Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability

Concurrent Oral 9 - Rheumatoid Arthritis: Aetiopathogenesis [OP59-OP64]: OP59. The Value of Interleukin-17 Serum Level in Rheumatoid Arthritis Immunopathogenesis

Background: Interleukin (IL)-17 is the main Th-1 cytokine, produced by activated T-lymphocytes. The potential IL-17 value in rheumatoid arthritis (RA) pathogenesis consists of its independent inflammatory response induction and mediated stimulation of proinflammatory factors synthesis resulting in joint destruction. The aim of study was to determine the role of IL-17 in immuno-inflammatory/autoimmune reactions development and to reveal IL-17 serum level associations with clinical and immunological characteristics of RA. Methods: 50 patients with early RA (disease duration >, Russia), anti-CCP antibodies (Axies-Shield Diagnostic, UK) were revealed using ELISA immunoassay. Results: On the base of IL-17 serum level patients were divided in two groups: group1 (n = 28) were patients with normal IL-17 serum level and group2 (n = 22) were those with high IL-17 serum level. In the group2, the rate of patients' pain assessment by visual analogue scale (67.3 ± 7.2 vs 32.8 ± 4.6; P < 0.001), tender (16.7 ± 2.0 vs 8.4 ± 1.1; P < 0.01) and swollen (12.3 ± 2.3 vs 3.9 ± 0.8; P < 0.01) joint count, DAS28 (5.0 ± 0.4 vs 2.8 ± 0.2 P < 0.01) were significantly higher compare to group1. It was found that in group2 the higher T-lymphocyte amount (CD3) was due to CD4 higher quantity, at the same time CD8 amount was significantly lower (22.2 ± 1.5% vs 28.4 ± 1.7%, P < 0.05) compare to group1. This caused the immunoregulative index increasing and indicated in the lost of autoimmune process regulation, including B-lymphocytes (CD19) activation. The CD154 expression was significantly lower in the group2 (3.4 ± 0.4% vs 10.8 ± 2.8%, P < 0.05) compare to group1. The difference in autoimmune reaction indices wasn't significant between groups except antibody-producing B-lymphocytes (13.7 ± 1.5% vs 8.5 ± 1.0%, P < 0.05) and IgM RF serum level (2.9 ± 0.3 U/ml vs 1.6 ± 0.5 U/ml, P < 0.05), which were significantly higher in group1. The IL-17 level had a positive correlative connections with DAS28 (r = 0.7; P < 0.05), circulative immune complex level (r = 0.38; P < 0.05), anti-CCP antibodies (r = 0.4; P < 0.05), IgM RF (r = 0.41; P < 0.05), CD4 (r = 0.38; P < 0.05) and negative correlative connection with CD8 (r = -0.39; P < 0.05). Conclusions: The importance of IL-17 value in immuno-inflammatory and autoimmune reactions development through T-lymphocytes activation in RA pathogenesis was confirmed. Thus the influence on T-depended immuno-inflammatory reaction products synthesis could be a new therapeutic target of RA patients' management. Disclosure statement: All authors have declared no conflicts of interes

An early history of T cell-mediated cytotoxicity.

After 60 years of intense fundamental research into T cell-mediated cytotoxicity, we have gained a detailed knowledge of the cells involved, specific recognition mechanisms and post-recognition perforin-granzyme-based and FAS-based molecular mechanisms. What could not be anticipated at the outset was how discovery of the mechanisms regulating the activation and function of cytotoxic T cells would lead to new developments in cancer immunotherapy. Given the profound recent interest in therapeutic manipulation of cytotoxic T cell responses, it is an opportune time to look back on the early history of the field. This Timeline describes how the early findings occurred and eventually led to current therapeutic applications

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Defective CD8(+)CD28(-) regulatory T cell suppressor function in rheumatoid arthritis is restored by tumour necrosis factor inhibitor therapy

Summary: Balanced immunoregulatory networks are essential for maintenance of systemic tolerance. Disturbances in the homeostatic equilibrium between inflammatory mediators, immune regulators and immune effector cells are implicated directly in the pathogenesis of autoimmune diseases, including rheumatoid arthritis (RA). In this study we characterize the peripheral blood CD8+CD28- regulatory T cells (Treg) contribution to the immunoregulatory network in health and in RA. In health, CD8+CD28- Treg are suppressive but, unlike CD4+Treg, they function predominantly through the action of soluble mediators such as interleukin (IL)-10 and transforming growth factor (TGF)-β. Neutralization of TGF-β consistently reduced CD8+CD28- Treg suppressor function in vitro. RA, CD8+CD28- Treg are increased numerically, but have reduced expression of inducible co-stimulator (ICOS) and programmed death 1 (PD-1) compared to healthy or disease controls. They produce more IL-10 but autologous T cells express less IL-10R. This expression was found to be restored following in-vitro addition of a tumour necrosis factor inhibitor (TNFi). Deficiencies in both the CD8+CD28- Treg population and reduced sensitivity of the T responder cells impact upon their regulatory function in RA. TNFi therapy partially restores CD8+CD28- Treg ability in vivo and in vitro, despite the defects in expression of functionally relevant molecules by RA CD8+CD28- Treg compared to healthy controls. This study places CD8+CD28- Treg cells in the scheme of immune regulation alongside CD4+ Treg cells, and highlights the importance of understanding impaired responsiveness to regulation that is common to these suppressor subsets and their restored function in response to TNFi therapy