Using language to identify a bladder pain component in women with Dysmenorrhoea-Related Pelvic Pain: A cross-sectional study.

BACKGROUND: Dysmenorrhoea-Related Pelvic Pain (DRPP) is a common condition, which may or may not include bladder-related symptoms. Primary health care practitioners (PHCP) rely heavily on language for diagnosis of DRPP-related conditions. However, there are no established pain descriptors to assist PHCP to determine whether an individual's DRPP may include a bladder component. AIMS: To identify differences in the use of pain descriptors in women with DRPP with and without a co-existing bladder pain component, through an exploratory study of the language of pelvic pain in women. MATERIALS AND METHODS: A cross-sectional online survey of Australian and New Zealand women (n = 750, ages 18-49) who have self-identified pelvic pain. Free text and predetermined pain descriptors used by women with a self-perceived bladder pain component (DRPPB+, n = 468) were compared to those without bladder pain (DRPPB-, n = 282). Statistical analysis included Pearson χ2, logistic regression and analysis of variance tests using StataCorp Stata Statistical Software combined with qualitative data from AntConc concordance software. RESULTS: Within free-form text, bloating (P = 0.014) and pressure (P = 0.031) were used more commonly to describe dysmenorrhoea in women with DRPPB+, while the word excruciating (P < 0.001) was more commonly used by women with DRPPB-. From a pre-determined list of descriptors, pounding (P < 0.001), tingling (P < 0.001), stabbing (P = 0.010), burning (P = 0.002) and cramping (P = 0.021) were more commonly used by women with DRPPB+, than women with DRPPB-. CONCLUSIONS: Systematic patterns of word use should encourage practitioners to further enquire about bladder symptoms that may co-exist with dysmenorrhoea. Knowledge of these words may be useful in targeting diagnostic and therapeutic interventions.

Experiences of living with juvenile idiopathic arthritis: a qualitative systematic review.

OBJECTIVE: The objective of this review was to investigate the available qualitative evidence to enhance understanding of the experiences of children and young adults living with juvenile idiopathic arthritis, and their carers, in any setting. INTRODUCTION: Juvenile idiopathic arthritis is the most common chronic rheumatic disease in childhood. Despite the availability of effective treatments, persistent pain, growth retardation, physical disability, and psychological problems can occur. This may reduce the quality of life for patients with juvenile idiopathic arthritis by negatively affecting their family, educational, and social well-being. Patient-centered management and care for patients with juvenile idiopathic arthritis requires increasing attention to their self-reported quality of life and experiences, in addition to clinically measured disease activity. Furthermore, caring for children with juvenile idiopathic arthritis may have negative impacts on the lives of their carers and families. The experiences of carers have been poorly understood and studied. This review describes experiences and perspectives from patients and carers in order to inform the needs of families throughout their juvenile idiopathic arthritis journey. INCLUSION CRITERIA: Studies describing the experiences of patients aged <21 years who have been diagnosed with juvenile idiopathic arthritis according to the International League of Associations for Rheumatology criteria, as well as the experiences of their carers, have been considered. METHODS: A comprehensive search using PubMed, CINAHL, Embase, PsycINFO, Web of Science, and Google Scholar, as well as relevant conference proceedings of the American College of Rheumatology (2018-2019), the European Pediatric Rheumatology Congress 2018, the European League Against Rheumatism (2018-2019), and the Asia Pacific League of Associations for Rheumatology (2018-2019), was undertaken in December 2020 to identify pertinent published and unpublished studies. Studies published in English from 2001 to 2020 were included. The JBI approach to study selection, critical appraisal, data extraction, and data synthesis was used. RESULTS: Ten studies were included in this review. A total of 61 findings were extracted and aggregated to form 12 categories. From the 12 categories, five synthesized findings were developed: i) Self-management of juvenile idiopathic arthritis requires pain management, medication management, and the acquisition of knowledge and professional support; ii) A promising relationship with health care professionals but unbalanced access to services; iii) Parental financial burden and their adjustment to maintain family happiness; iv) Patients and parents support the web-based approach to communicate and develop self-management skills and acknowledge the importance of clinical trials; v) Desire to live a normal life without prejudice from school, social settings, and the workplace. CONCLUSIONS: This review has provided a comprehensive overview of experiences and perceptions of patients juvenile idiopathic arthritis and their parents. It is important to understand what they need to know about the disease. This review also highlights the importance of appropriate web-based programs, career counseling, infrastructures, and school facilities. Findings in this review can guide future policy and practice in order to improve care for families and children with juvenile idiopathic arthritis. Further research is required to develop management strategies for medication intolerance and to evaluate the longitudinal benefits of relevant juvenile idiopathic arthritis programs. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42019133165.

