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1.  Oral mucosal involvement in bullous lupus 
Arthritis and rheumatism  2013;65(10):2622.
doi:10.1002/art.38051
PMCID: PMC4333153  PMID: 23780804
2.  The Clinical Continuum of Cryopyrinopathies 
Arthritis and rheumatism  2007;56(4):1273-1285.
Objective
The cryopyrinopathies are a group of rare autoinflammatory disorders that are caused by mutations in CIAS1, encoding the cryopyrin protein. However, cryopyrin mutations are found only in 50% of patients with clinically diagnosed cryopyrinopathies. This study was undertaken to investigate the structural effect of disease-causing mutations on cryopyrin, in order to gain better understanding of the impact of disease-associated mutations on protein function.
Methods
We tested for CIAS1 mutations in 22 patients with neonatal-onset multisystem inflammatory disease/chronic infantile neurologic, cutaneous, articular syndrome, 12 with Muckle-Wells syndrome (MWS), 18 with familial cold-induced autoinflammatory syndrome (FCAS), and 3 probands with MWS/FCAS. In a subset of mutation-negative patients, we screened for mutations in proteins that are either homologous to cryopyrin or involved in the caspase 1/interleukin-1β signaling pathway. CIAS1 and other candidate genes were sequenced, models of cryopyrin domains were constructed using structurally homologous proteins as templates, and disease-causing mutations were mapped.
Results
Forty patients were mutation positive, and 7 novel mutations, V262A, C259W, L264F, V351L, F443L, F523C, and Y563N, were found in 9 patients. No mutations in any candidate genes were identified. Most mutations mapped to an inner surface of the hexameric ring in the cryopyrin model, consistent with the hypothesis that the mutations disrupt a closed form of cryopyrin, thus potentiating inflammasome assembly. Disease-causing mutations correlated with disease severity only for a subset of known mutations.
Conclusion
Our modeling provides insight into potential molecular mechanisms by which cryopyrin mutations can inappropriately activate an inflammatory response. A significant number of patients who are clinically diagnosed as having cryopyrinopathies do not have identifiable disease-associated mutations.
doi:10.1002/art.22491
PMCID: PMC4321998  PMID: 17393462
3.  Complete and Sustained Remission of Juvenile Dermatomyositis Resulting From Aggressive Treatment 
Arthritis and rheumatism  2009;60(6):1825-1830.
Objective
To assess the time needed to achieve sustained, medication-free remission in a cohort of patients with juvenile dermatomyositis (DM) receiving a stepwise, aggressive treatment protocol.
Methods
Between 1994 and 2004, a cohort of 49 children with juvenile DM who were followed up at a single tertiary care children's hospital using disease activity measures according to a specific protocol received standardized therapy with steroids and methotrexate. If a patient's strength or muscle enzyme levels did not normalize with this initial therapy, additional medications were added in rapid succession to the treatment regimen. The primary outcome measure was time to complete remission. Additional outcome measures were onset of calcinosis, effect of treatment on height, and complications resulting from medications.
Results
Forty-nine patients were followed up for a mean ± SD of 48 ± 30 months. All but 1 patient received 2 or more medications simultaneously. Transient localized calcifications occurred in 4 patients (8%), and 2 additional patients (4%) had persistent calcinosis. Despite the aggressive therapy, complications associated with treatment were mild and were primarily attributable to steroids. No persistent effect on longitudinal growth was observed. A complete, medication-free remission was achieved in 28 patients; the median time to achievement of complete remission was 38 months (95% confidence interval 32–44 months). None of these patients experienced a disease flare that required resumption of medications during the subsequent period of observation (mean ± SD 36 ± 19.7 months).
Conclusion
Our findings suggest that aggressive treatment of juvenile DM aimed at achieving rapid, complete control of muscle weakness and inflammation improves outcomes and reduces disease-related complications. In more than one-half of the children whose disease was treated in this manner (28 of 49), a prolonged, medication-free remission was attained within a median of 38 months from the time of diagnosis.
doi:10.1002/art.24571
PMCID: PMC4254704  PMID: 19479872
4.  Recommendations for Screening and Detection of Connective-Tissue Disease Associated Pulmonary Arterial Hypertension 
Arthritis and rheumatism  2013;65(12):10.1002/art.38172.
Objectives
Pulmonary arterial hypertension (PAH) affects up to 15% of patients with connective tissue diseases (CTD). Previous recommendations developed as part of larger efforts in PAH did not provide detailed recommendations for patients with CTD-PAH. Therefore, we sought to develop recommendations for screening and early detection of CTD-PAH.
Methods
We performed a systematic review for the screening and diagnosis of PAH in CTD by searching the literature. Using the RAND/UCLA methodology, we developed case scenarios followed by 2 stages of voting—first international experts from a variety of specialties voted anonymously on the appropriateness of each case scenario and then the experts met in a face-to-face meeting to discuss and resolve discrepant votes to arrive at consensus recommendations.
Results
The key recommendations state that patients with systemic sclerosis (SSc) should be screened for PAH. In addition, mixed connective tissue diseases (MCTD) or other CTD’s with scleroderma features should also be screened for PAH (scleroderma-spectrum disorder). Initial screening evaluation in patients with SSc and scleroderma-spectrum disorders include pulmonary function test (PFT) including diffusion capacity carbon monoxide (DLCO), transthoracic echocardiogram (TTE), and NT- Pro BNP. In SSc and spectrum disorders, TTE and PFT should be performed on annual basis. The full screening panel (TTE, PFT, and NT-ProBNP) should be performed as soon as any new signs or symptoms are present.
