Use of forensic investigations in anti-doping

The fight against doping is mainly focused on direct detection, using analytical methods for the detection of doping agents in biological samples. However, the World Anti-Doping Code also defines doping as possession, administration or attempted administration of prohibited substances or methods, trafficking or attempted trafficking in any prohibited substance or methods. As these issues correspond to criminal investigation, a forensic approach can help assessing potential violation of these rules.In the context of a rowing competition, genetic analyses were conducted on biological samples collected in infusion apparatus, bags and tubing in order to obtain DNA profiles. As no database of athletes' DNA profiles was available, the use of information from the location detection as well as contextual information were key to determine a population of suspected athletes and to obtain reference DNA profiles for comparison.Analysis of samples from infusion systems provided 8 different DNA profiles. The comparison between these profiles and 8 reference profiles from suspected athletes could not be distinguished.This case-study is one of the first where a forensic approach was applied for anti-doping purposes. Based on this investigation, the International Rowing Federation authorities decided to ban not only the incriminated athletes, but also the coaches and officials for 2 years.

Use of forensic investigations in anti-doping.

The fight against doping is mainly focused on direct detection, using analytical methods for the detection of doping agents in biological samples. However, the World Anti-Doping Code also defines doping as possession, administration or attempted administration of prohibited substances or methods, trafficking or attempted trafficking in any prohibited substance or methods. As these issues correspond to criminal investigation, a forensic approach can help assessing potential violation of these rules. In the context of a rowing competition, genetic analyses were conducted on biological samples collected in infusion apparatus, bags and tubing in order to obtain DNA profiles. As no database of athletes' DNA profiles was available, the use of information from the location detection as well as contextual information were key to determine a population of suspected athletes and to obtain reference DNA profiles for comparison. Analysis of samples from infusion systems provided 8 different DNA profiles. The comparison between these profiles and 8 reference profiles from suspected athletes could not be distinguished. This case-study is one of the first where a forensic approach was applied for anti-doping purposes. Based on this investigation, the International Rowing Federation authorities decided to ban not only the incriminated athletes, but also the coaches and officials for 2 years.

Attentional networks efficiency in preterm children.

Recent studies have reported specific executive and attentional deficits in preterm children. However, the majority of this research has used multidetermined tasks to assess these abilities, and the interpretation of the results lacks an explicit theoretical backdrop to better understand the origin of the difficulties observed. In the present study, we used the Child Attention Network Task (Child ANT; Rueda et al. 2004) to assess the efficiency of the alerting, orienting and executive control networks. We compared the performance of 25 preterm children (gestational age < or = 32 weeks) to 25 full-term children, all between 5(1/2) and 6(1/2) years of age. Results showed that, as compared to full-term children, preterm children were slower on all conditions of the Child ANT and had a specific deficit in executive control abilities. We also observed a significantly higher correlation between the orienting and executive control networks in the preterm group, suggesting less differentiation of these two networks in this population.

The efficiency of a carbamazepine-mianserin combination scheme in opiate detoxification.

A recent comparative randomized double-blind study has suggested the utility of a carbamazepine/mianserin combination as a treatment for opiate withdrawal. The aim of the present study was to explore the feasibility and efficiency of this combination under naturalistic conditions. Five hundred and fifty mostly polysubstance abusing patients treated with a standardized scheme combining carbamazepine and mianserin were assessed with regard to deviations to the protocol, used co-medications and retention in treatment.Three hundred and sixty three patients (66.0%) received the carbamazepine/mianserin combination as specified by the standardized protocol. In 350 patients (63.7%) the whole 10 days was completed. The most frequently used p.r.n. medications were for anxiety (47.5%) and insomnia (54.5%).The treatment of opiate withdrawal with a carbamazepine/mianserin combination scheme in an inpatient setting seems to be feasible and applicable with few adaptations to most patients, and may represent an interesting treatment option for multidrug users.

Effects of fructose-containing caloric sweeteners on resting energy expenditure and energy efficiency: a review of human trials.

