Objective. To investigate the expression and function of Mohawk (MKX) in human adult anterior cruciate ligament (ACL) tissues and ligament cells from normal and osteoarthritis-affected knees.
Methods. Knee joints were obtained at autopsy within 24-48 hours postmortem from 13 normal donors (age 36.9±11.0 years), 16 OA donors (age 79.7±11.4 years) and 8 old donors without OA (age 76.9±12.9 years). All cartilage surfaces were graded macroscopically. MKX expression was analyzed by immunohistochemistry and quantitative PCR. ACL-derived cells were used to study regulation of MKX expression by IL-1β. MKX was knocked down by siRNA to analyze function of MKX in extracellular matrix (ECM) production and differentiation in ACL-derived cells.
Results. The expression of MKX was significantly decreased in ACL-derived cells from OA knees compared with normal knees. Consistent with this finding, immunohistochemistry showed that MKX positive cells were significantly reduced in ACL tissues from OA donors in particular in cells located in disorientated fibers. In ACL-derived cells, IL-1β strongly suppressed MKX gene expression and reduced ligament ECM genes, COL1A1 and TNXB. On the other hand, SOX9, chondrocyte master transcription factor, was up regulated by IL-1β treatment. Importantly, knock down of MKX expression by siRNA upregulated SOX9 expression in ACL-derived cells, whereas the expression of COL1A1 and TNXB were decreased.
Conclusion. Reduced expression of MKX is a feature of degenerated ACL in OA-affected joints and this may be in part mediated by IL-1β. MKX appears necessary to maintain the tissue specific cellular differentiation status and ECM production in adult human tendons and ligaments. © 2013 American College of Rheumatology.
Objective: To compare commonly-available tests for antibodies to citrullinated protein antigens (ACPAs) for diagnostic accuracy and assay agreement in established rheumatoid arthritis (RA) and subjects at elevated risk for RA.
Methods: ELISA testing for anti-cyclic citrullinated peptide (anti-CCP) antibodies was performed using CCP2 (Axis-Shield) and CCP3.1 (IgA/IgG INOVA) in the following subjects: 1) probands with established RA (N=340) from the Studies of the Etiology of RA (SERA), 2) first degree relatives (FDRs) without RA (family members of SERA RA probands; N=681), 3) Department of Defense Serum Repository (DoDSR) RA cases with pre-diagnosis samples (N=83; 47/83 also had post-diagnosis samples), and 4) blood-donor and DoDSR controls (N=283).
Results: In established RA, CCP2 was more specific (99.2% vs. 93.1%, p<0.01), but less sensitive (58.7% vs. 67.4%, p=0.01) than CCP3.1, with specificity of CCP3.1 increasing to 97.2% if levels ≥3 times the standard cut-off level were considered. In all subjects, at standard cut-off levels, CCP3.1 positivity was more prevalent. In DoDSR cases, CCP2 was more specific than CCP3.1 for a future diagnosis of RA, and higher CCP levels trended towards greater specificity for disease onset within 2 years. At standard cut-off levels, assay agreement was good in established RA (kappa=0.76), but poor in FDRs without inflammatory arthritis (kappa=0.25).
Conclusion: Anti-CCP assays differ to an extent that may be meaningful in diagnosing RA in patients with inflammatory arthritis, and in evaluating the natural history of RA development in subjects at-risk for future RA. Mechanisms underlying these differences in test performance need further investigation. © 2013 American College of Rheumatology.
Background: Sonography is a sensitive tool for evaluating synovitis in rheumatoid arthritis (RA) compared with clinical assessment. However, differences between these assessments may depend on how “joint activity” is defined. Therefore, we compared clinically vs. sonographically active joints in RA by different sonographic definitions.
Methods: Sonographic assessment of finger and wrist joints (11 joints) of each hand was performed in RA patients in remission (n=60; CDAI ≤ 2.8). Grey scale (GS) and power Doppler (PD) signals were evaluated on a 4-point (0=no, 3=severe) scale. We investigated sensitivity and specificity of swollen joint counts using increasingly stringent sonographic definitions of “active joint” as reference. Sonographic findings were also correlated to other clinical variables, including HAQ. Follow-up analyses were performed after 6 to 12 months.
Results: GS was positive in 67.2% of 1320 assessed joints and PD in 20.4%. Clinical joint swelling was 100% specific for sonographic activity independent of the stringency of definition; maximum sensitivity was 25% for the most stringent definition (i.e. GS=3 and PD=3).
Further, patients with higher PD signals (PD=3) showed higher HAQ levels (HAQ 0.45±0.62) compared to patients with lower PD signals (HAQ 0.20±0.35). Higher grades of PD signals at baseline were found in joints which were clinically swollen at the consecutive visit.
Conclusion: Low grade PD and GS signals may not necessarily reflect active synovitis, high grade PD correlates well with clinical joint swelling and clinical activity and high grade PD signals are associated with higher degrees of functional impairment. © 2013 American College of Rheumatology.
Objectives: CD8T cells lacking CD28 were originally reported by Wedderburn and colleagues as a characteristic feature of JIA, but the relevance of these unusual cells to JIA remains to be elucidated. Because of recent evidence that CD28 loss is typical of terminally differentiated lymphocytes, we examined for functional subsets of CD8T cells in JIA.
Methods: Following informed consent/assent, blood and/or waste synovial fluid were collected from children with definite diagnosis of JIA (n = 98). De-identified blood (n = 33) and cord blood (n = 13) samples from healthy donors were also collected. CD8T and CD4T cells were screened for novel receptors, and where indicated, bioassays were performed to determine functional relevance of the identified receptor.
Results: Patients had a naïve T cell compartment with shortened telomeres, and their entire T cell pool had reduced proliferative capacity. They had an over abundance of CD31+CD28null CD8T cells, which was a significant feature of oligoarticular JIA (n = 62) compared to polyarticular JIA (n = 36). CD31+CD28null CD8T cells had limited mitotic capacity, and expressed high levels of the senescence antigens γH2Ax and/or p16. Ligation of CD31, independent of the TCR, sufficiently induced tyrosine phosphorylation, vesicle exocytosis, and production of IFN-γ and IL-10.
Conclusion: These data provide the first evidence for cell senescence, represented by CD31+CD28null CD8T cells, in the pathophysiology of JIA. Activation of these unusual cells in a TCR-independent manner suggests they are maladaptive, and could be potential targets for immunotherapy. © 2013 American College of Rheumatology.
Objective: Methotrexate (MTX) monotherapy (MM) is recommended as the initial rheumatoid arthritis (RA) DMARD. We demonstrate for the first time in a blinded trial that initial MTX (IM) with the option to step-up to combination therapy results in similar outcomes to initial combination therapy (IC).
Methods: The TEAR trial randomized 755 early poor prognosis RA participants to MTX (IM) versus IC (MTX+etanercept or MTX+sulfasalazine +hydroxychloroquine,). IM participants stepped-up to combinations at 24 weeks if DAS28 was > 3.2.
Results: Attrition at 24 weeks was similar in IM and IC at 24 weeks. Of the 370 evaluable participants in the IM group, 28% achieved low disease activity and did not step-up to combination therapy (MM group). DAS28-ESR for participants remaining on MM at week 102 was 2.7±1.2, similar to participants treated with immediate combinations (2.9±1.2 ). Participants on MM had less radiographic progression at week 102 compared to participants who received immediate combination therapy (∆TSS 0.2±1.1 vs. 1.1±6.4). Participants assigned to IM requiring step-up at 24 weeks (72%, SU) demonstrated similar DAS28-ESR values (week 48: 3.5±1.3 vs. 3.2±1.3) and radiographic progression (week 102: ∆TSS1.2±4.1 vs. 1.1±6.4) as those treated with initial combination therapy. Results for either immediate combination approach, triple therapy or MTX+etanercept were similar.
Conclusions: These results in early, poor prognosis RA validate the strategy of starting with MTX mono-therapy. Approximately 30% of patients will not need combination therapy and the 70% who need it are clinically and radiographically indistinguishable from those that received immediate combination therapy. © 2013 American College of Rheumatology.
Studies in human volunteers have shown that vitamin C reduces serum urate (SU). The aim of this study was to determine effects of vitamin C on SU in patients with gout.
Patients with gout and SU >0.36mmol/L were recruited. 20 patients already receiving allopurinol were randomised to an increase in allopurinol dose or commenced on vitamin C 500mg/d. 20 patients not receiving allopurinol were randomised to start allopurinol or vitamin C 500mg/d. Plasma ascorbate, creatinine, and SU were measured at day 0 and week 8.
There was no significant difference in baseline SU or eGFR between those who received vitamin C and those who did not (SU 0.50±0.11mmol/l vs. 0.50±0.09mmol/l p=0.89; eGFR 65.5±15.7ml/min/1.73m2 vs. 67.9±20.7 ml/min/1.73m2 p=0.67). 30% in the vitamin C group and 25% in the no vitamin C group were receiving diuretics (p=0.72).
In the patients receiving vitamin C there was a significant increase between week 0 and 8 in plasma ascorbate. The reduction in SU was significantly less in those patients receiving vitamin C compared to those who started or increased the dose of allopurinol (0.014mmol/l vs. 0.118mmol/l p<0.001).
Modest dose vitamin C (500mg/d) for 8 weeks had no clinically significant urate lowering effect in patients with gout despite increasing plasma ascorbate. These results differ from findings in hyperuricaemic healthy controls. The uricosuric effect of modest dose vitamin C appears less in patients with gout both as monotherapy and in combination with allopurinol. © 2013 American College of Rheumatology.
Objectives: Hypocomplementemic urticarial vasculitis syndrome (HUVS) is characterized by recurrent urticaria along with dermal vasculitis, arthritis, and glomerulonephritis. Systemic lupus erythematosus (SLE) develops in more than half of the affected subjects, while etiopathogenesis is unknown.
Methods/Results: The aim of this study is to find the causative DNA mutations in two families with autosomal recessive HUVS to unveil the pathogenesis and to aid in the laboratory diagnosis. By combining autozygosity mapping with whole-exome sequencing in a family with three affected children, we identified a homozygous frameshift mutation, c.289_290delAC, in DNASE1L3. We subsequently found another homozygous DNASE1L3 mutation leading to exon skipping, c.320+4delAGTA, in an unrelated family. Detected mutations lead to the loss of function, either with nonsense mediated mRNA decay or abolished endonuclease activity demonstrated by a plasmid nicking assay.
Conclusion: These results show that HUVS is caused by mutations in DNASE1L3, encoding an endonuclease, which has been previously associated with SLE. © 2013 American College of Rheumatology.
Objective: 1) To use structural magnetic resonance imaging (MRI) to characterize changes in gray matter (GM) and white matter (WM) volumes 1) between patients with childhood-onset SLE (cSLE) and matched controls, 2) between cSLE patients with and without neurocognitive deficit (NCD) and 3) in relation to disease duration and treatment with steroids.
Methods: 22 cSLE patients and 19 healthy controls underwent high-resolution structural MRI. Probability density maps for GM and WM were compared between groups.
Results: Neuropsychological testing confirmed NCD to be present in eight cSLE patients (cSLE-NCD group). Multiple brain regions had reduced GM volume in the cSLE-NCD group vs. controls or cSLE patients without NCD. Neither disease duration nor cumulative oral or intravenous steroid doses accounted for decreases in GM. WM volume was also reduced in cSLE-NCD, and the reduction was positively associated with both disease duration and cumulative oral steroid dose. Conversely, higher cumulative intravenous steroid doses were associated with higher WM volumes.
Conclusions: NCD in cSLE patients is associated with multifocal decreases in both GM and WM volumes. Since only WM volume changes are related to disease duration and cumulative oral steroid use, this may suggest that GM and WM alterations relate to different underlying mechanisms. Further work is needed to understand the relationship between GM and WM alterations in cSLE, whether the underlying mechanisms relate to immunologic, vascular or other causes, and whether the changes are reversible or preventable. Likewise the protective properties of intravenous steroids in maintaining WM volumes require confirmation in larger cohorts. © 2013 American College of Rheumatology.
Objective: Systemic lupus erythematosus (SLE) is a prototype autoimmune disease, assumed to occur due to a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. Here, our objective was to identify the cause of an autosomal recessive form of SLE.
Methods: We investigated three siblings from one consanguineous kindred with juvenile-onset SLE and used next generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunological and functional assays to assess the impact of the identified mutations on B cell biology.
Results: We identified a homozygous missense mutation in PRKCD, encoding protein kinase delta (PKCδ) in all three affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ, involved in the deletion of autoreactive B cells, leading to a resistance to BCR- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increase of immature B cells in affected patients, and a developmental shift toward an immature phenotype of naïve B cells.
Conclusion: Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE. © 2013 American College of Rheumatology.
Objective: Although elevated IL-7 levels were reported in patients with primary Sjögren's syndrome (pSjS), the role of IL-7 in this disease remains unclear. Here we characterized the previously unexplored role of IL-7 in the development and onset of pSjS using C57BL/6.NOD-Aec1Aec2 (B6.NOD-Aec) mouse model that recapitulates human pSjS.
Methods: For gain-of-function studies, recombinant IL-7 or control PBS was injected intraperitoneally (i.p.) into 12-week old B6.NOD-Aec mice for 8 weeks. For loss-of-function studies, neutralizing anti-IL-7 receptor antibody or its isotype control IgG was administered i.p. into 16-week old B6.NOD-Aec mice. Salivary flow measurement, histological and flow cytometric analysis of salivary glands, and serum antinuclear antibody assay were performed to assess various disease parameters.
Results: Administration of exogenous IL-7 accelerated, whereas blockade of IL-7 receptor signaling almost completely abolished the development of pSjS, based on salivary gland inflammation and apoptosis, autoantibody production and secretory dysfunction. IL-7 positively regulated IFN-γ-producing Th1 and CD8 T cells in the salivary glands without affecting IL-17. Moreover, IL-7 enhanced the expression of CXCR3 ligands in a T cell- and IFN-γ-dependent fashion. Accordingly, IFN-γ induced a human salivary gland epithelial cell line to produce CXCR3 ligands. IL-7 also increased the level of TNF-α, another Th1-associated cytokine that can facilitate tissue destruction and inflammation.
Conclusion: IL-7 plays a pivotal pathogenic role in SjS, which is underpinned by an enhanced Th1 response and IFN-γ-CXCR3 ligand-mediated lymphocyte infiltration of target organs. These results suggest that targeting IL-7 pathway may be a potential future strategy to prevent and treat SjS. © 2013 American College of Rheumatology.
Primary Sjogren's syndrome (pSS) and mice with pSS-like disease are associated with increased IL-7 in salivary glands and serum, correlating with parameters of disease and inflammation. Through gain- and loss-of-function studies in both human and mouse experimental models it has been demonstrated that IL-7 plays a critical role in the immunopathological pathways observed in pSS. IL-7 can contribute to development and onset of pSS by enhancing T cell responses and T cell-dependent activation of glandular epithelial cells and immune cells such as B cells, DCs and macrophages. IL-7-driven immune activation is furthermore associated with disease phenomena characteristic of pSS, including increased apoptosis of epithelial cells, increased lymphocyte infiltration and neogenesis, production of ANA antibodies and reduced salivary output. These data justify careful evaluation of the clinical benefits, potentially achieved by interference with this pathway. © 2013 American College of Rheumatology.
Objective. To determine whether HLA-B27 expression alters the response of bone marrow monocytes (BMMo) from HLA-B27/human β2-microglobulin transgenic (B27-Tg) rats to tumor necrosis factor-α (TNFα), and whether this affects cells involved in bone homeostasis.
Methods. BMMo were treated with receptor activator of NF-κB ligand or TNFα to promote osteoclast formation. Osteoclasts were quantified by counting. Gene expression was measured using quantitative polymerase chain reaction, and protein was detected by enzyme-linked immunosorbent assay, immunoblotting, or immunofluorescence. Effects of endogenously produced cytokines on osteoclast formation were determined with neutralizing antibodies.
Results. TNFα enhanced osteoclast formation 2.5-fold in HLA-B27-expressing cells compared to either wild type or HLA-B7/human β2-microglobulin expressing monocytes. TNFα induced approximately 4-fold upregulation of HLA-B27, which was associated with accumulation of misfolded heavy chains, binding of the ER chaperone BiP, and activation of an ER stress response, which was not seen with HLA-B7. No differences were seen with RANKL-induced osteoclastogenesis. Enhanced interleukin-1α (IL-1α) production from ER stressed B27-Tg BMMo was found to be necessary and sufficient for enhanced osteoclast formation. However, B27-Tg BMMo also produced more interferon-β (IFNβ), which attenuated the effect of IL-1α on osteoclast formation.
Conclusions. HLA-B27-induced ER stress alters the response of BMMo from B27-Tg rats to TNFα, which is associated with enhanced production of IL-1α and IFNβ, cytokines that exhibit opposing effects on osteoclast formation. The altered response of cells expressing HLA-B27 to pro-inflammatory cytokines suggests that this MHC class I allele may contribute to the pathogenesis of spondyloarthritis and its unique phenotype through downstream effects involving alterations in bone homeostasis. © 2013 American College of Rheumatology.
Although articular cartilage has evolved to facilitate joint mobilization, severe loading can induce chondrocyte apoptosis, which is related to progression of osteoarthritis. To avoid apoptosis, chondrocytes synthesize heat shock proteins (Hsp). This study examines the role of Hsp70 and Hsp90 in biomechanically-induced osteoarthritis, and the possibility for Hsp90 inhibition (Hsp90i) as an intervention strategy for osteoarthritis management.
Osteoarthritis was biomechanically induced in rats by means of strenuous running. Disease progression was compared between running rats treated with Hsp90i and untreated running controls. From articular cartilage of both groups, Hsp70 and Hsp90 protein levels were determined using Western blots. Osteoarthritis progression was monitored with contrast-enhanced microCT to measure cartilage degradation and subchondral bone changes, with SPECT/CT to determine synovial macrophage activation and histology.
Chronic cartilage loading led to early osteoarthritis development, characterized by degeneration of cartilage ECM. In vivo Hsp90i resulted in increased Hsp70 synthesis, which suggests that Hsp90 activity limits Hsp70 production. Hsp90i treatment improved cartilage sGAG levels to concentrations even beyond baseline and (1) protected against cartilage degradation, (2) stimulated subchondral bone thickness, and (3) suppressed macrophage activation.
Hsp90 plays a pivotal role in biomechanically-induced chondrocyte stress responses. Intervention strategies that inhibit Hsp90 can potentially protect or improve cartilage health and might prevent osteoarthritis development. © 2013 American College of Rheumatology.
Elevated serum osteoprotegerin (OPG) represents an independent risk factor for atherosclerotic disease, although the underlying mechanism is not clear. Here, we investigated the association of serum OPG and circulating endothelial progenitor cells (EPCs) numbers, and explored the effect of OPG on EPCs apoptosis and its underlying mechanisms.
