Myths and methodologies: Considerations for evaluating the time course of thermoregulatory adaptation during heat acclimation.

Since the early 1900s, repeated heat exposure has been used as a method to induce physiological adaptations that enhance our ability to tolerate heat stress during athletic and occupational pursuits. Much of this work has been dedicated to quantifying the time course of adaptation and identifying the minimum duration of acclimation required to optimise performance or enhance safety. To achieve this, investigators have typically applied classical (constant load) heat acclimation, whereby 60-90 min exercise is performed at the same absolute or relative intensity in a hot environment for 3-24 days, with adaptations evaluated using an identical forcing function test before and after. This approach has provided a foundation from which to develop our understanding of changes in thermoregulatory function, but it has several, frequently overlooked shortcomings, which have resulted in misconceptions concerning the time course of adaptation. It is frequently suggested that most of the thermoregulatory adaptations during heat acclimation occur within a week, but this is an oversimplification and a predictable artefact of the experimental designs used. Consequently, the time course of complete human adaptation to heat remains poorly understood and appears to vary considerably due to numerous individual factors. The purpose of this communication is to highlight the key methodological considerations required when interpreting the existing literature documenting adaptation over time. We also propose potential means by which to improve the way we induce and quantify the magnitude of adaptation to expedite discovery.

IL-21 conditions antigen-presenting human γδ T-cells to promote IL-10 expression in naïve and memory CD4+ T-cells.

Direct interaction between T-cells exerts a major influence on tissue immunity and inflammation across multiple body sites including the human gut, which is highly enriched in 'unconventional' lymphocytes such as γδ T-cells. We previously reported that microbial activation of human Vγ9/Vδ2+ γδ T-cells in the presence of the mucosal damage-associated cytokine IL-15 confers the ability to promote epithelial barrier defence, specifically via induction of IL-22 expression in conventional CD4+ T-cells. In the current report, we assessed whether other cytokines enriched in the gut milieu also functionally influence microbe-responsive Vγ9/Vδ2 T-cells. When cultured in the presence of IL-21, Vγ9/Vδ2 T-cells acquired the ability to induce expression of the immunoregulatory cytokine IL-10 in both naïve and memory CD4+ T-cells, at levels surpassing those induced by monocytes or monocyte-derived DCs. These findings identify an unexpected influence of IL-21 on Vγ9/Vδ2 T-cell modulation of CD4+ T-cell responses. Further analyses suggested a possible role for CD30L and/or CD40L reverse signalling in mediating IL-10 induction by IL-21 conditioned Vγ9/Vδ2 T-cells. Our findings indicate that the local microenvironment exerts a profound influence on Vγ9/Vδ2 T-cell responses to microbial challenge, leading to induction of distinct functional profiles among CD4+ T-cells that may influence inflammatory events at mucosal surfaces. Targeting these novel pathways may offer therapeutic benefit in disorders such as inflammatory bowel disease.

The effects of combined action observation and motor imagery on corticospinal excitability and movement outcomes: Two meta-analyses.

Motor simulation interventions involving motor imagery (MI) and action observation (AO) have received considerable interest in the behavioral sciences. A growing body of research has focused on using AO and MI simultaneously, termed 'combined action observation and motor imagery' (AOMI). The current paper includes two meta-analyses that quantify changes in corticospinal excitability and motor skill performance for AOMI compared to AO, MI and control conditions. Specifically, the first meta-analysis collated and synthesized existing motor evoked potential (MEP) amplitude data from transcranial magnetic stimulation studies and the second meta-analysis collated and synthesized existing movement outcome data from behavioral studies. AOMI had a positive effect compared to control and AO but not MI conditions for both MEP amplitudes and movement outcomes. No methodological factors moderated the effects of AOMI, indicating a robust effect of AOMI across the two outcome variables. The results of the meta-analyses are discussed in relation to existing literature on motor simulation and skill acquisition, before providing viable directions for future research on this topic.

The Influence of a Competitive Field Hockey Match on Cognitive Function.

