = 0.67, 0.37 and 0.39, respectively). A multiple linear regression model predicted the three-year AMRICS (R2 = 0.78). Baseline radial osteitis predicted increased cartilage scores at the radiolunate and radioscaphoid joints, P = 0.0001 and 0.0012, respectively and synovitis at radioulnar, radiocarpal and intercarpal-carpometacarpal joints also influenced three-year cartilage scores (P-values of 0.001, 0.04 and 0.01, respectively).
Conclusions
MRI cartilage damage progression is preceded by osteitis and synovitis but is most influenced by pre-existing cartilage damage suggesting primacy of the cartilage damage pathway in certain patients.
doi:10.1186/ar4462
PMCID: PMC3978660  PMID: 24476340
33.  DMARD non-use in low-income, elderly rheumatoid arthritis patients: results of 86 structured interviews 
Introduction
Disease-modifying antirheumatic drugs (DMARDs) have become the treatment standard for patients with rheumatoid arthritis (RA). Although several general-population studies document that a large population of patients diagnosed with RA do not use DMARDs, little is known about this group. We explored the characteristics, experiences, and knowledge of a low-income, elderly RA population not currently using DMARDs, or receiving care from a rheumatologist.
Methods
We administered structured telephone interviews to participants enrolled in a large pharmacy benefits program for the elderly who had two diagnoses of RA ≥7 days apart and no DMARD prescriptions or rheumatologist visits in the prior year. The interview contained questions concerning each participant’s sociodemographic information, disease activity, DMARD experiences, and the Modified Health Assessment Questionnaire (MHAQ). We described responses and compared prior users with never users.
Results
A total of 86 people completed the interview. The mean age was 80 years and 89% were female. On average, disease duration was 20 years. Mean MHAQ score was 0.55 (SD = 0.55). Of 86 participants, 19 had previously used DMARDs, 10 of whom discontinued them because of side effects or safety concerns. Among 67 never-users, 35 (52.2%) reported that their physicians had never offered them DMARDs, 13 (19.4%) described fear of side effects, and 49 (73.1%) knew nothing about them. Prior-users reported experiencing more-severe RA symptoms than never-users.
Conclusions
We found that side effects or safety concerns were the primary cause for DMARD cessation among prior-users. Among never-users, most reported never discussing or being offered DMARDs, suggesting that an educational gap may deter patients with RA from using them.
doi:10.1186/ar4459
PMCID: PMC3978473  PMID: 24472640
34.  Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulfate-glycosaminoglycan depleted cartilage 
Introduction
Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration.
Methods
sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness.
Results
All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation.
Conclusions
Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage damage, but also resulted in pronounced subchondral sclerosis, synovial macrophage activation, and osteophyte formation.
doi:10.1186/ar4461
PMCID: PMC3978821  PMID: 24472689
35.  Notochordal cell disappearance and modes of apoptotic cell death in a rat tail static compression-induced disc degeneration model 
Introduction
The intervertebral disc has a complex structure originating developmentally from both the mesenchyme and notochord. Notochordal cells disappear during adolescence, which is also when human discs begin to show degenerative signs. During degeneration later in life, disc cells decline because of apoptosis. Although many animal models have been developed to simulate human disc degeneration, few studies have explored the long-term changes in cell population and phenotype. Our objective was to elucidate the time-dependent notochordal cell disappearance and apoptotic cell death in a rat tail static compression-induced disc degeneration model.
Methods
Twenty-four 12-week-old male Sprague–Dawley rat tails were instrumented with an Ilizarov-type device and loaded statically at 1.3 MPa for up to 56 days. Loaded and distal-unloaded discs were harvested. Changes in cell number and phenotype were assessed with histomorphology and immunofluorescence. Apoptosis involvement was determined with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and immunohistochemistry.
