6 Supplementary Tables from Gene Expression of Parathyroid Tumors

OBJECTIVE:To compare estimates by bioimpedance spectroscopy analysis (BIS) of extracellular water (ECW), fat mass (FM), and fat-free mass (FFM) against standard techniques of bromide dilution and dual energy X-ray absorptiometry (DXA) during intervention that causes significant changes in water compartments and body composition.METHODS:Body composition analysis using BIS, bromide dilution, and DXA was performed in 71 healthy recreational athletes (43 men, 28 women; aged 18-40 years; BMI 24 ± 0.4 kg/m(2)) who participated in a double-blinded, randomized, placebo-controlled study of GH and testosterone treatment. The comparison of BIS with bromide dilution and DXA was analyzed using linear regression and the Bland-Altman method.RESULTS:At baseline, there was a significant correlation between BIS and bromide dilution-derived estimates for ECW, and DXA for FM and FFM (P<0.001). ECW by BIS was 3.5 ± 8.1% lower compared with bromide dilution, while FM was 22.4 ± 26.8% lower and FFM 13.7 ± 7.5% higher compared with DXA (P<0.01). During treatment, the change in ECW was similar between BIS and bromide dilution, whereas BIS gave a significantly greater reduction in FM (19.4 ± 44.8%) and a greater increase in FFM (5.6 ± 3.0%) compared with DXA (P<0.01). Significant differences in body composition estimates between the BIS and DXA were observed only in men, particularly during the treatment that caused greatest change in water compartments and body composition.CONCLUSION:In healthy adults, bioimpedance spectroscopy is an acceptable tool for measuring ECW; however, BIS overestimates FFM and substantially underestimates FM compared with DXA.

Background: Immune mechanisms of achieving sustained virologic response among HCV infected subjects are unclear.Studies of spontaneous clearance suggest a role for adaptive immunity in effective viral clearance, however, such studies in standard of care therapy trials are confounded by exogenous interferon administration.The objective of this study was to assess changes in HCV specific immune responses associated with suppression of viral replication during interferon free anti-HCV direct acting antiviral (DAA) therapy.Methods: Sixty HCV GT-1 subjects were treated with the potent NS5B inhibitor GS-7977 and ribavirin for 24 weeks.Comprehensive peripheral blood mononuclear cell (PBMC) and liver infiltrating lymphocyte (LIL) T cell, B cell, and NK cell immunophenotyping was performed for 10 patients and 5 controls at baseline, day 10, and end of treatment (EOT).HCV specific immune responses were quantified by IFN-gamma ELISpot and intracellular flow cytometry (IL-2, TNFalpha, and IFN-gamma) after stimulation with overlapping peptides spanning the entire HCV genome.Results: Most patients had rapid decline of HCV VL within 7 days of therapy (mean 3.87 log 10 IU/mL decline), and all patients completing therapy had no detectable virus at EOT. Baseline CD57 + (9.6%±2.3%)and PD-1 + (0.82%±0.27%)T cell counts were higher in chronically HCV-infected individuals than uninfected individuals (7.59%±1.1%,p = 0.046; 0.29%±0.1%,p = 0.05).Exhausted CD57 + PD-1 + CD8 + T cells were also higher compared to uninfected (2.38%±0.8%vs 1.6%±0.2%,p = 0.049) at baseline.Fewer CD8 + T cells expressed the exhausted phenotype at day 10 (2.38%±1.7%vs 1.67%±0.50%;p = 0.05) and EOT.Tim-3 + (9.5%±2.8%vs 0.99%±0.3%,p = 0.0001), and PD-1 + (63.7%±2.0%vs 8.1%±1.8%,p = 0.0003) cells were more frequent in the liver than the periphery at EOT. Strikingly, there was an increase in peripheral HCV-specific T cell IFN-gamma responses at day 10 and EOT compared to baseline (3.69 spots/10 6 vs 7.4 spots/10 6 PBMC and 13.3 spots/10 6 PBMC; p = 0.005), coincident with HCV VL decline.Conclusions: Inhibition of HCV replication by a DAA regimen is associated with rapidly increased HCV specific immunity and improved peripheral immunophenotype.This is the first study demonstrating HCV specific immune modulation with interferon-free DAA therapy, suggesting immune enhancement is a consequence of viral inhibition not requiring exogenous immune stimulation.

