Age-associated decline in neural, endocrine, and immune responses in men and women: Involvement of intracellular signaling pathways.

The aim of this study was to investigate the alterations in the neuroendocrine-immune functions by using human peripheral blood mononuclear cells (hPBMCs) from three age groups (young, middle-aged, and old) of men and women for the analyses of lymphocyte proliferation and cytokine production, expression of cell signaling molecules, nitric oxide (NO) production, and expression of p-tyrosine hydroxylase (TH). Serum was examined for levels of testosterone in men, 17-ß-estradiol in women, and cortisol in both sexes. Lymphoproliferation, expression of p-ERK, p-CREB, p-Akt, and p-TH, and levels of serum sex steroid hormones declined with age in men and women. However, TNF-α production and serum cortisol level increased with age in men and women. mTOR expression was higher in older men while it was lower in older women. IFN-γ and IL-6 production and expression of p-TH and p-mTOR were differentially regulated in men and women. These results suggest that intracellular signaling mediators may be involved in the age-related alterations in the neuroendocrine-immune interactions in men and women.

Role of enhanced glucocorticoid receptor sensitivity in inflammation in PTSD: insights from computational model for circadian-neuroendocrine-immune interactions.

Although glucocorticoid resistance contributes to increased inflammation, individuals with posttraumatic stress disorder (PTSD) exhibit increased glucocorticoid receptor (GR) sensitivity along with increased inflammation. It is not clear how inflammation coexists with a hyperresponsive hypothalamic-pituitary-adrenal (HPA) axis. To understand this better, we developed and analyzed an integrated mathematical model for the HPA axis and the immune system. We performed mathematical simulations for a dexamethasone (DEX) suppression test and IC50-dexamethasone for cytokine suppression by varying model parameters. The model analysis suggests that increasing the steepness of the dose-response curve for GR activity may reduce anti-inflammatory effects of GRs at the ambient glucocorticoid levels, thereby increasing proinflammatory response. The adaptive response of proinflammatory cytokine-mediated stimulatory effects on the HPA axis is reduced due to dominance of the GR-mediated negative feedback on the HPA axis. To verify these hypotheses, we analyzed the clinical data on neuroendocrine variables and cytokines obtained from war-zone veterans with and without PTSD. We observed significant group differences for cortisol and ACTH suppression tests, proinflammatory cytokines TNFα and IL6, high-sensitivity C-reactive protein, promoter methylation of GR gene, and IC50-DEX for lysozyme suppression. Causal inference modeling revealed significant associations between cortisol suppression and post-DEX cortisol decline, promoter methylation of human GR gene exon 1F (NR3C1-1F), IC50-DEX, and proinflammatory cytokines. We noted significant mediation effects of NR3C1-1F promoter methylation on inflammatory cytokines through changes in GR sensitivity. Our findings suggest that increased GR sensitivity may contribute to increased inflammation; therefore, interventions to restore GR sensitivity may normalize inflammation in PTSD.

Exercise and the Kynurenine pathway: Current state of knowledge and results from a randomized cross-over study comparing acute effects of endurance and resistance training.

