Biological Variation Data in Triathletes for Metabolism and Growth-Related Biomarkers Included in the Athlete Biological Passport.

BACKGROUND: When using biological variation (BV) data, BV estimates need to be robust and representative. High-endurance athletes represent a population under special physiological conditions, which could influence BV estimates. Our study aimed to estimate BV in athletes for metabolism and growth-related biomarkers involved in the Athlete Biological Passport (ABP), by 2 different statistical models. METHODS: Thirty triathletes were sampled monthly for 11 months. The samples were analyzed for human growth hormone (hGH), insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein 3 (IGFBP-3), insulin, and N-terminal propeptide of type III procollagen (P-III-NP) by immunoassay. Bayesian and ANOVA methods were applied to estimate within-subject (CVI) and between-subject BV. RESULTS: CVI estimates ranged from 7.8% for IGFBP-3 to 27.0% for insulin, when derived by the Bayesian method. The 2 models gave similar results, except for P-III-NP. Data were heterogeneously distributed for P-III-NP for the overall population and in females for IGF-1 and IGFBP-3. BV components were not estimated for hGH due to lack of steady state. The index of individuality was below 0.6 for all measurands, except for insulin. CONCLUSIONS: In an athlete population, to apply a common CVI for insulin would be appropriate, but for IGF-1 and IGFBP-3 gender-specific estimates should be applied. P-III-NP data were heterogeneously distributed and using a mean CVI may not be representative for the population. The high degree of individuality for IGF-1, IGFBP-3, and P-III-NP makes them good candidates to be interpreted through reference change values and the ABP.

Long-Term Within- and Between-Subject Biological Variation Data of Hematological Parameters in Recreational Endurance Athletes.

BACKGROUND: Hematological parameters have many applications in athletes, from monitoring health to uncovering blood doping. This study aimed to deliver biological variation (BV) estimates for 9 hematological parameters by a Biological Variation Data Critical Appraisal Checklist (BIVAC) design in a population of recreational endurance athletes and to assess the effect of self-reported exercise and health-related variables on BV. METHODS: Samples were drawn from 30 triathletes monthly for 11 months and measured in duplicate for hematological measurands on an Advia 2120 analyzer (Siemens Healthineers). After outlier and homogeneity analysis, within-subject (CVI) and between-subject (CVG) BV estimates were delivered (CV-ANOVA and log-ANOVA, respectively) and a linear mixed model was applied to analyze the effect of exercise and other related variables on the BV estimates. RESULTS: CVI estimates ranged from 1.3% (95%CI, 1.2-1.4) for mean corpuscular volume to 23.8% (95%CI, 21.6-26.3) for reticulocytes. Sex differences were observed for platelets and OFF-score. The CVI estimates were higher than those reported for the general population based on meta-analysis of eligible studies in the European Biological Variation Database, but 95%CI overlapped, except for reticulocytes, 23.9% (95%CI, 21.6-26.5) and 9.7% (95%CI, 6.4-11.0), respectively. Factors related to exercise and athletes' state of health did not appear to influence the BV estimates. CONCLUSIONS: This is the first BIVAC-compliant study delivering BV estimates that can be applied to athlete populations performing high-level aerobic exercise. CVI estimates of most parameters were similar to the general population and were not influenced by exercise or athletes' state of health.

Longitudinal Profiling of Endogenous Steroids in Blood Using the Athlete Biological Passport Approach.

