Myths and Methodologies: Standardisation in human physiology research-should we control the controllables?

The premise of research in human physiology is to explore a multifaceted system whilst identifying one or a few outcomes of interest. Therefore, the control of potentially confounding variables requires careful thought regarding the extent of control and complexity of standardisation. One common factor to control prior to testing is diet, as food and fluid provision may deviate from participants' habitual diets, yet a self-report and replication method can be flawed by under-reporting. Researchers may also need to consider standardisation of physical activity, whether it be through familiarisation trials, wash-out periods, or guidance on levels of physical activity to be achieved before trials. In terms of pharmacological agents, the ethical implications of standardisation require researchers to carefully consider how medications, caffeine consumption and oral contraceptive prescriptions may affect the study. For research in females, it should be considered whether standardisation between- or within-participants in regards to menstrual cycle phase is most relevant. The timing of measurements relative to various other daily events is relevant to all physiological research and so it can be important to standardise when measurements are made. This review summarises the areas of standardisation which we hope will be considered useful to anyone involved in human physiology research, including when and how one can apply standardisation to various contexts.

Myths and Methodologies: Standardisation in Human Physiology Research-Should We Control the Controllables?

The premise of research in human physiology is to explore a multifaceted system whilst identifying one or a few outcomes of interest. Therefore, the control of potentially confounding variables requires careful thought regarding the extent of control and complexity of standardisation. One common factor to control prior to testing is diet, as food and fluid provision may deviate from participants' habitual diets, yet a self-report and replication method can be flawed by under-reporting. Researchers may also need to consider standardisation of physical activity, whether it be through familiarisation trials, wash-out periods, or guidance on levels of physical activity to be achieved before trials. In terms of pharmacological agents, the ethical implications of standardisation require researchers to carefully consider how medications, caffeine consumption and oral contraceptive prescriptions may affect the study. For research in females, it should be considered whether standardisation between- or within-participants in regards to menstrual cycle phase is most relevant. The timing of measurements relative to various other daily events is relevant to all physiological research and so it can be important to standardise when measurements are made. This review summarises the areas of standardisation which we hope will be considered useful to anyone involved in human physiology research, including when and how one can apply standardisation to various contexts.

A single bout of vigorous intensity exercise enhances the efficacy of rituximab against human chronic lymphocytic leukaemia B-cells ex vivo.

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal proliferation and accumulation of mature B-cells and is often treated with rituximab, an anti-CD20 monoclonal antibody immunotherapy. Rituximab often fails to induce stringent disease eradication, due in part to failure of antibody-dependent cellular cytotoxicity (ADCC) which relies on natural killer (NK)-cells binding to rituximab-bound CD20 on B-cells. CLL cells are diffusely spread across lymphoid and other bodily tissues, and ADCC resistance in survival niches may be due to several factors including low NK-cell frequency and a suppressive stromal environment that promotes CLL cell survival. It is well established that exercise bouts induce a transient relocation of NK-cells and B-cells into peripheral blood, which could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab in blood. In this pilot study, n = 20 patients with treatment-naïve CLL completed a bout of cycling 15 % above anaerobic threshold for âˆ¼ 30-minutes, with blood samples collected pre-, immediately post-, and 1-hour post-exercise. Flow cytometry revealed that exercise evoked a 254 % increase in effector (CD3-CD56+CD16+) NK-cells in blood, and a 67 % increase in CD5+CD19+CD20+ CLL cells in blood (all p < 0.005). NK-cells were isolated from blood samples pre-, and immediately post-exercise and incubated with primary isolated CLL cells with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab-mediated cell lysis increased by 129 % following exercise (p < 0.001). Direct NK-cell lysis of CLL cells - independent of rituximab - was unchanged following exercise (p = 0.25). We conclude that exercise improved the efficacy of rituximab-mediated ADCC against autologous CLL cells ex vivo and propose that exercise should be explored as a means of enhancing clinical responses in patients receiving anti-CD20 immunotherapy.

Harnessing the immunomodulatory effects of exercise to enhance the efficacy of monoclonal antibody therapies against B-cell haematological cancers: a narrative review.

