Recent animal studies suggest that activation of Wnt/β-catenin signaling in articular chondrocytes might be a driving factor in the pathogenesis of osteoarthritis (OA) by stimulating amongst others the expression of matrix metalloproteinases (MMPs). This study aimed to investigate the role of Wnt/β-catenin signaling in IL-1β-induced MMP expression in human chondrocytes.

Primary cultures of human, mouse and bovine articular chondrocytes as well as human mesenchymal stem cells (hMSCs) and mouse embryonic fibroblasts (MEFs) were used. Multiple strategies for activation and inhibition of signaling pathways were used. Reporter assays and co-immunoprecipitations were used to study the interaction between β-catenin and NF-κB.

In contrast to animal chondrocytes, in human chondrocytes Wnt/β-catenin is a potent inhibitor of MMP1, -3 and -13 expression and generic MMP activity both in basal conditions and after IL-1β stimulation. This effect is independent of TCF/LEF transcription factors but is due to an inhibitory protein-protein interaction between β-catenin and NF-κB. Furthermore we show that IL-1β indirectly activates β-catenin signaling by inducing canonical Wnt7B expression and by inhibiting the expression of canonical Wnt antagonists.

Our data reveal an unexpected anti-catabolic role of Wnt/β-catenin signaling in human chondrocytes by counteracting NF-κB-mediated MMP expression induced by IL-1β in a negative feedback loop.

The spondyloarthropathies, including ankylosing spondylitis, psoriatic arthritis, reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of large peripheral and axial joints, eyes, skin, ileum and colon. Genetic studies reveal common candidate genes for ankylosing spondylitis, psoriatic arthritis and Crohn's disease, including IL23R, IL12B, STAT3 and Card9, associated with IL-23 signaling downstream of the dectin-1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin-1-mediated fungal infection. We determined whether SKG mice injected with 1,3-D β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process.

SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly and organ pathology was determined. Anti-IL-23 mAb were injected into curdlan-treated SKG mice. CD4⁺ T cells were transferred from curdlan-treated to SCID mice and sera were analyzed for autoantibodies.

After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle and sacro-iliac arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell and IL-23-dependent and could be transferred to SCID recipients with CD4⁺ T cells. Spondyloarthritis was associated with collagen and proteoglycan-specific autoantibodies. Conclusion: The SKG ZAP-70^W163C mutation predisposes BALB/c mice to spondyloarthropathy after systemic β-glucan or mannan exposure resulting from innate and adaptiveautoimmunity.

Fibrosis in human diseases and animal models is associated with aberrant Wnt/β-catenin pathway activation. The regulation, activity, mechanism of action and significance of Wnt/β-catenin signaling in the context of systemic sclerosis (SSc) has not been characterized.

Expression of Wnt signaling pathway components in SSc skin biopsies was analyzed. The regulation of profibrotic responses by canonical Wnt/ß-catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied by proliferation, migration and gel contraction assays. The fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated.

Analysis of published genome-wide expression datasets revealed elevated expression of the Wnt receptor Fzd2 and the Wnt target Lef1, and decreased expression of Wnt antagonists Dkk2 and Wif1 in skin biopsies from subsets of dcSSc patients. Immunohistochemistry showed increased nuclear β-catenin expression in these biopsies. In vitro, Wnt3a induced ß-catenin activation, stimulated fibroblast proliferation, migration, gel contraction and myofibroblast differentiation, and profibrotic gene expression. Genetic and pharmacological approaches were used to demonstrate that these profibrotic responses involved autocrine TGF-β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes Wnt3a repressed adipogenesis and promoted myofibroblast differentiation.

Canonical Wnt signaling was hyperactivated in SSc skin biopsies, and in explanted mesenchymal cells Wnt3a stimulated fibrogenic responses while suppressing adipogenesis. Together, these results indicate that Wnts have potent profibrotic effects and canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.

To investigate the relationship of urinary biomarkers (UBM) and established measures of renal function (EMRF) to the histological findings with lupus nephritis (LN); and to test whether certain combinations of the above mentioned laboratory measures are diagnostic of specific histological features of LN.

Urine samples of 76 patients were collected within 2 months of a kidney biopsy and assayed for the UBM: lipocalin-like prostaglandin-D synthetase (L-PGDS), α1-acid-glycoprotein (AAG), transferrin (TF), ceruloplasmin (CP), neutrophil-gelatinase associated lipocalin (NGAL), and monocyte chemotactic factor 1 (MCP1). Using non-parametric analyses, UBM and EMRF levels were compared to histological features seen with LN: mesangial expansion, capillary proliferation, crescent formation, necrosis, wire loops, fibrosis, tubular atrophy, and epimembranous deposits. The area under the receiver operating characteristic (AUC) curve was calculated to predict LN activity, chronicity or membranous LN.

There was a differential increase of the UBM that formed a pattern reflective of specific histological features seen with active LN. The combination of MCP1, AAG, CP plus protein:creatinine ratio were excellent in predicting LN activity (AUC=0.85). NGAL together with creatinine clearance plus MCP1 was an excellent (AUC=0.83) and MCP1, AAG, creatinine clearance plus C4 (AUC=0.75) a good diagnostic test of LN chronicity and membranous LN, respectively.

Select UBM are associated with specific tissue changes observed with LN activity and chronicity. Especially in combination with select EMRF, UBM are well-suited to non-invasively quantify LN activity, LN chronicity, and the presence of membranous LN.

To compare associations of varus thrust and varus static alignment with pain in those with knee osteoarthritis (OA).

This was a cross-sectional study of participants from a randomized controlled trial of vitamin D for knee OA. Participants were video recorded walking and scored for presence of varus thrust. Standard PA knee X-rays were measured for static alignment. Pain questions from the Western Ontario McMasters Osteoarthritis (WOMAC) questionnaire assessed symptoms.

We calculated means for total WOMAC pain by varus thrust and varus alignment (i.e. corrected anatomic alignment < 178°). We performed ordinal logistic regressions; outcomes: individual WOMAC pain questions; predictors: varus thrust and varus alignment.

There were 82 participants, mean age 65.1 (±8.5), mean body mass index 30.2 (±5.4), and 60% female. Total WOMAC pain was 6.3 versus 3.9, p = 0.007 in those with versus without definite varus thrust. For varus alignment, total WOMAC pain was 5.2 versus 4.2, p = 0.30.

Odds ratios for pain with walking and standing were 5.5 (95%CI 2.0 – 15.1) and 6.0 (95%CI 2.2 – 16.2) in those with versus without definite varus thrust. There were no significant associations between varus alignment and individual WOMAC pain questions. Sensitivity analyses suggested a more stringent definition of varus might have been associated with walking and standing pain.

In those with knee OA, varus thrust and possibly varus static alignment, were associated with pain, specifically during weight-bearing activities. Treatment of varus thrust (e.g. via bracing or gait modification) may lead to improvement of symptoms.

Define the role indoleamine-2,3-dioxygenase (IDO) plays in driving pathogenic B cells responses leading to arthritis and determine if inhibitors of the IDO pathway can be used in conjunction with B cell depletion therapy to prevent the re-emergence of autoantibodies and arthritis following reconstitution of the B cell repertoire.

Immunoglobulin transgenic mice were treated with the IDO inhibitor 1-methyl-tryptophan (1MT) and followed for the extent of autoreactive B cell activation. Arthritic mice (K/BxN) were treated with B cell depletion therapy alone or in combination with 1MT. Mice were followed for the presence of autoantibody secreting cells, inflammatory cytokines, and joint inflammation.

1MT did not affect the initial activation or survival of autoreactive B cells, but did inhibit their ability to differentiate into autoantibody secreting cells. Treatment with anti-CD20 depleted the B cell repertoire and attenuated arthritis symptoms; however, arthritis symptoms rapidly returned as B cells repopulated the repertoire. Administration of 1MT prior to B cell repopulation prevented the production of autoantibodies, inflammatory cytokines, and flare in arthritis symptoms.

IDO activity is essential for the differentiation of autoreactive B cells into antibody secreting cells, but is not necessary for their initial stages of activation. Addition of 1MT to B cell depletion therapy prevents the differentiation of autoantibody secreting cells and recurrence of autoimmune arthritis following reconstitution of the B cell repertoire. These data suggest that IDO inhibitors could be used in conjunction with B cell depletion as an effective co-therapeutic strategy in the treatment of rheumatoid arthritis.

Blocking IL-1 with anakinra in the autoinflammatory syndrome NOMID reduces systemic and organ-specific inflammation. However, the impact of long term treatment is not established.

To evaluate the long-term effect of anakinra on clinical and laboratory outcomes, and safety, in NOMID.

Cohort study of 26 NOMID patients, ages 0.80 to 42.17 years followed at the NIH and treated with anakinra 1 to 5 mg/kg/day for at least 36 months. Disease activity was measured by daily diaries, questionnaires, and C-reactive protein (CRP). Central nervous system (CNS) inflammation, hearing, vision and safety were assessed.

Sustained improvements in diary scores, parent/patient and physician global scores, parent/patient pain scores, and inflammatory markers were observed (all p< 0.0001 at 36 and 60 months). At 36 and 60 months, CNS inflammation was suppressed with decreased cerebrospinal fluid (CSF) white blood cell (WBC) counts (p = 0.0026 and 0.0076, respectively), albumin levels, and opening pressures (p = 0.0012 and 0.0001, respectively). Most patients showed stable or improved hearing. Cochlear enhancement on magnetic resonance imaging (MRI) correlated with continued hearing loss. Visual acuity and peripheral vision were stable. Low optic nerve size correlated with poor visual field. Bony lesions progressed. Adverse events other than viral infections were rare and all patients remained on medication.

Anakinra provides sustained efficacy in the treatment of NOMID for up to five years, with the requirement of dose escalation. Damage progression in the CNS, ear, and eye – but not bone – is preventable. Anakinra is overall well tolerated.

WISP3/CCN6 is mutated in progressive pseudorheumatoid dysplasia and may have effects on cartilage homeostasis. In order to uncover further roles for WISP3/CCN6 its expression was explored in osteoarthritic cartilage. Effects of WISP3/CCN6 on cartilage-relevant metalloproteinase expression were investigated in immortalised (C-28/I2) and primary chondrocytes.

Cartilage steady state levels of WISP3/CCN6 mRNA and protein production were determined by quantitative RT-PCR and immunohistochemistry respectively. WISP3/CCN6 was over-expressed in C-28/I2 cells and resultant stable clones analysed by real time RT-PCR for metalloproteinase expression and signalling pathways involved explored with pharmacological inhibition. Effects of WISP3/CCN6 on metalloproteinase expression in primary chondrocytes were investigated by an siRNA approach.

WISP3/CCN6 was highly expressed in osteoarthritic cartilage compared to undamaged cartilage at RNA and protein levels. WISP3/CCN6 over-expression in C-28/I2 cells resulted in unexpected dual regulation of metalloproteinases: the expression of the potent aggrecanase, ADAMTS5, was down-regulated 9-fold, whilst MMP10 was up-regulated 14-fold, responses accentuated by suspension culture. MMP10 up-regulation was dependent on several MAP kinases but WISP3/CCN6-mediated ADAMTS5 repression was independent of these pathways and partially relieved by activation of β-catenin signalling. WISP3/CCN6 also suppressed ADAMTS5 expression in C-28/I2 cells treated with cytokines. In cytokine-treated primary chondrocytes gene silencing of WISP3/CCN6 resulted in enhanced ADAMTS5 expression whilst MMP10 expression was suppressed.

