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Test development (WP3)

Aims and Overview

We have identified two areas where new tests could be developed in order to improve high throughput phenotyping.  New tests will be developed to study cardiac arrhythmia which is a major contributor to human heart failure.  In addition Luminex Multiplex-Bead-Array technology will be further developed and tested for analysis of serum proteins.

Workplan

1. Development of Luminex Multiplex-Bead-Array platform for serum analysis

HZI, ICS & Ani.Rhône-Alpes (ENS-L)

Bastian Pasche, Andreas Lengeling, Werner Müller, Braunschweig, HZI, Laurent Monassier, ICS

Serum analysis tools gain increasing importance for the identification of biomarkers indicative for disease development and progression. One of the ultimate challenges is to gain more insight in systemic organismal responses by quantifying in parallel multiple proteins and peptides associated with abnormal physiology. This information can be subsequently be used to define the health status of an individual. In this workpackage, we will develop new, primary first-line phenotyping SOPs for mouse mutant lines that will allow measuring of multiple serum proteins and peptides in a suspension array system. To accomplish this we will use the Luminex Bead-Array System, which permits the multiplexing of up to 100 different assays within a single sample. This system uses a liquid suspension array of 100 sets of fluorophore-colour coded beads coupled with individual antibodies that can be used to bind specific analytes. Captured analytes are then subsequently quantified using a flow cytometer bead array reader. We will use the Luminex system to develop SOPs for the detection and quantification of proteins indicative for immune responses such as immunoglobulins, cytokines, chemokines and acute phase proteins but also for the measurement of other serum proteins that are important for secondary screens as suggested by EUMODIC partners. Particular attention will be given to adapt a number of hormonal assays to the Luminex technology. In fact endocrine parameters are at present rarely determined because a large amount of blood is required for adequate measurements. Multiplexing measurements will remedy this problem.

2. Development of an arrhythmia screen in EUMODIC

Mamas Mamas, Elizabeth J. Cartwright, Ludwig Neyses, Manchester; Laurent Monassier, ICS

Sudden cardiac death (SCD) contributes to significant mortality in both acute myocardial infarction and chronic cardiac failure, most commonly as a consequence of ventricular tachyarrhythmias. SCD accounts for approximately 60-70% of deaths in patients with NYHA class II congestive cardiac failure with rhythm disturbances being a major component. It is estimated that SCD accounts for 400,000-500,000 deaths in Europe each year. Screening for mutations which enhance the propensity to rhythm disorders would therefore add significant value to any large-scale mutational programme focussed on disease mechanisms.

At present, no thoroughly validated procedures for rhythm disorders are available for use in primary screening. Injection of pro-arrhythmic substances in mice has been used for years, but their use has largely been empirical and has not been standardised. We therefore suggest the development and standardisation of such tests in EUMODIC in order to identify mutations with high susceptibility to arrhythmias.

Screening techniques will be developed to measure QT duration, a major determinant of ventricular arrhythmias, by surface ECG. These will identify animals with 'long-QT syndrome', one of the hallmarks of both monogenic and polygenic arrhythmogenic diseases.  The screening test will be fully optimised and a standard operating procedure (SOP) will be instituted.  Analysis will be carried out in mice under basal conditions and under beta-blockade, which often unmasks latent QT prolongation, particularly in mice whose heart rate is mostly controlled by sympathetic drive.

Pharmacological screening methods will be developed; under ECG monitoring a variety of pro-arrhythmic substances including adrenaline (under parasympathetic blockade) and ouabain (a digitalis-like compound) will be injected to promote intracellular calcium overload and predispose mice to rhythm disturbances. Calcium overload is known to contribute to ventricular arrhythmias in cardiac failure, long-QT syndrome and other conditions. Adrenaline and ouabain have been used previously, however, careful dose-response curves and variability of sex- and strain-specific responses must be established prior to use in primary screening.  An SOP will be established for the procedure.

Overall, this development will lead to a highly standardised screening method for ventricular arrhythmias, which belong to the most frequent and dangerous manifestations of cardiovascular disease.