Christos Liaskos

B regulatory cells are decreased and functionally impaired in patients with systemic sclerosis

Breg cells, a regulatory cell subset that produces interleukin‐10 (IL‐10), play a significant role in suppressing autoimmune responses and preventing autoimmunity. This study was undertaken to examine the number and function of Breg cells in patients with systemic sclerosis (SSc), a disease with many autoantibodies.

IL-10-producing regulatory B cells (B10 cells), IL-17 + T cells and autoantibodies in systemic sclerosis

We aimed to analyze IL-10+ Breg (B10) cells, found to be reduced in systemic sclerosis (SSc), in relation to SSc-specific autoAbs and IL-17+ and IFNγ+ T cells in SSc. Peripheral blood B10 cells from 26 patients with SSc positive for anti-Topo I or anti-Cen autoAbs, and 12 healthy controls (HC) were studied by flow cytometry. IL-17+ and IFNγ+ T cells were also studied. B10 cells did not correlate with anti-Topo I or anti-Cen Ab levels but were inversely correlated with IL-17+ CD3+ cells and IFNγ+ CD3+ cells. IL-17+ CD3+ cells did not correlate with autoAb levels, but IFN-γ+ CD3+ cells were inversely correlated with anti-Topo I levels. In conclusion, in SSc, B10 cells did not correlate with SSc-specific autoAbs and exhibited an inverse correlation with IL-17+ T cells and IFNγ+ T cells.

Disease-related autoantibody profile in patients with systemic sclerosis

Abstract Background: Autoantibodies (autoAbs) help in diagnosis and predicting clinical phenotypes in systemic sclerosis (SSc). Aim of the study: To determine the clinical utility of 13 SSc-related autoAbs in SSc patients. Material and methods: A total of 131 consecutive patients with SSc (111 female, mean age 58.1u2009±u200914 years; 49 with diffused cutaneous SSc [dcSSc] and 82 with limited cutaneous SSc [lcSSc]) were analysed by a multiplex line immunoassay (Euroimmun) for autoantibodies (autoAbs) against 13 SSc-related antigens. A total of 22 patients with primary Raynaud phenomenon (RP), and 22 healthy controls were also analysed. Results: ANA by indirect immunofluorescence was present in 128 (97.7%) patients with SSc. Excluding anti-Ro52, 113 (89.3%) SSc patients were positive for at least one autoAb: anti-Topoisomerase I (anti-Topo) I abs in 54 (41.2%), anti-centromere proteins (anti-CENP) in 37 (28.2%, all reactive with centromere protein-A (CENPA) and centromere protein B (CENPB)), anti-RNA polymerase III(RP11) in 19 (14.5%), anti-RNA polymerase III(RP155) in 13 (9.9%), anti-fibrillarin in 4 (3.1%), anti-Ku in 6 (4.6%), anti-nucleolus-organizing region (anti-NOR90) in 8 (6.1%), anti-PM-Scl100 in 2 (1.5%), and anti-PM-Scl75 in 4 (3.1%). There was no immunoreactivity for Th/To or platelet-derived growth factor receptor (PDGFR). Overall, 102 (77.9%) SSc patients had autoAbs against Topo I, CENPA or CENPB, RP11 or RP155. Anti-Topo I abs were strongly associated with dcSSc, interstitial lung disease (ILD) (pu2009<u2009.001), pulmonary hypertension (PH) (pu2009=u2009.019) and ILD-PH (pu2009=u2009.003). Anti-CENPB abs were associated with lcSSc, and negatively associated with ILD. Anti-RP11 and anti-NOR90 abs were associated with male gender, and anti-NOR90 associated with ILD. Conclusions: Anti-Topo I, anti-CENP, and anti-RNA pol III are the most prevalent autoAbs in SSc. Anti-Topo I and anti-NOR90 abs are associated with ILD and/or PAH.

Anti-hsp60 antibody responses based on Helicobacter pylori in patients with multiple sclerosis: (ir)Relevance to disease pathogenesis

In view of published data suggesting that Helicobacter pylori (Hp) is a trigger of multiple sclerosis (MS), we assessed anti-heat shock protein 60 (hsp60)Hp antibody reactivity in 129 MS patients and 48 demograpically-matched healthy controls (HCs). Anti-Hp antibodies by ELISA were more elevated in MS than HCs but did not differ between different MS phenotypes. All anti-Hp-positive MS sera, irrespectively of their clinical phenotype, were anti-anti-hsp60 positive. Anti-hsp60 Hp seropositivity correlated with age at disease onset. In conclusion, anti-hsp60 Hp antibodies are present in all anti-Hp positive MS patients, and their relevance to disease pathogenesis is questionable.

