Plasma concentrations of thioredoxin, thioredoxin reductase and peroxiredoxin-4 can identify high risk patients and predict outcome in patients with acute coronary syndrome: A clinical observation.

BACKGROUND: Oxidative stress is a pathological feature of acute coronary syndrome (ACS), a complex disease with varying clinical outcomes. Surrogate biomarkers of oxidative stress including, peroxiredoxin-2 (PRDX2), PRDX4, thioredoxin (TRX) and thioredoxin reductase (TRXR) were measured in ACS patients at presentation and follow-up, to assess their clinical utility in diagnosis and risk stratification. METHODS: Plasma from 145 participants (80 ACS and 65 healthy) at diagnosis, 1-3 month (first) and 6-month follow-up (second) was analysed by ELISA. ACS patients were monitored for 12-months. RESULTS: ACS patients at diagnosis had significantly higher concentrations of TRX (p < 0.05), TRXR (p < 0.01) and PRDX4 (p < 0.01), compared to healthy donors. This was increase was driven by non-ST elevated myocardial infarction for TRX (p < 0.01) and PRDX4 (p < 0.05). For TRXR, ACS females were significantly higher than males (p < 0.05). TRX was also higher in older females (>55 years) at diagnosis (p < 0.05). At first follow-up, TRX had lowered, whereas PRDX4 remained significantly high (p < 0.05). Stratification of ACS patients according to percutaneous coronary intervention (PCI) revealed that TRXR was significantly higher in patients receiving PCI to the right coronary artery (p < 0.05). Whereas both TRXR (p < 0.01) and PRDX4 (p < 0.01) were significantly higher in patients receiving PCI to the left anterior descending (LAD) artery. ACS patients who had plasma TRX >13.40 ng/ml at second follow-up were at high risk of readmission (p < 0.05), as were patients with TRXR of <1000 pg/ml at diagnosis having PCI to the LAD (p < 0.05). CONCLUSION: This study indicates that TRX, TRXR and PRDX4 may have clinical utility for ACS stratification.

Adipose inflammation: South Asian ethnicity and central obesity are independently associated with higher immune cell recruitment to adipose-specific media: A pilot study in men.

A South Asian (SA) cardiovascular phenomenon exists whereby SAs have excess burden of cardiovascular disease (CVD) despite having low prevalence of recognized CVD risk factors. The aim of the current study was to determine whether perturbations in monocyte biology contribute to this phenomenon via higher circulating cell numbers, a more pro-inflammatory phenotype, and higher transmigration and adhesion. Adhesion is linked to vascular inflammation whereas transmigration is linked to tissue inflammation. SA men with (N = 10; SAs with central obesity [CO-SA]) and without (N = 10; lean SA [LE-SA]) central obesity, plus White European counterparts (N = 10; white Europeans with central obesity [CO-WE], N = 10; lean white Europeans [LE-WE]) participated. An ex vivo assay mimicking blood flow dynamics coupled to flow cytometry determined the adhesion and transmigration of monocyte subsets toward chemokine-rich media cultured from pre-adipocytes (absolute responses). Migration and adhesion were also standardized for differences in numbers of circulating monocytes between participants (relative responses). Metabolic and inflammatory markers were assessed. SAs had higher absolute (but not relative) adhesion and migration of monocytes than WEs. Central obesity was associated with higher absolute and relative adhesion and migration of monocytes. SAs had higher concentrations of all monocyte subsets compared with WEs coinciding with adverse cardiovascular-inflammatory profiles. LE-SAs had similar monocyte concentrations, transmigration, and adhesion compared with CO-WEs, corresponding with similar cardiovascular-inflammatory profiles. The study provides novel evidence for higher monocyte counts associated with higher transmigration and adhesion in SA compared with WE men. Importantly, similar monocyte biology and cardiovascular-inflammatory profiles were seen in LE-SAs compared with CO-WEs, which may contribute to the higher risk of CVD at lower body mass index experienced by SAs.

Effect of Acute Walking on Endothelial Function and Postprandial Lipemia in South Asians and White Europeans.