INTRODUCTION: The essential amino acid tryptophan (TRP) is primarily degraded through the kynurenine (KYN) pathway, which is dysregulated in several chronic diseases. KYN pathway metabolites have immune- and neuro-modulatory properties and are involved in th de novo synthesis of nicotinamide adenine dinucleotide (NAD+). Currently, little evidence exists demonstrating that physical exercise may influence this pathway. However, differences between acute and chronic stimuli as well as the influence of exercise modalities remain to be investigated. Here, we provide an overview of existing studies and present results of a randomized cross-over trial on acute effects of a single-bout of resistance and endurance exercise. METHODS: 24 healthy male adults conducted both an acute endurance exercise (EE) and resistance exercise (RE) session. Blood samples were collected before, immediately after and one hour after cessation of each exercise session. Outcomes comprised serum levels of TRP, KYN, kynurenic acid (KA), quinolinic acid (QA) and calculated ratios. Gene expression of the enzymes indoleamine 2,3 dioxygenase (IDO) 1 and kynurenine aminotransferase (KAT) 4 was measured in peripheral blood mononuclear cells (PBMCs). Moreover, serum concentrations of the potential KYN pathway mediators interleukin (IL)-6 and cortisol were determined. Finally, we investigated baseline correlations between immune cell subsets, potential mediators and initial KYN pathway activation outcomes. RESULTS: The KYN/TRP ratio correlated positively with IL-6 and CD56bright NK-cells and negatively with CD56dim NKcells. Expression of IDO1 in PBMCs correlated positively with IL-6, regulatory T-cells and CD56bright NK-cells, whereas negative correlations to cytotoxic T-cells and CD56dim NKcells were revealed. A significant time effect on KYN/TRP ratio was detected for RE. Regarding KA and KA/KYN ratio, an increase after exercise followed by a decrease at the follow- up measurement was revealed in EE. KAT4 expression also increased after exercise in EE. Moreover, elevated QA levels were observed after the EE session. CONCLUSIONS: In contrast to chronic exercise interventions, single-bouts of endurance exercise provoke acute alterations on KYN pathway outcomes in humans. Our results indicate that EE induces stronger alterations than RE. Enhanced conversion of KYN to both, KA and QA suggest a peripheral KYN clearance, thereby preventing pathological accumulation within the CNS. Future acute and chronic exercise studies are needed to examine the role of NAD+ synthesis starting with TRP and the interplay between KYN pathway activation and mid- to long-term immunological modulations.

Robot-assisted laparoscopic cystectomy with intracorporeal urinary diversion vs. open mini-laparotomy cystectomy: evaluation of surgical inflammatory response and immunosuppressive ability of CO2-pneumoperitoneum in an experimental porcine study.

OBJECTS: To compare surgical inflammatory response (SIR) after radical cystectomy (RC) in a porcine model using minimal invasive techniques. Additionally we aimed to investigate the potential immunosuppressive ability of preoperative CO2-pneumoperitoneum (CO2P). MATERIALS AND METHODS: Forty female landrace pigs were randomized to five groups: Three intervention groups all having a cystectomy and an ileal conduit either done by robot-assisted laparoscopic technique with intracorporeal urinary diversion (RALC) or an open mini-laparotomy with or without prior CO2P (OMC ± CO2P). Two control sham groups with or without prior CO2P (S ± CO2P). Serum samples were obtained preoperatively, immediately postoperative, 24, 48 and 72 hours postoperatively, and the inflammatory mediators CRP, Haptoglobin, Ceruloplasmin, Albumin, Cortisol, IL-4, IL-6, IL-12 and IFN-α were measured. RESULTS: Operative time was significantly longer in RALC compared to open groups (OMC ± CO2P) (p's < .0001). CRP and Haptoglobin levels were significantly higher for surgical intervention groups (SIG) compared to controls 24, 48 and 72 hours postoperatively (p's < .001). At 48 hours, CRP was higher for RALC vs OMC + CO2P (p = .029). At 72 hours, Haptoglobin was higher for RALC vs open groups (p's < .024). Ceruloplasmin, cortisol, albumin, IL-4, IL-6, IL-12 and IFN-α, revealed no significant differences between SIG. CONCLUSIONS: No major differences were found between RALC and OMC regarding the degree of tissue trauma quantified by inflammatory markers. Thirty minutes of CO2-insufflation preoperative appears to have a transient immunosuppressive effect of the innate postoperative SIR, whereas prolonged CO2P apparently diminishes this effect.

Cortisol inhibits CSF2 and CSF3 via DNA methylation and inhibits invasion in first-trimester trophoblast cells.

PROBLEM: Heightened maternal stress affects trophoblast function and increases risk for adverse pregnancy outcomes. METHODS OF STUDY: Studies were performed using the first-trimester trophoblast cell line, Sw.71. Cytokines were quantified using qPCR and ELISA. Epigenetic regulation of cytokines was characterized by inhibiting histone deacetylation (1 µmol/L suberoylanilide hydroxamic acid [SAHA]) or methylation (5 µmol/L 5-azacytidine), or with chromatin immunoprecipitation (ChIP) with a pan-acetyl histone-3 antibody. Invasion assays used Matrigel chambers. RESULTS: Cortisol inhibited expression of CSF2 (GM-CSF) and CSF3 (G-CSF) in trophoblast cells. Cortisol-associated inhibition was dependent on DNA methylation and was not affected by acetylation. There was also a modest decrease in trophoblast invasion, not dependent on loss of CSFs. CONCLUSION: In first-trimester trophoblast cells, the physiological glucocorticoid, cortisol, inhibited two cytokines with roles in placental development and decreased trophoblast invasion. Cortisol-associated changes in trophoblast function could increase the risk for immune-mediated abortion or other adverse pregnancy outcomes.