Despite the known positive effects of acute exercise on cognition, the effects of a competitive team sport match are unknown. In a randomized crossover design, 20 female and 17 male field hockey players (19.7 ± 1.2 years) completed a battery of cognitive tests (Visual Search, Stroop, Corsi Blocks, and Rapid Visual Information Processing) prior to, at half-time, and immediately following a competitive match (or control trial of seated rest); with effect sizes (ES) presented as raw ES from mixed effect models. Blood samples were collected prior to and following the match and control trial, and analyzed for adrenaline, noradrenaline, brain derived neurotrophic factor (BDNF), cathepsin B, and cortisol. The match improved response times for a simple perception task at full-time (ES = -14 ms; P < 0.01) and response times on the complex executive function task improved at half-time (ES = -44 ms; P < 0.01). Working memory declined at full-time on the match (ES = -0.6 blocks; P < 0.01). The change in working memory was negatively correlated with increases in cortisol (r = -0.314, P = 0.01; medium), as was the change in simple perception response time and the change in noradrenaline concentration (r = -0.284, P = 0.01; small to medium). This study is the first to highlight the effects a competitive hockey match can have on cognition. These findings have implications for performance optimization, as understanding the influence on specific cognitive domains across a match allows for the investigation into strategies to improve these aspects.

Antibody secreting cells are critically dependent on integrin α4ß7/MAdCAM-1 for intestinal recruitment and control of the microbiota during chronic colitis.

T and B cells employ integrin α4ß7 to migrate to intestine under homeostatic conditions. Whether those cells differentially rely on α4ß7 for homing during inflammatory conditions has not been fully examined. This may have implications for our understanding of the mode of action of anti-integrin therapies in inflammatory bowel disease (IBD). Here, we examined the role of α4ß7 integrin during chronic colitis using IL-10-/- mice, ß7-deficient IL-10-/-, IgA-deficient IL-10-/- mice, and antibody blockade of MAdCAM-1. We found that α4ß7 was predominantly expressed by B cells. ß7 deficiency and MAdCAM-1 blockade specifically depleted antibody secreting cells (ASC) (not T cells) from the colonic LP, leading to a fecal pan-immunoglobulin deficit, severe colitis, and alterations of microbiota composition. Colitis was not due to defective regulation, as dendritic cells (DC), regulatory T cells, retinaldehyde dehydrogenase (RALDH) expression, activity, and regulatory T/B-cell cytokines were all comparable between the strains/treatment. Finally, an IgA deficit closely recapitulated the clinical phenotype and altered microbiota composition of ß7-deficient IL-10-/- mice. Thus, a luminal IgA deficit contributes to accelerated colitis in the ß7-deficient state. Given the critical/nonredundant dependence of IgA ASC on α4ß7:MAdCAM-1 for intestinal homing, B cells may represent unappreciated targets of anti-integrin therapies.

An integrin αEß7-dependent mechanism of IgA transcytosis requires direct plasma cell contact with intestinal epithelium.

Efficient IgA transcytosis is critical for the maintenance of a homeostatic microbiota. In the canonical model, locally-secreted dimeric (d)IgA reaches the polymeric immunoglobulin receptor (pIgR) on intestinal epithelium via simple diffusion. A role for integrin αE(CD103)ß7 during transcytosis has not been described, nor its expression by intestinal B cell lineage cells. We found that αE-deficient (αE-/-) mice have a luminal IgA deficit, despite normal antibody-secreting cells (ASC) recruitment, local IgA production and increased pIgR expression. This deficit was not due to dendritic cell (DC)-derived retinoic acid (RA) nor class-switching defects, as stool from RAG-/- mice reconstituted with αE-/- B cells was also IgA deficient. Flow cytometric, ultrastructural and transcriptional profiling showed that αEß7-expressing ASC represent an undescribed subset of terminally-differentiated intestinal plasma cells (PC) that establishes direct cell to cell contact with intestinal epithelium. We propose that IgA not only reaches pIgR through diffusion, but that αEß7+ PC dock with E-cadherin-expressing intestinal epithelium to directly relay IgA for transcytosis into the intestinal lumen.

The effects of pre- and per-cooling interventions used in isolation and combination on subsequent 15-minute time-trial cycling performance in the heat.