INTRODUCTION: South Asians (SAs) have an elevated risk of cardiovascular disease (CVD) compared with White Europeans (WEs). Postprandial endothelial function (flow-mediated dilatation (FMD%)) in SA women and SA men with central obesity has not been investigated. Research in other populations has highlighted that a 1% higher FMD% is associated with a ~13% lower risk of future CVD events. We investigated whether FMD% and lipemia, two markers for CVD risk, were higher in SAs versus WEs, whether walking improved FMD% and lipemia, and if there were ethnic differences in the response. METHODS: Lean premenopausal women (study 1; 12 SA, 12 WE) and men with central obesity (study 2; 15 SA, 15 WE) completed two 2-d trials. On day 1, participants walked for 60 min at 60% of their peak oxygen uptake or rested. On day 2, participants rested and consumed two high-fat meals over 8 h. Repeated ultrasound assessments of endothelial function and venous blood samples for CVD risk markers were taken. RESULTS: Compared with WEs, SAs had lower postprandial FMD% (study 1, -1.32%; study 2, -0.54%) and higher postprandial triacylglycerol concentrations (study 1, 0.31 mmol·L -1 ·h -1 ; study 2, 0.55 mmol·L -1 ·h -1 ). Walking improved postprandial FMD% (study 1, 1.12%; study 2, 0.94%) and resulted in no significant change or small reductions in postprandial triacylglycerol concentrations (study 1, -0.01 mmol·L -1 ·h -1 ; study 2, -0.25 mmol·L -1 ·h -1 ). Exercise-induced changes in FMD% and triacylglycerol were consistent between ethnic groups. CONCLUSIONS: Walking mitigated the adverse postprandial effect of a high-fat diet on FMD% to a similar extent in SA and WE women and men, even with no/small improvements in triacylglycerol. This study highlights the importance of exercise to clinically improve FMD% in SAs and WEs.

Inactivity and obesity: consequences for macrophage-mediated inflammation and the development of cardiometabolic disease.

Obesity and dyslipidaemia are strongly associated with the development of cardiometabolic diseases including CVD, stroke, type 2 diabetes, insulin resistance and non-alcoholic fatty liver disease. While these conditions are preventable, they are leading causes of mortality globally. There is now overwhelming clinical and experimental evidence that these conditions are driven by chronic systemic inflammation, with a growing body of data suggesting that this can be regulated by increasing levels of physical activity and reducing sedentary time. In this review we address the role of macrophage-mediated inflammation on the development of cardiometabolic diseases in individuals with overweight and obesity and how reducing sedentary behaviour and increasing physical activity appears to lessen these pro-inflammatory processes, reducing the risk of developing cardiometabolic diseases. While loss of subcutaneous and visceral fat mass is important for reducing chronic systemic inflammation, the mediating effects of increasing physical activity levels and lowering sedentary time on the development of inflamed adipose tissue also occur independently of changes in adiposity. The message that weight loss is not necessary for the benefits of physical activity in lowering chronic inflammation and improving health should encourage those for whom losing weight is difficult. Additionally, while the health benefits of meeting the recommended physical activity guidelines are clear, simply moving more appears to lower chronic systemic inflammation. Reducing sitting time and increasing light physical activity may therefore provide an alternative, more approachable manner for some with overweight and obesity to become more active, reduce chronic inflammation and improve cardiometabolic health.

Higher levels of physical activity are associated with reduced tethering and migration of pro-inflammatory monocytes in males with central obesity.

Despite evidence that monocyte migration is accentuated by central adiposity, the impact of physical activity (PA) and exercise, particularly in the post-prandial state, on limiting migration are not established. We hypothesised that PA and a single bout of walking exercise would be associated with reduced ex vivo monocyte tethering and migration in middleaged males with central obesity (CO). Objective levels of PA were measured for 7 days in lean males (LE, N=12, mean (SD) age 39 (10) years, waist circumference 81.0 (6.3) cm) and males with CO (N=12, mean (SD) age 40 (9) years, waist circumference 115.3 (13.9) cm), followed by donation of a fasted blood sample. On the same day, CO undertook a bout of walking exercise, before donation of a second fasted blood sample. An ex vivo assay, coupled to flow cytometry, determined tethering and migration of classical, intermediate, and non-classical monocytes. C-C and CXC chemokine receptor (CCR2, CCR5 and CX3CR1) expression were also determined on total and classical monocytes. Monocyte subsets (total, classical, intermediate and CCR2+ monocytes), metabolic (glucose and lipids) and inflammatory (C-reactive protein) markers were greater in CO vs. LE (lower highdensity lipoprotein); however, adjustments for PA mitigated group differences for glucose, lipids, and monocyte subsets. Ex vivo tethering and migration (absolute and relative) of most monocyte subsets was greater in CO vs LE. Relative monocyte tethering and migration was largely not influenced by PA; however, higher PA was associated with reduced absolute migration and tethering of CD16 expressing monocytes in CO. Prior walking had no impact on these variables. These results highlight that regular PA, not single exercise bouts may limit the migration of pro-inflammatory monocytes in CO. These changes may relate to physiological parameters in blood (i.e. number of cells and their adhesion), rather than differences in chemokine receptor expression.

Acute Running and Coronary Heart Disease Risk Markers in Male Cigarette Smokers and Nonsmokers: A Randomized Crossover Trial.