Characterisation of the cancer-associated glucocorticoid system: key role of 11ß-hydroxysteroid dehydrogenase type 2.

BACKGROUND: Recent studies have shown that production of cortisol not only takes place in several non-adrenal peripheral tissues such as epithelial cells but, also, the local inter-conversion between cortisone and cortisol is regulated by the 11ß-hydroxysteroid dehydrogenases (11ß-HSDs). However, little is known about the activity of this non-adrenal glucocorticoid system in cancers. METHODS: The presence of a functioning glucocorticoid system was assessed in human skin squamous cell carcinoma (SCC) and melanoma and further, in 16 epithelial cell lines from 8 different tissue types using ELISA, western blotting and immunofluorescence. 11ß-HSD2 was inhibited both pharmacologically and by siRNA technology. Naïve CD8+ T cells were used to test the paracrine effects of cancer-derived cortisol on the immune system in vitro. Functional assays included cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical data of 11ß-HSD expression were generated using tissue microarrays of 40 cases of human SCCs as well as a database featuring 315 cancer cases from 15 different tissues. RESULTS: We show that cortisol production is a common feature of malignant cells and has paracrine functions. Cortisol production correlated with the magnitude of glucocorticoid receptor (GR)-dependent inhibition of tumour-specific CD8+ T cells in vitro. 11ß-HSDs were detectable in human skin SCCs and melanoma. Analyses of publicly available protein expression data of 11ß-HSDs demonstrated that 11ß-HSD1 and -HSD2 were dysregulated in the majority (73%) of malignancies. Pharmacological manipulation of 11ß-HSD2 activity by 18ß-glycyrrhetinic acid (GA) and silencing by specific siRNAs modulated the bioavailability of cortisol. Cortisol also acted in an autocrine manner and promoted cell invasion in vitro and cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical analyses using tissue microarrays showed that expression of 11ß-HSD2 was significantly reduced in human SCCs of the skin. CONCLUSIONS: The results demonstrate evidence of a cancer-associated glucocorticoid system and show for the first time, the functional significance of cancer-derived cortisol in tumour progression.

Dysfunctional Natural Killer Cells in the Aftermath of Cancer Surgery.

The physiological changes that occur immediately following cancer surgeries initiate a chain of events that ultimately result in a short pro-, followed by a prolonged anti-, inflammatory period. Natural Killer (NK) cells are severely affected during this period in the recovering cancer patient. NK cells play a crucial role in anti-tumour immunity because of their innate ability to differentiate between malignant versus normal cells. Therefore, an opportunity arises in the aftermath of cancer surgery for residual cancer cells, including distant metastases, to gain a foothold in the absence of NK cell surveillance. Here, we describe the post-operative environment and how the release of sympathetic stress-related factors (e.g., cortisol, prostaglandins, catecholamines), anti-inflammatory cytokines (e.g., IL-6, TGF-ß), and myeloid derived suppressor cells, mediate NK cell dysfunction. A snapshot of current and recently completed clinical trials specifically addressing NK cell dysfunction post-surgery is also discussed. In collecting and summarizing results from these different aspects of the surgical stress response, a comprehensive view of the NK cell suppressive effects of surgery is presented. Peri-operative therapies to mitigate NK cell suppression in the post-operative period could improve curative outcomes following cancer surgery.

Evidence for increased immune mobilization in First Episode Psychosis compared with the prodromal stage in males.

