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Background:Current concepts of cartilage destruction in inflammatory arthritis include pannus infiltration by inflamed synovial tissue as well as direct detrimental effects of inflammatory cytokines and proteinases. Fibrin maintains chronic inflammatory processes in arthritis but has never been shown to be directly involved in cartilage damage occurring in rheumatoid arthritis (RA).Objectives:To investigate fibrin-mediated cartilage degradation and the possible underlying mechanisms in arthritis.Methods:Human cartilage samples were obtained from patients with RA undergoing joint replacement and investigated by H.E. and immunohistochemistry for cartilage damage and fibrin deposition. Cartilage explants from RA patients were incubatedin vitrowith autologous synoviocytes and assessed by immunohistochemistry for cell-adhesion and colocalization with fibrin. Experimental RA was studied in the RA murine model of adjuvant-induced arthritis (AIA), in wildtype (WT) and fibrinogen deficient (Fg-/-) mice. Cartilage damage and chondro-synovial adhesion were analyzed by safranin-O staining and fibrin deposition by immunohistochemistry. Fibrinogen expression (Fgα, Fgβ, Fgɣ) was studied in murine primary chondrocytes by qRT-PCR. Cartilage explants were stained with alizarin-red staining and assessed for colocalization of calcific deposits and fibrin. Calcification of murine primary chondrocytes stimulated with secondary calciprotein particles (CPP) and treated with purified human plasma fibrinogen (100 µg/ml) was assessed by alizarin red staining and gene expression for chondrocytic differentiation (Agg, Coll2, Coll10, Sox9, Runx2), calcification (Alpl, Ank, Anx5, Pc1, Pit1, Pit2), and extracellular matrix remodeling (Adamts4, Adamts5, Mmp3, Mmp13, Comp) by qRT-PCR.Results:Abundant fibrin deposition on cartilage co-localized and positively correlated with cartilage damage in knee joints of patients with RA. In the AIA model, absence of fibrin deposition in Fg-/-mice was accompanied by significantly lower synovial inflammation, chondro-synovial adhesion and cartilage damage than in WT mice. Chondro-synovial adhesion correlated with cartilage damage in the WT and led to apparent mechanical stripping of the superficial cartilage, whilst this phenomenon was not observed in the Fg-/-mice.In vitro, autologous RA synoviocytes adhered to cartilage explants exclusively in the presence of fibrin deposition. Fibrinogen chains were not expressed by primary chondrocytes, indicating passive deposition from synovial fluid or tissue. In human RA cartilage explants, we found colocalization and a significant positive correlation between fibrin and calcific deposits. Fibrinogen caused exacerbated calcification in CPP-treated primary murine chondrocytes and induction of genes involved in chondrocyte calcification (Pc1, Pit1). Cartilage-oligomeric matrix protein (Comp) gene was also highly induced suggesting a pro-catabolic role of fibrinogen.Conclusion:Fibrin deposition is an active trigger of cartilage degeneration in RA via induction of chondro-synovial adhesion (mechanical aspect) and induction of calcification (catabolic aspect). Newer therapeutic approaches may not merely focus on fibrinolysis but protect cartilage from fibrin-induced adhesion or calcification e.g. by fibrin-targeted immunotherapy.Disclosure of Interests:Thomas Hügle Grant/research support from: Abbvie, Novartis, Consultant of: Abbvie, Pfizer, Novartis, Roche, Lilly, BMS, Sonia Nasi: None declared, Driss Ehirchiou: None declared, Alexander So Consultant of: Sobi, Grünenthal, Nathalie Busso: None declared

Background:Aggressiveness of synoviocytes and collective migration of organized synovial tissues play a key role in the pathogenesis of pannus invasion into adjacent joint structure. Interactions among synovial cells for grouped movement, however, have not been properly elucidated.Objectives:We hypothesized that cadherins which have functions on the synovial invasion in RA, may play a critical role in collective migration of rheumatoid synoviocytes.Methods:Cadherins expression patterns on the synoviocytes isolated from patients with RA were evaluated using RT-PCR, flow cytometry, and western blot analysis. Mesenchymal and epithelial phenotypes were examined in cadherin overexpressing cell line by flow cytometry. L-cells with overexpression of CDH2 (CDH2hi), CDH11 (CDH11hi), and combination of CDH2/CDH11 (CDH2hi/CDH11hi) were prepared. Migration of cells was observed by taking time-lapse images with laser confocal microscope.In vitrocollective migration and directional movement in response to inflammatory mediators and different matrix rigidity were evaluated.In vivohoming of CDH2hi/CDH11hi-L-cells into joint tissues was performed in collagen induced arthritis (CIA) mouse. In vivoandex vivomigration pattern of CDH11hi-L-cells were investigated in nude mice using optical imaging system.Results:In rheumatoid synovial tissues, CDH2 and CDH11 were highly expressed compared to synovial tissues from osteoarthritis. CDH2 and CDH11 were also highly expressed on synovial fibroblasts isolated from RA. Phenotype analysis of mesenchymal and epithelial cells in CDH11hi-L-cells and CDH2hi/CDH11hi-L-cells showed increased expression of α5β1, CD44s, vimentin, and α-SMA compared with MOCK-L-cells. We then analyzed the pattern of migration of MOCK, CDH2hi, CDH11hi, and CDH2hi/CDH11hi-L-cells using time lapse images. During migration over a hard ECM, CDH2hiand CDH11hi-L cells represented higher aspect ratio compare to a soft ECM. Aspect ratio relatively found lower in CDH2hi/CDH11hi-L-cell lines than MOCK cells. CDH2hi/CDH11hi-L-cells showed significantly higher migration velocity and Euclidean distance with narrower angle of migratory directions in a cytokine mediated migration. Compared to the MOCK cells, persistence ratio and aspect ratio of migration were also higher in CDH2hi, CDH11hi, and CDH2hi/CDH11hi-L-cells. CDH2hi/CDH11hi-L-cells collectively migrated with the formation of leader and follower cells. In a chemokine mediated hard stiffness of ECM, durotaxis was observed in CDH11hi-L-cells. After 24 hours of intraarticular knee injection in CIA mouse, higher number of CDH2hi/CDH11hi-L-cells invaded into the cartilage than MOCK cells.In vivomigration of CDH2hi/CDH11hi-L-cells was also found towards the chemokine and cartilage mixed matrigel plug in the subcutaneous space of mice.Conclusion:The expression of CDH2 and CDH11 promotes directional migration of synoviocytes, indicating the potential role of these cadherins on the pannus tissues in the invasion into adjacent joint structure in RA.References:[1]Noss EH, Chang SK, Watts GFM, Brenner MB. Cadherin-11 engagement modulates matrix metalloproteinase production by rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 2011 Dec; 63(12): 3768–3778Disclosure of Interests:None declared

Case history 1 A 47 year old housewife presented with a 3 year history of fleeting inflammatory pain and oedema affecting the small joints of the hands symmetrically. She had previously been given a diagnosis of rheumatoid arthritis (RA), and was receiving treatment with non-steroidal anti-inflammatory drugs (NSAIDs) and prednisolone (10 mg/day). For the past year she had experienced inflammatory neck pain with a recurrent sense of heaviness and muscle weakness in the upper and lower limbs. Her symptoms were present, especially, during prolonged flexion and were relieved in a favourable posture. Examination showed symmetrical arthritis and tenderness of wrist and metacarpophalangeal (MCP) joints in both hands. A reduction of about 30% was seen in the anterior and lateral flexion of the cervical spine. Neurological examination results were, however, normal. Laboratory tests showed high acute phase response and negative rheumatoid factor. Radiographs of the hands and feet disclosed osteopenia but no erosions. Further investigations included radiographs of the cervical spine (neutral and flexion position) (figs 1A and B) and an anterior atlantoaxial subluxation (aAAS) was seen. Magnetic resonance imaging (MRI; fig 1C) of the cervical spine showed the aAAS and also space-taking pathological soft tissue (synovitis pannus) around the dens of the axis. The pannus was in contact with the spinal cord, but there was still spinal fluid behind the cord. Figure 1 (A) Lateral view radiograph of the cervical spine (case 1) during flexion. Severe AAS is present (arrows show the anterior aspect of the dens of axis and the anterior arch of the atlas). (B) The AAS disappears during neutral posture. (C) Neutral position MRI examination of the cervical spine with the patient in a supine position. AAS is present, together with inflammatory tissue around the dens, but there is no compression of the spinal cord. The patient was referred to a neurosurgeon …

OBJECTIVES—Examination of synovial tissue (ST) obtained at surgery because of end stage destructive rheumatoid arthritis (RA) showed that macrophages and fibroblasts are the major cell types at the cartilage-pannus junction (CPJ). This study aimed at defining the cell infiltrate and mediators of joint destruction in ST selected at arthroscopy from the CPJ in patients with RA who did not require joint surgery. METHODS—Paired synovial biopsy specimens were obtained at arthroscopy from ST adjacent to the CPJ and the suprapatellar pouch from the knee joints of 17 patients with RA. Immunohistological analysis was performed using monoclonal antibodies to detect T cells, B cells, plasma cells, macrophages, fibroblast-like synoviocytes, mast cells, and granzyme B+ cytotoxic cells as well as the expression of metalloproteinase (MMP)-1, MMP-3, and MMP-13. The sections were evaluated by computer assisted image analysis and semiquantitative analysis. RESULTS—The cell infiltrate comprised mainly T cells, macrophages, and plasma cells. The ST was also infiltrated by the other cell types, but at lower numbers. Expression of MMPs was abundant, especially MMP-3. The features of ST at the CPJ were generally similar to those at the suprapatellar pouch. CONCLUSIONS—The synovium at the CPJ in patients with RA who did not require joint surgery exhibits, in general, the same type of cell infiltrate and expression of MMPs and granzymes as ST from the suprapatellar pouch. The pathological changes that have been described at the CPJ in patients with RA with end stage, destructive disease may well reflect the transition to a process in which macrophages, fibroblast-like synoviocytes, and other cell types become increasingly important.

