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Facet joint osteoarthritis may be a cause of low back pain in degenerative spine diseases including lumbar spinal stenosis. Subchondral bone is regarded as a potential therapeutic target for osteoarthritis treatment. The goal of this study was to characterize subchondral bone histopathology in osteoarthritic facet joints from lumbar spinal stenosis patients. Fifteen patients with degenerative spinal stenosis scheduled for transforaminal lumbar interbody fusion surgery were recruited for this study. Osteoarthritis severity was graded on T1- and T2-weighted MRI images using Weishaupt scoring system. Dissected osteoarthritic facet joints were subjected to histological and immunohistochemistry analyses to study relative abundance of osteoblast, osteoclasts, and macrophages using van Gieson's, tartrate-resistant acid phosphatase and CD68-antibody staining, respectively. Presence of nerve fibers was evaluated by PGP9.5-antibody staining. Differential bone histopathology, independent from radiological osteoarthritis grade, was observed in facet joints. Extensive de novo bone formation was found in subchondral bone tissues of eight of fifteen specimens. Regions of bone formation showed high abundance of blood vessels and CD68-positive macrophages, but were devoid of multinucleated osteoclasts. Additional pathological changes in subchondral marrow spaces, including inflammatory infiltration and enhanced osteoclast activity, were characterized by macrophage-rich tissues. PGP9.5-positive nerve fibers were detected near arterioles, but not in regions displaying bone pathology. Individual histopathological parameters did not associate with clinical features or radiological osteoarthritis severity. Subchondral bone histopathology of facet joint osteoarthritis in lumbar spinal stenosis is characterized by marrow infiltration by macrophage-rich tissues and enhanced de novo bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1475-1480, 2016.

Subchondral osteosclerosis, characterized by an increase of hypomineralized bone material, is a pathological hallmark of osteoarthritis. The cellular components in the subchondral marrow compartment that participate in this aberrant bone remodeling process remain to be elucidated. This study assessed the presence of marrow inflammatory cells and their relative abundance between nonsclerotic and sclerotic tissues in knee osteoarthritis. Bone samples from osteoarthritic knee tibial plateaus were stratified for histological analyses using computed tomography osteoabsorptiometry. Immunohistological analysis revealed the presence of CD20 (B-lymphocyte) and CD68 (macrophage), but not CD3 (T-lymphocyte) immunoreactive mononuclear cells in subchondral marrow tissues and their relative abundance was significantly increased in sclerotic compared with nonsclerotic bone samples. Multinucleated osteoclasts that stained positive for CD68 and tartrate-resistant acid phosphatase, predominantly associated with CD34-positive blood vessels and their abundance was strongly increased in sclerotic samples. Bone-specific alkaline phosphatase activity in outgrowth osteoblasts was induced by conditioned medium from nonsclerotic, but not sclerotic, bone pieces. These results suggest that an interaction between bone-resident cells and marrow inflammatory cells might play a role in aberrant bone remodeling leading to subchondral osteosclerosis. Elevated osteoclast activity in sclerotic bone suggests that bone formation and resorption activities are increased, yet uncoupled, in human knee osteoarthritis.

Multiple sclerosis (MS) is a debilitating disease characterized by demyelination of the central nervous system (CNS), resulting in widespread formation of white matter lesions (WMLs) and grey matter lesions (GMLs). WMLs are pathologically characterized by the presence of immune cells that infiltrate the CNS, whereas these immune cells are barely present in GMLs. This striking pathological difference between WMLs and GMLs raises questions about the underlying mechanism. It is known that infiltrating leukocytes contribute to the generation of WMLs; however, since GMLs show a paucity of infiltrating immune cells, their importance in GML formation remains to be determined. Here, we review pathological characteristics of WMLs and GMLs, and suggest some possible explanations for the observed pathological differences. In our view, cellular and molecular characteristics of WM and GM, and local differences within WMLs and GMLs (in particular, in glial cell populations and the molecules they express), determine the pathway to demyelination. Further understanding of GML pathogenesis, considered to contribute to chronic MS, may have a direct impact on the development of novel therapeutic targets to counteract this progressive neurological disorder.