WISP3/CCN6 was highly expressed in end-stage osteoarthritic cartilage suggesting a role for this growth factor in cartilage homeostasis. WISP3/CCN6 repression of ADAMTS5 expression and regulation of MMP10 expression suggests complex and context-dependent roles for WISP3/CCN6 in cartilage biology.

A major impediment toward the development of novel treatment strategies for fibromyalgia (FM) is the lack of an objective marker which tracks with spontaneous clinical pain report. Resting state intrinsic brain connectivity in FM has demonstrated increased insular connectivity to the default mode network (DMN), a network whose activity is increased during rest. Moreover increased insular connectivity to the DMN was associated with increased spontaneous pain levels. However as these analyses were cross-sectional in nature, they provided no insight to dynamic changes in connectivity and their relationship with variation in clinical pain report.

17 FM patients underwent resting state fMRI at baseline and following 4 weeks of a non-pharmacological intervention to diminish pain. Intrinsic DMN connectivity was evaluated using probabilistic independent component analysis. A paired analysis evaluated longitudinal changes in intrinsic DMN connectivity and a multiple linear regression investigated correlations between longitudinal changes in clinical pain and changes in intrinsic DMN connectivity. Changes in clinical pain were assessed with the Short Form of the McGill Pain Questionnaire (SF-MPQ).

Clinical pain was reduced following therapy (SF-MPQ sensory scale: p<0.02). Intrinsic DMN connectivity to the insula was reduced, and this reduction was correlated with reductions in pain (corrected p<0.05).

Our findings suggest that intrinsic brain connectivity can be used as a candidate objective marker that tracks intra-subject with changes in spontaneous chronic pain in FM. We propose that intrinsic connectivity measures could potentially be used either in research or clinical settings as a complementary, more objective outcome. © 2012 American College of Rheumatology.

Tissue glucocorticoid levels are regulated by the glucocorticoid activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). This enzyme is expressed in cells and tissues arising from mesenchymal stromal cells. Proinflammatory cytokines dramatically increase expression of 11β-HSD1 in stromal cells, an effect implicated in inflammatory arthritis, osteoporosis, obesity and myopathy. Additionally, glucocorticoids act synergistically with pro-inflammatory cytokines to further increase enzyme expression. The mechanisms underlying this regulation are unknown.

5′ RACE, gene reporter analyses, chemical inhibitors and genetic disruption of intracellular signalling pathways in mouse embryonic fibroblasts (MEFs) were used to define the molecular mechanisms underlying the regulation of 11β-HSD1 expression.

5′ RACE, gene reporter and chemical inhibitor studies demonstrated that the increase in 11β-HSD1 expression with TNFα/IL-1β?was via the proximal HSD11B1 gene promoter and depended on NF-κB signalling. These findings were confirmed using MEFs with targeted disruption of NF-κB signalling where RelA(p65) deletion prevented TNFα/IL-1β induction of 11β-HSD1? Glucocorticoids were unable to prevent TNFα-induced NF-κB nuclear translocation. The synergistic enhancement of TNFα-induced 11β-HSD1 expression with glucocorticoids was reproduced by specific inhibitors of p38MAPK. Inhibitor and gene deletion studies indicated that effects of glucocorticoids on p38MAPK activity were primarily through induction of dual specificity MAPK phosphatase-1 (DUSP1) expression.

The mechanism by which stromal cell expression of 11β-HSD1 is regulated is novel and distinct from that in other tissues. These findings open up new opportunities to inhibit or stimulate local glucocorticoid levels in cells of mesenchymal stromal lineage during inflammation. © 2012 American College of Rheumatology.

This study addressed the association of overweight and obesity to the presence, extent, and severity of lumbar disc degeneration on MRI in adults.

A population-based cross-sectional study of 2,599 Southern Chinese volunteers. Radiographic and clinical assessment, including weight and height, was conducted. Sagittal T2-weighted MRIs of the lumbar spine were obtained. The presence, extent, and severity of disc degeneration as well as additional radiographic and clinical findings were assessed. Asian-modified BMI (kg/m2) categories were utilized.

There were 1,040 males and 1,559 females (mean age= 41.9 years). Disc degeneration was noted in 1,890 (72.7%) subjects. BMI was significantly higher in subjects with disc degeneration (mean=23.3 kg/m²) compared to subjects without degeneration (mean=21.7 kg/m²) (p<0.001). A significant increase in the number of degenerated levels (p<0.001), global severity of disc degeneration (p<0.001), and end-stage disc degeneration with disc space narrowing (p<0.001) was noted with elevated BMI, in particular in overweight and obese individuals. In the adjusted multivariate logistic regression model, there was a positive linear trend (r²=0.99) between BMI categories and the overall presence of disc degeneration (normal weight (Ref); overweight, OR=1.30, 95% CI=1.03-1.62; obese, OR=1.79, 95% CI= 1.17-2.74). End-stage disc degeneration with disc space narrowing was significantly more pronounced in obese individuals (normal weight (Ref); adjusted OR=1.72, 95% CI=1.23 – 2.41).

In one of the largest studies to systematically assess lumbar disc degeneration on MRI, our study noted a significant association between the presence, increased extent, and global severity of disc degeneration in overweight and obese adults.

Emerging evidence suggests that genetic components contribute significantly to cartilage degeneration in osteoarthritis pathophysiology but little evidence is available on genetics of cartilage regeneration. Therefore, we investigated cartilage regeneration in genetic murine models using common inbred strains and a set of recombinant inbred lines generated from LG/J (healer of ear-wounds) and SM/J (non-healer) inbred strains.

An acute full-thickness cartilage injury was introduced through microsurgery in the trochlear groove of 8-weeks old mice (N=265). Knee joints were sagittally sectioned and stained with toluidine blue to evaluate regeneration. For ear-wound phenotype, a bilateral 2-mm through-and-through puncture was made (N=229) at 6-weeks and healing outcomes measured after 30-days. Broad-sense heritability and genetic correlations were calculated for both phenotypes.

Time-course studies from recombinant inbred lines show no significant regeneration until 16-weeks post-surgery; at that time, the strains can be segregated into three categories: good, intermediate and poor healers. Heritability (H²) showed that both cartilage regeneration (H²=26%; p=0.006) and ear-wound closure (H²=53%; p<0.00001) are significantly heritable. The genetic correlations between the two healing phenotypes for common inbred strains (r=0.92) and recombinant inbred lines (r=0.86) were found to be extremely high.

We report that i) articular cartilage regeneration is heritable, ii) the differences between the lines being due to genetic differences and iii) a strong genetic correlation between the two phenotypes exists indicating that they plausibly share a common genetic basis. We, therefore, surmise that LG/J by SM/J intercross can be used to dissect the genetic basis of variation in cartilage regeneration.

To examine the effect of hypoxia on Notch-1 signaling pathway components and angiogenesis in inflammatory arthritis.

Expression and regulation of Notch-1, its ligand DLL-4 and downstream signaling components (Hrt-1, Hrt-2) and HIF1α under normoxic and hypoxic conditions (1-3%) were assessed by immunohistology, dual-immunofluorescent staining (Notch-1/Factor VIII), Western blotting and Real-time PCR. In vivo synovial tissue oxygen levels (tpO₂) were measured under direct visualisation at arthroscopy. Microvascular endothelial cell (dHMVEC) activation under hypoxic conditions in the presence of Notch-1 siRNA, γ-secretase inhibitor (DAPT) or dimethyloxalylglycine (DMOG) was assessed by matrigel tube formation assay, migration assay, invasion assay and MMP2/9 zymography.

Notch-1, its ligand DLL-4 and Hrt-1 expression were demonstrated in synovial tissue, with strongest expression localised to perivascular/vascular regions. Localization of Notch-1 to synovial endothelium was confirmed by dual-immunofluorescent staining. Notch-1 intracellular domain (IC) expression was significantly higher in synovial tissue from patients with tpO₂ <20mmHg (<3% O₂) compared to those with tpO₂ >20mmHg (>3% O₂). Exposure of dHMVEC to 3% hypoxia induced HIF1α and Notch-1 IC protein expression and DLL-4, Hrt-1 and Hrt-2 mRNA expression. DMOG directly induced Notch-1 IC expression, while siRNA Notch-1 inhibited hypoxia-induced HIF1α expression, suggesting that Notch-1/HIF1α signaling is bi-directional. Finally 3% hypoxia-induced angiogenesis, EC migration, EC invasion, and pro-MMP-2 and-9 activities were inhibited by Notch-1 siRNA and/or the γ-secretase inhibitor DAPT.

Notch-1 is expressed in synovial tissue and increased Notch-1 IC expression is associated with low in vivo tpO₂. Furthermore Notch-1/HIF1α interactions mediate hypoxia-induced angiogenesis and invasion in inflammatory arthritis.

Calcineurin-binding protein 1 (Cabin1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLSs), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of Cabin1 in FLS apoptosis is not clear. The aim of this study was to define the regulatory role of Cabin1 in FLSs of mice with collagen-induced arthritis (CIA).

Transgenic mice overexpressing human Cabin1 in joint tissues, under the control of a type II collagen promoter, were generated. hCabin1 expression in joints and FLSs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLSs was determined by enzyme-linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were histologically examined after H&E and TRAP staining.

hCabin1-transgenic CIA mice had less severe arthritis than wild-type CIA mice, based on hind paw thickness and histology. This was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by significantly more TUNEL-positive cells in synovial tissues. The expression of inflammatory cytokines and MMPs was reduced, and the transgenic CIA mice exhibited decreased Akt activation and increased expression of p53, caspase-3, caspase-9, and Bax.

hCabin1 plays a critical role in promoting apoptosis of FLSs and in attenuating inflammation and the destruction of cartilage and bone in RA. These findings help elucidate the pathogenic mechanisms of RA and suggest that Cabin1 is a potential target for RA treatment.

This study addresses the role of the NR4A orphan nuclear receptors in synoviocyte transformation, hyperplasia, and regulation of MMPs and TIMPs in models of inflammatory arthritis.

NR4A mRNA levels were measured in synovial tissues and primary synoviocytes by RT-qPCR. NR4A2 was stably over-expressed in normal synoviocytes and cell proliferation, survival, anchorage-independent growth, migration, and invasion were monitored in vitro. MMP and TIMP expression levels were analyzed by RT-qPCR and MMP-13 promoter activity was measured by reporter assays. Stable depletion of endogenous NR4A levels was achieved by lentiviral transduction of NR4A shRNA and effects on proliferation, migration, and MMP-13 expression were analyzed.

