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Purpose: The capacity of whole blood cultures to produce cytokines in response to endotoxin (LPS) was studied in athletes before, 30 min after, 3 h after and 24 h after a half-marathon run., Methods: Eight well trained men and 8 well trained women (6 of them in the late luteal phase of their cycle) participated. EDTA blood was incubated with or without LPS for 1 h, and cytokine concentration and gene expression were determined. To quantify LPS-dependent release on a per monocyte basis (LDR), the mean values of the difference (delta) between cytokine concentration in stimulated and unstimulated cultures, normalized to monocyte numbers, were calculated., Results: LDR of TNF-alpha was significantly reduced by exercise with identical kinetic in men and women. TNF-alpha mRNA expression was slightly down-regulated following exercise (P < 0.05), but significantly so only in women. LDR of IL-6 was also reduced, but with a faster kinetic in women than in men. Similarly, 30 min post-exercise; LDR and spontaneous release of IL-1ra were significantly less in women than men. Concomitantly, IL-Ira mRNA was significantly elevated in unstimulated and in stimulated cultures in men only. IL-10 and IL-10 mRNA were significantly induced 30 min following exercise in absence of any detectable LDR. Women showed significantly lower levels than men. LDR and spontaneous release of IL-8 was enhanced in men and TGF-beta1 in women. A significant up-regulation was seen in unstimulated IL-8 mRNA for women and LPS-stimulated IL-8 mRNA expression for men following exercise., Conclusion: Altogether, LPS-dependent ex vivo cytokine release was strongly influenced by exercise and these changes could only in part be attributed to changes in messenger RNA. Results for IL-1ra, IL-6 and IL-10 pointed to a less pronounced anti-inflammatory response in women as compared with men. Our results also indicate an early production of IL-10 by peripheral blood cells in response to exercise.

Background: Exercise is an extreme physiological challenge for skeletal muscle energy metabolism and has notable health benefits. We aimed to identify and characterize metabolites, which are components of the regulatory network mediating the beneficial metabolic adaptation to exercise., Methodology and Principal Findings: First, we investigated plasma from healthy human subjects who completed two independent running studies under moderate, predominantly aerobic conditions. Samples obtained prior to and immediately after running and then 3 and 24 h into the recovery phase were analyzed by a non-targeted (NT-) metabolomics approach applying liquid chromatography-qTOF-mass spectrometry. Under these conditions medium and long chain acylcarnitines were found to be the most discriminant plasma biomarkers of moderately intense exercise. Immediately after a 60 min (at 93% V(IAT)) or a 120 min run (at 70% V(IAT)) a pronounced, transient increase dominated by octanoyl-, decanoyl-, and dodecanoyl-carnitine was observed. The release of acylcarnitines as intermediates of partial beta-oxidation was verified in skeletal muscle cell culture experiments by probing (13)C-palmitate metabolism. Further investigations in primary human myotubes and mouse muscle tissue revealed that octanoyl-, decanoyl-, and dodecanoyl-carnitine were able to support the oxidation of palmitate, proving more effective than L-carnitine., Conclusions: Medium chain acylcarnitines were identified and characterized by a functional metabolomics approach as the dominating biomarkers during a moderately intense exercise bout possessing the power to support fat oxidation. This physiological production and efflux of acylcarnitines might exert beneficial biological functions in muscle tissue.

The immunological reaction to exercise has been investigated with increasing intensity in the last 10-20 years, with most human studies performed in male subjects. Recently, gender-specific aspects have received growing attention, but studies carefully monitoring the influence of gender including the menstrual cycle, are rare. Here, we report gene expression patterns in response to a run at 93% of the individual anaerobic threshold of 9 women with regular menstrual cycles and no use of oral contraceptives who ran both at day 10 (follicular phase, F) and at day 25 (luteal phase, L) of their cycle. 12 male subjects (M) served as controls. The mRNA was pooled group wise and processed on a gene expression microarray encompassing 789 genes, including major genes of the inflammatory and anti-inflammatory reaction. The differences of gene expression between time points to (before run) and t1 (after run) were analyzed. Females in L showed a higher extent of regulation than females in F or men. Among those genes which were up-regulated above 1.5 fold change (log2) pro-inflammatory genes were significantly enriched (p = 0.033, after Bonferroni correction) in L, while this was not the case in F or M. Conversely, women in L showed a strong trend towards downregulation of anti-inflammatory genes. Some prominent genes like IL6 (coding for interleukin-6), and IL1RN (also termed IL1RA, coding for interleukin-1 receptor antagonist) were clearly regulated in opposite directions in L as opposed to F and M. In conclusion, women in L showed a distinctly different pattern of gene regulation in response to exercise, compared with women in F or M. The overall direction of gene expression changes of women in L is clearly pro-inflammatory. This finding accentuates a need for careful consideration of the female cyclic phase when investigating women in exercise immunology studies. Our results may also have implications relevant to other forms of stress in females.