There is anecdotal evidence that growth hormone (GH) is widely abused, reportedly often in combination with anabolic steroids at high doses for several months. Development of a robust test for GH has been challenging. There are wide physiological fluctuations in circulating GH concentrations and the recombinant human 22 kDa GH used in doping is indistinguishable analytically from endogenous GH. The GH-responsive markers approach to detect GH doping is based on the measurement of serum proteins, such as insulin-like growth factor (IGF) and collagen peptides, that have more stable concentrations and longer half-lives than GH. Age and gender are the major determinants of variability for IGF-I and the collagen markers. A test based on these markers must take age into account for men and women and ethnicity is unlikely to be a confounder for IGF-I and the collagen markers. Extensive data from administration studies are now available to validate the use of GH-responsive marker and a test based on this approach will likely use a combination of markers. This approach extends the time window of detection of GH and to detect abuse of other forms of GH and GH secretagogues. Implementation of a test based on GH-responsive markers is now largely dependent on overcoming hurdles including availability of standardized assays with assured supplies of antibodies.

Despite being banned by the World Anti-Doping Agency, growth hormone (GH) is abused by athletes in sport, reportedly often in combination with anabolic androgenic steroids (AASs). There is evidence for clear benefits of GH treatment for growth hormone deficiency, including normalisation of body composition by increasing lean body mass (LBM) and reducing fat mass due to its lipolytic and anabolic actions, increase in muscle mass but not muscle strength with short-term treatment, and improved aerobic exercise capacity (VO(2)max).There is only limited evidence available for benefit of GH use in healthy young adults and athletes. GH decreases fat mass and significantly increases LBM in young, healthy physically fit adults. Fluid retention accounts for most of the increase in LBM induced by GH, rather than an increase in the muscle mass. There has been debate as to whether the improved body composition and metabolic changes induced by GH actually translate into improved performance. We have recently demonstrated in a randomised controlled study an increase in anaerobic sprint capacity following treatment with GH for 8 weeks. There is no evidence that GH enhances muscle strength or power, or aerobic exercise capacity in healthy young adults.There is strong evidence of harm from GH abuse. Side effects of GH administration in healthy adults include swelling, arthralgias and paresthesias, fatigue, and changes in cardiac morphology and function. Side effects of GH such as myocardial hypertrophy are likely worsened by co-administration of AASs. The features of acromegaly, including cardiac complications, arthropathy, insulin resistance and increased risk of diabetes and malignancy, indicate potential health risks of chronic GH abuse.In conclusion, the evidence suggests that in healthy adults, GH may improve a selective aspect of performance, that of anaerobic exercise capacity; however, muscle strength, power and aerobic exercise capacity are not enhanced by GH administration. Athletes should be aware of the serious health risks from prolonged use of GH.

Background: Doping with growth hormone (GH) is banned; however, there is anecdotal evidence that it is widely abused. GH is reportedly often used in combination with anabolic steroids at high doses for several months. Development of a robust test for detecting GH has been challenging since recombinant human 22-kDa GH used in doping is indistinguishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentrations. Discussion: One approach to GH testing is based on measurement of different circulating GH isoforms using immunoassays that differentiate between 22-kDa and other GH isoforms. Administration of 22-kDa GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method is, however, limited by its short time window of detection. A second approach that extends the time window of detection is based on detection of increased levels of circulating GH-responsive proteins, such as the insulin-like growth factor (IGF) axis and collagen peptides. As age and gender are the major determinants of variability for IGF-I and the collagen markers, a test based on these markers must take these factors into account. Extensive data now validate the GH-responsive marker approach, and implementation is largely dependent on establishing an assured supply of standardized assays. Conclusions: Robust tests are available to detect GH and enforce the ban on its abuse in sports. Novel approaches that include gene expression and proteomic profiling must continue to be pursued to expand the repertoire of testing approaches available and to maintain deterrence of GH doping.