Therapeutic monoclonal antibodies (mAbs) are standard care for many B-cell haematological cancers. The modes of action for these mAbs include: induction of cancer cell lysis by activating Fcγ-receptors on innate immune cells; opsonising target cells for antibody-dependent cellular cytotoxicity or phagocytosis, and/or triggering the classical complement pathway; the simultaneous binding of cancer cells with T-cells to create an immune synapse and activate perforin-mediated T-cell cytotoxicity against cancer cells; blockade of immune checkpoints to facilitate T-cell cytotoxicity against immunogenic cancer cell clones; and direct delivery of cytotoxic agents via internalisation of mAbs by target cells. While treatment regimens comprising mAb therapy can lead to durable anti-cancer responses, disease relapse is common due to failure of mAb therapy to eradicate minimal residual disease. Factors that limit mAb efficacy include: suboptimal effector cell frequencies, overt immune exhaustion and/or immune anergy, and survival of diffusely spread tumour cells in different stromal niches. In this review, we discuss how immunomodulatory changes arising from exposure to structured bouts of acute exercise might improve mAb treatment efficacy by augmenting (i) antibody-dependent cellular cytotoxicity, (ii) antibody-dependent cellular phagocytosis, (iii) complement-dependent cytotoxicity, (iv) T-cell cytotoxicity, and (v) direct delivery of cytotoxic agents.

Immune cell status, cardiorespiratory fitness and body composition among breast cancer survivors and healthy women: a cross sectional study.

Methods: We examined whether immune cell profiles differ between healthy women (n = 38) and breast cancer survivors (n = 27) within 2 years of treatment, and whether any group-differences were influenced by age, cytomegalovirus infection, cardiorespiratory fitness and body composition. Using flow cytometry, CD4+ and CD8+ T cell subsets, including naïve (NA), central memory (CM) and effector cells (EM and EMRA) were identified using CD27/CD45RA. Activation was measured by HLA-DR expression. Stem cell-like memory T cells (TSCMs) were identified using CD95/CD127. B cells, including plasmablasts, memory, immature and naïve cells were identified using CD19/CD27/CD38/CD10. Effector and regulatory Natural Killer cells were identified using CD56/CD16. Results: Compared to healthy women, CD4+ CM were +Δ21% higher among survivors (p = 0.028) and CD8+ NA were -Δ25% lower (p = 0.034). Across CD4+ and CD8+ subsets, the proportion of activated (HLA-DR+) cells was +Δ31% higher among survivors: CD4+ CM (+Δ25%), CD4+ EM (+Δ32%) and CD4+ EMRA (+Δ43%), total CD8+ (+Δ30%), CD8+ EM (+Δ30%) and CD8+ EMRA (+Δ25%) (p < 0.046). The counts of immature B cells, NK cells and CD16+ NK effector cells were higher among survivors (+Δ100%, +Δ108% and +Δ143% respectively, p < 0.04). Subsequent analyses examined whether statistically significant differences in participant characteristics, influenced immunological differences between groups. Compared to healthy women, survivors were older (56 ± 6 y vs. 45 ± 11 y), had lower cardiorespiratory fitness (V˙O2max mL kg-1 min-1: 28.8 ± 5.0 vs. 36.2 ± 8.5), lower lean mass (42.3 ± 5.0 kg vs. 48.4 ± 15.8 kg), higher body fat (36.3% ± 5.3% vs. 32.7% ± 6.4%) and higher fat mass index (FMI kg/m2: 9.5 ± 2.2 vs. 8.1 ± 2.7) (all p < 0.033). Analysis of covariance revealed divergent moderating effects of age, CMV serostatus, cardiorespiratory fitness and body composition on the differences in immune cell profiles between groups, depending on the cell type examined. Moreover, across all participants, fat mass index was positively associated with the proportion of HLA-DR+ CD4+ EMRA and CD8+ EM/EMRA T cells (Pearson correlation: r > 0.305, p < 0.019). The association between fat mass index and HLA-DR+ CD8+ EMRA T cells withstood statistical adjustment for all variables, including age, CMV serostatus, lean mass and cardiorespiratory fitness, potentially implicating these cells as contributors to inflammatory/immune-dysfunction in overweight/obesity.