s / Osteoarthritis and Cartilage 24 (2016) S63eS534 S133 transcribed into cDNA and changes of expression levels of TGF-beta1-3 were analysed by quantitative Real-Time PCR using sequence-specific primers and probes (TaqMan 50-nuclease assays). Results: The WST-1 experiments showed no influence of CoCr particles on the viability of MG63 cells. Cobalt-chloride diminished the MG63 viability only at high concentrations >250 mM. The MG63 cells express all three TGF-beta isoforms, with TGF-beta1 as most abundently expressed form. A dose dependent reduction of all TGF-beta isoforms by Co(2þ) ions was observed. The strongest effect was found for TGF-beta2, showing a reduction to 16.6± 4.4%, 33.3± 10.9%, 49.5± 15.7%, 61.0± 13.0% (mean± SD) compared to unstimulated control for 250 mM, 100 mm, 50 mM, and 10 mM, resp. Similar effects were seen for TGF-beta3 with a reduction to 32.8± 5.0%, 40.4± 6.9%, 54.8± 5.0%, and 75.0± 6.0% vs. control. The gene expression level of TGF-beta1 was less affected, only for the highest concentration of 250 mM a reduction to mean 73.3± 1 2.9% was observed. CoCr wear debris particles did not influence the expression of TGFbeta1 in the concentration range of 1 x E5 1 x E7. However, stimulation of osteoblast-like MG63 cells with 1 x E6 and 1 x E7 particles resulted in the decrease of gene expression of the isoforms 2 and 3 by approximately 50%. Conclusions: The results of our study indicate that Co(2þ) ions and to a lesser degree CoCr wear debris particles affect the expression of the different TGF-beta isoforms in osteoblast-like cells. Interestingly, not only the well studied isoform 1 is affected, but also the less studied forms 2 and 3. These data clearly indicate the need for further detailed studies of these isoforms 2 and 3. 210 SUBCHONDRAL BONE MESENCHYMAL STROMAL CELLS FROM OSTEOARTHRITIC LESIONS GIVE RISE TO ABERRANT IN VITRO AND IN VIVO MINERALIZATION D. Bianco y, A. Todorov, Jr. y, T. Cengi c yz, G.I. Pagenstert y, T. Hugle y, C. Forster-Horvath y, I. Martin y, S. Sch€ aren y, J. Geurts y. yUniv. Hosp. Basel, Basel, Switzerland; zUniv. of Zagreb, Zagreb, Croatia Purpose: The subchondral bone tissue plays a key role in the onset and progression of osteoarthritis and alterations of the bony bed might precede cartilage lesions. Subchondral bone remodeling in osteoarthritis results in osteosclerosis, characterized by an increase of bone material that is hypomineralized. Whereas it is generally accepted that abnormal mineralization is caused by a dysregulated osteoblast phenotype, it is unknown when and how this phenotype is acquired. Here, we investigated the in vitro and in vivo osteogenic differentiation potential of subchondral bone-resident mesenchymal stromal cells/ osteoprogenitors from nonsclerotic and sclerotic osteoarthritic bone specimens. Methods: Five knee tibial plateaus and four femoral heads were obtained from patients undergoing total knee and hip arthroplasty, respectively. Subchondral bone tissues from nonsclerotic and sclerotic lesions were digested with collagenase and nucleated bone marrow cells were isolated. First passage cells were used for fibroblastic and osteogenic colony forming unit (CFU-f/-O) assays. Alkaline phosphatase activity and in vitro matrix mineralization were determined after osteogenic differentiation for three weeks. Nonsclerotic and sclerotic MSC from three donors were implanted subcutaneously using hydroxyapatite scaffold and explanted after eight weeks. De novo calcified tissue was determined using microCT and histological tissue sections were evaluated by tartrate-resistant acid phosphatase, bone sialoprotein (BSP) andMasson's trichrome staining and area fractions of stainings were quantified using ImageJ. Results: MSC clonogenicities, assessed by CFU-f counts were 20.1± 2.2% in nonsclerotic compared with 17.04± 3.5% in the sclerotic subchondral tissue, showing no statistical differences between regions. Osteogenic induction by CFU-O counts was highly efficient in the knee joint, without significant differences between nonsclerotic (91.81± 1.60%) and sclerotic (91.56± 3.41%) areas. MSC from the hip did not show significant differences in CFU-O counts between nonsclerotic (69.79± 5.62%) and sclerotic (56.03± 19.09%) regions; but their osteogenic differentiation potential was significantly lower compared with MSC from knee joints. Mineralization capacity, assessed by Alizarin Red staining was blunted in all donors. ALP activity was increased 5.4-fold in nonsclerotic and 4.16-fold in sclerotic regions under osteogenic stimulation. CT analysis revealed de novo calcified tissue in scaffold pores at eight weeks after implantation. Tissue volumewas equal betweenMSCs from nonsclerotic and sclerotic regions (area fraction: 0.029± 0.015 vs 0.020± 0.009, p1⁄4 0.81), respectively. BSP, a late osteogenic marker that is important to bone matrix mineralization, was found strongly expressed at the scaffold-MSC interface. Notably, BSP area fraction was higher in nonsclerotic MSC (13.9± 3.2) compared with sclerotic MSC (4.0± 0.7, p1⁄4 0.048). TRAP staining revealed no evidence of osteoclastmediated bone remodeling at 8 weeks. Masson trichrome provided histological evidence of bone formation in all donors. Conclusions: Our findings demonstrate that subchondral bone-resident MSC from osteoarthritic lesions maintain the capacity for osteogenic differentiation, but show aberrant mineralization in vitro and in vivo. Enhanced recruitment and osteogenic differentiation of MSC with an aberrant mineralization phenotype might play an important role in subchondral bone sclerosis in human osteoarthritis. 211 EVALUATION OF FEMALE ATHLETE TRIAD IN FEMALE UNIVERSITY ATHLETES A. Osawa y, S. Nakao y, Y. Maruyama y, N. Koikawa z, K. Sakuraba z. y Juntendo Univ. Urayasu Hosp., Urayasu, Japan; z Juntendo Univ., Inzai