The aim of the study was to gauge both the immune and neuroendocrine function in Ultra High Risk for psychosis (UHR) subjects and compare them with a cohort presenting with First Episode Psychosis (FEP). We recruited two groups, the first group consisted of 12 UHR males and the second of 25 males with FEP. We measured serum cortisol levels at 08:00, 12:00, 18:00 with their Area Under Curve with respect to the ground (AUCg) and the increase (AUCi) and we measured serum cytokines levels, Interleukin-1a, IL-1a, IL-2, IL-4,IL-5,IL-6,IL-8, IL-10,IL-12, IL-17a, Tumor Necrosis Factor-a (TNF-a), Interferon-γ (IFN-γ). Dexamethasone Suppression Test (DST) was also performed . The results suggest higher levels of both pro-inflammatory (TNF-a, IL-2, IL-12, IFN-γ) and anti-inflammatory (IL-10) cytokines in the FEP group compared with the UHR counterparts. Regarding the HPA axis function, the prodromal subjects showed a trend for higher AUCg and AUCi change/decrease cortisol levels. On the contrary, the DST results did not differ between the groups. No significant associations were demonstrated within each group among cytokines, cortisol and psychopathology. The findings favor a hypothesis of a relatively increased mobilization of both the pro- and anti-inflammatory cytokine networks, in FEP compared with that of UHR subjects.

Men and women differ in inflammatory and neuroendocrine responses to endotoxin but not in the severity of sickness symptoms.

Impaired mood and increased anxiety represent core symptoms of sickness behavior that are thought to be mediated by pro-inflammatory cytokines. Moreover, excessive inflammation seems to be implicated in the development of mood/affective disorders. Although women are known to mount stronger pro-inflammatory responses during infections and are at higher risk to develop depressive and anxiety disorders compared to men, experimental studies on sex differences in sickness symptoms are scarce. Thus, the present study aimed at comparing physiological and psychological responses to endotoxin administration between men and women. Twenty-eight healthy volunteers (14 men, 14 women) were intravenously injected with a low dose (0.4 ng/kg) of lipopolysaccharide (LPS) and plasma concentrations of cytokines and neuroendocrine factors as well as negative state emotions were measured before and until six hours after LPS administration. Women exhibited a more profound pro-inflammatory response with significantly higher increases in tumor necrosis factor (TNF)-α and interleukin (IL)-6. In contrast, the LPS-induced increase in anti-inflammatory IL-10 was significantly higher in men. The cytokine alterations were accompanied by changes in neuroendocrine factors known to be involved in inflammation regulation. Endotoxin injection induced a significant increase in noradrenaline, without evidence for sex differences. The LPS-induced increase in cortisol was significantly higher in woman, whereas changes in dehydroepiandrosterone were largely comparable. LPS administration also increased secretion of prolactin, but only in women. Despite these profound sex differences in inflammatory and neuroendocrine responses, men and women did not differ in endotoxin-induced alterations in mood and state anxiety or non-specific sickness symptoms. This suggests that compensatory mechanisms exist that counteract the more pronounced inflammatory response in women, preventing an exaggerated sickness response. Disturbance of these compensatory mechanisms by environmental factors such as stress may promote the development of affective disorders in women.

Diurnal cortisol and survival in epithelial ovarian cancer.

INTRODUCTION: Hypothalamic-pituitary-adrenal (HPA) deregulation is commonly observed in cancer patients, but its clinical significance is not well understood. We prospectively examined the association between HPA activity, tumor-associated inflammation, and survival in ovarian cancer patients prior to treatment. MATERIALS AND METHODS: Participants were 113 women with ovarian cancer who provided salivary cortisol for three days prior to treatment for calculation of cortisol slope, variability, and night cortisol. Cox proportional hazard regression analyses were used to examine associations between cortisol and survival in models adjusting for disease stage, tumor grade, cytoreduction and age. On a subsample of 41 patients with advanced disease ascites fluid was assayed for levels of interleukin-6 (IL-6) and correlated with cortisol variables. RESULTS: Each cortisol measure was associated with decreased survival time, adjusting for covariates (all p<.041). A one standard deviation increase in night cortisol was associated with a 46% greater likelihood of death. Patients in the high night cortisol group survived an estimated average of 3.3 years compared to 7.3 years for those in the low night cortisol group. Elevated ascites IL-6 was associated with each cortisol measure (all r>36, all p<.017). DISCUSSION: Abnormal cortisol rhythms assessed prior to treatment are associated with decreased survival in ovarian cancer and increased inflammation in the vicinity of the tumor. HPA abnormalities may reflect poor endogenous control of inflammation, dysregulation caused by tumor-associated inflammation, broad circadian disruption, or some combination of these factors. Nocturnal cortisol may have utility as a non-invasive measure of HPA function and/or disease severity.

The misleading nature of in vitro and ex vivo findings in studying the impact of stress hormones on NK cell cytotoxicity.