The term rheumatoid arthritis (RA) was first proposed by Garrod in 1859.1 By 1959, the histopathological features of synovitis, the proliferating pannus, and cartilage degradation in longstanding RA had been well described.2 Early histopathological studies were based on tissue samples obtained at surgery or at postmortem examinations. Occasionally, biopsy samples were obtained for analysis from patients with arthritis undergoing open arthrotomy. The initial interest in developing synovial biopsy techniques was to aid the differential diagnosis of joint diseases. In 1932 Forestier described a technique for obtaining synovial tissue with a dental nerve extractor that was introduced into the joint through a large calibre needle.3 He never published his results. Early experience with needle biopsy of the synovium was described in the 1950s.4 5 It was concluded that if strict aseptic techniques were employed, the procedure was safe and practical for use in both hospital wards and outpatient clinics. However, the biopsy needles tended to cause considerable trauma to the penetrated tissues owing to their wide bore and the requirement for an incision. In 1963 Parker and Pearson developed a simplified 14-gauge biopsy needle that did not require a skin incision.6 They described their experience of 125 procedures, almost all from the suprapatellar pouch of the knee joint, of which only five failed to yield adequate tissue for analysis. No serious complications were encountered. The potential of needle biopsy as a research tool in arthritis was highlighted in 1970 by Kinsella et al in their study of synovial lining layer cells in RA,7 and in 1972 by Schumacher and Kitridou in their clinicopathological study of the early features of synovitis.8 Arthroscopy was also initially developed as a diagnostic instrument. It was used primarily by orthopaedic surgeons.9 In the 1970s and 1980s a number …

Qualitative differences in synovitis between the cartilage-pannus junction (CPJ) region and the adjoining suprapatellar pouch (SPP) have been reported in rheumatoid arthritis and the spondyloarthropathies.To determine if the distribution of synovitis is the same in osteoarthritis (OA) using sensitive measures of inflammation derived from dynamic, contrast enhanced magnetic resonance imaging (DEMRI).20 subjects with established OA of the knee were recruited. Conventional MR images together with the DEMRI measurements were obtained. Areas of synovitis at the CPJ region and at a distant site in the SPP were calculated; differences in CPJ and SPP synovitis were determined using DEMRI parameters: the initial rate of contrast enhancement (IRE) and maximal enhancement (ME).The area of synovitis was significantly greater adjacent to the CPJ than in the SPP. IRE and ME measures were greater at the CPJ than the SPP.The magnitude of synovitis at the CPJ is not disease-specific and applies across the spectrum of degenerative disease as well as inflammatory diseases.

Correspondence to: Raimund W Kinne, Max-Planck-Society, Clinical Research Unit for Rheumatology/Immunology, Schwabachanlage 10, D-91054 Erlangen, Germany. Several reports have proposed the hypothesis that synovial fibroblasts play a major role in the crippling destruction of cartilage and bone in the inflamed joints of patients with rheumatoid arthritis (RA),la either autonomouslyl or in concert with T cells and macrophages. 1-3 4a 5 RA synovial fibroblasts are a major component of the invasive pannus,3 a vascular and fibrous granulation tissue arising from the joint recessus and extending onto the surface of the cartilage.6 A number of studies indicate that these fibroblasts are morphologically altered'-4 7 and, in addition, highly activated' 2 4a 8 9-phenotypic features that have been interpreted either as signs of irreversible cellular transformationl-4 7 or as the result of strong, but reversible stimulation originating from the surrounding inflammatory microenvironment.2 4a 5 Activation of fibroblasts in vitro is known to generate several functional responses, such as production of matrix components,'0 soluble mediators,"1 or enzymes' 2 4 8 11 that could contribute significantly to joint pathology in chronic RA in vivo. 1-4 811-15

Background Osteoclast-associated receptor [OSCAR] is expressed by monocyte, endothelial and dendritic cells. OSCAR is a co-stimulation receptor involved in transducing immunoreceptor tyrosine-based activation (ITAM) signals for differentiation from monocytes into active osteoclasts (OCs). Endothelial OSCAR expression may induce monocyte adhesion increasing monocyte extravasation available for osteoclastogenesis in affected joints. Higher expression of OSCAR by endothelial and pre-OCs, as detected by immunohistochemistry, is present in active RA compared with OA and healthy synovial samples (Crotti ART 2012). Inflammation in RA may increase OSCAR expression as supported by increased monocyte (Herman ART 2012) and endothelial cell (Crotti ART 2012) OSCAR expression in response to TNF-alpha in vitro. OSCAR expression in early RA (less than 6 months post diagnosis) and cartilage pannus junction (CPJ) and non-CPJ samples from established RA has not been reported. Objectives To examine OSCAR expression in the synovium of early RA patients at diagnosis and 6 months following treatment. OSCAR was also assessed in the CPJ and non-CPJ tissues of patients with established RA. Methods Samples included synovial biopsies from 15 patients newly diagnosed for RA at 0 and 6 months. CPJ and non-CPJ samples from established RA joints were also assessed (n=14). Serial sections for both cohorts were stained for OSCAR, TRAP (to identify pre-OCs/OCs) and von Willebrand factor (to identify vessels). Positive cells were counted (TRAP) or scored semiquantitatively (OSCAR) in 6x 2mm 2 fields. A paired T test was used to detect significance. Results OSCAR was associated with the synovial lining cells and distributed sparsely in association with monocytes in the subsynovial tissue or lymphoid aggregates in both early and established RA. A subset of samples from both cohorts showed a very mild staining associated with the vasculature and lumen. In early RA there was no significant difference in OSCAR expression within lining, sublining and vessels (p=0.217, 0.622, 0.906 respectively) between 0 and 6 months. The expression of OSCAR in the sublining reduced in 10/15 patients by 6 months. Very few TRAP positive cells were detected at either time point. The CPJ and non-CPJ established RA cohort had higher expression of OSCAR than the early cohort. The number of TRAP positive cells was significantly higher in the CPJ versus non-CPJ (p=0.015, mean=88.5 vs 44). Conclusions OSCAR positive cells were observed in all synovial tissues, particularly in the lining, in both early RA and established RA. This has not been previously reported. Expression of OSCAR by sublining cells including OC precursors, possibly dendritic cells, macrophages and endothelial cells is consistent with previous reports. Low numbers of TRAP positive cells may relate to early disease and/or site of biopsy as distant from the actual CPJ due to limitations of arthroscopic technique. In established RA, higher numbers of TRAP and OSCAR positive cells were present in both CPJ and non-CPJ tissues. The reduced sublining OSCAR expression in the majority of patients following treatment suggests modulation of OSCAR by DMARDS and may implicate OSCAR as an inflammation and bone erosion predictive biomarker. Disclosure of Interest None declared

