Projets de recherche

Code: 07A01JR

As autologous blood doping augments the athlete’s blood with their own cells, detecting the procedure is challenging. Blood cell shape and distribution are being explored as potential markers for; however, changes in the transcript (the mRNA intermediate in the DNA-to-protein pathway) complement of the blood may be a more informative indicator of reinfused blood cells in an athlete. The transcript complement of a tissue is extremely sensitive to the environment, so blood cells that have been removed, processed and stored may show changes in transcript frequencies that could be diagnostic for autologous blood doping. RNA is an excellent candidate for doping tests as it is stable when stored correctly, quantifiable using commercially supported techniques, and measurable in very small blood samples. This proposal describes a one year pilot project using high-resolution transcription profiling of blood to identify transcripts with the potential to be used as a diagnostic test for autologous blood doping. The project has three phases: 1) identification of transcripts in whole and leuko-depleted blood that represent genes that are activated or up-regulated in blood cells in response to post-withdrawal processing and/or short-term storage, 2) use of simulated autologous blood doping to determine if these transcripts could be detected in a “doped” athlete; and 3) characterization of the inter- and intra-individual variation of the transcripts shown to have potential test utility under a variety of conditions experienced by athletes (e.g. physical exertion, hypoxia/altitude, time of day, and time in menstrual cycle). The ultimate goal of the project is to identify transcripts that are characteristic of stored blood, that are of sufficient quantity as to be identifiable in a recipient’s circulation following reinfusion, and have patterns of expression that are consistent, predictable, and informative in athletes, which believe could form the basis for a blood-based, gene-expression test for autologous blood doping.

Main Findings

The aim of this study was to determine if there were genes overexpressed in blood in response to refrigerated storage that could be used as markers for the presence of stored blood in an athlete’s circulation. To be considered a potential transcript for follow-up, there had to be a minimum of a 10 fold expression difference (as this represented he the typical dilution that would occur in an autologous reintroduction of a unit of blood into the ‘doper’s’ circulation. We also felt that the overexpressed transcripts needed to be part of a unique pathway and not a variant of other commonly expressed pathways, as those would be unlikely sufficiently robust to serve as a diagnostic tool. Outcome: Transcriptional analysis identified only a single non-hemoglobin gene that met the criteria for being a candidate for a test for blood storage. The gene was GADD45G (encoding: Growth arrest and DNA-damage-inducible protein GADD45 gamma). This gene is a stress response gene that has been shown to be up-regulated in circulating cells in response to ‘behavioral stress’ in animal models (e.g. restraint, Flint at al., J Neuroimmunol. 2005; 167:34-44). This response would likely make it an unpromising candidate for a doping control assay as expression levels would likely fluctuate between athletes due to differential responses to perceived stress (e.g. doing control, imminent or recent competition). The only other genes that fit the candidacy profile were ‘rare’ hemoglobins (delta and zeta). As the adult blood transcriptome is overwhelmingly made up of hemoglobin alpha and beta transcripts, it is unlikely that an assay sufficiently specific and sensitive could be developed based on differentiating between the highly homologous hemoglobin transcripts against this background. Overall, the results of the project do not support the use of transcriptomic analysis of athletes' blood as a method for detecting the inclusion of stored blood (i.e. autologous blood doping).

Code: 07A04CR

Non-specific binding of the monoclonal anti-human EPO antibody (clone AE7A5; R&D Systems, Inc.) used for the detection of doping with recombinant human erythropoietin by the worldwide practised isoelectric focusing (IEF) and Western double blotting method has been in discussion for several years. Publications in peer-reviewed journals addressed this subject in 2005 and 2006 and led to some scientific argumentation on the specificity of the employed detection antibody. However, under experienced users of the Epo-method several additional bands (usually two to four) in the basic region of the pH 2-6 IEF-gel caused scientific interest, because theses bands could only be occasionally detected among the tested urinary samples. Their relationship to Epo remained unclear for years. Nevertheless, it was obvious that these bands did not interfere with the endogenous urinary and recombinant Epo-IEF profiles and thus the evaluation of the profiles was not disturbed or questioned at all. We were able to identify the protein causing these basic IEF-bands by means of carrier ampholyte IEF-PAGE, SDS-PAGE, Western blotting, and nano-ESI high resolution mass spectrometry and presented the results at the 2007 Cologne Workshop on Dope Analysis. The name of the protein is Zinc-alpha-2-glycoprotein. It is a high abundant urinary protein. The purpose of this project is a detailed study on the specificity of the clone AE7A5 antibody. According to the WADA technical document on the “Harmonization of the method for the identification of epoetin alfa and beta (Epo) and darbepoetin alfa (NESP) by IEF-double blotting and chemiluminescent detection” the IEF-method “relies on the particular specificity of the monoclonal antibody with which it was developed (clone AE7A5)” and “this antibody is considered a critical reagent and shall not be changed”. However, during SDS-PAGE experiments we were able to show that AE7A5 also binds to urinary proteins other than erythropoietin and Zinc-alpha-2-glycoprotein. These proteins might interfere with the interpretation of the Epo-IEF profiles.

