O. Meyer

Anti-heat shock protein 70 kDa and 90 kDa antibodies in serum of patients with rheumatoid arthritis

OBJECTIVES Stress proteins (HSPs) are highly conserved immunodominant antigens found in various species. The purpose of this study was to assess the prevalence and prognostic significance of antibodies to HSC 70 kDa and HSP 90 kDa in three groups of patients with longstanding rheumatoid arthritis (RA) defined based on the severity of articular erosions. METHODS 73 patients with longstanding (> 6 years) RA whose HLA-DR genotype was known were divided in three groups according to Larsen’s score and compared with 47 recent onset (<1 year) RA patients and with control groups composed of patients with other inflammatory diseases (n=137) or of normal controls (n=48). IgGs and IgMs to HSC 70 kDa and HSP 90 kDa were determined using an ELISA with purified bovine HSC 70kDa or HSP 90 kDa. RESULTS Concentrations of IgGs and IgMs to HSC 70 were significantly increased in 41.1% and 42.5% of longstanding RA patients, respectively. Corresponding figures for IgGs and IgMs to HSP 90 were 39.7% and 56%. IgMs to HSC 70 and HSP 90 were less frequent in recent onset RA (19% and 13% respectively). Among the groups with other inflammatory diseases, only the MCTD group exhibited high frequencies of IgGs to HSC 70 (80%) and HSP 90 (85%). DRB1*0401 positive RA patients (n=23) were not more likely to have increased concentrations of antibodies to HSC 70 kDa or HSP 90 kDa than other RA patients (DR4 positive but DRB1*0401 negative, or DR1 positive, n=31; or negative for both DR4 and DR1, n=14). IgGs to HSP 90 kDa were significantly more frequent (p<0.05) in longstanding RA patients whose Larsen’s score was 4 or more (57%) than in those whose Larsen’s score was 2 or 3 (39.4%) or less than 2 (16%). No associations were found between Larsen’s score and IgGs or IgMs to HSC 70 kDa or IgMs to HSP 90 kDa. A significant correlation was demonstrated between IgGs to HSP 90 kDa and two other serological markers for RA, rheumatoid factor, and anti-Sa antibody; there were no correlations with antikeratin antibody, antiperinuclear factor, or anti-RA 33. CONCLUSION IgGs to HSP 90 kDa are most common in longstanding RA patients with articular erosions, suggesting that they may be related to the articular prognosis in RA

Anti-oxidized low-density-lipoprotein (OxLDL) antibodies in systemic lupus erythematosus with and without antiphospholipid syndrome.

The aim of this study was to examine potential links between antiOxLDL antibodies and the clinical and biological features of secondary antiphospholipid syndrome (II APLS) associated with systemic lupus erythematosus (SLE). A cohort study was done of 98 SLE patients followed-up for 1 y, including 18 with definite II APLS and 13 patients with definite primary APLS (I APLS). IgG anticardiolipin, IgG antib2 GPI, lupus anticoagulant, VDRL and IgG antiOxLDL were measured in all 98 study subjects. High antiOxLDL titers were found in seven (39%) of the 18 patients with II APLS vs 10 (12.5%) of the 80 patients without APLS (P < 0.01; OR ‘ 4.45; 95% CI = 1.4–14.1) and none of the 13 patients with I APLS (P < 0.02). The mean antiOxLDL titer was not significantly higher in the SLE patients with than without II APLS (P > 0.05). A high antiOxLDL titer was correlated with deep venous thrombosis (P < 0.01; OR = 5.77; 95% CI = 0.54–61) but not with arterial thrombosis (P > 0.05; OR = 1; 95% CI = 0.29–3.09), thrombocytopenia, central nervous system involvement, livedo reticularis, or a positive Coombs test. The antiOxLDL antibody titer was correlated with the IgG anticardiolipin antibody titer (r = 0.235; P = 0.02) and with the IgG anti-b2 GPI antibody titer (r = 0.224; P = 0.026). AntiOxLDL elevation was found in 17% of SLE patients and was significantly associated with II APLS and venous thrombosis. We found no evidence suggesting that antiOxLDL may be associated with atherosclerosis.