EPCs were enumerated in peripheral blood of 91 patients with systemic lupus erythematosus (SLE) using flow cytometry. Cultured EPCs, isolated from peripheral blood, were challenged with OPG and apoptosis was evaluated by TUNEL staining. Apoptosis related proteins were measured by quantitative real-time PCR (qPCR) and Western blot analysis. Reactive oxygen species (ROS) was detected by flow cytometry, and the expression of NADPH oxidase (Nox) and mitogen-activated protein kinases (MAPK) was measured by qPCR and Western blot analysis.
Serum OPG level was independently associated with EPCs reduction in SLE patients. In vitro treatment of OPG significantly induced the apoptosis of EPCs. This effect was mediated by syndecan-4 on EPCs. OPG-induced apoptosis was abolished by ROS scavenger N-acetyl cysteine and Nox inhibitor diphenyleniodonium. OPG increased ROS production through activation of Nox2 and Nox4, and triggered phosphorylation of ERK1/2 and p38 MAPK. Quenching of ROS by knockdown of Nox2 or Nox4 transcripts inhibited phosphorylation of ERK1/2 and p38 MAPK. Moreover, inhibitors of ERK1/2 and p38 MAPK decreased ROS production and subsequent EPCs apoptosis, indicating a feed forward loop between Nox and MAPK to amplify ROS production related to apoptosis.
Elevated OPG increases the apoptosis of EPCs by induction of oxidative stress. © 2013 American College of Rheumatology.
Stiffening of the joint is a feature of knee osteoarthritis, which can be caused by fibrosis of the synovium. The infrapatellar fat pad (IPFP) present in the knee joint produces immune modulatory and angiogenic factors. The goal of the present study was to investigate whether IPFP can influence fibrotic processes in synovial fibroblasts and to determine the role of Transforming Growth Factor β and Prostaglandin F2α in these processes.
Batches of fat conditioned medium (FCM) were made by culturing pieces of IPFP obtained from 13 osteoarthritic knees. Passage 3 human OA fibroblast-like synoviocytes (FLS) were cultured in FCM with(out) TGFβ receptor/ALK5 or PGF2α inhibitors for 4 days and analyzed for collagen production, and Plod2 gene expression (encoding LH2b, an enzyme involved in collagen cross-linking), α-smooth muscle actin (Asma) and Col1. In parallel, proliferation and migration of the synoviocytes was analyzed.
FCM stimulated collagen production 1.8 times (p < 0.05), Plod2 gene expression 6.0 times (p < 0.01), migration, and proliferation of synoviocytes. Collagen production was positively associated with PGF2α levels in the FCM (R = 0.89, p < 0.05) and inhibition of PGF2α reduced the FCM-induced collagen production and Plod2 gene expression. Inhibition of TGFβ signaling did not alter profibrotic changes.
These results indicate that IPFP can contribute to the development of synovial fibrosis by increasing collagen production, Plod2 gene expression, cell proliferation and cell migration. Based on our results, not TGFβ but the more recently discovered pro-fibrotic factor PGF2α is partially involved in the regulation of profibrotic changes. © 2013 American College of Rheumatology.
In this study we focus on examining the impact of TLR5 on rheumatoid arthritis (RA) endothelial cell function and collagen induced arthritis (CIA) vascularization.
Endothelial migration and tube formation were employed to demonstrate the direct role of TLR5 ligation in angiogenesis. CIA mice were treated with TLR5 agonist, flagellin to document the effect of TLR5 ligation in RA pathology. CIA vascularization was examined by histology and joint cytokine levels and spleen TH-17 cells were quantified by ELISA and FACS analysis. Development of TH-17 cells by TLR5 ligation was validated in RA peripheral blood mononuclear cells.
Ligation of TLR5 to endogenous ligands expressed in RA synovial fluid contributes to endothelial infiltration and tube formation. Further, post onset treatment with flagellin, exacerbates CIA joint inflammation while in the control mice, disease activity reaches the plateau phase. We show that TLR5 enhanced disease severity is due to TH-17 cell differentiation and CIA joint vascularization. When the underlying mechanism was examined in RA peripheral blood mononuclear cells, we found that ligation of myeloid TLR5 and their production of TH-17 promoting cytokines was necessary for TH-17 cell polarization. Additionally we demonstrate that blockade of IL-17 cascade can markedly reduce endothelial migration activated by flagellin condition media suggesting that TLR5 ligation can mediate RA angiogenesis either directly through attracting endothelial cells or indirectly by fostering TH-17 cell development.
Our data demonstrate a novel role for TLR5 in RA angiogenesis hence TLR5 may be a promising new target for RA treatment. © 2013 American College of Rheumatology.
Lysophosphatidic acid (LPA) is a bioactive lipid that binds to a group of cell surface G protein-coupled receptors (LPA1-6) and has been implicated as an important mediator of angiogenesis, inflammation and cancer growth. In this study, we analyzed the effects of LPA1 on development of arthritis.
Expression of LPA receptors on the synovial tissue was analyzed by immunohistochemistry and quantitative RT-PCR. Effect of abrogation of LPA1 on collagen-induced arthritis (CIA) was evaluated using LPA1-deficient mice or LPA1 antagonist. Migrated fluorescence labeled-CD11b+ splenocytes, which were transferred into CIA, into the synovium were counted. CD4+ naïve T cells were incubated with T helper (Th)1-, Th2-, or Th17-polarizing condition and Th differentiation was analyzed. Osteoclast formation from bone marrow cells was examined.
LPA1 was highly expressed in the synovium of rheumatoid arthritis (RA) compared to osteoarthritis. LPA1-deficient mice did not develop arthritis following collagen type-II (CII) immunization. LPA1 antagonist also ameliorated murine CIA. Abrogation of LPA1 was associated with reduced cell infiltrates, bone destruction in the joints, and IL-17 production from CII-stimulated splenocytes. Infiltration of transferred LPA1-deficient CD11b+ macrophages into the synovium was suppressed compared with wild-type macrophages. LPA1 antagonist inhibited the infiltration of wild-type macrophages. Differentiation into Th17, but not Th1 or Th2, and osteoclast formation were also suppressed in LPA1-deficiency or LPA1 inhibition in vitro.
Collectively, the results indicate that LPA-LPA1 signaling contributes to the development of arthritis by cellular infiltration, Th17 differentiation and osteoclastogenesis, suggesting that LPA1 is a promising target molecule for RA therapy. © 2013 American College of Rheumatology.
The aetiology of osteoarthritis(OA) is complex with genetic, developmental, biochemical and biomechanical factors contributing to the disease process. Chondrocytes in articular cartilage must express appropriate genes to achieve tissue homeostasis and this is altered in OA. One facet of the aberrant gene expression in OA is the replay of chondrocyte differentiation with the expression of genes associated with chondrocyte hypertrophy. The pattern of gene expression and the transcription factors which control chondrogenesis are known in some detail. Mechanisms leading to altered gene expression in OA are less well understood.
MicroRNAs (miRNAs) are small non-coding RNAs that have recently been recognised as important regulators of gene expression in human cells. A number of miRNAs are regulated across chondrogenesis and their function is beginning to be delineated. Similarly miRNAs are differentially expressed in OA cartilage compared to normal tissue. MicroRNA-140, highly and selectively expressed in cartilage, has been the focus of much work to date, though the full gamut of its actions is still to be defined. Many other regulated miRNAs will likely act as a network to control cartilage homeostasis, catabolism and repair.
This review summarizes current knowledge in this area which will inform future research. © 2013 American College of Rheumatology.
The lipid mediator Sphingosine-1-phosphate (S1P) is found in the synovial fluid of osteoarthritis (OA) patients. S1P protects bovine cartilage by counteracting interleukin-1β (IL-1β). Here we examine the interaction of S1P and IL-1β in human OA chondrocytes.
Human cartilage was obtained from patients undergoing total knee joint replacement. Chondrocytes were cultured in monolayer and treated with IL-1β and S1P. Expression of S1P receptor sub-types and genes involved in cartilage degradation was evaluated using real-time PCR, immunohistochemistry and western blot. S1P signaling was evaluated using inhibitors of S1P receptors and siRNA knock-down of the S1P2 receptor. Phosphorylation of MAP kinases (MAPK) and NF-κB in response to IL-1β and S1P was detected by western blot.
S1P2 was the most prevalent S1P receptor subtype in human OA cartilage and chondrocytes in vitro. S1P reduced iNOS expression in IL-1β treated chondrocytes. Reduction of ADAMTS4 and MMP-13 expression by S1P correlated with S1P2 expression. Pharmacological inhibition of the S1P2 receptor but not S1P1 and S1P3 receptors abrogated inhibition of iNOS expression. Similar results were observed using siRNA knock-down. S1P signaling inhibited IL-1β induced phosphorylation of p38MAPK.
In human chondrocytes, S1P reduces the induction of catabolic genes in the presence of IL-1β. Activation of the S1P2 receptor counteracts the detrimental phosphorylation of p38MAPK by IL-1β. © 2013 American College of Rheumatology.
Low-amplitude whole-body vibration has been adopted for the treatment of back pain and spine pathologies; however, there is limited knowledge of the impact of vibration on the intervertebral disc (IVD). We examined the effects of acute vibration on anabolic and catabolic pathways in the IVD and characterized the dependence of these changes on time and frequency.
Custom-designed platforms were developed to apply acute vibration to ex vivo and in vivo mouse models. Spinal segments (ex vivo) or mice (in vivo) were subjected to vibration (30 min, 15-90 Hz, peak acceleration 0.3 g) and IVDs were examined at specific times post-vibration. Gene expression was quantified using real-time PCR and protein levels were examined by quantitative mass spectrometry and immunofluorescence.
In the ex vivo model, acute vibration at 15 Hz induced expression of anabolic genes (aggrecan, biglycan, decorin, type I collagen, Sox9) and suppressed expression of Mmp13, with the most pronounced changes detected 6 h following vibration. These beneficial effects were frequency dependent, no longer evident between 45-90 Hz. In vivo, the effects on anabolic gene expression were even more robust and were accompanied by decreased expression of Adamts4/5 and Mmp3. Moreover, significant increases in the protein levels of type I collagen biglycan, decorin and aggrecan were detected in vivo.
These findings demonstrate dramatic anabolic effects of acute vibration on IVD tissues, responses that are dependent on frequency. Similarity of responses in vivo and ex vivo indicates that at least some effects of vibration are tissue autonomous. © 2013 American College of Rheumatology.
Objective. Genetic and environmental factors may contribute to the etiology of the juvenile idiopathic inflammatory myopathies (JIIM), systemic autoimmune diseases characterized by muscle and skin inflammation. We investigated the association between ultraviolet radiation (UVR) exposure and the clinical and autoantibody expression of JIIM.
Methods. We assessed the relationship between UVR exposure in the month before symptom onset and prevalence of juvenile dermatomyositis (JDM) versus polymyositis (JPM) in 298 patients. Among JDM patients, the association between UVR exposure and myositis autoantibodies was assessed. Regression models were stratified by sex and race. The association between regional UV index in U.S. geoclimatic zones and the clinical and autoantibody subgroups was examined by weighted least squares regression analysis.
Results. We observed increasing odds of JDM compared with JPM per unit increase in the patients' highest UV index in the month before symptom onset in girls (OR = 1.18; 95% CI = 1.00-1.40). The average and highest UV indices were associated with increasing odds of anti-p155/140 autoantibodies, which was strongest in white males (OR 1.30 and 1.23, respectively). No association was observed between the UV index and anti-MJ autoantibodies or patients without myositis autoantibodies. Across US geoclimatic regions, the average UV index was associated with increasing odds of JDM and anti-p155/140 autoantibodies but decreasing odds of anti-MJ autoantibodies.
Conclusion. Short-term UVR exposure prior to illness onset may have a role in the clinical and serologic expression of juvenile myositis. Research examining mechanisms of UVR in JIIM pathogenesis is suggested from these findings. © 2013 American College of Rheumatology.
Meniscus tears are associated with a heightened risk for osteoarthritis. We aimed to advance our understanding of the metabolic state of human injured meniscus at the time of arthroscopic partial meniscectomy through transcriptome-wide analysis of gene expression in relation to patient age and degree of cartilage chondrosis.
The degree of chondrosis of knee cartilage was recorded at the time of meniscectomy in symptomatic patients without radiographic osteoarthritis. RNA preparations from resected menisci (N=12) were subjected to transcriptome-wide microarray and QuantiGene Plex analyses. The relative changes in gene expression variation with age and chondrosis were analyzed and integrated biological processes were investigated computationally.
We identified a set of genes in torn meniscus that were differentially expressed with age and chondrosis. There were 866 genes differentially regulated (≥1.5-fold; P<0.05) with age and 49 with chondrosis. In older patients, genes associated with cartilage and skeletal development and extracellular matrix synthesis were repressed while those involved in immune response, inflammation, cell cycle, and cellular proliferation were stimulated. With chondrosis, genes representing cell catabolism (cAMP catabolic process) and tissue and endothelial cell development were repressed and those involved in T cell differentiation and apoptosis were elevated.
Differences in age-related gene expression suggest that in older adults, meniscal cells might de-differentiate and initiate a proliferative phenotype. Conversely, meniscal cells in younger patients appear to respond to injury, but maintain the differentiated phenotype. Definitive molecular signatures identified in damaged meniscus could be segregated largely with age and, to a lesser extent, with chondrosis. © 2013 American College of Rheumatology.
The aim of this study was to identify epigenetic factors that are implicated in the pathogenesis of rheumatoid arthritis (RA) and to explore the therapeutic potential of the targeted inhibition of these factors.
PCR arrays were utilized to investigate the expression profile of genes that encod key epigenetic regulator enzymes. Mononuclear cells from RA patients and mice were monitored for gene expression changes, in association with arthritis development in murine models of RA. Selected genes were further characterized by quantitative real-time PCR, Western blot and flow cytometry methods. The targeted inhibition of the upregulated enzymes was studied in arthritic mice.
A set of genes with arthritis-specific expression was identified by the PCR arrays. Aurora kinase A and B, both of which were highly expressed in arthritic mice and treatment naïve RA patients, were selected for detailed analysis. Elevated Aurora kinase expression was accompanied with an increased phosphorylation of histone H3, which promotes proliferation of T lymphocytes. Treatment with VX-680, a pan-Aurora kinase inhibitor, promoted B cell apoptosis, provided significant protection against the onset, and attenuated the inflammatory reactions in arthritic mice.
Arthritis development is accompanied the changes in the expression of a number of epigenome-modifying enzymes. Drug-induced downregulation of the Aurora kinases, among other targets, seems to be sufficient to treat experimental arthritis. Development of new therapeutics that target the Aurora kinases can potentially improve RA management. © 2013 American College of Rheumatology.
Objective: To examine whether MRI-based 3D bone shape predicts the onset of radiographic knee osteoarthritis (OA).
Methods: We conducted a case-control study within the Osteoarthritis Initiative by identifying knees that developed incident tibiofemoral radiographic knee OA (case knees) over follow-up, and matching them to two random control knees. Using knee MRI's, we used active appearance modeling of the femur, tibia and patella and linear discriminant analysis to identify vectors that best classified knees having OA vs. not. Vectors were scaled such that -1 and +1 represented the mean non-OA and mean OA shapes, respectively. We examined the relation of 3D bone shape to incident OA (new onset Kellgren and Lawrence (KL) grade ≥2) occurring 12 months later using conditional logistic regression.
Results: 178 case knees (incident OA) were matched to 353 control knees. The whole joint (i.e., tibia, femur, and patella) 3D bone shape vector had the strongest magnitude of effect, with knees in the highest tertile having 3.0 times higher likelihood of developing incident radiographic knee OA 12 months later compared with those in the lowest tertile (95% CI 1.8-5.0, p<0.0001). The associations were even stronger among knees that showed completely normal radiographs before incidence (KL grade 0) (OR 12.5, 95% CI 4.0-39.3). Bone shape at baseline, often several years before incidence, predicted later OA.
Conclusions: MRI-based 3D bone shape predicted the later onset of radiographic OA. Further study is warranted to determine whether such methods can detect treatment effects in trials and provide pathophysiologic insight into OA development. © 2013 by the American College of Rheumatology
We examined the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium.
We utilized the RA synovial tissue (ST) severe combined immunodeficient (SCID) mouse chimera system to examine human dermal microvascular endothelial cell (HMVEC) and human endothelial progenitor cell (EPC) recruitment into engrafted human synovium injected intragraft with RA synovial fluid (SF) immunodepleted of CXCL16. CXCR6 deficient (CXCR6-/-) and wild-type (Wt) C57BL/6 mice were primed to develop K/BxN serum induced arthritis and evaluated for angiogenesis. HMVECs and EPCs from human cord blood were also examined for CXCR6 expression by immunofluorescence and signaling activity for CXCL16.
We found that CXCR6 is prominently expressed on human EPCs and HMVECs and can be upregulated by interleukin-1β (IL-1β). SCID mice injected intragraft with RA SF immunodepleted of CXCL16 showed a significant reduction in EPC recruitment. Using the K/BxN serum induced inflammatory arthritis model, CXCR6-/- mice showed profound reductions in hemoglobin (Hb) levels that correlated with reductions in monocyte and T-cell recruitment to arthritic joint tissue in CXCR6-/- compared to wildtype (Wt) mice. We also found that HMVECs and EPCs respond to CXCL16 stimulation but have unique signal transduction pathways and homing properties.
These results indicate that CXCL16 and its receptor CXCR6 may be a central ligand-receptor pair that can be highly correlated with EPC recruitment and blood vessel formation in the RA joint. © 2013 American College of Rheumatology.
Aging-associated changes in articular cartilage represent a main Osteoarthritis (OA) risk factor. Autophagy is an essential cellular homeostasis mechanism. Aging-associated or experimental defects in autophagy contribute to organismal and tissue specific aging while enhancement of autophagy may protect against certain aging related pathologies such as OA. The objective of this study was to determine whether glucosamine (GlcN) could activate autophagy.
Chondrocytes from normal human articular cartilage were treated with GlcN (0.1-10 mM). Autophagy activation and phosphorylation levels of Akt, FoxO3 and ribosomal protein S6 (prbS6) were determined by Western blotting. Autophagosome formation was analyzed by microscopy. Transgenic reporter mice with green fluorescent protein fused to LC3 (GFP-LC3 mice) were used to test changes in autophagy in response to starvation and GlcN administration.
GlcN treatment of chondrocytes activated autophagy as indicated by increased of LC3-II levels, formation of LC3 puncta and increased LC3 turnover. This was associated with GlcN-mediated inhibition of Akt, FoxO3 and mTOR pathway. Administration of GlcN to GFP-LC3 mice markedly activated autophagy in articular cartilage.