Main Findings

A comprehensive study on the non-specific binding behaviour of monoclonal anti-EPO antibody clone AE7A5, which has to be used according to the technical document on EPO-analysis of WADA (TD2009EPO) for the detection of EPO-doping, was performed. Human urine and serum were used as matrices, since both are also used for EPO-testing. Additionally, E. coli and S. cerevisiae lysates were investigated. Aside from zinc-alpha2-glycoprotein (ZAG) a weak interaction with transferrin was detected (urine, serum). However, the non-specific interaction of the four proteins, which were mentioned by Khan et al. (2005) [1] in the context of 2D-PAGE (Tamm Horsfall glycoprotein, alpha-1antichymotrypsin, alpha-2-thiol proteinase inhibitor, alpha-2-HS-glycoprotein precursor), could not be confirmed – neither on carrier ampholyte IEF- nor SDS-PAGE. Strong binding was observed for thioredoxin reductase of E. coli, but no interaction occured with human thioredoxin reductases 1 and 2. Another strong non-specific binding was found for enolase from S. cerevisiae. Contrary to E. coli thioredoxin reductase, human enolases (tested were two enolases from human brain) were also non-specifically detected by the antibody. E. coli thioredoxin reductase as well as human and S. cerevisiae enolases are also non-specifically bound on IEF-PAGE. However, due to their higher isoelectric points (pI) these proteins focus outside the region of the gel used for EPO evaluation and therefore have no influence on the analysis results. Due to the observed non-specific interactions of the clone AE7A5 anti-EPO antibody with proteins other than EPO, immunoaffinity purification is mandatory for SDS-PAGE analyses. This pre-cleaning step has already been performed in the past, since the high protein content of urinary retentates and serum samples impedes the accurate detection of EPO by SDS-PAGE. This step is mandatory due to the non-specific binding of the antibody to thioredoxin reductase of E. coli. The molecular mass of this protein is within the range of rhEPOs on SDS-PAGE. An interference with the detection of rhEPO-doping is excluded by the combined usage of (1) two monoclonal antibodies directed against different epitopes for the immunoaffinity purification step, (2) the Western double blotting procedure, and (3) the usage of IEF- and SDS-PAGE for the interpretation of analysis results. This strategy has already been implemented in TD2009EPO. Reference: [1] Khan A, Grinyer J, Truong ST, Breen EJ, Packer NH (2005) New urinary EPO drug testing method using two-dimensional gel electrophoresis. Clin Chim Acta 358:119-30.

Code: 07A14MA

Despite a committed antidoping movement, abuse of recombinant human erythropoietin (rHuEPO) continues. Clandestine use extends to the use of novel rHuEPO products in the hope these may be undetectable via the electrophoresis test which distinguishes endogenous EPO from the synthetic versions based on different electric charges on the respective molecules. Recombinant erythropoietins are the number one revenue-generating class of biological products on the market, with worldwide sales exceeding US $12 billion. The lapse of European patents in 2004 has opened the door for approval of biosimilar versions of rHuEPO, and multiple pharmaceutical companies are now embarked on the development of viable alternatives for this lucrative market. In order to maintain an effective screening process it is vital for laboratories to obtain pre-emptive information regarding the structure and detectability of these novel anaemia drugs. Several classes of products are being developed: biosimilar rHuEPOs (same molecular structure but produced using different procedures), long lasting EPOs (molecule modified to extend half-life in circulation), synthetic EPO receptor agonists (completely different molecular structure which nonetheless stimulates the EPO receptor) and EPO replacements (stimulate red cell production via non-EPO receptor pathways). The aim of this research is to validate the capacity for isoelectric focussing to discriminate between novel anaemia drugs and endogenous EPO, and where this proves impractical to develop new detection methodologies. We will enter collaborative arrangements with the pharmaceutical companies to seek access to their products prior to commercial release in order to thwart attempts to use novel drugs before an appropriate test has been introduced by antidoping authorities. The drugs will be screened at the WADA-accredited laboratory in Paris who developed the isoelectric test and are world leaders in its application to doping control.

