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Background: Our understanding of the biology of osteoblasts is important as they underpin bone remodelling, fracture healing and processes such as osseointegration. Osteoblasts isolated from human humeral samples display distinctive biological activity in vitro, which relates to the samples' bone types (subchondral (S), trabecular (T), cortical (C)). Our aim was to isolate primary osteoblast cultures from different bone types from the proximal femur of a clinical population of dogs presented for total hip replacement and compare the behaviour of the osteoblasts derived from different bone types, to identify a preferred bone type for isolation. Results: No differences were found for osteoblast doubling time (median for S = 2.9, T = 3.1 and C = 2.71 days, respectively; p = 0.33), final cell number (median for S = 54,849, T = 49,733, C = 61,390 cells/cm 2 ; p = 0.34) or basal tissue non-specific alkaline phosphatase (TNAP) activity (median for S = 0.02, T = 0.02, C = 0.03 U/min/mg protein; p = 0.81) between bone types after 6 days of culture in basal media. There were no differences in mineralizing TNAP activity (S = 0.02, T = 0.02, C = 0.03 U/min/mg protein, p = 0.84) or in mineralized area (S = 0.05, T = 0.04, C = 0.04%, p = 0.92) among cells from different bone types. Conclusions: There is no significant difference in mean doubling time, basal or mineralizing TNAP activity or mineralized area in osteoblasts derived from subchondral, cortical, or trabecular bone types from the canine femoral head. However, there appears to be a high level of inter-animal variability in the studied parameters, which was independent of age, body mass, and sex. Trabecular isolate osteoblasts have the least variation of the bone types studied, and therefore should be considered a preferred source for primary osteoblast cultures. The work here provides baselines for canine osteoblast function, which has utility for future comparative studies.

Copyright © 2017 Inês P. Perpétuo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16- monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6., This work was supported by Fundação para a Ciência e Tecnologia (SFRH/BD/70533/2010 to Inês P. Perpétuo) and by a research grant from Investigator-Initiated Studies Program of Merck Sharp & Dohme Corp. (Merck_P08574 to João E. Fonseca).

Background Surface topography is a key parameter in bone cells–biomaterials interactions. This study analyzed the behavior of human osteoclast precursor cells cultured over three hydroxyapatite (HA) surfaces ranging from a micro- to nanoscale topography. Methods HA surfaces were prepared with microsized HA particles, at 1300 °C (HA1), and with nanosized HA particles at 1000 °C (HA2) and 830 °C (HA3). Human osteoclast precursors were cultured in the absence or presence of M-SCF and RANKL. Results HA surfaces had similar chemical composition, however, HA1 and HA3 presented typical micro- and nanostructured topographies, respectively, and HA2 profile was between those of HA1 and HA3. The decrease on the average grain diameter to the nanoscale range (HA3) was accompanied by an increase in surface area, porosity and hydrophilicity and a decrease in roughness. Compared to HA1 surface, HA3 allowed a lower osteoclastic adhesion, differentiation and function. Differences in the cell response appeared to be associated with the modulation of relevant intracellular signaling pathways. Conclusions The decrease in HA grain size to a biomimetic nanoscale range, appears less attractive to osteoclastic differentiation and function, compared to the HA microsized topography. General significance This observation emphasizes the role of surface topography in designing advanced biomaterials for tailored bone cells response in regenerative strategies.

This chapter describes the isolation, culture, and staining of osteoblasts. The key advantages of this assay are that it allows direct measurement of bone matrix deposition and mineralization, as well as yielding good quantities of osteoblasts at defined stages of differentiation for molecular and histological analysis. An additional focus of this chapter will be the culture of osteoblasts from less conventional animal species.

