Mathieu Verdet

Recommendations for using TNFα antagonists and French Clinical Practice Guidelines endorsed by the French National Authority for Health.

The use of TNFα antagonists must follow specific guidelines to ensure optimal effectiveness and safety. The French Society for Rheumatology (SFR) and Task Force on Inflammatory Joint Diseases (CRI), in partnership with several French learned societies, asked the French National Authority for Health (HAS) to develop and endorse good practice guidelines for the prescription and monitoring of TNFα antagonist therapy by physicians belonging to various specialties. These guidelines were developed, then, validated by two multidisciplinary panels of experts based on an exhaustive review of the recent literature and in compliance with the methodological rules set forth by the HAS. They pertain to the initial prescription of TNFα antagonists and to a variety of clinical situations that can arise during the follow-up of patients receiving TNFα antagonists (infections, malignancies, pregnancy, vaccination, paradoxical adverse events, surgery, use in older patients, and vasculitides).

Soluble alpha-enolase activates monocytes by CD14-dependent TLR4 signalling pathway and exhibits a dual function

Rheumatoid arthritis (RA) is the most common form of chronic inflammatory rheumatism. Identifying auto-antigens targeted by RA auto-antibodies is of major interest. Alpha-enolase (ENO1) is considered to be a pivotal auto-antigen in early RA but its pathophysiologic role remains unknown. The main objective of this study was to investigate the in vitro effects of soluble ENO1 on peripheral blood mononuclear cells (PBMC) from healthy donors and RA patients in order to determine the potential pathogenic role of ENO1. ELISA, transcriptomic analysis, experiments of receptor inhibition and flow cytometry analysis were performed to determine the effect, the target cell population and the receptor of ENO1. We showed that ENO1 has the ability to induce early production of pro-inflammatory cytokines and chemokines with delayed production of IL-10 and to activate the innate immune system. We demonstrated that ENO1 binds mainly to monocytes and activates the CD14-dependent TLR4 pathway both in healthy subjects and in RA patients. Our results establish for the first time that ENO1 is able to activate in vitro the CD14-dependent TLR4 pathway on monocytes involving a dual mechanism firstly pro-inflammatory and secondly anti-inflammatory. These results contribute to elucidating the role of this auto-antigen in the pathophysiologic mechanisms of RA.

AB0278 Prolonging between-infusions interval is associated with positivity to anti-infliximab antibodies in rheumatoid arthritis and spondyloarthritis patients

Objectives To analyze association between the presence of antibodies to infliximab (ATI) and the clinical and biological characteristics in rheumatoid arthritis (RA) and spondyloarthritis (SpA) patients Methods Sera from RA (n = 22) or SpA (n = 23) patients treated with infliximab were analyzed. ATI and infliximab trough concentrations were measured using a commercial multiplex enzyme-linked immunosorbent assay kit (LISA-Tracker Infliximab Theradiag®, Marne la Vallée, France). Clinical and biological data were collected retrospectively from each patient’s medical file. Results Infliximab was given in combination with methotrexate (MTX) for 31 (69%) patients (RA patients 90%; SpA patients 43%). Fifteen patients were in remission at the time of analysis. The interval between two consecutive infliximab infusions exceeded 8 weeks for 15 (33%) patients; it was significantly longer for patients who had achieved remission (9.3 weeks) versus the other patients (6.8 weeks; Mann-Whitney U-test, P = 0.0005). Seven (15%) patients were ATI positive (4 RA and 3 SpA), 6 of them were treated in combination with MTX. Duration of infliximab intakedid not differ between ATI positive and negative patients. Their infliximab doses at the time of analysis were comparable, respectively: 4.29mg/kg and 4.13mg/kg (P = 0.74). A longer between-infusions interval was associated with the ATI-positivity (9.6 weeks versus ATI-negativity: 7.3 weeks; P = 0.02). Infliximab trough concentrations were significantly lower in ATI-positive patients (0.18 μg/ml) vs ATI-negative patients (2.1 μg/mL, P = 0.0005). A significant inverse correlation was found between ATI titers and infliximab trough concentrations (Spearman’s test, P = 0.0002, R2 = 0.29). No link was found between the methotrexate intake and ATI-positivity. Conclusions Immunogenicity of infliximab is related with a longer between-infusion interval, which could affect the treatment strategy, because prolonging those intervals for patients in clinical remission might favor ATI production. Disclosure of Interest None Declared

Prophylactic Injection of Recombinant Alpha-Enolase Reduces Arthritis Severity in the Collagen-Induced Arthritis Mice Model

