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The development of novel treatments for rheumatoid arthritis (RA) requires the interplay between clinical observations and studies in animal models. Given the complex molecular pathogenesis and highly heterogeneous clinical picture of RA, there is an urgent need to dissect its multifactorial nature and to propose new strategies for preventive, early and curative treatments. Research on animal models has generated new knowledge on RA pathophysiology and aetiology and has provided highly successful paradigms for innovative drug development. Recent focus has shifted towards the discovery of novel biomarkers, with emphasis on presymptomatic and emerging stages of human RA, and towards addressing the pathophysiological mechanisms and subsequent efficacy of interventions that underlie different disease variants. Shifts in the current paradigms underlying RA pathogenesis have also led to increased demand for new (including humanised) animal models. There is therefore an urgent need to integrate the knowledge on human and animal models with the ultimate goal of creating a comprehensive 'pathogenesis map' that will guide alignment of existing and new animal models to the subset of disease they mimic. This requires full and standardised characterisation of all models at the genotypic, phenotypic and biomarker level, exploiting recent technological developments in '-omics' profiling and computational biology as well as state of the art bioimaging. Efficient integration and dissemination of information and resources as well as outreach to the public will be necessary to manage the plethora of data accumulated and to increase community awareness and support for innovative animal model research in rheumatology.

/st> Molecular factors modulating angiogenic responses are dysregulated in patients with MCTD and SSc with increases of VEGF in MCTD and SSc and selective upregulation of endostatin in MCTD. Furthermore, high serum levels of VEGF might characterise patients with MCTD with a more severe course of the disease with increased prevalence of PAH and myositis.

OBJECTIVES: /st> To study the expression, regulation and function of the histone methyltransferase enhancer of zeste homologue 2 (EZH2) in synovial fibroblasts (SF) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: /st> SF were obtained from RA and OA patients undergoing joint surgery. Expression levels were assessed by quantitative real-time PCR and western blot. Kinase inhibitors and reporter gene assays were employed to study signalling pathways. Functional analyses included EZH2 overexpression by plasmid transfection and gene silencing by small interfering RNA. Chromatin immunoprecipitation assay was used to analyse histone methylation within distinct promoter regions. RESULTS: /st> By studying the expression and function of EZH2 in SF the authors found that EZH2 is overexpressed in rheumatoid arthritis synovial fibroblasts (RASF) and further induced by tumour necrosis factor alpha through the nuclear factor kappa B and Jun kinase pathways. As a target gene of EZH2 the authors identified secreted frizzled-related protein 1 (SFRP1), an inhibitor of Wnt signalling, which is associated with the activation of RASF, and show that SFRP1 expression correlates with the occupation of its promoter with activating and silencing histone marks. CONCLUSIONS: /st> These data strongly suggest that the chronic inflammatory environment of the RA joint induces EZH2 and thus might cause changes in the epigenetic programmes of SF.

Scientific interest in adipose tissue-derived peptides has increased dramatically in recent years (1). Several mediators known as adipo(cyto)kines were first associated with the pathophysiology of obesity-related complications; however a significant role for adipokines such as leptin, adiponectin, resistin and visfatin in regulating immune responses and inflammation has recently been discovered (1, 2). Several reports (3-6) have already demonstrated association of these adipokines with the severity of rheumatoid arthritis (RA). Vaspin, a member of the serine protease inhibitor family, and omentin (also known as intelectin) were recently identified in adipose tissue (7, 8). Vaspin is an adipokine with insulin-sensitizing effects that has been suggested to be a compensatory mediator for abrogating obesity and its inflammatory complications (7). Expression of the omentin gene was demonstrated in omental adipose tissue of patients with Crohn's disease, suggesting that it may be implicated in chronic inflammatory diseases (8). The aim of the present report was to compare local concentrations of vaspin and omentin in synovial fluid of RA patients with those in osteoarthritis (OA) patients and to characterize their potential association with the severity of the disease. Synovial fluid was obtained during therapeutic arthrocentesis from 33 patients with RA and 33 patients with knee OA. The disease activity of RA patients was assessed by DAS28. C-reactive protein (CRP), IgM-rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA) were routinely analyzed from peripheral blood obtained at the time of arthrocentesis. Characteristics of the patients are given in Table 1. Vaspin (AdipoGen Inc. Korea) and omentin (Apotech Corporation) were analyzed in synovial fluid by ELISA assays. Ethical approval was obtained from the local Ethics Committee and all patients provided informed consent. Statistical analysis was performed using GraphPad Prism 5.0 software. To meet a