Main Findings

An extensive network including pharmaceutical company representatives and their expert consultants was activated in order to identify and detect novel anaemia drugs. A comprehensive market appraisal and de novo interrogation of literature and websites dramatically expanded upon the initial estimate made in 2007 that around one dozen copy EPOs were available. The most recent estimate suggests this number to be in excess of 100 in 2010. A significant number of these products have been procured and analysed by an anti-doping laboratory; several unusual profiles, and in a small number of cases inexplicable characteristics, have been reported. An important emphasis of the project has been to facilitate collaboration between pharmaceutical manufacturers and the WADA. Introductions were made to different pharmaceutical and biotechnology companies which enabled WADA to commence discussions and in several cases in-depth collaboration to develop detection methods for novel ESAs considered of high interest. In 2009 the ESA industry experienced considerable upheaval following the publication of results (‘TREAT” study) indicating that administration of exogenous EPO posed previously unrecognised health risks. Subsequently it is anticipated that substantially greater research emphasis will be placed in the foreseeable future on development of novel ESA agents.

Code: 07C17JD

The project deals with the developpment of immunological methods for the screening and the confirmation analysis of tetracosactide in serum. In vitro and in vivo immunological studies will be performed to develop immunological tools and their performance will be compared to that of a spectrophysical method using a Q trap instrument. Immunoextraction from serum prior to immunological analysis will be scheduled for confirmation analysis but not for screening analysis. The aim is the specific detection of tetracosactide in serum at concentration level as low as 100 pg/ml or less in order to offer a relevant detection window.

Main Findings

Tetracosactide is a synthetic peptide which exercises the same biological effects as endogenous Adreno CorticoTropin Hormone (ACTH) produced by anterior pituitary gland. Its structure reproduces the first 24 amino acids of ACTH which is composed of 39 ones. A screening method by ELISA was developed and validated for detection of Tetracosactide in plasma. The principle of the method was to use an ELISA kit (Enzyme Immunossay Kit from Peninsula Laboratories, San Carlos, USA), that reacts with the part 1-24 of ACTH (and thus Tetracosactide), after total elimination of endogenous ACTH from the tested plasma samples. In such conditions, ELISA became specific for Tetracosactide. The sample preparation consisted in removing most of the proteins and any trace of ACTH from plasma. This was achieved by cation-exchange chromatography (CM Sephadex ®). The preanalytical conditions were shown to be very important. Only EDTA plasma samples must be analysed and if the plasma samples could not be frozen just after collection, it was essential to quickly ship them to the laboratory in refrigerated conditions. The detection limit of the method was 13 pg/mL. In parallel, a confirmation method by LC-MS/MS (Thermo) Triple Quad, ESI was developed and validated. The preparation of plasma included cation exchange chromatography (CM Sephadex ®) and solid-phase extraction (Oasis® HLB). The detection limit of the method was < 50 pg/mL.

Code: T07D01RB 

Many studies are being carried out to develop methods for identifying exposure to growth factors such as human growth hormone, IGF1 and other related factors with potential application in sport doping. A number of these studies are based on the hypothesis that identification of genes and their protein products abnormally expressed in tissues treated with these factors can provide definitive evidence – i.e., signatures - for exposure to such growth factors. A number of related parallel studies are being conducted under the sponsorship of WADA and an enormous amount of information is being accumulated. However, to date no concerted effort has been made to collate and compare these large data sets and results to identify factors common to these studies that would represent the markers most likely to be informative in a drug testing setting. We propose to carry out a pilot informatics study to compare results from several completed and on-going WADA-sponsored studies.