GlcN modulates molecular targets of the autophagy pathway in vitro and in vivo and the enhancement of autophagy was mainly dependent on the Akt/FoxO and mTOR pathway. These findings suggest that GlcN is an effective autophagy activator and motivate future studies on its efficacy in modifying aging-related cellular changes and supporting joint health. © 2013 American College of Rheumatology.
Ankylosing spondylitis (AS) is a highly heritable common inflammatory arthritis targeting the spine and sacroiliac joints of the pelvis, causing pain and stiffness leading eventually to joint fusion. While IL23Ris strongly associated with AS in white Europeans, previous studies in East Asian populations have shown no association with common variants of IL23R, suggesting they either play no role or rare genetic variants contribute. We therefore screened IL23R to identify rare variants associated with AS in Han Chinese.
A 170kb region containing IL23R and its flanking regions was sequenced in 50 Han Chinese cases and 50 ethnically matched controls, as well as the 30kb region of peak association in white Europeans in 650 cases and 1300 controls. Validation genotyping was undertaken in 872 cases and 1397 controls.
We identified 1047 variants, of which 729 were not found in dbSNP b130. Several potentially functional rare variants in IL23R were identified, including one non-synonomous (ns) SNP Gly149Arg (chr1:67421184 GA). Validation genotyping showed that the Gly149Arg variant is associated with AS (P=0.0054, OR=0.61).
This is the first study to implicate rare IL23R variants in AS aetiopathogenesis, and has identified a low frequency nsSNP with predicted loss of function effects that is protectively associated with AS in Han Chinese, suggesting that decreased IL-23R function protects against AS. These findings further support an important role for IL23-signalling in AS. © 2013 American College of Rheumatology.
To evaluate subchondral bone trabecular integrity (BTI) from a radiograph as a predictor of knee osteoarthritis (OA) progression.
Longitudinal (baseline, 12- and 24-month) knee radiographs were available from 60 female subjects with knee OA. OA progression was defined by 12- and 24-month change in radiographic medial compartment minimal joint space width (JSW) and medial joint space area (JSA), and medial tibial and femoral cartilage volume from magnetic resonance imaging. Bone Trabecular Integrity (BTI) of the medial tibial plateau was analyzed by fractal signature analysis with a commercially available software. Receiver Operating Characteristic curves of BTI were used to predict 5% change in OA progression parameters.
Individual terms (linear and quadratic) of baseline BTI of vertical trabeculae predicted knee OA progression based on 12- and 24- month change in JSA (p<0.01 for 24 months), 24-month change in tibial (p<0.05) but not femoral cartilage volume, and 24-month change in JSW (p=0.05). ROC utilizing both terms of baseline BTI predicted 5% change in the OA progression parameters over 24 months with high accuracy as reflected by the area under the curve (AUC) measures: JSW 81%, JSA 85%, tibial 75% and femoral 85% cartilage volume. Change in BTI was also significantly associated (p<0.05) with concurrent change in JSA over 12 and 24 months and change in tibial cartilage volume over 24 months.
BTI predicts structural OA progression as determined by radiographic and MRI outcomes. BTI may therefore be worthy of study as an outcome measure for OA studies and clinical trials. © 2013 American College of Rheumatology.
To examine cancer incidence in patients with systemic sclerosis (SSc) derived from population-based cohort studies by means of meta-analysis.
Five different databases (MEDLINE, Scopus, CINAHL, Web of Science and Cochrane Collaboration databases) were searched as well as reference lists of retrieved studies and review articles published between January 1966 and May 2012. Population-based cohort studies relevant for determining cancer risk for patients with SSc were included. All papers meeting strict inclusion criteria were scrutinized for data on population size, time of follow-up and observed-to-expected cancer ratios, also known as standardized incidence ratios (SIR).
Six articles were included. The pooled SIR for overall cancer was 1.41 (95% CI: 1.18-1.68) with significant heterogeneity, which is a consequence of variability in the participants, outcome, study design and risk of bias among the studies. The pooled SIR of 1.85 (95% CI: 1.49-2.31) for men was significantly higher than that of 1.33 (95% CI: 1.18-1.49) for women (p < 0.01) and stratification for sex eliminated heterogeneity, that means variability across the studies greatly contributed to sex. There were no differences between limited and diffuse SSc (p = 0.77). Significant increases were observed in risk of cancer of the lung, liver, hematological system and bladder as well as of non-Hodgkin lymphoma and leukemia.
SSc are associated with an increased risk of cancer, particularly lung, liver, hematological and bladder cancer, though absolute risk is relatively low. Men with SSc are at higher cancer risk than women. © 2013 American College of Rheumatology.
Objective. CRP is a serum pattern recognition molecule that binds to apoptotic cells and nucleoprotein autoantigens and FcγR. In SLE IC containing nucleoprotein autoantigens activate plasmacytoid dendritic cells (pDC) to produce type I IFN, which contributes to disease pathogenesis. Autoantibody IC are taken up by pDC through FcγRIIa into endosomes where the nucleic acid components activate TLR7 or TLR9. The objective of this study was to investigate the effect of CRP on pDC and monocyte responses to nucleoprotein autoantigens and IC.
Methods. Peripheral blood mononuclear cells (PBMC), purified monocytes and pDC were isolated from healthy volunteers and stimulated with autoantibody IC containing apoptotic cells, snRNPs, or DNA or direct TLR7 and TLR9 agonists. Supernatants were analyzed for IFN-α and cytokines by ELISA and multiplex assays. snRNPs were fluorescently labeled and the effect of CRP on binding, uptake and intracellular localization of autoantibody snRNP complexes was measured by flow cytometry and confocal microscopy.
Results. CRP bound to autoantigen did not induce IFN-α in PBMC or pDC, whereas complexes formed with autoantibody did. Significantly, CRP inhibited the IFN-α response to both α-U1 RNP-snRNPs and α-DNA-DNA, but not to other TLR7 and TLR9 agonists. CRP directly inhibited pDC IFN-α release, promoted pDC differentiation, and increased late endosome localization of autoantigen in pDC and monocytes.
Conclusion. CRP is a regulator of the type I IFN response to SLE IC. CRP increased the intracellular processing of IC in late endosomes, which is associated with decreased synthesis of type I IFN after intracellular TLR activation. © 2013 American College of Rheumatology.
Lupus flares when genetically predisposed people encounter appropriate environmental agents. Current evidence indicates that the environment contributes by inhibiting T cell DNA methylation, causing overexpression of normally silenced genes. DNA methylation depends on both dietary transmethylation micronutrients and Erk-regulated DNA methyltransferase 1 (Dnmt1) levels. We used transgenic mice to study interactions between diet, Dnmt1 levels and genetic predisposition on the development and severity of lupus.
A doxycycline-inducible Erk defect was bred into lupus-resistant (C57BL/6) or lupus-susceptible (C57BL/6xSJL) mouse strains. Doxycycline treated mice were fed a standard commercial diet for eighteen weeks then switched to diets supplemented (MS) or restricted (MR) in transmethylation micronutrients. Disease severity was assessed by anti-dsDNA antibodies, proteinuria, hematuria and histopathology of kidney tissues. Pyrosequencing was used to determine micronutrient effects on DNA methylation.
Doxycycline induced modest levels of anti-dsDNA antibodies in C57BL/6 mice and higher levels in C57BL/6xSJL mice. Doxycycline-treated C57BL/6xSJL mice developed hematuria and glomerulonephritis on the MR and standard but not the MS diet. In contrast C57BL/6 mice developed kidney disease only on the MR diet. Decreasing Erk signaling and methyl donors also caused demethylation and overexpression of the CD40lg gene in female mice, consistent with demethylation of the second X chromosome. Both the dietary methyl donor content and duration of treatment influenced methylation and expression of the CD40lg gene.
Dietary micronutrients that affect DNA methylation can exacerbate or ameliorate SLE disease in this transgenic murine lupus model, and contribute to lupus susceptibility and severity through genetic/epigenetic interactions. © 2013 American College of Rheumatology.
To identify miRNA in human T cells that can explain known anti-inflammatory properties of steroids.
Activated human CD4+ T cells from healthy donors were exposed to 1 μM of methylprednisolone in vitro were subjected to miRNA and mRNA microarray analysis and changes in expression profiles were recorded. Using qPCR, flow cytometry, and ELISA we confirmed suppression of predicted targets and through miRNA transfection experiments suggest mechanistic links.
We identified numerous steroid-responsive genes and miRNA — many known and some novel — including multiple previously unknown pro-inflammatory genes suppressed by methylprednisolone. Further studies using qPCR, flow cytometry, and ELISA demonstrated that methylprednisolone increased the expression of miR-98 and suppressed the levels of predicted targets including interleukin-13 and three TNF receptors FAS, FASL, and TNFRSF1B. Forced expression of miR-98 into T cells resulted in suppression of the same targets.
In this communication we demonstrate a link between miR-98 expression and the effects of methylprednisolone and provide evidence,which suggests that methylprednisolone acts through miR-98 to inhibit specific pro-inflammatory targets. Identification of this anti-inflammatory mechanism of glucocorticoids is important as it may pave the way toward the elusive goal of dissociating adverse from therapeutic effects. © 2013 American College of Rheumatology.
Broad spectrum histone deacetylase (HDAC) inhibitors repress metalloproteinase expression and cartilage degradation in vitro. We examine the ability of one such inhibitor to protect cartilage in vivo. The effects of class-selective HDAC inhibitors (HDACi) and siRNA knockdown of HDACs on metalloproteinase expression and cartilage degradation in vitro was explored.
Destabilisation of the medial meniscus (DMM) was used to assess in vivo activity of the HDACi trichostatin A (TSA). Human articular chondrocytes (HACs) and SW1353 chondrosarcoma cells were treated with cytokines and HDACi TSA, valproic acid (VPA), MS-275, or siRNA to determine the effect on metalloproteinase expression by qRT-PCR. HDACi activity was detected by western blotting. Bovine nasal cartilage (BNC) explant was used to measure cartilage resorption in vitro.
TSA, administered systemically, protected cartilage in the DMM model. TSA, VPA and MS-275 repressed cytokine-induced MMP1, MMP3 and MMP13 in HACs.Knockdown of each class I HDAC diminished IL-1-induced MMP13 expression.All inhibitors prevented degradation of BNC where TSA and MS-275 repressed cytokine-induced MMP expression.
The inhibition of class I HDACs (HDAC1, HDAC2, HDAC3) by MS-275 or by specific depletion is capable of repressing cytokine-induced metalloproteinase expression in cartilage cells and explants, resulting in inhibition of cartilage resorption. This indicates that specific inhibition of Class I HDACs provides a possible therapeutic strategy in the arthritides. © 2013 American College of Rheumatology.
Collagen type II (CII), post-translationally modified by reactive oxygen species (ROS-CII), present in an inflamed joint, is an auto-antigen in rheumatoid arthritis (RA). In this study we investigated the potential use of anti-ROS-CII auto-antibodies as a biomarker.
CII was exposed to oxidants that are present in the rheumatoid joint. Auto-reactivity to ROS-CII was tested by ELISA in synovial fluid (SF) and serum samples taken from various phases of RA including: a)disease modifying anti-rheumatic drug (DMARD) naïve patients with early RA (n=85 serum);b) patients with established RA (n=80 serum and 50 SF), both DMARD responders (DMARD-R) and non-responders (DMARD-NR). As controls we used c) anti-citrullinated peptide antibodies (ACPA) positive individuals with arthralgia (n=58 serum); d) samples from patients with osteoarthritis (OA, n=49 serum and 52 SF)and e)healthy individuals (n=51 serum).
Reactivity in DMARD naïve early RA to ROS-CII was significantly higher than in ACPA positive arthralgia, OA and healthy controls (p<0.0001), with 92.9% binders. There was no significant difference in anti-ROS-CII reactivity between ACPA positive and ACPA negative RA patients, with 93.8% and 91.6% binders, respectively. The sensitivity and specificity of binding to ROS-CII in early RA compared with HC was 92% and 98%, respectively. In established RA DMARD-NR serum reactivity was significantly higher than in DMARD-R (p<0.0001) with 58.3% and 70% binders compared to 7.6% and 60% in serum and SF, respectively. In longstanding RA,auto-reactivity to ROS-CII changed longitudinally.
Auto-antibodies to ROS-CII have the potential to become diagnostic biomarkers for RA. © 2013 American College of Rheumatology.
SIRT6, an NAD+-dependent deacetylase and mono-ADP-ribosyl transferase, is predominantly expressed in the nucleus. It is known to interfere with the NF-κB signaling pathway and thereby has an anti-inflammatory function. Due to the central role of NF-κB in rheumatoid arthritis (RA) development, we postulated that SIRT6 could have anti-arthritic effects.
An adenovirus containing SIRT6 cDNA (Ad-SIRT6) was used to deliver SIRT6 to human rheumatoid fibroblast-like synoviocytes (FLS) in vitro as well as to collagen-induced arthritis (CIA) mice in vivo via a bilateral intraarticular injection into the ankle joints.
In vitro experiments demonstrated that SIRT6 overexpression suppressed the NF-κB target gene expression induced by tumor necrosis factor-α. SIRT6 overexpression inhibited osteoclast differentiation induced by macrophage colony-stimulating factor and receptor activator of NF-κB ligand in bone marrow-derived macrophages. Mice with CIA had an increased incidence of disease and developed arthritis in the hind paws. In contrast, mice injected with Ad-SIRT6 showed attenuated severity of arthritis based on clinical scores, hind paw thickness, and radiological and pathological findings. Moreover, the injection of Ad-SIRT6 down-regulated local and systemic levels of proinflammatory cytokines. After the onset of arthritis, CIA mice injected with Ad-SIRT6 showed significantly decreased arthritis severity from the onset of clinical signs to the end of the study.
These results suggest that blocking the NF-κB pathway by SIRT6 in rheumatoid joints reduces both inflammatory response and tissue destruction. Therefore, the development of an immunoregulatory strategy based on SIRT6 may have therapeutic potential for the treatment of RA. © 2013 American College of Rheumatology.
Quantitative sensory testing (QST) and questionnaire-based assessment have been used to demonstrate features of neuropathic pain in those with musculoskeletal pain. However, their direct relationship has not been investigated in the community. We conducted an observational study to describe the characteristics of joint pain and examine the relationship between QST and the PainDETECT questionnaire.
Warm detection, heat pain, mechanical pain thresholds and mechanical pain sensitivity were determined over the sternum alongside PainDETECT scores in a cross-sectional study of 462 participants from the Chingford Study. Comparisons were made between those with and without joint pain. Logistic regression modelling was used to describe the association between neuropathic pain features, determined by the PainDETECT score, and each of the QST measures individually, adjusting for age, BMI and pain-modifying medication use.
66.2% reported recent joint pain with a median average pain severity of 5/10. There was increased sensitivity to painful stimuli in those with pain compared to the pain free group and this persisted after stratification by pain-modifying medication use. While only 6.7% had possible and 1.9% likely neuropathic pain features using standard PainDETECT thresholds, features of neuropathic pain were common and present in over 50%, with at least moderate severity, of those reporting pain. Heat pain thresholds and mechanical pain sensitivity were significantly associated with features of neuropathic pain identified using PainDETECT, OR 0.88(0.80-0.97), p=0.012 and 1.24(1.04-1.48), p=0.018 respectively.
QST and the PainDETECT questionnaire identified features of neuropathic pain in this community-based study, with significant overlap between the two techniques. © 2013 American College of Rheumatology.
Patients with rheumatoid arthritis (RA) are at increased risk for cardiovascular disease, an observation not explained by traditional cardiac risk factors and generally limited to those with RA-associated autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies (ACPA). We hypothesized that citrullinated proteins within the atherosclerotic plaque can be targeted by ACPA, forming stimulatory immune complexes which propagate the progression of atherosclerosis.
Protein lysates prepared from atherosclerotic segments of human aorta were investigated for the presence of citrulline-modified proteins and specifically citrullinated fibrinogen (cFb) by immunoprecipitation and/or immunoblotting followed by mass spectrometry. Immunohistochemistry was performed in coronary artery plaques for the presence of citrullinated proteins and the PAD4 enzyme. Serum levels of anti-cyclic citrullinated peptide (CCP), anti-citrullinated vimentin (cVim), and anti-cFb antibodies were measured in 134 women with seropositive RA previously characterized for the presence of subclinical atherosclerosis by electron beam CT scan (EBCT). Western analysis of atherosclerotic plaque lysates demonstrated several citrullinated proteins and the presence of cFb was confirmed by immunoprecipitation, and mass spectrometry. Immunohistochemistry demonstrated co-localization of citrullinated proteins and the PAD4 enzyme within the coronary artery plaque. In age-adjusted regression models, antibodies targeting cFb and cit-vimentin, but not CCP2, were associated with an increased aortic plaque burden.
Citrullinated proteins are prevalent within the atherosclerotic plaque, and certain ACPAs are associated with atherosclerotic burden. These observations suggest that targeting of citrullinated epitopes, specifically cFb, within the atherosclerotic plaque could provide a mechanism for accelerated atherosclerosis observed in patients with RA. © 2013 American College of Rheumatology.
Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy of several disorders among which osteo-articular diseases. For such clinical applications, intra-articular (IA) injection of MSCs may be favored for higher safety and specific joint targeting. Although safety of intravenous administration of MSCs has been reported in a number of clinical trials, safety and biodistribution of MSCs after IA injection has not been tested. Our objective was to assess the toxicity of clinical grade human adipose-derived stem cells (hASCs) as well as their biodistribution after IA injection in SCID mice.
SCID mice received IA or IV administration of 106 hASC. Several tissues were recovered at different time points and processed for histology or real-time PCR. We used a highly sensitive assay for monitoring hASC distribution, based on amplification of human-specific alu sequences.
Absence of toxicity was observed after ASC infusion. Alu PCR assay exhibits a high sensitivity (1 hASC/105 murine cells) with a large linear range (1–5x104/105 murine cells). Importantly, 15% of the IA injected hASCs were detectable in the joint for the first month and 1.5% of hASCs engrafted on the long-term, at least 6 months. They were observed in the injected joints and tissues referred as the stem cell niches namely, the bone marrow, the adipose tissue or the muscle.
These data argue for the feasibility and safety of using IA delivery of hASC for the treatment of rheumatic diseases affecting the joints. © 2013 American College of Rheumatology.
The development of pathogenic anti-neutrophil cytoplasmic autoantibodies (ANCAs) can result in systemic small vessel vasculitis. However, the breakdown in immune tolerance that results in the induction and persistence of ANCAs is not well-understood. We hypothesized that abnormal T cell regulation is central to disease pathogenesis and demonstrate here two separate abnormalities in T cell regulation in ANCA-associated vasculitis patients.
