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Code: 08C06CM

AIMS/SIGNIFICANCE: -differentiate blood from transfused and non-transfused individuals. - Correlate changes in protein pattern to Hb, physical performance and VO2 max. Finding markers of autologous blood doping is important in order to maintain the fundamental aspect of sports: fair play. BACKGROUND/HYPOTHESIS: Physical performance can be enhaced by blood boosting. Doping using the hormone EPO and homologous blood (non-self) can today be detected while autologous (self) blood transfusion is undetectable. Red blood cells (RBC) can be stored for up to 5 weeks in +4C and for several years in -80C. It is highly unlike that blood can be withdrawn from the body, treated and stored without change in any protein. METHODS: By using proteomic methods, thousands of proteins can be separated and identified. In combination with multivariate statistical methods protein markers to detect autologous blood transfusion can be found. Separation of proteins is done by 2D DIGE and proteon identification done by mass spectrometry. Wr can quantitatively detect changes in protein patterns, thereby separate blood from soped and un-soped individuals. RESULTS: A 10% difference in protein abundance can be detected (95%Cl). Over 2300 proteins protein spots can be separated from 100 uL of RBC. Fresh blood was comparared with blood stored for 5 weeks in -80C. 48 proteins were altered, including enzymes (e.g catalase) stress (E.G hsp 71) and structural (e.g actin) proteins. Ongoing experiments have detected -80 proteins changed by storage in +4 C for 5 weeks. STUDY DESIGN: After blood donation (10 subjects) and storage for 4 week at 4C, RBC will be reinfused. Blood samples will be taken from the subjects sevral times before and after donation and reinfusion. Control samples will be taken from a matched groups. Haemoglobin, Physical performance and VO2 max will be measured on 7 occasions.  

Main Findings: 

The specific aims of this study were: I. Differentiate blood from transfused and non-transfused individuals.
II. Correlate changes in protein pattern to Hb, physical performance and VO2max.       We have investigated the possibilities to use proteomics as a tool to screen the human Red blood cell (RBC) membrane proteome for novel and unique biomarkers useful for development of future diagnostic point-of-care tests. A comparison between fresh and freeze-stored (-80° C) RBC’s were performed using the 2D DIGE technique. From findings in freeze-stored blood, 20 candidate proteins were identified. 
A blood transfusion study was subsequently performed where 10 subjects underwent an autologous blood transfusion (2 x 450 mL donated whole was blood and 2 x 300 mL washed RBC’s re-infused) after 16 week freeze-storage of the RBC’s. Blood samples were drawn at 13 time points for hematological and proteomic analyses and physical performance testing done 9 times. 
Forty eight hours after blood transfusion, Hb increased by 5%, physical performance 
(Running time to exhaustion) was increased by 15% and VO2max by 16%. Only a weak correlation (R2 = 0.33) was seen between Running time and VO2max. 
Blood samples taken from the subjects as well as from the transfusion bags were analyzed by proteomic and standard clinical methods. There is a clear separation of blood taken from a freeze stored bag and fresh venous blood. Different protein profiles between blood taken before and after a transfusion can be visualized. Some of these results were confirmed by Western blot. 
Because no method is today available to directly detect an autologous blood transfusion, we believe that our method under development will provide a solution in a near future, and the current work-plan is to have a prototype (alpha-version) ready for testing within 18-24 months, pending funding and the speed of technical advancements. 

Code: 08C20TF 

AIMS OF THE PROJECT. This goal of this project has been to compare global patterns of gene expression as described in disparate WADA-sponsored studies of the effects of doping agents of methods such as erythropoietin or hypoxia, growth factors such as human growth hormone and IGF-1, steroids and others. To achieve that goal, we have continued to refine the informatics infrastructure and have developed protocols for the application of computational methods for large-scale meta analysis of gene expression data sets from three separate and independent doping studies. We approached the directors of a number of WADA-sponsored studies to obtain data bases that could all be subjected to uniform analytical procedures to identify those presumably few common features that might constitute rigorous markers of exposure to doping manipulation. We received extensive data sets from two other WADA-supported investigators – James Rupert of the University of British Columbia and Dr. Tejvir Khurana of the University of Pennsylvania – and have identified preliminary candidate signatures for further validation and comparison with results of additional data sets to be included in future analyses.   