Background Transforming growth factor-β-inducible gene-h3 (βig-h3) is an ECM protein, which consists of integrin-interacting structures including four homologous fas-1 domains and an Arg-Gly-Asp (RGD) motif, is abundantly found within synovial tissues and fluid of rheumatoid arthritis (RA). In the pathogenesis of RA, βig-h3 intensifies inflammatory processes by facilitating adhesion and migration of fibroblast-like synoviocyte (FLS) via binding to αvβ3 integrin. Based on these functions, fragmented peptides of βig-h3 have been developed as therapeutic agents for chronic inflammatory arthritis. Expression of MMP-2 is highly upregulated within the invasive pannus and associated with radiographic erosions in RA. Therefore, therapeutic agents that are transformable via introducing cleavable linkage by locally enriched MMP-2 within the pannus tissue would enhance targetability and therapeutic efficacy on chronic inflammatory arthritis. Objectives To investigate whether MMP-2 cleavable peptide complex consisted of fas-1 domain and RGD peptide blocks the interaction between βig-h3 and resident cells and leads to the amelioration of inflammatory arthritis. Methods We designed βig-h3-derivatives, including the fourth fas-1 domain truncated for H1 and H2 sequences of mouse (MFK00) and MMP-2-cleavable peptide complex (MFK902). MMP-2 selectivity was examined by treatment with a series of proteases. MFK902 efficacy was determined by the adhesion and migration assay with NIH3T3 cells in vitro and collagen-induced arthritis (CIA) model in vivo. The mice were treated intraperitoneally with MFK902 at different dosages (1, 10, or 30 mg/kg). Results MFK902 was specifically cleaved by active MMP-2 in a concentration-dependent manner. Both MFK00 and MFK902 revealed a dose-dependent inhibition of βig-h3-mediated adhesion of fibroblasts and were similarly effective at a lower concentration (1 μM), compared with RGD peptide. In the migration assay, a very high concentration of RGD peptide (500 μM) reduced fibroblast migration by less than 50%. The lowest effective concentration of MFK00 for inhibition was 1 μM with the IC50 of 13.56 ± 1.28 μM. The IC50 of cell migration for MFK902 was 4.56 ± 0.25 μM with the degree of inhibition higher than 2 fold at 1 μM and higher than 5 fold at 20 μM. In in vivo assay with a murine CIA model, treatment with all dosages of MFK902 resulted in a slower onset of arthritis, followed by less steep increase of clinical arthritis index up to day 50 with significantly lower scores, compared with controls. On day 50, the incidence of arthritic paws for the control group was 94%, whereas those for 1, 10, and 30 mg/kg groups with MFK902 treatment were 54, 41, and 38%, respectively. MFK902 ameliorated histopathologic deterioration and reduced the expression of inflammatory mediators in parallel with improvement of clinical features. In addition, a favorable safety profile of MFK902 was demonstrated in vivo. Conclusions The present study reveals that the MMP-2-cleavable peptide complex, based on βig-h3 structure, was a potent and safe therapeutic agent for chronic inflammatory arthritis and provides a reliable evidence for the MMP-2-cleavable mechanism as a tissue-targeted strategy to treat rheumatoid arthritis. Disclosure of Interest None declared

Background Antibodies to cyclic citrullinated peptide (anti-CCP) are valuable immunological marker for early diagnostic of rheumatoid arthritis (RA). They are highly sensitive and highly specific in RA. In addition, anti-CCP used as prognostic marker of severe RA. Arthroscopy is a minimally invasive technique, safe and efficient for visualization and biopsy of synovia (S) in rheumatology. Changes and structure of S are different for stages of RA. Detailed study of S changes features depending on the RA clinical course is an actual problem in rheumatology, as it allows early diagnostic of the disease, clinical course prognosis and individual therapy prescription. Objectives to define a peculiarities of S macro-assessment on arthroscopy in RA patients depending on the blood level of anti-CCP Methods knee arthroscopy was performed in 33 patients with RA (diagnosis is verified according to the criteria ACR/EULAR, 2010). Patients were divided into two groups depending on the blood level of anti-CCP. The first group (“−” anti-CCP), with negative or low positive level, included 13 patients (40%) (anti-CCP level was ≤40 IU/ml). The second group (“+” anti-CCP) positive and highly positive - 20 patients (60%) (anti-CCP level was >40 IU/ml). The patients of the two groups were not statistically different for clinical and demographic indicators. All arthroscopy was performed in the same sterile operating room via arthroscope (Karl Storz GmbH, Tuttlingen, Germany) of 2.4 mm with an angle of 30°. Arthroscopy was performed in accordance with Helsinki Declaration. All chambers of the joint were inspected. The joint lavage for visualization was perfomed throughout the procedure, but minimized (usually it is about 500 to 1000 ml). All the arthroscopy procedure was recorded on video for the further evaluation of the macroscopic picture of the knee joint changes. Parameters of the changes were estimated: the type of S hyperplasia, S vascularization, atrophic changes in S, presence of pannus and fibrin, shape of S villi Results Indices of inflammatory and of villous S hyperplasia were equally often (62% and 76%, 69% and 75%, respectively) in the patients of the two examined groups. S hyperemia and expressed vascular pattern predominated in “+” anti-CCP group of the patients - 76% of cases, compared with the group “−” anti-CCP - 36% (χ 2 =4.17, p=0.04). Growth of S with clavate villi tended to increase in the patients of “+” anti-CCP group. Presence of pannus significantly prevalent in patients with “+” anti-CCP group (75%) when compared with “−” anti-CCP groups - 31% (χ 2 =4.63, p=0.03). The presence of fibrin tended to increase in incidence in patients of “+” anti-CCP, but it was not statistically significant. The percentage of patients with atrophic changes of S was practically the same in the compared groups. Conclusions RA patients with anti-CCP blood level of >40 IU/ml have S hyperemia with increased vascular pattern twice more often, and the presence of pannus on S arthroscopy was 2.5 times more often in them. The data shows the intensity of inflammation and angiogenesis in S of high anti-CCP blood level patients. This contributes to an aggressive course of RA in these patients with the rapid development of cartillage-bone destruction Disclosure of Interest None declared

While evidence implicates both the adaptive and innate immune system in rheumatoid arthritis (RA) pathogenesis, accumulating data indicate that the synovial tissue itself actively participates in the destructive inflammatory process. Specifically, resident fibroblast-like synoviocytes (FLS), together with macrophages, re-organise to form an aggressive cell mass, called pannus, which destroys the articular cartilage and the subchondral bone. The exact molecular mechanisms of synovial pannus formation, FLS expansion and invasion into adjacent tissues are not yet known. Our data strongly suggest that the T-cell derived cytokine IFNγ is involved in FLS-mediated joint destruction. Migration and invasion assays revealed increased migratory activity for IFNγ-stimulated FLS, when compared to unstimulated FLS. Further, biochemical studies showed that IFNγ promotes the migratory and invasive activity of FLS via Janus kinase 2 (JAK2) and the focal adhesion kinase (FAK), a kinase known to integrate focal adhesion turnover and thus, regulates cell migration. In detail, IFNγ stimulation of FLS distinctly resulted in the phosphorylation of FAK-Y925, a phospho-site that has recently been demonstrated to be required for FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated the IFNγ-induced activation of FAK. Correspondingly, baricitinib, a JAK inhibitor that is currently successfully probed in RA clinical trials, abrogated IFNγ-stimulated activation of FAK. In conclusion, our studies contribute insight into the synovial response to IFNγ and reveal JAK2 and FAK as potential targets for synoviocyte-mediated joint destruction in arthritis, especially in RA.

Rheumatoid arthritis (RA) is characterised by the presence of an inflammatory synovitis accompanied by destruction of joint cartilage and bone. Destruction of cartilage matrix results predominantly from the action of connective tissue proteinases released by RA synovial tissues, chondrocytes, and pannus tissue. Several lines of evidence in RA and in animal models of arthritis support a role for osteoclasts in the pathogenesis of bone erosions. RA synovial tissues produce a variety of cytokines and growth factors that may increase osteoclast formation, activity, and/or survival. These include interleukin 1alpha (IL1alpha) and beta, tumour necrosis factor alpha (TNFalpha), IL11, IL17, and macrophage colony stimulating factor (M-CSF). Receptor activator of NFkappaB ligand (RANKL) is an essential factor for osteoclast differentiation and also functions to augment T cell-dendritic cell cooperative interactions. CD4+ T cells and synovial fibroblasts derived from RA synovium are sources of RANKL. Furthermore, in collagen induced arthritis (CIA), blockade with osteoprotegerin (OPG), a decoy receptor for RANKL, results in protection from bone destruction. To further evaluate the role of osteoclasts in focal bone erosion in arthritis, arthritis was generated in the RANKL knockout mouse using a serum transfer model. Despite ongoing inflammation, the degree of bone erosion in arthritic RANKL knockout mice, as assessed by microcomputed tomography and correlated histopathological analysis, was dramatically reduced compared with that seen in arthritic control mice. Cartilage damage was present in both the arthritic RANKL knockout mice and in arthritic control littermates, with a trend toward milder cartilage damage in the RANKL knockout mice. This study supports the hypothesis that osteoclasts play an important part in the pathogenesis of focal bone erosion in arthritis, and reveals distinct mechanisms of cartilage destruction and bone erosion in this animal model of arthritis. Future directions for research in this area include the further investigation of a possible direct role for the RANKL/RANK/OPG system in cartilage metabolism, and the possible role of other cell types and cytokines in bone erosion in arthritis.

Rheumatoid arthritis can be considered as one of the family of 'angiogenesis dependent diseases'. Angiogenesis in rheumatoid arthritis is controlled by a variety of factors found in the synovial fluid and pannus tissue. Modulation of the angiogenic component of the disease may alter the pathogenesis of the condition, and subsequent cartilage and joint destruction, by reducing the area of the endothelium in the pannus and restricting pannus growth. Current therapeutic strategies exert, to varying extents, an inhibitory effect on the angiogenic process. In particular, the mode of action of the slow acting antirheumatic drugs may be due to their effect on the angiogenic response. The development of novel angiostatic treatments for chronic inflammatory joint disease may lead to a new therapeutic approach in controlling disease progression.