Conclusion
We provide consensus-based, evidence-driven recommendations for screening and early detection of CTD-PAH. It is our hope that these recommendations will lead to earlier detection of CTD-PAH and ultimately improve patient outcomes.
doi:10.1002/art.38172
PMCID: PMC3883571  PMID: 24022584
Pulmonary hypertension; pulmonary arterial hypertension; connective tissue diseases; systemic sclerosis; recommendations; guidelines
5.  Genome-wide Association Study of Dermatomyositis Reveals Genetic Overlap with other Autoimmune Disorders 
Arthritis and rheumatism  2013;65(12):3239-3247.
Objective
To identify new genetic associations with juvenile and adult dermatomyositis (DM).
Methods
We performed a genome-wide association study (GWAS) of adult and juvenile DM patients of European ancestry (n = 1178) and controls (n = 4724). To assess genetic overlap with other autoimmune disorders, we examined whether 141 single nucleotide polymorphisms (SNPs) outside the major histocompatibility complex (MHC) locus, and previously associated with autoimmune diseases, predispose to DM.
Results
Compared to controls, patients with DM had a strong signal in the MHC region consisting of GWAS-level significance (P < 5x10−8) at 80 genotyped SNPs. An analysis of 141 non-MHC SNPs previously associated with autoimmune diseases showed that three SNPs linked with three genes were associated with DM, with a false discovery rate (FDR) < 0.05. These genes were phospholipase C like 1 (PLCL1, rs6738825, FDR=0.00089), B lymphoid tyrosine kinase (BLK, rs2736340, FDR=0.00031), and chemokine (C-C motif) ligand 21 (CCL21, rs951005, FDR=0.0076). None of these genes was previously reported to be associated with DM.
Conclusion
Our findings confirm the MHC as the major genetic region associated with DM and indicate that DM shares non-MHC genetic features with other autoimmune diseases, suggesting the presence of additional novel risk loci. This first identification of autoimmune disease genetic predispositions shared with DM may lead to enhanced understanding of pathogenesis and novel diagnostic and therapeutic approaches.
doi:10.1002/art.38137
PMCID: PMC3934004  PMID: 23983088
dermatomyositis; adult; juvenile; shared autoimmunity genes
6.  9G4+ auto-antibodies are an important source of apoptotic cell reactivity associated with high disease activity in systemic lupus erythematosus 
Arthritis and rheumatism  2013;65(12):3165-3175.
Objective
To determine the prevalence anti-apoptotic cell antibodies with the 9G4+ idiotype (9G4+) and the relationship between this reactivity and other known 9G4+ specificities and disease activity.
Methods
Sera from 60 SLE patients and 40 healthy control donors were incubated with apoptotic Jurkat cells and assayed for binding of 9G4+ antibodies by flow cytometry. These samples were also tested for 9G4+ reactivity against naïve B cells and total IgG and IgM anti-apoptotic cell antibody (AACA) reactivity.
Results
9G4+ antibodies bound late apoptotic cells in a majority of SLE patients (60%) but only 2 healthy control sera. Among samples with global IgM or IgG AACA, samples with 9G4+ AACA predominated. Patients with high levels of 9G4+ AACA were more likely to have active disease and this remained true even in patients with IgG AACA, or anti-dsDNA. Patients with lupus nephritis were also more likely to have 9G4+ AACA. While 9G4+ reactivity to apoptotic cells often coincided with anti-B cell reactivity, some samples had distinct anti-apoptotic cell or anti-B cell reactivity.
Conclusion
9G4+ antibodies represent a major species of anti-apoptotic cell antibodies in SLE serum and this autoreactivity is associated with disease activity. The anti-apoptotic cell reactivity of 9G4+ antibodies can be separated from the germline VH4-34 encoded anti-B cell autoreactivity. Our results indicate that apoptotic cells are an important antigenic source in SLE that positively select B cells with intrinsic autoreactivity against other self-antigens. This selection of 9G4+ B cells by apoptotic cells may represent an important step in disease progression.
doi:10.1002/art.38138
PMCID: PMC3981458  PMID: 23983101
7.  Overlapping structural and functional brain changes in patients with long-term exposure to fibromyalgia 
Arthritis and rheumatism  2013;65(12):3293-3303.
Objective
There is vast evidence for brain aberrations in patients with fibromyalgia (FM) and it is possible that central plasticity is critical for the transition from acute to chronic pain. However, the relationship between brain structure and function is poorly investigated.
Methods
The present study, including 26 FM patients and 13 age- and gender-matched healthy controls, investigated the differences between patients and controls regarding functional connectivity during intermittent pressure pain and measures of brain structure. Magnetic resonance imaging (MRI) was used to obtain high-resolution anatomical images and functional MRI scans for measures of pain-evoked brain activity.
Results
FM patients displayed a distinct overlap between decreased cortical thickness, brain volumes and measures of functional regional coherence in the rostral anterior cingulate cortex. The morphometric changes were more pronounced with longer exposure to FM pain. In addition, we found associations between structural and functional changes in the mesolimbic areas of the brain and comorbid depressive symptoms in FM patients.