Results
The number of disc nucleus pulposus and annulus fibrosus cells decreased with the loading period; particularly, the decrease was notable at day 7 in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, indicating notochordal origin. Subsequently, the proportion of cells positive for TUNEL and cleaved caspase-3, markers of apoptosis induction, increased from day 7 through day 56. Although the percentage of cells immunopositive for cleaved caspase-8, a marker of apoptosis initiation through the death-receptor pathway, increased only at day 7, the percentage of cells immunopositive for cleaved caspase-9 and p53-regulated apoptosis-inducing protein 1 (p53AIP1), markers of apoptosis initiation through the p53-mediated mitochondrial pathway, increased from day 7 through day 56. The percentage of cells immunopositive for B-cell lymphoma 2 (Bcl-2) and silent mating type information regulation 2 homolog 1 (SIRT1), antiapoptotic proteins, decreased consistently with compression.
Conclusions
This rat tail model mimics notochordal cell disappearance and apoptotic cell death in human disc aging and degeneration. Sustained static compression induces transient activation of apoptosis through the death-receptor pathway and persistent activation of apoptosis through the p53-mediated mitochondrial pathway in disc cells. The increased proapoptotic and decreased antiapoptotic proteins observed at all time points signify static compression-induced disc cell death and degeneration.
doi:10.1186/ar4460
PMCID: PMC3979117  PMID: 24472667
36.  Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis 
Introduction
Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT1R and anti-ETAR Abs on initiation of inflammation and fibrosis was analyzed.
Methods
Anti-AT1R and anti-ETAR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT1R and ETAR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy.
Results
Anti-AT1R and anti-ETAR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT1R and anti-ETAR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs.
Conclusions
We conclude that angiotensin and endothelin-receptor activation via anti-AT1R and anti-ETAR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT1R and anti-ETAR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.
doi:10.1186/ar4457
PMCID: PMC3978438  PMID: 24472528
37.  Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation 
Introduction
We previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.
Methods
Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.
Results
Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.
Conclusions
These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.
doi:10.1186/ar4456
PMCID: PMC3978694  PMID: 24467809
38.  The relationship between lateral meniscus shape and joint contact parameters in the knee: a study using data from the Osteoarthritis Initiative 
Introduction
The meniscus has an important role in force transmission across the knee, but a detailed three-dimensional (3D) morphometric shape analysis of the lateral meniscus to elucidate subject-specific function has not been conducted. The aim of this study was to perform 3D morphometric analyses of the lateral meniscus in order to correlate shape variables with anthropometric parameters, thereby gaining a better understanding of the relationship between lateral meniscus shape and its load-bearing function.
Methods
The lateral meniscus (LM) was manually segmented from magnetic resonance images randomly selected from the Osteoarthritis Initiative (OAI) non-exposed control subcohort. A 3D statistical shape model (SSM) was constructed to extract the principal morphological variations (PMV) of the lateral meniscus for 50 subjects (25 male and 25 female). Correlations between the principal morphological variations and anthropometric parameters were tested. Anthropometric parameters that were selected included height, weight, body mass index (BMI), femoral condyle width and axial rotation.
Results
The first principal morphological variation (PMV) was found to correlate with height (r = 0.569), weight (r = 0.647), BMI (r = 0.376), and femoral condyle width (r = 0.622). The third PMV was found to correlate with height (r = 0.406), weight (r = 0.312), and femoral condyle width (r = 0.331). The percentage of the tibial plateau covered by the lateral meniscus decreases as anthropometric parameters relating to size of the subject increase. Furthermore, when the size of the subject increases, the posterior and anterior horns become proportionally longer and wider.
Conclusion
The correlations discovered suggest that variations in meniscal shape can be at least partially explained by the levels of loads transmitted across the knee on a regular basis. Additionally, as the size of the subject increases and body weight rises, the coverage percentage of the meniscus is reduced, suggesting that there would be an increase in the load-bearing by the cartilage. However, this reduced coverage percentage is compensated by the proportionally wider and longer meniscal horn.
doi:10.1186/ar4455
PMCID: PMC3978753  PMID: 24467794
39.  The association between ANKH promoter polymorphism and chondrocalcinosis is independent of age and osteoarthritis: results of a case–control study 
Introduction
Chondrocalcinosis (CC) most commonly results from calcium pyrophosphate crystal deposition (CPPD). The objective of this study is to examine the association between candidate single-nucleotide polymorphisms (SNPs) and radiographic CC.