OBJECTIVES: To investigate the effects of pre- and per-cooling interventions on subsequent 15-min time-trial (TT) cycling performance in the heat. DESIGN: Randomized cross-over design. METHODS: Nine male athletes completed four experimental trials in the heat (40 °C, 50% rh): no-cooling (CON); warm-up per-cooling (PER: neck-cooling collar applied during the preload); pre-cooling (PRE: 30 min of cold water (22 °C) immersion [CWI]); and pre- and per-cooling combined (PRE + PER). In each trial, participants completed a 45-min preload exercise (50% V̇O2peak), followed by a 15-min TT. Physiological (rectal [Tre], skin [Tsk], and neck [Tneck] temperature, and heart rate [HR]) and perceptual data (ratings of perceived exertion [RPE], thermal comfort [TC] and thermal sensation [TS]) were measured throughout. RESULTS: Tre and Tsk were lower in PRE and PRE + PER at the start of the preload (p < 0.001). Tre remained lower throughout the preload following CWI although these differences were no longer present at the start of the TT (p = 0.22). Tneck was lowered throughout in PER and PRE + PER (p < 0.001). No other physiological or perceptual differences were observed at the start or end of the preload or TT. Participants covered a similar TT distance in all trials (15.7-15.9 km, p = 0.77). CONCLUSIONS: Pre-cooling induced thermoregulatory benefits for ~45 min and perceptual benefits for the same duration when supplemented with per-cooling. Neck per-cooling offered no such benefits when used in isolation. Neither pre- nor per-cooling, in isolation or combination, improved subsequent 15-min cycling time-trial performance in well-trained participants in the heat (40 °C).

Progressive hyperthermia elicits distinct responses in maximum and rapid torque production.

OBJECTIVES: To investigate the effect of progressive whole-body hyperthermia on maximal, and rapid voluntary torque production, and their neuromuscular determinants. DESIGN: Repeated measures, randomised. METHODS: Nine participants performed sets of neuromuscular assessments in HOT conditions (∼50°C, ∼35% relative humidity) at rectal temperatures (Tre) of 37, 38.5 and 39.5°C and in CON conditions (∼22°C, ∼35% relative humidity) at a Tre of ∼37°C and pre-determined comparative time-points. Electrically evoked twitch (single impulse) and octet (8 impulses at 300Hz) responses were measured at rest. Maximum voluntary torque (MVT), surface electromyography (EMG) normalised to maximal M-wave, and voluntary activation (VA) were measured during 3-5s isometric maximal voluntary contractions. Rate of torque development (RTD) and normalised EMG were measured during rapid voluntary isometric contractions from rest. RESULTS: All neuromuscular variables were unaffected by time in CON. In HOT, MVT, normalised EMG at MVT and VA were lower at 39.5°C compared to 37°C (p<0.05). Early- (0-50ms) and middle- (50-100ms) phase voluntary RTD were unaffected by increased Tre (p>0.05), despite lower normalised EMG at Tre 39.5°C (p<0.05) in rapid contractions. In contrast, late-phase (100-150ms) voluntary RTD was lower at 38.5°C and 39.5°C compared to 37°C (p<0.05) in HOT. Evoked twitch and octet RTD increased with increased Tre (p<0.05). CONCLUSIONS: Hyperthermia reduced late-phase voluntary RTD, likely due to reduced neural drive and the reduction in MVT. In contrast, early- and middle-phase voluntary RTD were unaffected by hyperthermia, likely due to the conflicting effects of reduced neural drive but faster intrinsic contractile properties.

Inherent Immune Cell Variation Within Colonic Segments Presents Challenges for Clinical Trial Design.