NADPH Oxidase Priming and p47phox Phosphorylation in Neutrophils from Synovial Fluid of Patients with Rheumatoid Arthritis and Spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor α(TNFα), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNFα, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNFα and IL-8 (but not GM-CSF) are detected in patients SF (compared to circulating blood levels). TNFα levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNFα and IL-8 but not GM-CSF.

Anti-proteinase-3 (PR3) antibodies (C-ANCA) recognize various targets on the human umbilical vein endothelial cell (HUVEC) membrane

Numerous studies suggest that C‐ANCA are directly pathogenic in vasculitis by activating leucocytes (oxidative burst, enzyme release, endothelial cytotoxicity, etc.). We and others have shown that C‐ANCA can also directly activate HUVEC, but the precise target on HUVEC is unknown. We show in this study that C‐ANCA recognize various targets on the HUVEC membrane (different from PR3 in our model), leading to secondary cell activation. Polyclonal affinity‐purified C‐ANCA recognized targets on the unfixed endothelial membrane in fluorescent ELISA, flow cytometry, and immunoprecipitation studies. C‐ANCA did not react with Fcγ receptors. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) experiments showed that HUVEC did not express PR3. The targets of polyclonal and monoclonal anti‐PR3 antibodies on the endothelial membrane were not the same. Some epitopes were lost after trypsin–EDTA digestion and formaldehyde fixation of cells, whereas anti‐PR3 targeted unfixed HUVEC. This suggests that anti‐PR3 react with the endothelial membrane and recognize conformational epitopes shared with PR3. Endothelial cells may thus participate in the inflammation associated with Wegeners granulomatosis and contribute to the emergence of clinical manifestations.

Five-year favorable outcome of patients with early rheumatoid arthritis in the 2000s: data from the ESPOIR cohort.

Objective. To report the 5-year outcome of a large prospective cohort of patients with very early rheumatoid arthritis (RA), and to identify factors predictive of outcome. Methods. Patients were recruited if they had early arthritis of < 6 months’ duration, had a high probability of developing RA, and had never been prescribed disease-modifying antirheumatic drugs (DMARD) or steroids. Logistic regression analysis was used to determine factors that predict outcome. Results. We included 813 patients from December 2002 to April 2005. Age was 48.1 ± 12.6 years, delay before referral 103.1 ± 52.4 days, 28-joint Disease Activity Score (DAS28) 5.1 ± 1.3, Health Assessment Questionnaire (HAQ) 1.0 ± 0.7; 45.8% and 38.7% had rheumatoid factor or antibodies to cyclic citrullinated peptide (anti-CCP), respectively; 22% had hand or foot erosions; 78.5% fulfilled the American College of Rheumatology/European League Against Rheumatism criteria for RA at baseline and 93.8% during followup. At 5 years, 573 patients were evaluated. The outcome was mild for most patients: disease activity (median DAS28 = 2.5) and HAQ disability (median 0.3) were well controlled over time; 50.6% achieved DAS28 remission and 64.7% low disease activity. Radiographic progression was low (2.9 Sharp unit/year) and only a few patients required joint surgery. Nevertheless, some patients developed new comorbidities. During the 5 years, 82.7% of patients had received at least 1 DMARD (methotrexate, 65.9%), 18.3% a biological DMARD, and about 60% prednisone at least once. Anti-CCP was the best predictor of remaining in the cohort for 5 years, of prescription of synthetic or biologic DMARD, and of radiographic progression. Conclusion. The 5-year outcome of an early RA cohort in the 2000s was described. Anti-CCP was a robust predictor of outcome. The generally good 5-year outcome could be related to early referral and early effective treatment, key processes in the management of early RA in daily practice.