Conclusion
The combined integration of structural and functional measures allowed for a unique characterization of the impact of FM pain on the brain. Our data may lead to the identification of early structural and functional brain alterations in response to pain, which could be used to develop markers to predict the development of FM and other pain disorders.
doi:10.1002/art.38170
PMCID: PMC3984030  PMID: 23982850
Magnetic Resonance Imaging; fibromyalgia; morphology; functional connectivity; ReHo
8.  P2X7 Blockade Attenuates Murine Lupus Nephritis by Inhibiting Activation of the NLRP3/ASC/Caspase 1 Pathway 
Arthritis and rheumatism  2013;65(12):3176-3185.
Objective
The NLRP3 inflammasome plays key roles in inflammation and autoimmunity, and puriner-gic receptor P2X7 has been proposed to be upstream of NLRP3 activation. The aim of the present study, using murine models, was to investigate whether the P2X7/ NLRP3 inflammasome pathway contributes to the pathogenesis of lupus nephritis (LN).
Methods
MRL/lpr mice were treated with the selective P2X7 antagonist brilliant blue G (BBG) for 8 weeks. Following treatment, the severity of renal lesions, production of anti-double-stranded DNA (anti-dsDNA) antibodies, rate of survival, activation of the NLRP3/ ASC/caspase 1 inflammasome pathway, and ratio of Thl7 cells to Treg cells were evaluated. P2X7-targeted small interfering RNA (siRNA) was also used for in vivo intervention. Similar evaluations were carried out in NZM2328 mice, a model of LN in which the disease was accelerated by administration of adenovirus-expressing interferon-α (AdIFNα).
Results
Significant up-regulation of P2X7/NLRP3 inflammasome signaling molecules was detected in the kidneys of MLR/lpr mice as compared with normal control mice. Blockade of P2X7 activation by BBG suppressed NLRP3/ASC/caspase 1 assembly and the subsequent release of interleukin-1β (IL-1β), resulting in a significant reduction in the severity of nephritis and circulating anti-dsDNA antibodies. The lifespan of the treated mice was significantly prolonged. BBG treatment reduced the serum levels of IL-1β and IL-17 and the Thl7:Treg cell ratio. Similar results were obtained by specific siRNA silencing of P2X7 in vivo. The effectiveness of BBG treatment in modulating LN was confirmed in NZM2328 mice with AdIFNα-accelerated disease.
Conclusion
Activation of the P2X7 signaling pathway accelerates murine LN by activating the NLRP3/ASC/caspase 1 inflammasome, resulting in increased IL-1β production and enhanced Thl7 cell polarization. Thus, targeting of the P2X7/NLRP3 pathway should be considered as a novel therapeutic strategy in patients with lupus.
doi:10.1002/art.38174
PMCID: PMC4038356  PMID: 24022661
9.  Value of Isolated IgA anti-β2GPI Positivity in the Diagnosis of the Antiphospholipid Syndrome 
Arthritis and rheumatism  2013;65(12):3186-3193.
Purpose
To examine the prevalence of isolated IgA anti-β2Glycoprotein I (anti-β2GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of β2GPI, with clinical manifestations of the Antiphospholipid Syndrome (APS) in three patients groups. The pathogenicity of IgA anti-β2GPI was also evaluated in a mouse model of thrombosis.
Methods
Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture [LUMINA]) (n=558), patients with SLE from the Hopkins Lupus Cohort (n=215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n=5,098) were evaluated. IgA anti-β2GPI titers and binding to domain IV/V of β2GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti- β2GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity.
Results
A total of 198 patients were found to be positive for IgA anti-β2GPI isotype, and 57 patients were positive exclusively for IgA anti-β2GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-β2GPI positive serum samples reacted with domain IV/V of anti-β2GPI, and 77% of those had clinical features of APS. Isolated IgA anti-β2GPI positivity was associated with an increased risk for arterial thrombosis (p<0.001), venous thrombosis (p=0.015) and all thrombosis (p<0.001). The association between isolated IgA anti-β2GPI and arterial thrombosis (p=0.0003) and all thrombosis (p=0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-β2GPI antibodies induced significantly larger thrombi and higher TF levels compared to controls.
Conclusion
Isolated IgA anti-β2GPI positive titers may identify additional patients with clinical features of APS. Testing for these antibodies when other antiphospholipid (aPL) tests are negative and APS is suspected is recommended. IgA anti-β2GPI antibodies directed to domain IV/V of β2GPI represent an important subgroup of clinically relevant antiphospholipids.
doi:10.1002/art.38131
PMCID: PMC4048705  PMID: 23983008
10.  Sulforaphane Represses Matrix-Degrading Proteases and Protects Cartilage From Destruction In Vitro and In Vivo 
Arthritis and Rheumatism  2013;65(12):3130-3140.
Objective
Sulforaphane (SFN) has been reported to regulate signaling pathways relevant to chronic diseases. The aim of this study was to investigate the impact of SFN treatment on signaling pathways in chondrocytes and to determine whether sulforaphane could block cartilage destruction in osteoarthritis.
Methods
Gene expression, histone acetylation, and signaling of the transcription factors NF-E2–related factor 2 (Nrf2) and NF-κB were examined in vitro. The bovine nasal cartilage explant model and the destabilization of the medial meniscus (DMM) model of osteoarthritis in the mouse were used to assess chondroprotection at the tissue and whole-animal levels.