BACKGROUND: Knee arthrocentesis is a commonly performed diagnostic and therapeutic procedure in rheumatology and orthopedic surgery. Classic teaching of arthrocentesis skills relies on hands-on practice under supervision. Video-based online teaching is an increasingly utilized educational tool in higher and clinical education. YouTube is a popular video-sharing Web site that can be accessed as a teaching source. OBJECTIVE: The objective of this study was to assess the educational value of YouTube videos on knee arthrocentesis posted by health professionals and institutions during the period from 2008 to 2012. METHODS: The YouTube video database was systematically searched using 5 search terms related to knee arthrocentesis. Two independent clinical reviewers assessed videos for procedural technique and educational value using a 5-point global score, ranging from 1 = poor quality to 5 = excellent educational quality. As validated international guidelines are lacking, we used the guidelines of the Swiss Society of Rheumatology as criterion standard for the procedure. RESULTS: Of more than thousand findings, 13 videos met the inclusion criteria. Of those, 2 contained additional animated video material: one was purely animated, and one was a check list. The average length was 3.31 +/- 2.28 minutes. The most popular video had 1388 hits per month. Our mean global score for educational value was 3.1 +/- 1.0. Eight videos (62 %) were considered useful for teaching purposes. Use of a "no-touch" procedure, meaning that once disinfected the skin remains untouched before needle penetration, was present in all videos. Six videos (46%) demonstrated full sterile conditions. There was no clear preference of a medial (n = 8) versus lateral (n = 5) approach. CONCLUSIONS: A discreet number of YouTube videos on knee arthrocentesis appeared to be suitable for application in a Web-based format for medical students, fellows, and residents. The low-average mean global score for over educational value suggests an improvement of future video-based instructional materials on YouTube would be necessary before regular use for teaching could be recommended.

IntroductionLumbar spinal stenosis (LSS) is a degenerative, age-related narrowing of the lower spinal canal that causes pressure on the nerves, leading to pain and reduced mobility. Hypertrophy of ...

