Terence Rooney
email: rooneyterence@gmail.com

III Carl K Edwards

Martina Gogarty

Laura Greenan

Douglas J Veale

Oliver Fitzgerald

Jean-Michel Dayer

Barry Bresnihan

Synovial tissue rank ligand expression and radiographic progression in rheumatoid arthritis: observations from a proof-of-concept randomized clinical trial of cytokine blockade

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

progressive joint erosion, and may be independent of the clinical response to targeted therapy. The potential therapeutic importance of modulating RANKL in RA is highlighted, if radiographic arrest is to be achieved.

INTRODUCTION

Rheumatoid arthritis (RA) is a disabling, inflammatory arthropathy, commonly associated with progressive articular cartilage loss, bone erosion and deformity [1]. In recent years, the use of biologic therapies targeting specific mediators of inflammation and matrix destruction has been associated with enhanced control of joint inflammation and retardation of progressive structural joint damage [START_REF] Bresnihan | Effects of anakinra monotherapy on joint damage in patients with rheumatoid arthritis. Extension of a 24week randomized, placebo-controlled trial[END_REF][START_REF] Lipsky | Infliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group[END_REF][START_REF] Bathon | A comparison of etanercept and methotrexate in patients with early rheumatoid arthritis[END_REF][START_REF] Keystone | Radiographic, clinical, and functional outcomes of treatment with adalimumab (a human anti-tumor necrosis factor monoclonal antibody) in patients with active rheumatoid arthritis receiving concomitant methotrexate therapy: a randomized, placebo-controlled, 52-week trial[END_REF][START_REF] Cohen | Rituximab for rheumatoid arthritis refractory to anti-tumor necrosis factor therapy: Results of a multicenter, randomized, double-blind, placebo-controlled, phase III trial evaluating primary efficacy and safety at twenty-four weeks[END_REF][START_REF] Kremer | Effects of abatacept in patients with methotrexate-resistant active rheumatoid arthritis: a randomized trial[END_REF].

A critical common pathway leading to localized articular bone erosion in RA involves the activation of RANK by RANKL, promoting differentiation of osteoclast progenitors into mature osteoclasts which effect bone resorption [START_REF] Goldring | Pathogenesis of bone and cartilage destruction in rheumatoid arthritis[END_REF]. RANKL is detectable in T-cells and fibroblasts in RA synovial tissue, and its expression is regulated by many proinflammatory cytokines and their inhibitors [START_REF] Schett | Mechanisms of Disease: the link between RANKL and arthritic bone disease[END_REF]. OPG is a naturally occurring inhibitor of RANK / RANKL interaction, is also expressed in RA synovial tissue and acts as a potent regulator of osteoclastogenesis [START_REF] Schett | Mechanisms of Disease: the link between RANKL and arthritic bone disease[END_REF]. Therapies modulating the OPG / RANKL axis have resulted in retardation of joint destruction in animal models of inflammatory arthritis, and recently in patients with RA [START_REF] Stolina | RANKL is a marker and mediator of local and systemic bone loss in two rat models of inflammatory arthritis[END_REF][START_REF] Redlich | Tumor necrosis factor alpha-mediated joint destruction is inhibited by targeting osteoclasts with osteoprotegerin[END_REF][START_REF] Zwerina | Single and combined inhibition of tumor necrosis factor, interleukin-1, and RANKL pathways in tumor necrosis factor-induced arthritis: effects on synovial inflammation, bone erosion, and cartilage destruction[END_REF][START_REF] Romas | Osteoprotegerin reduces osteoclast numbers and prevents bone erosion in collageninduced arthritis[END_REF][START_REF] Cohen | Denosumab treatment effects on structural damage, bone mineral density, and bone turnover in rheumatoid arthritis: a twelve-month, multicenter, randomized, double-blind, placebo-controlled, phase II clinical trial[END_REF].

Although a number of previous studies have examined synovial tissue OPG / RANKL expression in RA, few have examined the expression of OPG and RANKL in RA synovial tissue before and after biologic therapy [START_REF] Crotti | Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis[END_REF][START_REF] Crotti | Variability of RANKL and osteoprotegerin staining in synovial tissue from patients with active rheumatoid arthritis: quantification using color video image analysis[END_REF][START_REF] Haynes | Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls[END_REF][START_REF] Fonseca | Inflammatory cell infiltrate and RANKL/OPG expression in rheumatoid synovium: comparison with other inflammatory arthropathies and correlation with outcome[END_REF][START_REF] Makrygiannakis | Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis[END_REF][START_REF] Catrina | Anti-tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis[END_REF][START_REF] Kirkham | Synovial membrane cytokine expression is predictive of joint damage progression in rheumatoid arthritis: a two-year prospective study (the DAMAGE study cohort)[END_REF][START_REF] Haynes | 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[END_REF]. The aim of the present study was to evaluate the utility of synovial tissue levels of receptor RANKL and OPG as biomarkers of progressive structural joint damage, disease activity and therapeutic response, during a trial of selective cytokine blockade in RA. It was conducted as a component of the SPECTRA trial, a single centre, phase II open-label synovial tissue biopsy study, involving therapy with anakinra administered either alone or in combination with PEGylated soluble tumour necrosis factor (TNF) receptor type I (pegsunercept), for a planned duration of 52 weeks [START_REF] Rooney | Synovial Tissue and Serum Biomarkers of Disease Activity, Therapeutic Response and Radiographic Progression. Analysis of a Proof-of-Concept Randomized Clinical Trial of Cytokine Blockade[END_REF][START_REF] Edwards | Design of PEGylated soluble tumor necrosis factor receptor type I (PEG sTNF-RI) for chronic inflammatory diseases[END_REF].

PATIENTS AND METHODS

Patients

As previously described, eligible patients for the SPECTRA study were aged 18 years or older, had active RA of ≤ 10 years duration diagnosed according to the 1987 revised American College of Rheumatology (ACR) criteria, and had ≥ 9 tender joints (of 68 evaluated), ≥ 6 swollen joints (of 66 evaluated), a clinically inflamed knee joint, and at least 2 of the following 3 criteria: morning stiffness lasting ≥ 45 minutes, C-reactive protein (CRP) ≥ 1.5 mg/dl, erythrocyte sedimentation rate (ESR) ≥ 28mm/h [START_REF] Rooney | Synovial Tissue and Serum Biomarkers of Disease Activity, Therapeutic Response and Radiographic Progression. Analysis of a Proof-of-Concept Randomized Clinical Trial of Cytokine Blockade[END_REF][START_REF] Arnett | The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis[END_REF].