To identify the local factors in cartilage that are responsible for the induction of pannus invasion, a 14 day organ culture study in which rheumatoid synovium was grown in contact with cartilage pieces was carried out. Rheumatoid synovium preferentially extended over hyaluronidase treated cartilage pieces, but detached from untreated pieces. Rheumatoid synovium extended over hyaluronidase treated cartilage surfaces containing fibronectin more extensively than over surfaces treated with hyaluronidase only. Extension over hyaluronidase treated cartilage surfaces containing immune complexes was small. The adherence of synovial cells to hyaluronidase treated cartilage slices in vitro was specifically inhibited by the synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, which is the adhesive portion of the fibronectin molecule. Furthermore, synovial fibroblast-like cellular extension, morphologically similar to rheumatoid pannus, was observed in the organ culture experiments in which rheumatoid synovium grew over hyaluronidase treated cartilage surfaces containing fibronectin. Synovial tissue extension over fibronectin coated surfaces was inhibited when hyaluronic acid and chondroitin-4-sulphate, major components of cartilage proteoglycans, were present on the cartilage surface. These findings suggest that fibronectin present in the superficial region of cartilage potentiates rheumatoid synovial extension and proteoglycans and immune complexes inhibit rheumatoid synovial extension. It is likely that fibronectin deposited on the eroded surface of articular cartilage induces pannus formation in rheumatoid arthritis.

The cartilage-pannus junction has been studied in multiple sections from 23 rheumatoid joints. Changes suggesting a metaplastic reaction of the articular cartilage, termed transitional fibroblastic zone, were commonly found in hips and knees, but were rarely present in metatarsophalangeal joints, in which an invasive pannus with cartilage degradation in close association with inflammatory cells was seen. Thus when multiple sections from rheumatoid joints were examined a transitional fibroblastic zone was found in 1/15 (7%) sections from metatarsophalangeal joints compared with 29/57 (51%) and 15/48 (31%) sections from knee and hip joints respectively. In contrast, an invasive pannus occurred in 11/15 (73%) sections from metatarsophalangeal joints compared with 22/57 (39%) sections from knees and 19/48 (40%) sections from hips. These findings led to the suggestion that this pathological variation between different joints may explain the predominance of erosive change in small joints as compared with joint space narrowing with secondary osteoarthritis found in large joints in rheumatoid arthritis. Inappropriate comparisons between different joints may in part explain the variation in findings of previous histopathological studies.

Objectives:The eventual role of a disintegrin and a metalloproteinase 8 (ADAM8) in osteoclastogenesis was studied in erosive rheumatoid arthritis (RA) and in vitro.Methods:ADAM8 protein and mRNA expression was measured in RA pannus and synovitis and compared to osteoarthritic (OA) synovial membrane. Human monocytes were isolated and stimulated with proinflammatory cytokines and their ADAM8 expression and surface ADAM8 were measured. Human peripheral blood monocytes and RAW 264.7 mouse monocyte/macrophage cells were stimulated to osteclast like-cells, and their expression of ADAM8 and osteoclastic markers (calcitonin receptor, integrin β 3, cathepsin K, TRAP) were analysed. Transfection and small interfering RNA (siRNA) were used to assess the role of ADAM8 in formation of polykaryons.Results:Increased numbers of ADAM8 positive cells were shown particularly in the pannus-cartilage/bone junction close or adjoining to TRAP positive multinucleate cells under formation (60 (2)% in pannus, 47 (2)% in synovitis vs 10 (1)% in OA, pConclusions:ADAM8 may be involved in bone destruction in RA because it is upregulated in RA pannus adjacent to developing erosions and enhances maturation of osteoclast-like cells.

Background: Much interest has been shown recently in the pathogenic role of B cells in rheumatoid arthritis (RA) owing to the marked clinical responses to anti-CD20 treatment in RA. Case report: A patient with X linked agammaglobulinaemia (XLA) presented with an erosive symmetric polyarthritis with histological features of RA, including formation of a destructive pannus. Furthermore, the patient developed subcutaneous nodules that were histologically indistinguishable from rheumatoid nodules. Surprisingly, lymphocytic infiltrates in both the synovium and nodule consisted almost exclusively of CD8 + T cells. Discussion: Although some peculiar B cell subsets have been described in patients with XLA, no B cell subsets could be demonstrated in synovial tissue or the subcutaneous nodule. This case illustrates that classical RA can develop in the absence of mature B cells.

Objective: To investigate the intra-articular vascularisation of the synovial pannus in the knee of patients with rheumatoid arthritis (RA) with power Doppler ultrasonography (PDS) and an echo contrast agent and correlate the area under the time-intensity curves with the clinical findings and laboratory measures of disease activity. Method: Forty two patients with RA (31 women, 11 men) with history and signs of knee arthritis, classified according to a modified index of synovitis activity (active, moderately active, and inactive), were studied. Clinical and functional assessment (number of swollen joints, intensity of pain, general health—visual analogue scale, disability index—Health Assessment Questionnaire, Ritchie articular index) and a laboratory evaluation were made on all patients. Disease activity was evaluated using the disease activity score (DAS) and the chronic arthritis systemic index (CASI) for each patient. All patients were examined with conventional ultrasonography and PDS before injection of intravenous ultrasound contrast agent (Levovist). The quantitative estimation of the vascularisation of the synovial membrane was performed with time-intensity curves and calculation of the area under the curves. Results: The mean (SD) value of the area underlying time-intensity curves was 216.2 (33.4) in patients with active synovitis, 186.8 (25.8) in patients with moderately active synovitis, and 169.6 (20.6) in those with inactive synovitis. The mean value of the areas differed significantly between the patients with active and those with inactive synovitis (p

High resolution ultrasound (HRUS) was used to investigate the effects of tumour necrosis factor alpha (TNFalpha) blockade on pannus formation and vascularisation of small finger joints in patients with active rheumatoid arthritis (RA).Five patients with active RA were treated with etanercept, a soluble TNFalpha receptor protein, for one month. Before, during, and after treatment the patients were followed up by clinical rheumatological examination, determination of their subjective pain score, blood chemistry, and by HRUS of the second metacarpophalangeal (MCP) joint of the right hand.One month after treatment with etanercept, rheumatological examination showed a significant decrease in a modified single joint rheumatic disease activity index (from 2.9 (SD 0.2) to 1.2 (0.7); p0.05) in all patients. Moreover, a significant decrease in the general pain score (from 4.7 (0.4) to 1.8 (0.6); p0.05) and in C reactive protein (CRP) levels was seen (from 3.02 (0.9) to 0.24 (0.1); p0.05). Concordantly, HRUS showed a significant reduction in pannus vascularisation of the MCPII joints (from 23,602 (5339) to 2907 (1609) colour signals/region of interest, CS/ROI; p0.001). Pearson's correlation coefficient between the results obtained by HRUS and the clinical response was 0.85.HRUS is promising as an additional useful method in the assessment of RA activity, and probably also in monitoring therapeutic responses.

Background Endocan, previously called endothelial cell-specific molecule (ESM-1), has been studied as a pro-angiogenic factor in tumor tissues. Endocan binds CD11a/CD18 integrin (also called LFA-1 for Leukocyte Function-associated Antigen-1) on human leukocytes, consequently inhibiting its binding to ICAM-1 and preventing transendothelial migration. These physiological functions of endocan may also be involved in the angiogenesis of pannus in rheumatoid arthritic joints, which shows tumor-like growth. Here, we describe for the first time that endocan expression is increased in arthritic synovial tissues and that adiponectin is one of the important factors that increases endocan expression in synovial cells. Objectives This study was performed to evaluate whether endocan expression, which is known to be involved in tumor angiogenesis, is increased in rheumatoid arthritic tissues. In addition, we examined the contribution of adiponectin in the regulation of endocan expression in arthritic joints. Methods Arthritic synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were immunostained with antibodies to endocan and VEGF. Synovial cells and human umbilical vein endothelial cells (HUVEC) were cultured and stimulated with IL-1β and/or adiponectin. The mRNA and protein levels of endocan were measured by PCR and ELISA, respectively. Results Endocan expression was greatly increased in inflammatory sites of RA synovial tissues. In OA tissues, its expression was higher in tissues with moderate to severe inflammation than in those with mild inflammation. In vitro expression levels of endocan and VEGF in endothelial and synovial cells were differentially increased in response to IL-1β stimulation. Adiponectin was a more potent stimulator of endocan than was IL-1β at their respective physiological concentrations in synovial cells. Endocan silencing by siRNA transfection in synovial cells decreased in vitro cell migration and invasion. Conclusions Adiponectin is an important factor in the stimulation of endocan expression in synovial cells. Adiponectin-induced endocan expression in synovial cells may stimulate cell migration and invasion as well as angiogenesis in the pannus of arthritic joints. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3705