BACKGROUND:Growth hormone is widely abused by athletes, frequently with androgenic steroids. Its effects on performance are unclear. OBJECTIVE:To determine the effect of growth hormone alone or with testosterone on body composition and measures of performance. DESIGN:Randomized, placebo-controlled, blinded study of 8 weeks of treatment followed by a 6-week washout period. Randomization was computer-generated with concealed allocation. (Australian-New Zealand Clinical Trials Registry registration number: ACTRN012605000508673) SETTING:Clinical research facility in Sydney, Australia. PARTICIPANTS:96 recreationally trained athletes (63 men and 33 women) with a mean age of 27.9 years (SD, 5.7). INTERVENTION:Men were randomly assigned to receive placebo, growth hormone (2 mg/d subcutaneously), testosterone (250 mg/wk intramuscularly), or combined treatments. Women were randomly assigned to receive either placebo or growth hormone (2 mg/d). MEASUREMENTS:Body composition variables (fat mass, lean body mass, extracellular water mass, and body cell mass) and physical performance variables (endurance [maximum oxygen consumption], strength [dead lift], power [jump height], and sprint capacity [Wingate value]). RESULTS:Body cell mass was correlated with all measures of performance at baseline. Growth hormone significantly reduced fat mass, increased lean body mass through an increase in extracellular water, and increased body cell mass in men when coadministered with testosterone. Growth hormone significantly increased sprint capacity, by 0.71 kJ (95% CI, 0.1 to 1.3 kJ; relative increase, 3.9% [CI, 0.0% to 7.7%]) in men and women combined and by 1.7 kJ (CI, 0.5 to 3.0 kJ; relative increase, 8.3% [CI, 3.0% to 13.6%]) when coadministered with testosterone to men; other performance measures did not significantly change. The increase in sprint capacity was not maintained 6 weeks after discontinuation of the drug. LIMITATIONS:Growth hormone dosage may have been lower than that used covertly by competitive athletes. The athletic significance of the observed improvements in sprint capacity is unclear, and the study was too small to draw conclusions about safety. CONCLUSION:Growth hormone supplementation influenced body composition and increased sprint capacity when administered alone and in combination with testosterone. PRIMARY FUNDING SOURCE:The World Anti-Doping Agency.

Human growth hormone (GH) is widely abused by athletes; however, there is little evidence that GH improves physical performance. Replacement of GH in GH deficiency improves some aspects of exercise capacity. There is evidence for a protein anabolic effect of GH in healthy adults and for increased lean body mass following GH, although fluid retention likely contributes to this increase. The evidence suggests that muscle strength, power, and aerobic exercise capacity are not enhanced by GH administration, however GH may improve anaerobic exercise capacity. There are risks of adverse effects of long-term abuse of GH. Sustained abuse of GH may lead to a state mimicking acromegaly, a condition with increased morbidity and mortality.

CONTEXT:GH abuse is a significant problem in many sports, and there is currently no robust test that allows detection of doping beyond a short window after administration.OBJECTIVE:Our objective was to evaluate gene expression profiling in peripheral blood leukocytes in-vivo as a test for GH doping in humans.DESIGN:Seven men and thirteen women were administered GH, 2 mg/d sc for 8 wk. Blood was collected at baseline and at 8 wk. RNA was extracted from the white cell fraction. Microarray analysis was undertaken using Agilent 44K G4112F arrays using a two-color design. Quantitative RT-PCR using TaqMan gene expression assays was performed for validation of selected differentially expressed genes.RESULTS:GH induced an approximately 2-fold increase in circulating IGF-I that was maintained throughout the 8 wk of the study. GH induced significant changes in gene expression with 353 in women and 41 in men detected with a false discovery rate of less than 5%. None of the differentially expressed genes were common between men and women. The maximal changes were a doubling for up-regulated or halving for down-regulated genes, similar in magnitude to the variation between individuals. Quantitative RT-PCR for seven target genes showed good concordance between microarray and quantitative PCR data in women but not in men.CONCLUSION:Gene expression analysis of peripheral blood leukocytes is unlikely to be a viable approach for the detection of GH doping.

Application of methods for detecting GH doping depend on being able to discriminate between abnormal levels due to doping and normal physiological levels of circulating proteins that change in response to exogenous administration. Constituents of the IGF and collagen systems have been shown to be promising markers of GH abuse. Their ultimate utility, however, depends on identification of the factors that regulate their concentrations in blood. Among these are demographic factors that are known to influence these markers in the general population. In a large cross-sectional study of the GH-responsive markers in over 1000 elite athletes from 12 countries representing 4 major ethnic groups and 10 sport types, we have shown that there is a significant negative correlation between age and all the IGF and collagen markers we studied, with a rapid decrease in early adolescence. Age was the major contribution to the variability, equivalent to >80% of the attributable variation in IGF-I and the collagen markers. The IGF axis markers were all significantly higher in women, and the collagen markers significantly higher in men, however, the contribution of gender was smaller than that of age, except for IGFBP-3 and ALS. BMI had a minor contribution to variability of the GH-responsive markers. After adjustment for the confounding influences of age, gender and BMI, the effect of ethnicity in elite athletes was trivial except for IGFBP-3 and ALS, which were both lower in Africans and higher in Caucasians. Compared to age and gender, the contribution of sport type was also modest. Our findings on the influence of age, gender, BMI and sport type have also been confirmed in a study of mostly Caucasian elite athletes in the post-competition setting. In conclusion, age and gender are the major determinants of variability for IGF-I and the collagen markers, whereas ethnicity and sport type have a minor influence. Therefore, a test based on IGF-I and the collagen markers must take age into account for men and women, and ethnicity and sport type are unlikely to be confounders for these markers.