Results
SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes and in fibroblast-like synovial cells. SFN acted independently of Nrf2 and histone deacetylase activity to regulate metalloproteinase expression in human articular chondrocytes but did mediate prolonged activation of JNK and p38 MAPK. SFN attenuated NF-κB signaling at least through inhibition of DNA binding in human articular chondrocytes, with decreased expression of several NF-κB–dependent genes. Compared with cytokines alone, SFN (10 μM) abrogated cytokine-induced destruction of bovine nasal cartilage at both the proteoglycan and collagen breakdown levels. An SFN-rich diet (3 μmoles/day SFN versus control chow) decreased the arthritis score in the DMM model of osteoarthritis in the mouse, with a concurrent block of early DMM-induced gene expression changes.
Conclusion
SFN inhibits the expression of key metalloproteinases implicated in osteoarthritis, independently of Nrf2, and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo.
doi:10.1002/art.38133
PMCID: PMC4240673  PMID: 23983046
11.  Skin Gene Expression Correlates of Severity of Interstitial Lung Disease in Systemic Sclerosis 
Arthritis and rheumatism  2013;65(11):2917-2927.
Objective
We undertook this hypothesis-generating study to identify skin transcripts correlating with severity of interstitial lung disease (ILD) in systemic sclerosis (SSc).
Methods
Skin biopsy samples from 59 patients enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort or an open-label imatinib study (baseline visit) were examined by global gene expression analysis using Illumina HT-12 arrays. Skin transcripts correlating with concomitantly obtained forced vital capacity (FVC) values and the modified Rodnan skin thickness score (MRSS) were identified by quantitative trait analysis. Also, immunofluorescence staining for selected transcripts was performed in affected skin and lung tissue. Plasma levels of CCL2, soluble SELP, and soluble P-selectin glycoprotein ligand 1 (sPSGL-1) were examined in all patients enrolled in the GENISOS cohort (n = 266).
Results
Eighty-two skin transcripts correlated significantly with FVC. This gene list distinguished patients with more severe ILD (FVC <70% predicted) in unsupervised hierarchical clustering analysis (P < 0.001). These genes included SELP, CCL2, and matrix metalloproteinase 3, which are involved in extravasation and adhesion of inflammatory cells. Among the FVC correlates, 8 genes (CCL2, HAPLN3, GPR4, ADCYAP1, WARS, CDC25B, PLP1, and STXBP6) also correlated with the MRSS. Immunofluorescence staining revealed that SELP and CCL2 were also overexpressed in affected skin and lung tissue from SSc patients compared to those from controls. Plasma levels of CCL2 and sPSGL-1 correlated with concomitantly obtained FVC values (r = −0.22, P = 0.001 and r = 0.17, P = 0.015, respectively). This relationship was independent of potential confounders (age, sex, ethnicity, smoking status, anti–topoisomerase I positivity, treatment with immunosuppressive agents, MRSS, disease type, and disease duration).
Conclusion
A limited number of skin transcripts including genes involved in extravasation and adhesion of inflammatory cells correlate with severity of ILD.
doi:10.1002/art.38101
PMCID: PMC3898704  PMID: 23897225
12.  Insufficient autoantigen presentation and failure of tolerance in a mouse model of rheumatoid arthritis 
Arthritis and rheumatism  2013;65(11):2847-2856.
Objective
In the K/BxN mouse model of rheumatoid arthritis, T cells reactive for the self-antigen glucose-6-phosphate isomerase (GPI) escape negative selection even though GPI expression is ubiquitous. We sought to determine whether insufficient GPI presentation could account for the failure of negative selection and development of arthritis.
Methods
To increase the antigen presentation of GPI, we generated transgenic mice expressing a membrane-bound form of GPI (mGPI) and crossed them to the K/BxN mice. A monoclonal antibody specific for the alpha chain of the KRN TCR was generated to follow the fate of GPI-specific T cells.
Results
mGPI transgenic mice presented GPI more efficiently and showed a dramatic increase of negative selection and an inhibition of arthritis. Interestingly, thymic negative selection remained incomplete in these mice, and the escaped autoreactive T cells were anergic in the periphery, suggesting that enhanced antigen presentation also induces peripheral tolerance. Despite this apparent tolerance induction towards GPI, these mice did go on to develop a chronic wasting disease, characterized by colonic inflammation with epithelial dysplasia, as well as a dramatic reduction in Treg cells.
Conclusion
These data indicate that insufficient autoantigen expression or presentation results in defects of both central and peripheral tolerance in the K/BxN mice. It supports the idea that insufficient autoantigen levels may underlie the development of autoimmunity.
doi:10.1002/art.38085
PMCID: PMC3923054  PMID: 23840022
13.  Classification Criteria for Systemic Sclerosis: An ACR-EULAR Collaborative Initiative 
Arthritis and rheumatism  2013;65(11):2737-2747.
Background
The 1980 classification criteria for systemic sclerosis (SSc) lack sensitivity in early SSc and limited cutaneous SSc. A joint ACR-EULAR committee was established to develop new classification criteria for SSc.
