Investigation of the influence of CD4+CD25+ regulatory cells on the generation of tumour immunity in mice and human patients.

A population of T cells identified as CD4+CD25+FOXP3+ (Tregs) are present in rodents and humans. These cells are vital for suppressing the activity of conventional self- reactive T cells. Previous work conducted using tumour cell lines has indicated that Tregs also suppress effective anti-tumour immunity in mice. Existing data derived using immunocompromised strains of mice has strongly suggested that the immune system is capable of recognising malignant cells, a process termed immunosurveillance. It was reasonable to hypothesise therefore that Tregs impinge on this process, effectively hampering elimination of tumour cells by the immune system. This hypothesis was tested in this thesis by analysing the impact of Tregs on tumours induced in vivo using the chemical carcinogen, methylcholanthrene. Results of these studies indicated that chemically induced tumours are significantly infiltrated with Tregs and that partial depletion of theses cells prior to exposure to the carcinogen results in a marked reduction in tumour incidence. These findings support a role for Tregs in immunosurveillance. The role of Tregs during human malignancy was also addressed in this thesis using blood samples obtained from a cohort of patients with colorectal cancer. Specifically, studies were carried out to determine whether Treg frequencies were elevated in this group compared to healthy controls and to address whether these cells suppressed tumour antigen specific T cell responses. Results of these studies indicated that that colorectal carcinoma patients have an elevated frequency of Tregs in peripheral blood compared to age matched controls and that these Tregs suppress CD4+ T cell responses to tumour antigens. Collectively these data indicate that Tregs downmodulate immune responses to tumours and support strategies aimed at depleting or inactivating the cells for therapeutic purposes

Pectin-alginate does not further enhance exogenous carbohydrate oxidation in running:Hydrogel and exogenous carbohydrate oxidation

PURPOSE: Maximizing carbohydrate availability is important for many endurance events. Combining pectin and sodium alginate with ingested maltodextrin-fructose (MAL+FRU+PEC+ALG) has been suggested to enhance carbohydrate delivery via hydrogel formation but the influence on exogenous carbohydrate oxidation remains unknown. The primary aim of this study was to assess the effects of MAL+FRU+PEC+ALG on exogenous carbohydrate oxidation during exercise compared to a maltodextrin-fructose mixture (MAL+FRU). MAL+FRU has been well established to increase exogenous carbohydrate oxidation during cycling, compared to glucose-based carbohydrates (MAL+GLU). However, much evidence focuses on cycling, and direct evidence in running is lacking. Therefore, a secondary aim was to compare exogenous carbohydrate oxidation rates with MAL+FRU versus MAL+GLU during running. METHODS: Nine trained runners completed two trials (MAL+FRU and MAL+FRU+PEC+ALG) in a double-blind, randomised crossover design. A subset (n=7) also completed a MAL+GLU trial to address the secondary aim, and a water trial to establish background expired 13CO2 enrichment. Participants ran at 60% \dot{\mathrm{V}}\mathrm{O}_\mathrm{2}peak for 120 min while ingesting either water only, or carbohydrate solutions at a rate of 1.5 g carbohydrate·min-1. RESULTS: At the end of 120 min of exercise, exogenous carbohydrate oxidation rates were 0.9 (SD 0.5) g·min-1 with MAL+GLU ingestion. MAL+FRU ingestion increased exogenous carbohydrate oxidation rates to 1.1 (SD 0.3) g·min-1 (p=0.038), with no further increase with MAL+FRU+PEC+ALG ingestion (1.1 (SD 0.3) g·min-1; p=1.0). No time x treatment interaction effects were observed for plasma glucose, lactate, insulin or non-esterified fatty acids, nor for ratings of perceived exertion or gastrointestinal symptoms (all p>0.05). CONCLUSION: To maximise exogenous carbohydrate oxidation during moderate-intensity running, athletes may benefit from consuming glucose(polymer)-fructose mixtures over glucose-based carbohydrates alone, but the addition of pectin and sodium alginate offers no further benefit

Pectin-alginate does not further enhance exogenous carbohydrate oxidation in running:Hydrogel and exogenous carbohydrate oxidation