Major exclusion criteria included prior use of any TNFα antagonist or anakinra, a history of any other major chronic inflammatory disease, malignancy (besides nonmelanoma skin carcinoma within 5 years) or demyelinating disorder, a positive tuberculin skin test, history of tuberculosis (TB) infection, or chest radiograph suggestive of prior TB, any infection requiring systemic antibiotics within 12 weeks of screening, any uncontrolled medical condition, pregnancy or breast feeding, and ACR functional class IV disease [START_REF] Hochberg | The American College of Rheumatology 1991 revised criteria for the classification of global functional status in rheumatoid arthritis[END_REF]. Doses of concomitant RA therapies such as non-steroidal anti-inflammatory drugs (NSAIDs) and oral corticosteroids (≤ 10mg/day of prednisolone or equivalent) must have been stable for 4 weeks prior to screening.

Protocol

Patients were recruited for this 52-week, single-center, open-label study at St. Vincent's University Hospital (SVUH), Dublin. A total of 30 patients were to be recruited. The protocol was approved by the institution's ethics committee, and patients gave their written informed consent. Patients were randomly assigned to receive self-administered single subcutaneous injections of either anakinra 100 mg/day, or anakinra 100 mg/day plus pegsunercept 800μg/kg twice weekly.

Traditional DMARDs were discontinued at least 6 weeks prior to screening. Doses of concomitant NSAID therapy were kept constant throughout the study. If necessary, oral corticosteroid therapy could be temporarily increased for ≤ 2 weeks up to twice during the study. Up to 2 intra-articular corticosteroid injections were also permitted if required.

Clinical assessment

ACR20, ACR50, and ACR70 responses were assessed at week 4, and at the final timepoint [START_REF] Felson | American College of Rheumatology. Preliminary definition of improvement in rheumatoid arthritis[END_REF]. Patients who withdrew from the study prematurely due to active RA or adverse events were classified as non-responders. DAS28 scores were also calculated at baseline, week 4, and at the final timepoint [START_REF] Prevoo | Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis[END_REF].

Radiographic assessment

Radiographs of the hands and feet were obtained at baseline, and at weeks 24 and 52.

Radiographic damage was quantified by 2 blinded observers (TR, BB) using the van der Heijde modification of the Sharp scoring system [START_REF] Van Der Heijde | How to read radiographs according to the Sharp/van der Heijde method[END_REF]. Modified Sharp scores were then calculated as the mean of the scores obtained by the 2 observers. The minimum clinically important difference (MCID) in radiographic scores over time was calculated as the smallest detectable difference (SDD) for these 2 observers using the limits of agreement method of Bland and Altman [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF]. Progression scores below this threshold were classified as non-significant. Using an assumption of linear progression, inferred rates of annual radiographic progression prior to study entry were calculated using damage scores and disease duration at study baseline.

Acquisition and preparation of synovial tissue

Each patient underwent arthroscopy of a clinically inflamed knee joint at baseline, immediately prior to commencing therapy. Arthroscopy of the same knee was repeated following 4 and 52 weeks of therapy, or at the time of withdrawal from the study. Multiple synovial tissue biopsies (at least 6) were obtained throughout the knee joint under local anaesthesia using a 2.7mm arthroscope and 2.8mm universal biopsy forceps (Karl Storz GmbH & Co., Tuttlingen, Germany). The synovial tissue was prepared as previously described [START_REF] Rooney | Synovial tissue interleukin-18 expression and the response to treatment in patients with inflammatory arthritis[END_REF]. In brief, tissue samples were snap-frozen together en bloc in Tissue-Tek OCT (Miles Inc. Diagnostic Division, Elkhart, IN) by immersion in liquid nitrogen. Frozen blocks were stored in liquid nitrogen until sectioned for staining. Seven μm thick sections were cut in a cryostat and mounted on glass slides. The slides were fixed in acetone at room temperature for 10 minutes and stored at -70 0 C until immunohistochemical analysis was performed. After thawing for 20 minutes at room temperature, sections were stained with anti-OPG (MAB 805, R&D Systems, Minneapolis, USA) and anti-RANKL (sc-9073, Santa Cruz Biotechnology, Santa Cruz, USA). A standard three-stage immunoperoxidase method was used for each antibody (Vector Laboratories, Lumigen Inc., Southfiela, USA), and was followed by counterstaining with haematoxylin. Sections in which the relevant primary antibody was replaced with isotype-matched irrelevant primary antibodies served as negative controls.

Microscopic analysis of synovial tissue.

For each stained tissue section, a single 40X digital image was acquired as previously described [START_REF] Rooney | Microscopic measurement of inflammation in synovial tissue: inter-observer agreement for manual quantitative, semiquantitative and computerised digital image analysis[END_REF]. All slides were coded to maintain operator blinding. Image acquisition, modification and analysis were performed using AnalySIS software (Soft Imaging Systems, Denver, Colorado) and a personal computer with Intel Pentium III 800 MHz processor and Windows TM 2000 Professional Version 5.0.2195 environment. For each antibody, separate threshold RGB values were specified for total tissue (blue or brown) and for positive stain (brown). These threshold values remained constant throughout the analysis of sections stained for a given marker. The mean optical density (MOD) was calculated for the positively stained areas of each image and expressed as a % of the maximum possible density. The integrated optical density (IOD) was then calculated as the product of the % tissue occupied by positive stain and the MOD.

Study closure

In February 2003, while recruitment was ongoing, preliminary analyses of the results of 2 larger phase 3 clinical trials evaluating therapy with anakinra plus either etanercept or pegsunercept in RA indicated an increased incidence of serious infection in patients receiving combination therapy [START_REF] Genovese | Combination therapy with etanercept and anakinra in the treatment of patients with rheumatoid arthritis who have been treated unsuccessfully with methotrexate[END_REF]. All studies of combination anakinra plus anti-TNFα therapy were suspended. All patients involved in the present study were contacted and informed in person of the reasons behind the study's early closure.

Final assessments (including arthroscopic synovial tissue biopsy) were obtained, and patients receiving combination therapy were offered monotherapy by their supervising physician. Following study closure, week 26 and 52 radiographs were obtained, where possible, from all remaining patients who had participated in the study.(Terrance-you did a nice job of writing this---sometime let's discuss the behind the scenes discussions over this. There were actually 2-fold higher infections with the IL1ra+Enbrel versus the IL1ra+PEGsunercept therapy. Amgen did not want these data out there for obvious reasons.)

Statistical analysis

Cross-sectional differences between pairs of patient groups were evaluated using the Mann-Whitney U test. Proportional differences between groups were evaluated using Chi-squared tests. The Wilcoxon signed ranks test was employed to examine longitudinal differences in variables between baseline and subsequent timepoints.

Correlations were calculated using Spearman's Rho. For efficacy measures, intent-totreat analysis was performed for all patients who received at least one dose of study drug. The performance of baseline variables as predictors of radiographic progression (presence or absence of clinically significant progression) was evaluated using receiver operating characteristics (ROC) curves. A ROC curve plots sensitivity against 1-specificity for a range of values of a given independent variable, where that variable is being evaluated as a test for a dependent dichotomous variable, whose 2 possible values are typically taken as either "positive" or "negative". Area under the ROC curve (AUROC) values for a given predictor near 1 or 0 identify it as a good test for a specified outcome, while values near 0.5 indicate a poor test.