Objective To evaluate the ability of the glycolytic enzyme alpha-enolase (ENO1) or its immunodominant peptide (pEP1) to reduce the severity of CIA in DBA/1 mice when injected in a prophylactic way. Methods Mice were treated with mouse ENO1 or pEP1 one day prior to collagen II immunization. Clinical assessment was evaluated using 4 parameters (global and articular scores, ankle thickness and weight). Titers of serum anti-ENO1, anti-cyclic citrullinated peptides (anti-CCP) and anti-CII (total IgG and IgG1/IgG2a isotypes) antibodies were measured by ELISA at different time-points. Disease activity was assessed by histological analysis of both anterior and hind paws at the end of experimentation. Results Prophylactic injection of 100 μg of ENO1 reduced severity of CIA. Serum levels of anti-CII antibodies were reduced in ENO1-treated mice. Concordantly, ENO1-treated mice joints presented less severe histological signs of arthritis. ENO1 did not induce a shift toward a Th2 response since IgG1/IgG2a ratio of anti-CII antibodies remained unchanged and IL-4 serum levels were similar to those measured in the control group. Conclusions Pre-immunization with ENO1 or its immunodominant peptide pEP1 reduces CIA severity at the clinical, immunological and histological levels. Effects of pEP1 were less pronounced. This immunomodulatory effect is associated with a reduction in anti-CII antibodies production but is not due to a Th1/Th2 shift.

Buschke-Ollendorf syndrome in a patient with terminal renal failure.

Joint Bone Spine - In Press.Proof corrected by the author Available online since dimanche 3 juillet 2011

A1.32 Alpha-enolase activates monocytes by CD14-dependent TLR4 signalling pathway

Background and objectives Rheumatoid arthritis (RA) is the most common chronic inflammatory rheumatism. Identification of autoantigens targeted by RA autoantibodies has been of major interest. Alpha-enolase (ENO1) is considered as a pivotal autoantigen in early RA. Whatever the nature of this molecule, its role in RA pathophysiology remains unknown. The main objective of this study was to investigate the in vitro effects of soluble ENO1 on peripheral blood mononuclear cells (PBMCs) from healthy donors and RA patients in order to determine the potential pathogenic role of ENO1. Materials and methods PBMCs of healthy donors or RA patients were cultured with ENO1 or control. ELISA and transcriptomic analysis were performed to assess the pro- or anti-inflammatory effect of ENO1. Moreover, flow cytometry analysis, experiments of receptor inhibition and ELISA were performed to determine the target cell population and receptor of ENO1. Results Firstly, we showed that ENO1 has the ability to induce the production of a large amount of pro-inflammatory cytokines and chemokines, and to activate the innate immune system on PBMCs from healthy donors. ENO1 also exhibits this pro-inflammatory effect on cells from RA patients. Then, we demonstrated that ENO1, which exclusively bound monocytes, exhibited a LPS-like action mediated by TLR4. More precisely, the effects of ENO1 specifically involved the CD14-dependent TLR4 pathway. Conclusions Together, these findings illustrate for the first time that ENO1 triggers in vitro an inflammatory response mediated by monocytes through the CD14-dependent TLR4 pathway and contribute to elucidate the role of this autoantigenin the RA pathophysiological mechanisms.

THU0439 Bacterial Identification and Bad Prognostic Factors During Infectious Spondylodiscitis

Objectives Define the most relevant investigations to define the organism(s) involved in infectious spondylodiscitis and put in evidence the risk factors of poor outcome (death, relapse, recurrence) or after-effects at middle-term and long-term. Methods Retrospective bicentric study on 306 patients affected by an infectious spondylodiscitis and followed-up between 2000 and 2009. Study on medical records and systematic telephonic call of patients and/or general practitioner. An uni and multivariate statistical analysis was performed. Results The probability to isolate an organism was significantly lower when there was no fever (p<0,01; OR= 2,19 [1,23-3,94] or no inflammatory syndrome (p<0,01; OR=3,35 [1,27-8,81] as well as a recent spine surgery (p<0,001; OR=0,29 [0,16-0,55]), antibiotic exposure (p<0,01; OR=0,43 [0,23-0,78]) or malignancy past history (p<0,01; OR=0,42 [0,22-0,81]. A percutaneous discal and vertebral biopsy (PDVB) (p<0,0001; OR=0,29 [0,16-0,55]) even with PCR analysis (p<0,0001; OR=0,17 [0,08-0,36]) was not associated with a better probability to identify the germ. The diagnostic delay was significantly longer in the group of patients without isolated organism (p<0,01). An initial neurologic trouble (p<0,0001), an elevated age (p<0,0170), liver cirrhosis (p<0,01; OR=6,4 [1,74-19,05]) or epidural abscess (p<0,001; OR=3,07 [1,42-6,61]) were significant and independent factors of poor outcome. Back (p<0,01; OR=2,36 [1,28-4,34]) and radicular pains (p<0,01; OR=2,73 [1,46-5,10]), a recent spine surgery (p<0,01; OR=2,85 [1,50-5,37]) were associated to back pain sequelae at middle-term. They were significantly associated to spine stiffness (p<0,001; OR=2,85 [1,50-5,37]), persistant neurologic trouble (p=0,0365; OR= 2,62 [1,03-6,65]) or disc injury (p<0,001; OR=2,85 [1,50-5,37]). Long-term pain was significantly more frequent when the spondylodiscitis began after a back surgery (p<0,02; OR=3 [1,09-8,23]). Conclusions The probability to isolate an organism was lower when clinical and biological presentations were less severe. However, the antibiotic therapy allowed similar vital and functional prognostic even if the the organism was identified or not. Our results also showed that a second PDBV or even a chirurgical biopsy were not justified. References Spilf. Primary infectious spondylodiscites, and following intradiscal procedure, without prosthesis. Recommendations. Med Mal Infect2007;37:269-77. Zimmerli W. Clinical practice. Vertebral osteomyelitis. N England J Med2010;362:1022-9 O’Daly BJ. Long-term functional outcome in Pyogenic spinal infection. Spine (Phila Pa 1976) 2008;33:246-53 Disclosure of Interest None Declared