Background Rheumatoid arthritis (RA) is a systemic, immune mediated inflammatory disease that is associated with bone erosions and joint destruction. Methotrexate (MTX) slows bone damage but the mechanism by which it acts is still unknown. Objectives In this study we aimed to assess the effect of MTX and low dose prednisolone (MTX+PDN) on circulating osteoclast (OC) precursors and OC differentiation in RA patients. Methods RA patients before and at least 6 months after MTX therapy were analyzed and compared with healthy donors. A blood sample was collected in order to assess receptor activator of NF-κB (RANK) ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Serum quantification of bone turnover markers and cytokines and in vitro OC differentiation assays were performed. Results The number or RANKL+ neutrophils increased in RA patients when compared to healthy donors (p=0.006) and after treatment with MTX+PDN their count was reduced to healthy control numbers (p=0.0155). Classical activation markers of monocytes such as HLA-DR, CD86, CCR2 and CD11b, and also RANK were increased in RA patients at baseline, comparing to control healthy donors. After MTX+PDN exposure, expression decreased to healthy control levels. Serum RANKL levels were increased at baseline comparing to healthy donors (p=0.0164) and normalized after therapy. Although the number of OC was not different between groups, resorbed area and resorbed area/pit were elevated when compared to controls (p=0.0436 and 0.0249, respectively) and reduced after treatment (p Conclusions Our results suggest that MTX+PDN play an important role in downregulating OC function, which we believe occurs through a decrease in RANK surface expression in monocytes. Disclosure of Interest None declared

Our aim was to compare bone gene expression in rheumatoid arthritis (RA) and primary osteoporosis (OP) patients. Secondary aims were to determine the association of gene expression of the Wnt/β-catenin signaling pathway with inflammatory cytokines in the bone microenvironment and to assess the serum levels of Wnt/β-catenin proteins in both groups. RA patients referred for hip replacement surgery were recruited. Primary OP patients were used as controls. Gene expression of Wnt pathway mediators, matrix proteins, and pro-inflammatory cytokines were analyzed in bone samples. Bone turnover markers, inflammatory cytokines, and Wnt mediators were measured in serum. Twenty-two patients were included: 10 with RA and 12 with primary OP. The expressions of Wnt10b (p = 0.034), its co-receptor LRP6 (p = 0.041), and its negative regulator DKK1 (p = 0.008) were upregulated in RA bone. IL17 gene expression in bone was upregulated in RA patients (p = 0.031) and correlated positively with Wnt10b (r = 0.810, p = 0.015), DKK2 (r = 0.800, p = 0.010), and RANKL/OPG ratio (r = 0.762, p = 0.028). DKK2 (p = 0.04) was significantly decreased in RA serum compared with primary OP. In conclusion, bone fragility in RA patients is induced by an unbalanced bone microenvironment and is associated with a specific gene expression pattern, namely, the upregulation of IL17 and DKK1, suggesting that the modulation of these two pathways might prevent RA systemic bone loss.

Copyright: © 2017 Perpétuo, Caetano-Lopes, Vieira-Sousa, Campanilho-Marques, Ponte, Khmelinskii, Canhão, Ainola and Fonseca. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Introduction: Ankylosing spondylitis (AS) is typically characterized by focal bone overgrowth and also by systemic bone loss. We hypothesize that the increased osteoproliferation found in AS might be partially due to reduced ability of osteoclast precursors (OCPs) to differentiate into osteoclasts (OCs). Therefore, our aim was to characterize bone remodeling and pro-osteoclastogenesis inflammatory environment, monocytes’ phenotype, and in vitro osteoclast differentiation in AS patients. Methods: Patients with active AS without any ongoing therapy and age- and gender-matched healthy donors were recruited. Receptor activator of nuclear factor-κβ (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations were assessed. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and quantitative reverse transcription real-time PCR for OC-specific genes were performed. Results: Pro- and anti-inflammatory cytokine serum levels were higher in AS patients than in controls. RANKL neutrophil expression was higher in AS patients when compared to healthy donors, but CD51/CD61 expression was lower in the classical monocyte subpopulation. Concerning osteoclastogenesis, we found no differences in the in vitro osteoclast differentiating potential of these cells when compared to healthy donors. However, we observed low expression of CSF1R, RANK, and NFATc1 in AS OCPs. Conclusion: Despite the high levels of pro-inflammatory cytokines present in AS patients, no differences in the number of OC or resorbed area were found between AS patients and healthy donors. Moreover, we observed that OCPs have low OC-specific gene expression. These findings support our hypothesis of an impaired response of OCPs to pro-osteoclastogenic stimuli in vivo in AS patients.