Background and Objectives Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by synovial inflammation and osteoclast (OC) mediated bone erosions. AlphaVbeta3 integrin (αvβ3) is highly expressed in OCs and αvβ3 blocking antibodies reduce bone resorption. We have shown that Cilengitide, a synthetic Arginine-Glycine-Asparagine peptide (RGD-peptide), effectively inhibits osteoclastogenesis in vitro and reduces clinical signs of arthritis in Collagen Induced Arthritis (CIA). We aimed to further characterise the effect of cilengitide in arthritis. Materials and Methods CIA was induced in 6-8 week old male DBA/1 mice by immunisation with bovine type II collagen (CII) at day 1 and boosting at day 21. For CIA prevention mice received 1.5 mg/kg cilengitide (n = 15) or placebo (n = 15) subcutaneously (s.c.), 5 days/week, starting 1 day prior to CIA induction until day 53. For CIA treatment mice with established arthritis were randomised and received 1.5 mg/kg (low dose) or 75mg/kg (high dose) cilengitide or placebo s.c. 5 days/week until day 59. Incidence and severity of arthritis was assessed by weekly clinical scoring of paw swelling and grip strength. In the end histological staining was performed on hind paws with haematoxylin and eosin (H&E) for quantification of pannus, tartrate-resistant acid phosphatase (TRAP) for detection of bone erosions and OCs, toluidine blue (TB) for determination of cartilage breakdown. Blood vessel formation was assessed with endomucin staining. Cellular composition of joint infiltrates was determined with immunohistochemistry with anti-CD3, CD45RO, F4/80 and Neutrophil 7/4 antibodies. Results In the preventive experiment, cilengitide significantly reduced incidence (92.8% vs. 40%) and severity of CIA. Histological examination revealed significantly reduced numbers of synovial OCs, extent of bone destruction, cartilage damage as well as reduction of inflammatory pannus formation. In addition, blood vessel density in pannus was significantly reduced. Similarly, in the treatment experiment, low and high dose cilengitide effectively inhibited progression of established arthritis as evidenced by amelioration of clinical disease activity scores and histopathology. Conclusions Our findings demonstrate that cilengitide potently prevents and treats experimental CIA, reducing osteoclast-mediated bone erosions and inflammation. Further investigation of its mechanism of action in arthritis, in respect to neovascularisation and osteoclast function, may optimise its effect and provide a novel therapeutic target in RA.

OBJECTIVES To investigate the potential of quantitative magnetic resonance imaging (MRI) to differentiate between therapeutically induced changes in inflammation and synovial proliferation in rheumatoid arthritis (RA) of the knee. METHODS MRI of the knee was performed on patients with RA before and one week after injection with corticosteroid (triamcinolone acetonide, TA group, n=9) and before, four, and 12 weeks after injection with yttrium-90 plus TA (TA+Y group, n=7). MRI scans were analysed by subjective visual grading by a trained observer and by computer aided quantitation for three features: synovial fluid volume, synovial pannus volume, and synovial enhancement after intravenous contrast agent. RESULTS All TA subjects improved clinically at one week but the effects of TA+Y were more variable. TA significantly reduced synovial enhancement and effusion volume, whereas TA+Y at 12 weeks tended to increase synovial enhancement and decrease pannus volume. Quantitative MRI values agreed well with subjective assessment of scans. Comparison of calculated change on MRI scan before and immediately after aspiration with actual volume aspirated showed high correlation ( r =0.96). CONCLUSIONS Quantitative MRI correlates with subjective visual assessment and, at least for synovial fluid, is accurate. MRI can differentiate actions of two therapeutic modalities on various pathological processes and is sensitive enough to detect change after one week. With the additional advantage of lack of observer bias, it will probably become a useful tool in the development and assessment of existing and novel treatments.

Objective—To better understand the characteristics of synoviocytes located in the rheumatoid arthritis (RA) pannus. Methods—One cell line, termed PSC, was cloned from RA pannus lesions. Phenotypic analysis was done by contrast microscopy, indirect immunostaining, and safranin O staining. Transcription of several protooncogenes and matrix degrading enzymes was evaluated. The expression of mRNA for collagen II was detected by in situ hybridisation. The ability of anchorage independent growth was assessed by soft agarose culture. Results—PSCs showed a high transcription of protooncogenes c-fos, c-myc and c-jun. They also expressed mRNA for matrix degrading enzymes, such as collagenase, cathepsin B, and cathepsin L. Anchorage independent growth assay demonstrated that PSCs formed colonies in soft agar culture. Phenotypic analysis showed that this fibroblast-like PSC was stained intensely with anti-vimentin and anti-fibroblast antibody. In situ reverse transcriptase assay showed that the cell line expressed type II collagen mRNA. Conclusion—Alternative fibroblast-like cells were identified in the pannus lesion of RA sharing properties of fibroblasts and chondrocytes. These findings suggest that this fibroblast-like cell derived from pannus lesions may contribute to the destruction of the cartilage in RA. (Ann Rheum Dis 1997;56:262‐267)

Background Untreated gout transitions from an acute intermittent arthritis to a chronic inflammatory arthritis indicating at some point the inflammation associated with gout does not abate; it is unclear when this inflammatory process starts. Objectives The aim of this study was to determine the percentage of patients with inter-critical gout who have chronic synovial-based inflammation as evidenced by synovial pannus on a contrast-enhanced MRI of their most involved joint and determine if the presence and/or severity correlates with their serum urate levels. Methods All patients in this prospective trial had inter-critical gout and received a 3T MRI with and without gadolinium of their index joint (i.e. the joint most often involved with acute attacks of gout). Each subject also had a plain radiograph of the index joint as well as a serum urate, highly sensitive (hs)-CRP, and creatinine obtained on the same day. The MRI and radiograph were read by two musculoskeletal radiologists in an independent and blinded fashion. The primary endpoint was to determine the correlation of serum urate levels with the presence and/or severity (previously utilized monoarthritis MRI grading scale of 1-6) of the synovial pannus on the index joint. The MRI and radiographs were also assessed for erosions, intraosseous tophi, soft-tissue tophi, soft tissue swelling, joint effusions, and bone marrow reactive changes (MRI only). Secondary endpoints included the correlation with the presence and/or severity of synovial pannus with hs-CRP and estimated glomerular filtration rate (eGFR). The other MRI and plain radiograph findings were also assessed for correlation with their serum urate level. Results 74 subjects were screened; 72 completed the protocol. 65/72 (90%) participants were males (50 Caucasian, 10 African-American, 9 Hispanic, 3 other) with an average age and disease duration of 56.4 years (range 28-78) and 10.1 years (range 0.5-37), respectively. 53/72 (74%) index joints were the first metatarsalphalangeal joint; the average number of attacks in the index joint was 11.4 (+/- 13.6 SD) and 21.8 (+/- 25.8 SD) total attacks in any joint. 39 (54.2%) of the patients were on urate lowering therapy; 15 (20.8%) and 7 (9.7%) were taking colchicine or a NSAID daily, respectively. 63/72 (87.5%; 95% CI of 5.9%) of the subjects had synovial pannus on their MRI with good inter-reader agreement between the two radiologists (kappa 0.74). The mean serum urate level was 7.93 (+/- 2.13 SD). There was no correlation with the presence (p=0.33 [t-test]) or severity (Spearman correlation coefficient 0.12 [p=0.34]) of synovial pannus and serum urate levels. There was also no correlation with the presence (p=0.32) or severity (p=0.30) of synovial pannus and hs-CRP or the presence of pannus and eGFR (p=0.62), but there was a correlation with the severity of pannus and eGFR (p=0.02). Finally, the serum urate levels did not correlate with the presence of any of the other findings on MRI or plain radiograph of the index joint. Conclusions The overwhelming majority of patients with inter-critical gout have evidence of occult and chronic synovial-based inflammation. However, the presence and severity of this chronic synovial-based inflammation does not appear to correlate with serum urate levels. Acknowledgements Support provided by Takeda Pharmaceuticals USA, Inc. Disclosure of Interest J. Carter Grant/research support from: Takeda Pharmaceuticals, USA, Inc, Speakers bureau: Takeda Pharmaceuticals, USA, Inc, M. Patelli: None Declared, S. Anderson: None Declared, N. Prakash: None Declared, R. Aydelott: None Declared, E. Rodriguez: None Declared, H. Bateman: None Declared, A. Sterrett: None Declared, J. Valeriano: None Declared, L. Ricca: None Declared

Background Involment of the cervical spine in patients with RA is particulary important for the injury of the neurogical structures. Objectives The main aim of the present study was to determine the predisposing factors in the development of lesions of the cervical spine by magnetic resonance imaging (MRI) in patients with rheumatoid arthritis. Methods The study was conducted on 21 patients with definite rheumatoid arthritis (ACR criteria, 2010). Disease duration, age of onset, disease activity for 28 joint indices score (DAS-28), serology (RF and ACPA) were recorded. All patients underwent an MRI examination of the cervical spine as well as hands and feet X-ray examination. Bone oedema, MRI bone erosions, presence of pannus anterior and posterior atlantodental intervals were identifided. The Simple Erosion Narrowing Score (SENS) was applied to assess the number of eroded joints and the number of narrowed joints for hands and feet. Results In 6/21 (28.6%) cases MRI found signs indicative of lesion of the cervical spine. Presence of erosions was detected in 6/21; bone oedema – in 4/21; pannus – in 3/21; atlantoaxial subluxatiuon - in 3/21 patients. In 15/21 (71.4%) patients (comparison group) cervical spine injuries were not detected. There were no relationships between the detection of lesions of the cervical spine and age of onset, disease activity at the time of examination, evaluation of cervical rotation, serology.Duration of RA (13.9±7.8) in the group of the patients with lesions of the cervical spine exceed the duration of the disease among patients of comparison group (7.8±5.8), but not significantly: p=0.09. The SENS score was significantly higher in patients with RA and damage to the cervical spine than in the comparison group (52.5±27.9 and 21.0±11.8 respectively; p=0,016). As well the number of eroded joints for hands and feet showed higher score compared with patients without identified of spine lesion (25.8±16.7 and 9.87±7.7 respectively; p=0,038). Conclusions Our results indicate that clinical criteria are not sufficient to suggest damage in the cervical spine in patients with RA. While changes in the joints of hands and feet revealed by X-ray, more the number of eroded joints, may be predictive factor for the development of lesions in the cervical region. Disclosure of Interest None Declared

