M Bombardieri

Role of anti-cyclic citrullinated peptide antibodies in discriminating patients with rheumatoid arthritis from patients with chronic hepatitis C infection-associated polyarticular involvement

This study was performed to assess the utility of anti-cyclic citrullinated peptide (anti-CCP) antibodies in distinguishing between patients with rheumatoid arthritis (RA) and patients with polyarticular involvement associated with chronic hepatitis C virus (HCV) infection. Serum anti-CCP antibodies and rheumatoid factor (RF) were evaluated in 30 patients with RA, 8 patients with chronic HCV infection and associated articular involvement and 31 patients with chronic HCV infection without any joint involvement. In addition, we retrospectively analysed sera collected at the time of first visit in 10 patients originally presenting with symmetric polyarthritis and HCV and subsequently developing well-established RA. Anti-CCP antibodies and RF were detected by commercial second-generation anti-CCP2 enzyme-linked immunosorbent assay and immunonephelometry respectively. Anti-CCP antibodies were detected in 23 of 30 (76.6%) patients with RA but not in patients with chronic HCV infection irrespective of the presence of articular involvement. Conversely, RF was detected in 27 of 30 (90%) patients with RA, 3 of 8 (37.5%) patients with HCV-related arthropathy and 3 of 31 (9.7%) patients with HCV infection without joint involvement. Finally, anti-CCP antibodies were retrospectively detected in 6 of 10 (60%) patients with RA and HCV. This indicates that anti-CCP antibodies can be useful in discriminating patients with RA from patients with HCV-associated arthropathy.

Autoantibody profile in systemic lupus erythematosus with psychiatric manifestations: a role for anti-endothelial-cell antibodies

This study was performed to determine the correlation between psychiatric manifestations and several autoantibodies that might participate in the pathogenesis of psychiatric disorders in the course of systemic lupus erythematosus (SLE). Fifty-one unselected outpatients with SLE were enrolled. Psychiatric evaluation was performed according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition. The prevalence of antibodies against endothelial cells (AECA), cardiolipin, β2 glycoprotein I, Ro, Ro52, La, glial fibrillary acidic protein, ribosomal P protein, dsDNA, and nucleosomes was assessed by experimental and commercial enzyme-linked immunosorbent assays. According to the cutoff value, AECA were present in 11 of 17 (64.7%) SLE patients with psychosis and mood disorders and in 10 of 34 (29.4%) patients without psychiatric manifestations other than anxiety (P = 0.03). Moreover, the AECA binding index was significantly higher in the first group (P = 0.03). Conversely, no significant correlation was found between the presence of the other autoantibodies studied and psychiatric involvement. The results of this study suggest a relationship between AECA and psychosis and mood disorders in SLE, supporting the hypothesis of a biological origin of these disturbances.

Anti-tumour necrosis factor (TNF) α treatment of rheumatoid arthritis (infliximab) selectively down regulates the production of interleukin (IL) 18 but not of IL12 and IL13

Objective: To measure interleukin (IL)18 serum concentrations in patients with rheumatoid arthritis (RA) undergoing infliximab treatment (tumour necrosis factor (TNF) α blockade) and to evaluate the concomitant modification of IL12 and IL13 serum concentrations, two cytokines belonging to the Th1 and Th2 profile respectively and biologically related to IL18. Methods: Ten patients with RA not responding to disease modifying antirheumatic drugs (DMARDs) received intravenous infliximab at a dose of 3 mg/kg at baseline and after two and six weeks. Serum samples were collected from all patients before each infusion and assayed for IL18, IL12, and IL13 by enzyme linked immunosorbent assay (ELISA); IL18 was also measured eight weeks after the last infusion. Results: Serum concentrations of IL18 in all patients were already markedly reduced from baseline after two weeks (p<0.005). Serum IL18 was also decreased in a stable manner after six (p<0.01) and 14 weeks (p<0.01) compared with baseline concentrations. No significant modifications were found in serum concentrations of IL12 and IL13 at any time point. Conclusion: There was a rapid and persistent decrease in serum concentrations of IL18 in all the patients studied. This result provides evidence of an in vivo regulation of IL18 by TNFα and suggests that anti-TNFα therapy is likely to interrupt the synergistic effect between these two cytokines.