The polymorphism of the tumor necrosis factor (TNF) promoter gene at position -308 and that of the lymphotoxin alpha (LTA) gene at position 252 have been implicated as genetic risk factors for systemic lupus erythematosus (SLE) in some populations. In a nested case–control study, we investigated the possible association of these polymorphisms with susceptibility to SLE and with phenotypic disease features in Portuguese Caucasian patients. TNF-308 G>A and LTA 252 A>G polymorphisms were determined by restriction fragment length polymorphism analysis in a cohort of 115 SLE patients and 152 unrelated healthy controls, and the magnitude of the association between genotypes and SLE diagnosis was calculated. For SLE patients, we also tested the association between disease characteristics and genotypes. No significant differences in genotype or allele frequencies could be identified between SLE cases and controls. Lupus nephritis (OR = 2.84; 95%CI 1.14–7.03, P = 0.02) and the presence of anti-Sm antibodies (OR = 3.11; 95%CI 1.08–8.94; P = 0.03) were significantly more prevalent among lupus patients possessing the TNF-308 A allele. The occurrence of nephritis was also higher in LTA 252 G allele carriers (OR = 2.90; 95%CI 1.12–7.54; P = 0.02). Our results do not support a major role of either the TNF-308 G>A or the LTA 252 A>G polymorphisms as genetic risk factors for SLE. Nevertheless, these polymorphisms appear to associate with the risk of renal lupus and distinct immunological features.

Copyright: © 2015 Perpétuo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, Introduction: Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. Methods: 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. Results: RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-β levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. Conclusion: In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli., This work was supported by Fundação para a Ciência e Tecnologia (http://www.fct.pt/index.phtml.en) PhD grant (SFRH/BD/70533/2010) and in part by a research grant from Investigator-Initiated Studies Program of Merck Sharp & Dohme Corp (http://www.merck.com/index.html - Merck_P08574). The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp & Dohme Corp.

The identification of predictive factors of poor response to methotrexate (MTX) in juvenile idiopathic arthritis (JIA) patients could contribute to optimize the treatment strategy, namely by the earlier introduction of biological treatments.

Objectives Pachydermoperiostosis is a rare clinical entity characterized by skin thickening of the forehead, eyelids, and hands, digital clubbing, and periostosis. Two genes have been associated, HPGD and recently SLCO2A1. We present a detailed clinical and genetic description of an African pachydermoperiostosis patient with a SLCO2A1 mutation. Methods Standard clinical and laboratory evaluation was carried out. Genetic screening was done with PCR followed by direct sequencing. We discuss the clinical features and known mutations of previously reported cases identified through a PubMed literature review. Results The clinical findings showed special features, including exuberant knee effusions and an extraordinary good response on surgery of the blepharoptosis. We found a splice site mutation in the SLCO2A1 gene in homozygous form: c.940+1G>A. This mutation was previously reported only in 1 Chinese and 3 Japanese cases and was considered as a founder mutation in Japan. Beside our case, only one other patient in the literature carried this mutation in homozygous condition, but with different main clinical symptoms. Conclusions Our case demonstrates phenotypic heterogeneity of PDP even between homozygous carriers of the same mutation, suggesting further modifiers. Besides, it shows that this rare SLCO2A1 mutation is not exclusively present in East-Asia, but can occur in various ethnicities, with different origin, thus the incidence is probably underestimated.

Osteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization. We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event.In this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined. Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis.64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~0; p=0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p=0.029). ucOC levels were higher in the fracture group although not significantly (p=0.058). No differences were found regarding total OC (p=0.602), apoE (p=0.467) and Vitamin K (p=0.371). In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (β=0.607; p=0.013) and stiffness (β=0.693; p=0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (β=-0.411; p=0.030) and stiffness (β=-0.487; p=0.009).We demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.

Materials and methods RA patients submitted to hip replacement surgery were recruited. They were matched to a group of primary OP patients for bone mineral density and major clinical fracture risk factors (age, gender, BMI). Trabecular bone microarchitecture was assessed by micro-computed tomography and bone mechanical behavior by compression tests. Bone cell activity was analyzed by studying gene expression.