In vitro and ex vivo studies assessing the impact of stress hormones on immune competence commonly replace the natural milieu of leukocytes with an artificial medium, excluding plasma factors, hormones, and cytokines. Given prevalent inconsistencies between in vitro, ex vivo, and in vivo findings, we studied whether such procedures could yield misleading outcomes regarding the impact of stress hormones on NK cell cytotoxicity (NKCC), using fresh human whole blood samples. We found that in the presence of plasma 10-30-fold higher concentrations of cortisol, epinephrine, and prostaglandin-E2 (PGE2) were required to reach suppression levels evident in the context of artificial medium. Importantly, whereas the NK suppressive effects of PGE2 occurred immediately and remained stable upon prolonged exposure, the suppressive effects of cortisol slowly increased over time. Last, to simulate the exclusion of stress factors in the ex vivo approach, we subjected whole blood to stress hormones (as occurs in vivo), and abruptly removed them. We found that the effects of epinephrine and PGE2 quickly disappeared, while the effects of cortisol persisted. Overall, these findings demonstrate the potential misleading nature of in vitro and ex vivo procedures, and specifically suggest that (i) the common in vitro findings of profound suppression of NKCC by stress hormones are overestimation of their direct effects expected in vivo; and (ii) the common ex vivo approach cannot reflect the direct in vivo suppressive effects of epinephrine and PGE2 on NKCC, while inflating the effects of glucocorticoids. Some of these fallacies may be circumvented by using non-delayed whole blood NKCC assays in humans.

Variable neuroendocrine-immune dysfunction in individuals with unfavorable outcome after severe traumatic brain injury.

Bidirectional communication between the immune and neuroendocrine systems is not well understood in the context of traumatic brain injury (TBI). The purpose of this study was to characterize relationships between cerebrospinal fluid (CSF) cortisol and inflammation after TBI, and to determine how these relationships differ by outcome. CSF samples were collected from 91 subjects with severe TBI during days 0-6 post-injury, analyzed for cortisol and inflammatory markers, and compared to healthy controls (n=13 cortisol, n=11 inflammatory markers). Group-based trajectory analysis (TRAJ) delineated subpopulations with similar longitudinal CSF cortisol profiles (high vs. low cortisol). Glasgow Outcome Scale (GOS) scores at 6months served as the primary outcome measure reflecting global outcome. Inflammatory markers that displayed significant bivariate associations with both GOS and cortisol TRAJ (interleukin [IL]-6, IL-10, soluble Fas [sFas], soluble intracellular adhesion molecule [sICAM]-1, and tumor necrosis factor alpha [TNF]-α) were used to generate a cumulative inflammatory load score (ILS). Subsequent analysis revealed that cortisol TRAJ group membership mediated ILS effects on outcome (indirect effect estimate=-0.253, 95% CI (-0.481, -0.025), p=0.03). Correlational analysis between mean cortisol levels and ILS were examined separately within each cortisol TRAJ group and by outcome. Within the low cortisol TRAJ group, subjects with unfavorable 6-month outcome displayed a negative correlation between ILS and mean cortisol (r=-0.562, p=0.045). Conversely, subjects with unfavorable outcome in the high cortisol TRAJ group displayed a positive correlation between ILS and mean cortisol (r=0.391, p=0.006). Our results suggest that unfavorable outcome after TBI may result from dysfunctional neuroendocrine-immune communication wherein an adequate immune response is not mounted or, alternatively, neuroinflammation is prolonged. Importantly, the nature of neuroendocrine-immune dysfunction differs between cortisol TRAJ groups. These results present a novel biomarker-based index from which to discriminate outcome and emphasize the need for evaluating tailored treatments targeting inflammation early after injury.

Cortisol and proinflammatory cytokine profiles in type 1 (reversal) reactions of leprosy.