To evaluate the relationship between radiographic outcome and sustained expression of synovial OPG / RANKL during the course of the study, AUCs were calculated using baseline, week 4 and final timepoint tissue data. Linear change in these tissue biomarker levels between timepoints was assumed. Since the final timepoint differed between patients, time-averaged AUCs were calculated by dividing the AUC for each patient by the number of weeks from baseline to final biopsy, allowing inter-patient comparison. All statistical calculations were performed using SPSS software (version 11.0). Two-tailed significance testing was employed throughout, and p-values less than 0.05 were taken as significant.

RESULTS

Patient characteristics and outcomes

Twenty-two patients were randomized to the monotherapy or combination therapy groups and consented to serial arthroscopic synovial biopsies. Baseline demographic and clinical characteristics have been described previously [START_REF] Rooney | Synovial Tissue and Serum Biomarkers of Disease Activity, Therapeutic Response and Radiographic Progression. Analysis of a Proof-of-Concept Randomized Clinical Trial of Cytokine Blockade[END_REF]. In brief, the median age was 52 years and 18 patients (82%) were female. The median (range) disease duration was 23 (3-87) months, and 10 patients (46%) had symptoms of RA for less than 1 year. Sixteen patients (73%) were rheumatoid factor positive. The patient population had moderate to severe RA, and median tender and swollen joint counts were 20 (of 68) and 16 (of 66), respectively. Of the total patient group, 11 (50%) were DMARD naïve; the median number of previous DMARDs was 1. At the time of study closure, 3 patients (1 monotherapy, 2 combination) had completed 52 weeks of treatment, while 12 patients (6 in each treatment group) were receiving active treatment. These patients were withdrawn from the study at a median (range) timepoint of 31 weeks. All patients contributed a 2 nd synovial biopsy at 4 weeks and 16 contributed a 3 rd biopsy after a median (range) of 32 (12-52) weeks. A total of 17 patients contributed post-baseline radiographic scores.

The clinical and radiographic outcomes are presented in association with the levels of OPG and RANKL expression on an individual patient basis in Table 1. Of the total study group, 11 patients (50%) achieved an ACR20 response or better at the final timepoint. ACR20, 50 and 70 response rates at the final timepoint were 64%, 64% and 46% with combination therapy, and 36%, 9%, and 0% with monotherapy, respectively. Of the 15 patients with 12-month radiographic data, the proportions of responders (ACR20 or better) and non-responders who demonstrated clinically significant increases in total Sharp score were 28% and 40%, respectively (there were no statistically significant differences between response groups).

Baseline expression of OPG and RANKL in synovial tissue and progressive joint damage OPG and RANKL expression was observed in all synovial tissue sections evaluated.

OPG expression was largely confined to endothelial cells, whereas RANKL was widely expressed by inflammatory cells within the lining layer, sublining layer and in perivascular regions. Levels of expression of both biomarkers varied widely in the total patient group (Table 1) -baseline IODs ranged from 4.2 to 20.9 for OPG and from 3.6 to 35.2 for RANKL expression. At baseline, there was a statistically significant correlation between synovial tissue RANKL expression and serum CRP levels (Spearman's Rho 0.61, p<0.01), but not DAS28. There were no correlations between synovial tissue OPG expression and measures of disease activity.

In order to evaluate the potential utility of synovial tissue OPG and RANKL expression as predictors of progressive joint damage, correlations between baseline tissue biomarker levels, as well as the RANKL:OPG ratio, and changes in modified total Sharp, erosion and narrowing scores at 6 and 12 months were evaluated. ROC curve analysis was also employed to determine the ability of these baseline tissue signals to predict the occurrence of clinically meaningful progressive structural damage. For comparison, known predictors of radiographic outcome (ESR and serum CRP) were subjected to the same analysis. The results are presented in Table 2.

Baseline RANKL expression correlated significantly with erosive progression at both the 6 and 12 month time-points and also demonstrated predictive utility in ROC curve analysis. Similar results were observed for the baseline RANKL:OPG ratio and erosive progression at 1 year. As expected, both ESR and CRP also demonstrated predictive utility for erosive progression at 6 and 12 months. No significant relationship between OPG expression at baseline and radiographic change was observed. Linear regression could not be performed as sufficient variables could not be normalized using transformation. When logistic regression was used to examine the predictive value of multiple baseline variables simultaneously, no variable emerged as independently predictive of significant progression in any measure of joint damage. Neither early changes in, nor sustained expression (time-averaged AUC) of either biomarker demonstrated any significant correlations with radiographic outcome (data not shown).

Cross-sectional analysis of OPG and RANKL expression and disease activity

The relationship between measures of disease activity and synovial tissue OPG / RANKL expression are presented in Table 3. RANKL expression correlated significantly with ESR and CRP levels at baseline and with CRP and DAS28 scores at week 4. Strikingly however, at the final timepoint (median 32 weeks) this relationship was lost, and Spearman's Rho values for correlations between RANKL and disease activity were as low as 0.03. A similar trend was observed for the RANKL:OPG ratio. OPG expression did not correlate significantly with disease activity in cross-sectional analysis at any timepoint.

Effects of biologic therapy on OPG and RANKL expression in synovial tissue

Sixteen of the 22 study patients had baseline, 4 week and final biopsies. Table 4 highlights changes in tissue OPG and RANKL expression after treatment in the total group, by ACR response, and in the individual treatment groups. No significant changes in median OPG and RANKL IOD values were observed after treatment in the total group, or in either therapeutic subgroup. Ten of the 16 patients demonstrated an ACR20 response. Changes in tissue levels of OPG correlated significantly with DAS28 at both the early and late time-points (Table 3: Spearman's Rho 0.45 and 0.59, respectively, p<0.05). In ACR20 responders, a statistically significant decrease in OPG expression from baseline was observed, which exceeded the minimal, nonsignificant change observed in patients who did not respond to treatment (-33.7% vs 5.0%, respectively, p<0.05). RANKL expression did not change significantly from baseline following therapy in the total group or in any clinical response subgroup.

DISCUSSION

This paper is the first to describe the utility of OPG and RANKL expression, quantified using computerized digital image analysis, as synovial tissue biomarkers of radiographic progression, disease activity and therapeutic response, exclusively during targeted cytokine blockade. These 2 biomarkers were detected in all synovial tissue sections evaluated. The intensity with which they were expressed varied widely between individual patients and timepoints. Baseline RANKL expression correlated with erosive progression at 6 and 12 months with a high level of statistical significance. In cross-sectional analysis, RANKL expression correlated with measures of disease activity at baseline and week 4, but not at the final timepoint, while OPG levels did not reflect disease activity. In longitudinal analysis following treatment, tissue OPG expression decreased significantly in clinical responders and changed in proportion to the magnitude of the therapeutic response, whereas neither RANKL expression nor the ratio of RANKL:OPG in synovial tissue changed significantly in any response subgroup.