BACKGROUND AND AIMS: Intestinal biopsy sampling during IBD trials represents a valuable adjunct strategy for understanding drug responses at the tissue level. Given the length and distinctive embryonic origins of the proximal and distal colon, we investigated whether inherent regional differences of immune cell composition could introduce confounders when sampling different disease stages, or pre/post drug administration. Here, we capitalise on novel mass cytometry technology to perform deep immunophenotyping of distinct healthy colonic segments, using the limited numbers of biopsies that can be harvested from patients. METHODS: Biopsies [2.8 mm] were collected from the caecum, transverse colon, descending colon, and rectum of normal volunteers. Intestinal leukocytes were isolated, stained with a panel of 37 antibodies, and mass cytometry data acquired. RESULTS: Site-specific patterns of leukocyte localisation were observed. The proximal colon featured increased CD8+ T cells [particularly resident memory], monocytes, and CD19+ B cells. Conversely, the distal colon and rectum tissues exhibited enrichment for CD4+ T cells and antibody-secreting cells. The transverse colon displayed increased abundance of both γδ T cells and NK cells. Subsets of leukocyte lineages also displayed gradients of expression along the colon length. CONCLUSIONS: Our results show an inherent regional immune cell variation within colonic segments, indicating that regional mucosal signatures must be considered when assessing disease stages or the prospective effects of trial drugs on leukocyte subsets. Precise protocols for intestinal sampling must be implemented to allow for the proper interpretation of potential differences observed within leukocyte lineages present in the colonic lamina propria.

Sphingosine-1-Phosphate Lyase Inhibition Alters the S1P Gradient and Ameliorates Crohn's-Like Ileitis by Suppressing Thymocyte Maturation.

BACKGROUND: Lymphocytes recirculate from tissues to blood following the sphingosine-1-phosphate (S1P) gradient (low in tissues, high in blood), maintained by synthetic and degradative enzymes, among which the S1P lyase (SPL) irreversibly degrades S1P. The role of SPL in the intestine, both during homeostasis and IBD, is poorly understood. We hypothesized that modulation of tissue S1P levels might be advantageous over S1P receptor (S1PR) agonists (eg, fingolimod, ozanimod, etrasimod), as without S1PR engagement there might be less likelihood of potential off-target effects. METHODS: First we examined SPL mRNA transcripts and SPL localization in tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The in vivo effects of the SPL inhibitors 4-deoxypyridoxine hydrochloride (30 mg/L) and 2-acetyl-4 (tetrahydroxybutyl)imidazole (50 mg/L) were assessed through their oral administration to adult TNF∆ARE mice, which spontaneously develop Crohn's-like chronic ileitis. The effect of SPL inhibition on circulating and tissue lymphocytes, transcriptional regulation of proinflammatory cytokines, and on the histological severity of ileitis was additionally examined. Tissue S1P levels were determined by liquid chromatography-mass spectrometry. Mechanistically, the potential effects of high S1P tissue levels on intestinal leukocyte apoptosis were assessed via terminal deoxynucleotidyl transferase dUTP nick end-labeling assay and annexin 5 staining. Finally, we examined the ability of T cells to home to the intestine, along with the effects of SPL inhibition on cellular subsets within immune compartments via flow and mass cytometry. RESULTS: S1P lyase was ubiquitously expressed. In the gut, immunohistochemistry predominantly localized it to small intestinal epithelia, although the lamina propria leukocyte fraction had higher mRNA transcripts. Inhibition of SPL markedly increased local intestinal S1P levels, induced peripheral lymphopenia, downregulated proinflammatory cytokines, and attenuated chronic ileitis in mice. SPL inhibition reduced T and myeloid cells in secondary lymphoid tissues and the intestine and decreased naïve T-cell recruitment. The anti-inflammatory activity of SPL inhibition was not mediated by leukocyte apoptosis, nor by interference with the homing of lymphocytes to the intestine, and was independent of its peripheral lymphopenic effect. However, SPL inhibition promoted thymic atrophy and depleted late immature T cells (CD4+CD8+ double positive), with accumulation of mature CD4+CD8- and CD4-CD8+ single-positive cells. CONCLUSIONS: Inhibition of the S1P lyase alters the S1P gradient and attenuates chronic ileitis via central immunosuppression. SPL inhibition could represent a potential way to tame an overactive immune response during IBD and other T-cell-mediated chronic inflammatory diseases.

Short-term isothermic heat acclimation elicits beneficial adaptations but medium-term elicits a more complete adaptation.