PURPOSE: Cigarette smoking is an independent risk factor for coronary heart disease and is associated with impaired postprandial metabolism. Acute exercise reduces postprandial lipemia and improves other coronary heart disease risk markers in nonsmokers. Less is known about responses in cigarette smokers. METHODS: Twelve male cigarette smokers (mean ± SD; age = 23 ± 4 yr, body mass index = 24.9 ± 3.0 kg·m-2) and 12 male nonsmokers (age = 24 ± 4 yr, body mass index = 24.1 ± 2.0 kg·m-2) completed two, 2-d conditions (control and exercise) in a randomized crossover design. On day 1, participants rested for 9 h (0800-1700) in both conditions except a 60-min treadmill run (65% ± 7% peak oxygen uptake, 2.87 ± 0.54 MJ) was completed between 6.5 and 7.5 h (1430-1530) in the exercise condition. On day 2 of both conditions, participants rested and consumed two high-fat meals over 8 h (0900-1700) during which 13 venous blood samples and nine resting arterial blood pressure measurements were collected. RESULTS: Smokers exhibited higher postprandial triacylglycerol and C-reactive protein than nonsmokers (main effect group effect size [Cohen's d] ≥ 0.94, P ≤ 0.034). Previous day running reduced postprandial triacylglycerol, insulin, and systolic and diastolic blood pressure (main effect condition d ≥ 0.28, P ≤ 0.044) and elevated postprandial nonesterified fatty acid and C-reactive protein (main effect condition d ≥ 0.41, P ≤ 0.044). Group-condition interactions were not apparent for any outcome across the total postprandial period (0-8 h; all P ≥ 0.089), but the exercise-induced reduction in postprandial triacylglycerol in the early postprandial period (0-4 h) was greater in nonsmokers than smokers (-21%, d = 0.43, vs -5%, d = 0.16, respectively; group-condition interaction P = 0.061). CONCLUSIONS: Acute moderate-intensity running reduced postprandial triacylglycerol, insulin, and resting arterial blood pressure the day after exercise in male cigarette smokers and nonsmokers. These findings highlight the ability of acute exercise to augment the postprandial metabolic health of cigarette smokers and nonsmokers.

Short-term High-fat Overfeeding Does Not Induce NF-κB Inflammatory Signaling in Subcutaneous White Adipose Tissue.

CONTEXT: It is unclear how white adipose tissue (WAT) inflammatory signaling proteins respond during the early stages of overnutrition. OBJECTIVE: To investigate the effect of short-term, high-fat overfeeding on fasting abdominal subcutaneous WAT total content and phosphorylation of proteins involved in nuclear factor-κB (NF-κB) inflammatory signaling, systemic metabolic and inflammatory biomarkers. DESIGN: Individuals consumed a high-fat (65% total energy from total fat), high-energy (50% above estimated energy requirements) diet for 7 days. RESULTS: Fifteen participants (aged 27 ± 1 years; body mass index 24.4 ± 0.6 kg/m2) completed the study. Body mass increased following high-fat overfeeding (+1.2 ± 0.2 kg; P < 0.0001). However, total content and phosphorylation of proteins involved in NF-κB inflammatory signaling were unchanged following the intervention. Fasting serum glucose (+0.2 ± 0.0 mmol/L), total cholesterol (+0.4 ± 0.1 mmol/L), low-density lipoprotein cholesterol (+0.3 ± 0.1 mmol/L), high-density lipoprotein cholesterol (+0.2 ± 0.0 mmol/L), and lipopolysaccharide-binding protein (LBP; +4.7 ± 2.1 µg/mL) increased, whereas triacylglycerol concentrations (-0.2 ± 0.1 mmol/L) decreased following overfeeding (all P < 0.05). Systemic biomarkers (insulin, soluble cluster of differentiation 14 [CD14], C-reactive protein, interleukin-6, tumor necrosis factor-α and monocyte chemoattractant protein-1) and the proportion and concentration of circulating CD14+ monocytes were unaffected by overfeeding. CONCLUSION: Acute lipid oversupply did not impact on total content or phosphorylation of proteins involved in WAT NF-κB inflammatory signaling, despite modest weight gain and metabolic alterations. Systemic LBP, which is implicated in the progression of low-grade inflammation during the development of obesity, increased in response to a 7-day high-fat overfeeding period.

High intensity interval exercise increases the frequency of peripheral PD-1+ CD8+ central memory T-cells and soluble PD-L1 in humans.