Effects of Mild and Moderate Monoclonal Antibody Dose on Inflammation, Bone Loss, and Activation of the Central Nervous System in a Female Collagen Antibody-induced Arthritis Mouse Model.

Induction of severe inflammatory arthritis in the collagen antibody-induced arthritis (CAIA) murine model causes extensive joint damage and pain-like behavior compromising analysis. While mild models are less severe, their reduced, variable penetrance makes assessment of treatment efficacy difficult. This study aimed to compare macroscopic and microscopic changes in the paws, along with central nervous system activation between a mild and moderate CAIA model. Balb/c mice (n=18) were allocated to control, mild, and moderate CAIA groups. Paw inflammation, bone volume (BV), and paw volume (PV) were assessed. Histologically, the front paws were assessed for joint inflammation, cartilage damage, and pre/osteoclast-like cells and the lumbar spinal cord and the periaqueductal gray (PAG) region of the brain for glial reactivity. A moderate CAIA dose induced (1) significantly greater local paw inflammation, inflammatory cell infiltration, and PV; (2) significantly more osteoclast-like cells on the bone surface and within the surrounding soft tissue; and (3) significantly greater glial reactivity within the PAG compared with the mild CAIA model. These findings support the use of a moderate CAIA model (higher dose of monoclonal antibodies with low-dose lipopolysaccharide) to induce more consistent histopathological features, without excessive joint destruction.

Experiences of living with juvenile idiopathic arthritis: a qualitative systematic review protocol.

OBJECTIVE: The objective of this review is to identify, critically appraise and synthesize the available qualitative evidence to understand the experiences of children, young adults and their carers living with juvenile idiopathic arthritis in any setting. INTRODUCTION: Juvenile idiopathic arthritis is the most common rheumatic disease in childhood. Despite the availability of effective treatments, the disease still has negative impacts on patients' and carers' lives. Patients' and carers' experiences of living with juvenile idiopathic arthritis have been recognized as important in the measurement of health status and treatment implementation. Addressing these needs will facilitate more effective management and treatment of the disease. This protocol describes a method for a systematic review regarding the perspectives from patients and carers in order to highlight the needs of families throughout their juvenile idiopathic arthritis journey. INCLUSION CRITERIA: Studies on the experiences of patients aged <21 years who have been diagnosed with juvenile idiopathic arthritis according to the International League of Associations for Rheumatology criteria, as well as the experiences of their carers, will be considered. Papers included in this review will include, but not be limited to, designs such as phenomenology, grounded theory and ethnography. METHODS: A comprehensive search using PubMed, CINAHL, Embase, PsycINFO and Web of Science was undertaken in August 2019. Available studies published in English from 2001 to 2019 will be included. The recommended JBI method for study selection, critical appraisal, data extraction and data synthesis will be used. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO (CRD42019133165).

Osteocytes respond to particles of clinically-relevant conventional and cross-linked polyethylene and metal alloys by up-regulation of resorptive and inflammatory pathways.