PURPOSE: To investigate the effects of 60 min daily, short-term (STHA) and medium-term (MTHA) isothermic heat acclimation (HA) on the physiological and perceptual responses to exercise heat stress. METHODS: Sixteen, ultra-endurance runners (female = 3) visited the laboratory on 13 occasions. A 45 min sub-maximal (40% Wmax) cycling heat stress test (HST) was completed in the heat (40 °C, 50% relative humidity) on the first (HSTPRE), seventh (HSTSTHA) and thirteenth (HSTMTHA) visit. Participants completed 5 consecutive days of a 60 min isothermic HA protocol (target Tre 38.5 °C) between HSTPRE and HSTSTHA and 5 more between HSTSTHA and HSTMTHA. Heart rate (HR), rectal (Tre), skin (Tsk) and mean body temperature (Tbody), perceived exertion (RPE), thermal comfort (TC) and sensation (TS) were recorded every 5 min. During HSTs, cortisol was measured pre and post and expired air was collected at 15, 30 and 45 min. RESULTS: At rest, Tre and Tbody were lower in HSTSTHA and HSTMTHA compared to HSTPRE, but resting HR was not different between trials. Mean exercising Tre, Tsk, Tbody, and HR were lower in both HSTSTHA and HSTMTHA compared to HSTPRE. There were no differences between HSTSTHA and HSTMTHA. Perceptual measurements were lowered by HA and further reduced during HSTMTHA. CONCLUSION: A 60 min a day isothermic STHA was successful at reducing physiological and perceptual strain experienced when exercising in the heat; however, MTHA offered a more complete adaptation.

Targeting Cytokine Signaling and Lymphocyte Traffic via Small Molecules in Inflammatory Bowel Disease: JAK Inhibitors and S1PR Agonists.

The inflammatory Bowel diseases (IBDs) are a chronic, relapsing inflammatory diseases of the gastrointestinal tract with heterogeneous behavior and prognosis. The introduction of biological therapies including anti-TNF, anti-IL-12/23, and anti-integrins, has revolutionized the treatment of IBD, but these drugs are not universally effective. Due to the complex molecular structures of biologics, they are uniformly immunogenic. New discoveries concerning the underlying mechanisms involved in the pathogenesis of IBD have allowed for progress in the development of new treatment options. The advantage of small molecules (SMs) over biological therapies includes their lack of immunogenicity, short half-life, oral administration, and low manufacturing cost. Among these, the Janus Kinases (JAKs) inhibition has emerged as a novel strategy to modulate downstream cytokine signaling during immune-mediated diseases. These drugs target various cytokine signaling pathways that participate in the pathogenesis of IBD. Tofacitinib, a JAK inhibitor targeting predominantly JAK1 and JAK3, has been approved for the treatment of ulcerative colitis (UC), and there are other specific JAK inhibitors under development that may be effective in Crohn's. Similarly, the traffic of lymphocytes can now be targeted by another SM. Sphingosine-1-phosphate receptor (S1PR) agonism is a novel strategy that acts, in part, by interfering with lymphocyte recirculation, through blockade of lymphocyte egress from lymph nodes. S1PR agonists are being studied in IBD and other immune-mediated disorders. This review will focus on SM drugs approved and under development, including JAK inhibitors (tofacitinib, filgotinib, upadacitinib, peficitinib) and S1PR agonists (KRP-203, fingolimod, ozanimod, etrasimod, amiselimod), and their mechanism of action.

Impairment of Cycling Capacity in the Heat in Well-Trained Endurance Athletes After High-Intensity Short-Term Heat Acclimation.