Exercise can exert anti-inflammatory effects in an intensity-dependent manner; however, the mechanisms mediating these effects are continually being established. Programme Death Receptor-1 (PD-1) is a membrane bound receptor that maintains immune tolerance by dampening immune cell interactions, such as those mediated by cytotoxic T-cell lymphocytes (CD8+). The aim of this study was to characterise sub-populations of CD8+ T-cells with regards to their expression of PD-1 before and immediately after exercise. Interleukin (IL)-6, soluble PD-1 (sPD-1) and its ligand (sPD-L1) were also quantified in plasma. Eight individuals (mean â€‹± â€‹SD: age 29 â€‹± â€‹5 years; BMI 24.2 â€‹± â€‹3.4 â€‹kg â€‹m2; V ˙ O2max 44.5 â€‹± â€‹6.4 â€‹ml â€‹kg-1·min-1) undertook two time and energy-matched cycling bouts in a counterbalanced study design: one of moderate intensity (MOD) and a bout of high intensity interval exercise (HIIE). Both MOD and HIIE increased the number, but not the proportion of circulating CD8+ PD-1+ cells, with no differences between trials. Within the CD8+ PD-1+ pool, the expression of PD-1 increased on central memory cells following HIIE only (fold change: MOD 1.0 vs HIIE +1.4), as well the concentration of CD8+PD-1+ memory cells within the circulation (cells/uL: MOD -0.4 vs HIIE +5.8). This response composed a very small part of the exercise-induced CD8+ lymphocytosis (Pre-Ex: 0.38% to Post-Ex: 0.69%; p â€‹> â€‹0.05). sPD-L1 and IL-6 concentration increased in tandem following MOD and HIIE (r â€‹= â€‹0.57; P â€‹= â€‹0.021), with a reciprocal decline in sPD-1 observed. The current data demonstrate that PD-1+ CD8+ lymphocytes were mobilised following both MOD and HIIE. Both the number of central memory CD8+ T-cells expressing PD-1 and the expression level on these cells were increased following HIIE only. This intensity-dependent phenotypic response, in conjunction with increased circulatory sPD-L1 may represent an aspect of the anti-inflammatory response to exercise and warrants further investigation.

Sleep Deprivation: Cytokine and Neuroendocrine Effects on Perception of Effort.

INTRODUCTION: An increased perception of effort and subjective fatigue are thought to be central to decreased exercise performance observed after disrupted sleep. However, there is limited understanding of mechanisms that underpin these phenomena. We investigated the role of interleukin-6 (IL-6), the soluble IL-6 receptor, and neuroendocrine factors (cortisol, adrenaline, noradrenaline, and brain-derived neurotropic factor) in mediating these responses at rest and during exercise. METHODS: In a randomized order, 10 healthy active men completed three experimental trials following different sleep conditions: a single night of sleep deprivation, partial sleep deprivation equivalent to 4 h of sleep, and normal sleep. The experimental sessions consisted of physiological and perceptual measurements of exercise intensity throughout 45-min moderate intensity and 15-min maximal effort cycling. Cytokine and neuroendocrine factors were assessed at rest and in response to exercise. RESULTS: Sleep deprivation resulted in increased resting IL-6, lower blood glucose, increased perceived fatigue and perception of effort, lower free-living energy expenditure, and reduced maximal exercise performance. In contrast, sleep deprivation did not alter physiological, cytokine, or neuroendocrine responses to exercise. Variations in the resting concentration of IL-6 were associated with lowered blood glucose, an increased perception of effort, and impaired exercise performance. Resting concentrations of cortisol, adrenaline, noradrenaline, and BNDF showed subtle interactions with specific aspects of mood status and performance but were not affected by sleep deprivation. There were minimal effects of partial sleep deprivation. CONCLUSIONS: These findings demonstrate that cytokine and neuroendocrine responses to exercise are not altered by sleep deprivation but that changes in the resting concentration of IL-6 may play a role in altered perception of effort in this context.

The effects of a single night of complete and partial sleep deprivation on physical and cognitive performance: A Bayesian analysis.

This study investigated the effects of complete and partial sleep deprivation on multiple aspects of athletic performance. Ten males completed a cognitive function test, maximal handgrip strength, countermovement jump (CMJ) and a 15 min all out cycling test to assess aerobic performance. These tests were performed following 3 different sleep conditions; normal sleep (CON), a 4 hr sleep opportunity (PART) and complete sleep deprivation (DEP). Data were analysed using a Bayesian multi-level regression model to provide probabilities of impairment (p = %). Aerobic performance, CMJ and handgrip strength were impaired by 11.4% (p = 100%), 10.9% (p = 100%) and 6% (p = 97%) following DEP, while aerobic performance and CMJ were highly likely impaired by 4.1% (p = 90%) and 5.2% (p = 94%) following PART. Cognitive reaction time was not impacted by PART or DEP. In contrast the accuracy of responses was highly likely impaired by 2% (91) following DEP, while there was less certainty of impaired accuracy following PART (-1%, p = 73). Multiple aspects of physical and cognitive performance were impacted by sleep deprivation. The greatest detrimental effects were seen for aerobic performance and CMJ. Partial sleep deprivation equating to 4 hrs of sleep causes subtle, but potentially important negative impairments on athletic performance.