Periprosthetic osteolysis is a major cause of implant failure in total hip replacements. Aseptic loosening caused by osteolytic lesions is associated with the production of bioactive wear particles from the articulations of implants. Wear particles infiltrate the surrounding tissue of implants, promoting inflammation as well as bone resorption. Osteocytes have been shown to both regulate physiological osteoclastogenesis and directly remodel their perilacunar bone matrix by the process of osteocytic osteolysis. We hypothesise that osteocytes respond to wear debris of orthopaedic implant materials by adopting a pro-catabolic phenotype and thus contribute to periprosthetic osteolysis through the known pathways of bone loss. Osteocyte responses to particles derived from clinically relevant materials, ultra-high molecular weight polyethylene (UHMWPE), highly cross-linked polyethylene (XLPE) and metal alloys, Ti6Al4V and CoCrMo, were examined in vitro in human primary osteocyte-like cultures. Osteocyte-like cells exposed to both polyethylene and metal wear particle types showed upregulated expression of catabolic markers associated with osteocytic osteolysis, MMP13, carbonic anhydrase 2 (CA2) and cathepsin K (CTSK). In addition, pro-osteoclastogenesis markers RANKL and M-CSF were induced, as well as the expression of pro-inflammatory cytokines, IL-6 and TNFα, albeit with different kinetics. These findings suggest a previously unrecognised action of wear particles of multiple orthopaedic materials on osteocytes, and suggest a multifaceted role for osteocytes in periprosthetic osteolysis. STATEMENT OF SIGNIFICANCE: This study addresses periprosthetic osteolysis, a major clinical problem leading to aseptic loosening of orthopaedic implants. It is well accepted that wear particles of polyethylene and of other implant materials stimulate the activity of bone resorbing osteoclasts. Our recent work provided evidence that commercial particles of ultra-high molecular weight polyethylene (UHMWPE) stimulated osteocytes to adopt a bone catabolic state. In this study we demonstrate for the first time that particles derived from materials in clinical use, conventional UHMWPE, highly cross-linked polyethylene (XLPE), and Ti6Al4V and CoCrMo metal alloys, all stimulate human osteocyte activities of osteocyte-regulated osteoclastogenesis, osteocytic osteolysis, proinflammatory responses, osteocyte apoptosis, albeit to varying extents. This study provides further mechanistic insight into orthopaedic wear particle mediated bone disease in terms of the osteocyte, the most abundant and key controlling cell type in bone.

Intracellular apoptotic pathways: a potential target for reducing joint damage in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that results in both local and systemic bone erosion, causing significant joint deformities and functional disability. The increased number of synovial fibroblasts, inflammatory cells and osteoclasts in RA is associated with reduced apoptosis in these cells. The ability to modulate the cell proliferation or death (particularly apoptosis) is recognised for its immense therapeutic potential. Identifying new therapeutics to assist in stimulating apoptosis within the synovial joints therefore may be beneficial in reducing inflammation and bone loss in RA patients. In this review, the roles of anti-apoptotic proteins that are upregulated in RA synovial joints will be discussed in relation to their actions on bone destruction and inflammation. Evidence recently published suggests that intracellular apoptotic inhibitory molecules can be targeted by current or new therapeutics to reduce joint damage in RA. However, the therapeutics that target these molecules are yet to reach clinical trial stages. Even so it is evident that understanding the upregulation of anti-apoptotic molecules in RA is required to improve treatments currently available for RA patients.

Osteoclast-Associated Receptor (OSCAR) Distribution in the Synovial Tissues of Patients with Active RA and TNF-α and RANKL Regulation of Expression by Osteoclasts In Vitro.

Osteoclast-associated receptor (OSCAR) is a co-stimulatory receptor in osteoclastogenesis. Synovial tissues from active rheumatoid arthritis (RA) patients express higher levels of OSCAR compared with osteoarthritic and normal patients; however, the comparison of OSCAR levels in different regions of active RA synovium has not been reported. The regulation of OSCAR by TNF-α and receptor activator of NF kappa ß ligand (RANKL) in pre-osteoclasts/osteoclasts in vitro is unclear. OSCAR and tartrate-resistant acid phosphatase (TRAP) expression levels did not differ between the cartilage pannus junction (CPJ) and non-CPJ regions in active RA. We demonstrate a similar pattern of OSCAR expression in the CPJ and non-CPJ synovial tissue from patients with active RA. OSCAR was associated with mononuclear cells in both the lining and sub-lining and endothelial cells (von Willebrand factor positive). Pre-osteoclasts (TRAP-positive cells) were present in the lining and sub-lining of both regions. OSCAR messenger RNA (mRNA) expression and release by pre-oscteoclasts/osteoclasts was modulated by RANKL with/without TNF-α in vitro. Osteoclast resorption on dentine slices was significantly greater with TNF-α pre-treatment and RANKL (10 ng/ml) than RANKL 10 or 50 ng/ml alone or RANKL 10 ng/ml with TNF-α given from day 3 post-RANKL. The lower levels of OSCAR mRNA expression corresponded with high osteoclast activity levels.

Mixed effects of caffeic acid phenethyl ester (CAPE) on joint inflammation, bone loss and gastrointestinal inflammation in a murine model of collagen antibody-induced arthritis.