OBJECTIVE: In the present study we evaluated the decrease of cartilage destruction by a novel orally active and specific MMP-13 inhibitor in three different animal models of rheumatoid arthritis (RA). MATERIALS AND METHODS: The SCID mouse co-implantation model of RA, collagen-induced arthritis (CIA) model in mice and the antigen induced arthritis model (AIA) in rabbits were used. RESULTS: In the SCID mouse co-implantation model this inhibitor resulted in reduced cartilage destruction by 75%. In the CIA model of RA, the MMP-13 inhibitor resulted in a significant and dose dependent decrease in clinical symptoms as well as of cartilage erosion by 38% (30 mg/kg), 28% (10 mg/kg) and 21% (3 mg/kg). No significant effects were observed in the AIA model. No toxic effects were observed in all three animal models. CONCLUSION: Although several MMPs in concert with other proteinases play a role in the process of cartilage destruction, there is a need for highly selective MMP inhibitors to reduce severe side effects that occur with non-specific inhibitors. Significant inhibition of MMP-13 reduced cartilage erosions in two out of three tested animal models of RA. These results strongly support the development of this class of drugs to reduce or halt joint destruction in patients with RA.

BACKGROUND: Ankylosing spondylitis (AS) is a frequent largely genetically determined rheumatic disease that is characterised by spinal inflammation and new bone formation. However, the exact pathogenesis including pathology is still not clear. OBJECTIVE: To analyse tissue obtained at spinal surgery by immunohistochemistry and compare the specimen of patients with AS to those with degenerative disc disease (DDD). METHODS: Bony and soft tissue specimens of 30 AS and 20 DDD patients were obtained during spinal osteotomy from different anatomic regions including articular and spinous processes, interspinous ligaments and intervertebral disks. Immunohistolochemistry was performed with established markers for cathepsin-K, MMP-1, MMP-3 and RANK-ligand. RESULTS: Cathepsin-K and MMP-1-positive cells were only observed in AS specimen. Cathepsin-K-positive multinucleated cells were detected at articular processes adjacent to fibrous tissues. MMP-1 was expressed in smaller mononuclear cells attached to bone, Invasion of bone by MMP-1 cells was seen at entheseal sites. In the intervertebral disks, most mononuclear cells were cathepsin-K-positive. Isolated cells expressing these matrix degrading enzymes found in DDD never showed signs of invasion. No differences were found for MMP-3 between AS and DDD. Clear expression of RANK ligand was only detected in one AS patient. CONCLUSION: /B> Cathepsin-K is strongly expressed in different regions of the spine in AS. Mainly mononuclar cells, fibroblast-like cells, and cells attached to bone and at sites of bone remodelling expressed cathepsin K suggestive of high osteoclastic activity. This supports the role of persistent inflammation in the pathogenesis of AS. How these changes relate to osteoproliferation remains to be determined.