PURPOSE: Cortisol levels in the circulation and at the sites of peripheral inflammation regulate type 1 (Reversal) reactions in leprosy akin to delayed type hypersensitivity reactions (DTH). In this study we determine the extent to which the differential mRNA expression of genes encoding cortisone-cortisol shuttle enzymes (11 ß hydroxysteriod dehydrogenase I & II (11 ß HSD I & II)), circulatory levels of proinflammatory cytokines (IL-6, IL-7, IP-10, IL-17F, IL-23, TNF-α, IL-1ß, PDGF BB and CRP) and cortisol are associated with development of type 1 reactions in leprosy. METHODS: Urine, blood and incisional skin biopsy samples from site of lesions were collected from 49 newly diagnosed untreated leprosy cases in T1R and 51 cases not in reaction (NR). mRNA expression levels of genes encoding 11 ß HSD I & II in skin biopsy samples were determined by realtime PCR. Cortisol levels from the lesional skin biopsies, serum and urine samples and serum proinflammatory cytokine levels were measured using ELISA. RESULTS: The mean expression ratios of 11 ß HSD I & II are significantly lower in leprosy cases with T1R when compared to the NR leprosy cases. Cortisol levels in lesional skin biopsies and in urine are significantly lower (p=0.001) in leprosy cases with T1R. Serum cytokine levels of IP-10, IL-17F, IL-IL-6 and TNF-α are significantly higher (p<0.05) in leprosy cases with T1R when compared the NR leprosy cases. CONCLUSION: Our study indicated an association of urinary and lesional skin cortisol levels with the manifestation of T1R in leprosy. IP-10, IL-17F, IL-6 and TNF-α can be potential prognostic serological markers and gene expression markers for early detection of type 1 reactions in leprosy.

Relationship between the induction of inflammatory processes and infectious diseases in patients with ischemic stroke.

Pro-inflammatory cytokines participate in the induction of ischemic stroke. So far, their participation in the cerebral ischemia was proven for the tumor necrosis factor TNF-α, interleukin-1 (IL-1), and interleukin-6 (IL-6). The release of the pro-inflammatory cytokines into the extracellular space causes the enlargement of the brain damage region, and consequently increases the neurological deficit and negatively affects the survival rate prognoses. That is confirmed by the increased concentration of pro-inflammatory cytokines in blood and the cerebrospinal fluid of patients with brain stroke, as well as by the research on the induced/experimental cerebral ischemia in animals. The pro-inflammatory cytokines participate in the migration of the reactive T lymphocytes to the regions of brain ischemia where they enhance the nerve tissue damage by down-regulation of microcirculation, induce the pro-thrombotic processes and release other neurotoxic cytokines. Also, in the early stage of cerebral ischemia, cytokines activate the axis hypothalamus-pituitary gland-adrenal cortex and increase the cortisol concentration in blood, what results in the decreased resistance to infectious diseases. Administration of the inhibitor of the interleukin-1 receptor (IL-1Ra) inhibits the inflammatory processes in the region of brain ischemia, and subsequently improves the prognosis for the size of the neurological deficit and the survival rate, as well as resistance to infectious diseases.

The concept of the immune-pineal axis tested in patients undergoing an abdominal hysterectomy.

OBJECTIVE: Activation of the immune-pineal axis induces a transient reduction in nocturnal melatonin in the plasma during the proinflammatory phase of an innate immune response to allow the proper migration of leukocytes to the lesion site. This transient reduction should be regulated by inflammatory mediators, which are responsible for the fine-tuning of the process. In the present study, we measured the pre- and postoperative serum concentrations of melatonin, tumor necrosis factor (TNF) and cortisol in women who underwent an elective hysterectomy and correlated the variation in melatonin with postoperative pain. METHODS: We evaluated 12 women who had an abdominal hysterectomy. Blood was collected at 10.00 and 22.00 h 1 week and 1 day before the surgery, on the 1st and 2nd days after the surgery and at 22.00 h on the day of the surgery. RESULTS: On the night after the surgery, there was no melatonin detected at 22.00 h. High TNF levels were accompanied by a lower nocturnal melatonin output, higher postoperative pain according to a visual analog scale and the request of higher doses of analgesics. In addition, low cortisol levels were accompanied by a lower nocturnal melatonin output. CONCLUSION: Our results confirm that the same antagonistic pattern between TNF and glucocorticoids observed in cultured pineal glands also occurs in humans. This integrative pattern suggests that the cross talk between the immune and endocrine system orchestrates longitudinal changes in pineal activity, reinforcing the hypothesis of an immune-pineal axis.

Effect of post-exercise protein-leucine feeding on neutrophil function, immunomodulatory plasma metabolites and cortisol during a 6-day block of intense cycling.