Peripheral blood samples were obtained from patients with ANCA-associated vasculitis (n=63) and healthy controls (n=19) for flow cytometric analysis of CD4+ T cell populations. Functional T cell studies were performed with FACS sorted CD4+ T cell populations stimulated with anti-CD3/28.
First, we show that the Treg frequency in the peripheral blood of active disease patients is increased, but Tregs from patients with ANCA-associated vasculitis have decreased suppressive function. Tregs from active disease patients disproportionately utilize a FOXP3 isoform lacking exon 2, which may alter Treg function. Second, we identify a CD4+ T cell population with increased frequency that is resistant to Treg suppression, produces pro-inflammatory cytokines, and is antigen-experienced.
ANCA-associated vasculitis is associated with disruption of the suppressive Treg network and increased frequency of a distinct pro-inflammatory effector T cell subset which comprises the majority of peripheral CD4+ T cells. © 2013 American College of Rheumatology.
Macrophage Activation Syndrome (MAS) is a devastating cytokine storm syndrome complicating many inflammatory diseases and characterized by fever, pancytopenia, and systemic inflammation. It is clinically similar to Hemophagocytic Lymphohistiocytosis (HLH), which is caused by viral infection of a host with impaired cellular cytotoxicity. Murine models of MAS and HLH illustrate Interferon-γ (IFN-γ) as the driving stimulus for hemophagocytosis and immunopathology. We sought to understand the inflammatory contributors to a murine model of Toll-like Receptor 9 (TLR9)-induced fulminant MAS.
Wild-type (WT), transgenic, and cytokine-inhibited mice were treated with an IL-10 receptor blocking antibody and TLR9 agonist, and parameters of MAS were evaluated.
Fulminant MAS was characterized by dramatic elevations in IFN-γ, IL-12, and IL-6. Serum IFN-γ correlated with enhanced IFN-γ production within some hepatic populations, but fewer IFN-γ+ cells. Surprisingly, IFN-γKO mice developed immunopathology and hemophagocytosis comparably to WT mice. However, IFN-γKO mice did not become anemic and had greater numbers of splenic erythroid precursors. IL-12 neutralization phenocopied disease in IFN-γKO mice. Interestingly, Type I interferons contributed to the severity of hypercytokinemia and weight loss, but their absence did not otherwise affect MAS manifestations.
These data demonstrate that both fulminant MAS and hemophagocytosis can arise independently of IFN-γ, IL-12, or Type I interferons. They also suggest that IFN-γ-mediated dyserythropoiesis, not hemophagocytosis, is the dominant cause of anemia in fulminant TLR9-MAS. Thus, our data establish a novel mechanism for the acute anemia of inflammation, but suggest that a variety of triggers can result in hemophagocytic disease. © 2013 American College of Rheumatology.
We investigated the efficacy of different doses of the Fc-portion-conjugated soluble form of the receptor for advanced glycation end products (sRAGE) on nephritis in lupus-prone mice in comparison with the efficacy of combination therapy of mycophenolate mofetil plus prednisolone.
Twenty-eight female NZB/WF1 mice were divided into five groups (untreated; 0.5, 1, 2 μg sRAGE; mycophenolate mofetil plus prednisolone). Proteinuria and histological damage were evaluated. Immune-complex deposition and the nuclear translocation of NF-κB in kidney tissues were assessed by immunofluorescence staining. Serum concentrations of anti-dsDNA and IgG subclasses were also measured. The population of T cells was evaluated using a fluorescence-activated cell sorter and ICAM-1 and VCAM-1 expression in kidney tissues was assessed by immunohistochemical staining.
In comparison with untreated mice, mice treated with 1 or 2 μg sRAGE showed significantly reduced proteinuria and improved histological renal damage, with efficacy comparable to that of combination therapy. Treatment with 1 or 2 μg sRAGE significantly reduced immune-complex deposition and decreased the serum concentrations of anti-dsDNA, IgG2a, IgG2b and IgG3. Also sRAGE interrupted the nuclear translocation of NF-κB in kidney, resulting in reduction in the expression of downstream genes of NF-κB in vivo and in vitro. Furthermore, sRAGE effectively modified T cell populations.
sRAGE significantly improved nephritis in lupus-prone mice, with efficacy comparable to that of standard induction treatment for lupus nephritis. These data suggest that sRAGE have anti-inflammatory effects in lupus nephritis pathophysiology and could serve as a potent new therapy for lupus nephritis. © 2013 American College of Rheumatology.
To investigate the clinical relevance of grade 1 grey scale findings detected with arthrosonography in patients with rheumatoid arthritis (RA).
We examined wrists and small joints of 100 patients with early and established RA and of 30 healthy controls (HC) with grey scale and power Doppler ultrasound (GSUS and PDUS, respectively). All joints were independently assessed clinically for tenderness and swelling according to the EULAR examination technique. Joints with grade 1 GSUS findings were identified and evaluated for their association with swelling, pain and PDUS findings. Grade 1 GSUS findings of early RA patients were reassessed after 6 months of antirheumatic treatment.
Grade 1 GSUS findings represented the majority of all GSUS findings in RA patients and were also found frequently in HC. Grade 1 GSUS findings were not associated with tenderness, swelling and PD positivity. In comparison to grade 2 and grade 3 GSUS findings, grade 1 findings were less susceptible to treatment.
The clinical relevance of grade 1 GSUS findings appears to be rather limited. © 2013 American College of Rheumatology.
We investigated whether an increase in vitamin D levels in patients with systemic lupus erythematosus was associated with improvement in disease activity.
1006 SLE patients were followed over 128 weeks. SLE patients with low levels of 25-hydroxy Vitamin D (<40 ng/mL) were supplemented with 50,000 units Vitamin D2 weekly, with Ca/D3 200 units twice daily. Longitudinal regression models were used to estimate the association between levels of vitamin D and various measures of disease activity.
The SLE patients were 91% female, mean age 49.6, 54% Caucasian, 37% African-American and 8% other ethnicity. For those with low 25-hydroxy Vitamin D (<40 ng/mL), a 20 unit increase in 25-hydroxy Vitamin D was associated with a decrease in mean SELENA-SLEDAI by 0.22 (CI: -0.41, -0.02) (p= 0.032). This corresponded with a 21% decrease in the odds of having a SELENA-SLEDAI higher than 4 (CI: 1%, 37%). Mean urine protein-to-creatinine ratio decreased 2% (CI: -0.03, -0.01) (p=0.009), corresponding to a 15% decrease in the odds of having a ratio of 0.5 or greater (CI: 2%, 27%).
We found that a 20 ng/mL increase in vitamin D was associated with a 21% decrease in the odds of having a high activity score and a 15% decrease in the odds of having clinically important proteinuria. Though these associations were statistically significant, the clinical importance is relatively modest. There was no evidence of additional benefit beyond a level of 40 ng/mL. © 2013 American College of Rheumatology.
Mechanical stress plays important role in cartilage degradation and subchondral bone remodeling in osteoarthritis (OA). The remodeling of the subchondral bone could initiate cartilage loss in OA through the interplay of bone-cartilage. We aimed to identify soluble mediators released by loaded osteoblasts-osteocytes that could induce the release of catabolic factors by chondrocytes.
Murine osteoblasts-osteocytes underwent cyclic compression, then conditioned media from compressed (CM) or uncompressed (UCM) cells were used to stimulate mouse chondrocytes. Chondrocyte expression of MMP-3, MMP-13, type II collagen and aggrecan was assessed by RT-PCR, western blot and ELISA. Then, soluble mediators released by compressed osteoblasts-osteocytes were identified by iTRAQ®(isobaric tags for relative and absolute quantification), a differential secretome analysis. Subchondral bone and cartilage were isolated from OA patients and conditioned media were used to stimulate human chondrocytes.
CM strongly induced the mRNA expression and release of MMP-3 and -13 and inhibited the mRNA expression of type II collagen and aggrecan. Differential secretomic analysis between CM and UCM revealed 10 upregulated proteins. Among them, 14-3-3ε dose-dependently induced the release of catabolic factors by chondrocytes, mimicking the effects of CM. In addition, 14-3-3 blocking antibody greatly reduced CM-mediated induction of MMP-3 and -13 expression. Furthermore, s14-3-3ε was strongly released by human OA subchondral bone and stimulated MMP-3 expression in human OA chondrocytes.
Therefore, we identify s14-3-3ε as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. We speculate that s14-3-3ε may be a potential target for a future OA drug. © 2013 American College of Rheumatology.
Compare the efficacy of tanezumab versus placebo for reducing pain and improving physical function in patients with osteoarthritis (OA) of the hip.
This was a randomized, double-blind, placebo-controlled, phase 3 trial of 32 weeks total duration. Patients with baseline Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) Pain and Physical Function subscale scores of ≥5 and ≥4, respectively, and Patient's Global Assessment of OA (PGA)‘fair’, ‘poor’, or ‘very poor’ were treated at baseline, weeks 8 and 16. Co-primary efficacy endpoints were change from baseline to week 16 in WOMAC Pain and Physical Function subscales and PGA, analyzed using analysis of covariance. Adverse events (AEs) were monitored throughout.
Randomized patients (N=621, 1:1:1:1) were treated with intravenous tanezumab 2.5, 5, or 10 mg or placebo. Each tanezumab group showed significant improvement for the 3 co-primary endpoints versus placebo (P ≤ 0.001 for all); AE incidence ranged from 55–58%across tanezumab groups versus 44% for placebo. Safety findings were similar to those previously reported. The tanezumab OA clinical program was temporarily placed on hold because of AEs leading to joint replacement; here, total joint replacements were reported in 8 patients: 1 (10 mg), 2 (5 mg), 2 (2.5 mg), and 3 (placebo). A total of 9 joints were replaced (8 hips [7 were index joints] and 1 shoulder).
Tanezumab treatment provided superior pain relief, improvement in physical function and PGA versus placebo in patients with painful hip OA, and was generally well-tolerated in this study. © 2013 American College of Rheumatology.
To study left ventricular (LV) geometry in patients with rheumatoid arthritis (RA) who have no heart failure (HF) versus subjects without either RA or HF, and to determine the impact of RA on LV remodeling.
A cross-sectional, community-based study was conducted among adult (≥50 years) RA patients and age- and sex-matched non-RA subjects without a history of HF. All participants underwent a standard 2D/Doppler echocardiography. LV geometry was classified into four categories based on relative wall thickness and sex-specific cut-offs for LV mass index: concentric remodeling, concentric hypertrophy, eccentric hypertrophy, or normal geometry.
The study included 200 RA patients and 600 matched non-RA subjects (mean age 65; 74% female in both cohorts). RA patients were significantly more likely to have abnormal LV geometry than non-RA subjects (odds ratio [OR] 1.44; 95% confidence interval [CI] 1.03, 2.00), adjusting for cardiovascular risk factors and comorbidities. Among those with abnormal LV geometry, RA patients had significantly increased odds of concentric LV remodeling (OR 4.73; 95% CI 2.85, 7.83). In linear regression analyses, LV mass index appeared to be lower in RA patients currently using corticosteroids (Beta +/- standard error: -0.082 +/- 0.027; p=0.002), adjusting for cardiovascular risk factors and comorbidities.
RA was strongly associated with abnormal LV remodeling, particularly, with concentric LV remodeling, among patients without HF. This association was significant beyond adjustment for cardiovascular risk factors and comorbidities. RA disease related factors may promote changes in LV geometry. The biological mechanisms underlying LV remodeling warrant further investigation. © 2013 American College of Rheumatology.
Excessive glucocorticoid treatment increases the incidence of osteopenia and osteonecrosis. MicroRNAs reportedly target mRNA expression and regulate osteoblastogenesis and skeletal development. This study investigated whether miR-29a regulated glucocorticoid-mediated bone loss.
Rats were given methylprednisolone, lentivirus-mediated miR-29a precursor or inhibitor. Dual energy x-ray absorptiometry, micro-computed tomography, material testing, and ELISA were performed to quantify bone mass, micro-architecture, peak load, and serum Dkk-1 levels. Differential microRNA expression profiles were detected using PCR arrays. Abundances of signaling molecules were assessed using immunoblotting.
Glucocorticoid treatment induced loss of bone mineral density and trabecular microstructure in association with reduced miR-29a expression. Treatment with miR-29a precursor attenuated the adverse effects of glucocorticoid on bone mass, trabecular bone volume fraction, and mechanical property. Gain of miR-29a function alleviated the detrimental effects of glucocorticoid treatment on mineral acquisition and ex vivo osteoblast differentiation, as well as reduced osteoclast surface, ex vivo osteoclast differentiation, and RANKL expression in bone microenvironments. Knockdown of miR-29a accelerated osteoclast resorption, cortical bone porosity, bone fragility and loss of ex vivo osteogenic differentiation capacity. miR-29a regulated the abundances of Wnt signaling components (Wnt3a, GSK-3β and β-catenin), Wnt inhibitor Dkk-1, Akt, and phosphorylated ERK and the expression of osteogenic factors Runx2 and IGF-I in bone tissue.
miR-29a signaling protected against glucocorticoid-induced disturbance of Wnt and Dkk-1 actions and improved osteoblast differentiation and mineral acquisition. Promotion of miR-29a signaling is an alternative strategy for alleviating glucocorticoid-induced bone deterioration. © 2013 American College of Rheumatology.
To quantitatively evaluate position, size, and shape of the menisci in subjects with radiographic knee osteoarthritis (OA) compared to subjects free of OA using magnetic resonance (MR) imaging.
We studied right knees from 39 Osteoarthritis Initiative participants (24 women, 15 men; mean age 59.6±8.7 years) with medial compartment radiographic tibiofemoral OA (Kellgren Lawrence grade 2 or 3). We matched them individually by age, sex and body height with right knees of subjects without knee OA and without risk factors for knee OA as references. One observer performed manual segmentation of the tibial plateau and the medial and lateral meniscus based on coronally reconstructed DESSwe focusing on 5 central 3T MR images.
Meniscal coverage of the medial tibial plateau was less in OA knees (40.5% vs. 49.8%; p<0.0001), the medial meniscus body showed more extrusion (2.64 vs. 0.53mm; p<0.0001) and the peripheral margin had a more convex shape, i.e., bulged more (mean 0.61 vs. 0.27mm, p<0.0001). The thickness or volume of the medial meniscus body of OA knees did not differ substantially. In contrast the lateral meniscus body had a larger volume (mean 266 vs. 224mm3; p=0.0005), extruded more (mean 1.16 vs. -1.01mm; p<0.0001) and the external margin bulged more (mean 0.53 vs. 0.35mm; p<0.0001)in OA than in reference knees.
The study provides evidence for altered meniscal position and shape (i.e.,more bulging in OA knees) in both compartments in medial compartment knee OA. These changes may be important features of OA pathogenesis and/or disease consequences. © 2013 American College of Rheumatology.
Rheumatoid arthritis is a sexually dimorphic inflammatory autoimmune disease with both articular and extra-articular disease manifestations including rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Low levels of testosterone have been linked to disease severity in men with rheumatoid arthritis and supplemental testosterone has been shown to improve symptoms of rheumatoid arthritis in both postmenopausal women and men with low testosterone. The mechanisms by which sex and sex steroids affect the immune system and autoimmunity are poorly understood. In this study we examined the protective effect of testicular-derived sex hormones on both joint and lung disease development in an autoimmune mouse model.
Arthritis prevalence and severity were assessed in female, orchiectomized, sham orchiectomized, and intact male SKG mice over a 12 week period after intraperitoneal injection of zymosan. Lung tissues were evaluated by quantifying: cellular accumulation in bronchoalveolar lavage, collagen levels, and histology. An antigen microarray was used to evaluate autoantibody generation in each condition.
Female SKG mice developed arthritis and lung disease with increased prevalence and severity when compared to intact male mice. The absence of testosterone after orchiectomy led to increased arthritis, lung disease and autoantibody generation in orchiectomized male mice compared to intact male mice.
SKG mice represent an authentic sexually dimorphic mouse model of both the joint and lung disease seen in rheumatoid arthritis. Testosterone protects against the development of joint and lung disease in male SKG mice. © 2013 American College of Rheumatology.
Lupus nephritis (LN) trials have used many different primary outcome measures, ranging from complete response to time to end-stage renal disease. The objective of this study was to compare several possible outcome measures, using data from a large, multicenter trial of abatacept in LN (NCT00430677), and to gain insight into which outcome measure, if any, was best able to discern differences among treatment groups.
Study patients received either abatacept or placebo, with background mycophenolate mofetil and glucocorticosteroids. Using data from this trial, the following approaches to assessing outcome at 24 and 52 weeks were compared: complete response rate, major clinical response rate, total response rate (complete plus partial), improvement in proteinuria, improvement in glomerular filtration rate, and frequency of treatment failure. Time to complete response was also evaluated.
Complete response rate, major clinical response rate, and time to complete response discriminated best and were comparable in sensitivity. For these measures, group sizes of 50 patients would have been sufficient to demonstrate a statistically significant difference between treatment and control at 52 weeks. Each of the other measures also discriminated between treatment and control, but much larger group sizes would have been required to determine statistical significance.
The choice of primary outcome measure can substantially influence the ability to detect therapeutic benefit in LN trials. This study suggests that complete response rate and major clinical response rate at 52 weeks and time to complete response may be most sensitive in detecting differences among therapeutic regimens. © 2013 American College of Rheumatology.
To investigate joint counts in oligoarticular PsA for correlation with treatment decisions and proportion of patients with missed active disease using reduced joint counts.
The study utilised an international (GRACE) cohort. Oligoarthritis was defined as < 5 tender and/or swollen joints. At baseline, 66/68 joint counts were assessed. Reduced joint counts used for RA including 28 and 44 joint counts were analysed. In addition, new proposed joint counts for PsA were tested: PsA-44 (elbows, wrists, MCPs, PIPs, DIPs, knees and MTPs) and PsA-56 (as before plus ankles and PIP toes).
ROC analysis was used to assess prediction of treatment change. The proportion of patients with active disease missed by reduced joint counts was analysed.
266 of 503 (53%) of patients had oligoarthritis. No tender joint count predicted treatment change even a full 68/66 joint count (ROC AUC tender p=0.12, active p=0.16). A 66 SJC did predict treatment change (AUC 0.62, p=0.006) as did the SJC PsA44 and PsA56 (p<0.03). Neither of the RA reduced joint counts predicted treatment change.
Performing a RA 28 joint count missed 21% (n=29) of patients who had tender joints and 27% (n=23) of patients who had active swollen joints. PsA-44 and PsA-56 joint counts missed tender joints in 25 and 13 patients and swollen joints in 11 and 2 patients respectively.
Patients with oligoarticular PsA cannot be accurately assessed for active disease using reduced joint counts borrowed from RA. Full 66/68 joint counts should be performed to assess PsA patients. © 2013 American College of Rheumatology.