IL-10 producing Bregs are impaired in psoriatic arthritis and psoriasis and inversely correlate with IL-17- and IFNγ-producing T cells

Our aim was to study CD19(+)CD27(+)CD24(high) memory and CD19(+)CD24(high)CD38(high) transitional and IL-10+Breg cells, known to inhibit Th1 and Th17 cells in experimental arthritis, in psoriatic arthritis (PsA) and psoriasis (Ps). Peripheral blood Breg cells from 60 patients with PsA, 50 patients with Ps and 23 healthy controls were analyzed by flow cytometry. IL-17A-producing CD3(+) T cells and IFNγ-producing CD3(+) T cells and activation of p38 MAPK and STAT3 were also studied. CD19(+)CD27(+)CD24(high) and CD19(+)CD24(high)CD38(high) Breg cells were decreased in PsA and Ps. In Ps patients, CD19(+)CD27(+)CD24(high) Breg cells inversely correlated with PASI score. IL-10+Bcells were also decreased and inversely correlated with IL-17A+CD3+ and IFN-γ+CD3+ T cells. B cells from patients exhibited impaired activation of p38 MAPK and STAT3. In conclusion, IL-10+Breg cells are decreased PsA and Ps and inversely correlated with the severity of psoriasis and IL-17A+ and IFNγ+ T cells.

Immune responses against Helicobacter pylori -specific antigens differentiate relapsing remitting from secondary progressive multiple sclerosis

To assess whether Helicobacter pylori (Hp) antibody (ab) reactivity against individual Hp antigens is pathogenetically relevant to multiple sclerosis (MS), we systematically investigated prevalence and clinical significance of abs against 14 immunodominant and subdominant Hp antigens by ELISA and immunoblotting in 139 consecutive MS patients with relapsing-remitting (RRMS, nu2009=u2009102) or secondary progressive (SPMS, nu2009=u200937). Sera from 39 patients with Parkinson’s disease (PD), 21 with Alzheimer’s disease (ALZ) and 68 healthy controls (HCs), were also tested. Anti-flagellin (18.3%) and anti-p41 (25.0%) abs in MS were less frequent than in HCs (39.4%, 48.5%, respectively). Abs against 5 of the 14 antigens were less frequent in RRMS than HCs, including p41, p54-flagellin, p29-UreA, p67-FSH, and p120-CagA. Anti-VacA abs were more frequent in SPMS than in HCs (42.1 vs 12.1%, pu2009=u20090.019). Anti-p54, anti-p29-UreA and anti-p26 correlated with extended disability status scale (EDSS) (pu2009=u20090.017, pu2009=u20090.005, pu2009=u20090.002, respectively). Anti-p26 and anti-p17 correlated with the number of relapses (pu2009=u20090.037 and pu2009=u20090.047, respectively). This is the first comprehensive analysis of ab reactivities against most Hp antigens in MS patients. Ab responses differ between MS and HCs and between RRMS and SPMS, being more prevalent in SPMS than RRMS, thus suggesting an association between anti-Hp and the former type of MS.

Crohn's disease-specific anti-CUZD1 pancreatic antibodies are absent in ruminants with paratuberculosis.

BACKGROUNDnPancreatic autoantibodies (PABs) specifically recognizing GP2 and/or CUZD1 are present in more than 35% of patients with Crohns disease (CrD). We have recently provided evidence of the presence of GP2-specific PABs in ruminants with paratuberculosis (ptb), a Mycobacterium avium paratuberculosis (MAP)-induced disease resembling CrD.nnnOBJECTIVEnTo assess whether anti-CUZD1 antibodies are also present in ruminants with ptb.nnnMETHODSnA total of 110 samples (73 cattle/37 sheep) were studied including 40 with ptb (24 cattle/16 sheep; 20 anti-GP2 antibody pos) and 70 without ptb (49 cattle/21 sheep; 10 anti-GP2 antibody pos). The samples were pre-characterized for anti-MAP and anti-GP2 antibodies by ELISA. Evidence of MAP was confirmed by PCR. Anti-CUZD1 antibody testing was performed by indirect immunofluorescence (IIF) based on transfected HEK293 cells expressing CUZD1. Anti-sheep or anti-cattle specific antisera were used as revealing antibodies.nnnRESULTSnNone of the ruminant sera had anti-CUZD1 antibodies by IIF testing at dilutions varying from 1/10 to 1/160. Methodological flaws were prevented by a series of tests. Control sera from anti-CUZD1 positive CrD samples have shown anti-CUZD1 antibody reactivity at various concentrations. Antibody reactivity to GP2-expressing HEK293 cells has confirmed the reactivity to GP2 in ruminant sera found positive for anti-GP2 antibodies by ELISA.nnnCONCLUSIONnThe present study has found no evidence of anti-CUZD1 PABs in MAP-induced ptb. Our findings indicate that the induction of CUZD1-specific PABs is unrelated to MAP infection and that the mechanisms responsible for the loss of tolerance to GP2 and CUZD1 are probably quite distinct.