Epidemiological studies indicate that the consumption of fructose-containing caloric sweeteners (FCCS: mainly sucrose and high-fructose corn syrup) is associated with obesity. The hypothesis that FCCS plays a causal role in the development of obesity however implies that they would impair energy balance to a larger extent than other nutrients, either by increasing food intake, or by decreasing energy expenditure. We therefore reviewed the literature comparing a) diet-induced thermogenesis (DIT) after ingestion of isocaloric FCCS vs glucose meals, and b) basal metabolic rate (BMR) or c) post-prandial energy expenditure after consuming a high FCCS diet for > 3 days vs basal,weight-maintenance low FCCS diet. Nine studies compared the effects of single isocaloric FCCS and glucose meals on DIT; of them, six studies reported that DIT was significantly higher with FCCS than with glucose, 2 reported a non-significant increase with FCCS, and one reported no difference. The higher DIT with fructose than glucose can be explained by the low energy efficiency associated with fructose metabolism. Five studies compared BMR after consumption of a high FCCS vs a low FCCS diet for > 3 days. Four studies reported no change after 4-7 day on a high FCCS diet, and only one study reported a 7% decrease after 12 week on a high FCCS diet. Three studies compared post-prandial EE after consumption of a high FCCS vs a low FCCS diet for > 3 days, and did not report any significant difference. One study compared 24-EE in subjects fed a weight-maintenance diet and hypercaloric diets with 50% excess energy as fructose, sucrose and glucose during 4 days: 24-EE was increased with all 3 hypercaloric diets, but there was no difference between fructose, sucrose and glucose. We conclude that fructose has lower energy efficiency than glucose. Based on available studies, there is presently no hint that dietary FCCS may decrease EE. Larger, well controlled studies are however needed to assess the longer term effects of FCCS on EE.

Clinical evaluation of iron treatment efficiency among non-anemic but iron-deficient female blood donors: a randomized controlled trial.

ABSTRACT: Iron deficiency without anemia (IDWA) is related to adverse symptoms that can be relieved by supplementation. Since a blood donation can induce such an iron deficiency, we investigated the clinical impact of an iron treatment after blood donation. METHODS: One week after donation, we randomly assigned 154 female donors with IDWA aged <50 years to a 4-week oral treatment of ferrous sulfate vs. placebo. The main outcome was the change in the level of fatigue before and after the intervention. Also evaluated were aerobic capacity, mood disorder, quality of life, compliance and adverse events. Biological markers were hemoglobin and ferritin. RESULTS: Treatment effect from baseline to 4 weeks for hemoglobin and ferritin were 5.2 g/L (p < 0.01) and 14.8 ng/mL (p < 0.01) respectively. No significant clinical effect was observed for fatigue (-0.15 points, 95% confidence interval -0.9 to 0.6, p = 0.697) or for other outcomes. Compliance and interruption for side effects was similar in both groups. Additionally, blood donation did not induce overt symptoms of fatigue in spite of the significant biological changes it produces. CONCLUSIONS: These data are valuable as they enable us to conclude that donors with IDWA after a blood donation would not clinically benefit from iron supplementation. Trial registration: NCT00689793.

Human growth hormone doping in sport.

BACKGROUND AND OBJECTIVES: Recombinant human growth hormone (rhGH) has been on the list of forbidden substances since availability of its recombinant form improved in the early 1990s. Although its effectiveness in enhancing physical performance is still unproved, the compound is likely used for its potential anabolic effect on the muscle growth, and also in combination with other products (androgens, erythropoietin, etc.). The degree of similarity between the endogenous and the recombinant forms, the pulsatile secretion and marked interindividual variability makes detection of doping difficult. Two approaches proposed to overcome this problem are: the indirect method, which measures a combination of several factors in the biological cascade affected by administration of GH; and the direct method, which measures the difference between the circulating and the recombinant (represented by the unique 22 kD molecule) forms of GH. This article gives an overview of what is presently known about hGH in relation to sport. The available methods of detection are also evaluated. METHODS: Review of the literature on GH in relation to exercise, and its adverse effects and methods of detection when used for doping. RESULTS AND CONCLUSION: The main effects of exercise on hGH production and the use and effects of rhGH in athletes are discussed. Difficulties encountered by laboratories to prove misuse of this substance by both indirect and direct analyses are emphasised. The direct method currently seems to have the best reliability, even though the time window of detection is too short. hGH doping is a major challenge in the fight against doping. The effect of exercise on hGH and its short half-life are still presenting difficulties during doping analysis. To date the most promising method appears to be the direct approach utilising immunoassays.

The efficiency of a novel delivery system (PEI600-Tat) in development of potent DNA vaccine using HPV16 E7 as a model antigen.

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. The mode of plasmid DNA delivery is critical to make progress in DNA vaccination. Using human papillomavirus type 16 E7 as a model antigen, this study evaluated the effect of peptide-polymer hybrid including PEI600-Tat conjugate as a novel gene delivery system on the potency of antigen-specific immunity in mice model. At ratio of 10:50 PEI-Tat/E7DNA (w/w), both humoral and cellular immune responses were significantly enhanced as compared with E7DNA construct and induced Th1 response. Therefore, this new delivery system could have promising applications in gene therapy.