"Beet" the cold: beetroot juice supplementation improves peripheral blood flow, endothelial function, and anti-inflammatory status in individuals with Raynaud's phenomenon.

Raynaud's phenomenon (RP) is characterized by recurrent transient peripheral vasospasm and lower nitric oxide (NO) bioavailability in the cold. We investigated the effect of nitrate-rich beetroot juice (BJ) supplementation on 1) NO-mediated vasodilation, 2) cutaneous vascular conductance (CVC) and skin temperature (Tsk) following local cooling, and 3) systemic anti-inflammatory status. Following baseline testing, 23 individuals with RP attended four times, in a double-blind, randomized crossover design, following acute and chronic (14 days) BJ and nitrate-depleted beetroot juice (NDBJ) supplementation. Peripheral Tsk and CVC were measured during and after mild hand and foot cooling, and during transdermal delivery of acetylcholine and sodium nitroprusside. Markers of anti-inflammatory status were also measured. Plasma nitrite concentration ([nitrite]) was increased in the BJ conditions (P < 0.001). Compared with the baseline visit, thumb CVC was greater following chronic-BJ (Δ2.0 flux/mmHg, P = 0.02) and chronic-NDBJ (Δ1.45 flux/mmHg, P = 0.01) supplementation; however, no changes in Tsk were observed (P > 0.05). Plasma [interleukin-10] was greater, pan endothelin and systolic and diastolic blood pressure (BP) were reduced, and forearm endothelial function was improved, by both BJ and NDBJ supplementation (P < 0.05). Acute and chronic BJ and NDBJ supplementation improved anti-inflammatory status, endothelial function and blood pressure (BP). CVC following cooling increased post chronic-BJ and chronic-NDBJ supplementation, but no effect on Tsk was observed. The key findings are that beetroot supplementation improves thumb blood flow, improves endothelial function and anti-inflammatory status, and reduces BP in people with Raynaud's.NEW & NOTEWORTHY This is the first study to examine the effect of dietary nitrate supplementation in individuals with Raynaud's phenomenon. The principal novel findings from this study were that both beetroot juice and nitrate-depleted beetroot juice 1) increased blood flow in the thumb following a cold challenge; 2) enhanced endothelium-dependent and -independent vasodilation in the forearm; 3) reduced systolic and diastolic blood pressure, and pan-endothelin concentration; and 4) improved inflammatory status in comparison to baseline.

Characterization of extracellular redox enzyme concentrations in response to exercise in humans.

Redox enzymes modulate intracellular redox balance and are secreted in response to cellular oxidative stress, potentially modulating systemic inflammation. Both aerobic and resistance exercise are known to cause acute systemic oxidative stress and inflammation; however, how redox enzyme concentrations alter in extracellular fluids following bouts of either type of exercise is unknown. Recreationally active men (n = 26, mean ± SD: age 28 ± 8 yr) took part in either: 1) two separate energy-matched cycling bouts: one of moderate intensity (MOD) and a bout of high intensity interval exercise (HIIE) or 2) an eccentric-based resistance exercise protocol (RES). Alterations in plasma (study 1) and serum (study 2) peroxiredoxin (PRDX)-2, PRDX-4, superoxide dismutase-3 (SOD3), thioredoxin (TRX-1), TRX-reductase and interleukin (IL)-6 were assessed before and at various timepoints after exercise. There was a significant increase in SOD3 (+1.5 ng/mL) and PRDX-4 (+5.9 ng/mL) concentration following HIIE only, peaking at 30- and 60-min post-exercise respectively. TRX-R decreased immediately and 60 min following HIIE (-7.3 ng/mL) and MOD (-8.6 ng/mL), respectively. In non-resistance trained men, no significant changes in redox enzyme concentrations were observed up to 48 h following RES, despite significant muscle damage. IL-6 concentration increased in response to all trials, however there was no significant relationship between absolute or exercise-induced changes in redox enzyme concentrations. These results collectively suggest that HIIE, but not MOD or RES increase the extracellular concentration of PRDX-4 and SOD3. Exercise-induced changes in redox enzyme concentrations do not appear to directly relate to systemic changes in IL-6 concentration.NEW & NOTEWORTHY Two studies were conducted to characterize changes in redox enzyme concentrations after single bouts of exercise to investigate the emerging association between extracellular redox enzymes and inflammation. We provide evidence that SOD3 and PRDX-4 concentration increased following high-intensity aerobic but not eccentric-based resistance exercise. Changes were not associated with IL-6. The results provide a platform to investigate the utility of SOD3 and PRDX-4 as biomarkers of oxidative stress following exercise.

Using Flow Cytometry to Detect and Measure Intracellular Thiol Redox Status in Viable T Cells from Heterogeneous Populations.

