Search

Tumor necrosis factor α (TNF-α) is a key regulator of inflammation and rheumatoid arthritis (RA). TNF-α blocker therapies can be very effective for a substantial number of patients, but fail to work in one third of patients who show no or minimal response. It is therefore necessary to discover new molecular intervention points involved in TNF-α blocker treatment of rheumatoid arthritis patients. We describe a data analysis strategy for predicting gene expression measures that are critical for rheumatoid arthritis using a combination of comprehensive genotyping, whole blood gene expression profiles and the component clinical measures of the arthritis Disease Activity Score 28 (DAS28) score. Two separate network ensembles, each comprised of 1024 networks, were built from molecular measures from subjects before and 14 weeks after treatment with TNF-α blocker. The network ensemble built from pre-treated data captures TNF-α dependent mechanistic information, while the ensemble built from data collected under TNF-α blocker treatment captures TNF-α independent mechanisms. In silico simulations of targeted, personalized perturbations of gene expression measures from both network ensembles identify transcripts in three broad categories. Firstly, 22 transcripts are identified to have new roles in modulating the DAS28 score; secondly, there are 6 transcripts that could be alternative targets to TNF-α blocker therapies, including CD86 - a component of the signaling axis targeted by Abatacept (CTLA4-Ig), and finally, 59 transcripts that are predicted to modulate the count of tender or swollen joints but not sufficiently enough to have a significant impact on DAS28., Author Summary The collection and analysis of clinical data has played a key role in providing insights into the diagnosis, prognosis and treatment of disease. However, it is imperative that molecular and genetic data also be collected and integrated into the creation of network models, which capture underlying mechanisms of disease and can be interrogated to elucidate previously unknown biology. Bringing data from the clinic to the bench completes the cycle of translational research, which we demonstrate with this work. We built disease models from genetics, whole blood gene expression profiles and the component clinical measures of rheumatoid arthritis using a data-driven approach that leverages supercomputing. Genetic factors can be utilized as a source of perturbation to the system such that causal connections between genetics, molecular entities and clinical outcomes can be inferred. The existing TNF-α blocker treatments for rheumatoid arthritis are only effective for approximately 2/3 of the affected population. We identified novel therapeutic intervention points that may lead to the development of alternatives to TNF-α blocker treatments. We believe this approach will provide improved drug discovery programs, new insights into disease progression, increased drug efficacy and novel biomarkers for chronic and complex diseases.

BackgroundDirect cellular contact with stimulated T cells is a potent mechanism that induces cytokine production in human monocytes in the absence of an infectious agent. This mechanism is likely to be relevant to T cell-mediated inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Microparticles (MP) generated by stimulated T cells (MPT) display similar monocyte activating ability to whole T cells, isolated T cell membranes, or solubilized T cell membranes. We previously demonstrated that high-density lipoproteins (HDL) inhibited T cell contact- and MPT-induced production of IL-1beta but not of its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra).Methodology/principal findingsLabeled MPT were used to assess their interaction with monocytes and T lymphocytes by flow cytometry. Similarly, interactions of labeled HDL with monocytes and MPT were assessed by flow cytometry. In parallel, the MPT-induction of IL-1beta and sIL-1Ra production in human monocytes and the effect of HDL were assessed in cell cultures. The results show that MPT, but not MP generated by activated endothelial cells, bond monocytes to trigger cytokine production. MPT did not bind T cells. The inhibition of IL-1beta production by HDL correlated with the inhibition of MPT binding to monocytes. HDL interacted with MPT rather than with monocytes suggesting that they bound the activating factor(s) of T cell surface. Furthermore, prototypical pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL3 and CCL4 displayed a pattern of production induced by MPT and inhibition by HDL similar to IL-1beta, whereas the production of CCL2, like that of sIL-1Ra, was not inhibited by HDL.Conclusions/significanceHDL inhibit both MPT binding to monocytes and the MPT-induced production of some but not all cytokines, shedding new light on the mechanism by which HDL display their anti-inflammatory functions.

Etanercept, a TNF receptor 2-Fc fusion protein, is currently being used for the treatment of rheumatoid arthritis (RA). However, 25% to 38% of patients show no response which is suspected to be partially due to insufficient affinity of this protein to TNFα. By using computational protein design, we found that residue W89 and E92 of TNFR2 were critical for ligand binding. Among several mutants tested, W89Y/E92N displayed 1.49-fold higher neutralizing activity to TNFα, as compared to that of Etanercept. Surface plasmon resonance (SPR) based binding assay revealed that the equilibrium dissociation constant of W89Y/E92N to TNFα was 3.65-fold higher than that of Etanercept. In a rat model of collagen-induced arthritis (CIA), W89Y/E92N showed a significantly better ability than Etanercept in reducing paw swelling and improvement of arthritic joint histopathologically. These data demonstrate that W89Y/E92N is potentially a better candidate with improved efficacy in treating RA and other autoimmune diseases., Author Summary Rheumatoid arthritis (RA) is a frequently occurring, chronic, debilitating disease. TNFα plays a pivotal role in regulating its inflammatory response. TNFα inhibition with TNF receptor 2-Fc fusion protein (TNFR2-Fc) was effective in the treatment of RA. However, quite a few patients may not achieve good clinical outcomes after TNFR2-Fc therapy, and the high dosage of TNFR2-Fc in clinical treatment usually causes some side effects, such as injection site reactions. Enhancing the affinity of TNFR2-Fc to TNFα would be of benefit to its therapeutic effect on RA, and may reduce the clinical dosage. We modeled the interactions of TNFα and TNFR2, and figured out the sites that may be critical for ligand-receptor binding. A high affinity TNFR2-Fc variant (E92N/W89Y) was obtained by rational mutagenesis at residue 89 and 92. It shows significant improvements compared with wild type TNFR2-Fc in suppressing rat arthritis induced by collagen. This variant is more potent in neutralizing TNFα, and thereafter may offer a higher degree of RA symptom relief, and be in a much lower dosage.