Methods
SNPs in ankylosis human (ANKH), high ferritin (HFE), tissue non-specific alkaline phosphatase (TNAP), ecto-neucleotide pyrophosphatase 1 (ENPP1), and transferrin (TE) genes were genotyped in participants of the Genetics of Osteoarthritis and Lifestyle (GOAL) and Nottingham Osteoarthritis Case-Control studies. Adjusted genotype odds ratio (aORGENOTYPE), the OR for association between one additional minor allele and CC, was calculated and adjusted for age, gender, body mass index (BMI), and osteoarthritis (OA) by using binary logistic regression. Statistical significance was set at P ≤0.003 after Bonferroni correction for multiple tests.
Results
The -4bpG > A polymorphism in the 5′ untranslated region (5′ UTR) of ANKH associated with CC after Bonferroni correction. This was independent of age, gender, OA, and BMI; aORGENOTYPE (95% confidence interval, or CI) was 1.39 (1.14-1.69) (P = 0.001). rs3045 and rs875525, two other SNPs in ANKH, associated with CC; aORGENOTYPE (95% CI) values were 1.31 (1.09-1.58) (P = 0.005) and 1.18 (1.03-1.35) (P = 0.015), respectively; however, this was non-significant after Bonferroni correction.
Conclusions
This study validates the association between a functional polymorphism in the 5′ UTR of ANKH and CC and shows for the first time that this is independent of age and OA – the two key risk factors for CC. It shows that other SNPs in ANKH may also associate with CC. This supports the role of extracellular inorganic pyrophosphate in the pathogenesis of CC. The findings of this hospital-based study require replication in a community-based population.
doi:10.1186/ar4453
PMCID: PMC3978851  PMID: 24467728
40.  Expression of tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 in patients with polymyositis and dermatomyositis 
Introduction
The aim of this study was to investigate the expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) in patients with polymyositis (PM) and dermatomyositis (DM), and their relation to clinical manifestations.
Methods
Serum levels of TWEAK were detected in 98 PM/DM patients and 37 healthy controls by using the ELISA method. Total RNA isolated from fresh-frozen muscle tissue samples of 36 PM/DM patients and 10 healthy controls were used for analyzing the mRNA levels of TWEAK and Fn14 by quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunofluorescence staining of TWEAK and Fn14 was conducted on muscle biopsy specimens from 23 PM/DM patients and seven healthy controls.
Results
Serum levels of TWEAK were significantly decreased in the PM/DM patients compared to those in the healthy controls (P < 0.001), and serum TWEAK levels negatively correlated with serum CD163 levels in PM/DM patients (r = -0.49, P < 0.001). The expression of Fn14 mRNA was significantly increased in the muscle tissue of PM/DM patients than in the muscle tissue of healthy controls (P < 0.01), whereas the expression of TWEAK mRNA in PM/DM patients was not statistically different from that of the healthy controls (P > 0.05). Fn14 mRNA levels in muscle tissue positively correlated with muscle disease activity (r = 0.512, P < 0.01). Patients with oropharyngeal dysphagia had significantly higher Fn14 mRNA levels than patients without oropharyngeal dysphagia (P < 0.05). The results of immunofluorescence staining showed that 19 out of 23 PM/DM patients were TWEAK-positive, and 20 out of 23 PM/DM patients were Fn14-positive. No detectable expressions of TWEAK or Fn14 were observed in the healthy controls.
Conclusions
TWEAK-Fn14 axis may be involved in the pathogenesis of PM/DM. Further understanding of TWEAK-Fn14 function in PM/DM may help to define therapeutic targets for PM/DM.
doi:10.1186/ar4454
PMCID: PMC3978894  PMID: 24467773
41.  Safety and effectiveness of adalimumab in patients with rheumatoid arthritis over 5 years of therapy in a phase 3b and subsequent postmarketing observational study 
Introduction
Patients with active rheumatoid arthritis who had failed at least one disease-modifying anti-rheumatic drug (DMARD) were treated with adalimumab (ADA) in the ReAct study with the option to continue treatment for 5 years in ReAlise. The purpose of this study was to evaluate the long-term safety and effectiveness of ADA as prescribed from the first injection in ReAct to the last observation in ReAlise.