PURPOSE: To investigate the effects of short-term, high-intensity interval-training (HIIT) heat acclimation (HA). METHODS: Male cyclists/triathletes were assigned into either an HA (n = 13) or a comparison (COMP, n = 10) group. HA completed 3 cycling heat stress tests (HSTs) to exhaustion (60% Wmax; HST1, pre-HA; HST2, post-HA; HST3, 7 d post-HA). HA consisted of 30-min bouts of HIIT cycling (6 min at 50% Wmax, then 12 × 1-min 100%-Wmax bouts with 1-min rests between bouts) on 5 consecutive days. COMP completed HST1 and HST2 only. HST and HA trials were conducted in 35°C/50% relative humidity. Cycling capacity and physiological and perceptual data were recorded. RESULTS: Cycling capacity was impaired after HIIT HA (77.2 [34.2] min vs 56.2 [24.4] min, P = .03) and did not return to baseline after 7 d of no HA (59.2 [37.4] min). Capacity in HST1 and HST2 was similar in COMP (43.5 [8.3] min vs 46.8 [15.7] min, P = .54). HIIT HA lowered resting rectal (37.0°C [0.3°C] vs 36.8°C [0.2°C], P = .05) and body temperature (36.0°C [0.3°C] vs 35.8°C [0.3°C], P = .03) in HST2 compared with HST1 and lowered mean skin temperature (35.4°C [0.5°C] vs 35.1°C [0.3°C], P = .02) and perceived strain on day 5 compared with day 1 of HA. All other data were unaffected. CONCLUSIONS: Cycling capacity was impaired in the heat after 5 d of consecutive HIIT HA despite some heat adaptation. Based on data, this approach is not recommended for athletes preparing to compete in the heat; however, it is possible that it may be beneficial if a state of overreaching is avoided.

Cell Trafficking Interference in Inflammatory Bowel Disease: Therapeutic Interventions Based on Basic Pathogenesis Concepts.

After 20 years of successful targeting of pro-inflammatory cytokines for the treatment of IBD, an alternative therapeutic strategy has emerged, based on several decades of advances in understanding the pathogenesis of IBD. The targeting of molecules involved in leukocyte traffic has recently become a safe and effective alternative. With 2 currently approved drugs (ie, natalizumab, vedolizumab) and several others in phase 3 trials (eg, etrolizumab, ozanimod, anti-MAdCAM-1), the blockade of trafficking molecules has firmly emerged as a new therapeutic era for IBD. We discuss the targets that have been explored in clinical trials: chemokines and its receptors (eg, IP10, CCR9), integrins (eg, natalizumab, AJM300, vedolizumab, and etrolizumab), and its endothelial ligands (MAdCAM-1, ICAM-1). We also discuss a distinct strategy that interferes with lymphocyte recirculation by blocking lymphocyte egress from lymph nodes (small molecule sphingosine-phosphate receptor [S1PR] agonists: fingolimod, ozanimod, etrasimod, amiselimod). Strategies on the horizon include additional small molecules, allosteric inhibitors that specifically bind to the active integrin form and nanovectors that allow for the use of RNA interference in the quest to modulate pro-inflammatory leukocyte trafficking in IBD.

Passive Heat Exposure Alters Perception and Executive Function.

Findings regarding the influence of passive heat exposure on cognitive function remain equivocal due to a number of methodological issues including variation in the domains of cognition examined. In a randomized crossover design, forty-one male participants completed a battery of cognitive function tests [Visual Search, Stroop, Corsi Blocks and Rapid Visual Information Processing (RVIP) tests] prior to and following 1 h of passive rest in either hot (39.6 ± 0.4°C, 50.8 ± 2.3% Rh) or moderate (21.2 ± 1.8°C, 41.9 ± 11.4% Rh) conditions. Subjective feelings of heat exposure, arousal and feeling were assessed alongside physiological measures including core temperature, skin temperature and heart rate, at baseline and throughout the protocol. Response times were slower in the hot trial on the simple (main effect of trial, P < 0.001) and complex (main effect of trial, P < 0.001) levels of the Stroop test (Hot: 872 ± 198 ms; Moderate: 834 ± 177 ms) and the simple level of the visual search test (Hot: 354 ± 54 ms; Moderate: 331 ± 47 ms) (main effect of trial, P < 0.001). Participants demonstrated superior accuracy on the simple level of the Visual Search test in the hot trial (Hot: 98.5 ± 3.1%; Moderate: 97.4 ± 3.6%) (main effect of trial, P = 0.035). Participants also demonstrated an improvement in accuracy on the complex level of the visual search test following 1 h passive heat exposure (Pre: 96.8 ± 5.9%; Post: 98.1 ± 3.1%), whilst a decrement was seen across the trial in the moderate condition (Pre: 97.7 ± 3.5; Post: 97.0 ± 5.1%) (time*trial interaction, P = 0.029). No differences in performance were observed on the RVIP or Corsi Blocks tests (all P > 0.05). Subjective feelings of thermal sensation and felt arousal were higher, feeling was lower in the hot trial, whilst skin temperature, core temperature and heart rate were higher (main effects of trial, all P < 0.001). The findings of the present study suggest that response times for perception and executive function tasks are worse in the heat. An improvement in accuracy on perceptual tasks may suggest a compensatory speed-accuracy trade-off effect occurring within this domain, further highlighting the task dependant nature of heat exposure on cognition.