NR4A2 is expressed at elevated levels in normal, OA, and RA synovial tissues and in primary RA synoviocytes. TNF-α rapidly and selectively induces expression of NR4A2 in synoviocytes. Ectopic expression of NR4A2 in normal synoviocytes significantly increases proliferation and survival, promotes anchorage-independent growth, and induces migration and invasion. MMP-13 gene expression is synergistically induced by NR4A2 and TNF-α, while expression of TIMP-2 is antagonized. NR4A2 directly transactivates the proximal MMP-13 promoter and a point mutation in the DNA-binding domain of NR4A2 abolishes transcriptional activation. Depletion of endogenous NR4A receptors with shRNA reduces synoviocyte proliferation, migration, and MMP-13 expression.

The orphan nuclear receptor NR4A2 is a downstream mediator of TNF-α signaling in synovial tissue. NR4A2 transcriptional activity contributes to the hyperplastic and invasive phenotype of synoviocytes that leads to cartilage destruction, suggesting that this receptor may show promise as a therapeutic target in inflammatory arthritis.

Rheumatoid arthritis is characterized by persistent synovial inflammation and progressive joint destruction, mediated by innate and adaptive immune responses. Cytokine blockade successfully treats some patient subsets, however ˜50% do not respond to this approach. Targeting pathogenic T lymphocytes is emerging as an effective alternative/complementary therapeutic strategy. However, the factors that control T cell activation in joint disease are not well understood. Tenascin-C is an arthritogenic, extracellular matrix glycoprotein that is not expressed in healthy synovium but is elevated in the rheumatoid joint where high levels are produced by myeloid cells. Amongst these cells, tenascin-C expression is most highly induced in activated dendritic cells, prompting us to examine its role in this cell type.

We systematically compared the phenotype of dendritic cells isolated from wild type mice or mice with a targeted deletion in tenascin-C by assessing cell maturation, cytokine synthesis and T cell polarization.

Dendritic cells derived from tenascin-C null mice exhibit no defects in maturation; induction of MHC II and the co-stimulatory molecules CD40 and CD86 is unimpaired. However, dendritic cells that do not express tenascin-C produce lower levels of inflammatory cytokines than cells from wild type mice and exhibit specific defects in Th17 cell polarization. Moreover, tenascin-C null mice display ablated IL-17 levels in the joint during experimental arthritis.

These data demonstrate that tenascin-C is important in dendritic cell-mediated polarization of Th17 lymphocytes during inflammation and suggest a key role for this endogenous danger signal in driving adaptive immunity in erosive joint disease.

Which serologic and clinical findings predict adverse pregnancy outcome (APO) in patients with antiphospholipid antibody (aPL) is controversial.

PROMISSE is a multicenter, prospective observational study of risk factors for APO in patients with aPL (lupus anticoagulant [LAC], anticardiolipin antibody [aCL] and/or antibody to β₂ glycoprotein I [anti-β2-GP-I]). We tested the hypothesis that a pattern of clinical and serological variables can identify women at highest risk for APO.

Between 2003 and 2011 we enrolled 144 pregnant patients, of whom 28 had APO. Thirty-nine percent of patients with LAC had APO, compared to 3% who did not have LAC (p < 0.0001). Only 8% of women with IgG aCL ≥40 u/mL but not LAC suffered APO, compared to 43% of those with LAC (p = 0.002). IgM aCL or IgG or IgM anti-β2-GP-I did not predict APO. In bivariate analysis, APO occurred in 52% of patients with and 13% of patients without prior thrombosis (p = 0.00005), and in 23% with SLE compared to 17% without SLE (not significant); SLE was a predictor in multivariate analysis. Prior pregnancy loss did not predict APO, nor did maternal race. Simultaneous aCL, anti-β2-GP-I, and LAC did not predict APO better than did LAC alone.

LAC is the primary predictor of APO after 12 weeks gestation in aPL-associated pregnancies. ACL and anti-β2-GP-I, if LAC is not also present, do not predict APO.

Bone formation and destruction are usually tightly linked, however in disorders such as rheumatoid arthritis, periodontal disease and osteoporosis, elevated osteoclast activity leads to bone destruction. Osteoclast formation and activation are controlled by many signalling pathways including p38 MAPK. DUSP1 is a factor involved in the negative regulation of p38 MAPK. This article examines the effect of DUSP1 deficiency on bone destruction.

Penetrance, onset and severity of collagen-induced arthritis were recorded in Dusp1^+/+ and Dusp1^-/- mice. Bone destruction was assessed by histological and microCT inspection of joints. In vitro formation and activation of osteoclasts from Dusp1^+/+ and Dusp1^-/- precursors were assessed in the absence or presence of TNF.

Formation and activation of osteoclasts in vitro in the presence of TNF were enhanced by Dusp1 gene disruption. Dusp1^-/- mice exhibited higher penetrance, earlier onset and increased severity of experimental arthritis, accompanied by greater numbers of osteoclasts in inflamed joints and more extensive loss of bone. A Dusp1^-/- colony of mixed genetic background also demonstrated striking spontaneous osteolytic destruction of distal phalanges

DUSP1 is a critical regulator of osteoclast activity, and limits bone destruction in an experimental model of rheumatoid arthritis. Defects in the expression or activity of DUSP1 in humans may correlate with propensity to develop osteolytic lesions in arthritis.

To assess the association of industry funding with the characteristics, outcome, and reported quality of randomized controlled trials (RCTs) of drug therapy for rheumatoid arthritis (RA).

MEDLINE and Cochrane Central Register of Controlled Trials databases were searched to identify original RA drug therapy RCTs published in 2002-3 & 2006-7. Two reviewers independently assessed each RCT for the funding source, characteristics, outcome [positive (statistically significant result favoring experimental drug for the primary outcome) or not positive], and reporting of methodological measures whose inadequate performance may bias treatment effect assessment. RCTs registered at ClinicalTrials.gov and completed in the study years were assessed for publication bias.

103 eligible RCTs were identified with following funding sources: 58 (56.3%) industry; 19 (18.4%) non-profit; 6 (5.8%) mixed; and 20 (19.4%) unspecified. Industry funded RCTs had significantly more study centers and subjects; while non-profit funded RCTs had longer duration, and were more likely to study different treatment strategies. Outcome could be assessed for 86 (83.5%) RCTs. Funding source was not associated with higher likelihood of positive outcomes favoring the sponsored experimental drug [industry (75.5%), non-profit (68.8%), mixed (40%), and unspecified (81.2%); p = 0.37]. Industry funded RCTs had trend towards higher likelihood of non-publication (38.6% versus 16.7%, p = 0.093). Industry-funded RCTs reported more frequent performance of double-blinding, adequate participant flow description, and intention-to-treat analysis.

Industry funding was not associated with higher likelihood of positive outcomes of published drug therapy RCTs for RA, and reported better on some key RCT quality measures.

The aim of this study was to identify new biomarkers of osteoarthritis by proteomic analysis and to develop specific immunoassays to detect and quantify them.

Proteomic analysis was performed in urine from ten women (76.0 ± 5.0 years) undergoing knee replacement surgery due to severe osteoarthritis and five healthy women (25.6 ± 2.6 years). Protein content from each sample group was analyzed by differential in gel electrophoresis (DIGE). Protein spots that exhibit an abundance ratio greater than 1.5 between groups were identified by mass spectrometry. Specific ELISAs were developed and validated in serum population collected from 236 healthy subjects aged from 20 to 64 years and from 76 patients with severe radiological knee OA (68.8 ± 11.9 years). Immunohistochemistry analysis was performed on articular cartilage from tibial plateaus.

Thirteen proteins within spots significantly modified between groups were identified. Particular interest was focused on peptides of fibulin-3 named Fib3-1 and Fib3-2. Two antisera directed against these peptides were used to develop immunoassays. By comparison with age-matched healthy subjects, Fib3-1 (median, 54.6 pM vs 85.1 pM; p < 0.0001) and Fib3-2 (median, 144.4 pM vs 191.4 pM; p < 0.0001) serum levels were elevated in OA patients. Using ROC-AUC analysis, we have demonstrated that Fib3-1 and Fib3-2 discriminate OA and normal population. Immunostaining revealed the presence of Fib3-1 and Fib3-2 into chondrocytes and in the extracellular matrix of the superficial layer of the fibrillated cartilage.

Fib3-1 and Fib3-2 are potential biochemical markers for the diagnosis of OA.

To evaluate the efficacy, tolerability, and safety of multiple fixed doses of esreboxetine for the treatment of fibromyalgia.

Patients meeting American College of Rheumatology criteria for fibromyalgia were randomized to receive 4- (n = 277), 8- (n = 284), or 10-mg/d (n = 283) esreboxetine, or matching placebo (n = 278), for 14 weeks. Primary efficacy outcomes were weekly mean pain score and Fibromyalgia Impact Questionnaire (FIQ) total score at week 14. Secondary efficacy measures included Patient Global Impression of Change (PGIC), Global Fatigue Index (GFI), and 36-item Short-Form Health Survey (SF-36; Physical Functioning scale only) scores at week 14. Esreboxetine's safety profile was evaluated based on adverse events and other safety measures.

All doses of esreboxetine demonstrated statistically significant improvements over placebo on pain (P ≤ 0.025), FIQ (P ≤ 0.023), and PGIC (P ≤ 0.007). Additionally, the 4- and 8-mg/d esreboxetine demonstrated statistically significant improvements over placebo on the GFI (P = 0.001). No significant improvements over placebo were evident for any dose of esreboxetine on the SF-36 Physical Functioning scale. Adverse events were mostly mild to moderate in severity; insomnia, constipation, dry mouth, nausea, dizziness, hot flush, headache, hyperhidrosis, and palpitations were reported most frequently.

Esreboxetine was generally well tolerated and associated with significant improvements in pain, FIQ, PGIC, and fatigue scores compared with placebo. The lack of a dose-response relationship in both the efficacy and safety analyses suggests that 4-mg/d esreboxetine would offer clinical benefit with the least risk of drug exposure.

Th-17 cells are implicated in Rheumatoid Arthritis (RA). We hypothesized that interaction of T cells with mesenchymal cells derived from bone marrow (BM-MSC) or synovium (FLS) might promote Th-17 cells, with the help of T-cell-secreted inflammatory cytokines, i.e: IL-17A, TNFα, and/or IFNγ.

Healthy peripheral blood mononuclear cells (PBMC) were co-cultured with BM-MSC, or FLS from RA, or osteoarthritis (OA) patients. Co-cultures were exposed to PHA, +/- IL-17A, TNFα, or IFNγ. Q-RT-PCR, ELISA, and cytoflurometry were used to measure IL-17-A production.

Interaction of PBMC with BM-MSC inhibited Th-1 and Th-2 responses, but promoted Th-17 expansion, as soon as 24 hours, as assessed by increase of (i) ROR-C or IL-17A mRNA levels, (ii) IL-17A secretion levels, (iii) IL-17A-secreting cell frequency, and (iv) T-cell switch towards the Th-17 pathway after 2 rounds of stimulation with MSC. IL-17A production also increased when PBMC were stimulated with anti-CD3+CD28, or when CD3+, or CD45RO+ T cells were isolated, demonstrating thus the role of T-cell activation. Moreover, IL-6, IL-8, and IL-1β mRNA levels were further amplified when T-cell-secreted-inflammatory cytokines were added. Interestingly, OA- or RA-FLS also enhanced IL-17-A and IL-6 production, but only RA-FLS enhanced IFNγ and IL-1β production. Finally, we demonstrated that MSC-mediated Th-17 promotion requires caspase-1 activation, by using an inhibitory peptide and measuring its activity.

