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Background Primary Sjogren’s syndrome (pSS) is a systemic auto-immune disease, characterised by abnormalities of B cell homeostasis. Several studies demonstrated that the repartition of blood B cell subsets is altered during pSS. Objectives The aim of this prospective study was to determine the diagnostic value of blood B-cell subset profiling for pSS. Methods This cross-sectional study was conducted in a monocentric cohort of patients with suspected pSS prospectively included between November 2006 and April 2011. Clinical examination, basic biology, immunological tests and minor labial salivary gland biopsy were performed systematically. For blood B-cell subset profiling, the ratio (Bm2+Bm2’)/(eBm5+Bm5) was determined using flow cytometry. The gold standard for the analysis was a clinical diagnosis of pSS performed by a group of experts, blinded to the results of B-cell profiling. Results 170 patients have been included in the study. 72 patients had pSS, of whom 58 (80.6%) fulfilled AECG criteria. No differences were found between the 2 groups concerning age, disease duration, and sex ratio. All items of AECG criteria were significantly associated with the diagnosis of pSS except xerostomy. The mean (Bm2+Bm2’)/(eBm5+Bm5) ratio was significantly higher in the pSS group than in the non-pSS group (7.2±7.0 vs. 3.2±2.4, P Conclusions Blood B cell profiling using flow cytometry is a simple test that displays good diagnostic properties for pSS. Its inclusion into diagnostic criteria should be evaluated. Disclosure of Interest None Declared

Background Early arthritis is often undifferentiated and may resolve spontaneously, remain undifferentiated, or progress to rheumatoid arthritis or to another chronic joint disease such as SpA. There is a need for improving the early diagnosis of ankylosing spondylitis (AS) and other spondyloarthritis (SpAs). Objectives To 1- assess the level of agreement between a SpA at baseline and 10 years later in a cohort of patients with recent-onset arthritis and 2- evaluate the ability of baseline clinical, laboratory, and radiographic features to predict a final diagnosis of SpA. Methods Patients with swelling of at least one joint between 1995 and 1997 were included. Ten years later, the physicians of each patient were contacted to determine the final diagnosis. Baseline pelvic radiographs were subjected to centralized blinded review. The statistical analysis used the chi-square test, Mann-Whitney test, and Cohen’s kappa coefficient. Results The diagnosis at baseline and 10 years later was known for 164 patients. At baseline, a diagnosis of SpA was suggested in 25 (15.2%) patients, of whom only 10 had a diagnosis of SpA 10 years later, in addition to 10 patients not diagnosed with SpA at baseline (20/164, 12.2%, Kappa=0.36±0.1). The 10-year diagnoses were ankylosing spondylitis (n=3), psoriatic arthritis (n=7), reactive arthritis (n=4), undifferentiated SpA (n=4), SAPHO syndrome, and Crohn’s disease (n=1). Of the 20 patients with a 10-year diagnosis of SpA, only 4 had inflammatory back pain at baseline (versus 11/144 non-SpA patients, p =0.72), 5 had psoriasis (versus 9/144 non-SpA patients, p =0.005), and 1 had uveitis ( p =0.12); 10 of the 20 SpA patients were HLA-B27-positive versus 8/144 non-SpA patients ( p Conclusions SpA is relatively rare and extremely challenging to diagnose in patients with early peripheral arthritis. Disclosure of Interest None Declared

Joint Bone Spine - In Press.Proof corrected by the author Available online since lundi 8 octobre 2012