A number of previous cross-sectional studies have evaluated synovial tissue or fluid levels of RANKL and OPG in RA [START_REF] Crotti | Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis[END_REF][START_REF] Crotti | Variability of RANKL and osteoprotegerin staining in synovial tissue from patients with active rheumatoid arthritis: quantification using color video image analysis[END_REF][START_REF] Haynes | Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls[END_REF][START_REF] Fonseca | Inflammatory cell infiltrate and RANKL/OPG expression in rheumatoid synovium: comparison with other inflammatory arthropathies and correlation with outcome[END_REF][START_REF] Makrygiannakis | Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis[END_REF][START_REF] Catrina | Anti-tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis[END_REF][START_REF] Kirkham | Synovial membrane cytokine expression is predictive of joint damage progression in rheumatoid arthritis: a two-year prospective study (the DAMAGE study cohort)[END_REF][START_REF] Haynes | 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[END_REF][START_REF] Hein | sRANKL and OPG in serum and synovial fluid of patients with rheumatoid arthritis in comparison to non-destructive chronic arthritis[END_REF][START_REF] Feuerherm | Elevated levels of osteoprotegerin (OPG) and hepatocyte growth factor (HGF) in rheumatoid arthritis[END_REF][START_REF] Skoumal | Osteoprotegerin and the receptor activator of NF-kappa B ligand in the serum and synovial fluid. A comparison of patients with longstanding rheumatoid arthritis and osteoarthritis[END_REF]. While synovial fluid RANKL, but not OPG levels, correlated with the acute phase response in one study of patients with active RA, the present study provides the first observation of a significant crosssectional correlation between measures of disease activity and RANKL in synovial tissue [START_REF] Hein | sRANKL and OPG in serum and synovial fluid of patients with rheumatoid arthritis in comparison to non-destructive chronic arthritis[END_REF]. Several cross-sectional studies have highlighted differences in tissue RANKL and OPG expression between active and inactive RA or between RA and other diseases [START_REF] Crotti | Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis[END_REF][START_REF] Haynes | Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls[END_REF][START_REF] Fonseca | Inflammatory cell infiltrate and RANKL/OPG expression in rheumatoid synovium: comparison with other inflammatory arthropathies and correlation with outcome[END_REF]. In 1 study evaluating RANKL expression in RA, SpA, OA and healthy controls, the highest tissue RANKL levels were observed in RA patients with active disease [START_REF] Crotti | Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis[END_REF]. In a second study, OPG was absent from synovial tissue sections obtained from patients with active RA, in whom, conversely, the highest levels of RANKL expression were observed [START_REF] Haynes | Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls[END_REF]. A significant inverse relationship between fluid levels of RANKL and OPG has been reported in patients with RA and OA, and when these patients were treated as a signal disease group [START_REF] Skoumal | Osteoprotegerin and the receptor activator of NF-kappa B ligand in the serum and synovial fluid. A comparison of patients with longstanding rheumatoid arthritis and osteoarthritis[END_REF].

In the present study, OPG levels decreased following targeted therapy in proportion to the clinical response, while RANKL remained unchanged from baseline at the final timepoint, despite substantial reductions in disease activity. The effects of therapy on OPG / RANKL expression in RA synovial tissue have been evaluated in 3 previous studies [START_REF] Makrygiannakis | Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis[END_REF][START_REF] Catrina | Anti-tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis[END_REF][START_REF] Haynes | 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[END_REF]. In the first, tissue OPG and RANKL were quantified in 18 patients before and 8 weeks after commencement of infliximab or etanercept therapy [START_REF] Catrina | Anti-tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis[END_REF]. In keeping with the current findings, RANKL levels did not change significantly following treatment. However, in contrast to the observations arising from the present study, tissue OPG expression increased significantly following successful TNFα blockade. In a second study from the same center, synovial biopsies were obtained from the knees of patients before and 2 weeks after a single intra-articular corticosteroid injection [START_REF] Makrygiannakis | Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis[END_REF]. In this setting, OPG levels remained static, while RANKL expression decreased significantly following the therapeutic intervention. In a final study, patients receiving conventional DMARD therapy were followed with serial synovial biopsies for 3 years [START_REF] Haynes | 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[END_REF]. Longitudinal changes in tissue RANKL, but not OPG expression, reflected clinical and erosive change.

There are a number of possible reasons behind the discrepant OPG / RANKL signals observed among these studies. It is likely that differing therapies, designs, timeframes, staining, and quantification systems all contribute. In a study utilizing synovial tissue obtained from patients with spondylarthropathy, tissue OPG and RANKL signals differed substantially between differing antibodies [START_REF] Vandooren | The abundant synovial expression of the RANK/RANKL/Osteoprotegerin system in peripheral spondylarthritis is partially disconnected from inflammation[END_REF]. The present study involved anakinra therapy in all patients. No previous studies have evaluated the effects of anakinra on synovial tissue OPG. It has been shown that IL-1β can stimulate OPG production from RA fibroblast-like synoviocytes [START_REF] Yano | Synovial cells from a patient with rheumatoid arthritis produce osteoclastogenesis inhibitory factor/osteoprotegerin: reciprocal regulation of the production by inflammatory cytokines and basic fibroblast growth factor[END_REF]. It is biologically plausible, therefore, that therapy specifically antagonising this cytokine should result in OPG down-regulation.

A further novel aspect of the present study was the observation that high pretreatment synovial tissue RANKL levels and the ratio of tissue RANKL:OPG predicted progressive joint erosion, despite clinically effective targeted therapy. Two previous studies, both following observational cohorts, examined the potential utility of tissue RANKL as an early predictor of structural outcome in RA [START_REF] Fonseca | Inflammatory cell infiltrate and RANKL/OPG expression in rheumatoid synovium: comparison with other inflammatory arthropathies and correlation with outcome[END_REF][START_REF] Kirkham | Synovial membrane cytokine expression is predictive of joint damage progression in rheumatoid arthritis: a two-year prospective study (the DAMAGE study cohort)[END_REF]. In one, RANKL and OPG proteins were detected using immunofluorescence, while quantitative reverse transcription polymerase chain reaction was employed to detect RANKL mRNA in the second. In these studies, radiographic follow-up took place at timepoints between 2 and 3 years following the initial assessment. Unlike the present 1-year study, baseline tissue RANKL failed to predict subsequent progressive joint damage in either. It is likely that differing quantification methods, varying therapy, and the longer follow-up period account for the lack of predictive signal in these studies.