Background Polymorphism in the neuropeptide S receptor gene NPSR1 is associated with asthma and inflammatory bowel disease. NPSR1 is expressed in the brain, where it modulates anxiety and responses to stress, but also in other tissues and cell types including lymphocytes, the lungs, and the intestine, where it appears to be up-regulated in inflammation. We sought to determine whether genetic variability at the NPSR1 locus influences the susceptibility and clinical manifestation of rheumatoid arthritis (RA). Methodology/Principal Findings From the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) case-control study, 1,888 rheumatoid arthritis patients and 888 controls were genotyped for 19 single-nucleotide polymorphisms (SNPs) spanning the entire NPSR1 gene and 220 KB of DNA on chromosome 7p14. The association between individual genetic markers and their haplotypic combinations, respectively, and diagnosis of RA, presence of autoantibodies to citrullinated proteins (ACPA), and disease activity score based on 28 joints (DAS28) was tested. There was no association between diagnosis of RA and NPSR1 variants. However, several associations of nominal significance were detected concerning susceptibility to ACPA-negative RA and disease activity measures (DAS28). Among these, the association of SNP rs324987 with ACPA-negative RA [(p = 0.004, OR = 0.674 (95% CI 0.512–0.888)] and that of SNP rs10263447 with DAS28 [p = 0.0002, OR = 0.380 (95% CI 0.227–0.635)] remained significant after correction for multiple comparisons. Conclusions/Significance NPSR1 polymorphism may be relevant to RA susceptibility and its clinical manifestation. Specific alleles at the NPSR1 locus may represent common risk factors for chronic inflammatory diseases, including RA.

BackgroundComputational biology contributes to a variety of areas related to life sciences and, due to the growing impact of translational medicine--the scientific approach to medicine in tight relation with basic science--, it is becoming an important player in clinical-related areas. In this study, we use computation methods in order to improve our understanding of the complex interactions that occur between molecules related to Rheumatoid Arthritis (RA).MethodologyDue to the complexity of the disease and the numerous molecular players involved, we devised a method to construct a systemic network of interactions of the processes ongoing in patients affected by RA. The network is based on high-throughput data, refined semi-automatically with carefully curated literature-based information. This global network has then been topologically analysed, as a whole and tissue-specifically, in order to translate the experimental molecular connections into topological motifs meaningful in the identification of tissue-specific markers and targets in the diagnosis, and possibly in the therapy, of RA.SignificanceWe find that some nodes in the network that prove to be topologically important, in particular AKT2, IL6, MAPK1 and TP53, are also known to be associated with drugs used for the treatment of RA. Importantly, based on topological consideration, we are also able to suggest CRKL as a novel potentially relevant molecule for the diagnosis or treatment of RA. This type of finding proves the potential of in silico analyses able to produce highly refined hypotheses, based on vast experimental data, to be tested further and more efficiently. As research on RA is ongoing, the present map is in fieri, despite being--at the moment--a reflection of the state of the art. For this reason we make the network freely available in the standardised and easily exportable .xml CellDesigner format at 'www.picb.ac.cn/ClinicalGenomicNTW/temp.html' and 'www.celldesigner.org'.

Background Based on their capacity to suppress immune responses, multipotent mesenchymal stromal cells (MSC) are intensively studied for various clinical applications. Although it has been shown in vitro that the immunomodulatory effect of MSCs mainly occurs through the secretion of soluble mediators, the mechanism is still not completely understood. The aim of the present study was to better understand the mechanisms underlying the suppressive effect of MSCs in vivo, using cells isolated from mice deficient in the production of inducible nitric oxide synthase (iNOS) or interleukin (IL)-6 in the murine model of collagen-induced arthritis. Principal Findings In the present study, we show that primary murine MSCs from various strains of mice or isolated from mice deficient for iNOS or IL-6 exhibit different immunosuppressive potential. The immunomodulatory function of MSCs was mainly attributed to IL-6-dependent secretion of prostaglandin E2 (PGE2) with a minor role for NO. To address the role of these molecules in vivo, we used the collagen-induced arthritis as an experimental model of immune-mediated disorder. MSCs effectively inhibited collagen-induced inflammation during a narrow therapeutic window. In contrast to wild type MSCs, IL-6-deficient MSCs and to a lesser extent iNOS-deficient MSCs were not able to reduce the clinical signs of arthritis. Finally, we show that, independently of NO or IL-6 secretion or Treg cell induction, MSCs modulate the host response by inducing a switch to a Th2 immune response. Significance Our data indicate that MSCs mediate their immunosuppressive effect via two modes of action: locally, they reduce inflammation through the secretion of anti-proliferative mediators, such as NO and mainly PGE2, and systemically they switch the host response from a Th1/Th17 towards a Th2 immune profile.

BACKGROUND: Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact. METHODOLOGY/PRINCIPAL FINDINGS: To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNgamma, CCL2, CCL5, TNF, IL-1beta, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1beta, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E(2)-both involved in inflammatory response-were triggered by T cell contact and inhibited in the presence of HDL. CONCLUSIONS/SIGNIFICANCE: These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL.

Background: Several studies point to a role of Toll-like receptors (TLRs) in the development of rheumatoid arthritis (RA). We investigated if genetic variants in TLR genes are associated with RA and response to tumour necrosis factor blocking (anti-TNF) medication. Methodology and Principal Findings: 22 single nucleotide polymorphisms (SNPs) in seven TLR genes were genotyped in a Dutch cohort consisting of 378 RA patients and 294 controls. Significantly associated variants were investigated in replication cohorts from The Netherlands, United Kingdom and Sweden (2877 RA patients and 2025 controls). 182 of the Dutch patients were treated with anti-TNF medication. Using these patients and a replication cohort (269 Swedish patients) we analysed if genetic variants in TLR genes were associated with anti-TNF outcome. In the discovery phase of the study we found a significant association of SNPs rs2072493 in TLR5 and rs3853839 in TLR7 with RA disease susceptibility. Meta-analysis of discovery and replication cohorts did not confirm these findings. SNP rs2072493 in TLR5 was associated with anti-TNF outcome in the Dutch but not in the Swedish population. Conclusion: We conclude that genetic variants in TLRs do not play a major role in susceptibility for developing RA nor in anti-TNF treatment outcome in a Caucasian population. Original Publication:Marieke J H Coenen, Christian Enevold, Pilar Barrera, Mascha M V A P Schijvenaars, Erik J M Toonen, Hans Scheffer, Leonid Padyukov, Alf Kastbom, Lars Klareskog, Anne Barton, Wietske Kievit, Tim L Jansen, Dorine Swinkels, Piet L C M van Riel, Barbara Franke, Klaus Bendtzen and Timothy R D J Radstake, Genetic Variants in Toll-Like Receptors Are Not Associated with Rheumatoid Arthritis Susceptibility or Anti-Tumour Necrosis Factor Treatment Outcome, 2010, PLOS ONE, (5), 12.http://dx.doi.org/10.1371/journal.pone.0014326Licensee: Public Library of Science (PLoS)http://www.plos.org/