Background and Objectives Sjogren’s syndrome (SS) is an autoimmune exocrinopathy characterised by an epithelium injury surrounded by dense lymphocytic infiltrates composed of activated T and B cells. Present at the interface of genetic and environmental risk factors, epigenetic modifications are suspected to play a key role in SS. Accordingly, we decided to further characterise DNA methylation in SS. Materials and Methods We tested, using a 5 methyl cytosine (5MeCyt) ELISA, global DNA methylation in long-term cultured salivary gland epithelial cells (SGEC), peripheral T cells and B cells from eight SS patients. DNA methylation/demethylation partners were assessed by real time quantitative PCR (DNA methyl transferase (DNMT)1, DNMT3a/b, PCNA, UHRF1, MBD2, MBD4, and Gadd45-alpha). Immunofluorescence was conducted on labial salivary gland biopsy. Co-culture experiments were performed associating the human salivary gland cell line (HSG) and B cells. Results Global DNA methylation was reduced in SGEC from SS patients (5MeCyt: 36.3 ± 3.2% in SS versus 43.1 ± 3.3% in controls, P = 0.01), while no difference was observed in T and B cells. SGEC demethylation in SS patients was associated with a 7-fold decrease of DNMT1 and a 1.8-fold increase of Gadd45-alpha expression. The other DNA methylation/demethylation partners tested were not differently expressed when compared to controls. Interestingly, SGEC demethylation may be attributed to the B cell infiltrate as DNA methylation increased in salivary gland biopsy after rituximab (anti-CD20 antibody) treatment. Such hypothesis was confirmed using co-culture experiments (HSG cells and B cells) revealing an alteration of the PKC-delta/ERK/DNMT1 pathway. Finally, DNA methylation was associated with the overexpression of several SGEC genes such as ICAM-1 and human endogenous retrovirus (HERV). Conclusions SGEC dysfunction in SS may be linked to epigenetic modifications and this tissue specific defect may be ascribed in part to infiltrating B cells. This observation opens new therapeutic perspectives in SS.

Background and Objectives Recently described, the molecule STIM1 (stromal interaction molecule 1) acts as a key mediator of calcium influx by controlling cell proliferation after antigen stimulation, Erk phosphorylation, cytokine production and apoptosis. Although STIM1 mutations have been associated with severe immunodeficiency, no study has focused on the STIM1 molecule in autoimmune diseases. Materials and Methods T and B lymphocytes, purified by negative selection from peripheral blood of patients with systemic lupus erythematosus (SLE, n = 11), rheumatoid arthritis (RA, n = 7), primary Sjogren’s syndrome (pSS, n = 11) and healthy controls (HC, n = 12) were tested by flow cytometry and Western blotting to determine the expression of STIM1. Video microscopy using specific probes has been used to assess intracellular calcium levels. Results T cells from peripheral blood of HC express more STIM1 molecules (mean fluorescence intensity (MFI) 3.42 ± 0.13) than B cells (MFI 2.18 ± 0.20, P high CD38 high transitional B cells (MFI 4.83 ± 0.63) compared with CD24 low CD38 low mature B cells (MFI 2.47 ± 0.15, P high CD38 low memory B cells (MFI 3.64 ± 0.42, P An highest calcium influx and a constitutive Erk phosphorylation characterise T and B cells from SLE patients when compared with HC and disease controls. As suspected, STIM1 is over-expressed in SLE, when compared with HC and this expression is similar between T cells (MFI 8.70 ± 0.87) and B cells (MFI 9.00 ± 1.08). Within B cell subsets, STIM1 expression is 3.4 fold highest in transitional SLE B cells (MFI: 16.25 ± 2.18, P Conclusions These results suggest that the differential expression of STIM1 may be an important factor in the process of lymphocyte self-reactivity in SLE, which opens new pathophysiological and therapeutic perspectives.

Background Inappropriate toll-like receptor (TLR) 9 signaling contributes to the activation of anti-dsDNA antibody-producing B cells in systemic lupus erythematosus (SLE). IVIg, which modulates B cell response through a sialic acid-CD22 lectin interaction (Hillion, Blood 2010), might also acts by repressing the TLR9 signal transduction. Material and methods Blood and tonsil mononuclear cell suspensions were enriched in B cells. TLR4, 7–8 and 9 were engaged using LPS, CL097 and CpG oligonucleotides, respectively. The SHP-1 phosphatase was inhibited by methyl 3-4 dephostatin, and the MyD 88 adaptor of TLRs by a specific blocking peptide. Results IVIg prevented the in vitro activation of TLR7 and TLR9, as shown by CD25, CD40 and class II-MHC downregulation. For this to occur, IVIg has to be continuously present in the culture. Furthermore, IVIg suppresses the production of the proinflammatory IL-6 as well as the immunostimulatory sequence-induced production of the anti-inflammatory IL-10. There was also an IVIg-mediated reduction of CXCR4 and CXCR5, two chemokines receptors dependent on engagement of the TLRs by their agonists. Finally, there appeared that the SHP-1 phosphatase was recruited by IVIg, as a feature of the B cell modulation. Conclusion Taken together, our data suggest that IVIg could repress the TLR9-induced activation of B cells in autoimmunity, along with the expression of pro-inflammatory cytokines. This opens up a new therapeutic avenue in SLE and related diseases.