Background Synovial angiogenesis is considered to be an important early step in the pathogenesis of rheumatoid arthritis (RA) and in the perpetuation of disease.1 Because angiogenesis and formation of new blood vessels is a component of pannus in RA, we hypothesized that the balance between angiopoietin-1(Ang1) and angiopoietin-2 (Ang2) is in favor of Ang2 (represented by Ang2/Ang1 ratio) in RA pannus. Objectives To evaluate the serum levels of Ang1, Ang2 as well as the significance of Ang2/Ang1 ratio in RA in relation to blood flow signals in total of 10 joints and RA activity parameters. In addition, we studied their relevance to predict the state of angiogenesis. Methods This study was conducted on 65 consecutive very early RA patients. Power Doppler ultrasonography (PDUS) was performed in a total of 10 joints: the bilateral elbows, wrists, metacarpophalangeal, knees, and ankles. The blood flow signals at various sites of the synovial membrane of each joint were scored on a 3- grade scaling system. The score at the site with the strongest finding in each joint was adopted as the score of the joint, and the total of the scores of the 10 joints was defined as the total signal score (TSS). On the same day, Serum variables including serum VEGF, Ang1, and Ang2 levels as well as clinical disease activity were evaluated. All measured parameters were reassessed after one month. Results Serum VEGF and serum Ang2 levels as well as Ang2/Ang1 ratio were significantly correlated with CRP and DAS28-CRP at baseline and one month after. While, serum Ang1 level did not correlate with the indices of disease activity. Additionally, serum VEGF and Ang2 levels and Ang2/Ang1 ratio were significantly correlated with TSS at baseline and after one month. Moreover, significant correlations were noted between serum VEGF and level of Ang2 and Ang2/Ang1 ratio both at baseline and one month later. Serum Ang1 level was significantly inversely correlated with Ang2/Ang1 ratio in contrast to serum Ang2 level which correlated significantly with Ang2/Ang1 ratio at baseline and after one month. Interestingly baseline serum VEGF level was significantly correlated with TSS after one month. Also, baseline Ang2/Ang1 ratio was significantly correlated with TSS after one month. Conclusions Serum concentrations of VEGF and Ang2 as well as Ang2/Ang1 ratio were correlated with parameters of inflammation in early RA. Our results demonstrate that the elevated serum VEGF and Ang2/Ang1 ratio (in favor of Ang2) reflect a phase of vigorous angiogenesis. References Taylor PC. Serum vascular markers and vascular imaging in assessment of rheumatoid arthritis disease activity and response to therapy. Rheumatology 2005;44:721–728 Disclosure of Interest None Declared

Background Synovial neoangiogenesis is a key process in rheumatoid arthritis, contributing to pannus development and maintenance 1 . Anti-angiogenesis treatments targeting the VEGF/VEGF-R system ameliorate arthritis in several animal models 1,2 . Here we report the results of active immunization against VEGF on mice collagen induced arthritis (CIA). Mice were immunized with the VEGF kinoid, a construct of murine VEGF and a carrier protein, the keyhole limpet hemocyanine (KLH), in order to induce a polyclonal anti-VEGF immune response and reduce synovial angiogenesis. Objectives To determine: 1) the anti-VEGF immune response elicited by VEGF-K 2) the effect of VEGF-K on mice CIA. Methods Forty DBA/1 male mice received 4 intramuscular injections of one of the following: VEGF-K (30-30-5-5μg; 20 mice), KLH (15-15-2.5-2.5μg; 10mice), and PBS (10 mice) as an emulsion with ISA51vg. Three injections were performed before, one after collagen administration. Arthritides were induced by two subcutaneous injections of bovine type II collagen, the first at day 0 in complete Freund adjuvant, the second at day 21 in incomplete Freund adjuvant. All the animals were monitored for clinical scores of arthritis and were sacrificed at day 61. Right forepaws and hind paws were kept for histological analysis. Anti-VEGF antibody (Ab) titers were determined by ELISA. Results All VEGF-K treated mice produced anti-VEGF Ab. The VEGF group had lower mean clinical scores of arthritis vs. PBS and KLH groups and significantly lower scores of synovial inflammation (p Conclusions Angiogenesis targeting with active anti-VEGF immunization in CIA results in anti-VEGF Ab production and reduction of synovial inflammation and destruction. References Blood vessels, a potential therapeutic target in rheumatoid arthritis? Semerano L, Clavel G, Assier E, Denys A, Boissier MC. Joint Bone Spine. 2011 Mar;78(2):118-23. Peptides targeting inflamed synovial vasculature attenuate autoimmune arthritis. Yang YH, Rajaiah R, Ruoslahti E, Moudgil KD. Proc Natl Acad Sci U S A. 2011 Aug 2;108(31):12857-62. Disclosure of Interest L. Semerano: None Declared, E. Assier: None Declared, E. Duvallet: None Declared, A. Denys: None Declared, E. Adam Employee of: Neovacs S.A., E. Bernier Employee of: Neovacs S.A., G. Grouard-Vogel Employee of: Neovacs S.A., M.-C. Boissier Grant/Research support from: Neovacs S.A., Consultant for: Neovacs S.A.

Background Recent studies highlight the role of the EP4 prostanoid receptor in promoting autoimmune inflammation via T helper cells, namely Th1 and Th17. Objectives Aim of the present studies was to test CR6086, a selective antagonist at the EP4 receptor, for a potential activity in a widely recognized animal model for inflammatory arthritis. The Collagen Induced Arthritis (CIA) model in the Lewis rat was chosen. Methods The effect of CR6086 was studied in a dose-response paradigm, and in comparison to the Jak3 inhibitor DMARD Tofacitinib and to naproxen. For each experiment, 60 Lewis male rats were immunized by intradermal injection at the base of the tail of an emulsion containing 150 ug bovine collagen type II in complete Freund’s adjuvant (CFA), or were injected with incomplete Freund’s adjuvant (sham group). Three weeks after the first immunization, the same animals were boosted with the same method. Three days after booster injection, arthritis had developed in all animals, but not in the sham-injected; edema was assessed and the animals were then randomized and assigned to treatment groups (n=12). Treatments were thus started in a true curative protocol, after genuine arthritis had already developed, and were performed for 14 days. All compounds were administered orally. The doses of CR6086 were 1.5 mg/kg/b.i.d. and 15 mg/kg/b.i.d., or 15 mg/kg/o.a.d. Edema measurement was performed again after 7 and 14 days of treatment, and then all animals were blindly scored for clinical signs of arthritis (redness, swelling, pain of hindlimb joints) and sacrificed. Hind paws were explanted and processed for histology. At least three non-consecutive sections for each paw were blindly scored at the level of tarsus, metatarsus and calcaneus for the following histological features: edema; synovial hyperplasia (pannus); inflammatory cell infiltrate; cartilage damage; bone erosion; periosteal osteogenesis. Statistical analysis was performed with ANOVA followed by Dunn’s or Holm-Sidak tests comparing all treatment groups vs. vehicle. Results After 7 days of treatment, edema was significantly reduced in CR6086-treated groups at both doses, compared to vehicle. After 14 days of treatment, CR6086 15 mg/kg/b.i.d. completely abated the edema, while CR6086 1.5 mg/kg/b.i.d. significantly reduced the edema by over 60%. In all experiments, administration of CR6086 was associated with a profound, dose-dependent decrease of the arthritis clinical score compared to vehicle. All histological features of arthritis were significantly and dose-dependently ameliorated following treatment with CR6086. In later experiments, we observed that a dose of 15 mg/kg o.a.d. of CR6086 was sufficient to ensure maximum efficacy. Tofacitinib also showed dose-dependent efficacy in the CIA model, however with lower potency and a need for b.i.d. administration, likely due to the PK profile of the compound. Naproxen was active on inflammatory parameters, at the same doses as CR6086. Conclusions In conclusion, in a widely accepted animal model for inflammatory arthritis, in a curative protocol CR6086 demonstrated very good efficacy in all parameters examined, including edema, clinical arthritis score, and histological features. Disclosure of Interest None Declared