Implementation of Mass Cytometry as a Tool for Mechanism of Action Studies in Inflammatory Bowel Disease.

Background: Novel therapeutics for inflammatory bowel disease (IBD) are under development, yet mechanistic readouts at the tissue level are lacking. Techniques to assess intestinal immune composition could represent a valuable tool for mechanism of action (MOA) studies of novel drugs. Mass cytometry enables analysis of intestinal inflammatory cell infiltrate and corresponding molecular fingerprints with unprecedented resolution. Here, we aimed to optimize the methodology for isolation and cryopreservation of cells from intestinal tissue to allow for the potential implementation of mass cytometry in MOA studies. Methods: We investigated key technical issues, including minimal tissue requirements, cell isolation protocols, and cell storage, using intestinal biopsies and peripheral blood from healthy individuals. High-dimensional mass cytometry was employed for the analyses of biopsy-derived intestinal cellular subsets. Results: Dithiothreitol and mechanical dissociation decreased epithelial cell contamination and allowed for isolation of adequate cell numbers from 2 to 4 colonic or ileal biopsies (6 × 104±2 × 104) after a 20-minute collagenase digestion, allowing for reliable detection of most major immune cell subsets. Biopsies and antibody-labeled mononuclear cells could be cryopreserved for later processing and acquisition (viability > 70%; P < 0.05). Conclusions: Mass cytometry represents a unique tool for deep immunophenotyping intestinal cell composition. This technique has the potential to facilitate analysis of drug actions at the target tissue by identifying specific cellular subsets and their molecular signatures. Its widespread implementation may impact not only IBD research but also other gastrointestinal conditions where inflammatory cells play a role in pathogenesis.

Nitrate Supplement Benefits Contractile Forces in Fatigued but Not Unfatigued Muscle.

PURPOSE: Evidence suggests dietary nitrate supplementation enhances low-frequency (≤20 Hz) involuntary, but not voluntary, forces in unfatigued human muscle. We investigated the hypotheses that nitrate supplementation would also attenuate low-frequency fatigue and the loss of explosive voluntary forces in fatigued conditions. METHODS: In a counterbalanced double-blinded order, 17 male participants completed two experimental trials after 7 d of dietary supplementation with either nitrate-rich (NIT) or nitrate-depleted (PLA) beetroot juice. Each trial consisted of measuring isometric knee extension forces during a series of explosive maximal voluntary contractions (MVC) and involuntary tetanic contractions (at 10, 20 50, and 100 Hz) in unfatigued conditions, followed by a fatigue protocol of 60 MVC and a repeat of the tetanic contractions immediately after the 60 MVC. RESULTS: In unfatigued conditions, there was no effect of NIT on any of the measured dependent variables, including maximal voluntary force, explosive impulse, and tetanic peak forces or peak rate of force developments at any frequency. In contrast, the percentage decline in explosive voluntary impulse from the first to the last 6 MVC in the fatigue protocol was lower in NIT (51.1% ± 13.9%) than in PLA (57.3% ± 12.4%; P = 0.039; d = 0.51). Furthermore, low-frequency fatigue determined via the percentage decline in the 20/50-Hz ratio was attenuated in NIT for tetanic peak force (NIT: 12.3% ± 12.0% vs PLA: 17.0% ± 10.1%; P = 0.110; d = 0.46) and tetanic peak rate force development (NIT: 12.3% ± 10.4% vs PLA: 20.3% ± 9.5%; P = 0.011; d = 0.83). CONCLUSIONS: Nitrate supplementation reduced the decline in explosive voluntary forces during a fatiguing protocol and attenuated low-frequency fatigue, likely due to reduced disruption of excitation-contraction coupling. However, contrary to previous findings, nitrate supplementation had no effect on contractile performance in unfatigued conditions.