OBJECTIVE: To investigate the effect of caffeic acid phenethyl ester (CAPE) on local and systemic inflammation and bone loss in collagen antibody-induced arthritis (CAIA) mice. METHODS: Four groups of mice (n = 8 per group) were allocated; control, CAPE (1 mg/kg), CAIA and CAIA + CAPE (1 mg/kg). Local inflammation and bone loss were evaluated using clinical paw scores, in vivo micro-computed tomography (micro-CT), histological assessment and tartrate-resistant acid phosphatase (TRAP) staining. Serum levels of C-reactive protein (CRP) and C-terminal telopeptide (CTX-1) were measured by ELISA. Jejunum and colon sections were evaluated histopathologically for damage and toxicity. RESULTS: Greater paw scores and percentage change in paw volume were observed in CAIA + CAPE compared to the control groups (p < 0.05). Bone volume over time remained unchanged (p = 0.94) and the number of multinucleated TRAP-positive cells was greatest in CAIA + CAPE mice (p < 0.05). CRP and CTX-1 levels did not differ between groups. CAIA + CAPE mice exhibited lower colon toxicity scores and a reduced percentage of cavitated goblet cells in the colon crypts compared with CAIA mice (p = 0.026 and p = 0.003, respectively). Histopathology in the jejunum was not altered. CONCLUSION: CAPE did not reduce paw inflammation or bone loss in CAIA mice. CAPE reduced histopathological changes in the colon of CAIA mice.

The X-Linked Inhibitor of Apoptosis Protein Inhibitor Embelin Suppresses Inflammation and Bone Erosion in Collagen Antibody Induced Arthritis Mice.

OBJECTIVE: To investigate the effect of Embelin, an inhibitor of X-Linked Inhibitor of Apoptosis Protein (XIAP), on inflammation and bone erosion in a collagen antibody induced arthritis (CAIA) in mice. METHODS: Four groups of mice (n = 6 per group) were allocated: CAIA untreated mice, CAIA treated with Prednisolone (10 mg/kg/day), CAIA treated with low dose Embelin (30 mg/kg/day), and CAIA treated with high dose Embelin (50 mg/kg/day). Joint inflammation was evaluated using clinical paw score and histological assessments. Bone erosion was assessed using micro-CT, tartrate resistant acid phosphatase (TRAP) staining, and serum carboxy-terminal collagen crosslinks (CTX-1) ELISA. Immunohistochemistry was used to detect XIAP protein. TUNEL was performed to identify apoptotic cells. RESULTS: Low dose, but not high dose Embelin, suppressed inflammation as reflected by lower paw scores (P < 0.05) and lower histological scores for inflammation. Low dose Embelin reduced serum CTX-1 (P < 0.05) and demonstrated lower histological score and TRAP counting, and slightly higher bone volume as compared to CAIA untreated mice. XIAP expression was not reduced but TUNEL positive cells were more abundant in Embelin treated CAIA mice. CONCLUSION: Low dose Embelin suppressed inflammation and serum CTX-1 in CAIA mice, indicating a potential use for Embelin to treat pathological bone loss.

Osteoimmunology: Major and Costimulatory Pathway Expression Associated with Chronic Inflammatory Induced Bone Loss.

The field of osteoimmunology has emerged in response to the range of evidences demonstrating the close interrelationship between the immune system and bone metabolism. This is pertinent to immune-mediated diseases, such as rheumatoid arthritis and periodontal disease, where there are chronic inflammation and local bone erosion. Periprosthetic osteolysis is another example of chronic inflammation with associated osteolysis. This may also involve immune mediation when occurring in a patient with rheumatoid arthritis (RA). Similarities in the regulation and mechanisms of bone loss are likely to be related to the inflammatory cytokines expressed in these diseases. This review highlights the role of immune-related factors influencing bone loss particularly in diseases of chronic inflammation where there is associated localized bone loss. The importance of the balance of the RANKL-RANK-OPG axis is discussed as well as the more recently appreciated role that receptors and adaptor proteins involved in the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway play. Although animal models are briefly discussed, the focus of this review is on the expression of ITAM associated molecules in relation to inflammation induced localized bone loss in RA, chronic periodontitis, and periprosthetic osteolysis, with an emphasis on the soluble and membrane bound factor osteoclast-associated receptor (OSCAR).