Item does not contain fulltext OBJECTIVE: Activated synovial fibroblasts are key players in the pathogenesis of RA by driving inflammation and joint destruction. Numerous molecules including cytokines and Toll-like receptor (TLR) ligands induce pro-inflammatory signalling and gene expression through a hierarchical network of kinases. Upstream mitogen-activated protein kinase kinase kinases (MAP3Ks) represent an attractive target for RA treatment. In this study, we sought to determine the role of the MAP3K TGF-beta-activated kinase 1 (TAK1) in cytokine and TLR-mediated signalling. METHODS: TAK1 activity was inhibited using either a small molecule inhibitor or lentivirally overexpressed kinase-inactive TAK1-K63W mutant in murine embryonic and human dermal and synovial fibroblasts. Fibroblasts were stimulated with IL-1, TNF, TLR2 or TLR4 agonists and responses were evaluated using transcriptional reporters, western blotting and analysis of gene expression of collagenases (MMP3 and MMP13), cytokines (IL-1beta and IL-6) and chemokines (IL-8 and MCP-1). RESULTS: TAK1 inhibition abrogated cytokine- and TLR-induced nuclear factor-kappaB (NF-kappaB) and Saa3-promoter reporter activation in murine and human dermal fibroblasts. In synovial fibroblasts, TAK1 regulated IL-1 and TNF-mediated NF-kappaB, but not Saa3-promoter reporter activation. Inducible mRNA expression of cytokines, collagenases and chemokines, except MCP-1, was TAK1 dependent for IL-1, TNF and TLR2 signalling. Unexpectedly, TLR4-mediated NF-kappaB reporter activation and inducible mRNA expression was fully TAK1 independent. Accordingly, NF-kappaB p65 and p38 MAPK phosphorylation was unaffected by TAK1 inhibition. CONCLUSION: In general, TAK1 crucially regulates IL-1 and TNF signalling in fibroblasts. Interestingly, TLR4 signalling is specifically TAK1 independent in synovial fibroblasts. Consequently, therapeutic TAK1 inhibition in arthropathies may not dampen the damage-associated molecular pattern-mediated TLR4 activation of synovial fibroblasts.

Contains fulltext : 81602.pdf (Publisher’s version ) (Open Access) The promoter regions of genes that are differentially regulated in the synovial membrane during the course of rheumatoid arthritis (RA) represent attractive candidates for application in transcriptionally targeted gene therapy. In this study, we applied an unbiased computational approach to define proximal-promoters from a gene expression profiling study of murine experimental arthritis. Synovium expression profiles from progressing stages of collagen-induced arthritis (CIA) were classified into six distinct groups using k-means clustering. Using an algorithm based on local over-representation and comparative genomics, we identified putatively functional transcription factor-binding sites (TFBS) in TATA-dependent proximal-promoters. Applying a filter based on spacing between TATA box and transcription start site (TSS) combined with the presence of over-represented nuclear factor kappaB (NFkappaB), AP-1, or CCAAT/enhancer-binding protein beta (C/EBPbeta) sites, 382 candidate murine and human promoters were reduced to 66, corresponding to 45 genes. In vitro, 9 out of 10 computationally defined promoter regions conferred cytokine-inducible expression in murine cells and human synovial fibroblasts. Under these conditions, the serum amyloid A3 (Saa3) promoter showed the strongest transcriptional induction and strength. We applied this promoter for driving therapeutically efficacious levels of the interleukin-1 receptor antagonist (Il1rn) in a disease-regulated fashion. These results demonstrate the value of bioinformatics for guiding the selection of endogenous promoters for transcriptionally targeted gene therapy.

Contains fulltext : 53311.pdf (Publisher’s version ) (Closed access) The application of disease-regulated promoters in local gene therapy for rheumatoid arthritis potentiates the development of a sophisticated treatment that relies on a restricted and fine-tuned supply of biologicals. Although several studies have investigated regulated promoters for achieving effective transgene expression during arthritis, none have explored their potential for minimizing deleterious effects arising from constitutive overexpression of transgenes under naive conditions. Using naive and collagen-induced arthritic mice, we examined the applicability of a hybrid interleukin-1 enhancer/interleukin-6 proximal promoter for achieving efficacious murine interleukin-4 gene therapy under arthritic conditions, while minimizing interleukin-4-induced inflammation under naive conditions. We found strong upregulation of transgene expression in virally transduced knee joints under arthritic conditions compared to levels in naive animals. Besides its responsiveness, the promoter strength proved sufficient for generating therapeutically efficacious levels interleukin-4, as demonstrated by the successful protection against cartilage erosion in collagen-induced arthritis. Most importantly, promoter-mediated restriction of the potent chemotactic interleukin-4 in naive animals strongly reduced the amounts of inflammatory cell influx. This study suggests the suitability of the interleukin-1 enhancer/interleukin-6 proximal promoter for the development of a local gene therapy strategy for rheumatoid arthritis that requires fine-tuned and restricted expression of transgenes with a pleiotrophic nature.