Methods
Using consensus methods, 23 candidate items were arranged in a multi-criteria additive point system with a threshold to classify cases as SSc. The classification system was reduced by clustering items, and simplifying weights. The system was tested by: a) determining specificity and sensitivity in SSc cases and controls with scleroderma-like disorders; b) validating against the combined view of a group of experts on a set of cases with or without SSc.
Results
Skin thickening of the fingers extending proximal to the MCPs is sufficient to be classified as SSc, if that is not present, seven additive items apply with varying weights for each: skin thickening of the fingers, finger tip lesions, telangiectasia, abnormal nailfold capillaries, interstitial lung disease or pulmonary arterial hypertension, Raynaud's phenomenon, and SSc-related autoantibodies. Sensitivity and specificity in the validation sample were 0.91 and 0.92 for the new classification criteria and 0.75 and 0.72 for the 1980 ARA classification criteria. All selected cases were classified in accordance with consensus-based expert opinion. All cases classified as SSc by the 1980 ARA criteria were classified with the new criteria, and several additional cases were now considered to be SSc.
Conclusion
The ACR-EULAR classification criteria for SSc performed better than the 1980 ARA Criteria for SSc and should allow for more patients to be classified correctly as SSc.
doi:10.1002/art.38098
PMCID: PMC3930146  PMID: 24122180
Systemic Sclerosis; Scleroderma; Classification Criteria; Conjoint Analysis; Multi Criteria Additive Point System; Validation; ACR-EULAR
14.  Multiple Lupus Associated ITGAM Variants Alter Mac-1 Function on Neutrophils 
Arthritis and rheumatism  2013;65(11):2907-2916.
Objective
Multiple studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the ITGAM locus (including the non-synonymous SNPs rs1143679, rs1143678, rs1143683) are associated with SLE. ITGAM encodes the protein CD11b, a subunit of the β2 integrin Mac-1. The purpose of this study was to determine the effects of ITGAM genetic variation on the biological functions of neutrophil Mac-1.
Methods
Neutrophils from ITGAM genotyped and sequenced healthy donors were isolated for functional studies. The phagocytic capacity of neutrophil ITGAM variants was probed with complement coated erythrocytes, serum treated zymosan, heat treated zymosan and IgG coated erythrocytes. The adhesion capacity of ITGAM variants, in adhering to either purified intercellular adhesion molecule 1 or tumor necrosis factor α-stimulated endothelial cells was assessed in a flow chamber. Expression levels of total CD11b and activation of CD11b were assessed by flow cytometry.
Results
Mac-1–mediated neutrophil phagocytosis, determined in cultures with 2 different complement-coated particles, was significantly reduced in individuals with nonsynonymous variant alleles of ITGAM. This reduction in phagocytosis was related to variation at either rs1143679 (in the β-propeller region) or rs1143678/rs1143683 (highly linked SNPs in the cytoplasmic/calf-1 regions). Phagocytosis mediated by Fcγ receptors was also significantly reduced in donors with variant ITGAM alleles. Similarly, firm adhesion of neutrophils was significantly reduced in individuals with variant ITGAM alleles. These functional alterations were not attributable to differences in total receptor expression or activation.
Conclusion
The nonsynonymous ITGAM variants rs1143679 and rs1143678/rs113683 contribute to altered Mac-1 function on neutrophils. These results underscore the need to consider multiple nonsynonymous SNPs when assessing the functional consequences of ITGAM variation on immune cell processes and the risk of SLE.
doi:10.1002/art.38117
PMCID: PMC3969028  PMID: 23918739
15.  Most Patients With Cancer-Associated Dermatomyositis Have Antibodies to Nuclear Matrix Protein NXP-2 or Transcription Intermediary Factor 1γ 
Arthritis and rheumatism  2013;65(11):2954-2962.
Objective
Since dermatomyositis (DM) is associated with an increased risk of malignancy, accurate identification of patients likely to harbor cancers is important. Using immunoprecipitations from radiolabeled cell lysates, several groups recently showed that anti–transcription intermediary factor 1γ (anti–TIF-1γ) antibodies are associated with malignancy in DM. We undertook this study to develop sensitive, specific assays to detect antibodies against TIF-1γ and nuclear matrix protein NXP-2 and to evaluate their association with malignancy in DM.
Methods
To detect anti–TIF-1γ antibodies, immunoprecipitations were performed using lysates made from HeLa cells overexpressing TIF-1γ, with detection by immunoblotting. Anti–NXP-2 antibodies were assayed by immunoprecipitation using 35S-methionine–labeled NXP-2 generated by in vitro transcription/translation. We analyzed patient sera from DM cohorts seen at the Stanford University Dermatology Clinic (n = 111) and the Johns Hopkins Myositis Center (n = 102).
Results
A total of 17% and 38% of patients had antibodies against NXP-2 and TIF-1γ, respectively. Reactivity against either NXP-2 or TIF-1γ identified 83% of patients with cancer-associated DM. In addition to older age and male sex, cancer was associated with antibodies to NXP-2 or TIF-1γ on multivariate analysis (odds ratio 3.78 [95% confidence interval 1.33–10.8]). Stratification by sex revealed that anti–NXP-2 was specifically associated with cancer in males (odds ratio 5.78 [95% confidence interval 1.35–24.7]).