Interleukin (IL)-6 is a pleiotropic cytokine that promotes polyclonal activation of association of the transducing glycoprotein 130 with the membrane-anchored receptor (R) α, IL-6 can generate functionally distinct signals. Given these particular molecular aspects, numerous activities ascribed to this cytokine are, in fact, due to the insertion into soluble IL-6Rα. The system is instrumental in rheumatoid arthritis (RA), Sjogren’s syndrome and systemic lupus erythema-tosus (SLE). In this respect, it is interesting that the expression of the recombination-activating gene is sustained by IL-6 in B lymphocytes, and repressed by anti-IL-6R antibody (Ab) in RA and SLE. Agents that inhibit IL-6 signaling have now entered clinical trials. As expected, clinical benefits are reported in the treatment of autoimmunedisorders with anti-IL-6R Ab, but other perspectives remain open in the forthcoming biotherapies of immune-mediated disorders. Keywords: interleukin-6, B lymphocyte, rheumatoid arthritis, systemic lupus erythematosus, Sjogren’s syndrome, biotherapy

Abstract 1946 Poster Board I-969 STATEMENT OF PURPOSE: Rituximab (RTX) is a chimeric antibody targeting CD20, a membranous B-cell specific protein, currently used in the treatment of B-cell malignancies and various autoimmune diseases. Despite its clear efficacy in non-Hodgkin's lymphoma, clinical and biological responses are highly variable between patients. Therefore, predictive factors are necessary to adapt therapy to each individual as early as possible. MATERIAL AND METHODS: We studied a set of biomarkers in a cohort of 39 patients with indolent non-Hodgkin's B-cell lymphoma (low grade follicular lymphoma or marginal zone lymphoma) and a low tumour burden, treated with single agent RTX as follows: 4 weekly 375 mg/m2 infusions, associated with short-course oral steroids. All patients benefited from FDG-PET analysis before treatment and 10 weeks later, allowing their classification into 3 early response groups: complete isotopic remission, partial response and no response. Dosage of rituximabemia, BAFF and Human Anti-Chimeric Antibodies (HACA) was performed with in-house ELISAs before each infusion and 2 months later. Polymorphisms of FcγRIIIa[158], FcγRIIa[131] and C1qA[276] genes were determined using allele-specific PCR or direct sequencing. Results were compared using Student's t-Test or two-tailed Fischer's exact test when appropriated. RESULTS: Referring to PET analysis, 18 patients were classified as complete isotopic remission, and 21 as partial or no response. Mean age was significantly lower in the first group (54 y+/-19 vs 66+/-13, p=0.022), as well as initial SUVmax (6.3+/-3.4 vs 10.8+/-6.8, p=0.024). Serum levels of RTX were highly variable between patients at each timepoint, but were not statistically different between the different response groups. Basal levels of BAFF were not statistically different between lymphoma patients and a control population, and did not vary after RTX-induced B cell depletion. No patient developed a HACA response during the study. Homozygous FcγRIIIa158V/V genotype was significantly associated with a complete response (p=0.037). Conversely, FcγRIIa[131] and C1qA[276] polymorphisms were not associated with the response. Genetic polymorphisms seemed to modify RTX pharmacokinetics, since homozygous FcγRIIa[131]H/H, FcγRIIIa[158]V/V and C1qA[276]G/G subjects displayed lower levels of RTX than others, without reaching statistical significance. CONCLUSIONS: Younger age, lower initial SUVmax and FcγRIIIa[158]V/V genotype are associated with a better early response to RTX in indolent lymphoma patients, as assessed by PET analysis. FcγR polymorphisms affect RTX kinetics probably through a modification of the efficacy of antibody dependant cell cytotoxicity. Determination of these factors could help in adapting individually therapeutic protocols. Disclosures: No relevant conflicts of interest to declare.