Background Age is the most prominent risk factor for osteoarthritis and mitochondrial DNA dysfunction has been repeatedly described in human osteoarthritic chondrocytes. Mice expressing a proofreading-deficient mitochondrial DNA polymerase (POLG) mutant accumulate an excess of reactive oxygen species and apoptosis and develop a premature aging phenotype. While these mice display a reduction in bone density, it is unclear whether they are more prone to developing osteoarthritis. Objectives In this study, we assessed the histomorphometric properties of subchondral bone and cartilage tissues in premature aging mice. Methods Mice carrying heterozygous (wt/mut n= 5) or homozygous (mut/mut n= 4) D257A mutations in POLG were compared with wildtype littermates (wt n =7). Animals were aged between eleven and fifteen months. Sagittal histological sections from knee joints were stained with Safranin- O /Fast Green and cartilage degeneration was assessed using OARSI scores. Subchondral bone area fraction (B.Ar/T.Ar) and osteoclast numbers (Oc.N/mm perimeter) between epiphyses and articular cartilage was determined on tissues stained for tartrate-resistant acid phosphatase (TRAP) and methylene blue using bone histomorphometric analyses. Results Wild type mice revealed only low grade cartilage degeneration (OARSI score p Conclusions Mice with premature aging due to accumulation of mtDNA mutations display increased bone remodelling favouring subchondral bone resorption. Additional biomechanical factors might be required in this premature aging phenotype for development of osteoarthritis. Disclosure of Interest None declared

Rheumatoid arthritis (RA) is a systemic disease with polyarticular manifestation of chronic inflammation in the knees and small joints of hand and feet. The current systemic anti-tumor necrosis factor (TNF)-alpha therapies with biologics ameliorate disease in 60% to 70% of RA patients. However, biologics must be given systemically in relatively high dosages to achieve constant therapeutic levels in the joints, and side effects have been reported. To this end, local gene delivery can provide an alternative approach to achieve high, long-term expression of biologics, optimizing the therapeutic efficacy and minimizing systemic exposure. Evidence from animal models convincingly supports the application of local gene therapy in rheumatoid arthritis, but preclinical studies remain necessary to evaluate the merge of cell-specific targeting, viral vector development, and disease-regulated transgene expression to optimize efficacy and safety.

Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in synovial tissue from rheumatoid arthritis (RA) patients. Furthermore TLR ligands are found to be present in RA serum and synovial fluid and are significantly increased, compared with serum and synovial fluid from healthy volunteers and patients with systemic sclerosis and systemic lupus erythematosus. Identification of novel endogenous TLR ligands might contribute to the elucidation of the role of TLRs in RA and other autoimmune diseases. In this study, we investigated whether five members of the small heat shock protein (HSP) family were involved in TLR4-mediated DC activation and whether these small HSPs were present in RA synovial tissue. In vitro, monocyte-derived DCs were stimulated with recombinant αA crystallin, αB crystallin, HSP20, HSPB8, and HSP27. Using flow cytometry and multiplex cytokine assays, we showed that both αA crystallin and HSPB8 were able to activate DCs and that this activation was TLR4 dependent. Furthermore, Western blot and immunohistochemistry showed that HSPB8 was abundantly expressed in synovial tissue from patients with RA. With these experiments, we identified sHSP αA crystallin and HSPB8 as two new endogenous TLR4 ligands from which HSPB8 is abundantly expressed in RA synovial tissue. These findings suggest a role for HSPB8 during the inflammatory process in autoimmune diseases such as RA.