Conclusion
These studies demonstrate that anti–NXP-2 and anti–TIF-1γ antibodies are frequent DM specificities (found in 55% of patients) and are present in most patients with cancer-associated DM.
doi:10.1002/art.38093
PMCID: PMC4073292  PMID: 24037894
16.  The Mesenchymal Stem Cell Marker CD248 (Endosialin) Is a Negative Regulator of Bone Formation in Mice 
Arthritis and rheumatism  2012;64(10):3334-3343.
Objective
CD248 (tumor endothelial marker 1/endosialin) is found on stromal cells and is highly expressed during malignancy and inflammation. Studies have shown a reduction in inflammatory arthritis in CD248-knockout (CD248−/−) mice. The aim of the present study was to investigate the functional effect of genetic deletion of CD248 on bone mass.
Methods
Western blotting, polymerase chain reaction, and immunofluorescence were used to investigate the expression of CD248 in humans and mice. Micro-computed tomography and the 3-point bending test were used to measure bone parameters and mechanical properties of the tibiae of 10-week-old wild-type (WT) or CD248−/− mice. Human and mouse primary osteoblasts were cultured in medium containing 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid to induce mineralization, and then treated with platelet-derived growth factor BB (PDGF-BB). The mineral apposition rate in vivo was calculated by identifying newly formed bone via calcein labeling.
Results
Expression of CD248 was seen in human and mouse osteoblasts, but not osteoclasts. CD248−/− mouse tibiae had higher bone mass and superior mechanical properties (increased load required to cause fracture) compared to WT mice. Primary osteoblasts from CD248−/− mice induced increased mineralization in vitro and produced increased bone over 7 days in vivo. There was no decrease in bone mineralization and no increase in proliferation of osteoblasts in response to stimulation with PDGF-BB, which could be attributed to a defect in PDGF signal transduction in the CD248−/− mice.
Conclusion
There is an unmet clinical need to address rheumatoid arthritis–associated bone loss. Genetic deletion of CD248 in mice results in high bone mass due to increased osteoblast-mediated bone formation, suggesting that targeting CD248 in rheumatoid arthritis may have the effect of increasing bone mass in addition to the previously reported effect of reducing inflammation.
doi:10.1002/art.34556
PMCID: PMC4209224  PMID: 22674221
17.  MIF receptor CD74 mediates alphavirus-induced arthritis and myositis 
Arthritis and rheumatism  2013;65(10):2724-2736.
Objective
Arthrogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) circulate worldwide. This virus class causes debilitating illnesses characterised by arthritis, arthralgia, and myalgia. We previously identified macrophage migration inhibitory factor (MIF) as a critical inflammatory factor in the pathogenesis of alphaviral disease. Here, we characterise the role of CD74, a cell surface receptor of MIF, in both RRV- and CHIKV-induced alphavirus arthritides.
Methods
Mouse models of RRV and CHIKV infection were used to investigate the immunopathogenesis of arthritic alphavirus infection. The role of CD74 was assessed using histological analysis, real time PCR, flow cytometry and plaque assay.
Results
In comparison to wild-type mice, CD74−/− mice developed only mild clinical features and had low levels of tissue damage. Leukocyte infiltration, characterised predominantly by inflammatory monocytes and natural killer cells, was substantially reduced in infected tissues of CD74−/− mice, but production of pro-inflammatory cytokines and chemokines were not decreased. CD74 deficiency was associated with increased monocyte apoptosis, but had no effect on monocyte migratory capacity. Consistent with these findings, alphaviral infection resulted in a dose-dependent up-regulation of CD74 expression in human peripheral blood mononuclear cells and serum MIF levels were significantly elevated in humans with RRV or CHIKV infections.
Conclusion
We propose that CD74 regulates immune responses to alphaviral infection through effects on cellular recruitment and survival. These findings suggest that both MIF and CD74 play a critical role in mediating alphaviral disease and blocking these factors with novel therapeutic agents can substantially ameliorate pathology.
doi:10.1002/art.38090
PMCID: PMC3796577  PMID: 23896945
18.  High Body Mass Index is Associated with Increased Diurnal Strains in the Articular Cartilage of the Knee 
Arthritis and rheumatism  2013;65(10):2615-2622.
Objective
Obesity is an important risk factor for osteoarthritis and is associated with changes in both the biomechanical and inflammatory environments within the joint. However, the relationship between obesity and cartilage deformation is not fully understood. The goal of this study was to determine the effects of body mass index (BMI) on the magnitude of diurnal cartilage strain in the knee.
Methods
Three-dimensional maps of knee cartilage thickness were developed from 3T magnetic resonance images of asymptomatic age- and sex-matched subjects with normal (18.5–24.9 kg/m2) or high (25–31 kg/m2) BMI. Site-specific magnitudes of diurnal cartilage strain were determined using aligned images recorded at 8:00 AM and 4:00 PM on the same day.
Results
High BMI individuals had significantly thicker cartilage on the patella and femoral groove than the normal BMI individuals. Diurnal cartilage strains were dependent on location as well as BMI. Subjects with high BMI exhibited significantly higher compressive strain in tibial cartilage than did those with normal BMI. Cartilage thickness decreased significantly on both femoral condyles from the AM to PM time point; however, there was no significant effect of BMI on diurnal cartilage strain in the femur.