Oculomotor palsy in six patients with systemic lupus erythematosus. A possible role of antiphospholipid syndrome

The objective of this study was to describe clinical, laboratory,and radiologicalfeatures in systemic lupus erythematosus (SLE) patients with oculomotor palsy (OMP). Among a cohort of 750 SLE patients receiving follow-up at our unit between 1985 and 2000, all patients with OMP were studied. Clinical and laboratory data were recorded, as well as cerebral magnetic resonance imaging (MRI) findings where available. Immunological tests included tests for presence and specificity of antinuclear and antiphospholipidantibodies. Six patients had OMP, which occurred within the first 3 years in two patients and after more than 20 years in two patients. Four patients had focal neuropsychiatricSLE (NPSLE) manifestations and two had diffuse neurological involvement. The four patients with focal NPSLE were either anticardiolipin or lupus anticoagulant-positive. Cerebral fluid was abnormal in two of four patients who had this test. In four of the six patients, cerebral MRI showed evidence of vasculopathy. The therapeutic regimens varied, although all six patients initially received high-dose corticosteroids. OMP resolved or improved significantly in all six patients. OMP in SLE occurs in a broad spectrum of clinical situations but is an infrequent manifestation of NPSLE. This case series, together with literature review data, suggests various pathophysiologicalmechanisms in patients with focal or diffuse neurological symptoms. In patients with focal NPSLE, a possible cause of OMP is microthrombosis associated with presence of antiphospholipid antibodies.

Cholangite sclérosante compliquant une histiocytose langerhansienne

Resume Introduction. – L’histiocytose langerhansienne est une maladie rare, qui touche surtout le jeune enfant. L’atteinte hepatique represente un facteur de mauvais pronostic. Une cholangite sclerosante peut survenir chez 10 a 15 % des patients atteints de forme multiviscerale. Nous rapportons le cas d’un homme de 56 ans qui a developpe une cholangite sclerosante 12 ans apres le diagnostic d’histiocytose langerhansienne. Exegese. – Un homme de 56 ans est hospitalise pour alteration de l’etat general associee a des epigastralgies. Il est suivi pour une histiocytose langerhansienne revelee 12 ans auparavant par un diabete insipide, puis compliquee d’une atteinte fibrokystique pulmonaire et osteocondensante vertebrale. La maladie est cependant quiescente depuis 1 an. Il ne prend aucun traitement. Il existe une cholestase et un syndrome inflammatoire biologique. Les explorations radiologiques digestives (scanner, bili-IRM et echoendoscopie) revelent une hepatomegalie et un epaississement parietal regulier et diffus de l’arbre biliaire intra- et extrahepatique. La ponction–biopsie du foie confirme le diagnostic de cholangite sclerosante. Une corticotherapie orale est debutee, permettant une amelioration clinique et biologique (12 mois de recul). Conclusion. – La cholangite sclerosante est une complication classique de l’histiocytose langerhansienne, principalement en cas de forme multiviscerale. Elle survient en moyenne 2 ans apres le diagnostic chez l’enfant, mais parfois bien plus tard, notamment chez l’adulte, alors que l’histiocytose langerhansienne semble ne plus evoluer. Le diagnostic repose sur les explorations radiologiques et l’histologie hepatique. Habituellement, cette forme de cholangite sclerosante repond mal au traitement de l’histiocytose langerhansienne et la transplantation hepatique doit souvent etre envisagee.