Background and objective Activated synovial fibroblasts are key players in the pathogenesis of rheumatoid arthritis (RA) by driving inflammation and joint destruction. Numerous molecules including cytokines and toll-like receptor (TLR) ligands induce pro-inflammatory signalling and gene expression through a hierarchical network of kinases. Upstream mitogen-activated protein kinase kinase kinases (MAP3Ks) represent an attractive target for RA treatment. In this study the authors sought to determine the role of the MAP3K transforming growth factor β activated kinase1 (TAK1) in cytokine and TLR-mediated signalling. Materials and methods TAK1 activity was inhibited using either a small molecule inhibitor 5Z-7-oxozeaenol or lentivirally overexpressed kinase-inactive TAK1-K63W mutant in murine embryonic and human dermal and synovial fibroblasts. Fibroblasts were stimulated with interleukin 1 (IL-1), tumour necrosis factor (TNF), TLR2 or TLR4 agonists and responses were evaluated using transcriptional reporters and analysis of gene expression of collagenases (matrix metalloproteinases (MMP3, MMP13)), cytokines (IL-1β, IL-6) and chemokines (IL-8, MCP-1). Results TAK1 inhibition abrogated cytokine- and TLR-induced activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and Saa3 -promoter reporters in mouse and human dermal fibroblasts. In synovial fibroblasts, TAK1 crucially regulated IL-1 and TNF mediated NF-κB, but not Saa3 -promoter activation. Furthermore, TAK1 was required for inducible mRNA expression of IL-1β, IL-6, IL-8, MMP3 and MMP13, but not MCP-1, in response to IL-1, TNF and TLR2 agonist. Unexpectedly, TLR4-induced NF-κB activation and gene expression was fully TAK1-independent as found using the TAK1 inhibitor or kinase inactive mutant. Western blots demonstrated IL-1-inducible p38 and p65 phosphorylation and inhibition by the TAK1 inhibitor 5Z-7-oxozeaenol, whereas no effect of TAK1 inhibitor on lipopolysaccharides signalling in RA-SF. Conclusions In general, TAK1 plays a prominent role in regulation of IL-1- and TNF mediated signalling in fibroblasts. Interestingly, TLR4 signalling is specifically TAK1-independent in synovial fibroblasts. Consequently, therapeutic TAK1 inhibition in arthropaties may not dampen the damage-associated molecular pattern-mediated TLR4 activation of synovial fibroblasts.

Background Knee arthrocentesis is a commonly performed diagnostic and therapeutic procedure in rheumatology and orthopedic surgery. Classic teaching of arthrocentesis skills relies on hands-on practice under supervision. Video-based online teaching is an increasingly utilized educational tool in higher and clinical education. YouTube is a popular video-sharing Web site that can be accessed as a teaching source. Objectives The objective of this study was to assess the educational value of YouTube videos on knee arthrocentesis posted by health professionals and institutions during the period from 2008 to 2012. Methods The YouTube video database was systematically searched using 5 search terms related to knee arthrocentesis. Two independent clinical reviewers assessed videos for procedural technique and educational value using a 5-point global score, ranging from 1 = poor quality to 5 = excellent educational quality. As validated international guidelines are lacking, we used the guidelines of the Swiss Society of Rheumatology as criterion standard for the procedure. Results Of more than thousand findings, 13 videos met the inclusion criteria. Of those, 2 contained additional animated video material: one was purely animated, and one was a check list. The average length was 3.31±2.28 minutes. The most popular video had 1388 hits per month. Our mean global score for educational value was 3.1±1.0. Eight videos (62%) were considered useful for teaching purposes. Use of a “no-touch” procedure, meaning that once disinfected the skin remains untouched before needle penetration, was present in all videos. Six videos (46%) demonstrated full sterile conditions. There was no clear preference of a medial (n=8) versus lateral (n=5) approach. Conclusions A discreet number of YouTube videos on knee arthrocentesis appeared to be suitable for application in a Web-based format for medical students, fellows, and residents. The low-average mean global score for overall educational value suggests an improvement of future video-based instructional materials on YouTube would be necessary before regular use for teaching could be recommended. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1786