Main Findings: 

Most of the current methods to detect doping rely on the use of chemical tests to identify drugs in the body. These methods are very powerful and often highly effective. Recent advances have enabled scientists to develop entirely new and potentially even more effective kinds of tests that are based on the detection of the broad changes that drugs cause in the genes and the proteins of affected tissues. These new methods can examine the ways in which all of the 25,000 human genes are affected by exposure to drugs and how those changes in genes affect the content of the even larger number of proteins in human cells. WADA has mounted a major research effort to use these approaches to develop detection methods for doping and is supporting work in more than a dozen laboratories scattered around the world aimed at finding genetic and protein changes in blood, urine and other body fluids and tissues that can definitively identify exposure to given drugs. Very promising results are coming from this effort and potentially useful "signature" gene and protein changes are being identified. A feature of these approaches is that they produce enormous amounts of information that require unified methods of analysis that are generally beyond the computing and informatics capabilities of individual laboratories. To ensure that the data are being evaluated in a consistent and effective manner, WADA has established a centralized bioinformatics laboratory that uses the most modern computational and analytical methods to harmonize the results of all the separate research laboratories to ensure that tests coming from this approach are highly sensitive, accurate and specific. The facility has succeeded in establishing an effective infrastructure and operating procedures and is beginning to obtain and analyze data from the research laboratories. WADA is confident that this more unified and centralized approach to doping detection will lead to far more effective tests that are rigorous, accurate and sensitive and that provide an effective and fair approach to protect athletes and Sport from doping.

Code: 06B06MG

This is a highly interdisciplinary and coordinated project that is aimed at understanding the molecular modifications induced by prolonged IGF-1 gene expression in the skeletal muscle after viral mediated gene transfer in rodent animals. The project will exploit the availability of AAV vectors expressing different IGF-1 isoforms to transduce skeletal muscles, a unique system that permites the long-term evaluation of the effects induced by the growth factor in vivo. In the treated animals, the signatures of these modifications will be analyzed by detecting the presence of the delivered transgenes in serum by quantitative real-time PCR and by analyzing the modifications of the proteomic pattern in muscle by advanced proteomics and mass spectrometry. These studies will be complemented by a parallel evaluation of the proteomic modifications induced by IGF-1 gene transfer in skeletal muscle satellite cells in vitro.

The project will be divided into three tasks.

Task 1. The overall purpose of this task will be the identification of a proteomic signature of IGF-1 gene transfer in muscle cells. The project will involve the development of animal models for AAV-mediated IGF-1 gene transfer. In particular, viral vectors expressing different IGF-1 isoforms will be produced and used to inoculate the tibialis anterior and femoral quadriceps muscles of mice and rats. At different times after transduction, proteomic analysis of the transduced muscles will be performed. These studies will take advantage of state-of-the-art technology 2-DE DIGE and will have the ultimate purpose of identifying protein patterns specific to the IGF-1 expressing muscles. These studies will be paralleled by the analysis of proteomic changes in human skeletal myoblasts after ex vivo gene transfer of the IGF-1 cDNAs.

Task 2. This task is aimed at the identification of novel peptides markers that might be exploited for anti-doping purposes. In particular, the project is aimed at the indentifications, quantification and qualitative assessment of the proteins differential expressed in muscle transduced with IGF-1. Differentially expressed or modified proteins will be analyzed using advanced mass spectrometry instrumentation, including MALDI-TOF/TOF, ESI-linear quarupole ion trap and high resolution FT-MS instrumentation.

Task 3. This task is aimed at the assessment of gene doping by monitoring the presence of exogenous gene fragments in animal serum. This possibility is based on different experimental and clinical reports that indicate that muscle exercise determines signficant damage to muscle cells, with the release of intracellular content into the bloodstream. At different times after in vivo IGF-1 gene transfer, both in resting conditions and during muscle exercise, serum will be analyzed for the presence of promoter-, vectore backbone- or cDNA-specific gene fragments by TaqMan-based Real Time quantitative PCR.

Main findings

The main findings are not available due to the sensitivity of the information and results developed in this project.

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Publications connexes

• Reviewing research into attitudes towards doping in sport: Time to take stock
• Doping in sport: A review of medical practitioners’ knowledge, attitudes and beliefs
• Reviewing Coaches' Knowledge, Attitudes and Beliefs regarding Doping in Sport

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