Assaying Chemokines and Chemotaxis

Welwyn Garden City , Hertfordshire
Friday, 08 June 2007

Assaying Chemokines and Chemotaxis
Friday, 08 June 2007 09:00 - 17:00

BioPark Hertfordshire
Broadwater Road
Welwyn Garden City
Hertfordshire
AL7 3AX
United Kingdom

Map and Directions

The meeting will involve presentations from both academia and industry and will give an overview of current techniques employed in the assay of chemokines and chemotaxis in the context of current immunological research. Chair Dr James Pease, National Heart and Lung Institute, Imperial College London


09:00 – 09:45 Registration - Tea/Coffee

09:45 – 10:00 Introduction by the Chair
Dr James Pease, National Heart and Lung Institute, Imperial College London

10:00 – 10:30 Chemotactic factors involved in mast cell trafficking
Dr Charlotte Weller, Imperial College London, UK
Mast cells are tissue resident cells with important functions in allergy and inflammation. Knowledge is currently limited about factors that release mast cell progenitors from the bone marrow or recruit them to remote tissues. This talk will discuss some recent data obtained using a relatively high-throughput 96 well chemotaxis system, which has allowed us to identify chemoattractants produced by activated mast cells which recruit progenitor mast cells.

10:30 – 11:00 Use of the Gated-Autofluoresence Assay in determining eosinophil activation by
prostaglandins
Dr Hilary Sandig, Kings College London

Prostaglandin D2 (PGD2) is produced by activated mast cells and is believed to play an important role in allergic inflammation. In the last few years, a novel chemoattractant receptor for PGD2, CRTH2, has been described. CRTH2 is a promiscuous receptor and we have used assays of chemotaxis with eosionophils and a CRTH2 transfected cell line, and the Gated-Autofluorescence assay with eosinophils to investigate the ability of a panel of PGD2 metabolites to signal via CRTH2. The results show that the successive metabolism of PGD2 results in CRTH2 ligands of lower potency, but equal or in some cases enhanced efficacy.

11:00 – 11:25 Morning Tea/Coffee and poster viewing

11:25 – 11:55 Meso Scale Discovery: Multiplex Biomarker Platform for Immunology research
Mr Tuc Ahmad , Meso Scale Discovery
Meso Scale Discovery’s assays are based on a proprietary combination of electrochemiluminescence (ECL) detection and patterned arrays. As a result background signals are minimal because the stimulation mechanism (electricity) is decoupled from the signal (light). This enables MSD assays to offer greater sensitivity and dynamic range while conserving sample requirements.

MSD 96 well plate immunoassays can measure up to 10 analytes/25 ?l sample/well with a plate read speed of 1 minute per plate.

The MSD platform requires no fluidics, reservoirs, wavelength filters or calibration so it is exceedingly robust, simple to operate and can handle complex protein matrices.

11:55 – 12:25 Chemokine-induced leukocyte migration in vivo observed by intravital microscopy and confocal imaging
Dr Mathieu-Benoit
Voisin, Imperial College London
Chemokines are small molecules with pro-migratory properties generated during inflammatory reactions and are actively involved during the leukocyte extravasation process. In particular, they can induce firm adhesion and transmigration of immune cells through the vessel wall. In our studies we have investigated the direct effects of CXCL1/KC and CCL2/MCP-1 on leukocyte extravasation in vivo in a cremasteric inflammation model using intravital microscopy. This model allows direct observations of leukocyte/vessel wall interactions and has thus enabled us to study the mechanisms of chemokine-induced leukocyte migration in real-time in vivo.