Atypical EPO profiles in anti-doping analyses

Résumé : Erythropoietin (EPO) is a glycoprotein hormone endogenously produced by the kidney, whose main physiological role is the stimulation of erythropoiesis. Since the beginning of the nineties, recombinant human EPO (rhEPO), a potent anti-anaemia treatment drug, has been manufactured by pharmaceutical industries. However, the erythropoiesis stimulating power of rhEPO was rapidly misused by unscrupulous athletes in order to improve their performances in endurance sports. Endogenous EPO has the same amino-acid backbone as most of recombinant forms; the molecules however differ through their respective glycosylation patterns. This difference constitutes the basis of the usual EPO screening test (IEF) developed in 2000 and still currently used in all anti-doping laboratories of the world. Nowadays, 3 EPO generations have been commercialized. The fight against EPO abuse is a continuous challenge for anti-doping laboratories. The diversity of recombinant EPO forms and the continuous development of new ones considerably confuse the identification of EPO doping. Several facets of this fight were investigated in this work. One of the limiting aspects of doping agents screening is the availability of positive samples. Therefore, 2nd and 3rd generation EPOS, namely NESP and C.E.R.A., were injected to healthy subjects in the frame of pilot clinical studies. These latter allowed to review the current EPO identification criteria defined by the World Anti-Doping Agency (WADA) in the case of NESP and to validate and implement a new assay targeting C.E.R.A. in human serum. Both studies resulted in the determination of the respective detection windows of NESP and C.E.R.A. in biological fluids. Following that, Dynepo, a 1st generation EPO presenting similarities with the endogenous form, was also in the centre of a similar clinical study. Our work aimed to overcome the actual identification criteria, which are not adapted to Dynpeo, and to propose an alternative pattern classification method based on the discriminant analysis of IEF EPO profiles. This method might be validated for other EPO forms in the future. The detection window of this molecule was also determined. Under particular conditions, confounding effects can complicate the identification of EPO in biological matrices. For example, athletes having performed a strenuous physical effort can excrete modified isoforms of endogenous EPO, making it very similar to some recombinant forms. Such phenomena, called effort urines, were reproduced under controlled conditions and, after characterization of effort EPO, an urinary biochemical marker was proposed to unequivocally identify effort urines. It also happens that EPO analyses fail to detect endogenous levels of EPO. Such profiles were thoroughly investigated and potential causes identified. Natural reasons relying on urine properties and test specificity were underlined, but the possible addition of adulterant agents in urine samples was also considered. Therefore, a simple biochemical assay targeting the suspected substances was set up. Our work was based on the characterization of atypical EPO profiles from different origins. Therefore, 3 EPO molecules representing the 3 generations of the drug and 2 confounding effects confusing the results interpretation were studied. These studies resulted in tangible applications for the laboratory, the best example of which being the C.E.R.A. assay, but also in scientific findings allowing to improve our comprehension of EPO doping in sport.

Experimental manipulation of colony genetic diversity had no effect on short-term task efficiency in the Argentine ant Linepithema humile

Genetic diversity might increase the performance of social groups by improving task efficiency or disease resistance, but direct experimental tests of these hypotheses are rare. We manipulated the level of genetic diversity in colonies of the Argentine ant Linepithema humile, and then recorded the short-term task efficiency of these experimental colonies. The efficiency of low and high genetic diversity colonies did not differ significantly for any of the following tasks: exploring a new territory, foraging, moving to a new nest site, or removing corpses. The tests were powerful enough to detect large effects, but may have failed to detect small differences. Indeed, observed effect sizes were generally small, except for the time to create a trail during nest emigration. In addition, genetic diversity had no statistically significant impact on the number of workers, males and females produced by the colony, but these tests had low power. Higher genetic diversity also did not result in lower variance in task efficiency and productivity. In contrast to genetic diversity, colony size was positively correlated with the efficiency at performing most tasks and with colony productivity. Altogether, these results suggest that genetic diversity does not strongly improve short-term task efficiency in L. humile, but that worker number is a key factor determining the success of this invasive species.

Analytical aspects in doping control: challenges and perspectives.