OBJECTIVE: IL-23, composed of a p19 and a p40 subunit, is suggested to play key roles in rheumatoid arthritis (RA), dependent on the promotion and proliferation of IL-17-producing Th17 T-cells. However, previous studies on IL-23 expression in human tissues were based on p19 only. We aimed to study the expression and regulation of both IL-23 subunits, p19 and p40, in RA compared to osteoarthritis (OA) patients. METHODS: The expression of p19 and p40 in synovial tissues was analyzed by in-situ hybridization and immunohistochemistry. IL-23 in RA and OA synovial fluids and sera was determined by ELISA. TLR-dependent induction of p19, p40 and bioactive IL-23 was determined in RASF, monocytes and MDDC by RT-PCR, Real-time PCR, Western-blot and functional-assays. RESULTS: The p19 subunit was abundantly expressed in RA but not in OA synovial tissues. p19 was most prominently expressed by RASF in the synovial lining layer and at the site of invasion, but no heterodimeric IL-23 was detected at these sites. Correspondingly, soluble IL-23 was not detectable or found at very low levels in synovial fluids and sera of patients with RA. By in-vitro experiments, we confirmed that TLR-activated RASF expressed p19 but not p40, in contrast to monocytes, which produced IL-23 following TLR stimulation. CONCLUSION: The TLR-dependent induction of p19 but not p40 in RASF and the abundant expression of p19 along with the low or undetectable levels of IL-23 in RA patients, gives strong evidence that p19 does not necessarily indicate the presence of IL-23 as it has been proposed so far.

OBJECTIVES: The viral gene transfer of Interleukin-1 receptor antagonist (IL-1ra) and Interleukin-10 (IL-10) into rheumatoid arthritis (RA) synovial fibroblasts (RASF) showed protective effects on cartilage destruction in the SCID mouse model for RA. Nevertheless, side effects of viral transduction are possible and a number of cytokines or cytokine inhibitors are not available encoded in viral vehicles. As the production of viruses coding for bioactive proteins is cost- and time-intensive, we established an in vivo long-term release model using osmotic minipumps in the SCID mouse model for RA. METHODS: Isolated RASF were cultured for 4 passages and coimplanted together with human cartilage and an Alzet(R) Osmotic Miniature Pump Model 2004 containing 200 microl of IL-10 and IL-1ra for 40 days in SCID mice. Implants were removed after 40 days and evaluated histologically. The actual rates of IL-10 and IL-1ra in murine serum were measured by ELISA. RESULTS: Release of IL-10 and IL-1ra by the pumps was effective as both could be measured in significant amounts in the serum of the mice. IL-10 and IL-1ra release showed protective effects towards the co-implanted cartilage, similar to the adenovirally IL-10-/IL-1ra-transduced RASF. The invasion scores for the implants with the osmotic pumps were: invasion 0.7+/-0.5, degradation 0.5+/-0.3 (all parameters significant vs. controls, p<0.05). CONCLUSIONS: The results demonstrate that the combination of osmotic pumps with the SCID mouse model for RA can be used as approach for application and evaluation of cartilage-protective molecules. Furthermore, the effect of cartilage-protective cytokines is independent of the type of application.

We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-beta induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription-polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-beta induced significant increases in MMP-3 mRNA (12.35-fold, p < 0.001) and protein (mean 62 ng/ml, sixfold, p < 0.008) and in expression of MMP-10 mRNA (11.68-fold, p < 0.05) and protein (54 ng/ml, 20-fold, p > or = 0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-beta response. No phosphorylation of nuclear factor-kappaB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-beta suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-kappaB phosphorylation.

OBJECTIVE: To find previously unknown properties of ML3000, a competitive inhibitor of the cyclooxygenase and the lipoxygenase (LO) pathway. METHODS: Gene expression of ML3000 treated and untreated rheumatoid arthritis synovial fibroblasts were measured with Affymetrix gene arrays. Downregulation of chemokine (C-X-C motif) ligands CXCL9, CXCL10 and CXCL11 was verified with Real-time polymerase chain reaction, CXCL10 protein levels were determined with ELISA. Rheumatoid arthritis synovial fibroblasts were treated with the cyclooxygenase inhibitor naproxen, the 5-LO inhibitor BWA4C and the 5-lipoxygenase-activating protein (FLAP) inhibitor MK886, and consecutive changes in CXCL10 protein levels measured. 5-LO expression was determined by polymerase chain reaction and Western blot. RESULTS: In synovial fibroblasts and monocyte-derived macrophages ML3000 inhibited the tumour necrosis factor induced expression of CXCL9, CXCL10 and CXCL11, which are all ligands of the chemokine receptor CXCR3. No effect was observed in monocytes. Whereas inhibition of the cyclooxygenase pathway or the FLAP protein showed no effect, blockade of 5-LO significantly downregulated CXCL10 protein levels. 5-LO mRNA was detected in monocytes and in monocyte-derived macrophages. All tested cell types expressed 5-LO protein. CONCLUSIONS: ML3000 effectively downregulates CXCR3 ligands. This study confirms that a thorough analysis of the impact of a drug on its target cells cannot only reveal unexpected properties of a substance, but also helps to understand the underlying molecular mechanisms. Accordingly, our data provide the basis for further clinical studies testing the application of ML3000 in diseases such as rheumatoid arthritis or multiple sclerosis.