Whey protein and leucine ingestion following exercise increases muscle protein synthesis and could influence neutrophil function during recovery from prolonged intense exercise. We examined the effects of whey protein and leucine ingestion post-exercise on neutrophil function and immunomodulators during a period of intense cycling. In a randomized double-blind crossover, 12 male cyclists ingested protein/leucine/carbohydrate/fat (LEUPRO 20/7.5/89/22 g h(-1), respectively) or isocaloric carbohydrate/fat control (CON 119/22 g h(-1)) beverages for 1-3 h post-exercise during 6 days of high-intensity training. Blood was taken pre- and post-exercise on days 1, 2, 4 and 6 for phorbol myristate acetate (PMA)-stimulated neutrophil superoxide (O2 (-)) production, immune cell counts, amino acid and lipid metabolism via metabolomics, hormones (cortisol, testosterone) and cytokines (interleukin-6, interleukin-10). During recovery on day 1, LEUPRO ingestion increased mean concentrations of plasma amino acids (glycine, arginine, glutamine, leucine) and myristic acid metabolites (acylcarnitines C14, myristoylcarnitine; and C14:1-OH, hydroxymyristoleylcarnitine) with neutrophil priming capacity, and reduced neutrophil O2 production (15-17 mmol O2 (-) cell(-1) ± 90 % confidence limits 20 mmol O2 (-) cell(-1)). On day 2, LEUPRO increased pre-exercise plasma volume (6.6 ± 3.8 %) but haematological effects were trivial. LEUPRO supplementation did not substantially alter neutrophil elastase, testosterone, or cytokine concentrations. By day 6, however, LEUPRO reduced pre-exercise cortisol 21 % (±15 %) and acylcarnitine C16 (palmitoylcarnitine) during exercise, and increased post-exercise neutrophil O2 (-) (33 ± 20 mmol O2 (-) cell(-1)), relative to control. Altered plasma amino acid and acylcarnitine concentrations with protein-leucine feeding might partly explain the acute post-exercise reduction in neutrophil function and increased exercise-stimulated neutrophil oxidative burst on day 6, which could impact neutrophil-dependent processes during recovery from intense training.

Transcriptome analysis of neutrophils after endurance exercise reveals novel signaling mechanisms in the immune response to physiological stress.

Neutrophils serve as an intriguing model for the study of innate immune cellular activity induced by physiological stress. We measured changes in the transcriptome of circulating neutrophils following an experimental exercise trial (EXTRI) consisting of 1 h of intense cycling immediately followed by 1 h of intense running. Blood samples were taken at baseline, 3 h, 48 h, and 96 h post-EXTRI from eight healthy, endurance-trained, male subjects. RNA was extracted from isolated neutrophils. Differential gene expression was evaluated using Illumina microarrays and validated with quantitative PCR. Gene set enrichment analysis identified enriched molecular signatures chosen from the Molecular Signatures Database. Blood concentrations of muscle damage indexes, neutrophils, interleukin (IL)-6 and IL-10 were increased (P < 0.05) 3 h post-EXTRI. Upregulated groups of functionally related genes 3 h post-EXTRI included gene sets associated with the recognition of tissue damage, the IL-1 receptor, and Toll-like receptor (TLR) pathways (familywise error rate, P value < 0.05). The core enrichment for these pathways included TLRs, low-affinity immunoglobulin receptors, S100 calcium binding protein A12, and negative regulators of innate immunity, e.g., IL-1 receptor antagonist, and IL-1 receptor associated kinase-3. Plasma myoglobin changes correlated with neutrophil TLR4 gene expression (r = 0.74; P < 0.05). Neutrophils had returned to their nonactivated state 48 h post-EXTRI, indicating that their initial proinflammatory response was transient and rapidly counterregulated. This study provides novel insight into the signaling mechanisms underlying the neutrophil responses to endurance exercise, suggesting that their transcriptional activity was particularly induced by damage-associated molecule patterns, hypothetically originating from the leakage of muscle components into the circulation.

A comparison of mindfulness-based stress reduction and an active control in modulation of neurogenic inflammation.