CONTEXT: Detection of endogenous anabolic androgenic steroids (EAAS), like testosterone (T), as doping agents has been improved with the launch of the Steroidal Module of the Athlete Biological Passport (ABP) in urine samples. OBJECTIVE: To target doping practices with EAAS, particularly in individuals with low level of biomarkers excreted in urine, by including new target compounds measured in blood. DESIGN: T and T/androstenedione (T/A4) distributions were obtained from 4 years of anti-doping data and applied as priors to analyze individual profiles from 2 T administration studies in female and male subjects. SETTING: Anti-doping laboratory. Elite athletes (n = 823) and male and female clinical trials subjects (n = 19 and 14, respectively). INTERVENTION(S): Two open-label administration studies were carried out. One involved a control phase period followed by patch and then oral T administration in male volunteers and the other followed female volunteers during 3 menstrual cycles with 28 days of daily transdermal T application during the second month. MAIN OUTCOME MEASURE(S): Serum samples were analyzed for T and A4 and the performance of a longitudinal ABP-based approach was evaluated for T and T/A4. RESULTS: An ABP-based approach set at a 99% specificity flagged all female subjects during the transdermal T application period and 44% of subjects 3 days after the treatment. T showed the best sensitivity (74%) in response to transdermal T application in males. CONCLUSIONS: Inclusion of T and T/A4 as markers in the Steroidal Module can improve the performance of the ABP to identify T transdermal application, particularly in females.

Standardization of reticulocyte counts in the athlete biological passport: A practical update.

INTRODUCTION: The athlete biological passport monitors blood variables over time to uncover blood doping. With the phasing in of a new series of blood analyzers, the Sysmex XN series, it was necessary to examine the comparability of results with the previously employed XT/XE series. A previous comparison between XN and XT/XE series suggested a small but significant bias between the two instruments in the measurements of RET%. Here, we examined the comparability of RET% on the XN and XT/XE platform using data collected over the first year since the transition. METHODS: The comparability of results obtained from XN and XT/XE instruments was assessed using three datasets: (i) 767 blood samples measured on both instrument series in 22 WADA-accredited laboratories, (ii) 27 323 samples measured on either instrument across 31 laboratories, and (iii) 119 clinical samples and 110 anti-doping samples measured on both instruments in a single laboratory. RESULTS: Analysis of the three datasets confirms the previous observation of a bias toward higher RET% values for samples measured on Sysmex XN instruments compared with the XT/XE series. Using data across a larger number of XN instruments and a larger athlete population, the current work suggests that the bias is proportional and slightly higher than previously observed across most of the range RET% values. CONCLUSION: A model is proposed for the comparison of data across XN and XT/XE technologies whereby the instrument bias increases proportionally with RET% measured on Sysmex XN Series, but where the rate of increase is negatively related to IRF%.

Application of the Athlete Biological Passport Approach to the Detection of Growth Hormone Doping.

CONTEXT: Because of its anabolic and lipolytic properties, growth hormone (GH) use is prohibited in sport. Two methods based on population-derived decision limits are currently used to detect human GH (hGH) abuse: the hGH Biomarkers Test and the Isoforms Differential Immunoassay. OBJECTIVE: We tested the hypothesis that longitudinal profiling of hGH biomarkers through application of the Athlete Biological Passport (ABP) has the potential to flag hGH abuse. METHODS: Insulin-like growth factor 1 (IGF-1) and procollagen III peptide (P-III-NP) distributions were obtained from 7 years of anti-doping data in elite athletes (n = 11 455) and applied as priors to analyze individual profiles from an hGH administration study in recreational athletes (n = 35). An open-label, randomized, single-site, placebo-controlled administration study was carried out with individuals randomly assigned to 4 arms: placebo, or 3 different doses of recombinant hGH. Serum samples were analyzed for IGF-1, P-III-NP, and hGH isoforms and the performance of a longitudinal, ABP-based approach was evaluated. RESULTS: An ABP-based approach set at a 99% specificity level flagged 20/27 individuals receiving hGH treatment, including 17/27 individuals after cessation of the treatment. ABP sensitivity ranged from 12.5% to 71.4% across the hGH concentrations tested following 7 days of treatment, peaking at 57.1% to 100% after 21 days of treatment, and was maintained between 37.5% and 71.4% for the low and high dose groups 1 week after cessation of treatment. CONCLUSION: These findings demonstrate that longitudinal profiling of hGH biomarkers can provide suitable performance characteristics for use in anti-doping programs.

The Proteomic Signature of Recombinant Growth Hormone in Recreational Athletes.