OBJECTIVE: Biologicals have revolutionised the treatment of rheumatoid arthritis (RA). However, progressive joint destruction can still be observed in many patients and the search for novel molecular therapies targeting specific signalling pathways is ongoing. In the present study, we investigated the effects of GW282974, a novel compound directed against tyrosine kinase activity with respect to the potential suppression of inflammation and destruction. METHODS: Synovial tissue specimens were obtained from RA patients undergoing surgical joint replacement. Rheumatoid arthritis synovial fibroblasts (RASFs) were stimulated with cytokines and GW282974 was added in different concentrations. Gene expression was checked by TaqMan PCR, using 18S as housekeeping gene. Protein analysis was quantified by ELISA. Cell growth and proliferation was measured using the "ViaLight" proliferation assay. RESULTS: EGF had no effect on the gene expression profile of RASFs when used as single stimulatory agent. In combination with pro-inflammatory mediators however, EGF showed a synergistic effect. The expression of matrix metalloproteinases, inflammatory cytokines and cyclooxygenase-2 on mRNA levels was strongly increased, whereas the addition of GW282974 abrogated these effects in a dose-dependent manner. These data could be confirmed on protein/lipid levels analysing the supernatants of RASFs by ELISA. Similarly, cell growth and proliferation of RASFs were inhibited by GW282974 in a dose- and time-dependent manner. By contrast, no cytotoxic effects were seen within the concentrations used. DISCUSSION: GW282974 appears to interfere with the inflammatory and the destructive pathways in RASFs and might therefore be used as novel therapeutic strategy for the treatment of RA.

OBJECTIVES: Ki-67 is expressed in the nuclei of dividing cells and can be used to assess proliferation of synovial inflammatory and stromal cells. We evaluated subintimal Ki-67+ cell density as a tissue biomarker for inflammatory arthropathies and compared it to subintimal CD68, a synovial biomarker of RA. METHODS: Subintimal Ki-67+ and CD68+ cell densities were measured immunohistochemically in synovial specimens obtained from patients with rheumatoid arthritis (RA; n = 19), osteoarthritis (OA; n = 18), "non-inflammatory" orthopaedic arthropathies (avascular necrosis, meniscus injury, femur fracture; n = 16), chronic septic arthritis (n = 9), and histologically normal synovium (n = 10). RESULTS: were correlated with a histological synovitis score. Utilising the areas under receiver operating characteristic curves (AUCs), we compared the abilities of Ki-67 and CD68 to differentiate among these arthropathies. Results: Ki-67 was expressed widely in the subintimal of inflamed specimens and in RA pannus invading hard tissues. Compared to normal controls, it was highly overexpressed in RA (26.6-fold) and chronic septic arthritis (55-fold), and mildly elevated in OA (3.9-fold) and orthopaedic arthropathies (2.1-fold). Ki-67 and CD68 differentiated similarly well between RA and OA (AUC: Ki-67 = 0.91, CD68 = 0.94), Ki-67 better between chronic septic arthritis and RA, and CD68 better between OA and normal controls. Ki-67 (r = 0.80) and CD68 (r = 0.79) correlated positively with the synovitis score. CONCLUSIONS: Subintimal Ki-67 was overexpressed in inflammatory arthropathies, distinguished among differentially inflamed arthropathies, and correlated positively with the histological severity of synovitis. It may prove useful in synovial tissue classification and as a synovial marker of disease activity in clinical trials when biopsies are available.