Since the first anti-doping tests in the 1960s, the analytical aspects of the testing remain challenging. The evolution of the analytical process in doping control is discussed in this paper with a particular emphasis on separation techniques, such as gas chromatography and liquid chromatography. These approaches are improving in parallel with the requirements of increasing sensitivity and selectivity for detecting prohibited substances in biological samples from athletes. Moreover, fast analyses are mandatory to deal with the growing number of doping control samples and the short response time required during particular sport events. Recent developments in mass spectrometry and the expansion of accurate mass determination has improved anti-doping strategies with the possibility of using elemental composition and isotope patterns for structural identification. These techniques must be able to distinguish equivocally between negative and suspicious samples with no false-negative or false-positive results. Therefore, high degree of reliability must be reached for the identification of major metabolites corresponding to suspected analytes. Along with current trends in pharmaceutical industry the analysis of proteins and peptides remains an important issue in doping control. Sophisticated analytical tools are still mandatory to improve their distinction from endogenous analogs. Finally, indirect approaches will be discussed in the context of anti-doping, in which recent advances are aimed to examine the biological response of a doping agent in a holistic way.

Effects of the transition time between muscle-tendon stretch and shortening on mechanical efficiency.

The net mechanical efficiency of positive work (eta(pos)) has been shown to increase if it is immediately preceded by negative work. This phenomenon is explained by the storage of elastic energy during the negative phase and its release during the subsequent positive phase. If a transition time (T) takes place, the elastic energy is dissipated into heat. The aim of the present study was to investigate the relationship between eta(pos) and T, and to determine the minimal T required so that eta(pos) reached its minimal value. Seven healthy male subjects were tested during four series of lowering-raising of the body mass. In the first series (S (0)), the negative and positive phases were executed without any transition time. In the three other series, T was varied by a timer (0.12, 0.24 and 0.56 s for series S (1), S (2) and S (3), respectively). These exercises were performed on a force platform sensitive to vertical forces to measure the mechanical work and a gas analyser was used to determine the energy expenditure. The results indicated that eta(pos) was the highest (31.1%) for the series without any transition time (S (0)). The efficiencies observed with transition times (S (1), S (2) and S (3)) were 27.7, 26.0 and 23.8%, respectively, demonstrating that T plays an important role for mechanical efficiency. The investigation of the relationship between eta(pos) and T revealed that the minimal T required so that eta(pos) reached its minimal value is 0.59 s.

Efficiency and safety of bilateral contemporaneous pallidal stimulation (deep brain stimulation) in levodopa-responsive patients with Parkinson's disease with severe motor fluctuations: a 2-year follow-up review.

OBJECT: The aim of this study was to evaluate the long-term safety and efficacy of bilateral contemporaneous deep brain stimulation (DBS) in patients who have levodopa-responsive parkinsonism with untreatable motor fluctuations. Bilateral pallidotomy carries a high risk of corticobulbar and cognitive dysfunction. Deep brain stimulation offers new alternatives with major advantages such as reversibility of effects, minimal permanent lesions, and adaptability to individual needs, changes in medication, side effects, and evolution of the disease. METHODS: Patients in whom levodopa-responsive parkinsonism with untreatable severe motor fluctuations has been clinically diagnosed underwent bilateral pallidal magnetic resonance image-guided electrode implantation while receiving a local anesthetic. Pre- and postoperative evaluations at 3-month intervals included Unified Parkinson's Disease Rating Scale (UPDRS) scoring, Hoehn and Yahr staging, 24-hour self-assessments, and neuropsychological examinations. Six patients with a mean age of 55 years (mean 42-67 years), a mean duration of disease of 15.5 years (range 12-21 years), a mean "on/off' Hoehn and Yahr stage score of 3/4.2 (range 3-5), and a mean "off' time of 40% (range 20-50%) underwent bilateral contemporaneous pallidal DBS, with a minimum follow-up period lasting 24 months (range 24-30 months). The mean dose of levodopa in these patients could not be changed significantly after the procedure and pergolide was added after 12 months in five patients because of recurring fluctuations despite adjustments in stimulation parameters. All but two patients had no fluctuations until 9 months. Two of the patients reported barely perceptible fluctuations at 12 months and two at 15 months; however, two patients remain without fluctuations at 2 years. The mean improvements in the UPDRS motor score in the off time and the activities of daily living (ADL) score were more than 50%; the mean off time decreased from 40 to 10%, and the mean dyskinesia and complication of treatment scores were reduced to one-third until pergolide was introduced at 12 months. No significant improvement in "on" scores was observed. A slight worsening after 1 year was observed and three patients developed levodopa- and stimulation-resistant gait ignition failure and minimal fluctuations at 1 year. Side effects, which were controlled by modulation of stimulation, included dysarthria, dystonia, and confusion. CONCLUSIONS: Bilateral pallidal DBS is safe and efficient in patients who have levodopa-responsive parkinsonism with severe fluctuations. Major improvements in motor score, ADL score, and off time persisted beyond 2 years after the operation, but signs of decreased efficacy started to be seen after 12 months.