111Indium antimyosin antibody imaging of primary myocardial invovement in systemic diseases

OBJECTIVE The diagnosis of primary myocardial involvement in systemic diseases is clinically relevant but difficult in the absence of specific criteria. Whatever the underlying disease, myocytes degeneration is observed during the active phase of myocardial damage. The aim of this study was to assess the diagnostic value of scintigraphic imaging with111Indium antimyosin antibody (AM), a specific marker of the damaged myocyte, for ongoing myocardial damage related to systemic diseases. METHODS 40 patients with histologically confirmed systemic diseases were studied. They were classified into two groups according to the presence (group 1, n=30), or the absence (group 2, n=10) of clinical, electrocardiographic (ECG) or echocardiographic signs suggestive of myocardial involvement. Planar and tomographic acquisitions were obtained 48 hours after injection of AM (90 MBq). Rest 201thallium (Tl) scintigraphy was also performed to assess myocardial perfusion and scarring. Clinical, ECG, and echocardiographic ± scintigraphic evaluations were repeated during follow up (17 ±19 months) in 36 of 40 patients. RESULTS In group 1, 13 of 30 patients (43%) showed diffuse significant AM uptake throughout the left ventricle (LV), and no or mild Tl abnormality. Two of these were asymptomatic, four had normal ECG, and two had no clinical or echographic LV dysfunction. All patients in group 2 had negative AMA scintigraphy and normal Tl scintigraphy. During follow up of 12 AM positive patients, cardiac status improved after immunosuppressive treatment was intensified in nine cases, worsened in two cases, and remained stable in one. During follow up of 24 AM negative patients, cardiac status remained stable in 23 cases despite treatment not being increased in 20, including two patients with sequellary myocardial involvement. The last patient developed mild LV dysfunction after 36 months. CONCLUSION AM scintigraphy allows detection of active myocardial damage related to systemic diseases, with increased specificity compared with conventional methods, and increased sensitivity in some cases. Further studies are needed to assess the potential value of AM scintigraphy as a therapeutic guide.

1.60 Autoantibodies to human citrullinated fibrinogen (AhFibA) and their subfamilies directed to the fibrin peptides α36-50Cit38,42 and β60-7460,72,74 are prognostic markers of radiographic damage in the very early arthritides of the French ESPOIR cohort

Background and Objective In established RA, autoantibodies (AAB) to human citrullinated fibrinogen (AhFibA) were demonstrated to be mainly composed of two subfamilies of AAB directed to immunodominant epitopes borne by the fibrin peptides α36-50Cit38,42 and β60-74Cit60,72,74,respectively. Serum reactivity toward those peptides defines subgroups of patients. In the present study, we investigated whether AhFibA, anti-α36-50Cit38,42 and β60-74Cit60,72,74 AAB, have different predictive values for disease outcome in very early RA. We also analysed whether these AAB differentially associate with SE and tobacco exposure. Materials and Methods The French ESPOIR cohort is comprised of very early RA and of undifferentiated arthritides (UA) of less than 6-month duration. AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB were assayed by ELISA at baseline. After 3-year follow up, 701 patients were diagnosed RA according to the ACR/EULAR 2010 criteria. Relationships between SE HLA-DR alleles, tobacco exposure and the 3 AAB were analysed on those patients. Disease activity (DAS28, HAQ) and radiographic damage (SHS) were evaluated at baseline, and after 2- or 3-year follow up. Associations with clinical parameters and predictive value of each AAB were investigated. Results AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB were detected in 349/701 (50%), 203/701 (29%), and 257/701 (37%), RA sera, respectively. Their positive predictive values for RA (72%, 82%, and 79%, respectively) were not significantly different. The presence and titre of each AAB were associated with SE HLA-DR alleles without significant additional effect of tobacco exposure. When 2 vs 0 copies of SE alleles were present, the odds ratios for AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB presence reached 8.0, 6.1 and 9.5, respectively. Neither the presence nor the titres of AhFibA, α36-50Cit38,42 and β60-74Cit60,72,74 AAB were associated with DAS28 or HAQ at baseline and after 2 years. However, for the 3 AAB, patients whose sera contained one or several AAB had a progression of SHS during the first 3 years (medians from 6 to 8 depending on the subgroup), significantly higher than the AhFibA-negative patients (median = 4). Nevertheless, no significant correlation was observed between the titres of AAB and radiographic progression. Conclusion Not only AhFibA but also their anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB subfamilies, are prognostic markers for bone erosion in RA. Moreover, AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB are similarly associated to HLA-DR and to tobacco exposure.