Methods
Patients received ADA alone or in combination with DMARDs according to usual clinical care practices. Adverse events (AEs) were tabulated by five time windows after the first ADA injection. Effectiveness measures included achievement of low disease activity (LDA), defined as Simplified Disease Activity Index (SDAI) ≤11, or remission, (REM), defined as SDAI ≤3.3.
Results
Of the 6,610 ReAct patients, 3,435 (52%) continued in ReAlise. At baseline in ReAct, mean age was 54 years, mean DAS28 was 6.0 and mean HAQ DI was 1.64. The mean treatment duration was 1,016 days, representing 18,272 patient-years (PYs) of ADA exposure. Overall incidence rates of serious AEs and serious infections were 13.8 and 2.8 events (E)/100 PYs, respectively. Serious AEs occurred most frequently in the first 6 months and deceased thereafter. Standardised mortality ratio was 0.71 (95% CI 0.57 to 0.87) and standardised incidence ratio for malignancies was 0.64 (95% CI 0.53 to 0.76). LDA was achieved by 50% and REM by 21% of patients at last observation.
Conclusions
Results of this large observational study of ADA in routine clinical practice were consistent with controlled trials, with no new safety concerns during a follow-up of more than 5 years. Effectiveness of ADA was maintained during long-term observation.
Trial registration
NCT00448383, NCT00234884
doi:10.1186/ar4452
PMCID: PMC3979145  PMID: 24460746
42.  Nonsteroidal anti-inflammatory drugs and upper and lower gastrointestinal mucosal damage 
Arthritis Research & Therapy  2013;15(Suppl 3):S3.
NSAIDs are among the most commonly used drugs worldwide and their beneficial therapeutic properties are thoroughly accepted. However, they are also associated with gastrointestinal (GI) adverse events. NSAIDs can damage the whole GI tract including a wide spectrum of lesions. About 1 to 2% of NSAID users experienced a serious GI complication during treatment. The relative risk of upper GI complications among NSAID users depends on the presence of different risk factors, including older age (>65 years), history of complicated peptic ulcer, and concomitant aspirin or anticoagulant use, in addition to the type and dose of NSAID. Some authors recently reported a decreasing trend in hospitalizations due to upper GI complications and a significant increase in those from the lower GI tract, causing the rates of these two types of GI complications to converge. NSAID-induced enteropathy has gained much attention in the last few years and an increasing number of reports have been published on this issue. Current evidence suggests that NSAIDs increase the risk of lower GI bleeding and perforation to a similar extent as that seen in the upper GI tract. Selective cyclooxygenase-2 inhibitors have the same beneficial effects as nonselective NSAIDs but with less GI toxicity in the upper GI tract and probably in the lower GI tract. Overall, mortality due to these complications has also decreased, but the in-hospital case fatality for upper and lower GI complication events has remained constant despite the new therapeutic and prevention strategies.
doi:10.1186/ar4175
PMCID: PMC3890944  PMID: 24267289
43.  The use of H2 antagonists in treating and preventing NSAID-induced mucosal damage 
Arthritis Research & Therapy  2013;15(Suppl 3):S6.
Pain affects the quality of life for millions of individuals and is a major reason for healthcare utilization. As populations age, medical personnel will need to manage more and more patients suffering from pain associated with degenerative and inflammatory musculoskeletal disorders. Nonsteroidal anti-inflammatory drugs (NSAIDs) are an effective treatment for both acute and chronic musculoskeletal pain; however, their use is associated with potentially significant gastrointestinal (GI) toxicity. Guidelines suggest various strategies to prevent problems in those at risk for NSAID-associated GI complications. In this article, we review the data supporting one such strategy - the use of histamine type-2 receptor antagonists (H2RAs) - for the prevention of GI adverse events in NSAID users. Older studies suggest that high-dose H2RAs are effective in preventing upper GI ulcers and dyspepsia. This suggestion was recently confirmed during clinical trials with a new ibuprofen/famotidine combination that reduced the risk of ulcers by 50% compared with ibuprofen alone.
doi:10.1186/ar4178
PMCID: PMC3890976  PMID: 24267478
44.  The use of proton pump inhibitors in treating and preventing NSAID-induced mucosal damage 
Arthritis Research & Therapy  2013;15(Suppl 3):S5.