OBJECTIVE: Administration of human growth hormone (hGH) is prohibited in competitive sport and its detection in an athlete's sample triggers an adverse analytical finding. However, the biological processes that are modulated by recombinant hGH are not well characterized and associated blood serum proteins may constitute new biomarkers for hGH misuse. METHODS: Thirty-five recreational athletes were enrolled in a study to investigate the time- and dose-dependent response of serum protein levels to recombinant hGH administration. Participants were randomly assigned to 4 groups, receiving 1 of 3 different doses of recombinant hGH or a placebo. Bio samples were collected at 22 time points over a period of 13 weeks, starting 4 weeks before treatment, during 3 weeks of treatment, and at 6 weeks' follow-up. A total of 749 serum samples were analyzed for 1305 protein markers using the SOMAscan proteomics platform. RESULTS: We identified 66 proteins that significantly associated with recombinant hGH administration and dosage, including well known hGH targets, such as IGF1, but also previously unknown hGH-related proteins (eg, protease inhibitors, WFIKKN1, and chemokines, CCL2). Network analysis revealed changes in specific biological pathways, mainly related to the immune system and glucose metabolism. CONCLUSION: Our analysis suggests that hGH administration affects biological processes more strongly than previously acknowledged. Some of the proteins were dysregulated even after hGH treatment and could potentially be developed into biomarkers for hGH misuse. Moreover, our findings suggest new roles for hGH-associated proteins in the etiology of hGH-related diseases and may indicate new risks that may be associated with hGH misuse.

Biomarkers of doping: uses, discovery and validation.

A biomarker of doping indicates the biological response to the use of a prohibited substance or method. Uncovering novel biomarkers of doping is a key objective in order to improve antidoping outcomes such as the detection of doping and changing athlete behavior toward doping practices. While the antidoping field has been successful in validating novel metabolites of prohibited substances, there has been less success in developing new biomarkers of doping. Employing the most suitable study designs and analytical approaches is critical to successfully uncovering novel biomarkers of doping with a high potential for translation into routine analysis. Here we argue that the antidoping field is well positioned for biomarker discovery and outline considerations for the development of novel biomarkers of doping.

Standardization of reticulocyte counts in the athlete biological passport.

INTRODUCTION: The percentage of circulating reticulocytes (RET%) is a useful marker of blood doping in the context of the Athlete Biological Passport (ABP). The viability of the ABP depends on the comparability of sample data obtained across multiple laboratories for a given athlete. With the recent introduction of a different technology for the measurement of reticulocytes, the goal of this study was to compare currently employed Sysmex XT/XE analyzers to the recently introduced Sysmex XN analyzer. METHODS: RET% differences were searched in two independent data sets, the first consisting of 95 369 RET% values coming from 29 laboratories located in five continents as part of routine testing for the ABP, the second from a targeted study involving 510 samples analyzed on both a Sysmex XT and XN analyzers by two different laboratories. RESULTS: A relatively small but significant bias of 0.27 ([0.22-0.35] 95% CI) for the first data set and 0.19% ([0.16-0.22] 95% CI) for the second data set was observed with Sysmex XN analyzers returning higher values than Sysmex XT/XE analyzers. This bias appears constant over most of the range of RET% measured in elite athletes. CONCLUSION: When RET% values are obtained for the same athlete with different technologies (XT/XE vs XN), an adjustment of RET% emanating from the XT/XE instruments through a decrease of 0.22% within the ABP calculated ranges appears to be sufficient to integrate the results from the two technologies.

Decreased PCSK9 expression in human hepatocellular carcinoma.