Cytotoxic T-lymphocyte associated protein 4 (CTLA4) is a negative regulator of T-cell proliferation. Polymorphisms in CTLA4 have been inconsistently associated with susceptibility to rheumatoid arthritis (RA) in populations of European ancestry but have not been examined in African Americans. The prevalence of RA in most populations of European and Asian ancestry is ∼1.0%; RA is purportedly less common in black Africans, with little known about its prevalence in African Americans. We sought to determine if CTLA4 polymorphisms are associated with RA in African Americans. We performed a 2-stage analysis of 12 haplotype tagging single nucleotide polymorphisms (SNPs) across CTLA4 in a total of 505 African American RA patients and 712 African American controls using Illumina and TaqMan platforms. The minor allele (G) of the rs231778 SNP was 0.054 in RA patients, compared to 0.209 in controls (4.462×10−26, Fisher's exact). The presence of the G allele was associated with a substantially reduced odds ratio (OR) of having RA (AG+GG genotypes vs. AA genotype, OR 0.19, 95% CI: 0.13–0.26, p = 2.4×10−28, Fisher's exact), suggesting a protective effect. This SNP is polymorphic in the African population (minor allele frequency [MAF] 0.09 in the Yoruba population), but is very rare in other groups (MAF = 0.002 in 530 Caucasians genotyped for this study). Markers associated with RA in populations of European ancestry (rs3087243 [+60C/T] and rs231775 [+49A/G]) were not replicated in African Americans. We found no confounding of association for rs231778 after stratifying for the HLA-DRB1 shared epitope, presence of anti-cyclic citrullinated peptide antibody, or degree of admixture from the European population. An African ancestry-specific genetic variant of CTLA4 appears to be associated with protection from RA in African Americans. This finding may explain, in part, the relatively low prevalence of RA in black African populations., Author Summary Rheumatoid arthritis (RA) is a systemic autoimmune condition affecting the synovial membranes of diarthrodial joints. The etiology of RA is unclear but is thought to result from an environmental trigger in the context of genetic predisposition. We report that a single nucleotide polymorphism (SNP) (rs231778) in CTLA4, which encodes a negative regulator of T cell activation, is associated (p = 2.4×10−28) with protection from developing RA among African Americans. rs231778 is only polymorphic in populations of African ancestry. Protective alleles such as this one may contribute to the purported lower prevalence of RA in African Americans. Our finding appears to be independent from confounding by linkage with the HLA-DRB1 shared epitope or by genetic admixture. Furthermore, we did not replicate associations of CTLA4 SNPs with RA or other autoimmune diseases previously reported in Asians and Caucasians, such as rs3087243 (+60C/T) and rs231775 (+49A/G). The associations of different SNPs with RA susceptibility specific to different populations highlight the importance of CTLA4 in the pathogenesis of RA and demonstrate the ethnic-specific genetic background that contributes to its susceptibility.

Background Angiogenesis is considered an important factor in the pathogenesis of Rheumatoid Arthritis (RA) where it has been proposed as a therapeutic target. In other settings, active angiogenesis is characterized by pathologic, immature vessels that lack periendothelial cells. We searched for the presence of immature vessels in RA synovium and analyzed the dynamics of synovial vasculature along the course of the disease, particularly after therapeutic response to TNF antagonists. Methodology/principal findings Synovial arthroscopic biopsies from RA, osteoarthritis (OA) and normal controls were analyzed by double labeling of endothelium and pericytes/smooth muscle mural cells to identify and quantify mature/immature blood vessels. To analyze clinicopathological correlations, a cross-sectional study on 82 synovial biopsies from RA patients with variable disease duration and severity was performed. A longitudinal analysis was performed in 25 patients with active disease rebiopsied after anti-TNF-alpha therapy. We found that most RA synovial tissues contained a significant fraction of immature blood vessels lacking periendothelial coverage, whereas they were rare in OA, and inexistent in normal synovial tissues. Immature vessels were observed from the earliest phases of the disease but their presence or density was significantly increased in patients with longer disease duration, higher activity and severity, and stronger inflammatory cell infiltration. In patients that responded to anti-TNF-alpha therapy, immature vessels were selectively depleted. The mature vasculature was similarly expanded in early or late disease and unchanged by therapy. Conclusion/significance RA synovium contains a significant fraction of neoangiogenic, immature blood vessels. Progression of the disease increases the presence and density of immature but not mature vessels and only immature vessels are depleted in response to anti-TNFalpha therapy. The different dynamics of the mature and immature vascular fractions has important implications for the development of anti-angiogenic interventions in RA.

Finding an association between soluble B7-H4 and rheumatoid arthritis severity, Lieping Chen and colleagues use a mouse model to show that the soluble form blocks the inhibitory function of cell-surface B7-H4., Background A pathogenic hallmark of rheumatoid arthritis (RA) is persistent inflammatory responses in target tissues and organs. Immune responses mediated by T cells and autoantibodies are known to play pivotal roles. A possible interpretation for this observation is a loss of negative regulation of autoimmune responses. Here we sought to investigate whether B7-H4, a cell surface inhibitory molecule of the B7-CD28 signaling pathway, may play a role in the pathogenesis of RA. Methods and Findings In a cross-sectional study of a clinical convenience sample using monoclonal antibodies against human B7-H4 molecules, we detected high levels of the soluble form of B7-H4 (sH4) in the sera of 65% of patients with RA (n = 68) versus only 13% of healthy donors (n = 24). Elevated sH4 was associated with an increased disease severity score (DAS28) in a cross-sectional analysis. In a mouse model of RA, transgenic expression of sH4 or genetic deletion of B7-H4 accelerated the progression of collagen-induced arthritis, accompanied by enhanced T and B cell–mediated autoimmune responses as well as increased activity of neutrophils. Expression in vivo of an agonist, a B7-H4-immunoglobulin Fc fusion protein, profoundly suppressed disease progression in the mouse model. Conclusions Our findings in mice indicate that sH4 acts as a decoy molecule to block the inhibitory functions of cell-surface B7-H4, leading to exacerbation of collagen-induced arthritis. If the preliminary correlation between sH4 levels and disease activity in patients with RA can be confirmed to reflect a similar mechanism, these findings suggest a novel target for treatment approaches. Please see later in the article for the Editors' Summary, Editors' Summary Background Rheumatoid arthritis (RA) is a chronic disease caused by abnormal immune responses. In RA, the body's own immune system mainly attacks the joints, causing inflammation in their lining, but can affect other tissues and organs in the body. About 1% of the population in developed countries suffer from RA, and it can result in long-term joint damage, causing significant illness and disability. Sufferers have chronic pain, loss of function of the joint, and loss of mobility. The cause of RA is unknown and there is no known cure. However, neutrophils (an immune cell important for inflammation) are thought to contribute to the initiation of RA. Understanding the primary mechanisms behind the development of RA, and where the body's immune system goes wrong, is fundamental not only to find new treatments for the disease but also to aid diagnosis to help patients get treatment to help control their often debilitating symptoms. Why Was the Study Done? Regulation of the immune system is necessary to prevent overactivity. Interruptions to the normal signals that moderate the immune response can lead to destruction of normal tissues. Previous studies have shown that the B7 family of proteins, which interact with CD28 signaling proteins on the surface of immune cells, are important regulators of the immune response. B7 proteins have also been found to exist in soluble forms that have been implicated in the development of rheumatoid diseases, but their exact role is not well understood. In the current study, researchers examined a member of the B7 family, B7-H4, which normally acts as an inhibitor of the immune response, to find out whether this signaling molecule affects the immune response and has a role in the development of RA. What Did the Researchers Do and Find? The researchers collected blood from 68 patients with RA and 24 healthy volunteers, and measured levels of soluble B7-H4, also known as sH4. They found sH4 in blood from 65% of patients with RA, compared with only 13% of healthy people. The levels of sH4 were significantly higher in RA patients (96.1 ng/ml) compared to healthy people (