PURPOSE: Maximizing carbohydrate availability is important for many endurance events. Combining pectin and sodium alginate with ingested maltodextrin-fructose (MAL+FRU+PEC+ALG) has been suggested to enhance carbohydrate delivery via hydrogel formation but the influence on exogenous carbohydrate oxidation remains unknown. The primary aim of this study was to assess the effects of MAL+FRU+PEC+ALG on exogenous carbohydrate oxidation during exercise compared to a maltodextrin-fructose mixture (MAL+FRU). MAL+FRU has been well established to increase exogenous carbohydrate oxidation during cycling, compared to glucose-based carbohydrates (MAL+GLU). However, much evidence focuses on cycling, and direct evidence in running is lacking. Therefore, a secondary aim was to compare exogenous carbohydrate oxidation rates with MAL+FRU versus MAL+GLU during running. METHODS: Nine trained runners completed two trials (MAL+FRU and MAL+FRU+PEC+ALG) in a double-blind, randomised crossover design. A subset (n=7) also completed a MAL+GLU trial to address the secondary aim, and a water trial to establish background expired 13CO2 enrichment. Participants ran at 60% \dot{\mathrm{V}}\mathrm{O}_\mathrm{2}peak for 120 min while ingesting either water only, or carbohydrate solutions at a rate of 1.5 g carbohydrate·min-1. RESULTS: At the end of 120 min of exercise, exogenous carbohydrate oxidation rates were 0.9 (SD 0.5) g·min-1 with MAL+GLU ingestion. MAL+FRU ingestion increased exogenous carbohydrate oxidation rates to 1.1 (SD 0.3) g·min-1 (p=0.038), with no further increase with MAL+FRU+PEC+ALG ingestion (1.1 (SD 0.3) g·min-1; p=1.0). No time x treatment interaction effects were observed for plasma glucose, lactate, insulin or non-esterified fatty acids, nor for ratings of perceived exertion or gastrointestinal symptoms (all p>0.05). CONCLUSION: To maximise exogenous carbohydrate oxidation during moderate-intensity running, athletes may benefit from consuming glucose(polymer)-fructose mixtures over glucose-based carbohydrates alone, but the addition of pectin and sodium alginate offers no further benefit

Exogenous glucose oxidation during exercise is positively related to body size:Body size and carbohydrate metabolism

There is little evidence that body size alters exogenous glucose oxidation rates during exercise. This study assessed whether larger people oxidize more exogenous glucose during exercise than smaller people. Fifteen cyclists were allocated into two groups based on body mass (SMALL, <70 kg body mass, n = 9, two females) or (LARGE, >70 kg body mass, n = 6) matched for lactate threshold (SMALL: 2.3 ± 0.4 W/kg, LARGE: 2.3 ± 0.3 W/kg). SMALL completed 120 min of cycling at 95% of lactate threshold1. LARGE completed two trials in a random order, one at 95% of lactate threshold1 (thereby exercising at the same relative intensity [RELATIVE]) and one at an absolute intensity matched to SMALL (ABSOLUTE). In all trials, cyclists ingested 90 g/hr of 13C-enriched glucose. Total exogenous glucose oxidation was (mean ± SD) 33 ± 8 g/hr in SMALL versus 45 ± 13 g/hr in LARGE-RELATIVE (mean difference: 13 g/hr, 95% confidence interval [2, 24] g/hr, p = .03). Large positive correlations were observed for measures of exogenous carbohydrate oxidation versus body size (body mass, height, and body surface area; e.g., body surface area vs. peak exogenous glucose oxidation, r = .85, 95% confidence interval [.51, .95], p < .01). When larger athletes reduced the intensity from RELATIVE to ABSOLUTE, total exogenous glucose oxidation was 39 ± 7 g/hr (p = .43 vs. LARGE-RELATIVE). In conclusion, the capacity for exogenous glucose oxidation is, on average, higher in larger athletes than smaller athletes during exercise. The extent to which this is due to higher absolute exercise intensity requires further research, but body size may be a consideration in tailoring sports nutrition guidelines for carbohydrate intake during exercise

Suppression of tumour-specific CD4+ T cells by regulatory T cells is associated with progression of human colorectal cancer