Heritability studies have suggested an important role of genetic predisposition ito progression of joint destruction in Rheumatoid Arthritis (RA); the heritability is estimated at 45-58%. Several SNPs have been identified to associate with RA susceptibility. We studied the association of several of these loci with progression of joint destruction.
In total 1,750 RA-patients with 4,732 Sharp-van der Heijde scored X-rays of four independent data-sets were studied. Thirteen susceptibility SNPs that were not associated with joint destruction before were tested in 596 Dutch RA-patients. Subsequently, significant SNPs were studied in RA data-sets from North-America and Iceland. Data were summarized in inverse weighted variance meta-analyses. Further, the association with circulating protein levels was studied and the associated region was fine-mapped.
In stage-1, three loci (AFF3, IL2RA and BLK) were significantly associated with rate of joint destruction and were further analyzed in the additional data-sets. In the combined meta-analyses, the minor allele of IL2RA-rs2104286(C) was associated with less progression of joint destruction (P=7.2x10-4). Furthermore, the IL2RA-rs2104286 protective genotype was associated with lower circulating levels of soluble IL-2RA (0.85 95%CI 0.77-0.93, P=1.4x10-3). Additionally, lower sIL-2RA levels were associated with a lower rate of joint destruction (P=4.2x10-3). The association of IL2RA with rate of joint destruction was further focused to a region of 40kb encompassing the IL2RA intron 1 and the 5′ region of IL2RA and RBM17.
Present genetic and serologic data suggest that inherited altered genetic constitution at IL2RA locus may predispose to a less destructive course of RA. © 2013 American College of Rheumatology.
The interleukin (IL)-12 family of cytokines is suggested to play a critical role in auto-inflammatory diseases. Recent studies analysing peripheral blood and synovial fluid in patients with spondyloarthritides suggest that IL-23 might be a pro-inflammatory factor in spondyloarthritides.
The frequency of IL-23+ and IL-12+ cells was analysed within the subchondral bone marrow and within fibrous tissue replacing normal bone marrow in facet joints of AS patients by immunohistochemistry. The origin of IL-23+cells was determined by double staining of CD163+macrophages, CD68+macrophages, CD1a+dendritic cells, AA1+mast-cells, myeloperoxidase+cells, CD20+B-cells and CD3+ T-cells. We compared the results of 28 facet joints from 22 AS patients to 20 facet joints from 13 patients with osteoarthritis (OA) and 10 controls.
In subchondral bone marrow, a significantly higher frequency of IL-23+ cells (42.50+/-32.81 per high-power field was found in joints of AS patients compared to joints of osteoarthritis patients (OA 15.63+/-29.90,p=0.0017) and controls (19.36+/-16.8,p=0.03). Myeloperoxidase+cells and to a lesser extent macrophages and dendritic cells were the origin of IL-23 in the bone marrow. In AS and OA patients, the frequency of IL-23+cells was significantly higher compared to IL-12+cells (both p/0.001). Within fibrous tissue of OA and AS facet joints, IL-23 was predominantly produced by CD68+macrophages (AS:0.64+/-0.59, OA:4.36+/-3.4) and CD163+macrophages (AS:2.3+/-0.65, OA:6.54+/-4.1).
IL-23 is expressed in the subchondral bone marrow and in fibrous tissue replacing bone marrow in facet joints of AS joints. It might play a role in inflammatory processes and in chronic changes in AS joints, which makes it an interesting therapeutic target. © 2013 American College of Rheumatology.
Recent genome-wide association studies have revealed numerous genetic associations between specific single nucleotide polymorphisms (SNPs) and immune-mediated inflammatory diseases. The current challenge is to associate the genetic variants to effector mechanisms implicated in pathogenesis. We have investigated the link between genetic variation at loci associated with spondyloarthritis (SpA) and the effector function of CD4+ T lymphocyte subsets involved in chronic inflammatory disease.
We measured expression of Th17 and Th1 cytokines and transcription factors in CD4+ T cells isolated from SpA patients and correlated them with the patient's genotype at loci genetically linked to spondyloarthritis.
We show that the effector functions of Th17 and Th1 cells in SpA patients are under combinatorial control by multiple SNPs at genes associated with the IL-23/Th17 pathway. SpA patients carrying risk-associated alleles of genes in this pathway expressed the highest levels of genes involved in the differentiation and function of Th17 and Th1 cells, whereas the presence of protective alleles was associated with low-level expression of these genes. In contrast, variation at loci genetically linked to SpA, but not associated with the IL-23 pathway, did not affect the expression of Th17 and Th1 genes, suggesting that these SNPs may contribute to SpA pathogenesis through distinct cellular mechanisms.
Our results show that genetic variation at genes associated with the IL-23 signaling pathway affects the effector functions of Th17 and Th1 cells in SpA patients and provide a framework to delineate the mechanisms by which genetic variants contribute to pathology. © 2013 American College of Rheumatology.
Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disease due to mutations in MEFV. Descriptions of disease manifestations among patients carrying a single mutated MEFV allele are becoming more frequent although no data is available on the long-term outcome. Aim: To assess the accuracy of clinical diagnosis in children carrying a single mutated MEFV allele with symptoms of recurrent autoinflammatory disorder (AID). Methods: We performed a retrospective single-centre study of 33 AID patients younger than 6 years at disease onset with 1 mutated MEFV allele. The phenotype of the patients was investigated in detail and the clinical picture and outcome of 18 patients with an initial FMF diagnosis according to current clinical criteria were compared to those of 25 homozygotes or compound heterozygotes. Results: No major differences in presenting signs or initial response to colchicine were observed between patient groups. During follow-up, heterozygotes had a milder course of the disease and were less prone to experience new clinical signs of FMF than homozygotes. At puberty clinical signs of FMF completely disappeared in 5/18 heterozygotes allowing them to cease colchicine without recurrence of symptoms or raise of inflammatory markers. Conclusion: Our data suggest that the clinical diagnosis of FMF in very young heterozygous children should be made with caution; at this young age they can present with an FMF-like disease - similar to that seen in patients carrying two mutated alleles - that is not necessarily predictive of life-long illness. © 2013 American College of Rheumatology.
Rho-kinases (ROCKs) have been implicated in the pathogenesis of cardiovascular and renal disorders. We recently showed that ROCKs could regulate the differentiation of murine TH17 cells and production of IL-17 and IL-21, two cytokines associated with SLE. The goal of this study was to assess ROCK activation in human TH17 cells and evaluate ROCK activity in SLE patients.
An ELISA-based ROCK activity assay was employed to evaluate ROCK activity in human cord blood CD4+ T cells differentiated under TH0 or TH17 conditions. We then performed a cross-sectional analysis of 28 SLE patients and 25 healthy matched controls. ROCK activity in peripheral blood mononuclear cell (PBMC) lysates was assessed by ELISA. Cytokine and chemokine profiles were analyzed via ELISA.
Human cord blood CD4+T cells differentiated under TH17 conditions expressed higher levels of ROCK activity than CD4+T cells stimulated under TH0 conditions. Production of IL-17 and IL-21 was furthermore inhibited by addition of a ROCK inhibitor. SLE PBMCs expressed significantly higher levels of ROCK activity as compared to healthy controls, 1.25 vs. 0.56, respectively (p=0.0015). Sixteen (57%) SLE patients expressed high ROCK levels (OD450>1). Disease duration, lymphocyte count, and azathioprine use were significant independent predictors of ROCK activity in multivariable analyses.
Consistent with previous results in the murine system, increased ROCK activation was associated with TH17 differentiation. Enhanced ROCK activity was furthermore observed in a subgroup of SLE patients. These data support the concept that the ROCK pathway could represent an important therapeutic target for SLE. © 2013 American College of Rheumatology.
Sphingosine-1-phosphate (S1P) exerts a variety of activities in immune, inflammatory, and vascular systems. S1P plays an important role in systemic sclerosis (SSc) pathogenesis. Regulation of S1P on fibrotic diseases as well as systemic sclerosis was recently reported. FTY720, an oral S1P receptor modulator, has been shown to be a useful agent for the prevention of transplant rejection, and autoimmune diseases. Murine sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) is amodel for human Scl-cGVHD and SSc. In this study, the effects of FTY720 were investigated in Scl-cGVHD.
FTY720 was orally administered to allogeneic recipients from day 0 to day 20 (short-term, early-treatment group), day 0 to day 42 (full-term, early-treatment group), or day 22 to day 42 (delayed-treatment group) after bone marrow transplantation (BMT).
Delayed-treatment of FTY720 attenuated and early-treatment inhibited Scl-cGVHD severity and fibrosis. In early-treatment, FTY720 induced expansion of splenic myeloid-derived suppressor cells, regulatory T cells and regulatory B cells. Vascular damages in cGVHD were inhibited by FTY720 through downregulating serum levels of S1P and sE-Selectin. FTY720 inhibited infiltration of immune cells into skin. Moreover, FTY720 diminished expression of m RNA for MCP-1, MIP-1α, RANTES, TNF-α, IFN-γ, IL-6, IL-10, IL-17A, and TGF-β1 in the skin.
FTY720 suppressed the immune response by promoting the expansion of regulatory cells, reducing vascular damages and infiltration of immune cells into skin.Taken together, these results have important implications for the potential use of FTY720 in treatment of Scl-cGVHD and SSc in humans. © 2013 American College of Rheumatology.
TGFβ-inducible gene-h3 (βig-h3), abundantly expressed in rheumatoid synovium, and matrix metalloproteinases (MMP) play an important role in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the therapeutic efficacy of βig-h3-derived peptides using MMP-1-dependent target tissue delivery in chronic inflammatory arthritis.
The βig-h3 derivative-based peptides, including the 2nd and 4th YH peptides, 4th fas-1 domain, 4th fas-1 truncated for H1 and H2 (dhfas-1), and a MMP1-cleavable composite peptide (MFK24), were cloned. We confirmed the specificity of MFK24 cleavage by immunoblotting after treatment with different proteases.
The 4th YH18 peptide was weakly effective in suppressing arthritis severity in CIA mice. Treatment with higher dose (30mg/kg) of dhfas-1 showed a remarkable efficacy, whereas treatment with lower dose (10mg/kg) revealed only a partial improvement. A composite peptide (MFK24) consisted of dhfas-1 and RGD peptide linked by MMP1 substrate was cleaved by MMP1 specifically. βig-h3-mediated adhesion and migration of NIH3T3 cells were inhibited at low concentration of MFK24. MFK24 suppressed the adhesion of NIH3T3 cells more efficiently compared to either alone or in combination of MFK00 and RGD motif. Therapeutic efficacy of MFK24 in CIA mice was remarkably enhanced with a consistent reduction of expression in inflammatory mediators within joint tissues.
A proof of concept design of MMP-cleavable composite peptide, based on βig-h3 derivatives, revealed a marked improvement of therapeutic efficacy for chronic inflammatory arthritis, implicating a new expandable strategy for enhancement of efficacy of two different active molecules in RA. © 2013 American College of Rheumatology.
We sought to evaluate the longitudinal relationship of bone mineral density (BMD) and its change to knee osteoarthritis (OA) progression measured by cartilage outcomes.
We used observational cohort data from the Vitamin D for Knee Osteoarthritis trial. We obtained bilateral femoral neck BMDs as well as knee MRIs in each subject at baseline and subsequently at 12 and 24 months. We measured change in total cartilage volume, tibial and femoral cartilage thickness by manual cartilage segmentation of two sequential knee MRIs in each subject. Multivariable linear regression models were used to examine the associations of baseline BMD and BMD change with the cartilage outcomes, adjusting for baseline age, gender, BMI, malalignment and vitamin D treatment. We validated model fit and assumptions.
127 subjects were eligible for analysis. Longitudinal BMD loss was associated with loss of cartilage volume (β=1.25 per 0.1g/cm2, p=0.02) and tibial cartilage thickness (β=0.028, p=0.03). BMD loss of a magnitude greater than least significant change (≤ -4.7%) was associated with 1.02% cartilage volume loss per year (p=0.005), 0.014mm femoral cartilage thickness loss (p=0.04) and 0.021mm tibial cartilage thickness loss per year (p=0.009). There were no significant associations between baseline BMD and any of the cartilage outcomes.
Longitudinal BMD loss is associated with progressive cartilage loss in knees with OA. Further work to clarify the basis of this relationship could uncover novel therapeutic targets for knee OA. © 2013 American College of Rheumatology.
The cytokine TNF plays a central role in the pathogenesis of rheumatoid arthritis (RA), but its disease-specific effector mechanisms have not been fully elucidated. Goal of the study presented was to investigate the role of TNF in T cell accumulation and migration in synovitic joints of RA patients.
Vital tissue section from rheumatoid synovium were generated using a horizontally oscillating microtom, and were co-incubated with fluorescence-labelled CD4+ T cells. Migration was detected by fluorescence and confocal microscopy. Migrating T cells were recovered from the tissue and phenotypically analyzed. Chemokinesis of CD4+ T cells from RA patients in response to increasing concentration of TNF was analyzed in transwell experiments.
CD4+ T cells from RA patients migrated into the tissue sections in significantly higher numbers than T cells from healthy controls. Migrating CD4+ T cells differed from non-migrating ones in their increased expression of TNFR1, which is expressed on a fraction of circulating CD4+ T cells from RA patients, but not from controls. CD4+ T cells from the peripheral blood of RA patients were also found to migrate along TNF concentration gradients ex vivo. Accordingly, blockade of either TNF or of TNFR1 nearly abrogated in vitro T cell migration in synovial tissue.
The study shows, that the interaction of TNF with TNFR1 is pivotal for T cell migration in synovial tissue in vitro, and thereby suggests a relevant role for the cytokine for in vivo T cell trafficking to synovitic joints © 2013 American College of Rheumatology.
Identify susceptibility loci for rheumatoid arthritis (RA) in Latin American individualswith admixed European and Amerindian genetic ancestry.
Genotyping was performed on samples from 1475 patients with RA and 1213 controls using a custom BeadArray containing196,524 markers, covering loci previously associated with various autoimmune diseases. We used principal component analysis(EIGENSOFTpackage)and Structuresoftware to identify outliers and define population substructure, REAP to define cryptic relatedness and duplicates, and conducted genetic association analyses using PLINK.
A strong genetic association of RA with themajor histocompatibility complex region was observed, localized within BTNL2-HLA-DRA-DQB1-DQA2(p=7.6 × 10-10) with three independent effects. We identified an association in the PLCH2-HES5-TNFRSF14-MMEL1 region of chromosome 1 (p= 9.77 × 10-6) previously reportedin Europeans, Asians and Native Canadians. We identified onenovel putative association in ENOX1onchromosome 13 (p= 7.0 × 10-7).Previously reported RA associations were observed, with moderate p values (>x10-5), including STAT4, IRF5, IL2RA, SPRED2, CCL21 and PTPN22.Adjustment for Amerindian ancestryimproved the association of a novel locus in chromosome 12, atC12orf30 (NNA25) (p= 3.9 × 10-6). Associations with the HLA region, SPRED2 and PTPN22 improved in individuals positive for anti-CCP antibodies.
Our data define for the first time the contribution of Amerindian ancestry to the genetic architecture of RA in an admixed Latin American population by confirming the roleof the HLAregion, and supportinga locuson chromosome 1. In addition, we provide data for novel putative loci in chromosomes 12 and 13. © 2013 American College of Rheumatology.
Matrix fragments, including fibronectin fragments (Fnf), accumulate during the development of osteoarthritis (OA) stimulating chondrocyte matrix metalloproteinase (MMP) production. The objective of this study was to determine the role of the small GTPase Rac1 in chondrocyte signaling stimulated by Fnf that results in MMP-13 production.
Normal human cartilage was from tissue donors and OA cartilage from knee arthroplasty specimens. Rac1 activity was modulated with a chemical inhibitor, siRNA knock-down, constitutively active (CA)-Rac or dominant negative (DN)-Rac adenovirus. Cells were treated with Fnf or without known Rac activators, epidermal growth factor (EGF) or transforming growth factorα (TGFα). Rac1 activity was measured with a colorometric activity ELISA, pulldown assay, and immunostaining with a monoclonal antibody against active Rac.
Chemical inhibition of Rac1, as well as knockdown by siRNA and expression of DN-Rac blocked Fnf stimulated MMP-13 production while expression of CA-Rac increased MMP-13. Inhibition of Rho-associated kinase had no effect. EGF and TGFα, but not Fnf, increased Rac1 activity and promoted the increase in MMP-13 above that stimulated by Fnf alone. Active Rac was detected by immunostaining in OA cartilage.
Rac1 is required for Fnf induced signaling that results in increased MMP-13 production. EGF receptor ligands, which activate Rac, can promote this effect. The presence of active Rac in OA cartilage and the ability of Rac to stimulate MMP-13 production suggests that it could play a role in the cartilage matrix destruction seen in OA. © 2013 American College of Rheumatology.
Anti-TNF therapies are highly effective in rheumatoid (RA) and psoriatic (PsA) arthritis but a significant number of patients exhibit partial or no therapeutic response. Inflammation alters local and systemic metabolism and TNF plays a role in this. We sought to determine if the patient's metabolic fingerprint prior to therapy could predict responses to anti-TNF agents.
Urine was collected from 16 RA and 20 PsA patients before and during therapy with infliximab or etanercept. Urine metabolic profiles were assessed using NMR spectroscopy. Discriminating metabolites were identified, and the relationship between metabolic profiles and clinical outcomes was assessed.
Baseline urine metabolic profiles discriminated between RA patients who did or did not have a good response to anti-TNF therapy, according to EULAR criteria, with a sensitivity of 88.9% and specificity of 85.7%, with several metabolites (in particular histamine, glutamine, xanthurenic acid and ethanolamine) contributing. There was a correlation between baseline metabolic profiles and the magnitude of change in DAS 28 from baseline to 12 months in RA patients (p=0.04). In both RA and PsA urinary metabolic profiles changed between baseline and 12 weeks of anti-TNF therapy and within the responders, urinary metabolite changes distinguished between etanercept and infliximab treatment.
The clear relationship between urine metabolic profiles of RA patients at baseline and their response to anti-TNF therapy may allow development of novel approaches to the optimisation of therapy. Differences in metabolic profiles during treatment with infliximab and etanercept in RA and PsA may reflect distinct mechanisms of action. © 2013 American College of Rheumatology.
This study was undertaken to determine whether there is a neuropathic component in ankylosing spondylitis (AS) back pain and to delineate gray matter (GM) brain abnormalities associated with AS.
Seventeen patients with back pain secondary to AS, not on biologic agents and 17 age- and sex-matched controls consented to the study and were assessed with the PainDETECT (scores ≤ 12 indicating low probability of neuropathic pain) and McGill Pain Questionnaires. Mechanical and thermal thresholds were determined for all subjects, and 3T MRI used to assess brain GM.