We show that interaction of MSC, or FLS, with T cells promote Th-17 cells, through caspase-1 activation. Since pro- and T-cell-inflammatory cytokines are also amplified, this mechanism may participate to RA chronicity.

To examine the migratory properties of cytokine-activated T (Tck) cells.

Tcks were generated by culture of peripheral blood T cells in the presence of IL-6, TNFα and IL-2. Changes in cell surface phenotype were analysed by flow cytometry. Chemotactic responsiveness was measured using in vitro chemotaxis assays and transendothelial migration through HUVEC monolayers. VCAM-1 levels were measured by sandwich ELISA.

Cytokine stimulation upregulated expression of chemokine receptors and integrins on Tck including CXCR4, VLA-4 and LFA-1. Increased expression of CXCR4 and VLA-4 integrin resulted in concentration-dependent chemotaxis to their ligands, SDF-1 and VCAM-1, which could be selectively blocked using a specific CXCR4 inhibitor and antibodies against VLA-4. Increased expression of VLA-4 also resulted in increased transendothelial migration of Tck, which could be abrogated using blocking antibodies against VLA-4. Tcks also showed an increased chemotactic response to RA fibroblast- like synoviocytes cultured in vitro, which could be blocked using inhibitors against VLA-4 and CXCR4.

The activated phenotype of Tck results in increased migratory responsiveness to SDF-1 and soluble VCAM-1, which are among the chemokines and proteins found elevated in the RA synovial joint environment. Cytokine-dependant activation may contribute to RA pathogenicity by promoting T cell recruitment and retention to the joint perpetuating the inflammatory cascade in RA.

Single-nucleotide polymorphisms (SNPs) have been identified in several genes encoding Toll-like receptors (TLRs) that alter immune function, inflammatory responses and disease susceptibility. The SNPs with best evidence for affecting immune function are TLR1 (1805GG), TLR2 (2258GA) and TLR5 (1174CT).

We studied the frequency and functional outcome of these polymorphisms in 248 patients with Lyme disease. Cytokine and chemokine levels were determined in serum of patients with erythema migrans (EM), joint fluid of patients with Lyme arthritis, and supernatants of B. burgdorferi-stimulated PBMC from Lyme arthritis patients, using multiplex assays.

The frequency of TLR1-1805GG polymorphism was greater in patients with antibiotic-refractory arthritis compared with patients with EM or antibiotic-responsive arthritis. Early in the illness, EM patients with 1805GG, primarily those infected with B. burgdorferi RST1 strains, had higher serum levels of IFNγ, CXCL9 and CXCL10, and more severe infection than patients with 1805TG/TT. These inflammatory responses were amplified in patients with Lyme arthritis, and the highest responses were observed in antibiotic-refractory arthritis patients with 1805GG who had been infected with RST1 strains. When PBMC from Lyme arthritis patients were stimulated with a B. burgdorferi RST1 strain, the 1805GG group had significantly larger fold-increase in the levels of IFNγ, CCL2, CXCL9 and CXCL10, than the 1805TG/TT group. In contrast, the TLR2 and TLR5 polymorphisms did not vary among groups in frequency or function.

The TLR1-1805GG polymorphism in B. burgdorferi RST1-infected patients was associated with stronger T_H1-like inflammatory responses, which may set the stage for antibiotic-refractory arthritis.

Significant joint pain is usually widespread beyond the afflicted joint which results from the sensitization of nociceptive neurons in the central nervous system (central sensitization). In the present study we explored (a) whether the proinflammatory cytokine interleukin-6 (IL-6) in the joint induces central sensitization, (b) whether joint inflammation causes IL-6 release in the spinal cord, and (c) whether spinal IL-6 contributes to central sensitization.

In anesthetized rats electrophysiological recordings were made from spinal cord neurons with sensory input from the knee joint. Neuronal responses to mechanical stimulation of the knee and the leg were monitored. IL-6 and its soluble receptor sIL-6R were applied to the knee joint or the spinal cord. Spinal release of IL-6 was measured by ELISA. Sgp130 which neutralizes IL-6/sIL-6R was spinally applied during development of joint inflammation or during established inflammation.

A single injection of IL-6/sIL-6R into the knee joint as well as spinal application of IL-6/sIL-6R significantly increased the responses of spinal neurons to mechanical stimulation of the knee and ankle joint, i.e. induced central sensitization. Spinally applied sgp130 attenuated this IL-6 effect. Development of knee inflammation caused spinal release of IL-6. Spinal application of spg130 attenuated the development of inflammation-evoked central sensitization but did not reverse it.

Not only IL-6 in the joint is involved in the generation of joint pain but also IL-6 which is released in the spinal cord. Spinal IL-6 contributes to central sensitization and thus promotes the widespread hyperalgesia in the course of joint disease.

Many forms of arthritis are accompanied by significant chronic joint pain. Here we studied whether there is significant sprouting of sensory and sympathetic nerve fibers in the painful arthritic knee joint and whether nerve growth factor (NGF) drives this pathological reorganization.

A painful arthritic knee joint was produced by injection of complete Freund's adjuvant (CFA) into the knee joint of young adult mice. CFA-injected mice were then treated systemically with vehicle or anti-NGF antibody. Pain behaviors were assessed and at 28 days following the initial CFA injection, the knee joints were processed for immunohistochemistry using antibodies raised against calcitonin gene-related peptide (CGRP; sensory nerve fibers), neurofilament 200 kDa (NF200; sensory nerve fibers), growth associated protein-43 (GAP43; sprouted nerve fibers), tyrosine hydroxylase (TH; sympathetic nerve fibers), CD31 (endothelial cells) or CD68 (monocytes/macrophages).

In CFA-injected mice, but not vehicle-injected mice, there was a significant increase in the density of CD68⁺ macrophages, CD31⁺ blood vessels, CGRP⁺, NF200⁺, GAP43⁺, and TH⁺ nerve fibers in the synovium as well as joint pain-related behaviors. Administration of anti-NGF reduced these pain-related behaviors and the ectopic sprouting of nerve fibers, but had no significant effect on the increase in density of CD31⁺ blood vessels or CD68⁺ macrophages.

Ectopic sprouting of sensory and sympathetic nerve fibers occurs in the painful arthritic joint and may be involved in the generation and maintenance of arthritic pain.

To estimate the prevalence, types and sociodemographic and biobehavioral correlates of antinuclear antibodies (ANA) in the United States (U.S.).

Cross-sectional analysis of 4,754 individuals from the National Health and Nutrition Examination Survey (NHANES) 1999-2004. ANA by indirect immunofluorescence, including cellular staining patterns and specific autoantibody reactivities by immunoprecipitation in those with ANA.

ANA prevalence in the U.S. population ages 12 years and older was 13.8% (95% CI, 12.2% to 15.5%). ANA increased with age (P = 0.01) and were more prevalent among females than males (17.8% vs. 9.6%, P < 0.001), with the female to male ratio peaking at 40-49 years of age. ANA prevalence was modestly higher in African Americans than whites (adjusted prevalence odds ratio [POR], 1.30; 95% CI, 1.00 to 1.70). Remarkably, ANA were less common in overweight and obese (adjusted POR, 0.74; 95% CI, 0.59 to 0.94) individuals than persons of normal weight. No significant associations were seen with education, family income, alcohol use, smoking history, serum levels of cotinine or C-reactive protein. In ANA-positive individuals, nuclear patterns were seen in 84.6%, cytoplasmic patterns in 21.8%, and nucleolar patterns in 6.1%, and the most common specific autoantibodies were anti-Ro (3.9%) and anti-Su (2.4%).

These findings suggest that over 32 million persons in the U.S. have ANA and the prevalence is higher among females, older individuals, African Americans and those with normal weight. These data will serve as a useful baseline for future investigations of predictors and changes in ANA prevalence over time. © 2012 American College of Rheumatology.

To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in systemic lupus erythematosus (SLE) and to correlate this with clinical and serological parameters.

Sixty-eight clinically well-characterized SLE patients, 38 healthy controls (HC), 6 systemic sclerosis (SSc), and 6 rheumatoid arthritis (RA) patients were included. The numbers of annexin V-binding MPs displaying IgG, IgM or C1q were enumerated by flow cytometry. MP protein levels were determined by mass spectrometry in clinically defined subsets of SLE patients and controls. The MP-IgG load was determined by flow cytometry of all SLE and HC samples.

SLE patients had significantly increased total and relative numbers of IgG-positive MPs (p = 0.0004) with a much higher average IgG-load/MP (p < 0.0001) than HCs. Quantitative mass spectrometry of purified MPs verified significantly increased IgG, IgM, and C1q in SLE. In RA and SSc the average IgG/MP was significantly lower than in SLE (p = 0.006 and 0.05, respectively). Also, IgM/MP and C1q/MP were higher in SLE than in controls (p < 0.05) except for IgM in the RA-group. IgG-positive MPs were significantly associated with the presence of anti-dsDNA, anti-ENA, and anti-histone antibodies, with total IgG, and with decreased leukocyte counts. Average IgG/MP was associated with lower concentrations of MPs, the presence of anti-C1q antibodies, and with complement consumption.

Circulating cell-derived MPs in SLE carry increased loads of IgG, IgM, and C1q and IgG-MPs are associated with autoantibodies and complement activation. The findings link immunological reactions on MPs with the etiopathology of SLE. © 2012 American College of Rheumatology.

To evaluate performance of an interferon-gamma release assay (IGRA) versus the standard tuberculin skin test (TST) for latent tuberculosis (TB) infection screening prior to initiating anti-tumor necrosis factor therapy in patients with autoimmune inflammatory diseases.

This integrated analysis includes patients with rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis from Phase 3 trials of golimumab. Screening IGRA was the QuantiFERON®-TB Gold test In-Tube. Results: In this pooled analysis, 2282 patients had both IGRA and TST at screening; at least one test was positive in 13.8% of patients, including 9.4% with a positive TST, 7.0% with a positive IGRA, and 2.6% with both tests positive. The IGRA-indeterminate rate was 1.8%. Agreement of TST and IGRA measured by kappa coefficient was 0.22 (95% CI: 0.16- 0.28; p=0.021). Among patients with positive IGRA, 36.9% had positive TST; among those with positive TST, 27.4% had positive IGRA. Overall 34.2% (781/2282) of patients had prior Bacillus Calmette-Guérin (BCG) vaccine. For BCG-positive patients, the TST-positive rate was 15.2% (119/781) versus an IGRA-positive rate of 9.1% (71/781; p=0.0002). For BCG-negative patients, the TST-positive rate was 5.0% (62/1248) versus IGRA-positive rate of 5.8% (72/1248; p = 0.3745). By repeating initially indeterminate results, the IGRA indeterminate rate was much lower than in previous reports. Conclusion: In the absence of a true gold-standard test for latent TB infection, results of this large comparison of IGRA and TST in patients with rheumatic disease suggest that the IGRA provides greater specificity and possibly greater sensitivity than the TST. © 2012 American College of Rheumatology.