BACKGROUND: The management of hepatocellular carcinoma (HCC) is limited by the lack of adequate screening biomarkers and chemotherapy. In response, there has been much interest in tumor metabolism as a therapeutic target. PCSK9 stimulates internalization of the LDL-receptor, decreases cholesterol uptake into hepatocytes and affects liver regeneration. Thus, we investigated whether PCSK9 expression is altered in HCC, influencing its ability to harness cholesterol metabolism. METHODS: Thirty-nine patients undergoing partial hepatectomy or liver transplantation for HCC were consented for use of HCC tissue to construct a tissue microarray (TMA). The TMA was immunostained for PCSK9. Imagescope software was used to objectively determine staining, and assess for pathological and clinical correlations. PCSK9 and LDL receptor mRNA levels in flash-frozen HCC and adjacent liver tissue were determined by quantitative RT-PCR. Serum PCSK9 levels were determined by ELISA. RESULTS: By immunohistochemistry, there was significantly lower expression of PCSK9 in HCC as compared to adjacent cirrhosis (p-value < 0.0001, wilcoxon signed-rank test). Significantly greater staining of PCSK9 was present in cirrhosis compared to HCC (p value <0.0001), and positivity (percentage of positive cells) was significantly greater in cirrhosis compared to HCC (p-value < 0.0001). Conversely, significantly higher expression of LDL-R was present in HCC as compared to the adjacent cirrhosis (p-value < 0.0001). There was no significant correlation of PCSK9 staining with grade of tumor, but there were significant correlations between PCSK9 staining and stage of fibrosis, according to spearman correlation test. PCSK9 mRNA levels were relatively less abundant within HCC compared to adjacent liver tissue (p-value =0.08) and normal control tissue (p-value =0.02). In contrast, serum PCSK9 levels were significantly increased among patients with HCC compared to those with chronic liver disease without HCC (p-value =0.029). LDL receptor mRNA was consistantly greater in HCC when compared to normal control tissue (p-value = 0.06) and, in general, was significantly greater in HCC when compared to adjacent liver (p-value = 0.04). CONCLUSIONS: The decreased expression of PCSK9 and conversely increased LDL-R expression in HCC suggests that HCC modulates its local microenvironment to enable a constant energy supply. Larger-scale studies should be conducted to determine whether PCSK9 could be a therapeutic target for HCC.

Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and ß-cell protection.

Type 1 diabetes is due to destruction of pancreatic ß-cells. Lysine deacetylase inhibitors (KDACi) protect ß-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes in the nonobese diabetic (NOD) mouse model of type 1 diabetes and counteract inflammatory target cell damage by a mechanism of action consistent with transcription factor--rather than global chromatin--hyperacetylation. Weaning NOD mice received low doses of vorinostat and givinostat in their drinking water until 100-120 d of age. Diabetes incidence was reduced by 38% and 45%, respectively, there was a 15% increase in the percentage of islets without infiltration, and pancreatic insulin content increased by 200%. Vorinostat treatment increased the frequency of functional regulatory T-cell subsets and their transcription factors Gata3 and FoxP3 in parallel to a decrease in inflammatory dendritic cell subsets and their cytokines IL-6, IL-12, and TNF-α. KDACi also inhibited LPS-induced Cox-2 expression in peritoneal macrophages from C57BL/6 and NOD mice. In insulin-producing ß-cells, givinostat did not upregulate expression of the anti-inflammatory genes Socs1-3 or sirtuin-1 but reduced levels of IL-1ß + IFN-γ-induced proinflammatory Il1a, Il1b, Tnfα, Fas, Cxcl2, and reduced cytokine-induced ERK phosphorylation. Further, NF-κB genomic iNos promoter binding was reduced by 50%, and NF-κB-dependent mRNA expression was blocked. These effects were associated with NF-κB subunit p65 hyperacetylation. Taken together, these data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with autoimmune disease such as type 1 diabetes.

A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila.

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.

The stresses encountered during islet isolation and culture may have deleterious effects on beta-cell physiology. However, the biological response of human islet cells to isolation remains poorly characterized. A better understanding of the network of signaling pathways induced by islet isolation and culturing may lead to strategies aimed at improving islet graft survival and function. Laser capture microdissection (LCM) was used to extract beta-cell RNA from 1) intact pancreatic islets, 2) freshly isolated islets, 3) islets cultured for 3 days, and changes in gene expression were examined by microarray analysis. We identified a strong inflammatory response induced by islet isolation that continues during in-vitro culture manifested by upregulation of several cytokines and cytokine-receptors. The most highly upregulated gene, interleukin-8 (IL-8), was induced by 3.6-fold following islet isolation and 56-fold after 3 days in culture. Immunofluorescence studies showed that the majority of IL-8 was produced by beta-cells themselves. We also observed that several pancreas-specific transcription factors were down-regulated in cultured islets. Concordantly, several pancreatic progenitor cell-specific transcription factors like SOX4, SOX9, and ID2 were upregulated in cultured islets, suggesting progressive transformation of mature beta-cell phenotype toward an immature endocrine cell phenotype. Our findings suggest islet isolation and culture induces an inflammatory response and loss of the mature endocrine cell phenotype. A better understanding of the signals required to maintain a mature beta-cell phenotype may help improve the efficacy of islet transplantation.