Core and skin temperature influences on the surface electromyographic responses to an isometric force and position task.

The large body of work demonstrating hyperthermic impairment of neuromuscular function has utilized maximal isometric contractions, but extrapolating these findings to whole-body exercise and submaximal, dynamic contractions may be problematic. We isolated and compared core and skin temperature influences on an isometric force task versus a position task requiring dynamic maintenance of joint angle. Surface electromyography (sEMG) was measured on the flexor carpi radialis at 60% of baseline maximal voluntary contraction while either pushing against a rigid restraint (force task) or while maintaining a constant wrist angle and supporting an equivalent inertial load (position task). Twenty participants performed each task at 0.5°C rectal temperature (Tre) intervals while being passively heated from 37.1±0.3°C to ≥1.5°C Tre and then cooled to 37.8±0.3°C, permitting separate analyses of core versus skin temperature influences. During a 3-s contraction, trend analysis revealed a quadratic trend that peaked during hyperthermia for root-mean-square (RMS) amplitude during the force task. In contrast, RMS amplitude during the position task remained stable with passive heating, then rapidly increased with the initial decrease in skin temperature at the onset of passive cooling (p = 0.010). Combined hot core and hot skin elicited shifts toward higher frequencies in the sEMG signal during the force task (p = 0.003), whereas inconsistent changes in the frequency spectra occurred for the position task. Based on the patterns of RMS amplitude in response to thermal stress, we conclude that core temperature was the primary thermal afferent influencing neuromuscular response during a submaximal force task, with minimal input from skin temperature. However, skin temperature was the primary thermal afferent during a position task with minimal core temperature influence. Therefore, temperature has a task-dependent impact on neuromuscular responses.

Practical Torso Cooling During Soccer-Specific Exercise in the Heat.

CONTEXT: Precooling and midevent cooling of the torso using cooling vests can improve exercise performance in the heat with or without physiological changes; however, the effects of such cooling during intermittent exercise in the heat are unknown. OBJECTIVE: To investigate the effects of torso cooling during intermittent exercise in the heat (35°C, 50% relative humdity) on sprint performance and the physiological and perceptual responses to the exercise. DESIGN: Crossover study. SETTING: Walk-in environmental chamber. PATIENTS OR OTHER PARTICIPANTS: Ten non-heat-acclimated, male soccer players (age = 25 ± 2 years, height = 1.77 ± 0.06 m, mass = 72.9 ± 7.6 kg). INTERVENTION(S): Two 90-minute bouts of soccer-specific intermittent running in the heat: 1 trial with a cooling vest worn during the exercise and 1 trial without a cooling vest. Each trial comprised two 45-minute periods separated by approximately 15 minutes of seated rest in cool conditions (approximately 23°C, 50% relative humdity). MAIN OUTCOME MEASURE(S): Peak sprint speed, rectal temperature (Tr), mean-weighted skin temperature (Tsk), heart rate (HR), rating of perceived exertion (RPE), and thermal sensation (TS) were measured every 5 minutes. RESULTS: Peak sprint performance was largely unaffected by the cooling vest. The Tr, Tsk, HR, RPE, and TS were unaffected in the cooling-vest trial during the first 45 minutes, but Tr rose at a slower rate in the cooling-vest trial (0.026°C.min-1 ± 0.008°C.min-1) than in the no-vest trial (0.032°C.min-1 ± 0.009°C.min-1). During the second 45-minute period, Tr, Tr rate of rise, Tsk, RPE, and TS were lower in the cooling-vest trial (Hedges g range, 0.55-0.84), but mean HR was unaffected. CONCLUSIONS: Wearing a cooling vest during soccer-specific intermittent running in the heat reduced physiological and perceptual strain but did not increase peak sprint speed.