Parthenolide reduces empty lacunae and osteoclastic bone surface resorption induced by polyethylene particles in a murine calvarial model of peri-implant osteolysis.

The study aimed to determine the effects of parthenolide (PAR) on bone volume (BV) and bone surface resorption as assessed by live-animal microcomputed tomography (µCT) and possible osteocyte death as indicated by empty lacunae histologically in polyethylene (PE) particle-induced calvarial osteolysis in mice. Baseline µCT scans were conducted 7 days preimplantation of 2 × 10(8) PE particles/mL over the calvariae (day 0). PAR at 1 mg/kg/day was subcutaneously injected on days 0, 4, 7, and 10. At day 14, BV and surface resorption was analyzed with µCT. Calvarial tissue was processed for histomorphometric osteocyte evaluation. Serum was analyzed for type-1 carboxy-terminal collagen crosslinks (CTX-1) and osteoclast associated receptor (OSCAR) levels by ELISA. PE significantly decreased BV (p = 0.0368), increased surface bone resorption area (p = 0.0022), and increased the percentage of empty lacunae (p = 0.0043). Interestingly, PAR significantly reduced the resorption surface area (p = 0.0022) and the percentage of empty osteocyte lacunae (p = 0.0087) in the PE-calvariae, but it did not affect BV, serum CTX-1 or OSCAR levels. The ability of PAR to inhibit PE-induced surface bone erosion may better reflect the in vivo situation, where bone resorption occurs on the surface at the bone-implant interface and may also be related to the role of osteocytes in this pathology.

Comprehensive profiling analysis of actively resorbing osteoclasts identifies critical signaling pathways regulated by bone substrate.

As the only cells capable of efficiently resorbing bone, osteoclasts are central mediators of both normal bone remodeling and pathologies associates with excessive bone resorption. However, despite the clear evidence of interplay between osteoclasts and the bone surface in vivo, the role of the bone substrate in regulating osteoclast differentiation and activation at a molecular level has not been fully defined. Here, we present the first comprehensive expression profiles of osteoclasts differentiated on authentic resorbable bone substrates. This analysis has identified numerous critical pathways coordinately regulated by osteoclastogenic cytokines and bone substrate, including the transition from proliferation to differentiation, and sphingosine-1-phosphate signaling. Whilst, as expected, much of this program is dependent upon integrin beta 3, the pre-eminent mediator of osteoclast-bone interaction, a surprisingly significant portion of the bone substrate regulated expression signature is independent of this receptor. Together, these findings identify an important hitherto underappreciated role for bone substrate in osteoclastogenesis.

Dexamethasone increases αvß3 integrin expression and affinity through a calcineurin/NFAT pathway.

The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvß3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvß3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvß3 integrin was the result of an increase in the expression of the ß3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p<0.04) in ß3 integrin mRNA by day 2 compared to control and remained elevated for 6days of treatment and then an additional 10days once the DEX was removed. The increase in ß3 integrin mRNA levels required only 1day of DEX treatment to increase levels for 4days in the absence of DEX. In contrast, DEX did not alter ß1 integrin mRNA or protein levels. The DEX-induced upregulation of ß3 integrin mRNA was partly due to an increase in its half-life to 60.7h from 22.5h in control cultures (p<0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in ß3 integrin mRNA. In summary, the DEX-induced increase in ß3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvß3 integrin and the upregulation of the ß3 integrin subunit through the calcineurin/NFAT pathway.

Bone remodelling in inflammatory arthritis.

The inflammatory arthropathies that include rheumatoid arthritis, the seronegative spondyloarthropathies and systemic lupus erythematosus are characterised by marked alterations in the architecture and structural integrity of peri-articular bone; however, the pattern and natural history of the skeletal changes differs in these conditions. In part, this can be attributed to differences in the primary anatomical site of the inflammation, but also there is evidence that there are differences in the biological properties and products produced by inflammatory tissues. This review will focus on recent advances in the understanding of the cellular and molecular mechanisms that contribute to the differential pattern of articular bone remodelling in these prototypical inflammatory forms of arthritis.

The immunoreceptor tyrosine-based activation motif (ITAM) -related factors are increased in synovial tissue and vasculature of rheumatoid arthritic joints.