The effects of exercise training for eight weeks on immune cell characteristics among breast cancer survivors.

Methods: This study examined the effects of exercise training for 8 weeks on blood immune cell characteristics among 20 breast cancer survivors (age 56 ± 6 years, Body Mass Index 25.4 ± 3.0 kg m2) within two years of treatment. Participants were randomly allocated to a partly-supervised or a remotely-supported exercise group (n = 10 each). The partly supervised group undertook 2 supervised (laboratory-based treadmill walking and cycling) and 1 unsupervised session per week (outdoor walking) progressing from 35 to 50 min and 55% to 70% V˙O2max. The remotely-supported group received weekly exercise/outdoor walking targets (progressing from 105 to 150 min per week 55% to 70% V˙O2max) via weekly telephone calls discussing data from a fitness tracker. Immune cell counts were assessed using flow cytometry: CD4+ and CD8+ T cells (Naïve, NA; Central memory, CM; and Effector cells, EM and EMRA; using CD27/CD45RA), Stem cell-like memory T cells (TSCMs; using CD95/CD127), B cells (plasmablasts, memory, immature and naïve cells using CD19/CD27/CD38/CD10) and Natural Killer cells (effector and regulatory cells, using CD56/CD16). T cell function was assessed by unstimulated HLA-DR expression or interferon gamma (IFN-γ) production with Enzyme-linked ImmunoSpot assays following stimulation with virus or tumour-associated antigens. Results: Total leukocyte counts, lymphocytes, monocytes and neutrophils did not change with training (p > 0.425). Most CD4+ and CD8+ T cell subtypes, including TSCMs, and B cell and NK cell subtypes did not change (p > 0.127). However, across groups combined, the CD4+ EMRA T cell count was lower after training (cells/µl: 18 ± 33 vs. 12 ± 22, p = 0.028) and these cells were less activated on a per cell basis (HLA-DR median fluorescence intensity: 463 ± 138 vs. 420 ± 77, p = 0.018). Furthermore, the partly-supervised group showed a significant decrease in the CD4+/CD8+ ratio (3.90 ± 2.98 vs. 2.54 ± 1.29, p = 0.006) and a significant increase of regulatory NK cells (cells/µl: 16 ± 8 vs. 21 ± 10, p = 0.011). T cell IFN-γ production did not change with exercise training (p > 0.515). Discussion: In summary, most immune cell characteristics are relatively stable with 8 weeks of exercise training among breast cancer survivors. The lower counts and activation of CD4+ EMRA T cells, might reflect an anti-immunosenescence effect of exercise.

The impact of physical inactivity on glucose homeostasis when diet is adjusted to maintain energy balance in healthy, young males.

BACKGROUND & AIMS: It is unclear if dietary adjustments to maintain energy balance during reduced physical activity can offset inactivity-induced reductions in insulin sensitivity and glucose disposal to produce normal daily glucose concentrations and meal responses. Therefore, the aim of the present study was to examine the impact of long-term physical inactivity (60 days of bed rest) on daily glycemia when in energy balance. METHODS: Interstitial glucose concentrations were measured using Continuous Glucose Monitoring Systems (CGMS) for 5 days before and towards the end of bed rest in 20 healthy, young males (Age: 34 ± 8 years; BMI: 23.5 ± 1.8 kg/m2). Energy intake was reduced during bed rest to match energy expenditure, but the types of foods and timing of meals was maintained. Fasting venous glucose and insulin concentrations were determined, as well as the change in whole-body glucose disposal using a hyperinsulinemic-euglycemic clamp (HIEC). RESULTS: Following long-term bed rest, fasting plasma insulin concentration increased 40% (p = 0.004) and glucose disposal during the HIEC decreased 24% (p < 0.001). Interstitial daily glucose total area under the curve (tAUC) from pre-to post-bed rest increased on average by 6% (p = 0.041), despite a 20 and 25% reduction in total caloric and carbohydrate intake, respectively. The nocturnal period (00:00-06:00) showed the greatest change to glycemia with glucose tAUC for this period increasing by 9% (p = 0.005). CGMS measures of daily glycemic variability (SD, J-Index, M-value and MAG) were not changed during bed rest. CONCLUSIONS: Reduced physical activity (bed rest) increases glycemia even when daily energy intake is reduced to maintain energy balance. However, the disturbance to daily glucose homeostasis was much more modest than the reduced capacity to dispose of glucose, and glycemic variability was not negatively affected by bed rest, likely due to positive mitigating effects from the contemporaneous reduction in dietary energy and carbohydrate intake. CLINICAL TRIALS RECORD: NCT03594799 (registered July 20, 2018) (https://clinicaltrials.gov/ct2/show/NCT03594799).