© The Author 2010. Published by Oxford University Press on behalf of the British Society for Rheumatology., Objectives. B cells play an important role in the perpetuation of RA, particularly as autoantibody-producing cells. The ICs that further develop deposit in the joints and aggravate the inflammatory process. However, B-cell contribution in the very early stage of the disease remains unknown. The main goal of this work was to determine the concentration of cytokines potentially relevant for B-cell activation in serum from very early polyarthritis patients, with, Sociedade Portuguesa de Reumatologia/Schering-Plough 2005. R.A.M. and R.C. were funded by Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/30247/2006 and SFRH/BD/40513/2007, respectively. M.M.S.-C. was funded by Marie Curie Intra-European Fellowship PERG-2008-239422 and an EULAR Young Investigator Award.

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease of the axial skeleton, characterised by systemic osteoporosis along with new local bone formation. Previous studies have shown that serum levels of TNF, IL-6 and IL-17 are increased in AS patients and may be implicated in the development of secondary osteoporosis, since these cytokines are able to induce osteoclast (OC) differentiation and, therefore, bone resorption. In this work we aimed to assess the effects of TNF-blocking therapy in the systemic inflammatory environment of AS patients with active disease as well as in OC differentiation and activity. Patients with AS starting TNF-blocking therapy were recruited for this study and blood was collected at baseline and 6 months after the first treatment administration. We performed ELISAs in the serum of these patients to assess cytokine levels and bone turnover markers. We also characterised RANKL surface expression in circulating neutrophils, B and T lymphocytes and monocyte subpopulation frequency and phenotype by flow cytometry. We cultured circulating monocytes from AS patients, before and after therapy, and from healthy controls under osteoclastogenic conditions. We performed two functional assays (TRAP staining and resorption pit assay) and analysed the expression of osteoclast specific genes. We found no differences (before and after treatment) in any of the circulating monocytes’ subpopulations regarding frequency or cell death assessed by annexin staining. Phenotype analysis only revealed differences in the classical monocyte subpopulation where there was a significant decrease in HLA-DR surface expression after treatment. The frequency of RANKL positive B lymphocytes was reduced after treatment. No changes were observed on RANKL expression in neutrophils or T lymphocytes after treatment. Before TNF-blocking treatment AS patients have increased levels of pro-inflammatory cytokines when compared with healthy subjects. After TNF-blocking therapy IL-17, TGF-β and osteoprotegerin were significantly decreased. Interestingly, we observed that after TNF-blocking therapy, gene expression was favouring osteoclastogenesis and that differentiated OCs have increased resorption activity. Our results suggest that in AS patients there might be a paradoxical effect of TNF blocking therapy that induces OC activity.

Apolipoprotein E and undercaboxylated osteocalcin are associated with bone fragility but not with bone mineral density in osteoarthritis patients Ana Rodrigues, Joana Caetano-Lopes, Ana Lopes, Ana C Vale, Ines Aleixo, Ines P Perpetuo, Ana S Pena, Alexandra Faustino, Alexandre Sepriano, Joaquim Polido-Pereira, Elsa Vieira-Sousa, Bruno Vidal, Jose C Romeu, Pedro M Amaral, Luis G Rosa, Jose A Pereira da Silva, Jacinto Monteiro, Maria F Vaz, Joao E Fonseca, Helena Canhao

Background The reasons for the phenotypic differences between spondyloarthritis (SpA) and rheumatoid arthritis (RA) are still unclear. Slight divergences in cytokine networks driving the pathologies might contribute to the distinct clinical manifestations and may represent new treatment opportunities. Objectives The main goal of this work was to compare serum and synovial cytokines in established SpA and RA patients. Materials and methods The concentration of a panel of cytokines was measured in the serum of SpA and RA patients, as well as in synovial fluid from SpA, RA and osteoarthritis patients. Results The authors found that SpA and RA patients shared a similar pattern of cytokine concentration, with the exception of higher levels of interleukin-21 in the synovial fluid of RA patients. Conclusions Our results suggest that although SpA and RA are chronic inflammatory diseases with clearly distinct clinical manifestations, both pathologies share a similar cytokine profile, including Th17-related cytokines.