Background The expansion of synovial epithelium in Rheumatoid Arthritis (RA), and the subsequent pannus invasion of underlying cartilage and bone, needs an increase in the vascular supply to the synovium. Angiogenesis is recognised as a key event in the formation and maintenance of the pannus in RA. The pro-angiogenic cytokine, vascular endothelial growth factor (VEGF), has been demonstrated to have a central involvement in the angiogenic process in RA. Objectives We evaluated gene expression levels of a set of genes associated with angiogenesis in Synovial Fluid (SF) from RA patients compared with osteoarthritis patients. Methods Knee SF samples were classified in two groups, Group I: RA with DAS 28 score > 5.1, high disease activity (n=10, 7F/3M, age: 56,3 ± 20,9, range: 17-84) and Group II: Osteoarthritis (OA, n=18, 13F/5M, age: 70,5 ± 6,6, range: 58-86). Levels of gene expression of three genes previously associated with angiogenesis, including pro-angiogenic factors: VEGF and Angiopoietin 1 (ANGPT-1) and anti-angiogenic factor, Thrombospondin I (TSP1) were evaluated using Quantitative Real Time PCR (QPCR). All amplifications were carried out in duplicate and threshold cycle (C t ) scores were averaged for calculations of relative expression values. The C t scores were normalized against C t scores by subtracting the corresponding β2Microglobuline (β2M) control, or ΔCt=C t,gene - C t,B2M . To test for differential gene expression between groups, a two sample t-test was performed to compare the ΔCt in the two groups. Results Gene expression level is showed in table 1. Conclusions In the present cross-sectional study, increased levels of VEGF and decreased levels of TSP-1 were observed in RA patients. These gene expression results might be associated with increased angiogenesis in RA patients with high disease activity. References Paleolog EM. Angiogenesis in rheumatoid arthritis. Supplement Review. Arthritis Res 2002, 4 (3): S81-S90. Afuwape AO et al. The role of the angiogenic molecule VEGF in the pathogenesis of rheumatoid arthritis. Histol Histopathol 2002, 17: 961-972. Disclosure of Interest None Declared

Chronic synovitis in rheumatoid arthritis results in the formation of pannus that invades the joint cartilage and the underlying bone. Matrix metalloproteinases (MMPs) can degrade cartilage, bone, and connective tissue matrix.1 Of these matrix degrading enzymes, MMP-1 is the most important and is present in the synovial lining of patients with rheumatoid arthritis (RA).2 In synovial tissue, the level of tissue inhibitor of MMP (TIMP) is reduced, tilting the balance towards tissue destruction. The effect of soluble tumour necrosis factor receptor (sTNFr) on these mediators is not known, because the only study available had pooled data of patients treated with sTNFr alone and in combination with methotrexate.3 Angiogenesis is central to the maintenance of pannus and is controlled by many factors. Identification of vascular endothelial growth factor (VEGF) as the major angiogenic factor, demonstration of its presence in …

Background In rheumatoid arthritis (RA), progressive joint destruction is a hallmark of disease and results from both increased bone resorption and the lack of repair mechanisms. tumour necrosis factor α (TNFα) contributes to both aspects of pathologic joint remodelling by increasing the number of bone-resorbing osteoclasts and decreasing the number of bone-forming osteoblasts. Sclerostin is a potent inhibitor of osteoblast development by antagonising the Wnt/β-catenin signalling pathway. Based on recent data that have shown increased expression of sclerostin under inflammatory conditions, the authors studied its expression in human RA and in human TNF transgenic (hTNFtg) mice, which develop a RA-like destructive arthritis. Moreover, the authors analysed the effects of sclerostin deficiency on the development and severity of the arthritis in these mice. Methods Expression of sclerostin was determined by immunohistochemistry, western blot and reverse transcriptase PCR. To assess the functional role of sclerostin in vivo, sclerostin knockout ( SOST −/− ) mice were crossed with hTNFtg mice. In addition to determining the clinical severity of disease in SOST −/− /hTNFtg and hTNFtg mice, histological changes including bone erosion, cartilage destruction and inflammation were evaluated by histomorphometric analyses. The number of osteoclasts was quantified using tartrate-resistant acid phosphatase staining. Immunohistochemistry was performed to analyse the expression of molecules of the Wnt pathway. Results Immunohistochemistry and western blot analyses revealed a strong overexpression of sclerostin in synovial tissue of RA compared to osteoarthritis patients. Likewise, ankle joints of hTNFtg mice showed high levels of sclerostin, especially in the infiltrating pannus, whereas only negligible staining was observed in wild-type animals. In vitro, expression of sclerostin was only found in osteoblasts and osteoclasts, but could be induced in RA synovial fibroblasts by TNFα. Surprisingly, the lack of sclerostin not only increased the clinical severity of arthritis in hTNFtg mice but most dramatically accelerated joint damage in this mouse model of RA. SOST −/− /hTNFtg mice displayed significant higher bone erosion, synovial hyperplasia and osteoclast numbers compared to hTNFtg mice. Moreover, immunohistochemistry revealed higher levels of dickkopf 1 (DKK-1) and Wnt-5a in joints of SOST-deficient hTNFtg mice. Conclusions The authors hypothesise that under inflammatory conditions, higher levels of DKK-1 and Wnt-5a in joints of sclerostin-deficient arthritic mice counteract the beneficial effect of sclerostin deficiency by increasing osteoclast development through enhanced blockade of the Wnt-3a pathway by DKK-1 as well as by promoting synovial hyperplasia through Wnt-5a-mediated synoviocyte activation. These results may have an impact on the use of sclerostin inhibitors in inflammatory joint diseases.

Background and objectives After antigenic stimulation, T helper lymphocytes have to expand clonally in order to generate a large pool of specific effector cells. This process is positively regulated by the interleukin-2 induced microRNA-182 (miR-182), which inhibits the expression of the forkhead transcription factor Foxo1, a suppressor of T helper cell proliferation. Specific inhibition of miR-182 in T helper cells limits their expansion in vitro and in vivo. Therefore, the authors examined whether specific inhibition of miR-182 by antagomirs affects the ability of T helper cells to induce inflammation in a mouse model of OVA-induced arthritis. Material and methods The authors treated OVA-specific T helper lymphocytes from DO11.10 mice with antagomir-182 ex vivo and transferred them adoptively into Balb/c mice. One day after transfer the authors immunised with OVA. On day 14 after transfer, the authors injected OVA into the left knee joint and assessed knee swelling as a sign of inflammation. Knee joints were processed for histological examination. Results Knee swelling was about 40% less after transfer of antagomir-182-treated T helper cells. Knockdown of miR-182 with antagomirs resulted in significantly lower histological scores for inflammation and tissue destruction. Infiltration of granulocytes and mononuclear cells into the inflamed tissue of the knee joint, as well as bone and cartilage destruction and the formation of pannus tissues, were lower in the mice given antagomir-182-treated cells than in the control group. Conclusions The results showing less disease severity in a transfer model of OVA-induced arthritis open a new therapeutic avenue for the control of unwanted T helper cell expansion in immune-mediated diseases by antagomirs.

Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease. In RA, a systemic autoimmune response translates into an inflammatory attack on the synovium that yields the formation of an aggressive cell mass called pannus which attaches to, encroaches over and destroys the articular cartilage. Cartilage destruction is mediated by fibroblast-like synoviocytes (FLS) which are the prevailing cell type of the …

Cytokines play a key role in the development of rheumatoid arthritis (RA), allowing hypertrophy of the pannus by neovascularisation, articular inflammation and destruction. We recently demonstrated in experimental models the efficacy of vaccination against cytokines, using a heterocomplex of keyhole limpet hemocyanin (KLH) and human tumour necrosis factor (TNF), called TNF kinoid (K). Vascular endothelial growth factor (VEGF) is a …

To compare the macroscopic and microscopic characteristics of synovial tissue in rheumatoid arthritis (RA), spondyloarthropathy (SpA), and osteoarthritis (OA) after exclusion of possible biases induced by disease duration or activity, or both.Synovial biopsy specimens were obtained by needle arthroscopy in patients with early RA (n=16), late RA (n=14), early SpA (n=23), and OA (n=12). Macroscopic and microscopic features were scored on a four point scale and analysed as a function of disease duration (early versus late RA), local and systemic disease activity, and diagnosis.Except for the maximal synovial lining thickness, no significant differences were seen between early and late RA. For disease activity, synovial histology was only weakly correlated with C reactive protein in RA, but seemed to be strongly dependent on effusion of the biopsied joint in all disease groups. After stratification for local disease activity, no disease related differences were found in patients without joint effusion. In contrast, important differences were found between patients with RA and SpA with active joint effusion. Synovial vascularity was macroscopically increased in SpA versus RA (p=0.017). A straight vessel pattern was only seen in RA, while tortuous vessels were preferentially seen in SpA. Vascularity was also microscopically increased in SpA compared with RA (p=0.031), and correlated with the macroscopic vascularity (r(s)=0.36, p=0.036). CD3+ (p=0.008), CD4+ (p=0.008), and CD20+ (p=0.024) lymphocytes were overrepresented in RA compared with SpA. The integrin expression in RA was characterised by a decrease of alphaVbeta3 in the synovial lining (p=0.006) and an increase of alphaVbeta5 in the sublining (p0.001).The immune architecture of the synovial membrane is more dependent on local disease activity than on disease duration. Synovium obtained from clinically affected joints shows important histological differences between RA and SpA.

