Whole-Blood and Peripheral Mononuclear Cell Transcriptional Response to Prolonged Altitude Exposure in Well-Trained Runners.

BACKGROUND: Recombinant human erythropoietin (rHuEpo) abuse by athletes threatens the integrity of sport. Due to the overlap in physiological response to rHuEpo and altitude exposure, it remains difficult to differentiate changes in hematological variables caused by rHuEpo or altitude, and therefore, other molecular methods to enhance anti-doping should be explored. OBJECTIVE: To identify the hematological and transcriptomic response to prolonged altitude exposure typical of practices used by elite athletes. DESIGN: Longitudinal study. SETTING: University of Cape Town and Altitude Training Centre in Ethiopia. PARTICIPANTS AND INTERVENTION: Fourteen well-trained athletes sojourned to an altitude training camp in Sululta, Ethiopia (∼2400-2500 m above sea level) for 27 days. Blood samples were taken before arrival, 24 hours, and 9, 16, and 24 days after arrival at altitude in addition to 24 hours and 6, 13, and 27 days upon return to sea level. MAIN OUTCOME MEASURES: Blood samples were analyzed for hemoglobin concentration, hematocrit, and reticulocyte percentage. The transcriptomic response in whole blood and peripheral blood mononuclear cells (PBMC) were analyzed using gene expression microarrays. RESULTS: A unique set of 29 and 10 genes were identified to be commonly expressed at every altitude time point in whole blood and PBMC, respectively. There were no genes identified upon return to sea level in whole blood, and only one gene within PBMC. CONCLUSIONS: The current study has identified a series of unique genes that can now be integrated with genes previously validated for rHuEpo abuse, thereby enabling the differentiation of rHuEpo from altitude exposure.

The MMAAS Project: An Observational Human Study Investigating the Effect of Anabolic Androgenic Steroid Use on Gene Expression and the Molecular Mechanism of Muscle Memory.

OBJECTIVE: It remains unknown whether myonuclei remain elevated post anabolic-androgenic steroid (AAS) usage in humans. Limited data exist on AAS-induced changes in gene expression. DESIGN: Cross-sectional/longitudinal. SETTING: University. PARTICIPANTS: Fifty-six men aged 20 to 42 years. INDEPENDENT VARIABLES: Non-resistance-trained (C) or resistance-trained (RT), RT currently using AAS (RT-AS), of which if AAS usage ceased for ≥18 weeks resampled as Returning Participants (RP) or RT previously using AAS (PREV). MAIN OUTCOME MEASURES: Myonuclei per fiber and cross-sectional area (CSA) of trapezius muscle fibers. RESULTS: There were no significant differences between C (n = 5), RT (n = 15), RT-AS (n = 17), and PREV (n = 6) for myonuclei per fiber. Three of 5 returning participants (RP1-3) were biopsied twice. Before visit 1, RP1 ceased AAS usage 34 weeks before, RP2 and RP3 ceased AAS usage ≤2 weeks before, and all had 28 weeks between visits. Fiber CSA decreased for RP1 and RP2 between visits (7566 vs 6629 µm 2 ; 7854 vs 5677 µm 2 ) while myonuclei per fiber remained similar (3.5 vs 3.4; 2.5 vs 2.6). Respectively, these values increased for RP3 between visits (7167 vs 7889 µm 2 ; 2.6 vs 3.3). CONCLUSIONS: This cohort of past AAS users did not have elevated myonuclei per fiber values, unlike previous research, but reported AAS usage was much lower. Training and AAS usage history also varied widely among participants. Comparable myonuclei per fiber numbers despite decrements in fiber CSA postexposure adheres with the muscle memory mechanism, but there is variation in usage relative to sampling date and low numbers of returning participants.

Altitude Training and Recombinant Human Erythropoietin: Considerations for Doping Detection.

The benefit of training at altitude to enhance exercise performance remains equivocal although the most widely accepted approach is one where the athletes live and perform lower-intensity running at approximately 2300 m with high-intensity training at approximately 1250 m. The idea is that this method maintains maximal augmentations in total hemoglobin mass while reducing the performance impairment of high-intensity sessions performed at moderate altitude and thus preventing any detraining that can occur when athletes live and train at moderate altitude. This training regimen, however, is not universally accepted and some argue that the performance enhancement is due to placebo and training camp effects. Altitude training may affect an athlete's hematological parameters in ways similar to those observed following blood doping. Current methods of detection appear insufficient to differentiate between altitude training and blood doping making the interpretation of an athlete's biological passport difficult. Further research is required to determine the optimal method for altitude training and to enhance current detection methods to be able to differentiate better blood doping and altitude exposure.

The Fluidity of Gender and Implications for the Biology of Inclusion for Transgender and Intersex Athletes.