Background TNF alpha blockade agents like infliximab are actually the treatment of choice for those rheumatoid arthritis (RA) patients who fail standard therapy. However, a considerable percentage of anti-TNF alpha treated patients do not show a significant clinical response. Given that new therapies for treatment of RA have been recently approved, there is a pressing need to find a system that reliably predicts treatment response. We hypothesized that the analysis of whole blood gene expression profiles of RA patients could be used to build a robust predictor to infliximab therapy. Methods and Findings We performed microarray gene expression analysis on whole blood RNA samples from RA patients starting infliximab therapy (n = 44). The clinical response to infliximab was determined at week 14 using the EULAR criteria. Blood cell populations were determined using flow cytometry at baseline, week 2 and week 14 of treatment. Using complete cross-validation and repeated random sampling we identified a robust 8-gene predictor model (96.6% Leave One Out prediction accuracy, P = 0.0001). Applying this model to an independent validation set of RA patients, we estimated an 85.7% prediction accuracy (75–100%, 95% CI). In parallel, we also observed a significantly higher number of CD4+CD25+ cells (i.e. regulatory T cells) in the responder group compared to the non responder group at baseline (P = 0.0009). Conclusions The present 8-gene model obtained from whole blood expression efficiently predicts response to infliximab in RA patients. The application of the present system in the clinical setting could assist the clinician in the selection of the optimal treatment strategy in RA.

Background Large-scale gene expression profiling of peripheral blood mononuclear cells from Rheumatoid Arthritis (RA) patients could provide a molecular description that reflects the contribution of diverse cellular responses associated with this disease. The aim of our study was to identify peripheral blood gene expression profiles for RA patients, using Illumina technology, to gain insights into RA molecular mechanisms. Methodology/Principal Findings The Illumina Human-6v2 Expression BeadChips were used for a complete genome-wide transcript profiling of peripheral blood mononuclear cells (PBMCs) from 18 RA patients and 15 controls. Differential analysis per gene was performed with one-way analysis of variance (ANOVA) and P values were adjusted to control the False Discovery Rate (FDR

ObjectiveC-reactive protein (CRP) levels>3 mg/L and>10 mg/L are associated with high and very high cardiovascular risk, respectively, in the general population. Because rheumatoid arthritis (RA) confers excess cardiovascular mortality, we determined the prevalence of these CRP levels among RA patients stratified on the basis of their RA disease activity.MethodsWe evaluated physician and patient global assessments of disease activity, tender and swollen 28 joint counts, erythrocyte sedimentation rate (ESR), and CRP measured in a single clinic visit for 151 RA patients. Disease activity was calculated using the Clinical Disease Activity Index (CDAI) and the Disease Activity Score 28 Joints (DAS28-ESR and DAS28-CRP).ResultsMedian CRP level was 5.3 mg/L. 68% of patients had CRP>3 mg/L, and 25% had CRP>10 mg/L. Of those with 0-1 swollen joints (n = 56), or 0-1 tender joints (n = 81), 64% and 67%, respectively, had CRP>3 mg/L, and 23% and 20%, respectively, had CRP>10 mg/L. Of those with remission or mildly active disease by CDAI (n = 58), DAS28-ESR (n = 39), or DAS28-CRP (n = 70), 49-66% had CRP>3 mg/L, and 10-14% had CRP>10 mg/L. Of patients with moderate disease activity by CDAI (n = 51), DAS28-ESR (n = 78), or DAS28-CRP (n = 66), 67-73% had CRP>3 mg/L, and 25-33% had CRP>10 mg/L.ConclusionEven among RA patients whose disease is judged to be controlled by joint counts or standardized disease scores, a substantial proportion have CRP levels that are associated high or very high risk for future cardiovascular events in the general population.

Background The anti-inflammatory capacity of heat shock proteins (HSP) has been demonstrated in various animal models of inflammatory diseases and in patients. However, the mechanisms underlying this anti-inflammatory capacity are poorly understood. Therefore, the possible protective potential of HSP70 and its mechanisms were studied in proteoglycan (PG) induced arthritis (PGIA), a chronic and relapsing, T cell mediated murine model of arthritis. Methodology/Principal Findings HSP70 immunization, 10 days prior to disease induction with PG, inhibited arthritis both clinically and histologically. In addition, it significantly reduced PG-specific IgG2a but not IgG1 antibody production. Furthermore, IFN-γ and IL-10 production upon in vitro restimulation with HSP70 was indicative of the induction of an HSP70-specific T cell response in HSP70 immunized mice. Remarkably, HSP70 treatment also modulated the PG-specific T cell response, as shown by the increased production of IL-10 and IFN-γ upon in vitro PG restimulation. Moreover, it increased IL-10 mRNA expression in CD4+CD25+ cells. HSP70 vaccination did not suppress arthritis in IL-10−/− mice, indicating the crucial role of IL-10 in the protective effect. Conclusions/Significance In conclusion, a single mycobacterial HSP70 immunization can suppress inflammation and tissue damage in PGIA and results in an enhanced regulatory response as shown by the antigen-specific IL-10 production. Moreover, HSP70 induced protection is critically IL-10 dependent.