NSAIDs are prescribed widely but have rare serious gastrointestinal side effects. More recently, adverse cardiovascular effects of these drugs have also been recognized, leading to the withdrawal of some agents and continuing uncertainty about the best approach for patients requiring NSAID therapy. Proton pump inhibitors (PPIs) provide potent and long-lasting inhibition of gastric acid secretion and have proven efficacy in healing NSAID-associated ulcers, including those with continued exposure to NSAIDs. PPIs have also shown efficacy in reducing the risk of ulcerations due to NSAID use compared with NSAIDs alone in randomized controlled trials (RCTs) where endoscopic ulcers are used as the primary endpoint, albeit a surrogate marker for clinical ulcers and complications. Large RCT outcome trials comparing patients exposed to NSAIDs with and without PPI co-therapy have not been performed, but adequately powered RCTs in high-risk patients demonstrate that PPI + nonselective NSAID provides similar rates of symptomatic ulcer recurrence rates as the use of a cyclooxygenase (COX)-2 selective inhibitor. A RCT in high-risk patients with previous ulcer complications supports the additive bene3 t of two risk-reducing strategies, as ulcer complication recurrence was eliminated in high-risk patients who were given a COX-2 selective agent with a PPI. Helicobacter pylori, an independent risk factor for ulcers, should be sought out and eradicated in patients at increased gastrointestinal risk, typically those with an ulcer history. Following H. pylori eradication, however, patients remain at risk and co-therapy with a PPI is recommended. NSAID medication selection should consider both the individual patients' gastrointestinal and cardiovascular risks.
doi:10.1186/ar4177
PMCID: PMC3891010  PMID: 24267413
45.  Endoscopic ulcers as a surrogate marker of NSAID-induced mucosal damage 
Arthritis Research & Therapy  2013;15(Suppl 3):S4.
The characteristic of a biomarker that makes it a useful surrogate is the ability to identify a high risk of clinically important benefits or harms occurring in the future. A number of definitions or descriptions of surrogate definition have been put forward. Most recently the Institute of Medicine of the National Academy of Sciences in the USA has put forward an evaluation scheme for biomarkers, looking at validation (assay performance), qualification (assessment of evidence), and utilisation (the context in which the surrogate is to be used). This paper examines the example of endoscopy as a surrogate marker of NSAID-induced mucosal damage using the Institute of Medicine criteria. The article finds extensive evidence that the detection of endoscopic ulcers is a valid marker. The process of qualification documents abundant evidence showing that endoscopic ulcers and serious upper gastrointestinal damage are influenced in the same direction and much the same magnitude by a variety of risk factors and interventions. Criticisms of validation and qualification for endoscopic ulcers have been examined, and dismissed. Context is the key, and in the context of serious NSAID-induced upper gastrointestinal harm, endoscopic ulcers represent a useful surrogate. Generalisability beyond this context is not considered.
doi:10.1186/ar4176
PMCID: PMC3891314  PMID: 24267380
46.  Use of NSAIDs in treating patients with arthritis 
Arthritis Research & Therapy  2013;15(Suppl 3):S2.
Patients with rheumatic diseases, including rheumatoid arthritis and osteoarthritis, almost universally describe pain and stiffness as important contributors to reduced health-related quality of life. Of the treatment options available, NSAIDs are the most widely used agents for symptomatic treatment. NSAIDs are effective anti-inflammatory and analgesic drugs by virtue of their ability to inhibit biosynthesis of prostaglandins at the level of the cyclooxygenase enzyme. However, many of the adverse effects of NSAIDs are also related to inhibition of prostaglandin production, making their use problematic in some patient populations. For the clinician, understanding the biology of prostaglandin as it relates to gastrointestinal, renal, and cardiovascular physiology and the pharmacologic properties of specific NSAIDs is key to using these drugs safely. Of particular importance is the recognition of co-morbid conditions and concomitant drugs that may increase the risk of NSAIDs in particular patients. In patients with risk factors for NSAID toxicity, using the lowest dose of a drug with a short half-life only when it is needed is likely to be the safest treatment option. For those patients whose symptoms cannot be managed with intermittent treatment, using protective strategies is essential.