The physiological interstitial tonicity of healthy articular cartilage (350-480 mOsm) is lowered to 280-350 mOsm in osteoarthritis. This results in loss of tissue pre-stress, altered compressive behavior and thus inferior tissue properties. We aimed at determining potential synergistic effects of physiological tonicity in combination with inhibiting calcineurin activity by FK506 on human articular chondrocytes and explants in vitro.

Osteoarthritic chondrocytes and explants and non-osteoarthritic chondrocytes were cultured in cytokine-free medium of 280 and 380 mOsm with or without calcineurin inhibition by FK506. Chondrogenic, hypertrophic and catabolic marker expression was evaluated on mRNA, protein and activity level.

Culturing osteoarthritic chondrocytes at 380 mOsm increased mRNA expression of chondrogenic markers (e.g. COL2, ∼13-fold, p<0.001) compared to 280 mOsm, while suppressing COL1 (∼0.5-fold, p<0.01). Inhibiting calcineurin activity under physiological tonicity further enhanced expression of anabolic markers on mRNA (COL2, ∼50-fold, p<0.001; AGC1, ∼2-fold, p<0.001; SOX9, ∼3.5-fold, p<0.001) and protein (COL2, ∼6-fold, p<0.001) level. Calcineurin inhibition suppressed relevant collagenases as well as hypertrophy and mineralization markers on mRNA and activity level. Aggrecanase-1 and -2 expression was not influenced by tonicity or FK506 alone, but the combination suppressed both, by ∼50% (p<0.05) and ∼40% (p<0.001) respectively. Generally, similar anabolic and anti-hypertrophic effects were observed in ex-vivo cartilage explant cultures and non-osteoarthritic chondrocytes.

Calcineurin inhibition at physiological tonicity exerts a superior effect than physiological tonicity or FK506 alone: increasing anabolic, while suppressing hypertrophic and catabolic markers. Our data may aid in developing improved cell-based chondral repair and osteoarthritis treatment strategies.

To assess temporal changes in cartilage and bone morphology, reactive oxygen species (ROS), and vascularization in mono-iodoacetate (MIA)-induced osteoarthritis (OA) in rats via advanced imaging methodologies.

Right knees of male 8-week old Wistar rats were injected with 1mg MIA in 50μl saline and left knee controls injected with 50μl saline. After 1, 2, and 3 weeks (n=5 each), changes in cartilage morphology and composition were quantified using equilibrium partitioning of an ionic contrast agent microcomputed tomography (EPIC-μCT) and changes in subchondral and trabecular bone were assessed by standard μCT. ROS were characterized via in vivo fluorescence imaging at 1, 11, and 21 days (n=5 each). At 3 weeks, after fluorescence imaging, alterations in knee joint vascularity were quantified with μCT after vascular contrast agent perfusion (n=5).

Femoral cartilage volume, thickness, and proteoglycan content were significantly decreased in MIA injected knees compared to controls, accompanied by loss of trabecular bone and erosion of subchondral bone surface. ROS quantities were significantly increased 1 day after MIA injection and were gradually relieved by 21 days. Vascularity in the whole knee and distal femora was significantly increased at 3 weeks after MIA injection.

Contrast-enhanced μCT and fluorescence imaging were combined to characterize articular cartilage, subchondral bone, vascularization, and ROS, providing unprecedented 3-D joint imaging and quantification for multiple tissues during OA progression. These advanced imaging techniques have the potential to become standardized methods for comprehensive evaluation of articular joint degeneration and characterization of therapeutics.

Reactive oxygen species (ROS) produced by cytokines induced expression of inflammatory mediators in rheumatoid arthritis (RA). Heme oxygenase-1 (HO-1) exerts as an anti-inflammatory effect. However, the mechanisms underlying IL-1β-induced cytosolic phospholipase A₂ (cPLA₂) expression through ROS generation modulated by HO-1 remains unknown in RA synovial fibroblasts (RASFs).

IL-1β-induced ROS generation was determined by a flow cytometer. The involvement of MAPKs and NADPH oxidase (Nox)/ROS in IL-1β-induced cPLA₂ expression was investigated using pharmacological inhibitors and transfection with small interfering RNAs (siRNAs), and analyzed by Western blot and promoter assay. Overexpression of HO-1 was performed by transfection of RASFs with a recombinant adenovirus containing human HO-1 plasmid. Severe combined immune-deficient (SCID) mice with inflammation caused by IL-1β were infected with adenovirus-HO-1. The joint histologic characterization of inflammation and local expression of cPLA₂ were evaluated after treatment.

IL-1β-induced cPLA₂ expression was mediated through NOX activation/ROS production, which was attenuated by N-acetylcysteine (NAC, a scavenger of ROS), the inhibitors of NOX (diphenyleneiodonium chloride and apocynin), MEK1/2 (U0126), JNK1/2 (SP600125), transfection with respective siRNAs, and overexpression of HO-1 in RASFs. IL-1β-induced cPLA₂ expression was mediated through recruitment of AP-1 to the cPLA₂ promoter region which was attenuated by NAC and overexpression of HO-1. Furthermore, HO-1 overexpression inhibited IL-1β-mediated cPLA₂ expression in SCID mice.

In RASFs, IL-1β induced cPLA₂ expression via activation of p42/p44 MAPK and JNK1/2 leading to phosphorylation of p47^phox and ROS production, and AP-1. The induction of HO-1 exerted protective effects in the pathogenesis of RA.

To investigate the inhibitory effect and possible mechanism of a novel influenza virus haemagglutinin (HA)-derived peptide in collagen-induced arthritis (CIA).

CIA was induced in DBA/1 mice by immunization with type II collagen (CII). Altered HA308-317 peptide (aHAP 308-317), wild HA308-317 peptide (wHAP 308-317), wild CII263-272 peptide (wCIIP 263-272) and irrelevant peptide (IP) were administered intranasally beginning from arthritis onset. Clinical and histologic scores were assessed, and cytokine levels in the serum or supernatants from splenocytes were determined. Characteristics of T cell subsets in response to different peptides were analyzed both in vivo and in vitro.

Intranasal administration of wCIIP, wHAP or aHAP could significantly ameliorate CIA, but aHAP offered greater therapeutic effects than wCIIP and wHAP. The effect of aHAP was associated with a substantial decrease in production of interleukin (IL)-17 and interferon (IFN)-γ, and an markedly increase in production of IL-10 and transforming growth factor (TGF)-β both in serum or supernatants from splenocytes treated with aHAP. Both the number and function of CD4⁺ regulatory T cells (Tregs) were significantly upregulated by aHAP with a decreased induction of Th1 (CD4⁺INF-γ⁺) / Th17 (CD4⁺IL-17⁺) cells. Adoptive transfer of CD4⁺CD25⁺ T cells from aHAP-treated mice displayed greater suppressive capacity in ameliorating CIA severity than those from wHAP, wCIIP or IP-treated mice.

Intranasal administration of aHAP potently suppressed the severity of CIA by increasing the number and function of CD4⁺ Tregs, suggesting that aHAP 308-317 might be a promising candidate for RA treatment.

To determine cut-off values for remission, minimal disease activity, and parent and child acceptable symptom state in juvenile idiopathic arthritis (JIA) using the Juvenile Arthritis Disease Activity Score (JADAS).

For the selection of cut-offs, data from a clinical database including 618 children with JIA were used. ‘Optimal’ cut-offs were determined against external criteria by calculating the 75^th percentile of cumulative score distribution and through receiver operating characteristic curve analysis. External criteria included formal definitions of inactive disease (ID) and minimal disease activity (MDA), subjective rating of remission by physicians, parents and children, and rating of acceptable symptom state by parents and children. The choice of cut-offs was made on clinical and statistical grounds. Cross-validation was performed in 4 samples, which included a total of around 1400 patients, and was based on assessment of construct, discriminant and predictive validity.

The cut-off for ID was 1 for all JADAS versions, whereas the cut-off for MDA was 2 for oligoarthritis and 3.8 for polyarthritis. Cut-offs for physicians', parents' and children's subjective rating of remission ranged from 2 to 2.3. Cut-offs for acceptable symptom state ranged from 3.2 to 5.4 for parents and from 3 to 4.5 for children. Results of cross-validation analyses strongly supported the cut-offs.

Cut-off values for disease states using the JADAS were developed. In cross-validation analyses, they proved to have good construct and discriminant validity and ability to predict disease outcome.

The aims of this prospective study were to: 1) determine the prevalence of delayed gastric emptying in unselected SSc patients, using ¹³C octanoic acid breath test; 2) evaluate whether the findings of ¹³C octanoic acid breath test are associated with: (i) digestive clinical manifestations, gastric mucosal abnormalities on gastroscopy and antro-duodenal motor activity dysfunction on manometry, and (ii) esophageal motor impairment and extra-digestive manifestations of SSc; and 3) develop a risk prediction score of gastric emptying in SSc.

57 consecutive patients with SSc underwent ¹³C octanoic acid breath test. All SSc patients also completed a questionnaire for digestive symptoms, and a global symptomatic score was calculated.

The prevalence of delayed gastric emptying was 47.8% in our SSc patients. We observed a marked correlation between values of global symptomatic score of digestive symptoms ≥ 5 and the presence of delayed gastric emptying (p<10⁻⁶); both sensibility and specificity of global symptomatic score ≥ 5 to predict delayed gastric emptying were as high as 0.93 and 0.73, respectively. Moreover, global symptomatic score ≥ 5, mucosal gastric abnormalities, severe esophageal motor impairment and interstitial lung disease were: 1) independently associated with the presence of delayed gastric emptying; and 2) used to create a predictive score. The area under the ROC curve was 0.9; both sensitivity and specificity of this latter score were 0.93 and 0.77, respectively.

Our study underscores that delayed gastric emptying often occurs in SSc patients. Interestingly, using routine clinical observations, we identified a simple score which predicted the presence of delayed gastric emptying in SSc patients.

To identify candidate genes which are regulated by human pregnancy and bear the potential to modulate rheumatoid arthritis (RA) disease activity.

Peripheral blood mononuclear cells (PBMC) of healthy volunteers (ND) were analyzed using Affymetrix GeneChips at four time points (1^st, 2^nd and 3^rd trimester and 6 weeks postpartum). Based on the GeneChip data, target genes were further analyzed via quantitative real-time PCR (qPCR) using PBMC from ND and RA patients. In order to determine the cellular source, monocytes and lymphocytes from ND and RA patients were positively selected using magnetic beads and their mRNA was analyzed by qPCR.

One-way ANOVA analysis identified 1286 mRNAs differentially expressed with regard to the four time points. The changes became more pronounced as pregnancy progressed and they were reverted postpartum. A subsequent pathway analysis suggested a regulatory role of pregnancy on the adipocytokine pathway as well as the PPAR-signaling pathway. Of 19 pre-selected candidate genes, AKT3, SOCS3, FADS2, STAT1 and CD36 proofed to be differentially regulated by pregnancy. In samples of RA patients the differences were concordant but more pronounced. Both, T lymphocytes and monocytes contributed to the regulated expression of these genes.

Normal human pregnancy leads to changes in the expression level of several molecular pathways in PBMC which are reverted postpartum. Changes in RA, though concordant, exceed levels observed in ND. Genes of the adipocytokine- and the PPAR-signaling pathways qualify as candidates for the modulation of RA disease activity during pregnancy.