A2.14 Potential in Vitro Immunomodulatory Effects of the Recombinant Human Alpha-Enolase on Peripheral Blood Mononuclear Cells (PBMCs) from Healthy Donors

Background Identification of autoantibodies associated with rheumatoid arthritis has been of major interest. In this context, we have previously identified for the first time α-enolase (ENO) as a new auto-antigen in early RA. ENO is an evolutionary conserved protein involved both in glycolysis pathway and as a plasminogen receptor which confer it a role in anti-infectious inflammatory response. In vivo, preliminary studies showed that ENO had immunomodulatory effect in the collagen induced arthritis mouse model. [1] To better understand the immunological mechanisms of ENO, the aim of this in vitro study was to determine the effects of ENO on PBMCs from healthy donors. Methods In one hand, PBMCs or different cell types (monocytes, B and T cells, and immature dendritic cells [iDC]) (n = 3) were cultured with ENO (20 µg/mL) or Bovine Serum Albumin (20 µg/mL). TNFα and IL-10 production was measured in the supernatants by ELISA at different times. On the other hand, TNFα and IL-10 production were evaluated in PBMCs, monocytes or B and T cells after LPS stimulation and pre-incubation with ENO for 24 h (n = 3). Cytometric analyses have evaluated the ability of ENO to inhibit the differentiation of monocytes into iDC. Before differentiation into iDC (GM-CSF and IL-4), monocytes (1.106 cells/mL) were incubated with ENO (20 or 50 µg/mL) for 24 h. Results In cultures of PBMCs, monocytes or iDC, ENO induces, dose dependently, an early production of TNFα followed by extended secretion of IL-10. PBMCs or individual cells (monocytes, B and T cells) stimulated by LPS secreted successively TNFα and IL-10, while PBMCs or individual cells, stimulated by LPS but previously incubated with ENO for 24 h did not secrete these cytokines. In contrast to LPS, ENO did not induce differentiation of immature dendritic cells into mature cells. But ENO has not the capacity to inhibit differentiation from monocytes to iDC. Conclusions This study suggests that ENO has no pro-inflammatory effect unlike LPS. Indeed, ENO might have immunomodulatory properties via IL-10 production. Others studies focused on an extended cytokine panel and different signalling pathways are underway to better understand the immunological mechanisms induced by ENO. Reference C Guillou et al, Arthritis and Rheumatism 2011; 63:S815.

Prophylactic injection of non-citrullinated α-enolase has immunomodulatory effects in collagen-induced arthritis mice

Background Identification of autoantibodies associated with rheumatoid arthritis (RA) has been of major interest. In this context, the authors have previously identified for the first time α-enolase as a new auto-antigen in early RA. Moreover, subsequent studies have shown that citrullination of α-enolase is crucial for its autoantigenicity. α-enolase is an evolutionary conserved protein implicated both in glycolysis pathway and as a plasminogen receptor. Here, the authors have evaluated, in the well-known collagen induced arthritis model, the clinical, immunological and histological effects of both recombinant non-citrullinated α-enolase and immunodominant peptides from human and bacterial species. Methods Different doses of α-enolase (10 and 100 µg) or immunodominant enolase peptide 1 from human (hEP1) or porphyromonas Gingivalis (pEP1) (10 or 100µg) were intraperitoneally injected to 6 week-old DBA/1 mice one day prior to collagen II arthritis induction (CIA). Both clinical (weight, arthritis score, tarsal thickness) and biological (anticollagen II and anti-α-enolase antibodies) were assessed during the 90 days follow-up period. Four histological score were also assessed: inflammation, syniovial thickening, cartilage resorption and bone resorption. Results Prophylactic injection of recombinant α-enolase was able to significantly prevent weight loss and to decrease the severity of arthritis evaluated by the arthritis score as well as the tarsal thickness. There was a dose-effect since 100 µg led to better results. Levels of anticollagen II antibodies were significantly lower whereas titers of anti-α-enolase antibodies were significantly higher in mice treated with 100 µg of α-enolase compared to control mice. Moreover, histological score were in agreement with clinical score. As regards to hEP1 and pEP1, the authors etablished a dose-dependant protective effect in CIA which is significant for pEP1. This protective effect is not due to once again a decrease of anti-collagen II antibodies titer. Conclusion Prophylactic treatment with recombinant α-enolase suggest a protective role of this molecule. The clinical effect is not due to an imunological response mediated by anti-α-enolase antibodies. Prophylactic injection could induce either an immune deviation or an emergence of regulatory lymphocyte population, responsible of a decrease of anti-CII antibodies production. These results suggest α-enolase has an immunomodulatory effect in CIA mice. Those results suggest that non-citrullinated α-enolase could constitute a potential new therapeutic approach in RA.