The painDETECT scores were >12 in 11/17 AS patients. The patients had decreased mechanical and cold sensitivity on their dorsal feet but did not have altered pain thresholds. Compared to controls, the AS patients showed a) cortical thinning in the primary sensory (S1), insular, anterior cingulate (ACC) and anterior mid-cingulate cortices (aMCC) and supplemental motor area, b) increased grey matter volume in the thalamus and putamen, and c) a correlation between painDETECT scores and decreased GM in S1 and increased GM in the motor cortex, ACC, prefrontal cortex, thalamus and striatum.
These findings indicate that AS patients have signs of neuropathic pain. Furthermore, abnormal brain gray matter and neural correlates of neuropathy are concordant with the clinical picture of AS having sensorimotor and mood deficits as well as neuropathic pain symptoms. These data suggest that back pain in AS is a mixed pain condition that includes a neuropathic pain component. © 2013 American College of Rheumatology.
To study changes in lipid profiles at 24 weeks among early rheumatoid arthritis (RA) patients participating in the Treatment of Early Rheumatoid Arthritis (TEAR) Trial randomized to initiate methotrexate plus etanercept (MTX+ETA), triple therapy (TT) [MTX plus sulfasalazine plus hydroxychloroquine] or aggressively-titrated MTX monotherapy.
The TEAR biorepository study had 459 participating patients. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured in serum plasma at 0 and 24 weeks.
At 24 weeks, there were statistically significant mean increases in cholesterol levels in the MTX + ETA, TT, and MTX monotherapy arms, the observed increases were 31.4, 28.7 and 30 mg/dL in LDL-C; 19.3, 22.3 and 20.6 mg/dL in HDL-C and 56.8, 53 and 57.3 mg/dL values in TC (p < 0.001 all compared to baseline). There was a statistically significant decrease in TC/HDL-C ratio at 24 weeks in all 3 treatment groups from baseline. There was no difference in any lipid changes between the 3 treatment arms. After multivariable adjustment, change in C-reactive protein was associated with change in LDL-C (p=0.03), HDL-C (p=0.09), and TC (p=0.01), but disease activity score in 28-joints was not. Baseline glucocorticoid use was associated with changes in HDL-C (p=0.03) and TC (p=0.02).
Levels of TC, LDL-C, and HDL-C increased equivalently shortly after initiation of MTX + ETA, TT and MTX monotherapy among early RA patients with active disease participating in a clinical trial. The clinical relevance of short term changes in traditional lipids on cardiovascular outcomes remains to be determined. © 2013 American College of Rheumatology.
The mechanisms involved in breaking immunological tolerance against nuclear autoantigens in systemic lupus erythematosus (SLE) are not fully understood. Our recent studies in non-autoimmune mice provided evidence of an important role of Toll-like receptor (TLR) 2 in anti-chromatin autoantibody induction by High Mobility Group Box protein 1 (HMGB1)-nucleosome complexes derived from apoptotic cells. Here we asked if TLR2 signaling is also required for the induction of autoantibodies and the development of SLE-like disease in murine pristane-induced lupus.
Lupus-like disease in C57BL/6 and TLR2-/- mice was induced by pristane. The numbers of immune cells and serum cytokine concentrations were determined by flow cytometry. Renal disease was assessed by quantification of proteinuria, histological analyses and ELISPOT.
Pristane-injected TLR2-/- mice generated reduced numbers of splenic CD138+/ cytoplasmic κ/λ-L chain+ plasma cells and displayed diminished IgG responses against dsDNA, histones, nucleosomes, some extractable nuclear autoantigens (ENAs), and cardiolipin when compared with wild type controls. TLR2 deficiency prevented the pristane-induced systemic release of IL-6 and IL-10. Absence of TLR2 attenuated peritoneal recruitment of CD11c+ cells and formation of lipogranulomas. Importantly, pristane-treated TLR2-/- mice developed less severe renal disease compared to controls, reflected by milder proteinuria, reduced glomerular depositions of IgG and complement as well as decreased renal infiltration of autoantibody-secreting cells.
TLR2 is required for the production of prototypical lupus autoantibodies and the development of renal disease in pristane-induced murine lupus. Interference with TLR2 signaling may be a promising novel strategy for the treatment of SLE. © 2013 American College of Rheumatology.
Children with rheumatoid-factor or anti-citrullinated peptide antibody positive juvenile idiopathic arthritisrepresent the childhood onset of RA (CORA). To test the hypothesis that adult-onset RA-associated variants are also associated with CORA, we investigated RA-associated variants at five loci in ourCORA cohort. We also assessedthe cumulative association of these variants in the susceptibility to CORA using aweighted genetic risk score (wGRS).
155 children with CORA and 684healthy controls were genotyped for fivevariants in PTPN22, TRAF1/C5, STAT4, and TNFAIP3 loci. High-resolution HLA-DRB1 genotypes were available for149 cases and 373 controls. We testedeach locus for associationwith CORA via logistic regression. Wealso computed a wGRS for each subject, with weights based on the natural log of the published odds ratios for the alleles investigated, and used logistic regression to test the wGRS for association with CORA.
CORA was associated withTNFAIP3-rs10499194 [OR 0.60 (95%CI 0.44-0.83)], PTPN22-rs2476601 [OR 1.61 (1.11-2.31)], and STAT4-rs7574865 [OR 1.41 (1.06-1.87)] variants. The wGRS was significantly different between cases and controls (P<2×10-16). Individuals in the third to fifth quintiles of wGRS had a significantly increased disease risk compared to the baseline.Higher wGRSassociated with increased risk of CORA, especially among males.
TNFAIP3, STAT4 and PTPN22 variants are associated with CORA in a similar magnitude and direction as in RA,suggesting that adult-onset RA and CORA share common genetic risk factors. Utilizing a wGRS, we have demonstrated the cumulative associationof RA-associated variants in the susceptibility to CORA. © 2013 American College of Rheumatology.
To investigate the reason why the level of LYN is significantly decreased in B cell from a majority of patients with systemic lupus erythematosus (SLE) and to determine the role of microRNA-30a (miR-30a) in B cell hyperactivity of patients with SLE.
Luciferase reporter gene assays were performed to identify the interaction between miR-30a and three prime untranslated region (3′UTR) of LYN. The level of miR-30a in B cell was determined by TaqMan quantitative polymerase chain reaction (qPCR) analysis. LYN messenger RNA level was tested by real-time qPCR. The protein level of LYN was determined by Western blotting. The quantity of IgG was determined by enzyme-linked immunosorbent assay. The proliferation of B cell was measured by [3H]-thymidine incorporation.
We demonstrated that miR-30a, but not miR-30b/c/d/e, could specifically bind the 3′UTR of LYN in B cell lines. Meanwhile, overexpression of miR-30a could inhibit the level of LYN. The level of miR-30a was significantly higher in B cell from SLE patients than that in B cell from health donor. The level of miR-30a was negatively associated with the level of LYN in B cell. Overexpression of miR-30a could promote the proliferation of B cell lines and the production of IgG antibodies. The effect of miR-30a could be recovered by overexpression of LYN in B cell.
We herein reveal that the elevated miR-30a is responsible for the reduction of LYN in B cell from the patients of SLE and consequently plays an important role in B cell hyperactivity. © 2013 American College of Rheumatology.
First Degree Relatives (FDR) of Rheumatoid Arthritis (RA) patients sharing genetic and environmental risk factors for RA may represent a pre-RA state. Anti-cyclic citrullinated protein/peptide antibodies (ACPA) appear years before the onset of RA. We determined the prevalence of various ACPA in RA FDR.
Subjects were RA patients (n=88), unaffected FDR (n=50) and healthy controls (n=20). Six different types of ACPA were determined by ELISA. Joint and periodontal disease symptoms were self-reported. Patients and FDR were HLA-typed for the Shared Epitope (SE) and the RA-protective alleles, HLA-DRB*1301/1302.
FDR had a high prevalence of ACPA (48%) compared to controls (10%). The prevalence of the SE and smoking in FDR were also high (62% and 49% respectively). 13/32 (41%) of all ACPA in FDR was of the IgA isotype. The most commonly expressed IgG ACPA targeted citrullinated vimentin, occurring in 20% of FDR. FDR had an average of 1 type of ACPA, whereas, RA patients expressed a median of 5 different ACPA. The only FDR to later develop RA expressed 4 different ACPA. Joint and PD symptoms in FDR were significantly associated with smoking (OR 5.714; 95%CI: 1.151-28.3 and OR 12.25; 95%CI: 2.544-58.99 respectively), but not with ACPA.
The rate of ACPA positivity in unaffected FDR of RA patients with a high prevalence of the SE and smoking was 48%; whereas, ACPA were rare in healthy controls. ACPA in FDR was most commonly of the IgA isotype, but IgG ACPA targeting citrullinated vimentin was also frequently found. © 2013 American College of Rheumatology.
To explore the role of immune dysregulation in antibiotic-refractory Lyme arthritis, the phenotype, frequency and function of CD4+ Teff and Treg cells were compared in patients with antibiotic-responsive or antibiotic-refractory arthritis. In the latter condition, infection-induced autoimmunity is thought to have a pathogenic role.
Matched peripheral blood (PB) and synovial fluid (SF) samples from 15 patients with antibiotic-responsive arthritis were compared with those from 16 patients with antibiotic-refractory arthritis using flow cytometry, suppression and cytokine assays.
Critical differences between the 2 patient groups were found in the SF CD4+CD25hi+ populations, a subset of cells usually composed of FOXP3-positive Treg cells. In patients with antibiotic-refractory arthritis, this cell population often had fewer FOXP3-positive cells, and greater frequencies of FOXP3-negative (Teff) compared to patients with antibiotic-responsive arthritis. Moreover, in the refractory group, CD4+CD25hi+ cells had significantly greater expression of GITR and OX-40, two co-receptors that augment T cell function. Suppression assays showed that CD4+CD25hi+ cells in patients with refractory arthritis did not effectively suppress proliferation of CD4+CD25- cells, or secretion of IFN-γ or TNF-α, whereas those from patients with responsive arthritis did. Finally, in the refractory group, higher ratios of CD25hi+FOXP3-/CD25hi+FOXP3+ cells correlated directly with longer post-treatment durations of arthritis.
Patients with antibiotic-refractory Lyme arthritis often had lower frequencies of Treg, higher expression of activation co-receptors, and less effective inhibition of pro-inflammatory cytokines. This suggests that immune responses in these patients were excessively amplified leading to immune dysregulation and refractory arthritis. © 2013 American College of Rheumatology.
Alterations in the mechanical loading environment in joints may have both beneficial and detrimental effects on articular cartilage and subchondral bone and subsequently influence the development of osteoarthritis (OA). We used an in vivo tibial loading model to investigate the adaptive responses of cartilage and bone to mechanical loading and to assess the influence of load level and duration.
We applied cyclic compression of 4.5 and 9.0N peak loads to the left tibia via the knee joint of adult (26-week-old) C57Bl/6 male mice for 1, 2, and 6 weeks. Only 9.0N loading was utilized in young (10-week-old) mice. The changes in articular cartilage and subchondral bone were analyzed by histology and microcomputed tomography.
Loading promoted cartilage damage in both age groups, with increased damage severity dependent upon the duration of loading. Metaphyseal bone mass increased in the young mice, but not in the adult mice, whereas epiphyseal cancellous bone mass decreased with loading in both young and adult mice. Articular cartilage thickness decreased, and subchondral cortical bone thickness increased in the posterior tibial plateau in both age groups. Both age groups developed periarticular osteophytes at the tibial plateau in response to the 9.0N load, but no osteophyte formation occurred in adult mice subjected to 4.5N peak loading.
This non-invasive loading model permits dissection of temporal and topographical changes in cartilage and bone and will enable investigation of the efficacy of treatment interventions targeting joint biomechanics or biological events that promote OA onset and progression. © 2013 American College of Rheumatology.
(Objective): We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of RA. Herein, we evaluated the effects of blockade of CTGF pathway on the development of arthritis in collagen-induced arthritis (CIA) mice. (Methods): Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen and complete Freund's adjuvant (CFA). We evaluated the efficacy of prevention of development of arthritis in the CIA mice treated with or without neutralizing anti-CTGF monoclonal antibody (mAb). (Results): Inhibition of the CTGF functions in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to that in the non-treated CIA mice. Serum levels of matrix metalloproteinase 3 (MMP-3) were reduced by anti-CTGF mAb treatment. Moreover, blockade of the CTGF decreased interleukin 17 (IL-17) expression on purified CD4+ T lymphocytes. Although the expression of retinoic-acid-receptor-related orphan receptors γt (RORγt) gene was not suppressed by anti-CTGF mAb treatment, those of interferon regulatory factor 4 (IRF4) and IkappaBzeta (Nfkbiz), which are other important molecules for differentiation of Th-17 cells, were suppressed. In addition, blockade of CTGF inhibited pathological proliferation of T lymphocytes against type II collagen restimulation in vitro. Moreover, aberrant osteoclastogenesis in CIA mice was restored by anti-CTGF mAb treatment. (Conclusions): The present study showed that blockade of CTGF prevented progression of arthritis in CIA mice. Anti-CTGF mAb treatment suppressed the pathological T cells function and restored aberrant osteoclastogenesis in CIA mice. CTGF may become a new therapeutic target for treatment of RA. © 2013 American College of Rheumatology.
To explore the mechanisms of osteoarthritis (OA), gene expression profiling was doneon cartilage from mice with surgically-induced OA. We used wild type mice (WT), and mice lacking ADAMTS-5 activity (Adamts5Δcat) where aggrecan loss and cartilage erosion is inhibited, to distinguish betweengene expression changes that are independent of ADAMTS-5 activity and cartilage breakdown.
Mechanical instability was introduced into 10 week old male mouse knee joints by surgical destabilization of the medial meniscus (DMM).Cartilage from the developing lesion in DMM and corresponding regions in sham-operated joints at 1, 2 and 6 weeks post-surgery was harvested by microdissection, RNA extracted, amplified and hybridized to whole genome microarrays.
We show for the first time thatOA-related genes including Ptgs2, Crlf1 and Inhba, and novel genes, such asPhdla2 and Il11are upregulated in WT and Adamts5Δcatmice and are thusindependent of ADAMTS-5 activity, identifying them as possible OA initiation candidates. The altered expression of other genes requires ADAMTS-5 activity including Col10a1,the sentinel marker of cartilage hypertrophy and Wnt/β-catenin pathway genes. Cell death pathway genes are dysregulated and TP53, FOXO4 and XBP1 ER-stress transcriptional networks activated. Analysis of degradome genes identified the upregulation of many proteases, including Mmp3, Capn2 and novel cartilage proteasesPrss46 and Klk8. Comparison with other studies identified 16 genes also dysregulated in rat and human OA as priorities for study.
This work provides new insights into the sequence of gene dysregulation and molecular basis of cartilage destruction in OA. © 2013 American College of Rheumatology.
To describe the spatial distribution of incident cases of systemic lupus erythematosus (SLE) using Geographic Information Systems (GIS).
Spatial analyses were carried on 890 SLE and 541 PsA patients as controls. Age and gender adjusted rates for SLE/PsA for each census tract were calculated using denominator population values from the Canadian census. Spatial variations in relative risk were estimated by modeling risk as the product of a time effect, age effect, and a spatially auto-correlated risk surface to identify hot spots. Patients within the hot spot detected were compared to those outside of the hot spot to identify explanatory factors.
SLE patients were predominantly female (87.5%) and the incidence rate was highest among those aged 15-19 (2.4 cases/100 000 person-years). In a SLE hot spot containing 59 patients, 100% (n=59) of patients were female and 49.1% (n=29) were Caucasian while outside of the hot spot, 86.9% (n=722) were female and 68.4% (n=568) Caucasian. The proportion of cases of Chinese race was significantly greater within the hot spot. An interaction was found between Chinese race and residence within the hot spot, with the risk of SLE to the Chinese population found to be twice that of the non-Chinese population.
GIS was used to map SLE cases and identified a hot spot after adjusting for age and gender. Race by itself did not confer an increased risk for SLE but the interaction of race with location of residence significantly increased the risk of SLE. © 2013 American College of Rheumatology.
To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis.
DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively.
Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor α–stimulated FLS, in a dose-dependent manner. The 4-MU–induced decreases in the serum HA concentration in mice with CIA and in “medium” and “pericellular” HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression.
Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis.
The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS.
A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain–containing phosphatase 2 (SHP-2), a PTP that was up-regulated in RA, was then analyzed by knockdown using cell-permeable antisense oligonucleotides.
PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP-2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP-2 knockdown. RA FLS that were deficient in SHP-2 exhibited decreased activation of focal adhesion kinase and mitogen-activated protein kinases.
These findings indicate that SHP-2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP-2 to be a candidate therapeutic target for RA.
Current approaches offer no cures for rheumatoid arthritis (RA). Accumulating evidence has revealed that manipulation of bone marrow–derived mesenchymal stem cells (BM-MSCs) may have the potential to control or even prevent RA, but BM-MSC–based therapy faces many challenges, such as limited cell availability and reduced clinical feasibility. This study in mice with established collagen-induced arthritis (CIA) was undertaken to determine whether substitution of human gingiva-derived mesenchymal stem cells (G-MSCs) would significantly improve the therapeutic effects.
CIA was induced in DBA/1J mice by immunization with type II collagen and Freund's complete adjuvant. G-MSCs were injected intravenously into the mice on day 14 after immunization. In some experiments, intraperitoneal injection of PC61 (anti-CD25 antibody) was used to deplete Treg cells in arthritic mice.
Infusion of G-MSCs in DBA/1J mice with CIA significantly reduced the severity of arthritis, decreased the histopathology scores, and down-regulated the production of inflammatory cytokines (interferon-γ and interleukin-17A). Infusion of G-MSCs also resulted in increased levels of CD4+CD39+FoxP3+ cells in arthritic mice. These increases were noted early after infusion in the spleens and lymph nodes, and later after infusion in the synovial fluid. The FoxP3+ Treg cells that were increased in frequency mainly consisted of Helios-negative cells. When Treg cells were depleted, infusion of G-MSCs partially interfered with the progression of CIA. Pretreatment of G-MSCs with a CD39 or CD73 inhibitor significantly reversed the protective effect of G-MSCs on CIA.
The role of G-MSCs in controlling the development and severity of CIA mostly depends on CD39/CD73 signals and partially depends on the induction of CD4+CD39+FoxP3+ Treg cells. G-MSCs provide a promising approach for the treatment of autoimmune diseases.
Through its location on nociceptors, acid-sensing ion channel 3 (ASIC-3) is activated by decreases in pH and plays a significant role in musculoskeletal pain. We recently showed that decreases in pH activate ASIC-3 located on fibroblast-like synoviocytes (FLS), which are key cells in the inflammatory process. The purpose of this study was to test whether ASIC-3–deficient mice with arthritis have altered inflammation and pain relative to controls.