The presence of fibronectin (Fn) in rheumatoid pannus was investigated by an immunoelectron microscopic method using horseradish peroxidase-conjugated antibody to stain Fn. Large amounts of Fn were demonstrated in the interstitial collagenous tissue of invasive rheumatoid pannus. Fn was also observed on the surface of both fibroblast-like and macrophage-like pannus cells, suggesting that it was secreted by these cells and that the secreted Fn might facilitate the spreading of the pannus cells over the cartilage matrix. However, decreased amounts of Fn were observed in areas of contact between pannus cells and the cartilage matrix. The reduction in the amount of Fn demonstrable at the cartilage-pannus junction may be a consequence either of proteolysis of Fn by enzymes secreted at the junction, inhibition of Fn synthesis in cells in contact with cartilage matrix, or transfer of Fn from the pannus cell surface to collagen of the cartilage matrix.

Cervical myelopathy is a rare but potentially dangerous complication of rheumatoid arthritis and presents considerable therapeutic problems. A conservative approach carries high mortality and surgical intervention is not without serious risks. Reduction of subluxation and posterior fusion is widely practised but may require prolonged bed rest and continuous skull traction, sometimes for many weeks. When anterior decompression has been attempted prolonged immobilisation and external fixation have created problems. In this series 23 rheumatoid patients with cervical myelopathy were investigated over a four-year period. Seventeen underwent anterior decompression of the cervical cord, of whom 14 had a transoral removal of the odontoid peg and pannus and posterior occipitocervical fusion during the same anaesthetic without mortality or serious postoperative complications; all but one have improved. The authors believe that early mobilisation after a combined cord decompression and internal fixation has reduced the mortality and morbidity. Management of cervical myelopathy in rheumatoid arthritis and indications for operation are discussed.

Introduction I. Structure of articular cartilage Microscopical appearance and organization of articular cartilage surfaces Scanning electron microscope study of normal human articular cartilage Sequential changes in superficial defects in articular cartilage studied with the scanning electron microscope Cartilage fibrillation versus ink-staining patterns: Scanning electron microscope study Stain distribution with alcian blue in articular cartilage I. Short communications Some new morphological data on histogenesis of articular cartilage Matrix structure in articular cartilage Three-dimensional observations of articular cartilage matrix Ultrastructural aspects of normal and osteoarthrotic cartilage Scanning electron microscopy of human osteoarthrotic cartilage II. Chemistry of the matrix Structure and stability of proteoglycan aggregates Structure of cartilage proteoglycans Interaction of cartilage collagen fibrils and proteoglycans: Application of fluorescent labelling techniques for assay of enzymes degrading cartilage Chain composition of pepsin-solubilized collagen from normal bovine and osteoarthrotic human articular cartilage Passive agglutination method using collagen-coated tanned sheep erythrocytes to demonstrate collagen-glycosaminoglycan interaction II. Short communications Procollagen of chick xyphoid cartilage Nature and location of crosslinks in cartilage collagen Discontinuous nature of the protein-keratan sulphate core of cartilage proteoglycan Concentration, distribution, and metabolism of glycosaminoglycans in different layers of normal human articular cartilage Variations in glycosaminoglycans with surface site in human knee joint cartilage Charged group behaviour in cartilage proteoglycans in relation to pH Equilibria of calcium and phosphate ions in human articular cartilage III. Metabolism Nucleotide sugar metabolism in glycosaminoglycan biosynthesis Biosynthesis of proteoglycans in cartilage Cartilage turnover III. Short communications Effect of in vitro incubation on distribution and synthesis of glycosaminoglycans of articular cartilage Effect of antiproteoglycan serum and Hyaluronidase on metabolic functions of calf cartilage cells in vitro Overlabelling of chondroitin sulphate (ChS) chains after application of specific inhibitors of their biosynthesis Synthesis of cerebroside sulphatides in human articular cartilage Circadian rhythms in the excretion of urinary acid glycosaminoglycans IV. Enzymes and degradation Enzymes involved in degradation of cartilage in osteoarthrosis Breakdown of protein-polysaccharide complexes by cartilage proteases The possible function of lysozyme in cartilage metabolism IV. Short communications Evidence for secretion of a proteolytic enzyme from rheumatoid pannus and synovium Action of rheumatoid synovial collagenase on cartilage collagen Mechanism of cartilage and synovial fluid hyaluronate degradation by polysaccharidases Response of articular cartilage in organ culture to growth hormone Release and degradation of sulphated proteoglycans and collagen from the cell layer of human chondrocytes Studies on cartilage vascularization V.A. Functional properties Fluid transport in cartilage Fundamental fluid transport mechanisms through articular cartilage An approximate equation for weeping lubrication, solved with an electrical analogue Purification and properties of articular lubricant An overall view of synovial joint lubrication V.A. Short communications Proteins associated with hyaluronic acid Formation of lubricating monolayers at the cartilage surface Lubrication defect of immobilized connective tissue matrices: Study of the biomechanical and biochemical characteristics of normal and immobilized rabbit knee V.B. Functional properties Some aspects of the mechanical behaviour of articular cartilage In vitro fatigue testing of articular cartilage Mechanical properties of articular cartilage and their relationship to matrix degradation and age Contact pressures in the loaded cadaver human hip V.B. Short communications A simple mechanical model of articular cartilage A new approach to the determination of the elastic modulus of articular cartilage Wear properties of articular cartilage Load bearing function of the menisci of the knee joint VI. Pathogenesis of osteoarthrosis Pathogenesis of osteoarthrosis: An hypothesis Articular cartilage lesions in the Liverpool population Contribution to the study of the initial stage of cartilage degeneration Bone density in osteoarthrosis Mechanical factors in the aetiology of osteoarthrosis VI. Short communications Histological changes in osteoarthrotic femoral head cartilage Cartilage of the patella and its degeneration: Topographical variation of the glycosaminoglycan content Ultrastructural and histological studies on articular cartilage in experimental osteoarthrosis Proteoglycan aggregation in early experimental osteoarthrosis Experimentally-induced osteoarthrosis in the dog: Collagen biosynthesis in control and fibrillated articular cartilage Experimental evidence for an injury threshold for articular cartilage Early loss of glycosaminoglycans in experimental haemarthrosis in dogs Osteocyte death in rheumatic diseases Differentiation and maintenance of articular (secondary) cartilage on avian membrane bones Immunocytochemical and historical studies of a reversible transformation of chondrocytes in degraded cartilage matrix to a fibroblast-like morphology Conclusions of a working party of morbid anatomists Bibliography

Erosions on the triquetrum and pisiform are frequent in early rheumatoid arthritis and occur characteristically at 3 sites. (a) A shallow erosion on the proximal medial triquetral "bare" area is related to synovial proliferation at the margin of the radiocarpal cavity, although compressive forces from an adjacent long ulnar styloid may be contributory; (b) an erosive abnormality on the distal medial triquetrum is related to synovial proliferation along the margin of the midcarpal joint; and (c) ring-like erosions on apposing surfaces of the triquetrum and pisiform are produced by pannus within the pisiform-triquetral compartment.

The early changes (five weeks) in the structure of newly synthesised and endogenous articular cartilage sulphated proteoglycans were studied in lapine IgG induced experimental immune synovitis. Rabbits with immune synovitis (IS-IgG) were compared with animals with a developed hypersensitivity to IgG (I-IgG) and with non-treated normal weight matched controls. Medial and lateral femoral condyle and tibial plateau cartilage was pooled and radiolabelled for 24 h in vitro with 35SO4. The samples constituted tissue from regions underlying pannus and from pannus free sites. Cartilage from animals with IS-IgG showed a significantly diminished amount of newly synthesised and endogenous proteoglycan aggregate and an increased amount of hydrodynamically small proteoglycans. Newly synthesised (obtained by in vivo radiosulphate labelling) and endogenous proteoglycans showed a similar profile. The proteoglycan monomer fraction from animals with IS-IgG failed to form proteoglycan aggregates in the presence of excess hyaluronic acid. In the group with IS-IgG linear regression analysis showed a statistically significant relationship between the synovial pathology scores (but not cartilage pathology score) and diminished newly synthesised and endogenous proteoglycan aggregate.

The inflammatory destruction of cartilage in rat adjuvant arthritis has been studied by histochemistry and autoradiography. Naphthol-AS-D-chloroacetate esterase has been used as a marker for polymorphs. The evidence presented here shows that polymorphs accumulate at the cartilage-pannus border and in areas of cartilage loss. These cells appear therefore to be of decisive importance for the destruction of cartilage. Proteoglycans were demonstrated by safranin-O staining: there is a loss of PG that is particularly prominent in zones where pannus had invaded cartilage. By means of 35S labelling of proteoglycans it was possible to show that pannus containing polymorphs can invade living cartilage.

Hyaline cartilage was obtained from patients undergoing synovectomy of the knee joint for rheumatoid arthritis. Eroded cartilage from beneath the invading pannus and relatively normal cartilage from the same joint were maintained in organ culture for three days. During the first 48 hours in culture the explants were exposed to 35SO4 in the medium. The equivalent layers of normal and eroded cartilage were analysed for DNA uronic acid and 35SO4 incorporation. There was a decrease in the DNA and uronic acid of the eroded cartilage, although only the latter reached statistical significance. The uptake of radioactive sulphate was significantly greater in explants taken from the eroded site than from normal areas. This increase in metabolic activity could well be a protective phenomenon.