Increased production of reactive oxygen species (ROS) and deficiencies in cellular antioxidant defenses are the principal causes of cellular oxidative stress. ROS can react with a variety intracellular molecules, including redox active cysteine thiols (-SH) within proteins. Cysteine thiols can occupy several redox states and conversion between them is highly dynamic during, for example, cell growth, resulting in modification and subsequent loss of the "reduced thiol" form (-SH or -S-). The challenge lies with detecting and measuring thiol redox status inside viable heterogeneous cell populations (e.g., peripheral blood mononuclear cells (PBMCs)). Here we describe a flow cytometric approach for the evaluation of intracellular thiol redox status in human CD3+ T cells within a viable PBMC preparation. Using the thiol reactive probe, fluorescein-5 maleimide (F5M), we demonstrate that loss of reduced intracellular thiol correlates with a decrease in F5M fluorescence. We also detected a loss of F5M fluorescence in Jurkat cell cultures exposed to exogenous H2O2 generated by glucose oxidase. Since F5M binds irreversibly to reduced cysteine thiols, cells may be sorted based on F5M fluorescence intensity and redox active proteins can subsequently be extracted and separated using SDS-PAGE. This final step facilitates identification of redox active proteins from individual cell populations in live heterogeneous cell mixes using proteomic analysis.

Impact of aerobic exercise and fatty acid supplementation on global and gene-specific DNA methylation.

Lifestyle interventions, including exercise and dietary supplementation, can modify DNA methylation and exert health benefits; however, the underlying mechanisms are poorly understood. Here we investigated the impact of acute aerobic exercise and the supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) and extra virgin olive oil (EVOO) on global and gene-specific (PPARGC1A, IL6 and TNF) DNA methylation, and DNMT mRNA expression in leukocytes of disease-free individuals. Eight trained male cyclists completed an exercise test before and after a four-week supplementation of n-3 PUFA and EVOO in a double-blind, randomised, repeated measures design. Exercise triggered global hypomethylation (Pre 79.2%; Post 78.7%; p = 0.008), alongside, hypomethylation (Pre 6.9%; Post 6.3%; p < 0.001) and increased mRNA expression of PPARGC1A (p < 0.001). Associations between PPARGC1A methylation and exercise performance were also detected. An interaction between supplement and trial was detected for a single CpG of IL6 indicating increased DNA methylation following n-3 PUFA and decreased methylation following EVOO (p = 0.038). Global and gene-specific DNA methylation associated with markers of inflammation and oxidative stress. The supplementation of EVOO reduced DNMT1 mRNA expression compared to n-3 PUFA supplementation (p = 0.048), whereas, DNMT3a (p = 0.018) and DNMT3b (p = 0.046) mRNA expression were decreased following exercise. In conclusion, we demonstrate that acute exercise and dietary supplementation of n-3 PUFAs and EVOO induce DNA methylation changes in leukocytes, potentially via the modulation of DNMT mRNA expression. Future studies are required to further elucidate the impact of lifestyle interventions on DNA methylation.

Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: An optimised protocol for digital flow cytometers.

Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 (Durand and Olive [1]). There has been little development in these methodologies since that time, a period that has witnessed huge technological advances, particularly with the emergence of digital multi-parameter flow cytometric systems. Concurrent advancement in our understanding of redox regulation within eukaryotic cellular systems has also followed, whereby it is now accepted that cysteine thiols partake in redox reactions, which regulate protein activity and function (Groitl and Jakob (2014), Won et al. (2012)). Moreover, we are at the dawn of a new era in redox biology whereby the importance of 'reductive stress' in eukaryotic cellular systems is gathering momentum (Wadley et al. (2018) [4]). It is therefore critical that methods be continually advanced to better understand these concepts in more detail at the cellular level. Flow cytometry is a powerful technique that may be used for this purpose. Henceforth we have rejuvenated these methods to address modern scientific questions. In this paper, essential detail is provided on: •The adaption of a protocol initially described by Durand and Olive [1] for use with modern digital flow cytometer configurations. Here we provide optimal conditions for labelling intracellular thiols with F5M for detection using digital flow cytometers. Our modifications avoid the use of methanol fixation thus preserving cell viability in single cell suspension cultures.•Demonstration that flow cytometry can detect the gain and loss of reduced intracellular thiols in cells exposed to physiological doses of hydrogen peroxide mediated by glucose oxidase (Hole et al. (2013) [5]).•Validation of F5M protein labelling by coupling method to confocal microscopy and downstream proteomics, thus permitting a powerful experimental platform for potential use with next generation flow cytometry e.g. CyTOF (Lin and Maecker (2018) [6]).

Acute aerobic exercise induces a preferential mobilisation of plasmacytoid dendritic cells into the peripheral blood in man.