Onset of physical activity induces a wide variety of rapid biophysical and biochemical responses that act on cells and lead to a wide range of phenotypic adaptations. Here we elucidate the role of immediate early gene (IEG) expression as a first line of transcriptional response that mediates protein neosynthesis and leads to adaptation of immune function. New posttranscriptional mechanisms have been described that speed up transcriptional responses. These include RNA-RNA interactions such as those exploited by miRNAs and stimulus-dependent cytoplasmic polyadenylation. We describe these shortcuts that modulate expression and discuss the challenges of accurately measuring them using various transcriptomic screening and quantification approaches. Although there is high complexity of the upstream as well as the downstream pathways that lead to IEG expression, IEG expression itself may only show a limited number of response patterns. Focusing transcriptomic approaches in exercise immunology at the IEG-level may facilitate the discovery of exercise-specific transcriptional signatures.

Accidental trauma and heavy endurance exercise, both induce a kind of systemic inflammatory response, also called systemic inflammatory response syndrome (SIRS). Exercise-related SIRS is conditioned by hyperthermia and concomitant heat shock responses, whereas trauma-induced SIRS manifests concomitantly with tissue necrosis and immune activation, secondarily followed by fever. Inflammatory cytokines are common denominators in both trauma and exercise, although there are marked quantitative differences. Different anti-inflammatory cytokines may be involved in the control of inflammation in trauma- and exercise-induced stress. Exercise leads to a balanced equilibrium between inflammatory and anti-inflammatory responses. Intermittent states of rest, as well as anti-oxidant capacity, are lacking or minor in trauma but are high in exercising individuals. Regular training may enhance immune competence, whereas trauma-induced SIRS often paves the way for infectious complications, such as sepsis.

It is generally accepted that exhausting endurance exercise exhibits strong effects on the immune system. Such effects have been attributed to changes in the cellular composition of peripheral blood as well as to changes in the expression of plausible candidate genes. The list of candidate genes is far from being complete, since this issue has not yet been investigated in a systematic way. In this study, we used a custom-made cDNA microarray focused on inflammation as a screening approach to study gene expression in eight one-half marathon runners before, immediately after, and 24 h after exercise. Significant differential gene expression was verified by quantitative real-time PCR. Linear regression analysis showed that microarray expression analysis of cell type-specific surface molecules reflects the observed individual cellular shifts in peripheral blood cells with high statistical significance. In line with the results of former studies, we observed an upregulation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2), L-selectin, and IL-1 receptor antagonist (IL-1ra) after exhaustive exercise. The main results of this study report, for the first time, the downregulation of CD81; the upregulation of thioredoxin, which may play an important part in anti-oxidative defense; and, surprisingly, the downregulation of the anti-carcinogenic gene glutathione-S-transferase-3 (GSTM3) in peripheral blood. The study shows cDNA microarray expression analysis as a reliable systematic instrument to complete the list of candidate genes that may play a role in exhaustive exercise-induced modulation of the immune response.

Exhausting endurance exercise has strong effects on the immune system. Changes have been shown in the cellular composition of peripheral blood and in gene expression within those cells. In this study, custom-made cDNA microarrays focused on inflammation were used to analyze gene expression blood cells obtained from eight half-marathon runners before (t0), immediately after (t1) and 24 hours after exercise (t2). The microarrays that were used contained 384 different cDNAs spotted in triplicate. Differentially-regulated gene expression was analyzed using a simple rule-based clustering. Comparing t1 vs. t0, and t2 vs. t0, 36 and 21 sequences respectively, showed a consistent pattern of changes in all eight athletes. Taken together, the pattern of these modified genes can be viewed as a "gene expression fingerprint" for each time point in response to a half marathon. The known and novel genes identified here represent targets for further molecular characterization of the complex reaction of the body to an exhaustive challenge. These data suggest that gene expression fingerprints can serve as a powerful research tool to design novel strategies for diagnosis and treatment of exercise related injury and stress.

Elevated ambient temperature may exert an additional impact on the exercise-induced expression of heat shock proteins (HSP) and DNA damage in leukocytes. The protective functions of HSP include antioxidative and antiapoptotic effects and may prevent damage to DNA. Twelve athletes completed a continuous run (75% VO2max) on the treadmill, six at 28 degrees C and six at 18 degrees C room temperature. Leukocyte expression of HSP27 and inducible HSP70 was analyzed on mRNA- (RT-PCR) and protein-level (flow cytometry), while DNA damage was quantified by the comet assay. High ambient temperature induced an additional accumulation of HSP-mRNA and -protein in leukocytes compared with the exercise-induced expression at 18 degrees C. HSP27 showed a special heat sensitivity. Surprisingly, the increase of DNA damage was less pronounced after exercise at 28 degrees C compared to 18 degrees C although heat shock in vitro clearly induced DNA damage. The inverse relation between HSP and DNA damage may indicate functions of HSP which protect against exercise-induced DNA-damage in terms of thermotolerance or apoptosis.