In keeping with a previous study, high baseline ESR and CRP levels, which correlated with RANKL expression, were also associated with subsequent radiographic progression in univariate analysis [START_REF] Scott | Prognostic factors in early rheumatoid arthritis[END_REF]. Multiple linear regression analysis would be required to examine whether the relationship between RANKL and erosive progression was independent of its correlation with the acute phase response. This was not possible in the present study as the data could not be sufficiently normalized by transformation. A larger sample size would serve to clarify this issue. The association observed in this study between intense RANKL expression at baseline and progressive articular erosion is, however, entirely consistent with the biologic role of this mediator. Failure of RANKL to decrease following clinically efficacious therapy is a possible explanation for the discordance between radiographic progression and the clinical response to targeted therapy that has been observed previously [START_REF] Smolen | Evidence of radiographic benefit of treatment with infliximab plus methotrexate in rheumatoid arthritis patients who had no clinical improvement: a detailed subanalysis of data from the anti-tumor necrosis factor trial in rheumatoid arthritis with concomitant therapy study[END_REF].

Moreover, in studies using animal models of RA to evaluate novel therapies targeting the RANKL pathway, radiographic benefit without clinical improvement was observed, in keeping with the hypothesis that distinct, though related mechanisms may underlie synovitis and joint destruction [START_REF] Redlich | Tumor necrosis factor alpha-mediated joint destruction is inhibited by targeting osteoclasts with osteoprotegerin[END_REF][START_REF] Zwerina | Single and combined inhibition of tumor necrosis factor, interleukin-1, and RANKL pathways in tumor necrosis factor-induced arthritis: effects on synovial inflammation, bone erosion, and cartilage destruction[END_REF][START_REF] Romas | Osteoprotegerin reduces osteoclast numbers and prevents bone erosion in collageninduced arthritis[END_REF][START_REF] Van Den Berg | Uncoupling of inflammation and destruction in rheumatoid arthritis: myth or reality?[END_REF]]. These observations have recently been replicated in humans with denosumab, a monoclonal antibody which targets RANKL [START_REF] Cohen | Denosumab treatment effects on structural damage, bone mineral density, and bone turnover in rheumatoid arthritis: a twelve-month, multicenter, randomized, double-blind, placebo-controlled, phase II clinical trial[END_REF].

A number of important limitations should prompt caution in interpreting the findings from this study: the patient numbers were small, the study was terminated prematurely, and an anti-TNF monotherapy arm was not included. The study was not specifically powered to detect particular outcomes or inter-group differences. It would be desirable to employ multivariate analysis to evaluate whether the relationship between RANKL and erosive progression was independent, or reflecting other interactions. The sample size in this study precluded such an approach. Single primary antibodies were used to stain for RANKL and OPG. Ideally the signals observed in the study should be validated using other antibodies. Nevertheless, this is the first study in which a direct relationship between intense baseline synovial tissue expression of RANKL, a pivotal mediator of osteoclastogenesis, and progressive bone erosion has been described in RA. Excellent clinical responses to cytokine-targeted therapy were not necessarily accompanied by RANKL suppression in tissue. The potential therapeutic importance of modulating RANKL in RA is therefore highlighted, if radiographic arrest is to be achieved [START_REF] Schett | Mechanisms of Disease: the link between RANKL and arthritic bone disease[END_REF]

Table 2 .

 2 . = osteoprotegerin, RANKL = receptor activator of NF-B. ACR responses are at final timepoint. Treatment group 1 received anakinra, group 2 received anakinra + pegsunercept. IOD = integrated optical density. Patients with clinically significant radiographic progression are highlighted in grey. See text for further definitions. Baseline synovial tissue RANKL, OPG and progressive structural damage All values are Spearman's Rho. * P < 0.05, ** P < 0.01 for correlations. † P < 0.05, † † P < 0.01 for areas under ROC curves. ROC curves were constructed using baseline biomarkers as test variables and presence or lack of clinically significant progression in structural damage as outcome. See text andTable 1 for further definitions.

	Patient ACR response Treatment group		OPG, IOD		RANKL, IOD
				Baseline Week 4 Final	Baseline Week 4 Final
	1	ACR70	2	15.1	7.0	5.2	23.6	8.6	6.8
	2	ACR70	2	6.2	3.5	.	11.1	5.4	.
	3	ACR70	2	5.7	7.4	5.1	13.6	8.2	14.7
	4	ACR70	2	20.9	20.4	8.5	17.4	26.2	26.6
	5	ACR70	2	12.7	15.8	14.3	16.7	8.5	16.6
	6	ACR50	1	9.5	8.9	6.3	12.7	10.4	9.8
	7	ACR50	2	6.9	9.4	1.7	3.6	5.0	5.4
	8	ACR50	2	7.1	7.8	5.7	14.6	15.8	4.8
	9	ACR20	1	19.4	7.0	9.7	4.4	11.4	5.0
	10	ACR20	1	6.6	4.1	.	17.6	17.2	4.7
	11	ACR20	1	11.9	9.1	11.7	31.1	23.7	33.0
	12	-	2	8.5	1.4	.	18.6	5.5	.
	13	-	1	6.0	8.6	.	8.4	6.3	.
	14	-	1	10.0	9.1	.	13.9	11.5	.
	15	-	2	8.5	6.4	10.1	9.5	8.9	10.5
	16	-	1	8.5	12.6	7.3	8.5	20.2	13.6
	17	-	1	4.2	10.2	9.0	4.5	14.1	18.7
	18	-	1	14.5	10.3	7.5	24.0	19.9	6.0
	19	-	2	9.2	17.2	8.5	11.2	20.7	7.4
	20	-	2	6.8	4.5	8.0	9.5	13.7	13.8
	21	-	1	7.0	7.8	.	22.6	19.5	.
	22	-	1	18.0	22.0	.	35.2	19.0	.

OPG

Table 3 .

 3 Synovial tissue OPG/RANKL expression and disease activity IOD = integrated optical density. * P < 0.05, ** P < 0.01 for change from baseline. ‡ P < 0.05 vs. non-responders. See Table1for further definitions.

	OPG
	Baseline IOD	8.5 (6.8, 13.1)	9.5 (6.6, 15.1)	8
	Week 4 % change	-4.3 (-35.7, 31.3)	-6.4 (-43.7, 24.7)	3.2
	Final % change	-13.6 (-50.3, 13.3)	-33.7 (-62.4, -6.6)* ‡	-33.
	RANKL
	Baseline IOD	13.7 (9.2, 19.6)	14.6 (11.1, 17.6)	14
	Week 4 % change	-15.4 (-41.3, 45.7)	-18.3 (-49.1, 38.9)	-29
	Final % change	7.0 (-58.8, 48.8)	2.8 (-68.1, 23.7)	-0.
	RANKL:OPG ratio
	Baseline	1.4 (1.1, 2.1)	1.6 (0.8, 2.4)	1
	Week 4 % change	-0.4 (-21.2, 55.6)	-0.5 (-20.6, 54.3)	-12
	Final % change	16.1 (-16.6, 94.9)	16.1 (-14.4, 203.9)	16.1

ACKNOWLEDGMENTS

This study was supported by research grants from Amgen inc. The authors would like to thank Evelyn Murphy and Ann Madigan for laboratory advice and assistance and for data collection.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.