OBJECTIVE:The goal of this study was to determine how the choice of the primary endpoint influenced sample size estimates in randomised controlled trials (RCTs) of treatments for primary Sjögren's syndrome (pSS). METHODS:We reviewed all studies evaluating biotechnological therapies in pSS to identify their inclusion criteria and primary endpoints. Then, in a large cohort (ASSESS), we determined the proportion of patients who would be included in RCTs using various inclusion criteria sets. Finally, we used the population of a large randomised therapeutic trial in pSS (TEARS) to assess the impact of various primary objectives and endpoints on estimated sample sizes. These analyses were performed only for the endpoints indicating greater efficacy of rituximab compared to the placebo. RESULTS:We identified 18 studies. The most common inclusion criteria were short disease duration; systemic involvement; high mean visual analogue scale (VAS) scores for dryness, pain, and fatigue; and biological evidence of activity. In the ASSESS cohort, 35 percent of patients had recent-onset disease (lower than 4 years), 68 percent systemic manifestations, 68 percent high scores on two of three VASs, and 52 percent biological evidence of activity. The primary endpoints associated with the smallest sample sizes (nlower than 200) were a VAS dryness score improvement higher to 20 mm by week 24 or variable improvements (10, 20, or 30 mm) in fatigue VAS by week 6 or 16. For patients with systemic manifestations, the ESSDAI change may be the most logical endpoint, as it reflects all domains of disease activity. However, the ESSDAI did not improve significantly with rituximab therapy in the TEARS study. Ultrasound score improvement produced the smallest sample size estimate in the TEARS study. CONCLUSION:This study provides valuable information for designing future RCTs on the basis of previously published studies. Previous RCTs used inclusion criteria that selected a small part of the entire pSS population. The endpoint was usually based on VASs assessing patient complaints. In contrast to VAS dryness cut-offs, VAS fatigue cut-offs did not affect estimated sample sizes. SGUS improvement produced the smallest estimated sample size. Further studies are required to validate standardised SGUS modalities and assessment criteria. Thus, researchers should strive to develop a composite primary endpoint and to determine its best cut-off and assessment time point.