Background Subchondral bone sclerosis is a well known and potentially reversible feature of knee osteoarthritis (OA). The infiltration of immune cells into the subchondral bone in OA has been demonstrated previously. However, the exact assocation between immune cell infiltration and osteosclerosis, as well as their functional connection, remain elusive. Objectives To investigate whether the interaction between bone and immune systems might be involved in the regulation of subchondral osteosclerosis in human OA. Methods Computed tomography osteoabsorptiometry (CT-OAM) mapping of subchondral bone mineralization density (BMD) distribution was used to guide tissue preparation from nonsclerotic and sclerotic areas of explanted OA tibial plateaus. Cartilage degeneration and subchondral bone area fraction (B.Ar./T.Ar.) were evaluated using histomorphometric analyses. Presence of lymphocytes, macrophages and osteoclasts in subchondral bone marrow tissue was investigated using (immuno)histological and flow cytometry analyses for expression of CD3, CD20, CD68 and tartrateresistant acid phosphatase (TRAP). Alkaline phosphatase (ALP) activity was assessed in primary OA osteoblasts stimulated with conditioned medium from nonsclerotic and sclerotic subchondral bone. Results Subchondral BMD distribution was heterogeneous and displayed focal areas of high density that macroscopically showed severe cartilage degeneration. Histomorphometry demonstrated a strong positive correlation between Mankin score and subchondral B.Ar/T.Ar. Immunohistological and flow cytometry analyses of subchondral bone marrow tissue showed a highly specific increase in CD68pos macrophages and multinucleated cells and CD20pos B-lymphocytes in sclerotic compared with nonsclerotic subchondral bone. Correspondingly, increased numbers of functional TRAPpos osteoclasts associating with CD34pos vascular structures were detected. Sclerotic OA osteoblasts showed increased basal alkaline (ALP) phosphate activity. Conditioned medium from sclerotic bone pieces, unlike nonsclerotic bone pieces, failed to induce osteoblastic ALP activity. Conclusions Enhanced marrow immune cell infiltration and osteoclast activity, along with phenotypic alterations in osteoblasts are involved in uncoupled and aberrant bone remodeling. This indicates that immune cells induce a 9resorbtive state9 as a potential repair mechanism. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2863

Currently, gene therapy in rheumatoid arthritis is predominantly based on viral transfer of a transgene under control of a constitutively active promoter, e.g. the CMV promoter. Although constitutive production of therapeutic proteins has shown efficacy in experimental arthritis models, production of excessive amounts of these biologicals may cause serious side effects. More ideally, the promoter activity must parallel the severity of inflammation, thereby providing auto-regulatory therapeutic protein expression. Previously, we have described inflammation-inducible upregulation of transgenes under control of a hybrid IL-1 enhancer/IL-6 (IL-1E/IL-6P) promoter in both the collagen type II- (CIA), and zymosan-induced arthritis (ZIA) model. However, the absolute protein level reached with the IL-1E/IL-6P promoter limited its application in arthritis utilizing the IL-1 receptor antagonist. Therefore, we designed a strategy to identify a new candidate promoter from gene expression profiling in murine synovial tissue that meets the following requirements: i) low basal activity, ii) inflammation-inducible, and iii) capable of high transgene expression.