doi:10.1186/ar4174
PMCID: PMC3891482  PMID: 24267197
47.  Reduced levels of CCL2 and CXCL10 in systemic lupus erythematosus patients under treatment with prednisone, mycophenolate mofetil, or hydroxychloroquine, except in a high STAT1 subset 
Introduction
Our recent data showed that signal transducers and activators of transcription 1 (STAT1), adenosine deaminase acting on RNA (ADAR), C-C motif chemokine ligand 2 (CCL2), and C-X-C motif chemokine 10 (CXCL10) were significantly elevated in a systemic lupus erythematosus (SLE) cohort compared to healthy donors. High and low STAT1 subsets were identified in SLE patient visits. The present study analyzed the correlation of common treatments used in SLE with the levels of these biomarkers.
Methods
Peripheral blood leukocytes were collected from 65 healthy donors and 103 SLE patients, of whom 60 had samples from two or more visits. Total RNA was isolated and analyzed for the expression of mRNA and microRNA using Taqman real-time polymerase chain reaction (PCR) assays. Relative expression of interferon signature genes, CCL2, and CXCL10 were determined by the ΔΔCT method. Results were correlated with therapy using prednisone, mycophenolate mofetil, and hydroxychloroquine and analyzed by Wilcoxon/Kruskal-Wallis test and Fisher’s exact test.
Results
CCL2 and CXCL10 were significantly higher in untreated patients compared to treated patients, however, in high STAT1 patient visits there is no significant difference between treated and untreated patients’ visits. When comparing linear regression fits of interferon (IFN) score with CCL2 and CXCL10, untreated patients and high STAT1 patients displayed significantly higher slopes compared to treated patients. There was no significant difference between the slopes of high STAT1 and untreated patients indicating that CCL2 and CXCL10 were correlated with type-I IFN in high STAT1 patients similar to that in untreated patients. CCL2 and CXCL10 levels in the high STAT1 subset remained high in treated patient visits compared to those of the low STAT1 subset.
Conclusions
Among the biomarkers analyzed, only CCL2 and CXCL10 showed significantly reduced levels in treated compared to untreated SLE patients. STAT1, CCL2, and CXCL10 are potentially useful indicators of therapeutic action in SLE patients. Further work is needed to determine whether high STAT1 levels convey resistance to therapies commonly used to treat SLE and whether STAT1 inhibitors may have therapeutic implication for these patients.
doi:10.1186/ar4451
PMCID: PMC3978465  PMID: 24460726
48.  Elevated signal transducers and activators of transcription 1 correlates with increased C-C motif chemokine ligand 2 and C-X-C motif chemokine 10 levels in peripheral blood of patients with systemic lupus erythematosus 
Introduction
The present study examines the levels of recently reported biomarkers, adenosine deaminase acting on RNA (ADAR), C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine 10 (CXCL10), signal transducers and activators of transcription 1 (STAT1), and miR-146a in systemic lupus erythematosus (SLE) patients over multiple visits.
Methods
Peripheral blood leukocytes were collected from 65 healthy donors and 103 SLE patients, 60 of whom had samples from 2 or more visits. Total RNA was isolated and analyzed for the expression of mRNA and microRNA using Taqman real time PCR assays. Relative expression of I-IFN signature genes, chemokines, and miR-146a were determined by the ΔΔCT method. Results were correlated with clinical data and analyzed by Wilcoxon/Kruskal-Wallis test and Fisher’s exact test.
Results
Levels of ADAR, CCL2, CXCL10, and STAT1 in SLE were significantly elevated compared with the healthy controls (P <0.0001). ADAR, CCL2, and CXCL10 showed significant correlation with IFN score in both healthy donors (P <0.0033) and SLE patients (P <0.0001). In SLE patients, miR-146a level was not significantly different from healthy controls nor correlated to the IFN score. Two STAT1 populations were identified: a low STAT1 and a high STAT1 group. High STAT1 patient visits displayed higher (P ≤0.0020) levels of CCL2 and CXCL10 than the low STAT1 patient visits. STAT1 levels correlated with IFN score in low STAT1 group but not in high STAT1 group. More importantly, high STAT1 levels appeared as an enhancer of CCL2 and CXCL10 as indicated by the significantly stronger correlation of CCL2 and CXCL10 with IFN score in high STAT1 patient visits relative to low STAT1 patient visits.