Osteoporosis is a systemic skeletal disease characterised by low bone mass, deterioration in microarchitecture, increased bone fragility and susceptibility to fracture. Besides structural properties, biological phenomena driven by differential gene expression could also contribute to the individual diversity of the structural and mechanical properties of bone that ultimately are reflected in different risks of fracture. The fracture risk assessment tool (FRAX) is an algorithm that takes into account clinical risk factors for fracture and dual energy x-ray absorptiometry results. It has been found …

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are systemic, immune-mediated diseases. In RA the main target are the peripheral joints while in AS the axial skeleton and enthesis are affected. RA is characterised by bone repair impairment and AS by exaggerated repair, leading to joint ankylosis. The cause for these differences is not yet understood, however we hypothesize that monocytes from AS patients exhibit reduced responses to osteoclastogenic and/or migratory stimulus. The aim of this work was to characterise bone remodelling regulators and the inflammatory signals that promote osteoclastogenesis and the circulating monocyte subpopulations to understand their response to inflammation and osteoclastogenic signals in untreated AS patients and compare them to RA patients and healthy controls. Untreated RA or AS patients with active disease and age and gender-matched healthy donors were recruited for this study. Blood was collected for flow cytometry measurements of RANKL expression and monocyte subpopulation characterisation. Serum was collected for cytokine and bone turnover marker quantification. We found that RANKL lymphocyte expression is higher in AS and RA patients when compared to healthy donors. When analyzing the monocyte sub-populations we found that both in the classical and the intermediate subpopulations, RANK expression was decreased in AS patients as compared to RA patients and healthy controls. In accordance, CTX-I serum level was also lower in AS patients than in healthy controls. In conclusion, and despite comparable osteoclastogenic stimuli in AS and RA patients, RANK expression is reduced in AS circulating monocytes which may contribute to the bone forming phenotype observed in AS patients.

Background The identification of predictive factors of poor response to methotrexate (MTX) in juvenile idiopathic arthritis (JIA) patients could contribute to optimize the treatment strategy, namely by the earlier introduction of biological treatments. Objectives To identify baseline clinical or laboratorial predictive factors of MTX poor response at 6 months in patients with JIA. Methods In a cohort of 141 JIA patients registered in Reuma.pt (the Portuguese Register of Rheumatic Diseases)we selected patients diagnosed after 2000 and that were treated at least for 6 months with MTX (at least 10mg/m2). Bivariate and multivariate logistic analyses were used to identify the predictors for non-response to MTX treatment during the first 6 months of treatment. Non-response to MTX was defined according the American College of Rheumatology pediatric 70 criteria. Concomitant treatment with NSAIDs and corticosteroids up to 10mg was allowed for all treatment groups. For this analysis we included: sex, age, age at disease onset, subtype, disease duration, rheumatoid factor (RF), ANA, HLA-B27, uveitis, age at MTX onset, disease duration until the start of MTX, baseline physician and patient/parents VAS, baseline active and limited joints and ESR. Results From the 58 patients identified, 23 were excluded (2 started biological therapy before 6 months, 3 were on concomitant DMARD’s, 12 had insufficient data, 1 developed criteria for systemic lupus erythematosus, 3 had interrupted MTX and 2 were loss for follow-up). From our population 43% have achieved ACR-ped 70 response and the remaining 57% did not reach this response criterion. We found no differences in the parameters analyzed between the 2 groups, except the age of MTX onset (p=0.03; responders 5 [3-11.5], non-responders 17.5 [11-22.6]) and parents/patients VAS (p=0.047; responders 50 [40.9-61.6], non-responders 20 [13-53.6]). We observed that older age at the beginning of MTX and lower patient/parents VAS were independently associated with poor response to MTX therapy. Accordingly, we have introduced in a logistic regression model both of these parameters. After multivariate analysis, age at the beginning of MTX was the only determinant independently associated with ACR-ped 70 non responders (p=0.05; OR 0.83). Conclusions In our cohort of JIA patients, older age at MTX onset was correlated with poorer response to a 6-month MTX course. Disclosure of Interest None Declared