INTRODUCTION: The immunoreceptor tyrosine-based activation motif (ITAM) pathway provides osteoclast co-stimulatory signals and regulates proliferation, survival and differentiation of effector immune cells. In the osteoclast, the receptors Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Osteoclast Associated Receptor (OSCAR) and their respective adaptor proteins, DAP12 and FcRγ mediate ITAM signals and induce calcium signaling and the crucial transcription factor, NFATc1. In rheumatoid arthritis (RA), OSCAR expression by monocytes is inversely correlated with disease activity. Additionally, serum levels of OSCAR are reduced in RA patients versus healthy controls suggesting that expression and secretion or cleavage of soluble (s) OSCAR is immune modulated. Recent data suggest that endothelial cells may also be a source of OSCAR. METHODS: ITAM receptors, their adaptor proteins, and NFATc1 and cathepsin K were detected in human synovial tissues by immunohistochemistry. Synovial tissues from patients with active RA were compared with tissue from patients in remission, osteoarthritis (OA) patients and healthy individuals. OSCAR was measured by immunoassay in synovial fluids recovered from active RA and OA patients. Endothelial cells were cultured with or without 5 ng/mL TNF-α or IL-1ß over 72 hours. Temporal expression of OSCAR mRNA was assessed by qRT PCR and OSCAR protein in the supernatant was measured by ELISA. RESULTS: Significantly higher (P < 0.05) NFATc1-positive inflammatory cell aggregates were found in active RA tissues than in healthy synovial tissue. Similarly, the percentage of OSCAR, FcRγ, DAP12 and TREM2 positive cells was significantly higher in active RA tissues compared to the healthy synovial tissue. Notably, OSCAR was strongly expressed in the microvasculature of the active RA tissues (9/9), inactive RA (8/9) weakly in OA (4/9) but only in the lumen of healthy synovial tissue (0/8). OSCAR levels were detected in synovial fluids from both RA (47 to 152 ng/mL) and OA (112 to 145 ng/mL) patients. Moreover, OSCAR mRNA expression and soluble OSCAR release was stimulated by TNF-α and IL1-ß in cultured endothelial cells. CONCLUSIONS: Increased levels of ITAM related factors were present in synovial tissue from active RA joints compared to OA and healthy joints. OSCAR was strongly expressed by the vasculature of active RA patients and membrane bound and soluble OSCAR was stimulated by inflammatory mediators in endothelial cells in vitro.

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis.

INTRODUCTION: TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro. METHODS: TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry. RESULTS: TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts. CONCLUSIONS: The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.

Bone matrix regulates osteoclast differentiation and annexin A8 gene expression.

While attachment to bone is required for optimal osteoclast function, the molecular events that underlie this fact are unclear, other than that the cell requires adhesion to mineralized matrix to assume a fully differentiated phenotype. To address this issue, we cultured murine bone marrow-derived osteoclasts on either cell culture plastic or devitalized mouse calvariae to identify the distinct genetic profile induced by interaction with bone. Among a number of genes previously unknown to be expressed in osteoclasts we found that Annexin A8 (AnxA8) mRNA was markedly up-regulated by bone. AnxA8 protein was present at high levels in osteoclasts present in human tissues recovered from sites of pathological bone loss. The presence of bone mineral was required for up-regulation of AnxA8 mRNA since osteoclasts plated on decalcified bone express AnxA8 at low levels as did osteoclasts plated on native or denatured type I collagen. Finally, AnxA8-regulated cytoskeletal reorganization in osteoclasts generated on a mineralized matrix. Thus, we used a novel approach to define a distinct bone-dependent genetic program associated with terminal osteoclast differentiation and identified Anxa8 as a gene strongly induced late in osteoclast differentiation and a protein that regulates formation of the cell's characteristic actin ring.

Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins.