International audience; BackgroundInterleukin-32 (IL-32) is a newly described cytokine produced after stimulation by IL-2 or IL-18 and IFN-γ. IL-32 has the typical properties of a pro-inflammatory mediator and although its role in rheumatoid arthritis has been recently reported its effect on the osteoclastogenesis process remains unclear. Methodology/Principal Findings In the present study, we have shown that IL-32 was a potent modulator of osteoclastogenesis in vitro, whereby it promoted the differentiation of osteoclast precursors into TRAcP+ VNR+ multinucleated cells expressing specific osteoclast markers (up-regulation of NFATc1, OSCAR, Cathepsin K), but it was incapable of inducing the maturation of these multinucleated cells into bone-resorbing cells. The lack of bone resorption in IL-32-treated cultures could in part be explain by the lack of F-actin ring formation by the multinucleated cells generated. Moreover, when IL-32 was added to PBMC cultures maintained with soluble RANKL, although the number of newly generated osteoclast was increased, a significant decrease of the percentage of lacunar resorption was evident suggesting a possible inhibitory effect of this cytokine on osteoclast activation. To determine the mechanism by which IL-32 induces such response, we sought to determine the intracellular pathways activated and the release of soluble mediators in response to IL-32. Our results indicated that compared to RANKL, IL-32 induced a massive activation of ERK1/2 and Akt. Moreover, IL-32 was also capable of stimulating the release of IL-4 and IFN-γ, two known inhibitors of osteoclast formation and activation. Conclusions/Significance This is the first in vitro report on the complex role of IL-32 on osteoclast precursors. Further clarification on the exact role of IL-32 in vivo is required prior to the development of any potential therapeutic approach.

Background: Mouse AA-amyloidosis is a transmissible disease by a prion-like mechanism where amyloid fibrils act by seeding. Synthetic peptides with no amyloid relationship can assemble into amyloid-like fibrils and these may have seeding capacity for amyloid proteins. Principal Findings: Several synthetic peptides, designed for nanotechnology, have been examined for their ability to produce fibrils with Congo red affinity and concomitant green birefringence, affinity for thioflavin S and to accelerate AA-amyloidosis in mice. It is shown that some amphiphilic fibril-forming peptides not only produced Congo red birefringence and showed affinity for thioflavin S, but they also shortened the lag phase for systemic AA-amyloidosis in mice when they were given intravenously at the time of inflammatory induction with silver nitride. Peptides, not forming amyloid-like fibrils, did not have such properties. Conclusions: These observations should caution researchers and those who work with synthetic peptides and their derivatives to be aware of the potential health concerns. © 2009 Westermark et al. Original Publication:P. Westermark, Katarzyna Lundmark and Gunilla Westermark, Fibrils from designed non-amyloid-related synthetic peptides induce AA-amyloidosis during inflammation in an animal model, 2009, PLoS ONE, (4), 6, e6041.http://dx.doi.org/10.1371/journal.pone.0006041

Background: One of the hallmarks of rheumatoid arthritis (RA) is hyperplasia and inflammation of the synovial tissue being characterized by in situ occurrence of highly differentiated leukocytes. Fms-like tyrosine kinase 3 (Flt3) has a crucial role in hematopoiesis, regulation of cell proliferation, differentiation and apoptosis. Typically, Flt3 is expressed on early myeloid and lymphoid progenitors and is activated by its soluble ligand (Flt3-L). The highly differentiated cellular pattern in the synovium of the RA patients made us hypothesize that Flt3-L, with its ability to induce proliferation and differentiation, could be of importance in induction and/or progression of arthritis.\ud \ud Methodology/Principal Findings: To investigate occurrence of Flt3-L in RA we have measured its levels in matched serum and synovial fluid samples from 130 patients and 107 controls. To analyse the pro-inflammatory role of Flt3-L, we continuously overexpressed this protein locally in healthy mouse joints using homologous B-cell line transfected with Flt3-L gene. Additionally, recombinant Flt3-L was instillated intra-articularly in combination with peptidoglycans, a Toll Like Receptor 2-ligand with stong arthritogenic properties. Our results show significantly higher levels of Flt3-L in the synovial fluid as compared to serum levels in RA subjects (p = 0.0001). In addition, RA synovial fluid levels of Flt-3-L were significantly higher than these obtained from synovial fluids originating from non-inflammatory joint diseases (p = 0.022). Intra-articular administration of B-cell line transfected with Flt3-L gene resulted in highly erosive arthritis while inoculation of the same B-cell line without hyperexpression of Flt3-L did not induce erosivity and only in a minority of cases caused synovial proliferation! Flt3-ligand potentiated peptidoglycan induced arthritis as compared to mice injected with peptidoglycan alone (p

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFalpha levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Epicutaneous immunization is a potential non-invasive technique for antigen-specific immune-modulation. Topical application of protein antigens to barrier-disrupted skin induces potent antigen-specific immunity with a strong Th2-bias. In this study, we investigate whether the autoimmune inflammatory response of chronic collagen-induced arthritis (CCIA) in DBA/1-TCR-beta Tg mice can be modified by epicutaneous immunization. We show that epicutaneous immunization with type II collagen (CII) inhibited development and progression of CCIA and, importantly, also ameliorated ongoing disease as indicated by clinical scores of disease severity, paw swelling and joints histology. Treated mice show reduced CII-driven T cell proliferation and IFN-gamma production, as well as significantly lower levels of CII-specific IgG2a serum antibodies. In contrast, CII-driven IL-4 production and IgE antibody levels were increased consistent with skewing of the CII response from Th1 to Th2 in treated mice. IL-4 production in treated mice was inversely correlated with disease severity. Moreover, T cells from treated mice inhibited proliferation and IFN-gamma production by T cells from CCIA mice, suggesting induction of regulatory T cells that actively inhibit effector responses in arthritic mice. The levels of CD4(+)CD25(+) T cells were however not increased following epicutaneous CII treatment. Together, these results suggest that epicutaneous immunization may be used as an immune-modulating procedure to actively re-programme pathogenic Th1 responses, and could have potential as a novel specific and simple treatment for chronic autoimmune inflammatory diseases such as rheumatoid arthritis.

The characteristics of human resistin (RETN) are unclear and controversial despite intensive adipose-focused research. Its transcriptional and functional similarity with the murine myeloid-specific and CCAAT/enhancer binding protein epsilon (Cebpe)-dependent gene, resistin-like gamma (Retnlg), is unexplored. We examined the human CEBPE-regulatory pathway by unbiased reference and custom gene expression assays. Real-time RT-PCR analysis demonstrated lack of both the transcriptional factor CEBPE and RETN expression in adipose and muscle cells. In contrast, primary myelocytic samples revealed a concerted CEBPE-RETN transcription that was significantly elevated in inflammatory synoviocytes relative to intact peripheral blood mononuclear cells (PBMC). Mouse Cebpe and Retnlg were predictably expressed in macrophages, whereas Retn was abundant in adipocytes. Quite the opposite, a low and inconsistent RETN transcription was seen in some human white adipose tissue (WAT) biopsies without any relationship to body mass index, insulin sensitivity, or fat depot. However, in these cases, RETN was co-detected with CEBPE and the leukocyte-specific marker, EMR1, indicating the presence of inflammatory cells and their possible resistin-mediated effect on adipocytes. Indeed, addition of human resistin to WAT in culture induced, like in PBMC, the inflammatory cytokines IL6, IL8 and TNF. Importantly, the expression of the adipose-specific markers CEBPA, FABP4 and SLC2A4 was unchanged, while the expected inhibitory effect was seen with TNF. Both cytokines increased the mRNA level of CCL2 and MMP3, which may further promote inflammation in WAT. Thus, the myeloid-restricted nature of CEBPE precludes the expression of RETN in human adipocytes which, however, are targeted by this innate immune-derived proinflammatory cytokine.