AB0751 The epstein-barr virus infection in systemic sclerosis

Background Epstein-Barr virus (EBV) infection has been considered trigger of various autoimmune diseases, including systemic sclerosis (SSc), mainly due to studies investigating cross-reactive responses amongst EBV and disease-specific antigens. Meticulous assessment of antibody reactivities to the most immunodominant EBV antigens in SSc has not been performed. Objectives To assess ab reactivity against EBV viral capsid antigens (VCA), early antigens (EA) and EBNA-1 in SSc, and investigate their clinical relevance. Methods Sera from 59 SSc patients, including 31 diffuse SSc (dcSSc) and 28 limited SSc (lcSSc), 43 matched multiple sclerosis (MS) as controls and 32 matched healthy controls (HC) were tested for IgG anti-EBV VCA, EA and EBNA-1 abs by immunoblotting, using EBV whole SDS extract as antigen substrate. Results Percentages of EA and EBNA-1 reactivities were significantly higher in SSc patients compared to HC (EA: 33.9% vs 3.1%, p=0.001; EBNA-1: 89.8% vs 68.8%, p=0.012), but were comparable between SSc and MS. These differences remained when SSc was divided in dcSSc and lcSSc (EA: 32.3% in dcSSc and 35.7% in lcSSc, p dcSSc vs HC=0.002, p lcSSc vs HC=0.001; EBNA: 92.9% in lcSSc, p lcSSc vs HC=0.020). VCA positivity was comparable between SSc or its two subgroups and MS or HCs. Also, triple positivity for all three antigen categories was observed more frequently in SSc, dcSSc and lcSSc compared to HCs (32.2% in SSc, 29% in dcSSc and 35.7% in lcSSc vs 3.1% in HC, p=0.001, p=0.004u2009and p=0.001, respectively). Anti- EA was present more frequently in SSc patients with calcinosis compared to those without (75% vs 27.5%, p=0.014) and tended to be more frequent in patients with pulmonary fibrosis compared to those without (47.8% vs 25%, p=0.071). Conclusions Antibodies against EBV appear to be more frequent in SSc than in healthy controls, and equally prevalent with MS, a disease known to be associated with anti-EBV antibody responses and a known risk factor for MS. Whether an EBV-specific response is also an initiating trigger of SSc remains to be investigated. Disclosure of Interest G. Efthymiou: None declared, C. Liaskos: None declared, E. Marou: None declared, E. Dardiotis: None declared, V. Tsimourtou: None declared, T. Scheper Employee of: EUROIMMUN, W. Meyer Employee of: EUROIMMUN, D. Bogdanos: None declared, G. Hadjigeorgiou: None declared, L. Sakkas: None declared

AB0019 IL-10-Producing Bregs Are Decreased in Psoriatic Arthritis and Inversely Correlate with Th17 Cells

Background B cells may form ectopic lymphoid-like structures in the synovium of patients with psoriatic arthritis (PsA) but the significance of this finding is not clear. A recent study found high frequency of autoantibodies in PsA1. Whether regulatory B cells (Bregs) are decreased or not in PsA is not clear. Objectives To assess the phenotypic and functional characteristics of Breg subsets, CD19(pos)CD27(pos)CD24(high) memory and CD19(pos)CD24(high)CD38(high) transitional Bregs in patients with PsA. Methods Peripheral blood mononuclear cells, isolated from 61 PsA patients, 21 patients with psoriasis (Ps) and 15 normal controls (NCs), were assessed. The expression of surface CD19, CD24, CD27 and CD38 and cytoplasmic IL-10 by Bregs was examined by flow cytometry following 24 hour bacterial CpG (ODN2006) stimulation using fluorochrome-conjugated monoclonal antibodies. We also measured IL-17 expression from T cells (Th17 cells) stimulated with PMA plus Ionomycin for 5 hours followed by intracellular staining (BD Biosciences). Results Transitional Bregs were significantly decreased in PsA patients compared to NCs (0.72±0.54 vs1.56±0.58, p=0.000) and in Ps compared to NCs (1.08±0.38 vs 1.56±0.58, p=0.016, as well as in PsA compared to Ps patients (p=0.002). Memory Bregs were significantly decreased in PsA patients compared to NCs (2.93±2.31vs 6.94±2.03, p=0.000) and in Ps patients (2.97±1.83) compared to NCs (p=0.000) but did not differ between PsA and Ps patients. The impairment of Bregs in PsA was not associated with number of swollen or tender joints, CRP, ESR, or treatment with biologics. IL-10-producing Bregs inversely correlated with Th17 cells in PsA and in Ps. Conclusions PsA exhibits numerical decrease and functional impairment of Bregs, especially of transitional Bregs. IL-10-producing Bregs inversely correlate with Th17 cells. The impairment of IL-10-producing Bregs may contribute to the pathogenesis of psoriatic disease. References Dolcino M et al. PLoSone 2014;9:e115424 Acknowledgement Financial support by ELKE, University of Thessaly Disclosure of Interest None declared