Snail, a transcriptional repressor of E-cadherin expression, plays a role in the process of epithelial-mesenchymal transition. However, the molecular basis of the role of snail in epithelial-mesenchymal transition has not been fully clarified. Here we show that the expression of snail in epithelial Madin-Darby canine kidney (MDCK) and A431 cells enhances both cell detachment and attachment. Snail did not confer resistance to anoikis induced by loss of contact but instead enhanced cell attachment to extracellular matrices such as fibronectin. This attachment was inhibited by Arg-Gly-Asp (RGD) peptides. Up-regulation of the promoter activity of integrin alphaV was observed in snail-expressing MDCK (MDCK/snail) cells. Snail also enhanced MDCK cell migration toward osteopontin that is a ligand for integrin alphaVbeta3. We confirmed the reduction of basement membrane proteins such as laminin (LN) alpha3, beta3, and gamma2 (laminin-5/LN-5) and of receptors for LN-5 such as integrins alpha3, alpha6, or beta4 in MDCK/snail or in snail-expressing A431 (A431/snail) cells. Nevertheless, suppression of LN-alpha3 chain by transient transfection of small interference RNAs resulted in no enhancement of cell detachment. We also found an induction of matrix metalloproteinase-3 in MDCK/snail and A431/snail cells. However, the inhibition of matrix metalloproteinase-3 showed no significant effect on the detachment of MDCK/snail cells. These results suggest that snail enhances cell detachment by multiple mechanism and leads to cell migration and reattachment at a second site, at least in part, by changing the expression of integrins in the cells.

Modulation of RANKL and osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis in response to disease-modifying antirheumatic drug treatment and correlation with radiologic outcome.

OBJECTIVE: To demonstrate the effect of treatment with disease-modifying agents on the expression of osteoprotegerin (OPG) and RANKL in the synovial tissue from rheumatoid arthritis (RA) patients and to correlate these changes with radiologic damage measured on sequential radiographs of the hands and feet. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 25 patients with active RA (16 of whom had a disease duration <12 months) before and at 3-6-month intervals after starting treatment with a disease-modifying agent. Immunohistologic analysis was performed using monoclonal antibodies to detect OPG and RANKL expression, with staining quantitated using computer-assisted image analysis and semiquantitative analysis techniques. Serial radiographs of the hands and feet were analyzed independently by 2 radiologists and a rheumatologist using the van der Heide modification of the Sharp scoring method. RESULTS: Thirteen patients achieved a low disease state as defined by a disease activity score <2.6 while 19 patients achieved an American College of Rheumatology response >20% after disease-modifying antirheumatic drug (DMARD) treatment. Successful DMARD treatment resulted in an increase in OPG expression and a decrease in RANKL expression at the synovial tissue level, which correlated with a reduction in erosion scores measured on annual radiographs of the hands and feet. CONCLUSION: Successful treatment-induced modulation of OPG and RANKL expression at the synovial tissue level, resulting in a reduction in the RANKL:OPG ratio, is likely to have a significant impact on osteoclast formation and joint damage in patients with active RA.

PU.1 and NFATc1 mediate osteoclastic induction of the mouse beta3 integrin promoter.

Expression of the alpha(v)beta(3) integrin is required for normal osteoclast function. We previously showed that an evolutionary conserved NFATc1 binding site is required for RANKL induction and NFATc1 transactivation of the human beta(3) promoter. The mechanism conferring specificity for RANKL induction and NFATc1 transduction of the beta(3) gene in osteoclast differentiation is unclear since NFATc1 is expressed and activated in numerous cell types that do not express the beta(3) gene. PU.1 is an ETS family transcription factor in myeloid cells associated with expression of various osteoclast genes. The present study investigates the role of NFATc1 in concert with PU.1 in osteoclast-specific transcription of the mouse beta(3) integrin gene. The mouse beta(3) promoter was transactivated by NFATc1 in RAW264.7 cells and deletion or mutation of either of the conserved NFAT and PU.1 binding sites abrogated transactivation. NFATc1 transactivation of the mouse beta(3) promoter was specifically dependent on co-transfected PU.1 in HEK293 cells, to the exclusion of other ETS family members. Direct binding of NFATc1 and PU.1 to their cognate sequences was demonstrated by EMSA and NFATc1 and PU.1 occupy their cognate sites in RANKL-treated mouse marrow precursors in chromatin immuno-precipitation (ChIP) assays. TAT-mediated transduction with dominant-negative NFATc1 dose-dependently blocked endogenous expression of the mouse beta(3) integrin and the formation of TRAP positive multinucleated cells in RANKL-treated mouse macrophages. These data provide evidence that NFATc1, in concert with PU.1, are involved in regulation of beta(3) integrin expression during osteoclast differentiation and suggest that PU.1 confers specificity to the NFATc1 response to macrophage lineage cells.