B cells play a central role in primary Sjögren's syndrome (pSS), but little is known about the autoreactive B cells that secrete autoantibodies against the autoantigen SSA. Here, we aimed to characterize the circulating SSAspecific B cells (SSA+ B cells), in order to better understand the mechanisms leading to the breakdown of self-tolerance in patients with pSS.Using a flow-cytometry-based method, we detected and phenotypically characterized SSA+ B cells among peripheral blood mononuclear cells (PBMCs) collected from patients with pSS and from healthy controls (HCs). We tested their ability to secrete SSA-specific immunoglobulins in vitro following stimulation. Then, we performed a first experience of transcriptional profiling of these cells by single-cell RNA seq to understand further the characteristics of these autoreactive B cells in healthy subjects.Phenotype analysis showed an expansion of a CD19+IgD+CD27-CD21low/-CD11c-SSA+ naïve B-cell subset with a downregulated B cell receptor in pSS patients.SSA+ B cells were also enriched within the IgMlow unswitched memory B-cell subset in patients, as well as in double negative (IgD-CD27-) and switched memory B cells of the different isotypes. Sorted SSA+ B cells from HCs were able to secrete in vitro IgM but not IgG anti-SSA immunoglobulins contrary to SSA+ B cells from pSS patients, that were able to secrete IgG autoantibodies. From the scRNA-seq analysis in a healthy subject, we obtained 6 clusters with 2 enriched in SSA+ B cells. DEG from clusters enriched with SSA+ B cells, showed upregulation of HLA genes implicated in the antigen presentation and negative regulation of BCR.Our results show that autoreactive SSA+ B cells in pSS patients maturate and reach the memory compartment, suggesting that they escape to the different layers of peripheral tolerance checkpoints and differentiate in vivo into anti-SSA antibody-secreting cells., Les LB jouent un rôle central dans le syndrome de Sjögren primitif (SSp), cependant très peu d’études ont été réalisées sur les caractéristiques des LB auto-réactifs qui sécrètent des autoanticorps contre l'auto-antigène SSA.Nous avons cherché à caractériser les LB circulants spécifiques de la SSA (LB SSA+), afin de mieux comprendre les mécanismes conduisant à la rupture de la tolérance chez les patients atteints du SSp.En utilisant une méthode de cytométrie en flux, basée sur la spécificité du BCR, nous avons détecté et caractérisé phénotypiquement les LB SSA+ circulants chez des patients atteints de SSp et chez des contrôles sains (HCs). Nous avons testé leur capacité à sécréter des immunoglobulines spécifiques de la SSA in vitro après stimulation. Ensuite, nous avons réalisé une première expérience de profilage transcriptionnel de ces cellules par RNA seq unicellulaire afin de mieux comprendre le rôle des LB auto-réactifs chez les sujets sains.L'analyse du phénotype a montré une expansion d'un sous-ensemble de LB naïfs CD19+IgD+CD27-CD21low/-CD11c-SSA+ avec un BCR régulé à la baisse chez les patients atteints de SSp.Les LB SSA+ étaient également enrichis parmi les LB mémoires non commutés IgMlow chez les patients, ainsi que dans les LB mémoires DN (IgD-CD27-) et commutés des différents isotypes. Les LB SSA+ triés, provenant d'un sujet sain, étaient capables de sécréter in vitro des immunoglobulines IgM mais pas IgG anti-SSA, contrairement aux LB SSA+ provenant de patients atteints de SSp, qui sont capables de sécréter des autoanticorps IgG spécifiques de SSA. Sur le plan transcriptomtique, les DEG du cluster enrichi en LB SSA+ ont montré une régulation positive des gènes HLA impliqués dans la présentation de l'antigène ainsi qu’une régulation négative du BCR par la surexpression de CD72.Nos résultats suggèrent que les LB SSA+ auto-réactifs, chez les patients atteints de SSP, sont enrichis dans le compartiment mémoire, ce qui suggère qu'ils échappent aux différents points de contrôle de la tolérance périphérique et se différencient in vivo encellules sécrétant des anticorps anti-SSA.

Le questionnement général de cette étude porte sur les pratiques des sages-femmes face aux lombalgies. L’objectif principal est de faire un état des lieux des pratiques des sages-femmes dans le traitement et la prévention des lombalgies pendant la grossesse.Matériel et méthode : Cette étude qualitative a eu lieu dans le Finistère, de septembre et novembre 2017. Nous avons contacté par téléphone des sages-femmes correspondant aux critères d’inclusion. Nous avons procédé à des entretiens semi-directifs. Nous les avons ensuite retranscrits intégralement par écrit puis nous les avons analysés. Les thèmes abordés au cours des entretiens concernent les données socio-démographiques, le traitement des lombalgies, la prévention des lombalgies et le travail en réseau.Résultats : En prénatal, le traitement des lombalgies fait partie des pratiques courantes de la sage-femme. Elle sait comment prendre en charge cette pathologie dans le respect de ses compétences. Elle adresse les patientes et travaille en réseau avec d’autres professionnels de santé. Cependant la prévention de ces maux n’est que trop peu mise en place. Elle n’est pas faite de manière systématique lors du suivi-prénatal.Conclusion : Les sages-femmes pourraient davantage favoriser la prévention des maux de dos et ainsi rendre la grossesse plus épanouissante. Nous préconisons d’inclure au sein des consultations, une prévention systématique des lombalgies par de simples recommandations : le maintien d’une activité physique quotidienne mais également une éducation posturale appropriée.