A genetic association study by Timothy Vyse and colleagues suggests that there is a significant association between CRP variants and acute-phase serum CRP concentrations in patients with rheumatoid arthritis, including those with chronic inflammation., Background The acute-phase increase in serum C-reactive protein (CRP) is used to diagnose and monitor infectious and inflammatory diseases. Little is known about the influence of genetics on acute-phase CRP, particularly in patients with chronic inflammation. Methods and Findings We studied two independent sets of patients with chronic inflammation due to rheumatoid arthritis (total 695 patients). A tagSNP approach captured common variation at the CRP locus and the relationship between genotype and serum CRP was explored by linear modelling. Erythrocyte sedimentation rate (ESR) was incorporated as an independent marker of inflammation to adjust for the varying levels of inflammatory disease activity between patients. Common genetic variants at the CRP locus were associated with acute-phase serum CRP (for the most associated haplotype: p = 0.002, p, Editors' Summary C-reactive protein (CRP) is a serum marker for inflammation or infection and acts by binding to a chemical (phosphocholine) found on the surface of dead or dying cells (and some types of bacteria) in order to activate the immune system (via the complement system). Fat cells release factors that stimulate the liver to produce CRP, and serum levels greater than 10 mg/l are generally considered indicative of an infectious or inflammatory process. After an inflammatory stimulus, serum CRP levels may exceed 500 times baseline, so CRP is used in all medical specialities to help diagnose inflammation and infection. Although patients with chronic inflammatory diseases, such as rheumatoid arthritis, have raised levels of CRP, levels of CRP are still highly variable. Some studies have suggested that there may be genetic variations of CRP (CRP variants) that determine the magnitude of the acute-phase CRP response, a finding that has important clinical implications: CRP thresholds are used as a diagnostic component of formal clinical algorithms and play an important role in a clinician's decision-making process when diagnosing inflammatory disease and choosing treatment options. Therefore, it is possible that false reassurance could be given to a patient with disease, or optimal treatment withheld, because some patients are genetically predisposed to have only a modest increase in acute-phase CRP. Why Was This Study Done? Although some studies have looked at the CRP gene variant response, few, if any, studies have examined the CRP gene variant response in the context of chronic inflammation, such as in rheumatoid arthritis. Therefore, this study aimed to determine whether CRP gene variants could also influence CRP serum levels in rheumatoid arthritis. What Did the Researchers Do and Find? The authors studied two independent sets of patients with chronic inflammation due to rheumatoid arthritis (total 695 patients): one patient set used a cohort of 281 patients in the UK, and the other patient set (used for replication) consisted of 414 patients from New Zealand and Australia. A genetic technique (a tagSNP approach) was used to capture common variations at the CRP locus (haplotype association analysis) at both the population and the individual level. The relationship between genotype and serum CRP was explored by linear modeling. The researchers found that common genetic variants at the CRP locus were associated with acute-phase serum CRP in both patient sets translating into an approximate 3.5-fold change in expected serum CRP between carriers of two common CRP variants. For example, when ESR = 50 mm/h the expected CRP serum level for one common CRP variant was 43.1 mg/l and for another CRP variant was 14.2 mg/l. What Do These Findings Mean? The findings of this study raise questions about the interpretation of acute-phase serum CRP, as they suggest that there is a significant association between CRP variants and acute-phase serum CRP concentrations in a group of patients with rheumatoid arthritis, including those with chronic active inflammation. The size of the genetic effect may be large enough to have a clinically relevant impact on the assessment of inflammatory disease activity, which in turn may influence therapeutic decision making. Failure to take into account the potential for genetic effects may result in the inappropriate reassurance or undertreatment of patients simply because they carry low-CRP–associated genetic variants. CRP is increasingly being incorporated into clinical algorithms to compare disease activity between patients and to predict future clinical events, so these findings impact on the use of such algorithms. The accuracy and utility of these algorithms might be improved by using a genetically adjusted CRP measurement. Additional Information Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000341 Lab Test Online provides information on CRP The Wellcome Trust provides a glossary of genetic terms Learn.Genetics provides access to the Genetic Science Learning Center, which is part of the human genome project

OBJECTIVES: To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a. METHODS: We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology. RESULTS: Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered. CONCLUSIONS: Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.