12:25– 12:45 *Tour of the BioPark

12:45 – 13:45 Lunch and poster viewing

13:45 – 14:25  The Chemical Synthesis of Chemokines
Dr Alastair Hay, Almac Sciences, Scotland, UKThe chemical synthesis of peptides has for, many practitioners, been limited to short sequences of up to 40 or 50 amino acids. Through academic research and customer demand, we have developed methods of chemically synthesising much longer peptides than otherwise thought to be accessible, enabling the many benefits of peptide synthesis to be realised in larger proteins and protein domains. In particular, chemokines have proven to be an attractive synthetic target, and chemical synthesis comes to the fore in enabling the introduction of unnatural amino acids, or labels that are useful aids in research. Recently, we have begun offering a range of chemokine products that cover ligands for a range of chemokine receptors, and incorporate site-specifically labelled biotin or AlexaFluor®647. We describe some of the successes we have had in overcoming the challenges faced in the synthesis of modified chemokines, and give an indication of how we strive to make these products more accessible.

14:15 – 14:45  L-Arginine-Threonine-Arginine (RTR) inhibits Proline-Glycine-Proline(PGP)-Induced
Neutrophil Chemotaxis and Development of Lung Emphysema
Dr Aletta D Kraneveld Utrecht University The Netherlands

COPD-associated lung matrix degradation results in the generation of collagen-derived peptides such as N-acetyl-proline-glycine-proline (PGP) that have been reported to have chemotactic activities. Recently, we have demonstrated that the CXCreceptors mediate PGP-induced chemotaxis and that enhanced levels of PGP are present in lungs of COPD patients (Nat Med, 2006, 12:317). The complementary peptide, arginine-threonine-arginine (RTR), has been shown to bind PGP sequences and impede neutrophil infiltration. In the presented study, we have investigated the role of PGP and the possible effects of RTR in neutrophil chemotaxis in vitro and the development of lung emphysema in vivo.

Our results have provided evidence that PGP may be of importance in the pathogenesis of COPD especially in patients in which the disease is associated to a neutrophilic inflammation. The delineation of a population of patients with PGP associated COPD has potential therapeutic relevance in that administration of RTR or other PGP antagonists may prevent or ameliorate the adverse manifestation in patients with this incapacitating and fatal pulmonary disease.

14:45 – 15:15 Chemokine receptor internalisation: pharmacological characterisation of CXCR3
Dr Louise Jopling UCB Pharma, UK
Pharmacokinetic/pharmacodynamic (PK/PD) models are essential to the drug discovery process (Chien et al., 2005) and enable discrimination between compounds and dosing regimens and the nature of the PK/PD relationship. Here we describe the use of a murine CXCR3 internalisation assay (Jopling et al., 2006) and its utility for analysing the PK/PD relationship of the small molecule CXCR3 antagonist, NBI-74330 (Heise et al., 2005).

15:15 – 15:45 Afternoon Tea/Coffee and last poster viewing 

15:45 – 16:25 Chemokines and the regulation of leukocyte-endothelial cell adhesion
Professor Dorian Haskard, Imperial College, London, UK
Leukocyte interaction with endothelial cells is the first step in their passage into inflamed tissues and may be rate-limiting for the course of inflammatory responses. The talk will focus on the role of chemokines in modulating leukocyte adhesion molecule function under dynamic conditions, drawing from recent work investigating how antibodies and immune-complexes enhance leukocyte recruitment.

16:15 – 16:45 Context-dependent signaling and T lymphocyte navigation
Professor Stephen Ward,
University of Bath, UK
Hence, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad-spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3K? was the major contributor to CXCL12-induced migration and PI3K/Akt signaling. However, ex vivo maintenance/activation of T cells alone, resulted in the directed migratory response of T cells becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294402. Hence, the role of PI3K in directional migration is context-dependent and influenced by ex vivo maintenance/activation conditions.

16:45 - 17:00 Chairman’s summing up & close.

 


Abstract guidelines can be found at
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Abstracts can be submitted for poster presentations

All presentations will be published in the proceedings

Early registration deadline 20th April 2007

Before the early registration deadline

After the early registration deadline

Standard

£249

£485

Academic

£149

£298

Student

£99

£298

IBMS

£149

£198

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