The primary screening method for the detection of doping by athletes using synthetic versions of endogenous steroids such as testosterone relies on measurement of the ratio of testosterone (T) to epitestosterone (E) in urine. In 2005 the World Anti-Doping Agency (WADA) lowered the T/E value at which samples undergo further investigation from six to four. This has resulted in a large increase in the number of athletes with naturally elevated T/E ratios undergoing investigation without a corresponding increase in the number of proven doping offences involving testosterone.Our objective was to develop a new simple screening protocol that can, with high probability, not only distinguish athletes whose natural T/E values exceed four from those whose T/E values have been elevated by testosterone doping but also detect those athletes with naturally low T/E values that do not exceed four despite being administered testosterone.Testosterone (250 mg Sustanon) was administered weekly to a group of 47 young adult males for five weeks in a double-blind placebo controlled study and urine samples collected. The samples were analysed for steroid concentrations using GC/MS and for luteinizing hormone (LH) by immunoassay.The elevation of T/E that occurred in all subjects was accompanied by a significant reduction in urinary LH concentrations to levels that are rare in normal subjects.The appropriate measurement of urinary LH, with the measurement of T/E values, can markedly improve the efficiency of detection of doping with testosterone by male athletes, particularly those who have low natural T/E ratios.

Context: Parafibromin, encoded by HRPT2, is the first marker with significant benefit in the diagnosis of parathyroid carcinoma. However, because parafibromin is only involved in up to 70% of parathyroid carcinomas and loss of parafibromin immunoreactivity may not be observed in all cases of HRPT2 mutation, a complementary marker is needed. Objective: We sought to determine the efficacy of increased expression of protein gene product 9.5 (PGP9.5), encoded by ubiquitin carboxyl-terminal esterase L1 (UCHL1) as an additional marker to loss of parafibromin immunoreactivity for the diagnosis of parathyroid carcinoma. Design: In total, 146 parathyroid tumors and nine normal tissues were analyzed for the expression of parafibromin and PGP9.5 by immunohistochemistry and for UCHL1 by quantitative RT-PCR. These samples included six hyperparathyroidism-jaw tumor syndrome-related tumors and 24 sporadic carcinomas. Results: In tumors with evidence of malignancy, strong staining for PGP9.5 had a sensitivity of 78% for the detection of parathyroid carcinoma and/or HRPT2 mutation and a specificity of 100%. Complete lack of nuclear parafibromin staining had a sensitivity of 67% and a specificity of 100%. PGP9.5 was positive in a tumor with the HRPT2 mutation L64P that expressed parafibromin. Furthermore, UCHL1 was highly expressed in the carcinoma/hyperparathyroidism-jaw tumor syndrome group compared to normal (P < 0.05) and benign specimens (P < 0.001). Conclusion: These results suggest that positive staining for PGP9.5 has utility as a marker for parathyroid malignancy, with a slightly superior sensitivity (P = 0.03) and similar high specificity to that of parafibromin.

CONTEXT GH is a known modulator of the immune system, but the effect of exogenous GH administration on white blood cell proteins has not been investigated. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a powerful platform for the study of GH effects on immune system proteins. OBJECTIVE Our objective was to explore a novel approach for the detection of GH-responsive proteins in human leukocytes by proteomic analysis using SELDI-TOF MS. DESIGN We conducted a randomized double-blind, placebo-controlled GH administration study of 8 wk treatment followed by 6 wk washout. Pre- and posttreatment samples from 30 subjects were used for biomarker discovery. SETTING The study was performed at a clinical research facility. PARTICIPANTS We studied 30 recreationally trained healthy athletes. INTERVENTION Subjects received either recombinant human GH (2 mg/d sc; n = 22) or placebo (n = 8) for 8 wk. MAIN OUTCOME MEASURES Proteomic profiles were determined using CM10 weak cation-exchange protein chips, and some GH-regulated proteins were purified and identified by mass spectrometry and/or immunoblotting. RESULTS SELDI-TOF analysis revealed a number of GH-regulated peptides/proteins in the 3- to 22-kDa range that are either up- or down-regulated by GH. Several of these may be useful as biomarkers of GH action. The calcium-binding, proinflammatory calgranulins S100A8, S100A9, and S100A12 were all significantly down-regulated in response to GH treatment. CONCLUSION This study illustrates the novel use of human leukocyte proteomic profiling by SELDI-TOF MS and reveals the negative regulation of proinflammatory S100 proteins by GH in human white blood cells.