Anti-lysobisphosphatidic acid antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus

Lyso(bis)phosphatidic acid (LBPA) is a novel antigenic target in anti‐phospholipid syndrome (APS) and antibodies directed against LBPA (aLBPA) have been detected in sera from APS patients. In this study we first evaluated aLBPA in comparison with the most widely used methods (i.e. anticardiolipin [(aCL)‐enzyme‐linked immunosorbent assay (ELISA)] and antibeta‐2‐glycoprotein‐I antibodies (aβ2‐GPI‐ELISA) utilized to detect antiphospholipid antibodies in patients with primary or secondary APS, systemic lupus erythematosus, chronic HCV infection and healthy subjects. We then assessed the relationship between aLBPA, lupus anticoagulant (LAC) and the main clinical manifestations of APS. Finally, we evaluated the presence of ‘pure’ (i.e. β2‐GPI‐independent) aLBPA in patients with APS and controls. The results indicate that aLBPA as well as aβ2‐GPI display higher specificity but lower sensitivity for APS compared to aCL. Moreover, serum aLBPA correlate closely with aCL and aβ2‐GPI in APS patients and are strictly associated with LAC positivity. We demonstrate that β2‐GPI binds to LBPA with affinity similar to CL, and antibodies able to react with phosholipid‐protein complex exist; however, ‘pure’ aLBPA can also be detected in sera of APS patients. Altogether these data confirm that LBPA may be an antigenic target in APS and that aLBPA are serological markers of APS with similar sensitivity and specificity compared to aβ2‐GPI. However, the clinical utility of aLBPA detection alone or in combination with aCL and/or aβ2‐GPI remains to be elucidated in larger and longitudinal studies.

Anti-carbonic anhydrase II antibodies in systemic sclerosis: Association with lung involvement

Carbonic anhydrase II (CAII) is expressed on alveolar epithelium and participates to CO 2 elimination, fluid secretion and post-capillary pH regulation. CAII is overexpressed in animal models of lung fibrosis in sites of epithelial injury. Autoantibodies directed against CAII (anti-CAII) have been described in sera from patients affected by systemic sclerosis (SSc), but no study focused on their clinical associations in this disease. The aim of this study was to assess the presence of anti-CAII in sera of SSc patients and to investigate their association with lung involvement. We performed ELISA to detect anti-CAII in 34 SSc patients who underwent pulmonary function tests (PFT) and Doppler echocardiography. We found increased prevalence and significantly elevated serum levels of anti-CAII in SSc patients affected by restrictive lung disease (RLD) compared to SSc patients without lung involvement and healthy controls. These findings suggest both a possible pathogenic role of anti-CAII in the development of lung damage and a potential clinical utility as serological marker of pulmonary involvement in SSc patients.

A6.2 Inducible Tertiary Lymphoid Structures and Autoimmunity in a Novel model of Sialoadenitis in Wild-Type Mice