Systemic sclerosis (SSc) is an autoimmune disease with a predilection for females. Although the CD40-CD154 (CD40 ligand) interaction is known to be involved in the development of SSc and the CD40 ligand (CD40L) is overexpressed in SSc patients, the mechanisms leading to CD40L overexpression in SSc are not well understood. Elsewhere we demonstrated that DNA demethylation reactivates the silent X chromosome resulting in CD40L overexpression in healthy women. We hypothesized that CD40L upregulation by DNA demethylation and the subsequent reactivation of the silent X chromosome in female SSc patients explain the susceptibility of females to this disorder. We undertook this study to investigate the effect of DNA demethylation on CD40L expression in CD4⁺ T-cells from females with SSc.

CD40L expression in CD4⁺ T-cells from SSc patients and healthy controls was measured by flow cytometry and real-time RT-PCR. Bisulphite sequencing was performed to determine the methylation status of the CD40L regulatory region.

In female SSc patients CD40L expression was significantly elevated; it was correlated with a decrease in the DNA regulatory sequence methylation level compared with healthy women. There was a significant inverse correlation between the methylation level and CD40L mRNA expression. In contrast, in male SSc patients and healthy male controls there was no significant difference in the expression of CD40L. The DNA regulatory regions from both patients and controls were largely unmethylated.

Demethylation of the CD40L regulatory elements on the inactive X chromosome contributes to CD40L overexpression in CD4⁺ T-cells from female SSc patients.

Mutations in LMNA encoding the A-type lamins cause several diseases including those with features of premature aging and skeletal abnormalities. We therefore examined the expression of lamin A in cartilage from humans with osteoarthritis (OA) and studied the effects of its overexpression on chondrocyte senescence and apoptosis.

Human chondrocyte-like cells (SW1353) were utilized in this study. RNA isolated from human OA and non-OA cartilages were used for profiling mRNA expression using Affymetrix microarray. Effects of lamin A overexpression on mitochondrial function and apoptosis were examined by assessing mitochondrial membrane potential, ATP levels, cytochrome C release and TUNEL assay. Western blots were performed for protein expression.

Lamin A expression was markedly elevated in OA cartilage samples compared with non-OA controls. Western analysis confirmed elevated levels of lamin A expression in OA compared to non-OA cartilage. IL-1β treatment inhibited, whereas PGE₂ caused a marked increase in lamin A accumulation. These effects of exogenous PGE₂ on lamin A expression were mediated via EP2/4 receptor. Transfected chondrocytes that expressed lamin A displayed markers of early senescence/apoptosis.

Our results suggest that lamin A is upregulated in OA chondrocytes, and increased nuclear accumulation of lamin A in response to catabolic stress may account for the premature aging phenotype and apoptosis of chondrocytes in OA.

To utilize proteomic analysis to identify protein biomarkers associated with pro-inflammatory HDL in patients with active rheumatoid arthritis.

Liquid chromatography-mass spectrometry (LC-MS) was used to analyze proteins associated with immunoaffinity purified HDL from plasma of two sets of RA patients carrying distinct HDL (anti- or pro-) inflammatory properties. Proteins were fractionated by Offgel electrophoresis and analyzed by LC-MS/MS equipped with a high capacity high performance liquid chromatography chip (HPLC-Chip) incorporating C18 reverse phase trapping and analytical columns. Sandwich enzyme-linked immunosorbent assays were used to validate select HDL-associated proteins in a second RA cohort.

Seventy-eight proteins were identified in the HDL complexes. Twelve proteins were significantly increased in RA patients with pro-inflammatory HDL compared to RA patients with anti-inflammatory HDL. These proteins included acute phase proteins, including apolipoprotein J, fibrinogen, haptoglobin, serum amyloid A, and complement factors (B, C3, C9). Four of the proteins associated with HDL were validated in a second RA cohort.

Pro-inflammatory HDL in patients with RA contains a significantly altered proteome including increased amounts of acute phase proteins and proteins involved in the complement cascade. These findings suggest that HDL is significantly altered in the setting of chronic inflammation from active RA with resultant loss of its anti-inflammatory function. The characterization of the biomarkers reported here may identify novel molecular connections that contribute to the higher risk of CVD in RA patients.

It remains unknown whether and how B lymphocytes contribute to the pathogenesis of spondyloarthritis (SpA), a seronegative arthritis associated with gut inflammation. As innate-like CD5⁺ B lymphocytes with regulatory functions have been identified in colitis models, we analyzed the presence and function of CD5⁺ B cells in human SpA.

Peripheral blood B cells were analyzed by flow cytometry in SpA, healthy controls (HC) and rheumatoid arthritis (RA). Synovial biopsies were analyzed by immunohistochemistry. Sorted CD5⁺ and CD5⁻ B cells were analyzed for somatic hypermutation, the expression of costimulatory molecules, and cytokine production.

The naïve, marginal zone-like, and to a lesser extend memory B cell compartments of SpA but not RA displayed a clear and specific increase of CD5⁺ B cells. This increase was neither due to B-cell activation nor to preferential migration of CD5⁻ B cells to the inflamed synovium. In line with their phenotype and the low affinity, poly-reactive immunoglobulins produced by their murine counterparts, SpA CD5⁺ B cells showed low levels of somatic hypermutation. As to antigen presentation, CD5⁺ B cells expressed slightly increased HLA-DR but low CD80 and CD86 levels. In vitro activation failed to upregulate these costimulatory molecules but induced significant production of IL-10 and IL-6 by CD5⁺ B cells.

CD5⁺ B cells are specifically increased in SpA. Analysis of somatic hypermutation, expression of antigen presenting and costimulatory molecules, and cytokine production indicate that this B-cell subset has regulatory capacities. These data warrant further investigation of their potential role in SpA.

The induction of Rheumatoid Arthritis (RA) by active and passive immunization of mice results in the development of pain at the same time as swelling and inflammation, with both peripheral and central sensitization contributing to joint pain. Here we examined the development of pain in the rat Collagen Induced Arthritis (CIA) model and evaluated the contribution of neuro-immune interactions to established arthritis pain.

Mechanical hypersensitivity was assessed in female Lewis rats before and up to 18 days after induction of CIA by immunization with type II collagen. The effect of selective inhibitors of microglia were then evaluated by prolonged intrathecal delivery of a Cathepsin S (CatS) inhibitor and a Fractalkine (FKN) neutralizing antibody, from days 11 to 18 following immunization.

CIA rats developed significant mechanical hypersensitivity which started on day 9, before the onset of clinical scores. Mechanical hypersensitivity peaked with the severity of the disease, when significant microglial and astrocytic response, alongside T-cell infiltration were observed in the spinal cord. Intrathecal delivery of microglial inhibitors, a CatS inhibitor or FKN neutralizing antibody, attenuated mechanical hypersensitivity and spinal microglial response in CIA rats.

The inhibition of microglial targets by centrally-penetrant CatS inhibitors and FKN/CX3CR1 receptor antagonists represents a potential therapeutic avenue for the treatment of pain in RA.

To provide an intermediate step between classical arthritis models and clinical trials, the RA synovium SCID mouse model is a valuable tool during preclinical research. In this study, the validity of this humanized mouse model was investigated using anti-TNF and anti-IL-1 treatment. In addition, the direct effect of T and B cell-related therapies on the transplanted RA synovial tissue was investigated.

SCID.CB17 mice were engrafted with human RA synovial tissue, and systemically treated with anti-TNF, anti-IL-1, anti-IL-17, CTLA4-Ig, anti-CD20, or isotype control antibodies.

Validation of the model with anti-TNF treatment significantly reduced serum cytokine levels and decreased histological inflammation, whereas anti-IL-1 therapy did not show any effect on the RA synovial grafts. In mice engrafted with B cell-rich synovial tissue, anti-CD20 treatment showed clear therapeutic effects. Surprisingly, CTLA4-Ig treatment did not show any effects in this transplantation model, even despite pre-screening of the synovial tissue for the presence of CD3+ T cells and the co-stimulatory molecules CD80/86. In contrast, great therapeutic potential was observed for anti-IL-17 treatment, however only when CD3+ T cells were abundantly present in the RA synovial tissue.

This human RA synovium SCID mouse model enabled us to show that CTLA4-Ig lacks direct effects on T cell activation processes in the synovial tissue. Further evidence was obtained that IL-17 might indeed be an interesting therapeutic target in RA patients with CD3-rich synovial tissue. Further characterization of the RA patients' individual synovial profile is of great importance to achieve tailor-made therapy.

Aim was to assess several baseline risk factors that may predict patello-femoral and tibio-femoral cartilage loss during a 6 month period.

For 177 subjects with chronic knee pain, 3T MRI of both knees was performed at baseline and follow-up. Knees were semiquantitatively assessed evaluating cartilage morphology, subchondral bone marrow lesions (BMLs), meniscal morphology/extrusion, synovitis and effusion. Age, gender and body mass index (BMI), BMLs, meniscal damage/extrusion, synovitis, effusion and prevalent cartilage damage in the same subregion were evaluated as possible risk factors for cartilage loss. Logistic regression models were applied to predict cartilage loss. Models were adjusted for age, gender, treatment, and BMI.

79 (1.6%) subregions showed incident or worsening cartilage damage at follow-up. None of the demographic risk factors were predictive of future cartilage loss. Predictors for patello-femoral cartilage loss were effusion (adjusted odds ratio [aOR] 3.5, 95% confidence interval [CI] 1.3-9.4) and prevalent cartilage damage in the same subregion (aOR 4.3, 95% CI 1.3-14.1). Risk factors for tibio-femoral cartilage loss were baseline meniscal extrusion (aOR 3.6, 95% CI 1.3-10.1), prevalent bone marrow lesions (BMLs) (aOR 4.7, 95% CI 1.1-19.5) and prevalent cartilage damage (aOR 15.3, 95% CI 4.9-47.4)

Cartilage loss over 6 months is rare, but may be detected semiquantitatively at 3T MRI and is most commonly observed in Kellgren-Lawrence 3 knees. Predictors of patello-femoral cartilage loss were effusion and prevalent cartilage damage in the same subregion. Predictors of tibio-femoral cartilage loss were prevalent cartilage damage, BMLs and meniscal extrusion.

Systemic sclerosis (SSc) is characterized by fibrosis of skin and visceral organs, vascular dysfunctions and immunological dysregulation. Platelet-derived growth factors (PDGFs) are implicated in the development of fibrosis and dysregulation of vascular function. We investigated the effect of sunitinib and sorafenib, two tyrosine kinase inhibitors that interfere with PDGF signalling, in a mouse model of diffuse SSc.

SSc was induced in BALB/c mice by daily subcutaneous injections of HOCl for 6 weeks. Mice were randomized to treatment with either sunitinib or sorafenib or vehicle. The levels of native and phosphorylated PDGFR-β and VEGFR were assessed in the skin by western blot analysis and immunohistochemistry. Skin and lung fibrosis were evaluated by histological and biochemical methods. Auto-antibodies were detected by ELISA, and spleen cell populations analyzed by flow cytometry.