Main Findings: 

 We have successfully used the WADA Informatics facility to down-load and analyze several large transcriptomic datasets, including the one generated in our own laboratory for the IGF-1 study (Bhasker and Friedmann, 2008), as well as datasets rom the WADA-supported studies of James Rupert at the University of British Columbia and Dr. Tejvir Khurana of the University of Pennsylvania. The purpose of these preliminary studies has been to identify and solve the up-loading difficulties that outside users might encounter. The results of that exercise are presented in detail in the attached figures. Briefly, we have demonstrated that a comparison of studies using disparate methods of creating hypoxic conditions in mice reveal similar patterns of transcriptional dysregulation, despite many major differences in experimental design. These similarities include established categories of biological processes, molecular function and specific gene aberrations (slides 4-6) of Powerpoint summary. Those similarities may constitute the beginnings of a rudimentary molecular “signature” for metabolic and gene expression responses to hypoxia and/or to possibly related manipulations such as artificially augmented blood production in a sport setting (Slide 7). In contrast, a comparison of hypoxia conditions with the expected “negative control” effects of IGF-1 exposure of muscle stem cells reveals fewer transcriptional changes in common with the hypoxic conditions, as expected. We emphasize that these results require extensive validation and corroboration with other related and unrelated data sets from other WADA investigators. That will be the emphasis for future studies with this system.

Code: 08C04MM

Different analytical approaches can be foreseen for direct analysis and for the identification of a characteristic signature pattern following gene doping. The pilot project will evaluate the proof of principle of Affinity Based Biosensors (ABB) integrated with bioinformatics and biomolecular approaches for gene doping detection. Our challenge is to provide a total analytical process for the evaluation of the presence of the gene doping event. The heart of the project is a new multi-screening and real time bioanalytical protocol, based on an affinity sensing platform to be used both in direct and indirect based approaches for gene doping detection. We believe affinity-based biosensors (ABBs), flanking conventional and profiling methodologies, can contribute to gene doping detection as fast, low cost and easy to use instrumental approach. In this context, a flexible platform, consisting of a biochip coupled to a label free technology for simultaneous measurements in short time could represent an innovative approach for selectively detecting gene doping markers (direct approach) or secondary effects induced by gene transfer (indirect approach). The feasibility of this project is assured by the high interdisciplinarity of the partners of the proponent team which will contribute with their specific competences to the definition of a total analytical process using bioanalytical, bioinformatics, biomolecular and immunological competences. The pilot project outcome, will be transferred to the Italian reference anti-doping laboratory (Federazione Italiana Medico Sportiva, Roma), accredited by WADA.