Conclusions
Increased BMI is associated with increased diurnal strains in the articular cartilage of both the medial and lateral compartments of the knee. The increased cartilage strains measured in high BMI individuals may, in part, explain the elevated OA risk associated with obesity or may reflect altered cartilage mechanical properties in subjects with high BMI.
doi:10.1002/art.38062
PMCID: PMC3954747  PMID: 23818303
obesity; MRI; stress; adipokine; chondrocyte; biomechanics
19.  Accelerated Aging Influences Cardiovascular Disease Risk in Rheumatoid Arthritis 
Arthritis and rheumatism  2013;65(10):2562-2566.
OBJECTIVE
To determine whether the impact of aging on cardiovascular disease (CVD) risk in the general population (as estimated by the Framingham risk score [FRS]) differs in patients with rheumatoid arthritis (RA).
METHODS
A population-based inception cohort of Olmsted County, Minnesota residents aged ≥30 years who fulfilled 1987 ACR criteria for RA in 1988–2008 was assembled and followed until death, migration, or 7-1-2012. Data on CVD events were collected by medical record review. The 10-year FRS for CVD was calculated. Cox models adjusted for FRS were used to examine the influence of age on CVD risk.
RESULTS
The study included 563 patients with RA without prior CVD (mean age: 55 years, 72% women; 69% seropositive [i.e., rheumatoid factor and/or anti-citrullinated protein antibody positive]). During a mean follow-up of 8.2 years, 98 patients developed CVD (74 seropositive and 24 seronegative), but FRS predicted only 59.7 events (35.4 seropositive and 24.3 seronegative). The gap between observed and predicted CVD risk increased exponentially across age, and the age effect on CVD risk in seropositive RA was nearly double its effect in the general population with additional log(age) coefficients of 2.91 for women (p=0.002) and 2.06 for men (p=0.027).
CONCLUSION
Age exerts an exponentially increasing effect on CVD risk in seropositive RA, but no increased effect among seronegative patients. The causes of accelerated aging in patients with seropositive RA deserve further investigation.
doi:10.1002/art.38071
PMCID: PMC3966134  PMID: 23818136
accelerated aging; rheumatoid arthritis; cardiovascular disease
20.  Extension of the Germinal Center Stage of B-cell Development Promotes Autoantibodies in BXD2 Mice 
Arthritis and rheumatism  2013;65(10):2703-2712.
Objective
Regulator of G-protein Signaling (RGS) proteins inhibit chemokine signaling by desensitizing G-protein coupled receptor signals. The mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in germinal centers (GC) were determined using BXD2-Rgs13−/− mice.
Methods
Confocal and light microscopy imaging was used to determine the location of cells that express RGS13 and activation-induced cytidine deaminase (AID) in the spleen and the number of plasmablasts. The levels of GC and plasma cell program transcripts in GC B cells were determined by quantitative real-time PCR. Differential IL-17-mediated expression of Rgs13 in GC versus non-GC B cells was analyzed using A20 versus 70Z/3 B cells.
Results
In spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 upregulated Rgs13 in A20 GC but not 70Z/3 non-GC B cells. BXD2-Rgs13−/− mice exhibited smaller GCs, lower AID levels, suggesting lower somatic hypermutation and affinity maturation. There were, however, increased IgMbright plasmablasts, upregulation of plasma program genes Irf4, Blimp1, Xbp1 and pCREB target genes Fosb and Obf1, with down-regulation of GC program genes Aicda, Pax5 and Bach2 in GC B cells of BXD2-Rgs13−/− mice. BXD2-Rgs13−/− mice showed lower titers of IgG autoantibodies and IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity.
Conclusion
RGS13 deficiency is associated with reduction in GC program genes and exit of less pathogenic IgM plasmablasts in BXD2 mice. Prolonged GC program, mediated by upregulation RGS13, enhanced AID expression and enabled generation of pathogenic autoantibodies in autoreactive GCs.
doi:10.1002/art.38059
PMCID: PMC3979745  PMID: 23818250
21.  RBPjκ-dependent Notch signaling is required for articular cartilage and joint maintenance 
Arthritis and rheumatism  2013;65(10):2623-2633.
Objective
Osteoarthritis (OA) is a degenerative disease resulting in severe joint cartilage destruction and disability. While the mechanisms underlying the development and progression of OA are poorly understood, gene mutations have been identified within cartilage-related signaling molecules implicating impaired cell signaling in OA and joint disease. The Notch pathway has recently been identified as a crucial regulator of growth plate cartilage development and components are expressed in joint tissues. Therefore, we set out to investigate a novel role for Notch signaling in joint cartilage development, maintenance, and the pathogenesis of joint disease.
Methods
We performed the first mouse genetic studies in which the core Notch signaling component, RBPjκ, was tissue-specifically deleted within joints. The Prx1Cre transgene removed Rbpjκ floxed alleles in mesenchymal joint precursor cells, while the Col2CreERT2 transgene specifically deleted Rbpjκ in postnatal chondrocytes. Articular chondrocyte cultures were also utilized to examine Notch regulation of gene expression.
Results
Loss of Notch signaling in mesenchymal joint precursor cells does not affect embryonic joint development, but rather results in an early, progressive OA-like pathology. Additionally, partial loss of Notch signaling in postnatal cartilage results in progressive joint cartilage degeneration and an age-related OA-like pathology. Inhibition of Notch signaling alters expression of the ECM-related factors: COL2A1, PRG4, COL10A1, MMP13, and ADAMTSs.