The phosphorylation of PDGFR-β and VEGFR was higher in the fibrotic skin from HOCl-mice than from PBS-mice. Injections of HOCl induced cutaneous and lung fibrosis, increased the proliferation rate of fibroblasts in fibrotic skin areas, increased splenic B and T cell counts and anti-DNA topoisomerase-1 autoantibody levels in BALB/c mice. All of those features were reduced by sunitinib but not by sorafenib. Sunitinib significantly reduced the phosphorylation of both PDGF and VEGF receptors.

Inhibition of the hyperactivated PDGF and VEGF pathways by sunitinib prevented the development of fibrosis in HOCl-induced murine SSc and may represent a new treatment for SSc to be tested in clinical trials.

Intestinal inflammation observed in ankylosing spondylitis (AS) patients has been demonstrated to be characterized by an over-expression of IL-23. IL-23 is known to regulate IL-22 production by lamina propria NKp44⁺ NK cells, which are thought to be involved in protective mucosal mechanisms. This study was undertaken to evaluate the frequency of NKp44⁺ NK cells, and to evaluate IL-22 expression in the ileum of AS patients.

Tissue NKp44⁺, NKp46⁺ NK and IL-22 producing cells were analyzed by flow cytometry. Quantitative gene expression analysis, by rt-PCR of IL-22, IL-23, IL-17, STAT3 and MUC-1 was performed on ileal samples from 15 AS, 15 Crohn's Disease (CD) patients, and 15 healthy controls. NKp44, phospho-STAT3 (p-STAT3) and IL-22 expression was analyzed by immunohistochemistry.

NKp44⁺ but not NKp46⁺ NK cells were increased in the inflamed ileum of AS compared to CD patients and controls. On the other hand NKp46⁺NK cells were significantly increased only in CD patients. Among CD4⁺ lymphocytes and Nkp44⁺NK cell subsets, the last were the major source of IL-22 on lamina propria mononuclear cells (LPMC) of AS patients. A strong and significant up-regulation of IL-22, IL-23p19, MUC-1 and STAT3 transcripts in the terminal ileum of patients with AS was observed. Immunohistochemical analysis confirmed the increased IL-22 and p-STAT3 expression in inflamed mucosa from AS and CD patients.

Our findings indicate that overexpression of IL-22 together with an increased number of IL-22-producing-NKp44⁺ NK cells occurs in the gut of AS patients where appear to play a tissue-protective role.

To assess defects in expression of critical cell cycle checkpoint genes and proteins in subjects with rheumatoid arthritis relative to presence or absence of methotrexate medication and assess the role of Jun N-terminal kinase in methotrexate induction of these genes.

Flow cytometry analysis was used to quantify changes in intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) was employed to determine changes in cell cycle checkpoint target genes.

RA subjects express lower baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and total JNK protein. MAPK9, TP53, CDKN1A, and CDKN1B mRNA expression, but not CHEK2, and RANGAP1, is higher in patients on low-dose MTX therapy. Further, JNK levels inversely correlate with CRP levels in RA patients. In tissue culture, MTX induces expression of both p53 and p21 by JNK2 and JNK1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 are not induced by MTX. MTX also induces ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocks these MTX-mediated effects.

Our findings support the notion that MTX restores some, but not all of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway.

To evaluate the presence of pulmonary abnormalities in subjects with rheumatoid arthritis (RA)-related autoantibody (Ab) positivity without inflammatory arthritis (IA).

42 subjects without IA but with elevations of anti-cyclic citrullinated peptide antibodies and/or 2 or more rheumatoid factor isotypes (a profile that is 96% specific for RA), 15 Ab(-) controls and 12 patients with early established seropositive RA (<1 year duration) underwent spirometry and high-resolution computed tomographic (HRCT) lung imaging.

The median age of Ab(+) subjects was 54 years-old, 52% were female and 38% were smokers (not significantly different than Ab(-) controls). No Ab(+) subject had IA on joint examination. On HRCT, 76% of Ab(+) subjects had airways abnormalities including bronchial wall thickening, bronchiectasis, centrilobular opacities and air trapping, compared to 33% of Ab(-) controls (p=0.005). The Ab(+) subjects had similar prevalence and type of lung abnormalities compared to patients with early RA. Two Ab(+) subjects with airways disease developed IA classifiable as articular RA ∼13 months after lung evaluation.

Airways abnormalities that are consistent with inflammation are common in Ab(+) subjects without IA, and similar to airways abnormalities seen in early RA. These findings suggest that the lung may be an early site of autoimmune-related injury, and potentially a site of generation of RA-related autoimmunity. Further studies are needed to define the mechanistic role of lung inflammation in the development of RA.

We hypothesized that the Transient Receptor Potential Vanilloid 4 (TRPV4) activation induces inflammation in the Temporomandibular Joint (TMJ). We studied the expression of TRPV4 in the inflamed joint and the effects of TRPV4 agonists in TMJ. Additionally, we investigated the sensitizing effects of inflammatory mediators such as Protease-Activated Receptor 2 (PAR₂) agonist on TRPV4 responses.

TRPV4 and PAR₂ expression in the rat TMJ and trigeminal ganglion (TG) neurons were evaluated by real-time RT-PCR and immunofluorescence followed by confocal microscopy, 4 h after intra-articular injection of carrageenan. The functionality of TRPV4 and its sensitization by PAR₂ activating peptide (PAR₂-AP) were analyzed by measurements of intracellular Ca²⁺ in TMJ fibroblast-like synovial cells or TG neurons. Plasma extravasation, myeloperoxidase activity and head-withdrawal threshold (index of mechanical allodynia) were evaluated after intra-articular injection of selective TRPV4 agonists, either alone or co-injected with PAR₂-AP.

In the TMJ, TRPV4 and PAR₂ expressions were up-regulated after carrageenan-induced inflammation. Two TRPV4 agonists specifically activated calcium influx in TMJ fibroblast-like synovial cells or TG neurons. In vivo, they triggered dose-dependent increases in plasma extravasation, myeloperoxidase activity and mechanical allodynia. In synovial cells or TG neurons, pre-treatment with PAR₂-AP potentiated TRPV4 agonist-induced increase in [Ca²⁺]_i. In vivo, TRPV4 agonist-induced inflammation was potentiated by PAR₂-AP.

TRPV4 is expressed and functional in TG neurons and synovial cells. In vivo, activation of TRPV4 causes inflammation in the TMJ. Pro-inflammatory mediators such as PAR₂ agonists sensitize TRPV4 activity. These results identify TRPV4 as an important inflammatory signal in the joint.

Expansion of auto-reactive, CD4⁺CD28 negative T cells (CD4⁺CD28^null) is associated with extra–articular disease manifestations including rheumatoid vasculitis and it could be demonstrated recently that expansion of these T cells was associated with anti-cytomegalovirus (CMV) seropositivity. The aim of our study was to investigate a possible link between latent CMV infection and rheumatoid arthritis.

In a retrospective analysis, anti-CMV antibodies were determined in 202 RA patients and in 272 healthy controls together with clinical, serological, and radiological parameters of joint destruction. In addition, frequencies of CD4⁺CD28 negative T cells, concentrations of the cytokines MCP–1, IFNα, and IP-10, as well as anti-CMV specific T cell responses were analyzed in RA patients.

Overall, no significant difference in the frequency of anti-CMV seropositivity between RA patients and healthy controls was observed. In individuals older than 55 years, however, anti-CMV IgG antibodies were significantly more frequent in RA patients (65.3% vs. 54.7%, p=0.05). Anti-CMV seropositivity in RA patients was associated with increased frequency of CD4⁺CD28 negative T cells and increased serum concentrations of MCP–1. The frequency of anti-CMV specific CD4⁺ T cells producing IFNγ was increased in RA patients compared to controls. Most importantly, anti-CMV seropositive RA patients showed radiographic evidence of more advanced joint destruction and had increased frequencies of surgical joint procedures, indicating more severe joint disease.

Latent CMV infection aggravates the clinical course of rheumatoid arthritis and is associated with increased frequencies of CD4⁺ CD28 negative and of CMV specific IFNγ secreting CD4⁺ T cells.

To describe the temporal patterns of knee pain in a community-based cohort over 12-years.

Self-reported knee pain data at four time points over 12 years were analysed in participants from the Chingford Study. Pain was defined by any pain in the preceding month and pain on most days in the preceding month. This was used to classify participants into asymptomatic, persistent, incident and intermittent groups. Multinomial logistic regression was used to identify baseline predictors for each group.

At baseline, 489 women had a median (IQR) age of 52 years (48, 58), BMI 24.39 kg/m² (22.46, 27.20) and 11.7% had at least KL grade 2 in at least one knee. For any pain vs. pain on most days in the preceding month, 9% and 2% had persistent pain, 24% and 16% had incident pain and 29% and 18% had intermittent pain respecitively. Higher BMI predicted persistent (OR 1.14 (1.05,1.25) and incident pain patterns (OR 1.10 (1.02,1.18). Radiographic OA predicted persistent pain (OR 3.82 (1.37,10.68), p=0.010) and reported knee injury predicted both persistent (OR 4.25 (1.38,1.12),p=0.012) and intermittent (OR 4.21 (1.80,9.89),p=0.001) pain patterns.

Significant variability in the temporal fluctuation of self reported knee pain was seen in this community-based prospective study over a period of 12 years with few women consistently reporting knee pain at each time point. Distinct baseline predictors for each pain pattern were identified and may explain the observed heterogeneity of reported predictors of pain when pain is measured at only one time point.

Proliferating pannus is in many aspects similar to placental tissue: Both fibroblast-rich tissues have high vascularity, and tissues demonstrate conversion from androgenic prehormones to downstream estrogens. To investigate similarities, the angiogenic placental growth factor – 1 (PlGF 1), was in the focus of this study in OA and RA patients.

In order to study the presence of PlGF-1, its synovial distribution, and the PlGF-1 expressing synovial cell type immunohistochemistry was used. The relationship between PlGF-1 and conversion of the biologically inactive placental prehormone DHEAS to the biologically active DHEA was investigated in mixed synovial cells. Effects of DHEA on PlGF-1 expression were studied by intracellular FACS analysis.

PlGF-1+ cells were detected in the lining and sublining area of RA/OA patients with a similar cellular density. Double staining revealed that PlGF-1+ cells were macrophages. In RA/OA, density of PlGF-1+ cells positively correlated with density of macrophages and density of collagen type IV-positive vessels. Only in OA, supernatant concentration of [³H]DHEA after conversion from [³H]DHEAS and density of aromatase-positive cells were positively correlated to density of PlGF-1+ cells. Low DHEA concentrations (≤ 10⁻⁹M) had stimulatory effects on PlGF-1 when compared to serum concentrations (10⁻⁷M) in the monocytic cell line THP-1 and in primary mixed synovial cells.

PlGF-1 in inflamed synovium plays similar roles as compared to its function in the placenta: It is related to vessel formation and, in OA patients, to androgen/estrogen conversion. Evolutionarily conserved functions of PlGF-1 for placental phenomena are obviously also present in synovial inflammation.

To investigate safety, tolerability, pharmacokinetics, and efficacy of apilimod mesylate, an oral interleukin (IL)-12/IL-23 inhibitor, in patients with rheumatoid arthritis (RA).