Main Findings

The present project aimed to develop an innovative analytical approach or delivering sampling and analytical protocol to be applied to gene doping detection, eventually setting up a database. A new multi-screening and real time bioanalytical protocol, based on an affinity sensing platform for gene doping detection was developed using an integrated multidisciplinary approaches based on bioanalytical, bioinformatics, biomolecular and immunological competences. In particular, we developed a bioinformatics supported study for the identification of suitable markers for gene doping tracing. The initial purpose of the Gene Doping Detection Database (GDDDB) is to provide functionality for the design of primers on sequences that can be potentially used as Vectors during gene doping. Since it is currently the most commonly used gene therapy vector, the pilot study GDDDB contains Adenovirus sequences only. The database scheme is designed so that it can be interfaced by a biomart engine (www.biomart.org). Primer design is done using the primer-BLAST web service provides by NCBI (www.ncbi.nlm.nih.gov/tools/primer-blast/) which is a web-based graphical interface to the Primer3 and BLAST algorithms. Here we describe the entity relationship diagram of the database, and the show how it is adjusted to a data warehouse scheme as required for use by the biomart engine. The GDDDB can be accessed and tested via the GDD portal at http:/aragon.cl.cam.ac.uk/GDD/dbportal.html. Furthermore a simple discrete Bayesian analysis is done to calculate the posterior probabilities if gene doping. These results are shown and are given for each probe used in the developed assay. The conditional posterior probabilities are also shown, depending on whether a high or low affinity has been observed from samples. The project also developed an animal model (in vivo approach). The in vivo approach has first used transgenic mice to for the EGFP reporter gene to validate the molecular analysis of the marker in different tissue. Once the applicability of the develop method for the analysis of the selected marker has been proved in this first transgenic model, then a second model system based on the injection of the vector, containing the same reporter gene EGFP, in the tibialis anterior and of the femoral quadriceps muscles was developed. The sampling has been executed at different times after transfection and from different tissues: muscles, liver, spleen, kidney, lung, heart, right quadriceps (site of injection) and left quadriceps. Moreover body fluids (urine, blood, tears) have also been used to evaluate the presence oof viral vector signature. To trace the marker gene dedicated approaches have been developed and applied to these animal models. The EGFP expression has also monitored in different tissue of transgenic mice. In order to unambiguously detect the presence of recombinant vector, a protocol for construct-specific sequences was also developed. For this purpose new primers pairs were designed respectively on 3' promoter and 5' end of the EGFP sequences. Finally different region of the EGFP marker was amplified for tracing the marker in the mice after gene-doping event mimicking. The target sequences were found in all the sampled tissue. Surface plasmon Resonance imaging (SPRi)-based sensing for the detection of the gene-doping event was achieved. In particular Affinity Based Biosensors (ABB) have been developed. Both for DNA target sequence detection (DNA sensing) and antibody detection in human serum (immunosensor) are reported. Immobilization chemistries for molecular robe surface binding, analytical protocol were first optimized using standard solutions and further applied to complex samples (PCR mixture for DNA sensing - direct approach and serum for indirect approach) The analytical platform (biochip) allows simultaneous and real-time detection of sequences belonging to the vector.

Code: 08C19YP 

Living at altitude increases haemoglobin and haematocrit, hence altitude-training is popular among endurance athletes. Since increases in haematocrit can be attained using illicit means like blood-doping or erythropoietin (Epo) use and are potentially hazardous, high haematocrit levels are used to exclude athletes from competition, also without evidence of doping. While sea-level athletes can choose to train at altitude, for others living at altitude has been a way of life for generations (e.g. east-Africans). Recent approaches developed to distinguish the effects of altitude on haematological profiles from those of blood-doping are approximately 20-80% successful. When applying these approaches to the haematological profiles of elite athletes, we found indications of systematic blood-doping or cases of naturally elevated blood markers. There is therefore an urgent need for these methods to be revised to remove any possibility of athletes being incorrectly banned from competition or, conversely, avoiding sanction due to broad definition of legal limits. In this project we will investigate standard red cell indices and contrast these following Epo administration in athletes not involved in competition. Gene-expression profiles will be assessed using the very latest gene-microarray technology. These results will be used to formulate new methods with improved discriminatory power relative to current detection protocols and in doing so eliminate the possibility of naturally elevated blood markers due to athletes living and/or training at altitude and unidentified doping due to inadequate detection. The use of gene-arrays, validated in this context, may provide gene-expression profiles relevant to other illegitimate approaches to improving oxygen carriage that may have been, or will be in the future, devised.