THU0118 25 hydroxyvitamin D interfere with the clinical response to rituximab in rheumatoid arthritis

Background Vitamin D (Vit D) is increasingly recognized as an important immune modulating agent. No data has been reported on how Vit D status can interfere with the biotherapy response in RA patients. Objectives The main objective was to determine the Vit D status in a cohort of RA patients and to evaluate the 6 month response to rituximab according to the level of plasmatic 25OHVit D at baseline. Methods 111 RA consecutive patients (ARA 1987 criteria) were recruited from one single institution between 2005 and 2010 prior to initiation of rituximab. Patients characteristics including ethnic origin, BMI, smoking status, disease duration, autoantibody status (RF, ACPA), number of previous DMARDs, corticosteroid therapy status, Vit D supplementation, season of enrolment were documented. 25OHVit D was measured using baseline serum samples and a commercial ELISA kit (IDS immunodiagnostic system). Patients were classified in 3 categories according to 25OHVit D level as adequate (≥30 ng/ml), insufficient (between 10 and 30 ng/ml), or deficient (<10 ng/ml). Treatment response was evaluated according to DAS28 variation between M0 and M6 (Δ DAS28) (EULAR classification: good, moderate, none). We identified factors associated with the treatment response by univariate analysis (Chi-square or Fisher’s test for categorical variables, and analysis of variance for continuous variables). Factors with p-value less than 20% were entered into the multinomial logistic regression. Results The cohort included 83% F, mean age 53±10.3 y, 86.5% were Caucasians; mean BMI 27.2±6.8; mean disease duration 12.1±9.7 y; RF positive 85.6%; ACPA positive 80%; mean number of previous biotherapies 1.4±1.1. The mean DAS28 at M0 was 5.2±1.3.The mean dose of prednisone at M0 was 8.6±7.7 mg/d. Season for enrolment was autumn (20.7%), winter (29.7%), spring (27.9%) and summer (21.6%). 64% received daily supplementation with Vit D (800 units/d). The mean level of 25OH Vit D was 17.1±8.7 ng/ml for the entire group. According to 25OH Vit D dosage, 9 patients were classified as adequate, 79 (71.2%) as insufficient and 23 (20.7%) as deficient. At M6 20.7% were in remission (DAS28 <2.6); 9% were in low disease activity (DAS28 <3.2); 47.7% moderate activity (DAS28 ≤5.1) and 22.5% were in high disease activity (DAS 28 >5.1).Response to treatment with rituximab was considered as good for 24.3%, moderate for 38.7% and 36.9% had no response. According to Vit D status at M0, the proportion of no response was higher in patients with 25OHVit D deficiency, 52.2% vs 31.6% for 25OH Vit D insufficiency and 44.4% for 25OH Vit D adequacy (p=0.0459; Fisher test). No correlations were seen between 25OHVit D level and Δ DAS28 or DAS28 at M0. The model of multinomial logistic regression analysis showed an association between a significant response and the season of mabthera initiation (better response frequency if initiated in automn) (p global value =0.0166). Conclusions Whatever the season, inadequate Vit D status was observed in 92% of RA patients in this series, despite daily supplementation for 64% of them. The effect of normalization of Vit D status prior to the initiation of rituximab should be evaluated in order to minimize the rate of treatment failure. Disclosure of Interest R. M’Barek: None Declared, T. Dupré: None Declared, F. Tubach: None Declared, P. Dieudé: None Declared, E. Palazzo: None Declared, G. Hayem: None Declared, K. Dawidowicz: None Declared, S. Ottaviani: None Declared, T. Alfaiate: None Declared, V. Leçon-Malais: None Declared, A. Boutten: None Declared, O. Meyer Grant/Research support from: ROCHE