Collagen antibody–induced arthritis (CAIA) was generated by injection of an anti–type II collagen antibody cocktail. Inflammation and pain parameters in ASIC-3−/− and ASIC-3+/+ mice were assessed. Disease severity was assessed by determining clinical arthritis scores, measuring joint diameters, analyzing joint histology, and assessing synovial gene expression by quantitative polymerase chain reaction analysis. Cell death was assessed with a Live/Dead assay of FLS in response to decreases in pH. Pain behaviors in the mice were measured by examining withdrawal thresholds in the joints and paws and by measuring their physical activity levels.
Surprisingly, ASIC-3−/− mice with CAIA demonstrated significantly increased joint inflammation, joint destruction, and expression of interleukin-6 (IL-6), matrix metalloproteinase 3 (MMP-3), and MMP-13 in joint tissue as compared to ASIC-3+/+ mice. ASIC-3+/+ FLS showed enhanced cell death when exposed to pH 6.0 in the presence of IL-1β, which was abolished in ASIC-3−/− FLS. Despite enhanced disease severity, ASIC-3−/− mice did not develop mechanical hypersensitivity of the paw and showed greater levels of physical activity.
Our findings are consistent with the hypothesis that ASIC-3 plays a protective role in the inflammatory arthritides by limiting inflammation through enhanced synoviocyte cell death, which reduces disease severity, and through the production of pain, which reduces joint use.
Glucocorticoid-induced leucine zipper (GILZ) has effects on inflammatory pathways that suggest it to be a key inhibitory regulator of the immune system, and its expression is exquisitely sensitive to induction by glucocorticoids. We undertook this study to test our hypothesis that GILZ deficiency would exacerbate experimental immune-mediated inflammation and impair the effects of glucocorticoids on inflammation and, correspondingly, that exogenous GILZ would inhibit these events.
GILZ−/− mice were generated using the Cre/loxP system, and responses were studied in delayed-type hypersensitivity (DTH), antigen-induced arthritis (AIA), K/BxN serum–transfer arthritis, and lipopolysaccharide (LPS)–induced cytokinemia. Therapeutic expression of GILZ via administration of recombinant adeno-associated virus expressing the GILZ gene (GILZ-rAAV) was compared to the effects of glucocorticoid in collagen-induced arthritis (CIA).
Increased T cell proliferation and DTH were observed in GILZ−/− mice, but neither AIA nor K/BxN serum–transfer arthritis was affected, and GILZ deficiency did not affect LPS-induced cytokinemia. Deletion of GILZ did not impair the effects of exogenous glucocorticoids on CIA or cytokinemia. In contrast, overexpression of GILZ in joints significantly inhibited CIA, with an effect similar to that of dexamethasone.
Despite effects on T cell activation, GILZ deficiency had no effect on effector pathways of arthritis and was unexpectedly redundant with effects of glucocorticoids. These findings do not support the hypothesis that GILZ is central to the actions of glucocorticoids, but the efficacy of exogenous GILZ in CIA suggests that further evaluation of GILZ in inflammatory disease is required.
To describe the frequency of treatment switching and outcomes among patients with psoriatic arthritis (PsA) who switched tumor necrosis factor α inhibitor (TNFi) agents in routine care.
We conducted an observational cohort study based on the Danish nationwide DANBIO registry. Treatment outcomes were evaluated using the American College of Rheumatology criteria for 20% improvement (ACR20)/ACR50/ACR70, European League Against Rheumatism (EULAR) response criteria for good response, and the 28-joint count Disease Activity Score (DAS28) (remission). Kaplan-Meier and regression analyses were used for drug survival analyses and to identify predictors of outcome after treatment switching.
Of 1,422 patients starting TNFi agents, 548 patients (39%) switched to a second biologic drug during up to 10 years of followup. Median followup was 2.3 years (interquartile range [IQR] 1.0–4.3 years). Switchers were more frequently women (56% versus 45%), had a shorter disease duration (3 versus 4 years), a higher median Health Assessment Questionnaire (HAQ) score (1.1 [IQR 0.6–1.6] versus 0.9 [IQR 0.5–1.4]), DAS28 (4.8 [4.0–5.7] versus 4.4 [3.6–5.2]), pain score on a visual analog scale (VAS) (65 mm [46–77] versus 62 mm [40–75]), and fatigue score on a VAS (69 mm [50–83] versus 64 mm [42–80] mm) (all P < 0.05 at start of first TNFi). During the first and second treatment, HAQ, DAS28, and VAS scores and C-reactive protein levels had decreased after 6 months (all P < 0.05), and median drug survival was 2.2 versus 1.3 years (P < 0.001). Lower fatigue score increased survival of the second TNFi. After switching, the proportions of patients achieving a sustained ACR20, ACR50, ACR70, EULAR good response, and DAS28 remission after 3–6 months were 22% (number needed to treat [NNT] 4.5), 13% (NNT 7.9), 5% (NNT 20), 19% (NNT 5.3), and 34% (NNT 2.9), respectively. Response rates were lower during the second treatment (all P < 0.01 versus first TNFi). At the 2-year visit, 47% of switchers had achieved an ACR20 response. No differences between drug–drug combinations were found.
Thirty-nine percent of the patients with PsA switched TNFi agents. Response rates and drug survival were lower after switching; however, half of the switchers had an ACR20 response 2 years after starting the first TNFi.
Psoriatic arthritis (PsA) is a common inflammatory joint disease distinct from other chronic arthritides and frequently accompanied by psoriasis vulgaris. In a first genome-wide association study (GWAS), we were able to identify several genetic risk factors. However, even combined with previously identified factors, the genetic contribution to disease was not fully explained. Therefore, we undertook this study to investigate further 17 loci from our GWAS that did not reach genome-wide significance levels of association in the initial analysis.
Twenty-one of 22 single-nucleotide polymorphisms were successfully genotyped in independent cohorts of 1,398 PsA patients and 6,389 controls and in a group of 964 German patients with psoriasis vulgaris.
Association with a RUNX3 variant, rs4649038, was replicated in independent patients and controls and resulted in a combined P value of 1.40 × 10−8 by Cochran-Mantel-Haenszel test and an odds ratio (OR) of 1.24 (95% confidence interval [95% CI] 1.15–1.33). Further analyses based on linkage disequilibrium (LD) at RUNX3 refined the most significant association to an LD block located in the first intron of one isoform. Weaker evidence for association was detected in German patients with psoriasis vulgaris (P = 5.89 × 10−2; OR 1.13 [95% CI 1.00–1.28]), indicating a role in the skin manifestations of psoriasis.
Our analyses identified variants in RUNX3 as susceptibility factors for PsA. RUNX3 has already been implicated in susceptibility to ankylosing spondylitis, another spondyloarthritis, although its risk allele is independent from the one for PsA. RUNX-3 is involved in CD8+ T lymphocyte differentiation and is therefore a good candidate for involvement in PsA and psoriasis vulgaris as T cell–mediated diseases.
Lyme arthritis (LA) is characterized by infiltration of inflammatory cells, mainly neutrophils (polymorphonuclear cells [PMNs]) and T cells, into the joints. This study was undertaken to evaluate the role of the neutrophil-activating protein A (NapA) of Borrelia burgdorferi in eliciting inflammation and in driving the adaptive immune response.
Levels of NapA, interferon-γ (IFNγ), interleukin-17 (IL-17), and T cell–attracting chemokines were assessed by enzyme-linked immunosorbent assay in synovial fluid from patients with LA. The profile of T cells recruited into the synovia of patients with LA was defined by fluorescence-activated cell sorting analysis. NapA was intraarticularly injected into rat knees, and the cells recruited in synovia were characterized. The role of NapA in recruiting immune cells was confirmed by chemotaxis assays using a Transwell system.
NapA, IFNγ, IL-17, CCL2, CCL20, and CXCL10 accumulated in synovial fluid from patients with LA. Accordingly, T cells obtained from these patients produced IFNγ or IL-17, but notably, some produced both cytokines. NapA promoted neutrophil and T lymphocyte recruitment both in vitro and in vivo. Interestingly, the infiltration of T cells not only resulted from the chemotactic activity of NapA but also relied on the chemokines produced by PMNs exposed to NapA.
We provide evidence that NapA functions as one of the main bacterial products involved in the pathogenesis of LA. Accordingly, we show that, at very early stages of LA, NapA accumulates and, in turn, orchestrates the recruitment of inflammatory cells into the joint cavity. Thereafter, with the contribution of recruited cells, NapA promotes the infiltration of T cells producing IL-17 and/or IFNγ.
To evaluate patient predictors of good outcome following total joint arthroplasty (TJA).
A population cohort with hip/knee arthritis (osteoarthritis [OA] or inflammatory arthritis) ages ≥55 years was recruited between 1996 and 1998 (baseline) and assessed annually for demographics, troublesome joints, health status, and overall hip/knee arthritis severity using the Western Ontario and McMaster Universities OA Index (WOMAC). Survey data were linked with administrative databases to identify primary TJAs. Good outcome was defined as an improvement in WOMAC summary score greater than or equal to the minimal important difference (MID; 0.5 SD of the mean change). Logistic regression and Akaike's information criterion were used to determine the optimal number of predictors and the best model of that size. Log Poisson regression was used to determine the relative risk (RR) for a good outcome.
Primary TJA was performed in 202 patients (mean age 71.0 years; 79.7% female; 82.7% with >1 troublesome hip/knee; 65.8% knee replacements). Mean improvement in WOMAC summary score was 10.2 points (SD 18.05; MID 9 points). Of these patients, 53.5% experienced a good outcome. Four predictors were optimal. The best 4-variable model included pre-TJA WOMAC, comorbidity, number of troublesome hips/knees, and arthritis type (C statistic 0.80). The probability of a good outcome was greater with worse (higher) pre-TJA WOMAC summary scores (adjusted RR 1.32 per 10-point increase; P < 0.0001), fewer troublesome hips/knees (adjusted RR 0.82 per joint; P = 0.002), OA (adjusted RR for rheumatoid arthritis versus OA 0.33; P = 0.009), and fewer comorbidities (adjusted RR per condition 0.88; P = 0.01).
In an OA cohort with a high prevalence of multiple troublesome joints and comorbidity, only half achieved a good TJA outcome, defined as improved pain and disability. A more comprehensive assessment of the benefits and risks of TJA is warranted.
African American patients are significantly less likely to undergo knee replacement for the management of knee osteoarthritis (OA). Racial difference in preference (willingness) has emerged as a key factor. This study was undertaken to examine the efficacy of a patient-centered educational intervention on patient willingness and the likelihood of receiving a referral to an orthopedic clinic.
A total of 639 African American patients with moderate-to-severe knee OA from 3 Veterans Affairs primary care clinics were enrolled in a randomized, controlled trial with a 2 × 2 factorial design. Patients were shown a knee OA decision-aid video with or without brief counseling. The main outcome measures were change in patient willingness and receipt of a referral to an orthopedic clinic. Also assessed were whether patients discussed knee pain with their primary care provider or saw an orthopedic surgeon within 12 months of the intervention.
At baseline, 67% of the participants were definitely/probably willing to consider knee replacement, with no difference among the groups. The intervention increased patient willingness (75%) in all groups at 1 month. For those who received the decision aid intervention alone, the gains were sustained for up to 3 months. By 12 months postintervention, patients who received any intervention were more likely to report engaging their provider in a discussion about knee pain (92% versus 85%), to receive a referral to an orthopedic surgeon (18% versus 13%), and for those with a referral, to attend an orthopedic consult (61% versus 50%).
An educational intervention significantly increased the willingness of African American patients to consider knee replacement. It also improved the likelihood of patient–provider discussion about knee pain and access to surgical evaluation.
To evaluate the relevance of ongoing nociceptive joint inputs to the maintenance of widespread pain hypersensitivity in patients with hip osteoarthritis (OA) and to determine whether a reversal in the widespread pressure hypersensitivity together with an improvement in pain and function occurs after total hip replacement in these patients.
Forty patients with hip OA participated. Twenty patients underwent total hip replacement, and the other 20 patients were assigned to a waiting list. Pressure–pain thresholds (PPTs) over the second metacarpal bone and the gluteus medius, vastus medialis, vastus lateralis, and tibialis anterior muscles were assessed bilaterally with a pressure algometer before and 3 months after total hip replacement surgery. Assessments of pain intensity (by visual analog scale [VAS]), physical function (by the Western Ontario and McMaster Universities Osteoarthritis Index), and health status (by the Short Form 12 health survey and the EuroQol 5-domain index) were also performed.
Patients who underwent total hip arthroplasty exhibited a reduction in widespread pressure pain hyperalgesia (increases in PPTs) over local and distant pain-free areas, as compared with before surgery and as compared with the patients assigned to the waiting list. PPTs were related to hip pain intensity, and significant correlations were found between higher VAS scores and lower average PPTs over all points assessed (−0.409 < r < −0.306, P < 0.05). Patients who underwent total hip arthroplasty exhibited a greater decrease in pain intensity and greater increases in function and health status than did those who were on the waiting list. Changes in the intensity of hip pain were moderately associated with changes in pressure pain sensitivity in the hip arthroplasty group.
Normalization of widespread pressure pain hyperalgesia was found after successful hip joint replacement in patients with hip OA. Altered pain processing seems to be driven by ongoing peripheral joint pathology, which stresses the importance of reducing pain in OA.
To examine the effect of different sources of Good Manufacturing Practice clinical grade adipose-derived mesenchymal stem cells (AD-MSCs) on inflammatory factors in osteoarthritic (OA) chondrocytes and synoviocytes.
AD-MSCs from infrapatellar Hoffa fat, subcutaneous (SC) hip fat, and SC abdominal fat were cocultured in Transwells with chondrocytes or synoviocytes. Inflammatory factors (interleukin-1β [IL-1β], tumor necrosis factor α, IL-6, CXCL1/growth-related oncogene α, CXCL8/IL-8, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α, and CCL5/RANTES) were evaluated by quantitative reverse transcription–polymerase chain reaction or multiplex bead–based immunoassay. The role of different immunomodulators was analyzed.
All the inflammatory factors analyzed were down-modulated at the messenger RNA or protein level independently by all 3 AD-MSC sources or by allogeneic AD-MSCs used in coculture with chondrocytes or synoviocytes. Inflammatory factor down-modulation was observed only when AD-MSCs were cocultured with chondrocytes or synoviocytes that produced high levels of inflammatory factors, but no effect was observed in cells that produced low levels of those factors, thus highlighting a dependence of the AD-MSC effect on existing inflammation. The immunomodulators IL-10, IL-1 receptor antagonist, fibroblast growth factor 2, indoleamine 2,3-dioxygenase 1, and galectin 1 were not involved in AD-MSC effects, whereas the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway exerted a role in the mechanism of antiinflammatory AD-MSC action.
The antiinflammatory effects of AD-MSCs are probably not dependent on AD-MSC adipose tissue sources and donors but rather on the inflammatory status of OA chondrocytes and synoviocytes. AD-MSCs seem to be able to sense and respond to the local environment. Even though a combination of different molecules may be involved in AD-MSC effects, the COX-2/PGE2 pathway may play a role, suggesting that AD-MSCs may be useful for therapies in osteoarticular diseases.
Biomechanical interventions for knee osteoarthritis (OA) aim to improve pain and retard disease progression by decreasing knee loading. This study was undertaken to evaluate the effects of 6 months of use of flat, flexible footwear (the mobility shoe) on knee loading in OA.
Subjects with knee OA underwent baseline gait analyses under conditions of walking in their own shoes, walking in mobility shoes, and walking barefoot. Thereafter, subjects wore the mobility shoes at least 6 hours per day for 6 days per week. Gait evaluations were repeated at 6, 12, and 24 weeks. An intent-to-treat analysis was performed to assess the longitudinal effects on knee loading with the shoe intervention.
Compared to knee loading at baseline with the participants' own shoes, there was an 18% reduction in the knee adduction moment (KAM) by 24 weeks with the mobility shoes (P < 0.001) and no significant differences in the KAM by 24 weeks between mobility shoe and barefoot walking (P = 0.192). Over the 6 months of followup, participants also experienced an 11% reduction in the KAM when walking in their own shoes (P = 0.002) and a 10% reduction in the KAM when walking barefoot (P = 0.002 for the whole followup), as compared to these values at baseline under the same conditions.
This study suggests that use of flat, flexible footwear results in significant reductions in knee loading in subjects with OA. By 24 weeks, there is evidence of a gait adaptation with sustained load reduction even when the mobility shoes are removed, suggesting that footwear may serve as a biomechanical training device to achieve beneficial alterations in gait mechanics for knee OA.
To investigate the mechanism of matrix metalloproteinase 13 (MMP-13) expression in chondrocytes via pattern-recognition receptors (PRRs) for double-stranded RNA (dsRNA).
Differential expression of PRRs was determined by real-time reverse transcription–polymerase chain reaction (RT-PCR) of RNA from patients with osteoarthritis (OA) and patients with femoral neck fracture (as normal control). Isolated human articular chondrocytes and the chondrosarcoma cell line SW-1353 were activated with poly(I-C) of different molecular weights as a dsRNA mimic, and changes in gene and protein expression were monitored by real-time RT-PCR and immunoblotting, respectively.
The dsRNA signaling moieties Toll-like receptor 3 (TLR-3), retinoic acid–inducible gene 1 (RIG-1), and nucleotide-binding oligomerization domain–like receptor X1 were all differentially expressed in OA cartilage compared to normal cartilage, as determined by gene expression screening. Depletion of the dsRNA-sensing receptors TLR-3, RIG-1, or melanoma differentiation–associated gene 5 (MDA-5) suppressed the induction of MMP13 messenger RNA (mRNA) expression by poly(I-C), regardless of its mode of delivery. In addition, depletion of the downstream transcription factor interferon regulatory factor 3 resulted in reduced induction of MMP13 mRNA expression by poly(I-C).
Signaling by dsRNA in chondrocytes requires a range of PRRs, including TLR-3, RIG-1, and MDA-5, for the full-induction of MMP13, thus providing tight regulation of a gene critical for maintenance of cartilage integrity. Our data add to the understanding of MMP13 regulation, which is essential before such mechanisms can be exploited to alleviate the cartilage destruction associated with OA.
To determine the effects of hypoxia on both anabolic and catabolic pathways of metabolism in human articular cartilage and to elucidate the roles played by hypoxia-inducible factors (HIFs) in these responses.
Normal human articular cartilage from a range of donors was obtained at the time of above-the-knee amputations due to sarcomas not involving the joint space. Fresh cartilage tissue explants and isolated cells were subjected to hypoxia and treatment with interleukin-1α. Cell transfections were performed on isolated human chondrocytes.