Background Recent investigations have provided substantial evidence that distinct molecular and morphological changes in subchondral bone tissue, most notably sclerosis, play an active and important role in the pathogenesis of OA. The cellular and molecular regulation of this pathological process remains poorly understood. Objectives We investigated whether osteoimmunology, the reciprocal signaling between cells from the immune and bone system, is involved in OA subchondral bone sclerosis. Methods Tibial plateaus and informed consent were obtained from patients undergoing total knee arthroplasty due to end-stage OA. Subchondral bone mineralization distribution was analyzed using computed tomography osteoabsoptiometry (CT-OAM) and standardized cryosections of low (non-sclerotic) and high (sclerotic) bone mineralization were prepared ( n =18 each). Cartilage degeneration was graded in Safranin- O -stained sections using the Mankin scoring system. The presence of T-lymphocytes, B-cells and macrophages was assessed using immunohistochemical staining of their respective surface markers CD3, CD20 and CD68. Osteoclast activity was visualized by staining of the enzyme marker tartrate-resistant acid phosphatase (TRAP). Cellular characterization of ex vivo subchondral bone outgrowth cultures was performed using alkaline phosphatase (ALP), TRAP staining. Correlation between histological parameters was assessed using Spearman´s rank correlation. Statistical differences were calculated using Wilcoxon signed rank test or paired t -test, where appropriate. Results CT-OAM revealed a heterogeneous distribution of subchondral bone mineralization in OA tibial plateaus, displaying focal areas of sclerosis that overlapped macroscopically with areas of cartilage damage. These data were confirmed at the histological level by a strong correlation between Mankin score and grade of sclerosis ( r =0.7, p + , but not CD3 + , lymphocytes and CD68 + mononuclear (macrophage) and multinucleated (osteoclast) cells were present in subchondral marrow spaces. Notably, the number of CD20 + lymphocytes and CD68 + cells was significantly ( p p + cells in sclerotic bone. Finally, the number of CD68 + cells was strongly correlated ( p r =0.7), grade of sclerosis ( r =0.8), CD20 + lymphocytes ( r =0.8), and TRAP-positive cells ( r =0.9). Outgrowth cultures of subchondral bone showed cells of different morphologies including fibroblast-shaped osteoblasts and macrophage-like cells. Expression of ALP was detected in the prior, while TRAP expression was evident in the latter. Corresponding with histological analyses, the number of TRAP + cells was increased in ex vivo outgrowth cultures of sclerotic compared to non-sclerotic subchondral bone. Conclusions Together, our data suggest that osteoimmunological mechanisms, specifically the interaction of CD68 + macrophages with bone-resident cells, play a - previously unknown - role in regulating subchondral bone sclerosis in progressive OA. Targeting osteoimmunology might hold potential as a disease-modifying treatment for OA. Disclosure of Interest None Declared

Introduction Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that mainly affects synovial joints. Biologics directed against tumor-necrosis-factor (TNF)-α are efficacious in the treatment of RA. However, the role of TNF receptor-1 (TNFR1) in mediating the TNFα effects in RA has not been elucidated and conflicting data exist in experimental arthritis models. The objective is to investigate the role of TNFR1 in the synovial lining cells (SLC) and the reticuloendothelial system (RES) during experimental arthritis. Methods Third generation of adenovirus serotype 5 were either injected locally in the knee joint cavity or systemically by intravenous injection into the retro-orbital venous sinus to specifically target SLC and RES, respectively. Transduction of organs was detected by immunohistochemistry of the eGFP transgene. An adenoviral vector containing a short hairpin (sh) RNA directed against TNFR1 (HpTNFR1) was constructed and functionally evaluated in vitro using a nuclear factor-kappaB (NF-κB) reporter assay and in vivo in streptococcal cell wall-induced arthritis (SCW) and collagen-induced arthritis (CIA). Adenoviruses were administered before onset of CIA, and the effect of TNFR1 targeting on the clinical development of arthritis, histology, quantitative polymerase chain reaction (qPCR), cytokine analyses and T-cell assays was evaluated. Results Systemic delivery of Ad5.CMV-eGFP predominantly transduced the RES in liver and spleen. Local delivery transduced the synovium and not the RES in liver, spleen and draining lymph nodes. In vitro, HpTNFR1 reduced the TNFR1 mRNA expression by three-fold resulting in a 70% reduction of TNFα-induced NF-κB activation. Local treatment with HpTNFR1 markedly reduced mRNA and protein levels of interleukin (IL)-1β and IL-6 in SLC during SCW arthritis and ameliorated CIA. Systemic targeting of TNFR1 in RES of liver and spleen by systemic delivery of Ad5 virus encoding for a small hairpin RNA against TNFR1 markedly ameliorated CIA and simultaneously reduced the mRNA expression of IL-1β, IL-6 and Saa1 (75%), in the liver and that of Th1/2/17-specific transcription factors T-bet, GATA-3 and RORγT in the spleen. Flow cytometry confirmed that HpTNFR1 reduced the numbers of interferon (IFN)γ (Th1)-, IL-4 (Th2)- and IL-17 (Th17)-producing cells in spleen. Conclusions TNFR1-mediated signaling in both synovial lining cells and the reticuloendothelial system independently played a major pro-inflammatory and immunoregulatory role in the development of experimental arthritis.