MD, PhD Fernando Martinez-Valle

PhD. JOSEP Eva Balada

Dr Josep Ordi-Ros

Segundo Bujan-Rivas

Agustin Sellas-Fernandez

Miquel Vilardell-Tarres

MD Ordi-Ros

PhD Segundo Bujan-Rivas

PhD Agustin Sellas-Fernandez

MD Miquel Vilardell-Tarres

DNase1 activity in systemic lupus erythematosus patients with and without nephropathy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Objective: To determine whether the activity of DNase1 is associated to the presence of nephropathy in patients with SLE.

Methods: Forty-five patients affected with SLE and renal involvement were analyzed. The type of renal involvement was type III or IV glomerulonephritis. At least two serum samples were withdrawn from each patient, one obtained in a renal flare and the other obtained in a period of clinical stability. C3 and C4 complement levels and anti-DNA antibodies were determined. DNase1 activity was measured by using a radial enzyme diffusion method.

Results: When comparison of DNase1 activity was established between samples obtained during a phase of active renal involvement and those obtained in the clinically stable phase, we did not find statistically significant differences. When the comparison was performed with matched samples of the same patient, DNase1 activity was lower when patients had active renal involvement than when samples were taken in clinically stable phase (

Introducction

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology characterized by the production of a wide range of autoantibodies. More than 100 different autoantibodies have been identified in the sera of patients with SLE [START_REF] Sherer | Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients[END_REF]. In particular, anti-DNA antibodies are a hallmark of SLE and may induce renal failure. It has been shown that anti-DNA antibodies and DNA-anti-DNA antibodies complexes are deposited on many organs, including the kidney, which leads to tissue inflammation [START_REF] Bruneau | Circulating DNA:anti-DNA complexes in systemic lupus erythematosus. Detection and characterization by ultracentrifugation[END_REF][START_REF] Brinkman | Anti-DNA antibodies and lupus nephritis: the complexity of crossreactivity[END_REF]. The incidence of clinical immune complex-mediated glomerulonephritis in SLE patients range between 30-50% [START_REF] Cervera | Morbidity and mortality in systemic lupus erythematosus during a 5-year period. A multicenter prospective study of 1,000 patients. European Working Party on Systemic Lupus Erythematosus[END_REF][START_REF] Yang | Lupus nephritis in children--a review of 167 patients[END_REF][START_REF] Cervera | Systemic lupus erythematosus: clinical and immunologic patterns of disease expression in a cohort of 1,000 patients. The European Working Party on Systemic Lupus Erythematosus[END_REF][START_REF] Mak | Renal damage in systemic lupus erythematosus: a comparative analysis of different age groups[END_REF]. Different studies suggest a strong correlation between increasing levels of anti-DNA antibodies and subsequent activation of the disease [START_REF] Bootsma | Prevention of relapses in systemic lupus erythematosus[END_REF][START_REF] Ter Borg | Measurement of increases in anti-double-stranded DNA antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus. A long-term, prospective study[END_REF]. An abrupt, marked increase of these autoantibodies is usually followed by exacerbations of the disease. Exacerbations of glomerulonephritis, vasculitis, or both are the manifestations most likely to be heralded by rising titters of antibodies to ds-DNA.

Auto-antigens from apoptotic cells released during apoptosis are thought to drive the auto-immune response in human SLE [START_REF] Pickering | Systemic lupus erythematosus, complement deficiency, and apoptosis[END_REF][START_REF] Graham | Sources of autoantigens in systemic lupus erythematosus[END_REF][START_REF] Walport | Lupus, DNase and defective disposal of cellular debris[END_REF]. During apoptosis, chromatin is cleaved by specific DNases and nucleosomes (the fundamental units of chromatin), are released [START_REF] Koutouzov | Nucleosomes in the pathogenesis of systemic lupus erythematosus[END_REF]. Nucleosomes contain structures (DNA and histones) that are targeted in lupus and it provides a link between apoptosis and SLE.

DNase1, a Ca 2+ /Mg 2+ -dependent enzyme, is the major nuclease found in body fluids such as serum and urine [START_REF] Laskowski | Deoxyribonuclease I. 3rd ed. The enzymes[END_REF][START_REF] Lacks | Deoxyribonuclease I in mammalian tissues. Specificity of inhibition by actin[END_REF]. DNase1 has also been regarded as a candidate apoptotic endonuclease [START_REF] Mannherz | A new function for an old enzyme: the role of DNase I in apoptosis[END_REF][START_REF] Napirei | Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system[END_REF]. We have previously measured Dnase1 activity in different clinical and immunological settings in SLE patients [START_REF] Martinez-Valle | DNase 1 activity in patients with systemic lupus erythematosus: relationship with epidemiological, clinical, immunological and therapeutical features[END_REF]. In this work, we set out to test the hypothesis that DNase1 activity is related to the presence of lupus nephritis.

MATERIALS AND METHODS

The study was performed by the Department of Systemic Autoimmune Diseases at the Vall d'Hebron Hospital, a tertiary hospital in Barcelona, Spain, with a referral population of 400,000 people. Between September 2000 and October 2007 we collected 45 SLE patients with renal involvement (10 men -22.2%-and 35 women -77.8%-). All the patients fulfilled at least four of the American College of Rheumatology (ACR) criteria. The type of renal involvement was type III or IV glomerulonephritis (according to the World Health Organization (WHO) classification), and it was diagnosed by altered urinoanalysis, proteinuria, decreased complement levels, and biopsy. Clinical stability was defined if SLEDAI was < 4 and no changes in BILAG score.

Overall, we collected a total of 125 samples, 49 (39.2%) being obtained at renal flare and 76 (60.8%) being withdrawn during the stable phase. Serums were frozen and stored at -80ºC. When more than one sample were obtained from a patient during a phase of stability or renal flare, we calculate a mean value. The study was approved by the ethical committee of our centre.

Anti-dsDNA antibodies levels were measured by an enzyme-linked immunosorbent assay (Quanta Lite TM dsDNA ELISA from Inova Diagnostics, Inc. San Diego, USA). Positivity was considered when achieving > 15 U/mL. Complement levels were measured by a nephelometric method (normal levels: 85.0 -180.0 mg/dL for C3, and 10.0 -40.0 mg/dL for C4).