Using chromatin immunoprecipitation, we found that hypoxia induced cartilage production in human tissue explants through direct binding of HIF-2α to a specific site in the master-regulator gene SOX9. Importantly, hypoxia also suppressed spontaneous and induced destruction of human cartilage in explant culture. We found that anticatabolic responses were predominantly mediated by HIF-1α. Manipulation of the hypoxia-sensing pathway through depletion of HIF-targeting prolyl hydroxylase–containing protein 2 (PHD-2) further enhanced cartilage responses as compared to hypoxia alone. Hypoxic regulation of tissue-specific metabolism similar to that in human cartilage was observed in pig, but not mouse, cartilage.
We found that resident chondrocytes in human cartilage are exquisitely adapted to hypoxia and use it to regulate tissue-specific metabolism. Our data revealed that while fundamental regulators, such as SOX9, are key molecules both in mice and humans, the way in which they are controlled can differ. This is all the more important since it is upstream regulators such as this that need to be directly targeted for therapeutic benefit. HIF-specific hydroxylase PHD-2 may represent a relevant target for cartilage repair.
To investigate whether attention deficit hyperactivity disorder (ADHD) may serve as a marker of neuropsychiatric disease and as a target for N-acetylcysteine (NAC) treatment in patients with systemic lupus erythematosus (SLE).
The ADHD Self-Report Scale (ASRS) was used to assess 49 patients with SLE and 46 matched healthy control subjects. Twenty-four of the patients with SLE were randomized to receive either placebo, NAC at a dosage of 2.4 gm/day, or NAC at a dosage of 4.8 gm/day. Disease activity was evaluated monthly using the British Isles Lupus Assessment Group (BILAG) index, the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), the Fatigue Assessment Scale (FAS), and the ASRS, before and during the 3-month treatment period and after a 1-month washout period.
The cognitive/inattentive (ASRS part A), hyperactivity/impulsive (ASRS part B), and combined (total) ASRS scores were increased in patients with SLE compared with control subjects (mean ± SEM 17.37 ± 1.03 [P = 3 × 10−7], 14.51 ± 0.89 [P = 2 × 10−4], and 31.92 ± 1.74 [P = 8 × 10−7], respectively, versus 10.41 ± 1.02, 9.61 ± 1.21, and 20.02 ± 1.98, respectively. ASRS part A scores correlated with SLEDAI (r = 0.53, P < 0.0001) and BILAG scores (r = 0.36, P = 0.011). ASRS total scores also correlated with SLEDAI (r = 0.45, P = 0.0009) and BILAG scores (r = 0.31, P = 0.025). ASRS part A (r = 0.73, P < 0.0001), ASRS part B (r = 0.47, P = 0.0006), and ASRS total scores (r = 0.67, P < 0.0001) correlated with the FAS score. Relative to the scores in placebo-treated patients, ASRS total scores were reduced in SLE patients treated with NAC dosages of 2.4 gm/day and 4.8 gm/day combined (P = 0.037). ASRS part A scores were reduced by NAC dosages of 2.4 gm/day (P = 0.001) and 4.8 gm/day (P < 0.0001) as well as by NAC at dosages of 2.4 gm/day and 4.8 gm/day combined (P = 0.001).
In patients with SLE, elevated ASRS scores reveal previously unrecognized and clinically significant symptoms of ADHD that respond to NAC treatment.
Patients with systemic lupus erythematosus (SLE) have a higher rate of premature death compared to the general population, suggesting a phenotype of premature senescence in SLE. Telomere length can be used to assess overall biologic aging. This study was undertaken to address the hypothesis that patients with SLE have reduced telomere length.
Telomere length was measured cross-sectionally in whole blood from SLE patients and age-matched healthy female controls, using real-time quantitative polymerase chain reaction. SLE-related and cardiovascular risk factors were assessed.
We compared telomere length in 63 SLE patients and 63 matched controls with a median age of 50.8 years (interquartile range [IQR] 37–59 years) and 49.9 years (IQR 32–60 years), respectively. The median relative telomere length in SLE patients was 0.97 (IQR 0.47–1.57), compared to 1.53 (IQR 0.82–2.29) in controls (P = 0.0008). We then extended our cohort to measure telomere length in 164 SLE patients. Shorter telomere length was associated with Ro antibodies (β ± SE −0.36 ± 0.16; P = 0.023), and longer telomere length was associated with steroid therapy (0.29 ± 0.14; P = 0.046). We also noted an association of longer telomere length with increasing body mass index (β ± SE 0.07 ± 0.01; P < 0.0001) and tobacco smoking (0.64 ± 0.26; P = 0.016), as well as with the presence of carotid plaque (0.203 ± 0.177; P = 0.032).
Telomere length is shortened in SLE patients compared to controls and does not appear to be a reflection of disease activity or immune cell turnover. Subsets of patients such as those positive for Ro antibodies may be particularly susceptible to premature biologic aging. The predictive value of telomere length as a biomarker of future risk of damage/mortality in SLE requires longitudinal evaluation.
To evaluate the specificity of expression patterns of cell-free circulating microRNAs (miRNAs) in systemic lupus erythematosus (SLE).
Total RNA was purified from plasma, and 45 different specific, mature miRNAs were determined using quantitative reverse transcription–polymerase chain reaction assays. A total of 409 plasma samples were obtained from 364 different patients with SLE, healthy control subjects, and control subjects with other autoimmune diseases. The results in the primary cohort of 62 patients with SLE and 29 healthy control subjects were validated in 2 independent cohorts: a validation cohort comprising 68 patients with SLE and 68 healthy control subjects, and a disease control cohort comprising 20 patients with SLE (19 of whom were from the other validation cohort), 46 healthy control subjects, 38 patients with vasculitis, 18 patients with rheumatoid arthritis, and 20 immunosuppressed patients.
Seven miRNAs were statistically significantly differentially expressed in plasma from patients with SLE. The expression of miRNA-142-3p (miR-142-3p) and miR-181a was increased, and the expression of miR-106a, miR-17, miR-20a, miR-203, and miR-92a was decreased. In addition, the expression of miR-342-3p, miR-223, and miR-20a was significantly decreased in SLE patients with active nephritis. A predictive model for SLE based on 2 or 4 miRNAs differentiated patients with SLE from control subjects (76% accuracy) when validated independently (P < 2 × 10−9). Use of the 4-miRNA model provided highly significant differentiation between the SLE group and disease controls, except for those with vasculitis.
Circulating miRNAs are systematically altered in SLE. A 4-miRNA signature was diagnostic of SLE, and a specific subset of miRNA profiles was associated with nephritis. All of the signature miRNAs target genes in the transforming growth factor β signaling pathways. Other targets include regulation of apoptosis, cytokine–cytokine receptors, T cell development, and cytoskeletal organization. These findings highlight possible dysregulated pathways in SLE and suggest that circulating miRNA patterns distinguish SLE from other immunoinflammatory phenotypes.
To explore the expression of thymic stromal lymphopoietin (TSLP) in patients with diffuse cutaneous systemic sclerosis (dcSSc) and compare its effects in vivo and in vitro with those of interleukin-13 (IL-13) and transforming growth factor β (TGFβ).
Skin biopsy specimens from patients with dcSSc (n = 14) and healthy controls (n = 13) were analyzed by immunohistochemistry and immunofluorescence for TSLP, TSLP receptor, CD4, CD8, CD31, and CD163 markers. Wild-type, IL-4Rα1–, and TSLP-deficient mice were treated with TGFβ, IL-13, poly(I-C), or TSLP by osmotic pump. Human fibroblasts and peripheral blood mononuclear cells (PBMCs) were stimulated with TGFβ, IL-13, poly(I-C), or TSLP. Microarray analysis and quantitative polymerase chain reaction were performed to determine gene expression, and protein levels of phospho-Smad2 and macrophage marker CD163 were tested.
TSLP was highly expressed in the skin of dcSSc patients, more strongly in perivascular areas and in immune cells, and was produced mainly by CD163+ cells. The skin of TSLP-treated mice showed up-regulated clusters of gene expression that overlapped strongly with those in IL-13– and TGFβ-treated mice. TSLP up-regulated specific genes, including CXCL9, proteasome, and interferon (IFN)–regulated genes. TSLP treatment in IL-4Rα1–deficient mice promoted similar cutaneous inflammation as in wild-type mice, though TSLP-induced arginase 1, CCL2, and matrix metalloproteinase 12 messenger RNA levels were blocked. In PBMCs, TSLP up-regulated tumor necrosis factor α, Mx-1, IFNγ, CXCL9, and mannose receptor 1 gene expression. TSLP-deficient mice treated with TGFβ showed less fibrosis and blocked expression of plasminogen activator inhibitor 1 and osteopontin 1. Poly(I-C)–treated mice showed high levels of cutaneous TSLP.
TSLP is highly expressed in the skin of dcSSc patients and interacts in a complex manner with 2 other profibrotic cytokines, TGFβ and IL-13, strongly suggesting that it might promote SSc fibrosis directly or indirectly by synergistically stimulating profibrotic genes, or production of these cytokines.
Levels of interleukin-17A (IL-17A) have been found to be increased in synovial fluid from individuals with systemic sclerosis (SSc). This study was undertaken to investigate whether IL-17A–producing cells are present in affected SSc skin, and whether IL-17A exerts a role in the transdifferentiation of myofibroblasts.
Skin biopsy samples were obtained from the involved skin of 8 SSc patients and from 8 healthy control donors undergoing plastic surgery. Immunohistochemistry and multicolor immunofluorescence techniques were used to identify and quantify the cell subsets in vivo, including IL-17A+, IL-4+, CD3+, tryptase-positive, α-smooth muscle actin (α-SMA)–positive, myeloperoxidase-positive, and CD1a+ cells. Dermal fibroblast cell lines were generated from all skin biopsy samples, and quantitative polymerase chain reaction, Western blotting, and solid-phase assays were used to quantify α-SMA, type I collagen, and matrix metalloproteinase 1 (MMP-1) production by the cultured fibroblasts.
IL-17A+ cells were significantly more numerous in SSc skin than in healthy control skin (P = 0.0019) and were observed to be present in both the superficial and deep dermis. Involvement of both T cells and tryptase-positive mast cells in the production of IL-17A was observed. Fibroblasts positive for α-SMA were found adjacent to IL-17A+ cells, but not IL-4+ cells. However, IL-17A did not induce α-SMA expression in cultured fibroblasts. In the presence of IL-17A, the α-SMA expression induced in response to transforming growth factor β was decreased, while MMP-1 production was directly enhanced. Furthermore, the frequency of IL-17A+ cells was higher in the skin of SSc patients with greater severity of skin fibrosis (lower global skin thickness score).
IL-17A+ cells belonging to the innate and adaptive immune system are numerous in SSc skin. IL-17A participates in inflammation while exerting an inhibitory activity on myofibroblast transdifferentiation. These findings are consistent with the notion that IL-17A has a direct negative-regulatory role in the development of dermal fibrosis in humans.
Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage in peripheral blood mononuclear cells (PBMCs). Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been implicated in autoimmune and inflammatory diseases. The goal of this study was to assess whether markers of ER stress and the UPR are present in PBMCs from lcSSc patients with PAH.
PBMCs were purified from 36 healthy controls, 32 lcSSc patients with PAH, and 34 lcSSc patients without PAH. Gene expression in healthy control PBMCs stimulated with thapsigargin was analyzed by DNA microarray. Genes were validated by quantitative real-time reverse transcription–polymerase chain reaction in PBMCs from healthy controls and lcSSc patients.
Several ER stress/UPR genes, including BiP, activating transcription factor 4 (ATF-4), ATF-6, and a spliced form of X-box binding protein 1, were up-regulated in PBMCs from lcSSc patients, with the highest levels in patients with PAH. Thapsigargin up-regulated heat-shock proteins (HSPs) and interferon (IFN)–regulated genes in PBMCs from healthy controls. Selected HSP genes (particularly DnaJB1) and IFN-related genes were also found at significantly elevated levels in PBMCs from lcSSc patients, while IFN regulatory factor 4 expression was significantly decreased. There was a positive correlation between DnaJB1 and severity of PAH (measured by pulmonary artery pressure) (r = 0.56, P < 0.05) and between ER stress markers and interleukin-6 levels (r = 0.53, P < 0.0001) in PBMCs from lcSSc patients.
This study demonstrates an association between select ER stress/UPR markers and lcSSc with PAH, suggesting that ER stress and the UPR may contribute to the altered function of circulating immune cells in lcSSc.
Systemic sclerosis (SSc) is characterized by microvascular damage, fibrosis of skin and visceral organs, and autoimmunity. Previous studies have shown that angiotensin II is involved in the synthesis of type I collagen. We investigated whether the blockade of angiotensin II receptor type I (AT1) by irbesartan reduces skin and lung fibrosis in 2 murine models of SSc.
SSc was induced by daily intradermal injection of HOCl into the backs of BALB/c mice (HOCl-induced SSc). Mice were treated daily with irbesartan by oral gavage.
Irbesartan reduced dermal thickness, collagen concentration, Smad2/3, and α-smooth muscle actin expression, as well as fibroblast proliferation and H-Ras expression in the skin of mice with HOCl-induced SSc. Mice treated with irbesartan also displayed less lung fibrosis, less inflammation, and a lower concentration of collagen in the lungs than untreated mice. Exhaled nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine expression in the lungs were decreased following irbesartan treatment. Moreover, irbesartan reduced the number and the proliferation of splenic B and T cells and the serum levels of anti–DNA topoisomerase I autoantibodies.
Irbesartan, an AT1 antagonist, prevents fibrosis and inflammation and inhibits nitric oxide production in HOCl-induced models of systemic fibrosis. Our findings extend the indication of an AT1 antagonist to SSc patients with diffuse fibrosis, especially those with lung involvement.
Transforming growth factor β (TGFβ) is a profibrotic cytokine, and its aberrant function is implicated in several types of fibrotic pathologies including scleroderma (systemic sclerosis [SSc]). Multiple lines of evidence show that increased TGFβ signaling contributes to progressive fibrosis in SSc by promoting fibroblast activation, excessive extracellular matrix (ECM) deposition, and dermal thickening. We have previously identified CD109 as a TGFβ coreceptor and have shown that it antagonizes TGFβ signaling and TGFβ-induced ECM expression in vitro in human keratinocytes and fibroblasts. The aim of the present study was to examine the ability of CD109 to prevent skin fibrosis in a mouse model of bleomycin-induced SSc.
Transgenic mice overexpressing CD109 in the epidermis and their wild-type (WT) littermates were injected with bleomycin in phosphate buffered saline (PBS) or with PBS alone every other day for 21 days or 28 days. Dermal thickness and collagen deposition were determined histologically using Masson's trichrome and picrosirius red staining. In addition, collagen and fibronectin content was analyzed using Western blotting, and activation of TGFβ signaling was examined by determining phospho-Smad2 and phospho-Smad3 levels using Western blotting and immunohistochemistry.
Transgenic mice overexpressing CD109 in the epidermis showed resistance to bleomycin-induced skin fibrosis, as evidenced by a significant decrease in dermal thickness, collagen crosslinking, collagen and fibronectin content, and phospho-Smad2/3 levels, as compared to their WT littermates.
Our findings suggest that CD109 inhibits TGFβ signaling and fibrotic responses in experimental murine scleroderma. They also suggest that CD109 regulates dermal–epidermal interactions to decrease extracellular matrix synthesis in the dermis. Thus, CD109 is a potential molecular target for therapeutic intervention in scleroderma.
To compare incidence rates of selected opportunistic infections among children with and children without juvenile idiopathic arthritis (JIA).
Using US national Medicaid administrative claims data from 2000 through 2005, we identified a cohort of children with JIA based on physician diagnosis codes and dispensed medications. We also identified a non-JIA comparator cohort of children diagnosed as having attention deficit hyperactivity disorder (ADHD). We defined 15 types of opportunistic infection using physician diagnosis or hospital discharge codes; criteria for 7 of these types also included evidence of treatment with specific antimicrobial agents. We calculated infection incidence rates. The rates in the ADHD comparator cohort were standardized to the age, sex, and race distribution of the JIA cohort. We calculated incidence rate ratios (IRRs) with 95% confidence intervals (95% CIs) to compare infection rates.
The JIA cohort included 8,503 children with 13,990 person-years of followup. The ADHD comparator cohort included 360,362 children with 477,050 person-years of followup. When all opportunistic infections were considered together as a single outcome, there were 42 infections in the JIA cohort (incidence rate 300 per 100,000 person-years; IRR 2.4 [95% CI 1.7–3.3] versus ADHD). The most common opportunistic infections among children with JIA were 3 cases of Coccidioides (incidence rate 21 per 100,000 person-years; IRR 101 [95% CI 8.1–5,319] versus ADHD), 5 cases of Salmonella (incidence rate 35 per 100,000 person-years; IRR 3.8 [95% CI 1.2–9.5]), and 32 cases of herpes zoster (incidence rate 225 per 100,000 person-years; IRR 2.1 [95% CI 1.4–3.0]).
Opportunistic infections are rare among children with JIA. Nevertheless, children with JIA had a higher rate of opportunistic infections, including an increased rate of Coccidioides, Salmonella, and herpes zoster compared to children with ADHD.
To investigate the role of the antiinflammatory neuropeptide cortistatin in chronic pain evoked by joint inflammation.
Thermal and mechanical hyperalgesia was evoked in mouse knee joints by intraplantar injection of tumor necrosis factor α and intraarticular infusion of Freund's complete adjuvant, and the analgesic effects of cortistatin, administered centrally, peripherally, and systemically, were assessed. In addition, the effects of cortistatin on the production of nociceptive peptides and the activation of pain signaling were assayed in dorsal root ganglion cultures and in inflammatory pain models. The role of endogenous cortistatin in pain sensitization and perpetuation of chronic inflammatory states was evaluated in cortistatin-deficient mice. Finally, the effect of noxious/inflammatory stimuli in the production of cortistatin by the peripheral nociceptive system was assayed in vitro and in vivo.
Expression of cortistatin was observed in peptidergic nociceptors of the peripheral nociceptive system, and endogenous cortistatin was found to participate in the tuning of pain sensitization, especially in pathologic inflammatory conditions. Results showed that cortistatin acted both peripherally and centrally to reduce the tactile allodynia and heat hyperalgesia evoked by arthritis and peripheral tissue inflammation in mice, via mechanisms that were independent of its antiinflammatory action. These mechanisms involved direct action on nociceptive neurons and regulation of central sensitization. The analgesic effects of cortistatin in murine arthritic pain were linked to binding of the neuropeptide to somatostatin and ghrelin receptors, activation of the G protein subunit Gαi, impairment of ERK signaling, and decreased production of calcitonin gene-related peptide in primary nociceptors.
These findings indicate that cortistatin is an antiinflammatory factor with potent analgesic effects that may offer a new approach to pain therapy in pathologic inflammatory states, including osteoarthritis and rheumatoid arthritis.