Dendritic cells (DCs) are important sentinel cells of the immune system responsible for presenting antigen to T cells. Exercise is known to cause an acute and transient increase in the frequency of DCs in the bloodstream in humans, yet there are contradictory findings in the literature regarding the phenotypic composition of DCs mobilised during exercise, which may have implications for immune regulation and health. Accordingly, we sought to investigate the composition of DC sub-populations mobilised in response to acute aerobic exercise. Nine healthy males (age, 21.9 ±â€¯3.6 years; height, 177.8 ±â€¯5.4 cm; body mass, 78.9 ±â€¯10.8 kg; body mass index, 24.9 ±â€¯3.3 kg·m2; V̇O2 MAX, 41.5 ±â€¯5.1 mL·kg·min-1) cycled for 20 min at 80% V̇O2 MAX. Blood was sampled at baseline, during the final minute of exercise and 30 min later. Using flow cytometry, total DCs were defined as Lineage- (CD3, CD19, CD20, CD14, CD56) HLA-DR+ and subsequently identified as plasmacytoid DCs (CD303+) and myeloid DCs (CD303-). Myeloid DCs were analysed for expression of CD1c and CD141 to yield four sub-populations; CD1c-CD141+; CD1c+CD141+; CD1c+CD141- and CD1c-CD141-. Expression of CD205 was also analysed on all DC sub-populations to identify DCs capable of recognising apoptotic and necrotic cells. Total DCs increased by 150% during exercise (F(1,10) = 60; p < 0.05, η2 = 0.9). Plasmacytoid DCs mobilised to a greater magnitude than myeloid DCs (195 ±â€¯131% vs. 131 ±â€¯100%; p < 0.05). Among myeloid DCs, CD1c-CD141- cells showed the largest exercise-induced mobilisation (167 ±â€¯122%), with a stepwise pattern observed among the remaining sub-populations: CD1c+CD141- (79 ±â€¯50%), followed by CD1c+CD141+ (44 ±â€¯41%), with the smallest response shown by CD1c-CD141+ cells (23 ±â€¯54%) (p < 0.05). Among myeloid DCs, CD205- cells were the most exercise responsive. All DC subsets returned to resting levels within 30 min of exercise cessation. These results show that there is a preferential mobilisation of plasmacytoid DCs during exercise. Given the functional repertoire of plasmacytoid DCs, which includes the production of interferons against viral and bacterial pathogens, these findings indicate that exercise may augment immune-surveillance by preferentially mobilising effector cells; these findings have general implications for the promotion of exercise for health, and specifically for the optimisation of DC harvest for cancer immunotherapy.

Preliminary evidence of reductive stress in human cytotoxic T cells following exercise.

This study investigated immunophenotypic differences in intracellular thiol redox state of peripheral blood mononuclear cells (PBMCs) isolated from trained [ n = 9, means ± SD: age 28 ± 5 yr; (body mass index) BMI 23.2 ± 2.6 kg/m2; V̇o2max (maximal oxygen intake)56.9 ± 6.1 ml·kg-1·min-1] and recreationally active (RA, n = 11, means ± SD: age 27 ± 6 yr; BMI 24.2 ± 3.7 kg/m2; V̇o2max 45.1 ± 6.4 ml·kg-1·min-1) participants before and after a maximal aerobic exercise tolerance test. Blood samples were taken before (Pre), during (sample acquired at 70% maximum heart rate), immediately after (Post + 0), and 15 min postexercise (Post + 15). PBMCs were isolated, and reduced thiol analysis [fluorescein-5 maleimide (F5M)] by immunophenotype [cluster of differentiation (CD)3+, CD4+, and CD8+] was performed using flow cytometry. A significant increase in cellular F5M fluorescence was observed in CD3+ T cells at Post + 0, with changes driven to a greater extent by CD8+ T cells (fold change in both groups CD4: +2.3, CD8: +2.8; P < 0.05). Further analysis revealed a population of highly reduced CD8+ T cells (CD8+T-reduced+) that significantly increased from Pre to Post + 0 in RA participants only (RA: +272 cell/µl, P < 0.05). To understand these results further, CD8+T-reduced+ and CD8+T-reduced- cells were analyzed for immunophenotype in response to the same exercise protocol ( n = 6, means ± SD: age 24 ± 5 yr; BMI 25.7 ± 4.1 kg·m-2; V̇o2max 41.33 ± 7.63 ml·kg-1·min-1). CD8+T-reduced+ had significantly less lymphoid homing potential (chemokine receptor type 7) Post + 0 compared with Pre. This study is the first, to our knowledge, to demonstrate that lymphocyte populations become more reductive in response to acute exercise. NEW & NOTEWORTHY The study presented provides the first evidence to suggest that cytotoxic T cells become transiently reductive in healthy individuals following a single bout of cycling. Detection of these cells was enabled via the use of a flow cytometric assay that incorporates the thiol reactive probe fluorescein-5 maleimide. Using this method, transient reductive stress in viable T cells is permissible and provides the basis for further research in the area of exercise immunology.