Main findings: 

The use of recombinant human erythropoietin (rHuEpo) is prohibited by the World Anti-Doping Agency. An OMICS-based longitudinal screening approach has the potential to improve further the performance of current detection methods such as the Athlete Biological Passport. For this project, we successfully used gene expression profiling in whole blood to identify genes that are differentially regulated following rHuEpo administration in Caucasian trained males and Kenyan endurance runners living at sea-level and moderate altitude (~2150 m), respectively. Relative to baseline, the expression of hundreds of genes were found to be altered by rHuEpo. In particular, 15 transcripts were profoundly up-regulated during the 4 weeks of rHuEpo administration and subsequently down-regulated up to 4 weeks post administration in both groups. Importantly, the same pattern was observed in all subjects. Furthermore, 30 transcripts were already differentially expressed two days after the first injection and are therefore promising candidate genes to detect microdose rHuEpo doping. The functions of the discovered genes were mainly related to either the functional or structural properties of the erythrocyte or to the cell cycle and its regulation. In summary, this research project successfully identified the blood “molecular signature” of rHuEpo administration and provided a set of candidate genes with potential to be robust biomarkers of rHuEpo doping. These preliminary results provide the strongest evidence to date that OMICS technologies such as gene expression have the potential to substantially improve and add a new dimension to the current anti-doping methods such the Athlete Biological Passport for rHuEpo detection.

Code: 08B01RG 

Protein hormones represent an extremely challenging analytical problem in terms of anti-doping control. The main reasons are that most of these proteins are produced by humans and that the concentrations in body fluids, such as blood or urine, are very low. Whereas the second condition puts stringent demands on the analytical instrument in terms of sensitivity the first condition does similar to the scientist’s ingenuity in order to enable differentiation between like and non-like. Thus far, all protocols addressing protein hormone doping are based on immunological techniques only and suffer from the problems like the unknown specificity of the antibodies in the assays, the potential cross-reactivity under different conditions, and the fact that all sample handlings cannot be monitored and only an end-stage reading is provided. Still, regardless the analytical measurement immunoglobulins will be required to address specifically a particular category of proteins in complex mixtures.  
This project aims at the development of a single step purification of all protein hormones from plasma through a multi-antibody platform followed by in-situ solid-phase proteolysis and nano-LC chip mass spectrometric identification and quantification. Three different phases can be distinguished for each protein hormone:  A- the characterisation of antibodies addressing a particular protein (or category) by means of surface Plasmon resonance (SPR). From this study the best immunoglobulin (in terms of surface bound properties, specificity and cross reactivity, thermodynamic parameters of the interaction, compatibility in a mixed antibody setting) will be selected.
B- extrapolation of the SPR results to a LC-compatible immunoaffinity stationary phase. For this purpose monoliths functionalised to use similar immobilisation chemistry as in SPR will be employed. Again, first single antibody monoliths shall be characterised and subsequently multi antibody monoliths will be build to finally address all protein hormones in a single step.
C- Identification and quantification of the IAC purified material. This will be accomplished by -1- eluting the IAC captured material onto a protease-containing stationary phase with a short (min) stop-flow setting to allow proteolysis -2- elution of the generated peptides into a reversed phase column to allow conventional chromatography -3- identification of the peptides from each hormone. Quantification shall be achieved using diagnostic peptides with specific isotopic labelling that will be injected as internal standards in each analysis.
The outcome should be a “all-in-one” single injection system that reduces sample manipulation/loss and addresses all hormones employing the same sample, recovering the remainder of the sample for other analytical procedures.

Main findings: 

The project AntiProDo aimed at the development of a single instrumental set-up that included purification of four protein hormones from plasma through a multi-antibody platform followed by in-situ solid-phase proteolysis and nano-LC chip mass spectrometric identification and quantification. 
Through a meticulous characterisation of the binding characteristics of multiple antibodies, one was selected for each of the four initial target analytes: hGH, EPO, hCG and IGF-I. Subsequently, customised monolith solid supports in silica capillaries were produced and functionalised with the antibodies. The functional behaviour was verified. Simultaneously, the same capillary support was developed to house proteolytic enzymes and the activity, efficiency and durability established. At another front, the target proteins were submitted to mass spectrometric analysis to establish the proteotypic peptides to target in a final setting. Heavy isotope labelled peptides were produced as internal standards and the analytical method based on nano-LC ms designed and validated. Ultimately, all elements were hyphenated to demonstrate the proof of concept for this approach.  
Further optimisation of the individual steps, particularly at the reproducibility in the manufacture of the functionalised capillaries, is required before this approach can be taken further.