We performed a phase IIa, randomized, double-blind, placebo-controlled proof of principle study of apilimod, in combination with methotrexate, in 29 patients with active RA (3:1, apilimod to placebo) in three stages. Patients received apilimod 100 mg QD or placebo for 4 weeks (stage I) or 8 weeks (stage II). Stage III consisted of apilimod 100 mg BID or placebo for 8 weeks, with an optional extension of 4 weeks. Clinical response (DAS28 and ACR criteria) was assessed throughout; synovial tissue samples collected at baseline and day 29 (I/II) or 57 (III) were stained for cellular markers and cytokines by immunohistochemistry.

While only mild adverse events were observed in stages I/II, all stage III patients experienced headache and/or nausea. Apilimod-treated patients (100 mg QD) showed a small, but significant, reduction in DAS28 at day 29 and 57 compared with baseline. ACR20 was reached in only 6% at day 29 and 25% at day 57, similar to responses in placebo group. Increasing the dosage (100 mg BID) did not improve clinical efficacy. Consistent with clinical results, there was no effect of apilimod on expression of synovial biomarkers. Of importance, there was also no effect of apilimod on synovial IL-12 and IL-23 expression.

Our results do not support the notion that IL-12/IL-23 inhibition by apilimod is able to induce robust clinical improvement of RA.

Global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts (RASF) contributes to their intrinsic activation. The aim is to explore that an increased polyamine metabolism is associated with a decreased level of S-adenosylmethionine (SAM), causing the global DNA hypomethylation.

RASF (n = 12) and osteoarthritis synovial fibroblasts (OASF, n = 6) were isolated from synovial tissues. The cells were stained for adenosylmethionine decarboxylase (AMD), spermine/spermidine N1-acetyltransferase (SSAT1), polyamine modulated factor binding protein 1 (PMFBP1), solute carrier3A2 (SC3A2), DNA methyltransferase1 (Dnmt1), integrin α9, and β1, were analyzed by flow cytometry. Nuclear 5-methylcytosine (5-MeC) was measured by flow cytometry, diacetylspermine (DASp) in cell culture supernatants and cell extracts were determined by ELISA and SAM was measured in cell extracts by fluorometry.

SSAT1, AMD and PMFBP1 were significantly (p < 0.05) increased in RASF, compared to OASF. DASp in cell culture supernatants and SC3A2 were significantly elevated (p < 0.01) in RASF. The levels of SAM in cell culture extracts, as well as of Dnmt1 protein and 5-MeC were significantly reduced (p < 0.001) in RASF. Parameters of the polyamines metabolism negatively correlated with SAM, Dnmt1 and 5-MeC. Supplementation of RASF with SAM increased Dnmt1 and 5-MeC, whereas integrin β1 was decreased.

These data clearly show that intrinsic elevations of PMFBP1 and SSAT1 enhance the catabolism and recycling of polyamines in RASF and suggest that a high consumption of SAM by this pathway is an important factor contributing to the global DNA hypomethylation in these cells.

Rheumatoid arthritis (RA) is an inflammatory and angiogenic disease. However, the molecular mechanisms promoting angiogenesis in RA are not clearly identified. Our objective was to study the role of CD147 in angiogenesis and whether the strategy to suppress CD147 could be useful in reducing angiogenesis in RA.

The correlation among CD147, vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 α (HIF-1 α) was determined by immunohistochemistry staining. After RA fibroblastlike synoviocytes (FLS) cells were cultured at different conditions, the production of VEGF and HIF-1 α was examined by Realtime PCR and ELISA. The SCID mouse coimplantation model of RA (SCID-HuRAg) was established and treated separately with CD147 mAb, Infliximab or combination of both, and the volume of engrafts and average vascular density were measured and analyzed. Western blot analyses were performed to examine the potential signaling pathways.

The expression levels of CD147 showed significantly positive correlations with VEGF and HIF-1 a as well as vascular density in RA synovium. After siRNA transfection or specific antibodies for CD147 were added, the production of VEGF and HIF-1 α reduced significantly. Expressions of VEGF and HIF-1 α decreased more after CD147 inhibition than after Infliximab treatment in the grafted tissues on SCID-HuRAg. PI3K-Akt pathway may be involved in this process.

CD147 induces upregulation of VEGF and HIF-1 α in RA FLS, further promotes angiogenesis and leads to the persistence of synovitis. Inhibition of CD147 may be a promising target for novel therapeutic strategies.

Pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome is a rare autoinflammatory disease. We identified five new cases from 4 kindreds and sought to describe their genotypes, phenotypes, immunophenotypes, and treatments.

Clinical information was gathered from medical records and patient interviews. PSTPIP1 (CD2BP1) exon 10 and 11 sequencing was done in each patient. Neutrophil granule content and cytokine levels were determined in plasma and from stimulated peripheral blood mononuclear cells (PBMC) in available patients and controls.

We identified two previously described PAPA associated PSTPIP1 mutations, A230T, E250Q, and a novel change, E250K. Disease penetrance was incomplete with variable expressivity. The cutaneous manifestations included pathergy, cystic acne, and pyoderma gangrenosum (PG). IL-1β and circulating neutrophil granule enzymes were markedly elevated in patients compared to controls. PBMC stimulation studies demonstrated impaired IL-10 and enhanced GM-CSF production. Good resolution of PG was achieved in three patients using TNF-α blockade.

Mutations in PSTPIP1 are incompletely penetrant and variably expressed. Neutrophil granule proteins were markedly elevated in the plasma and ex vivo and might be helpful in suggesting the diagnosis of PAPA syndrome. TNF-α blockade was effective in treating the cutaneous manifestations in three patients with PAPA syndrome.

We investigated the expression and the impact of the microRNA-34 (miR-34) family on apoptosis in synovial fibroblasts (SF) of rheumatoid arthritis (RA) patients.

Expression of the miR-34 family was analyzed by real-time PCR in SF with or without stimulation with Toll-like receptor (TLR) ligands, TNF-α, IL-1β, hypoxia and 5-azacytidine. Promoter methylation was studied by combined bisulfite restriction analysis. Overexpression and silencing of miR-34a and miR-34a* was used to analyze their effect on apoptosis by annexin V/PI staining. Production of XIAP (X-linked inhibitor of apoptosis protein) was analyzed by real-time PCR and immunohistochemistry. Reporter gene assay was used to study the signaling pathways of miR-34a*.

Basal expression levels of miR-34a*, but not of miR-34a, miR-34b/b* and miR-34c/c*, were found to be reduced in SF from RA compared to osteoarthritis. Neither TNF-α, IL-1β, TLR ligands nor hypoxia altered miR-34a* expression. However, we identified the promoter of miR-34a/34a* to be methylated and showed that transcription of the miR-34a duplex is induced upon treatment with demethylating agents. Enforced expression of miR-34a* led to an increased rate of FasL and TRAIL mediated apoptosis in RASF. Moreover, levels of miR-34a* highly correlated with the expression of XIAP, which was found to be upregulated in RA synovial cells. Finally, our study identified XIAP as a direct target of miR-34a*.

Our data provide evidence for a methylation specific downregulation of pro-apoptotic miR-34a* in RASF. Decreased expression of miR-34a* results in upregulation of its direct target XIAP, thereby contributing to apoptosis resistance of RASF.

Psoriasis is associated with ischemic heart diseases (IHD). Prior studies suggest that methotrexate (MTX) may improve vascular disease in psoriasis and rheumatoid arthritis. The aim of this study was to compare the risk of new ischemic heart diseases (IHDs) among patients of psoriasis taking MTX and other nonbiologic anti-psoriatic drugs.

A retrospective cohort study among 179,200 patients with a diagnosis of psoriasis or psoriatic arthritis on at least 3 visits. We conducted the analyses by using the National Health Insurance Research Database of Taiwan. The risk of new IHD hospitalizations was compared between psoriasis patients taking MTX monotherapy (MTX cohort) and those taking nonbiologic anti-psoriatic drugs other than MTX (reference cohort). Additional adjustments were made for cardiovascular risk factors, number of hospital visits, Charlson score and use of other anti-inflammatory drugs. The study cohorts consisted of 6,578 patients in MTX cohort and 5,471 subjects in reference cohort between January 1996 and December 2008.

The incidence rates of IHDs were 666 and 830 cases per 100,000 person-years in MTX-treated cohort and reference cohort, respectively (p=0.027, unadjusted). Increasing age, males, hypertension, diabetes, and use of phototherapies were independent risk factors for new IHD hospitalizations in study cohorts. However, the multivariate adjusted hazard ratio for hospitalized IHD was 0.97 (95% confidence interval 0.79-1.19) for MTX, in comparison with other nonbiologic anti-psoriatic drugs after adjustment of age, gender, comorbidity index, hospital visits and treatments of psoriasis.

Among patients with psoriasis and psoriatic arthritis, the adjusted risk of hospitalized IHD for individuals starting MTX was comparable with those starting with other nonbiologic anti-psoriatic drugs.

Platelet-derived growth factor (PDGF) and its receptor (PDGFR) promote fibrosis in scleroderma (SSc) dermal fibroblasts, which produce excessive reactive oxygen species (ROS). PDGFR is phosphorylated upon PDGF stimulation, and dephosphorylated by protein tyrosine phosphatases (PTPs), including PTP1B. In this study we determine whether the thiol-sensitive PTP1B is affected by ROS, thus enhancing PDGFR phosphorylation (p-PDGFR) and collagen I (Col I) synthesis. The effect of a thiol antioxidant, n-acetylcysteine (NAC), was also investigated.

Fibroblasts were isolated from skin. A phosphate release assay was used for PTP1B activity.

ROS and Col I were significantly higher in SSc fibroblasts, accompanied by significantly lower amounts of free thiols compared to normal fibroblasts. After PDGF stimulation, not only were the PDGFR and ERK_1/2 phosphorylated to a greater extent, but the ability to produce PTP1B was also hampered in SSc fibroblasts. PTP1B activity was significantly inactivated in SSc fibroblasts, which resulted from cysteine oxidation by higher levels of ROS, since oxidation of multiple PTPs, including PTP1B, was observed. Decreased PTP1B expression in normal fibroblasts led to increased Col I. NAC restored the low PTP1B activity, improved the profile of p-PDGFR, decreased the numbers of tyrosine-phosphorylated proteins and Col I, and scavenged ROS in SSc fibroblasts.

We introduce a new mechanism by which ROS promote a profibrotic phenotype in SSc fibroblasts through oxidative inactivation of PTP1B leading to pronounced PDGFR activation. Our study also provides a novel molecular mechanism by which NAC therapy may act on ROS and PTP1B to benefit SSc patients.

PKCδ activation was found to be a principal rate-limiting step in matrix-degrading enzyme production in human articular chondrocytes. However, the role of the PKC pathways, specifically PKCδ, has not yet been assessed in intervertebral disc tissue homeostasis.

Using in vitro, ex vivo, and in vivo techniques, we evaluated the pathophysiological role of the PKCδ pathway by examining (i) proteoglycan deposition; (ii) matrix-degrading enzyme production and activity; (iii) downstream signaling pathways regulated by PKCδ; and (iv) the effect on in vivo models of disc degeneration in genetically-engineered PKCδ knockout mice.