DNase1 activity was measured by means of a radial enzyme-diffusion method described by Chitrabamrung et al. [START_REF] Chitrabamrung | A radial diffusion assay for plasma and serum deoxyribonuclease I[END_REF][START_REF] Chitrabamrung | Serum deoxyribonuclease I and clinical activity in systemic lupus erythematosus[END_REF], modified by Macanovic et al. [START_REF] Macanovic | Measurement of deoxyribonuclease I (DNase) in the serum and urine of systemic lupus erythematosus (SLE)-prone NZB/NZW mice by a new radial enzyme diffusion assay[END_REF] and adapted by us. The method is based on the hydrolysis of DNA in a DNAagar plate. To perform the DNA-agar plate we used calf thymus DNA (Sigma, St Louis, USA) and low melting point agarose (GIBCO-BRL, United Kingdom). We also used recombinant DNase1 (Pulmozyme Dornasa Alfa, Roche Farma, Madrid, Spain) in order to create the standard curve. The area of hydrolysed DNA was identified after staining with Sibr Green I (Sigma, St Louis, USA).

Digital images were adquired with a BioDoc-itTM UVP system. Data were analyzed by the statistical program SPSS 12. Statistical analyses were performed by using the Kolmogorov-Smirnov test and the Mann-Whitney U test. To analyze matched samples we use the Wilcoxon test. We performed the Spearman test to determine the correlation between complement levels and DNase1 activity.

RESULTS

Twenty-six samples (21.66%) were negative for anti-DNA antibodies, whereas 94 samples (78.33%) were positive. Data from 5 samples were not available.

The clinical stability phase was not always associated to an anti-DNA negative seroconversion.

With regard to the C3 complement factor we found that 72 samples had low levels (57.6%), whereas 53 (42.4%) samples had normal levels. Forty-five samples (36%) had low levels of C4 complement factor, whereas 80 (64%) samples had normal levels.

When comparison of DNase1 activity was established between the overall samples obtained during an active renal involvement and those obtained in the clinically stable phase, we did not find statistically significant differences (20.78 ± 16.16 μg/ml vs. 22.17 ± 17.20 μg/ml) (p=0.524).

However, when the comparison was performed with matched samples of the same patient, we observed that DNase1 activity was lower when patients had active renal involvement than when they were clinically stable (21.21 ± 16.47 μg/ml vs. 25.62 ± 18.81 μg/ml; p<0.05).

None difference was observed in DNase1 activity of samples that were anti-DNA positive with respect to those which were negative (20.85

DISCUSSION

The exact aetiology of SLE is unknown but antibodies against nucleosomes and anti-DNA are a serological hallmark of this disease and have been clearly involved in the immune complex-mediated lesion in the kidneys [START_REF] Tan | Deoxybonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus[END_REF]; they have also been widely used to evaluate the clinical activity in SLE [START_REF] Schur | Immunologic factors and clinical activity in systemic lupus erythematosus[END_REF][START_REF] Miniter | Reassessment of the clinical significance of native DNA antibodies in systemic lupus erythematosus[END_REF]. The formation of the SLE autoantibodies seems to be a T-cell dependent and an autoantigen-driven process [START_REF] Berden | Lupus nephritis[END_REF].

Apoptotic cells are the unique source of nucleosomes, which are formed through cleavage of chromatin by nucleases [START_REF] Bell | Immunogenic DNA-related factors. Nucleosomes spontaneously released from normal murine lymphoid cells stimulate proliferation and immunoglobulin synthesis of normal mouse lymphocytes[END_REF][START_REF] Dieker | Triggers for anti-chromatin autoantibody production in SLE[END_REF][START_REF] Licht | Decreased phagocytosis of apoptotic cells in diseased SLE mice[END_REF]. At physiological condition, apoptotic cells, which maintain their membrane integrity for a certain time, have to be cleared quickly and efficiently by phagocytes in order to prevent the release of tissue-damaging intracellular constituents [START_REF] Gaipl | Inefficient clearance of dying cells and autoreactivity[END_REF].

Disturbances in either apoptosis or the phagocytosis of apoptotic cells have been proposed in the development of SLE [START_REF] Denny | Accelerated macrophage apoptosis induces autoantibody formation and organ damage in systemic lupus erythematosus[END_REF][START_REF] White | Apoptosis in systemic lupus erythematosus[END_REF][START_REF] Savill | A blast from the past: clearance of apoptotic cells regulates immune responses[END_REF].

DNase1, the major serum nuclease, is responsible for the degradation of the chromatin released by inappropriately cleared dead cells [START_REF] Mannherz | A new function for an old enzyme: the role of DNase I in apoptosis[END_REF][START_REF] Peitsch | Genomic organisation and expression of mouse deoxyribonuclease I[END_REF]. Furthermore, DNase1 in cooperation with the plasminogen system guarantees a fast and an effective breakdown of chromatin during necrosis by the combined cleavage of DNA as well as DNA binding proteins [START_REF] Napirei | Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system[END_REF]. DNase1 and C1q also cooperate in the degradation of chromatin from necrotic cells [START_REF] Gaipl | Cooperation between C1q and DNase I in the clearance of necrotic cellderived chromatin[END_REF].

Observations in vivo and in vitro have pointed out that DNase1 and SLE have a close relationship. Chitrabamrung et al [START_REF] Chitrabamrung | Serum deoxyribonuclease I and clinical activity in systemic lupus erythematosus[END_REF] described that DNase1 activity was low in the serum of patients with SLE. These authors also found that DNase1 activity was particularly low in patients with renal disease. Macanovic et al [START_REF] Macanovic | Measurement of deoxyribonuclease I (DNase) in the serum and urine of systemic lupus erythematosus (SLE)-prone NZB/NZW mice by a new radial enzyme diffusion assay[END_REF] observed that mean serum concentrations of DNase1 in the murine lupus model NZB/NZW were lower than in normal mice.

In agreement with the results obtained by Chitrabamrung et al. [START_REF] Chitrabamrung | Serum deoxyribonuclease I and clinical activity in systemic lupus erythematosus[END_REF], we also found that the mean DNase1 activity of samples obtained through active renal disease was significantly lower than those obtained in the inactive renal disease period. Some authors have demonstrated that the low DNase1 activity observed in these samples is not due to the lost of DNase1 in the urine [START_REF] Chitrabamrung | Serum deoxyribonuclease I and clinical activity in systemic lupus erythematosus[END_REF][START_REF] Macanovic | Measurement of deoxyribonuclease I (DNase) in the serum and urine of systemic lupus erythematosus (SLE)-prone NZB/NZW mice by a new radial enzyme diffusion assay[END_REF]. None relationship between proteinuria and DNase1 activity has been detected by our group either [START_REF] Martinez-Valle | DNase 1 activity in patients with systemic lupus erythematosus: relationship with epidemiological, clinical, immunological and therapeutical features[END_REF].