In a retrospective cohort study, Gabriel Chodick and colleagues find a significant association between persistence with statin therapy and reduced risk of developing rheumatoid arthritis, but only a modest decrease in risk of osteoarthritis., Background The beneficial effects of statins in rheumatoid arthritis (RA) have been suggested previously, but it is unclear whether statins may prevent its development. The aim of this retrospective cohort study was to explore whether persistent use of statins is associated with onset of RA. Methods and Findings The computerized medical databases of a large health organization in Israel were used to identify diagnosed RA cases among adults who began statin therapy between 1998 and 2007. Persistence with statins was assessed by calculating the mean proportion of follow-up days covered (PDC) with statins for every study participant. To assess the possible effects of healthy user bias, we also examined the risk of osteoarthritis (OA), a common degenerative joint disease that is unlikely to be affected by use of statins. A total of 211,627 and 193,770 individuals were eligible for the RA and OA cohort analyses, respectively. During the study follow-up period, there were 2,578 incident RA cases (3.07 per 1,000 person-years) and 17,878 incident OA cases (24.34 per 1,000 person-years). The crude incidence density rate of RA among nonpersistent patients (PDC level of, Editors' Summary Background The role of statins in the management of diseases that have an inflammatory component is unclear. There is some evidence that statins may have anti-inflammatory and immunumodulatory properties, demonstrated by reducing the level of C-reactive protein that may play an important role in chronic inflammatory diseases, such as rheumatoid arthritis—a chronic condition that is a major cause of disability. Some small studies have suggested a modest effect of statins in decreasing disease activity in patients with rheumatoid arthritis, but a recent larger study involving over 30,000 patients with rheumatoid arthritis showed no beneficial effect. Furthermore, it has been suggested that statins may have a role in the primary prevention of rheumatoid arthritis, but so far there has been no solid evidence base to support this hypothesis. Before statins can potentially be included in the treatment options for rheumatoid arthritis, or possibly prescribed for the prevention of this condition, there needs to be a much stronger evidence base, such as larger studies with longer follow-up periods, which clearly demonstrates any significant clinical benefits of statin use. Why Was This Study Done? This large study (more than 200,000 patients) with a long follow-up period (average of 10 years) was conducted to discover whether there was any kind of association between persistent use of statins and the onset of rheumatoid arthritis. What Did the Researchers Do and Find? The researchers conducted a retrospective cohort study among the members of Maccabi Healthcare Services (a health maintenance organization [HMO]) in Israel, which has 1.8-million enrollees and covers every section of the Israeli population, to identify statin users who were at least 18 years of age and did not have RA or a related disease at study entry. The cohort covered the period 1998–2007 and included members who were continuously enrolled in the HMO from 1995 to 1998. The researchers then analyzed the incidence of newly diagnosed rheumatoid arthritis, recording the date of first diagnostic codes (International Classification of Diseases, 9th revision [ICD-9]) associated with rheumatoid arthritis during the study follow-up period. To assess any potential effects of “healthy adherer” bias (good adherence to medication in patients with a chronic illness may be more likely to lead to better health and improved survival), the researchers also examined any possible association between persistent statin use and the development of osteoarthritis, a common degenerative joint disease that is unlikely to be affected by statin use. During the study follow-up period, there were 2,578 incident cases of rheumatoid arthritis and 17,878 incident cases of osteoarthritis. The crude incidence density rate of rheumatoid arthritis among patients who did not persistently take statins was 51% higher than that of patients who used statins for at least 80% of the follow-up period. Furthermore, patients who persistently used statins had a risk ratio of 0.58 for rheumatoid arthritis compared with patients who did not persistently use statins. In the osteoarthritis cohort analysis, high persistence with statin use was associated with a modest decrement in risk ratio (0.85) compared to patients who did not persist with statins. What Do These Findings Mean? This study suggests that there is an association between persistence with statin therapy and reduced risk of developing rheumatoid arthritis. Although the researchers took into account the possibility of healthy adherer bias (by comparing results with the osteoarthritis cohort), this study has other limitations, such as the retrospective design, and the nonrandomization of statin use, which could affect the interpretation of the results. However, the observed associations were greater than those that would be expected from methodological biases alone. Larger, systematic, controlled, prospective studies with high efficacy statins, particularly in younger adults who are at increased risk for rheumatoid arthritis, are needed to confirm these findings and to clarify the exact nature of the biological relationship between adherence to statin therapy and the incidence of rheumatoid arthritis. Additional Information Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000336. Arthritis Research UK provides a wide range of information on arthritis research The American College of Rheumatology provides information on rheumatology research Patient information on rheumatoid arthritis is available at Patient UK Extensive information about statins is available at statin answers

BackgroundPower Doppler ultrasound (PDUS) is increasingly used to assess synovitis in Rheumatoid Arthritis (RA). Prior studies have shown correlations between PDUS scores and vessel counts, but relationships with T cell immunopathology have not been described.Methodology/principal findingsPBMC were isolated from healthy controls (HC) or RA patients and stimulated ex vivo with PMA and ionomycin for 3 hours in the presence of Golgistop. Paired synovial fluid (SF) or synovial tissue (ST) were analysed where available. Intracellular expression of IL-17, IFNgamma, and TNFalpha by CD4+ T cells was determined by flow cytometry. Synovial blood flow was evaluated by PDUS signal at the knees, wrists and metacarpophalangeal joints of RA patients. Serum, SF and fibroblast culture supernatant levels of vascular endothelial growth factor-A (VEGF-A) were measured by ELISA. The frequency of IL17+IFNgamma-CD4+ T cells (Th17 cells) was significantly elevated in peripheral blood (PB) from RA patients vs. HC (median (IQR) 0.5 (0.28-1.59)% vs. 0.32 (0.21-0.54)%, p = 0.005). Th17 cells were further enriched (mean 6.6-fold increase) in RA SF relative to RA PB. Patients with active disease had a higher percentage of IL-17+ T cells in ST than patients in remission, suggesting a possible role for Th17 cells in active synovitis in RA. Indeed, the percentage of Th17 cells, but not Th1, in SF positively correlated with CRP (r = 0.51, p = 0.04) and local PDUS-defined synovitis (r = 0.61, p = 0.002). Furthermore, patients with high levels of IL-17+CD4+ T cells in SF had increased levels of the angiogenic factor VEGF-A in SF. Finally, IL-17, but not IFNgamma, increased VEGF-A production by RA synovial fibroblasts in vitro.Conclusions/significanceOur data demonstrate a link between the presence of pro-inflammatory Th17 cells in SF and local PDUS scores, and offer a novel immunological explanation for the observation that rapid joint damage progression occurs in patients with persistent positive PDUS signal.

BACKGROUND: It is known that genetic predisposition to rheumatoid arthritis (RA) is associated with the MHC class II allele HLA-DR4 and that residues 261-273 of type II collagen (huCollp261) represent an immunodominant T cell epitope restricted by the DR4 molecule. Despite recent advances in characterization of MHC and T cell receptor (TCR) contacts to this epitope, the atomic details of TCR/huCollp261/HLA-DR4 ternary complex are not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we have used computational modeling to get insight into this interaction. A three-dimensional model of the TCR Vbeta domain from a DR4(+) patient affected by RA has been derived by homology modeling techniques. Subsequently, the structure of the TCR Vbeta domain in complex with huCollp261/HLA-DR4 was obtained from a docking approach in conjunction with a filtering procedure based on biochemical information. The best complex from the docking experiments was then refined by 20 ns of molecular dynamics simulation in explicit water. The predicted model is consistent with available experimental data. Our results indicate that residues 97-101 of CDR3beta are critical for recognition of huCollp261/HLA-DR4 by TCR. We also show that TCR contacts on p/MHC surface affect the conformation of the shared epitope expressed by DR alleles associated with RA susceptibility. CONCLUSIONS/SIGNIFICANCE: This work presents a three-dimensional model for the ternary complex TCR-Vbeta/collagenII(261-273)/HLA-DR4 associated with rheumatoid arthritis that can provide insights into the molecular mechanisms of self reactivity.