Evidence of endoplasmic reticulum stress mediating cell death in transplanted human islets.

A key limitation to the success of islet transplantation is islet cell exhaustion and cell death during islet isolation and following transplantation. Endoplasmic reticulum (ER) stress has been identified as an important mechanism in the development of ß-cell dysfunction, cell death, and diabetes. This study investigated the role of ER stress in islet loss during human islet isolation and posttransplantation in a diabetic athymic mouse model. Islets were isolated from human organ donor pancreata using intraductal enzymatic dissociation and continuous density gradient purification. ER stress mediators were assessed by Western blot and by RT-PCR. Caspase-3 activity was quantified by a bioluminescent peptide cleavage assay. Normal and streptozotocin-treated diabetic nude mice were transplanted with 2,000 IEQ of human islets under the kidney capsule and the grafts were harvested 3 or 28 days after transplantation. The grafts were analyzed for the presence for ER stress signals by immunohistochemistry. Isolated islets demonstrated higher levels of ER chaperone Bip, ER stress mediators eIF2α, ATF, spliced XBP-1, and CHOP, and also ER stress-associated apoptotic signals like JNK, caspase-3/7, and cleaved PARP. Donor pancreatic tissue did not show expression of any of these ER stress mediators. After transplantation, low expression of only protective ER stress mediators was evident in the grafts from the normal recipients. In contrast, both protective and apoptotic ER stress mediators were highly expressed in the grafts of hyperglycemic mice. ER stress mediators were induced during islet isolation and may contribute to islet apoptosis and cell death. Islet isolation activates ER stress and apoptotic pathways in isolated islets. Hyperglycemia may prolong this ER stress signal in engrafted islets, converting the protective aspects of the ER stress response to a proapoptotic response and thus contribute to deterioration of ß-cell function and survival.

Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells.

OBJECTIVE: Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells. RESEARCH DESIGN AND METHODS: Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis factor-alpha). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively. RESULTS: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling. CONCLUSIONS: Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

The role of kinases in the Hedgehog signalling pathway.

The Hedgehog (Hh) signalling pathway has a crucial role in several developmental processes and is aberrantly activated in a variety of cancers. In Drosophila, many of the canonical Hh pathway components are phosphorylated, yet the precise role of these phosphorylation events in the regulation of Hh signal transduction is unclear. Furthermore, the Hh pathway receives input from several kinases that have well-described roles in other cellular functions, some of which have both positive and negative effects on Hh signalling. Several recent studies have characterized the role of specific phosphorylation events in the Hh pathway, and have begun to shed light on how phosphorylation of Hh signalling components affects their subcellular location, stability and activity to mediate the transcriptional response to the Hh gradient.

Islet neogenesis: a potential therapeutic tool in type 1 diabetes.

Current therapies for type 1 diabetes, including fastidious blood glucose monitoring and multiple daily insulin injections, are not sufficient to prevent complications of the disease. Though pancreas and possibly islet transplantation can prevent the progression of complications, the scarcity of donor organs limits widespread application of these approaches. Understanding the mechanisms of beta-cell mass expansion as well as the means to exploit these pathways has enabled researchers to develop new strategies to expand and maintain islet cell mass. Potential new therapeutic avenues include ex vivo islet expansion and improved viability of islets prior to implantation, as well as the endogenous expansion of beta-cell mass within the diabetic patient. Islet neogenesis, through stem cell activation and/or transdifferentiation of mature fully differentiated cells, has been proposed as a means of beta-cell mass expansion. Finally, any successful new therapy for type 1 diabetes via beta-cell mass expansion will require prevention of beta-cell death and maintenance of long-term endocrine function.