Psychological stress is a major provocative factor of symptoms in chronic inflammatory conditions. In recent years, interest in addressing stress responsivity through meditation training in health-related domains has increased astoundingly, despite a paucity of evidence that reported benefits are specific to meditation practice. We designed the present study to rigorously compare an 8-week Mindfulness-Based Stress Reduction (MBSR) intervention to a well-matched active control intervention, the Health Enhancement Program (HEP) in ability to reduce psychological stress and experimentally-induced inflammation. The Trier Social Stress Test (TSST) was used to induce psychological stress and inflammation was produced using topical application of capsaicin cream to forearm skin. Immune and endocrine measures of inflammation and stress were collected both before and after MBSR training. Results show those randomized to MBSR and HEP training had comparable post-training stress-evoked cortisol responses, as well as equivalent reductions in self-reported psychological distress and physical symptoms. However, MBSR training resulted in a significantly smaller post-stress inflammatory response compared to HEP, despite equivalent levels of stress hormones. These results suggest behavioral interventions designed to reduce emotional reactivity may be of therapeutic benefit in chronic inflammatory conditions. Moreover, mindfulness practice, in particular, may be more efficacious in symptom relief than the well-being promoting activities cultivated in the HEP program.

Non-genomic inhibitory effect of glucocorticoids on activated peripheral blood basophils through suppression of lipid raft formation.

We investigated the non-genomic effects of glucocorticoids (GCs) on inhibition of plasma membrane lipid raft formation in activated human basophils. Human basophils obtained from house dust mite (HDM)-sensitive volunteers were pretreated with hydrocortisone (CORT) or dexamethasone (Dex) for 30 min and then primed with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) or HDM (10 µg/ml). The expression of CD63, a basophil activation marker, was assessed by flow cytometry. Membrane-bound GC receptors (mGCRs) were analysed by flow cytometry and confocal laser microscopy. Lipid rafts were assessed using a GM1 ganglioside probe and visualization by confocal laser microscopy. Pretreatment of basophils with CORT (10(-4) M and 10(-5) M) and Dex (10(-7) M) significantly inhibited CD63 expression 20 min after addition of PMA or HDM. The inhibitory effects of GCs were not altered by the nuclear GC receptor (GCR) antagonist RU486 (10(-5) M) or the protein synthesis inhibitor cycloheximide (10(-4) M) (P < 0·05). CORT coupled to bovine serum albumin (BSA-CORT) mimicked the rapid inhibitory effects of CORT, suggesting the involvement of mGCRs. mGCRs were detectable on the plasma membrane of resting basophils and formed nanoclusters following treatment with PMA or HDM. Pretreatment of cells with BSA-CORT inhibited the expression of mGCRs and nanoclustering of ganglioside GM1 in lipid rafts. The study provides evidence that non-genomic mechanisms are involved in the rapid inhibitory effect of GCs on the formation of lipid raft nanoclusters, through binding to mGCRs on the plasma membrane of activated basophils.

T-cell numbers and antigen-specific T-cell function follow different circadian rhythms.

PURPOSE: Circadian rhythms play an important role in modulating cellular immune responses. The present study was performed to characterise circadian variations in lymphocyte numbers and antigen-specific T-cell functionality in healthy individuals under physiological conditions. METHODS: Blood leukocyte populations of six healthy volunteers were quantified over 24 h. In addition, antigen-specific T-cell functionality was analysed directly ex vivo from whole blood using flow cytometry based on intracellular cytokine induction after a 6-hour stimulation with adenovirus antigen and Staphylococcus aureus enterotoxin B (SEB), respectively. RESULTS: T-cell numbers and reactivity were stable during daytime, whereas a significant increase was observed during late evening and early morning hours. The percentage of T cells reacting towards adenovirus antigen and SEB showed a 1.76 ± 0.55-fold (p = 0.0002) and a 1.42 ± 0.33-fold (p = 0.0002) increase, respectively. Dynamics in T-cell reactivity were independent of the mode of antigen stimulation and inversely correlated with plasma levels of endogenous cortisol. Interestingly, peak frequencies of reactive T cells occurred late in the evening and did not directly coincide with peak numbers of bulk T cells that were observed in the early morning hours. CONCLUSIONS: Taken together, our data reveal a circadian regulation of T-cell immune responses in the peripheral blood of humans under physiological conditions. This knowledge may be of practical consequence for the timing of blood sampling for functional T-cell assays as well as for immunosuppressive drug intake after organ transplantation, where T-cell function may be influenced not only by drug-mediated inhibition but also by circadian fluctuations in T-cell reactivity.