Microarray analysis offers a set of analytical platforms that provide rapid, affordable and substantial information at the DNA, RNA or protein level. It enables the analysis of thousands of genes simultaneously in a parallel manner across samples derived from various biological sources and treatment regimens. This development became possible as a result of the combination of three technological advances achieved at the beginning of the 1990's, namely parallelism, miniaturization and automation. In regular physical checkups the microarray technology could be used to supplement the current spectrum of tests and therefore enhance the quality of the data obtained. Arrays for analyses of RNA expression will allow gene expression profiling also in exercise physiology. DNA chips may also be used for genetic screening and diagnostics to analyze polymorphisms and mutations which may underlie genetic diseases or interindividual variations between subjects. Using microarrays in exercise physiology will provide new insights into the complex molecular mechanisms of the exercise-induced stress response, adaptation to training and modulation of immune function. Gene expression profiling and genetic screening will probably help to characterize and predict the individually variable response to and efficiency of training.

Previous research revealed an increased expression of HSP72 in leukocytes after vigorous endurance exercise. We questioned whether more intensive but shorter exercise also induces leukocyte HSP72 synthesis. To delineate the role of reactive oxygen species (ROS) in exercise-related HSP72 induction, we additionally examined the effect of RRR-alpha-tocopherol (alpha-toc) on HSP72 expression using a double-blind placebo (P) controlled cross-over design. After supplementation with alpha-toc (500 I.U. daily) or P for 8 days, 9 male subjects performed a combined exhaustive treadmill protocol (total duration 29.4 +/- 2.0 min). HSP72 was assessed on mRNA (RT-PCR) and protein levels (flow cytometry). HSP72 mRNA rose 3 h after exercise only in the P group, but individual differences (alpha-toc - P) did not reveal significant treatment effects. A moderate but significant rise of HSP72 protein occurred in granulocytes up to 48 h after exercise. Three hours post-exercise, granulocyte HSP72 protein was lower when subjects received alpha-toc, but this effect vanished 24 and 48 h post-exercise. Exhaustive treadmill exercise augments HSP72 mRNA in leukocytes and induced a moderate but prolonged response of granulocyte HSP72 protein. These exercise effects are lower when compared to earlier findings obtained after vigorous endurance exercise. ROS seem to be involved, but do not play the major role in the induction of granulocyte HSP72 synthesis after exhaustive exercise.

Overexpression of the heat shock protein HSP72 provides thermotolerance. We asked if two consecutive endurance runs 1 week apart (CR1, CR2) and additional environmental heat stress affect HSP72-expression in leukocytes of nonheat-acclimated endurance athletes. Twelve subjects were allocated randomly into two groups. Group HH completed both runs at 28 degrees C ambient temperature, and group NH performed CR1 at 18 degrees C and CR2 at 28 degrees C. HSP72-expression was determined by flow cytometry and RT-PCR before and 0, 24, and 48 h after exercise. Additionally, post-exercise cells were exposed to in vitro heat shock (HS; 2 h, 42 degrees C). The prolonged, high HSP72 protein level after CR1 in HH compared with NH may reflect thermotolerance induced by endurance exercise at high ambient temperature. Adaptation of cardiocirculatory/thermoregulatory capacity after CR2 in HH went along with a more rapid down-regulation of HSP72 compared with CR1. HSP72 mRNA demonstrated temperature-related changes after exercise. The reduced HS response in vitro after CR2 may represent exercise-related adaptation mechanisms. HSP72 concentrations in leukocytes may indicate previous exercise- and temperature-related stress conditions and adaptation in immunocompetent cells.