One of the most contentious issues in modern day sport arises when sports are divided into male and female categories. The International Association of Athletics Federations' (IAAF) previous policy regulating intersex athletes was suspended by the Court of Arbitration for Sport (CAS), resulting in a new policy. The challenge faced by the governing body of athletics is to formulate a policy that upholds both international law and the Olympic charter that stipulates athletes compete without discrimination of any kind. Implementation of the policy has been delayed until after a verdict, expected no later than March 26, 2019, in the Semenya versus IAAF trial in the Court of Arbitration for Sport. If the policy is enacted, it will restrict athletes from competing in the female athletics category with specific differences of sex development (DSD) in races from 400 m up to the mile in international level competitions unless they lower their natural testosterone (T) levels below 5 nmol·L. To thoroughly assess this new IAAF policy, one needs to appreciate its legal, sociological, and scientific underpinnings but also the history of previous policies attempting to define precisely how athletes should be divided into male and female categories. We previously proposed a system to deal with gender variant athletes that relied on a determination of an "athlete/athletic gender." The concept of "athlete gender" was presented to multiple audiences, and the resulting survey is included. A large majority of participants (71% of 153) who answered the survey agreed with the idea of an athlete gender. This position also was accompanied by the request for more studies (20% of those who agreed) and concern over the process of hormone monitoring (32% of those who agreed) to avoid doping misuse. The primary argument of those participating in the survey that disagreed with the position (23% of 153) was that biological differences between males and females remained even after the transition (47% of opposing comments). Mixed gender/sex competitions provide unique opportunities for athletes to compete against one another outside of the traditional male/female divide and pave the way for a more flexible approach for dealing with gender variant athletes.

An observational human study investigating the effect of anabolic androgenic steroid use on the transcriptome of skeletal muscle and whole blood using RNA-Seq.

BACKGROUND: The effects of Anabolic Androgenic Steroids (AAS) are largely illustrated through Androgen Receptor induced gene transcription, yet RNA-Seq has yet to be conducted on human whole blood and skeletal muscle. Investigating the transcriptional signature of AAS in blood may aid AAS detection and in muscle further understanding of AAS induced hypertrophy. METHODS: Males aged 20-42 were recruited and sampled once: sedentary controls (C), resistance trained lifters (RT) and resistance trained current AAS users (RT-AS) who ceased exposure ≤ 2 or ≥ 10 weeks prior to sampling. RT-AS were sampled twice as Returning Participants (RP) if AAS usage ceased for ≥ 18 weeks. RNA was extracted from whole blood and trapezius muscle samples. RNA libraries were sequenced twice, for validation purposes, on the DNBSEQ-G400RS with either standard or CoolMPS PE100 reagents following MGI protocols. Genes were considered differentially expressed with FDR < 0.05 and a 1.2- fold change. RESULTS: Cross-comparison of both standard reagent whole blood (N = 55: C = 7, RT = 20, RT-AS ≤ 2 = 14, RT-AS ≥ 10 = 10, RP = 4; N = 46: C = 6, RT = 17, RT-AS ≤ 2 = 12, RT-AS ≥ 10 = 8, RP = 3) sequencing datasets, showed that no genes or gene sets/pathways were differentially expressed between time points for RP or between group comparisons of RT-AS ≤ 2 vs. C, RT, or RT-AS ≥ 10. Cross-comparison of both muscle (N = 51, C = 5, RT = 17, RT-AS ≤ 2 = 15, RT-AS ≥ 10 = 11, RP = 3) sequencing (one standard & one CoolMPS reagent) datasets, showed one gene, CHRDL1, which has atrophying potential, was upregulated in RP visit two. In both muscle sequencing datasets, nine differentially expressed genes, overlapped with RT-AS ≤ 2 vs. RT and RT-AS ≤ 2 vs. C, but were not differentially expressed with RT vs. C, possibly suggesting they are from acute doping alone. No genes seemed to be differentially expressed in muscle after the long-term cessation of AAS, whereas a previous study found long term proteomic changes. CONCLUSION: A whole blood transcriptional signature of AAS doping was not identified. However, RNA-Seq of muscle has identified numerous differentially expressed genes with known impacts on hypertrophic processes that may further our understanding on AAS induced hypertrophy. Differences in training regimens in participant groupings may have influenced results. Future studies should focus on longitudinal sampling pre, during and post-AAS exposure to better control for confounding variables.

Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples.

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes. Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments. Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

Anti-nociceptive effect of kinin B1 and B2 receptor antagonists on peripheral neuropathy induced by paclitaxel in mice.

BACKGROUND AND PURPOSE: In the current study, we investigated the role of both kinin B1 and B2 receptors in peripheral neuropathy induced by the chronic treatment of mice with paclitaxel a widely used chemotherapeutic agent. EXPERIMENTAL APPROACH: Chemotherapy-evoked hyperalgesia was induced by i.p. injections of paclitaxel (2 mg·kg⁻¹) over 5 consecutive days. Mechanical and thermal hyperalgesia were evaluated between 7 and 21 days after the first paclitaxel treatment. KEY RESULTS: Treatment with paclitaxel increased both mechanical and thermal hyperalgesia in mice (C57BL/6 and CD1 strains). Kinin receptor deficient mice (B1, or B2 receptor knock-out and B1B2 receptor, double knock-out) presented a significant reduction in paclitaxel-induced hypernociceptive responses in comparison to wild-type animals. Treatment of CD1 mice with kinin receptor antagonists (DALBK for B1 or Hoe 140 for B2 receptors) significantly inhibited both mechanical and thermal hyperalgesia when tested at 7 and 14 days after the first paclitaxel injection. DALBK and Hoe 140 were also effective against paclitaxel-induced peripheral neuropathy when given intrathecally or i.c.v. A marked increase in B1 receptor mRNA was observed in the mouse thalamus, parietal and pre-frontal cortex from 7 days after the first paclitaxel treatment. CONCLUSIONS AND IMPLICATIONS: Kinins acting on both B1 and B2 receptors, expressed in spinal and supra-spinal sites, played a crucial role in controlling the hypernociceptive state caused by chronic treatment with paclitaxel.