BACKGROUND:The utility of insulinlike growth factor (IGF) axis and collagen markers for a growth hormone (GH) doping test in sport depends on their stability and reproducibility. We sought to determine short-term within-subject variability of these markers in a large cohort of healthy individuals.METHODS:We measured IGF-I, IGF binding protein 3 (IGFBP-3), acid labile subunit (ALS), and the collagen markers N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), and N-terminal propeptide of type III procollagen (PIIINP) in serum samples obtained on multiple occasions (median 3 per participant) over a 2- to 3-week period from 1103 elite athletes (699 men, 404 women) ages 22.2 (5.2) years [mean (SD)]. We estimated between-subject and within-subject variances by mixed-effects ANOVA.RESULTS:Within-subject variance accounted for 32% to 36% and 4% to 13% of the total variance in IGF markers and collagen markers, respectively. The within-subject CV ranged from 11% to 21% for the IGF axis markers and from 13% to 15% for the collagen markers. The index of individuality for the IGF axis markers was 0.66-0.76, and for the collagen markers, 0.26-0.45. For each marker, individuals with initial extreme measured values tended to regress toward the population mean in subsequent repeated measurements. We developed a Bayesian model to estimate the long-term probable value for each marker.CONCLUSIONS:These results indicate that in healthy individuals the within-subject variability was greater for IGF-I than for the collagen markers, and that where a single measurement is available, it is possible to estimate the long-term probable value of each of the markers by applying the Bayesian approach. Such an application can increase the reliability and decrease the cost of detecting GH doping.

Although doping with growth hormone (GH) is banned, there is anecdotal evidence that it is widely abused. GH is reportedly used often in combination with anabolic steroids at high doses for several months. Development of a robust test for GH has been challenging because recombinant human 22 kDa (22K) GH used in doping is indistinguishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentrations. One approach to GH testing is based on measurement of different circulating GH isoforms using immunoassays that differentiate between 22K and other GH isoforms. Administration of 22K GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method has been implemented; however, its utility is limited because of the short window of opportunity of detection. The second approach, which will extend the window of opportunity of detection, is based on the detection of increased levels of circulating GH-responsive proteins, such as insulin-like growth factor (IGF) axis and collagen peptides. Age and gender are the major determinants of variability for IGF-I and the collagen markers; therefore, a test based on these markers must take age into account for men and women. Extensive data is now available that validates the GH-responsive marker approach and implementation is now largely dependent on establishing an assured supply of standardized assays. Future directions will include more widespread implementation of both approaches by the World Anti-Doping Agency, possible use of other platforms for measurement and an athlete's passport to establish individual reference levels for biological parameters such as GH-responsive markers. Novel approaches include gene expression and proteomic profiling.

Context: IGF axis proteins and collagen peptides are promising markers of GH abuse.Objective: Our objective was to investigate whether responses of serum IGF axis and collagen markers to GH differ between men and women, and are influenced by testosterone (T).Design: This was a randomized, double-blind, placebo-controlled study of 8-wk treatment followed by 6-wk washout.Setting: The study was performed at a clinical research facility.Participants: A total of 96 recreationally trained healthy athletes (63 men, 33 women), aged 18-40 yr, were studied.Intervention: All subjects received GH (2 mg/d sc) or placebo for 8 wk; men also received T (250 mg/wk im) or placebo for 5 wk.Main Outcome Measures: Serum IGF axis proteins (IGF-I, IGF binding protein-3, and acid labile subunit) and collagen peptides (N-terminal propeptide of type I procollagen, C-terminal telopeptide of type I collagen, and N-terminal propeptide of type III procollagen) were measured.Results: GH induced significant increases in IGF axis and collagen markers that were greater in men than women (P < 0.001). Of the IGF axis markers, IGF-I showed the greatest increase. The relative incremental responses of the collagen markers in general were greater than the IGF markers, especially for PIIINP. The collagen markers increased and decreased more slowly with most remaining elevated (P < 0.01) after 6 wk, in comparison to IGF markers, which returned to baseline within 1 wk. Addition of T to GH amplified the response of PIIINP by more than 1.5-fold but did not affect any other marker. T alone did not affect IGF axis markers but modestly increased collagen markers.Conclusions: These markers of GH abuse are less responsive in women. The increases in collagen markers have a different time course to the IGF markers and extend the window of detection in both sexes. The response of PIIINP is increased by coadministration of T.