Free radicals are an integral part of metabolism and are formed continuously in the body. Many sources of stress heat, irradiation, hyperoxia, inflammation and any increases in metabolism including exercise, injury, and even repair processes lead to increased production of free radicals and associated reactive oxygen or nitrogen species (ROS/RNS). Evidence is accumulating that free radicals have important functions in the signal network of cells, including induction of growth and apoptosis and as killing tools of immunocompetent cells. Endogenous and nutritional antioxidant systems have to be adjusted to ensure adequate removal of radicals during stress to prevent damage to membranes, proteins, or nucleic acids. Excessive stress will induce DNA damage in the form of oxidized nucleosides, strand breaks, or DNA-protein crosslinks. Possible consequences of DNA damage are repair, apoptosis/necrosis, or defective repair leading to DNA sequence alterations and possibly to the development of cancer or, in case of mitochondrial DNA, to metabolic dysfunction. Excessive exercise will also induce DNA damage in peripheral leukocytes. The good message is that moderate stress in form of regular exercise/training may have protective effects against exercise-induced DNA damage. Up-regulation of endogenous antioxidant defense systems and complex regulation of repair systems such as heat shock proteins (HSP 70, HSP 27, HO 1) are seen in response to training and exercise. Up-regulation of antioxidants and modulation of the repair response may be mechanisms by which exercise can beneficially influence our health. Massive intervention into the redox state by pharmaceutical doses of exogenous antioxidants should be regarded with caution due to the ambiguous role of free radicals in regulation of growth, apoptosis, and cytotoxicity by immunocompetent cells.

Heat shock proteins (HSP) represent cell-protective and antioxidant systems that may be induced by reactive oxygen species, cytokines, and hyperthermia. In the present study, we evaluated the influence of heavy endurance exercise and training on HSP27 and HSP70 in peripheral leukocytes of 12 athletes (before and at 0, 3, and 24 h after a half-marathon) and 12 untrained controls on protein and mRNA levels by flow cytometry and RT/PCR, respectively. HSP transcripts increased significantly immediately after acute exertion accompanied by elevated levels of corresponding proteins. HSP protein expression remained high until 24 h postexercise. Significant increases of plasma interleukin-8, myeloperoxidase, and creatine kinase occurred after exercise. Basal HSP expression was usually lower in trained compared with untrained subjects. Applying in vitro heat shock to resting blood samples of all subjects significantly stimulated HSP mRNA, showing higher increases in trained individuals. The exercise-induced alterations indicate that immunocompetent cells became activated. In addition to heat stress, other exercise-associated stress agents (oxidants, cytokines) may have also participated in stimulation of HSP expression in leukocytes. The expression pattern of HSP due to training status may be attributed to adaptive mechanisms.

This study evaluated the effects of RRR-alpha-tocopherol (500 IU/day, 8 days) on in vivo cytokine response and cytoplasmic expression of inducible nitric oxide synthase (iNOS) and the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes after exhaustive exercise. Thirteen men were investigated in a double-blind, placebo-controlled, cross-over study with a wash-out period of 28 days. The exercise procedure consisted of an incremental treadmill test followed by a continuous run until exhaustion at 110% of the individual anaerobic threshold (total duration 28.5 +/- 0.8 min). HO-1 and iNOS protein were assessed in mono- (M), lympho-, and granulocytes (G) using flow cytometry. Plasma interleukin-6 (IL-6) and IL-8 were measured by ELISA. IL-6 rose significantly whereas IL-8 did not exhibit significant changes after exercise. Changes of IL-6 were not affected by RRR-alpha-tocopherol. Exercise induced an increase of iNOS protein primarily in M and G. A small, but significant, increase of HO-1 protein was measured in M and G. RRR-alpha-Tocopherol did not show any significant effects on cytoplasmic expression of iNOS and HO-1 at rest and after exercise. In conclusion, exhaustive exercise induces expression of iNOS and HO-1 in human leukocytes by a mechanism that is not sensitive to RRR-alpha-tocopherol supplementation.

Reactive oxygen (ROS) and nitrogen species (RNS) are continuously generated in the biological system and play an important role in a variety of physiological and pathological processes. There is evidence that physical exercise augments the generation of ROS/RNS. The present review discusses and compares insights into the generation and function of ROS/RNS such as superoxide, hydrogen peroxide, hypochloric acid, and nitric oxide released by leukocytes in response to exercise. Emphasis is placed on: (a) mechanisms and regulation of ROS/RNS generation in immunocompetent cells with respect to acute exercise and regular training; (b) damaging effects of ROS/RNS in terms of oxidative stress which may be causally involved in features such as exercise-induced damage to muscle tissue and leukocyte DNA; (c) (immuno-) modulating effects of ROS/RNS which include activation of transcription factors; (d) responses of antioxidant stress proteins to acute exercise and regular training; and (e) effects of antioxidants on exercise-induced changes in immune function. Available data suggests that ROS/RNS are involved in the inflammatory response to heavy exercise and therefore exert damaging effects. Several immune functions are influenced by actions of ROS/RNS, and it is hypothesized that adaption to regular training is also modulated in part by free radicals. Furthermore, regular training seems to reduce the capacity of leukocytes for oxidant release and leads to an adaptation of antioxidative mechanisms, which may contribute to a limitation of exercise-induced oxidative stress.