Background and Objectives Rheumatoid arthritis (RA) is characterised by bone resorption and joint destruction. The receptor activator of NF-kB ligand (RANKL) plays a major role in bone loss because it is responsible for osteoclast differentiation and it is known that hyperactive immune system cells express surface RANKL. Several therapies commonly used for RA treatment have been shown to stop RA joint destruction. One of the hypothetical mechanisms explaining this effect could be an interference with the RANKL system. The aim of this work was to assess the effects of RA therapies in RANKL surface expression in different leukocyte populations by flow cytometry. Methods Forty-nine patients diagnosed with RA were recruited for this study. Seventeen patients were naive to any therapy, 14 were under methotrexate (MTX) – 8 of them at baseline of treatment with TNF blockers – and 18 patients were treated with TNF blockers. Blood was collected and total leukocytes were used for flow cytometry staining with anti human-CD66b for neutrophils, CD3 for T lymphocytes, CD19 for B lymphocytes and RANKL. Results There were no differences regarding gender distribution, age, disease activity, C-reactive protein (CRP) levels or erythrocyte sedimentation rate (ESR). Patients treated with MTX or TNF blockers have reduced RANKL expression in neutrophils, T and B lymphocytes (p = 0.0027, p = 0.0003 and p = 0.0032, respectively) when compared to untreated patients. However the number of circulating RANKL + T and B lymphocytes was increased in patients treated with TNF blockers when compared to naive patients (p = 0.0070 and p = 0.0183 respectively). No differences were found between groups regarding circulating number of leukocytes. We found no correlation of the studied parameters with CRP, ESR or DAS28. Conclusions RANKL surface expression on T and B lymphocytes decreases and RANKL + cells tend to accumulate in the circulation of patients treated with TNF blockers. The reasons for this effect are not clear but might be related to disturbances induced by TNF blockage in gene expression, cell activation and migration.

© 2010 Cascão et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by sustained synovitis. Recently, several studies have proposed neutrophils and Th17 cells as key players in the onset and perpetuation of this disease. The main goal of this work was to determine whether cytokines driving neutrophil and Th17 activation are dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration and before treatment (VERA). Methods: Cytokines related to neutrophil and Th17 activation were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed. Results: VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1b and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to induce neutrophil-mediated inflammation. In established RA this pattern is more evident within the SF. Early treatment with methotrexate or corticosteroids led to clinical improvement but without an impact on the cytokine pattern. Conclusions: VERA patients already display increased levels of cytokines related with Th17 polarization and neutrophil recruitment and activation, a dysregulation also found in SF of established RA. 0 Thus, our data suggest that a cytokine-milieu favoring Th17 and neutrophil activity is an early event in RA pathogenesis., This work was supported by a grant from Sociedade Portuguesa de Reumatologia/Schering-Plough 2005. RAM and RC were funded by Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/30247/2006 and SFRH/BD/40513/2007, respectively. MMS-C was funded by Marie Curie Intra-European Fellowship PERG-2008-239422 and a EULAR Young Investigator Award.

extremely useful. Methods: We have measured the trend of characteristic parameters of bone metabolism (serum calcium, osteocalcin, β-Ctx) in 31women with severe postmenopausal osteoporosis (mean age 69,2±8,1 years) treated with the anabolic drug PTH 1-84 at the baseline and after 6, 12 and 18 months of therapy. Indeed, we have measured hip and spine T-score at baseline and at the end of therapy. Results: In the 31 patients treated for 18 months, while serum calcium values remained on normal range (9,44±0,5 mg/dl at the baseline, 9,60±0,6 mg/dl after 6 months, 9,60±0,5 after 12 months and 9,41±0,4 at the end of treatment), after 18 months of treatment the T-score raised compaired with the baseline both at the hip (-2,27±0,7 at baseline, -2,0±0,7 at the end of the treatment) and at the spine (-3,57±0,8 at the baseline, -3,0±0,7 at the end of the treatment). Osteocalcin values raised 4,14 times after 6 months compared with the baseline, undergoing a further slight increase after 12 months of therapy and, although they falled down after 18 months, they still maintained at a level equal to 2,88 times above the baseline, testifying the strong anabolic effect of PTH. The levels of β-Ctx showed a trend similar to osteocalcin: after 6 months of treatment with PTH, they raised 2,55 times compared with the baseline, they still increased after 12 months (2,79 times compared with the baseline) and after 18 months, while resulting falled down compared with 12 months, they showed an increase of 1,81 times compared with the baseline. Conclusions: Results obtained in our clinical experience confirm the efficacy of treatment with PTH 1-84, testified by the statistically significant increase of T-score both at the spine and at the hip, besides the increase of β-Ctx and osteocalcin levels, indicating strong osteoanabolic activity. Disclosure of Interest: None declared