Tumor necrosis factor alpha (TNF-alpha) is a major inflammatory mediator that exhibits actions leading to tissue destruction and hampering recovery from damage. At present, two antibodies against human TNF-alpha (hTNF-alpha) are available, which are widely used for the clinic treatment of certain inflammatory diseases. This work was undertaken to identify a novel functional epitope of hTNF-alpha. We performed screening peptide library against anti-hTNF-alpha antibodies, ELISA and competitive ELISA to obtain the epitope of hTNF-alpha. The key residues of the epitope were identified by means of combinatorial alanine scanning and site-specific mutagenesis. The N terminus (80-91 aa) of hTNF-alpha proved to be a novel epitope (YG1). The two amino acids of YG1, proline and valine, were identified as the key residues, which were important for hTNF-alpha biological function. Furthermore, the function of the epitope was addressed on an animal model of collagen-induced arthritis (CIA). CIA could be suppressed in an animal model by prevaccination with the derivative peptides of YG1. The antibodies of YG1 could also inhibit the cytotoxicity of hTNF-alpha. These results demonstrate that YG1 is a novel epitope associated with the biological function of hTNF-alpha and the antibodies against YG1 can inhibit the development of CIA in animal model, so it would be a potential target of new therapeutic antibodies.

Background Dendritic cells (DCs) are highly specialized cells, which capture antigen in peripheral tissues and migrate to lymph nodes, where they dynamically interact with and activate T cells. Both migration and formation of DC-T cell contacts depend on cytoskeleton plasticity. However, the molecular bases governing these events have not been completely defined. Methodology/principal findings Utilizing a T cell-dependent model of arthritis, we find that PLCgamma2-/- mice are protected from local inflammation and bone erosion. PLCgamma2 controls actin remodeling in dendritic cells, thereby affecting their capacity to prime T cells. DCs from PLCgamma2-/- mice mature normally, however they lack podosomes, typical actin structures of motile cells. Absence of PLCgamma2 impacts both DC trafficking to the lymph nodes and migration towards CCL21. The interaction with T cells is also affected by PLCgamma2 deficiency. Mechanistically, PLCgamma2 is activated by CCL21 and modulates Rac activation. Rac1/2-/- DCs also lack podosomes and do not respond to CCL21. Finally, antigen pulsed PLCgamma2-/- DCs fail to promote T cell activation and induce inflammation in vivo when injected into WT mice. Conversely, injection of WT DCs into PLCgamma2-/- mice rescues the inflammatory response but not focal osteolysis, confirming the importance of PLCgamma2 both in immune and bone systems. Conclusions/significance This study demonstrates a critical role for PLCgamma2 in eliciting inflammatory responses by regulating actin dynamics in DCs and positions the PLCgamma2 pathway as a common orchestrator of bone and immune cell functions during arthritis.

BackgroundFc gamma receptors (FcγRs) play a crucial role in immunity by linking IgG antibody-mediated responses with cellular effector and regulatory functions. Genetic variants in these receptors have been previously identified as risk factors for several chronic inflammatory conditions. The present study aimed to investigate the presence of copy number variations (CNVs) in the FCGR3B gene and its potential association with the autoimmune disease rheumatoid arthritis (RA).Methodology/principal findingsCNV of the FCGR3B gene was studied using Multiplex Ligation Dependent Probe Amplification (MLPA) in 518 Dutch RA patients and 304 healthy controls. Surprisingly, three independent MLPA probes targeting the FCGR3B promoter measured different CNV frequencies, with probe#1 and #2 measuring 0 to 5 gene copies and probe#3 showing little evidence of CNV. Quantitative-PCR correlated with the copy number results from MLPA probe#2, which detected low copy number (1 copy) in 6.7% and high copy number (≥3 copies) in 9.4% of the control population. No significant difference was observed between RA patients and the healthy controls, neither in the low copy nor the high copy number groups (p-values = 0.36 and 0.71, respectively). Sequencing of the FCGR3B promoter region revealed an insertion/deletion (indel) that explained the disparate CNV results of MLPA probe#1. Finally, a non-significant trend was found between the novel -256A>TG indel and RA (40.7% in healthy controls versus 35.9% in RA patients; P = 0.08).Conclusions/significanceThe current study highlights the complexity and poor characterization of the FCGR3B gene sequence, indicating that the design and interpretation of genotyping assays based on specific probe sequences must be performed with caution. Nonetheless, we confirmed the presence of CNV and identified novel polymorphisms in the FCGR3B gene in the Dutch population. Although no association was found between RA and FCGR3B CNV, the possible protective effect of the -256A>TG indel polymorphism must be addressed in larger studies.

BACKGROUND:Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man. METHODS/PRINCIPAL FINDINGS:We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (99mTc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4 x 10(-3) (0.95-5.1 x 10(-3)) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion. CONCLUSIONS/SIGNIFICANCE:The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention.

Background The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality. Methods and Findings PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed. Conclusions Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.

BackgroundCD23 is a differentiation/activation antigen expressed by a variety of hematopoietic and epithelial cells. It can also be detected in soluble forms in biological fluids. Initially known as the low-affinity receptor for immunoglobulin E (Fc epsilonRII), CD23 displays various other physiologic ligands such as CD21, CD11b/c, CD47-vitronectin, and mannose-containing proteins. CD23 mediates numerous immune responses by enhancing IgE-specific antigen presentation, regulating IgE synthesis, influencing cell differentiation and growth of both B- and T-cells. CD23-crosslinking promotes the secretion of pro-inflammatory mediators from human monocytes/macrophages, eosinophils and epithelial cells. Increased CD23 expression is found in patients during allergic reactions and rheumatoid arthritis while its physiopathologic role in these diseases remains to be clarified.Methodology/principal findingsWe previously generated heptapeptidic countrestructures of human CD23. Based on in vitro studies on healthy and arthritic patients' cells, we showed that CD23-specific peptide addition to human macrophages greatly diminished the transcription of genes encoding inflammatory cytokines. This was also confirmed by significant reduction of mediator levels in cell supernatants. We also show that CD23 peptide decreased IgE-mediated activation of both human and rat CD23(+) macrophages. In vivo studies in rat model of arthritis showed that CD23-blocking peptide ameliorates clinical scores and prevent bone destruction in a dose dependent manner. Ex-vivo analysis of rat macrophages further confirmed the inhibitory effect of peptides on their activation. Taken together our results support the role of CD23 activation and subsequent inflammatory response in arthritis.ConclusionCD23-blocking peptide (p30A) prevents the activation of monocytes/macrophages without cell toxicity. Thus, targeting CD23 by antagonistic peptide decreases inflammatory markers and may have clinical value in the treatment of human arthritis and allergic reactions involving CD23.