Background. There is indirect evidence that T cell responses can control the metastatic spread of colorectal cancer (CRC). However, an enrichment of CD4(+)Foxp3(+) regulatory T cells (Tregs) has also been documented. Objective. To evaluate whether CRC promotes Treg activity and how this influences anti-tumour immune responses and disease progression. Methods. A longitudinal study of Treg activity on a cohort of patients was performed before and after tumour resection. Specific CD4(+) T cell responses were also measured to the tumour associated antigens carcinoembryonic antigen (CEA) and 5T4. Results. Tregs from 62 preoperative CRC patients expressed a highly significant increase in levels of Foxp3 compared to healthy age-matched controls (p=0.007), which returned to normal after surgery (p=0.0075). CD4(+) T cell responses to one or both of the tumour associated antigens, CEA and 5T4, were observed in approximately two-thirds of patients and one third of these responses were suppressed by Tregs. Strikingly, in all patients with tumour recurrence at 12 months, significant preoperative suppression was observed of tumour-specific (p=0.003) but not control CD4(+) T cell responses. Conclusion. These findings demonstrate that the presence of CRC drives the activity of Tregs and accompanying suppression of CD4(+) T cell responses to tumour-associated antigens. Suppression is associated with recurrence of tumour at 12 months, implying that Tregs contribute to disease progression. These findings offer a rationale for the manipulation of Tregs for therapeutic intervention

Preexercise Breakfast Ingestion versus Extended Overnight Fasting Increases Postprandial Glucose Flux after Exercise in Healthy Men

Aims To characterize postprandial glucose flux after exercise in the fed versus overnight fasted-state and to investigate potential underlying mechanisms. Methods In a randomized order, twelve men underwent breakfast-rest (BR; 3 h semi-recumbent), breakfast-exercise (BE; 2 h semi-recumbent before 60-min of cycling (50% peak power output) and overnight fasted-exercise (FE; as per BE omitting breakfast) trials. An oral glucose tolerance test (OGTT) was completed post-exercise (post-rest on BR). Dual stable isotope tracers ([U-13C] glucose ingestion and [6,6-2H2] glucose infusion) and muscle biopsies were combined to assess postprandial plasma glucose kinetics and intramuscular signaling, respectively. Plasma intestinal fatty acid binding (I-FABP) concentrations were determined as a marker of intestinal damage. Results Breakfast before exercise increased post-exercise plasma glucose disposal rates during the OGTT, from 44 g•120 min-1 in FE [35 to 53 g•120 min-1] (mean [normalized 95% CI]) to 73 g•120 min-1 in BE [55 to 90 g•120 min-1; p = 0.01]. This higher plasma glucose disposal rate was, however, offset by increased plasma glucose appearance rates (principally OGTT-derived), resulting in a glycemic response that did not differ between BE and FE (p = 0.11). Plasma I-FABP concentrations during exercise were 264 pg•mL-1 [196 to 332 pg•mL-1] lower in BE versus FE (p = 0.01). Conclusion Breakfast before exercise increases post-exercise postprandial plasma glucose disposal, which is offset (primarily) by increased appearance rates of orally-ingested glucose. Therefore, metabolic responses to fed-state exercise cannot be readily inferred from studies conducted in a fasted state

Interventions targeting glucocorticoid-Krüppel-like factor 15-branched-chain amino acid signaling improve disease phenotypes in spinal muscular atrophy mice

The circadian glucocorticoid-Krüppel-like factor 15-branched-chain amino acid (GC-KLF15-BCAA) signaling pathway is a key regulatory axis in muscle, whose imbalance has wide-reaching effects on metabolic homeostasis. Spinal muscular atrophy (SMA) is a neuromuscular disorder also characterized by intrinsic muscle pathologies, metabolic abnormalities and disrupted sleep patterns, which can influence or be influenced by circadian regulatory networks that control behavioral and metabolic rhythms. We therefore set out to investigate the contribution of the GC-KLF15-BCAA pathway in SMA pathophysiology of Taiwanese Smn−/−;SMN2 and Smn2B/− mouse models. We thus uncover substantial dysregulation of GC-KLF15-BCAA diurnal rhythmicity in serum, skeletal muscle and metabolic tissues of SMA mice. Importantly, modulating the components of the GC-KLF15-BCAA pathway via pharmacological (prednisolone), genetic (muscle-specific Klf15 overexpression) and dietary (BCAA supplementation) interventions significantly improves disease phenotypes in SMA mice. Our study highlights the GC-KLF15-BCAA pathway as a contributor to SMA pathogenesis and provides several treatment avenues to alleviate peripheral manifestations of the disease. The therapeutic potential of targeting metabolic perturbations by diet and commercially available drugs could have a broader implementation across other neuromuscular and metabolic disorders characterized by altered GC-KLF15-BCAA signaling