Conclusions
These data have identified the RBPjκ-dependent Notch pathway as: 1) a novel pathway involved in joint maintenance and articular cartilage homeostasis, 2) a critical regulator of articular cartilage ECM-related molecules, and 3) a potentially important therapeutic target for OA-like joint disease.
doi:10.1002/art.38076
PMCID: PMC4038327  PMID: 23839930
RBPjκ; Notch; chondrocyte; articular cartilage; osteoarthritis
22.  Detection of anti-Ro, La, Smith and RNP autoantibodies by autoantigen microarray analysis and interferon-alpha induction in juvenile dermatomyositis 
Arthritis and rheumatism  2013;65(9):2424-2429.
Objective
To evaluate serum interferon-α (IFNα) activity in the context of autoantibody profiles in patients with juvenile dermatomyositis (JDM).
Methods
Sera from 36 JDM patients were analyzed. Autoantibody profiles were determined by probing microarrays, fabricated with ~80 distinct autoantigens, with serum and a Cy3-conjugated secondary antibody. Arrays were scanned and analyzed to determine antigen reactivity. Serum IFNα activity was measured using a functional reporter cell assay. Sera were assayed alone or in combination with cellular material released from necrotic U937 cells to stimulate peripheral blood mononuclear cells from healthy donors in vitro, and IFNα production in culture was measured by a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA).
Results
Reactivity against at least 1 of 41 autoantigens on the microarray, including Ro 52, Ro 60, La, Sm, and RNP, was observed in 75% of the serum samples from patients with JDM. IFNα activity was detected in 7 samples by reporter cell assay. The reporter cell assay showed a significant association of reactivity against Ro, La, Sm, and proliferating cell nuclear antigen with serum IFNα activity (P = 0.005). Significance Analysis of Microarrays (SAM) identified increased reactivity against Sm, RNP, Ro 52, U1-C, and Mi-2 in these sera. Sixteen samples induced IFNα production as measured by DELFIA, and there was a significant association of reactivity against Ro, La, Sm, and RNP with the induction of IFNα by serum and necrotic cell material (P = 0.034). SAM identified increased reactivity against Ro 60 in these sera.
Conclusion
These data support the hypothesis that nucleic acid–associated autoantibodies, including the Ro/La and Sm/RNP complexes, may stimulate the production of active IFNα in children with JDM.
doi:10.1002/art.38038
PMCID: PMC4169271  PMID: 23740815
23.  Host-derived CD4+ T cells attenuate stem cell mediated transfer of autoimmune arthritis in lethally irradiated B6.g7 mice 
Arthritis and rheumatism  2013;65(3):681-692.
Objective
In the KBxN mouse model of inflammatory arthritis, T cells carrying a transgenic TCR initiate disease by helping B cells to produce arthritogenic anti-GPI (glucose -6-phosphate isomerase) autoantibodies. We found that lethally-irradiated lymphocyte-deficient B6.g7 (I-Ag7+) Rag-/- mice reconstituted with KBxN hematopoetic stem and progenitor cells (HPSC) exhibit arthritis by week 4. In contrast, healthy B6.g7 recipients of KBN HSPC show only mild arthritis, with limited extent and duration. The objective of this study was to investigate the factors responsible for the attenuation of arthritis in the B6.g7 recipients.
Methods
Antibody responses were measured by ELISA. FACs analyses were performed for testing chimerism, expression of markers of activation and suppression, tetramer binding and intracellular cytokines in CD4+ T cell. Suppressive activity of CD4+ T cells was studied by adoptive transfer.
Results
Titers of anti-GPI Abs in reconstituted B6.g7 mice are ∼60 fold lower than in reconstituted B6.g7 Rag-/- mice. Examination of chimerism in the reconstituted B6.g7 mice showed that B and myeloid cells in these mice are donor-derived, but CD4+ T cells are primarily host-derived and enriched for cells expressing the conventional regulatory markers, CD25+FoxP3+. Notably, CD25-Foxp3- CD4+ T cells express markers of suppressive function, CD73 and FR4, and delay disease after adoptive transfer. Activation of donor-derived CD4+ T cells is reduced, and thymic deletion of these cells appears increased.
Conclusion
Despite myeloablation, host CD4+ T cells having a regulatory phenotype emerge in these mice and attenuate autoimmunity.
doi:10.1002/art.37800
PMCID: PMC4157728  PMID: 23233229
24.  Subchondral Bone Trabecular Integrity Predicts and Changes Concurrently with Radiographic and MRI Determined Knee Osteoarthritis Progression 
Arthritis and rheumatism  2013;65(7):1812-1821.
Objective
To evaluate subchondral bone trabecular integrity (BTI) from a radiograph as a predictor of knee osteoarthritis (OA) progression.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1002/art.37970
PMCID: PMC4152231  PMID: 23576116
25.  Anti-Cyclic Citrullinated Peptide Assays Differ in Subjects at Elevated Risk for Rheumatoid Arthritis and Subjects with Established Disease 
Arthritis and rheumatism  2013;65(9):2243-2252.
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.
doi:10.1002/art.38017
PMCID: PMC3776020  PMID: 23686569
Rheumatoid arthritis; autoantibodies; CCP; ACPA; preclinical

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