Rheumatoid arthritis (RA) is a systemic disease characterised by hyperactivation of the immune system leading to chronic inflammation and joint damage. The inflammatory environment potentiates bone resorption, modulating the balance between RANKL and osteoprotegerin (OPG). Prednisone (PDN), methotrexate (MTX), anti-tumour necrosis factor (TNF) and anti-interleukin 6 (IL6) receptor therapies reduce disease activity, but their differential biological impact …

Rheumatoid arthritis (RA) is a systemic disease characterised by hyperactivation of the immune system and by stimulation of bone resorption by activation of osteoclasts (OC) leading to joint destruction. To assess the activity of OC differentiated from circulating monocytes in patients with RA treated with methotrexate (MTX), anti-tumour necrosis factor (anti-TNF) drugs and tocilizumab. 19 women with RA were recruited, 6 of whom were …

To identify the cytokines that are present at higher levels in the serum and synovial fluid (SF) of patients with established rheumatoid arthritis (RA). Interleukin (IL)1β, IL2, IL4, IL6, IL8, IL10, IL12 (p70), IL17A, IL18, IL22, IL23, interferon (IFN)γ, leptin, MCP-1, MIP-1α, OPG, transforming growth factor β (TGFβ) and …

© 2010 Caetano-Lopes et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Introduction: In this study we used a mice model of chronic arthritis to evaluate if bone fragility induced by chronic inflammation is associated with an imbalance in bone turnover and also a disorganization of the bone type I collagen network. Methods: Serum, vertebrae and femur bones were collected from eight-month-old polyarthritis SKG mice and controls. Strength of the femoral bones was evaluated using three-point bending tests and density was assessed with a pycnometer. Bone turnover markers carboxy-terminal collagen cross-linking telopeptides (CTX-I) and amino-terminal propeptide of type I procollagen (PINP) were measured in serum. The organization and density of bone collagen were analyzed in vertebrae using second-harmonic generation (SHG) imaging with a two-photon microscope and trabecular bone microstructure was assessed by scanning electron microscope (SEM). Results: Femoral bones of SKG mice revealed increased fragility expressed by deterioration of mechanical properties, namely altered stiffness (P = 0.007) and reduced strength (P = 0.006), when compared to controls. Accordingly, intertrabecular distance and trabecular thickness as observed by SEM were reduced in SKG mice. PINP was significantly higher in arthritic mice (9.18 ± 3.21 ng/ml) when compared to controls (1.71 ± 0.53 ng/ml, P < 0.001). Bone resorption marker CTX-I was 9.67 ± 3.18 ng/ml in arthritic SKG mice compared to 6.23 ± 4.11 ng/ml in controls (P = 0.176). The forward-to-backward signal ratio measured by SHG was higher in SKG animals, reflecting disorganized matrix and loose collagen structure, compared to controls. Conclusions: We have shown for the first time that chronic arthritis by itself impairs bone matrix architecture, probably due to disturbed bone remodeling and increased collagen turnover. This effect might predispose patients to bone fragility fractures., This work was supported by the PTDC/SAU-BEB/65992/2006 grant from Fundação para a Ciência e Tecnologia.

Methods Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were stratified according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n=13); between the 4 and 7 day (n=33); and after one week from the fracture (n=10). Inflammationand bone metabolism-related genes were assessed in trabecular bone.