Patients with type III and IV glomerulonephritis classically have an increase of anti-DNA antibodies levels, which play a major role in the development of human lupus nephritis [START_REF] Suzuki | Possible pathogenic role of cationic anti-DNA autoantibodies in the development of nephritis in patients with systemic lupus erythematosus[END_REF]. Decreased DNase1 activity is related to an inappropriately clearing of dead cells, which leads to an increase in the number of cells that undergo secondary necrosis, that furthermore will promote the maturation of dendritic cells to full antigen-presenting capacity [START_REF] Savill | A blast from the past: clearance of apoptotic cells regulates immune responses[END_REF][START_REF] Rovere | Bystander apoptosis triggers dendritic cell maturation and antigen-presenting function[END_REF][START_REF] Albert | Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs[END_REF]. The maturation of dendritic cells to full antigen-presenting cells and an increase of chromatin fragments that escape from non-ingested apoptotic cells (and that can be directly immunogenic [START_REF] Savill | A blast from the past: clearance of apoptotic cells regulates immune responses[END_REF]) are two mechanisms that can promote autoimmunity. Since nucleosomes, anti-nucleosomes antibodies, and anti-DNA antibodies are hallmarks of some types of glomerulonephritis in SLE patients, decreased Dnase1 activity can initiate the process that drive toward the apparition of nephropathy in SLE patients.

  Abbreviations: systemic lupus erythematosus (SLE), double-stranded DNA

  μg/ml ± 16.80 vs. 26.77 μg/ml ± 17.09; p=0.063). None difference in DNase1 activity was observed between patients with normal or decreased C3 levels (25.09 µg/ml ± 17.78 versus 20.01 µg/ml ± 16.15 respectively; p=0.073). Similar results were obtained when considering C4 levels (23.52 µm/ml ± 16.60 versus 19.62 µg/ml ± 17.54; p=0.060). No correlation could be established between C3 or C4 levels and DNase1 activity.


Athina Pyrpasopoulou

Stella Douma

Themistoklis Vassiliadis

Sofia Chatzimichailidou

Areti Triantafyllou

MD Consultant Rheumatologist Spyros Aslanidis

Reactivation of chronic hepatitis B virus infection following

INTRODUCTION

In chronic hepatitis B virus infection, immune suppression has been linked with potential activation and, in a number of cases, acute liver failure and fatal outcome; patients with haematologic or other malignancies under chemotherapy were mostly affected [START_REF] Yeo | Frequency of hepatitis B virus reactivation in cancer patients undergoing cytotoxic chemotherapy: a prospective study of 626 patients with identification of risk factors[END_REF]. In the same cohort of patients the incidence of HBV reactivation rose significantly in the past decade, after the introduction of rituximab, a monoclonal antibody targeted against the CD20(+) lymphocytes [START_REF] Tsutsumi | Reactivation of hepatitis B virus with rituximab[END_REF][START_REF] Yeo | Hepatitis B virus reactivation in lymphoma patients with prior resolved hepatitis B undergoing anticancer therapy with or without rituximab[END_REF]. In this setting, reactivation was recorded even in HBsAb (+) patients (4). In patients with rheumatic diseases, analogous cases are still mainly anecdotal. Taking into account the increasing application of rituximab in the treatment of autoimmune diseases, and the global prevalence of chronic hepatitis B virus infection, cases of treatment-induced reactivation need to be recorded, in order to formulate prophylaxis and treatment guidelines, and to safely establish the safety profile of these agents in analogous circumstances [START_REF] Furst | The risk of infections with biologic therapies for rheumatoid arthritis[END_REF].

CASE REPORT

A 56-yr-old female patient with a 21-yr-history of severe rheumatoid arthritis (cervical fusion surgery, hip and knee arthroplasty) and arterial hypertension received the 1 st cycle of rituximab in 7/08. The patient had received etanercept (monoclonal antibody targeted against the soluble TNFa receptor) in 2002 for 1 year with good response; treatment was discontinued after setting the diagnosis of chronic HBV infection (HBsAg+, HBeAg -, HBeAb +) and the patient was started on lamivudine, steroids and DMARDs (cyclosporine) with moderate response in terms of the primary disease until 5/07. In 5/07 infliximab, and adalimumab were initiated sequentially (HBV DNA PCR <6 IU/ml). Both were discontinued shortly after initiation, due to failure of response. The patient developed an allergic reaction to abatacept and gave her informed consent to receive rituximab (at initiation DAS28 was 6.28, the biochemical profile was normal, and PCR for HBV DNA was 9.6x10 2 IU/ml).

One month after administration of rituximab (2x 1000mg) the patient presented with fever (38 o C) and elevated transaminases (SGOT 110, SGPT 150U/l). HBV DNA was >1.1x10 8 IU/ml and antibodies for HDV were negative. Tenofovir was added to the concomitant lamivudine. Transaminases normalised 1 month after initiation of tenofovir, and, 10 months later, HBV DNA is 46 IU/ml. The patient is in good clinical condition and liver morphology (by imaging studies) and function remain normal.

DISCUSSION

An unrecognised HBV infection may lead to serious complications in patients receiving DMARDs and/ or biologics (e,g TNFa antagonists, rituximab). On the other hand, a diagnosed chronic HBV infection may cause delay to or undertreatment of patients with rheumatic diseases. Reactivation of chronic HBV infection has been reported with anti-TNF agents [START_REF] Zingarelli | Prophylaxis and therapy of HBV infection in 20 patients treated with disease modifying antirheumatic drugs or with biological agents for rheumatic disases[END_REF][START_REF] Nathan | Hepatitis B and C virus infections and anti-tumor necrosis factor-alpha therapy: guidelines for clinical approach[END_REF]. However, under prophylactic antiviral treatment these agents seem, at least in a restricted number of patients and under short-term follow up, to be relatively safe [START_REF] Calabrese | Safety of antitumour necrosis factor (anti-TNF) therapy in patients with chronic viral infections: hepatitis C, hepatitis B, and HIV infection[END_REF][START_REF] Roux | Safety of anti-TNF-alpha therapy in rheumatoid arthritis and spondyloarthropathies with concurrent B or C chronic hepatitis[END_REF]. Longer follow-up of the patients involved is needed to safely extrapolate this conclusion. Experience with rituximab in analogous patients is scarce [START_REF] Salliot | Risk of serious infections during rituximab, abatacept and anakinra treatments for rheumatoid arthritis: meta-analyses of randomized placebo-controlled trials[END_REF]. Current guidelines suggest avoidance of this biologic in patients with chronic HBV infection (HBsAb -), based on experience from oncological patients and its interference with B-lymphocyte biology. In cases in which all other therapeutic options have been excluded and the patient consents to its use, treatment requires close monitoring of the patient (clinical and laboratory) under concomitant antiviral treatment, and prompt reevaluation of the latter with early signs of viral reactivation.