Factors influencing post-exercise plasma protein carbonyl concentration.

Exercise of sufficient intensity and duration can cause acute oxidative stress. Plasma protein carbonyl (PC) moieties are abundant, chemically stable, and easily detectable markers of oxidative stress that are widely used for the interpretation of exercise-induced changes in redox balance. Despite many studies reporting acute increases in plasma PC concentration in response to exercise, some studies, including those from our own laboratory have shown decreases. This review will discuss the differences between studies reporting increases, decreases, and no change in plasma PC concentration following exercise in humans; highlighting participant physiology (i.e. training status) and study design (i.e. intensity, duration, and novelty of the exercise bout) as the main factors driving the direction of the PC response to exercise. The role of the 20S proteasome system is proposed as a possible mechanism mediating the clearance of plasma PC following exercise. Resting and exercise-induced differences in plasma protein composition and balance between tissues are also discussed. We suggest that exercise may stimulate the clearance of plasma PC present at baseline, whereas simultaneously increasing reactive oxygen species production that facilitates the formation of new PC groups. The balance between these two processes likely explains why some studies have reported no change or even decreases in plasma PC level post-exercise when other biomarkers of oxidative stress (e.g. markers of lipid peroxidation) were elevated. Future studies should determine factors that influence the balance between PC clearance and formation following acute exercise.

Intensive Exercise Does Not Preferentially Mobilize Skin-Homing T Cells and NK Cells.

PURPOSE: This study investigated whether natural killer (NK) cells and CD8+ T cells expressing cutaneous lymphocyte antigen (CLA)-a homing molecule for endothelial cell leukocyte adhesion molecule 1, which enables transmigration to the skin-are selectively mobilized in response to acute exercise. METHODS: Nine healthy men (mean ± SD age: 22.1 ± 3.4 yr) completed two exercise sessions: high-intensity continuous cycling ("continuous exercise" at 80% V˙O2max for 20 min) and low-volume high-intensity interval exercise (at 90% V˙O2max 10 × 1 min repetitions with 1 min recovery intervals). Blood was collected before, immediately and 30 min postexercise for cryopreservation of peripheral blood mononuclear cells. CLA+ and CLA- cells were quantified within NK subpopulations (CD56 "regulatory" and CD56 "cytotoxic" cells) as well as the following CD8+ T cell subpopulations: naive ("NA"; CD45RA+ CCR7+), central memory ("CM"; CD45RA- CCR7+), effector-memory ("EM"; CD45RA- CCR7-), and CD45RA-expressing effector-memory cells ("EMRA"; CD45RA+ CCR7-). RESULTS: CLA+ NK cells and CD8+ memory T cells increased in response to both exercise bouts, but, overall, their numerical contribution to the exercise lymphocytosis was inferior to CLA- cells, which increased to a much greater extent during exercise. Tellingly, the most exercise-responsive cells-effector memory CD8+ cells and CD56 cells-were CLA-. CONCLUSIONS: A small subset of CLA+ lymphocytes are mobilized into blood during acute intensive exercise, but CLA+ cells are not major contributors to exercise lymphocytosis, thus providing preliminary evidence that the skin is not a major origin, or homing destination, of exercise-sensitive lymphocytes.

An unexplored role for Peroxiredoxin in exercise-induced redox signalling?

Peroxiredoxin (PRDX) is a ubiquitous oxidoreductase protein with a conserved ionised thiol that permits catalysis of hydrogen peroxide (H2O2) up to a million times faster than any thiol-containing signalling protein. The increased production of H2O2 within active tissues during exercise is thought to oxidise conserved cysteine thiols, which may in turn facilitate a wide variety of physiological adaptations. The precise mechanisms linking H2O2 with the oxidation of signalling thiol proteins (phosphates, kinases and transcription factors) are unclear due to these proteins' low reactivity with H2O2 relative to abundant thiol peroxidases such as PRDX. Recent work has shown that following exposure to H2O2 in vitro, the sulfenic acid of the PRDX cysteine can form mixed disulphides with transcription factors associated with cell survival. This implicates PRDX as an 'active' redox relay in transmitting the oxidising equivalent of H2O2 to downstream proteins. Furthermore, under oxidative stress, PRDX can form stable oxidised dimers that can be secreted into the extracellular space, potentially acting as an extracellular 'stress' signal. There is extensive literature assessing non-specific markers of oxidative stress in response to exercise, however the PRDX catalytic cycle may offer a more robust approach for measuring changes in redox balance following exercise. This review discusses studies assessing PRDX-mediated cellular signalling and integrates the recent advances in redox biology with investigations that have examined the role of PRDX during exercise in humans and animals. Future studies should explore the role of PRDX as a key regulator of peroxide mediated-signal transduction during exercise in humans.