Conclusion
Our data indicate a novel role for STAT1 in the pathogenesis of SLE as an expression enhancer of CCL2 and CXCL10 in SLE patients with high levels of STAT1. Future study is needed to examine the exact role of STAT1 in the etiology of SLE.
doi:10.1186/ar4448
PMCID: PMC3978614  PMID: 24451065
49.  High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain 
Introduction
Excessive mechanical loading of intervertebral discs (IVDs) is thought to alter matrix properties and influence disc cell metabolism, contributing to degenerative disc disease and development of discogenic pain. However, little is known about how mechanical strain induces these changes. This study investigated the cellular and molecular changes as well as which inflammatory receptors and cytokines were upregulated in human intervertebral disc cells exposed to high mechanical strain (HMS) at low frequency. The impact of these metabolic changes on neuronal differentiation was also explored to determine a role in the development of disc degeneration and discogenic pain.
Methods
Isolated human annulus fibrosus (AF) and nucleus pulposus (NP) cells were exposed to HMS (20% cyclical stretch at 0.001 Hz) on high-extension silicone rubber dishes coupled to a mechanical stretching apparatus and compared to static control cultures. Gene expression of Toll-like receptors (TLRs), neuronal growth factor (NGF) and tumour necrosis factor α (TNFα) was assessed. Collected conditioned media were analysed for cytokine content and applied to rat pheocromocytoma PC12 cells for neuronal differentiation assessment.
Results
HMS caused upregulation of TLR2, TLR4, NGF and TNFα gene expression in IVD cells. Medium from HMS cultures contained elevated levels of growth-related oncogene, interleukin 6 (IL-6), IL-8, IL-15, monocyte chemoattractant protein 1 (MCP-1), MCP-3, monokine induced by γ interferon, transforming growth factor β1, TNFα and NGF. Exposure of PC12 cells to HMS-conditioned media resulted in both increased neurite sprouting and cell death.
Conclusions
HMS culture of IVD cells in vitro drives cytokine and inflammatory responses associated with degenerative disc disease and low-back pain. This study provides evidence for a direct link between cellular strain, secretory factors, neoinnervation and potential degeneration and discogenic pain in vivo.
doi:10.1186/ar4449
PMCID: PMC3979109  PMID: 24457003
50.  Adiposity and hand osteoarthritis: the Netherlands Epidemiology of Obesity study 
Introduction
Obesity, usually characterized by the body mass index (BMI), is a risk factor for hand osteoarthritis (OA). We investigated whether adipose tissue and abdominal fat distribution are associated with hand OA.
Methods
The Netherlands Epidemiology of Obesity (NEO) study is a population-based cohort aged 45 to 65 years, including 5315 participants (53% women, median BMI 29.9 kg/m2). Fat percentage and fat mass (FM) (kg) were estimated using bioelectrical impedance analysis. The waist-to-hip ratio (WHR) was calculated. In 1721 participants, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) (cm2) were assessed using abdominal MR imaging. Hand OA was defined according to the ACR criteria.
Odds ratios (OR) with 95% confidence intervals (CI) were calculated for the association of fat percentage, FM, WHR, VAT and SAT with hand OA using logistic regression analyses per standard deviation, stratified by sex and adjusted for age.
Results
Hand OA was present in 8% of men and 20% of women. Fat percentage was associated with hand OA in men (OR 1.34 (95% CI 1.11 to 1.61)) and women (OR 1.26 (1.05 to 1.51)), as was FM. WHR was associated with hand OA in men (OR 1.45 (1.13 to 1.85)), and to a lesser extent in women (OR 1.17 (1.00 to 1.36)). Subgroup analysis revealed that VAT was associated with hand OA in men (OR1.33 (1.01 to 1.75)). This association increased after additional adjustment for FM (OR 1.51 (1.13 to 2.03)).
Conclusions
Fat percentage, FM and WHR were associated with hand OA. VAT was associated with hand OA in men, suggesting involvement of visceral fat in hand OA.
doi:10.1186/ar4447
PMCID: PMC3978723  PMID: 24447395

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