CD4(+)CD25(+)FOXP3(+) Regulatory T Cells Suppress Anti-Tumor Immune Responses in Patients with Colorectal Cancer

BACKGROUND: A wealth of evidence obtained using mouse models indicates that CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4(+) T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients. METHODOLOGY AND PRINCIPAL FINDINGS: Treg were identified and characterized as CD4(+)CD25(+)FOXP3(+) using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4(+) T cell responses, as observed by IFNγ release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group. CONCLUSIONS/SIGNIFICANCE: Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy

Lipid Metabolism Links Nutrient-Exercise Timing to Insulin Sensitivity in Men Classified as Overweight or Obese

Context Pre-exercise nutrient availability alters acute metabolic responses to exercise, which could modulate training responsiveness. Objective To assess acute and chronic effects of exercise performed before versus after nutrient ingestion on whole-body and intramuscular lipid utilization and postprandial glucose metabolism. Design (1) Acute, randomized, crossover design (Acute Study); (2) 6-week, randomized, controlled design (Training Study). Setting General community. Participants Men with overweight/obesity (mean ± standard deviation, body mass index: 30.2 ± 3.5 kg⋅m-2 for Acute Study, 30.9 ± 4.5 kg⋅m-2 for Training Study). Interventions Moderate-intensity cycling performed before versus after mixed-macronutrient breakfast (Acute Study) or carbohydrate (Training Study) ingestion. Results Acute Study—exercise before versus after breakfast consumption increased net intramuscular lipid utilization in type I (net change: –3.44 ± 2.63% versus 1.44 ± 4.18% area lipid staining, P 0.05). However, postprandial insulinemia was reduced with exercise training performed before but not after carbohydrate ingestion (P = 0.03). This resulted in increased oral glucose insulin sensitivity (25 ± 38 vs –21 ± 32 mL⋅min-1⋅m-2; P = 0.01), associated with increased lipid utilization during exercise (r = 0.50, P = 0.02). Regular exercise before nutrient provision also augmented remodeling of skeletal muscle phospholipids and protein content of the glucose transport protein GLUT4 (P < 0.05). Conclusions Experiments investigating exercise training and metabolic health should consider nutrient-exercise timing, and exercise performed before versus after nutrient intake (ie, in the fasted state) may exert beneficial effects on lipid utilization and reduce postprandial insulinemia

International Consensus Based Review and Recommendations for Minimum Reporting Standards in Research on Transcutaneous Vagus Nerve Stimulation (Version 2020)

Given its non-invasive nature, there is increasing interest in the use of transcutaneous vagus nerve stimulation (tVNS) across basic, translational and clinical research. Contemporaneously, tVNS can be achieved by stimulating either the auricular branch or the cervical bundle of the vagus nerve, referred to as transcutaneous auricular vagus nerve stimulation(VNS) and transcutaneous cervical VNS, respectively. In order to advance the field in a systematic manner, studies using these technologies need to adequately report sufficient methodological detail to enable comparison of results between studies, replication of studies, as well as enhancing study participant safety. We systematically reviewed the existing tVNS literature to evaluate current reporting practices. Based on this review, and consensus among participating authors, we propose a set of minimal reporting items to guide future tVNS studies. The suggested items address specific technical aspects of the device and stimulation parameters. We also cover general recommendations including inclusion and exclusion criteria for participants, outcome parameters and the detailed reporting of side effects. Furthermore, we review strategies used to identify the optimal stimulation parameters for a given research setting and summarize ongoing developments in animal research with potential implications for the application of tVNS in humans. Finally, we discuss the potential of tVNS in future research as well as the associated challenges across several disciplines in research and clinical practice