Introduction Salivary glands of patients with Sjögren’s syndrome (SS) develop ectopic lymphoid structures (ELS) characterised by B/T cell compartmentalisation, the formation of high endothelial venules (HEV), follicular dendritic cell networks (FDCs), functional B cell activation with expression of activation-induced cytidine deaminase (AID) as well as local differentiation of autoreactive plasma cells. The mechanisms triggering ELS formation, autoimmunity and exocrine dysfunction in SS are largely unknown. Here we present a novel model of inducible ectopic lymphoid tissue formation, breach of humoral self-tolerance and salivary hypofunction following delivery of a replication-deficient adenovirus-5 (AdV5) in submandibular glands of C57BL/6 mice through retrograde excretory duct cannulation. Materials and Methods Luciferase- or LacZ-encoding Ad5 were delivered in C57BL/6 mice salivary glands (SG) through retrograde cannulation. SGs were collected at various time-points 1, 2 and 3 weeks post-cannulation (pc) and frozen sections were graded for infiltration and stained for T/B cell segregation, FDCs and HEV markers. Submandibular salivary flow was induced by pilocarpine stimulation and the amount of saliva measured. Expression of TLS-related genes was investigated by TaqMan-PCR. Anti-viral antibodies and autoantibodies were detected by IF and western blot. Results In this model, inflammation rapidly and consistently evolves from diffuse infiltration towards the development of SS-like periductal lymphoid aggregates within 2 weeks from AdV delivery. These infiltrates progressively acquire ELS features and support functional GL7+/AID+ germinal centres. Formation of ELS is preceded by ectopic expression of lymphoid chemokines CXCL13, CCL21 and CCL19 as well as lymphotoxin-β and is associated with development of anti-nuclear antibodies in 75% of the mice. Finally, reduction in salivary flow was observed over 3 weeks post AdV infection consistent with exocrine gland dysfunction as a consequence of the inflammatory response. Conclusions This novel model has the potential to unravel the cellular and molecular mechanisms regulating ELS formation and their role in exocrine dysfunction and autoimmunity in SS.

AB0099 Aberrant basal and TLR-stimulated expression of TSLP in rheumatoid synovial fibroblasts

Background Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine, which strongly activated dendritic cells for Th2 polarization. Previous collagen-induced arthritis model demonstrated its role in exacerbating disease severity via T-cell dependent mechanism. Objectives Here we investigated TSLP and its receptor (TSLPR) expression in the synovium of rheumatoid arthritis (RA) patients and in rheumatoid synovial fibroblasts (RASF), osteoarthritis fibroblast (OASF, to dissect disease specificity) as well as paired RA dermal fibroblasts (RADF, to investigate site specificity) in basal conditions and upon stimulation with Toll-like receptors (TLR) ligands. Methods mRNA and protein (cytoplasmic and soluble) expression of TSLP in RASF, OASF and RADF, stimulated with or without TLR agonists: bLP (TLR2 ligand), PIC (TLR3 ligand) and LPS (TLR4 ligand), was assessed by Taqman PCR (QT-PCR), immunocytochemistry and ELISA. TSLP and TSLPR expression in 40 synovium of RA patients was investigated by QT-PCR and immunohistochemistry. Results RASF and, to a lesser extent OASF, constitutively displayed higher TSLP mRNA (∼8-16 fold) compared to RADF. In vitro stimulation of TLR3 and TLR4, but not TLR2 on RASF led to strong induction of TSLP mRNA expression (∼20-fold increase with TLR3), which peaked early at 8h. Cytoplasmic staining of TSLP was increased in TLR3-activated RASF but not RADF, while soluble TSLP was time-dependently released in the supernatant of TLR3-stimulated RASF (∼100pg/ml) and undetectable in RADF. TSLP mRNA was observed in all the RA samples examined while TSLPR was significantly increased in patients with follicular synovitis. Conclusions Overall, our data strongly support a pivotal role for RASF in the dysregulated production of pro-arthritogenic/inflammatory TSLP in the rheumatoid synovium, suggesting that the TSLP/TSLPR pathway contributes to chronic inflammation in RA. References Koyama K, Ozawa T, Hatsushika K, Ando T, Takano S, Wako M, et al. A possible role for TSLP in inflammatory arthritis. Biochem Biophys Res Commun2007 May 25;357(1):99-104. Ozawa T, Koyama K, Ando T, Ohnuma Y, Hatsushika K, Ohba T, et al. Thymic stromal lymphopoietin secretion of synovial fibroblasts is positively and negatively regulated by Toll-like receptors/nuclear factor-kappaB pathway and interferon-gamma/dexamethasone. Mod Rheumatol2007;17(6):459-63. Disclosure of Interest None Declared