Lifetime Stressor Exposure and Psychophysiological Reactivity and Habituation to Repeated Acute Social Stressors.

This study addressed whether lifetime stressor exposure was associated with psychophysiological reactivity and habituation to a novel laboratory-based stressor. Eighty-six participants (Mage = 23.31 years, SD = 4.94) reported their exposure to lifetime non-sport and sport-specific stressors before completing two consecutive trials of the Trier Social Stress Test, while cardiovascular (i.e., heart rate) and endocrine (i.e., salivary cortisol) data were recorded. Exposure to a moderate number of lifetime non-sport and sport-specific stressors was associated with adaptive cardiovascular reactivity, whereas very low or very high stressor exposure was related to maladaptive reactivity. Moreover, experiencing a very low number of lifetime non-sport (but not sport-specific) stressors was associated with poorer habituation. In contrast, lifetime stressor severity was unrelated to cardiovascular reactivity. Finally, greater lifetime non-sport and sport-specific stressor counts were associated with blunted cortisol reactivity and poorer habituation. These results suggest that lifetime stressor exposure may influence sport performers' acute stress responses.

The effects of exercise on complement system proteins in humans: a systematic scoping review.

BACKGROUND: The complement system is comprised of the classical, lectin and alternative pathways that result in the formation of: pro-inflammatory anaphylatoxins; opsonins that label cells for phagocytic removal; and, a membrane attack complex that directly lyses target cells. Complement-dependent cytotoxicity (CDC) - cell lysis triggered by complement protein C1q binding to the Fc region of antibodies bound to target cells - is another effector function of complement and a key mechanism-of-action of several monoclonal antibody therapies. At present, it is not well established how exercise affects complement system proteins in humans. METHODS: A systematic search was conducted to identify studies that included original data and investigated the association between soluble complement proteins in the blood of healthy humans, and: 1) an acute bout of exercise; 2) exercise training interventions; or, 3) measurements of habitual physical activity and fitness. RESULTS: 77 studies were eligible for inclusion in this review, which included a total of 10,236 participants, and 40 complement proteins and constituent fragments. Higher levels of exercise training and cardiorespiratory fitness were commonly associated with reduced C3 in blood. Additionally, muscle strength was negatively associated with C1q. Elevated C3a-des-Arg, C4a-des-Arg and C5a, lower C1-inhibitor, and unchanged C3 and C4 were reported immediately post-laboratory based exercise, compared to baseline. Whereas, ultra-endurance running and resistance training increased markers of the alternative (factor B and H), classical (C1s), and leptin (mannose binding lectin) pathways, as well as C3 and C6 family proteins, up to 72-h following exercise. Heterogeneity among studies may be due to discrepancies in blood sampling/handling procedures, analytical techniques, exercise interventions/measurements and fitness of included populations. CONCLUSIONS: Increased anaphylatoxins were observed immediately following an acute bout of exercise in a laboratory setting, whereas field-based exercise interventions of a longer duration (e.g. ultra-endurance running) or designed to elicit muscle damage (e.g. resistance training) increased complement proteins for up to 72-h. C3 in blood was mostly reduced by exercise training and associated with increased cardiorespiratory fitness, whereas C1q appeared to be negatively associated to muscle strength. Thus, both acute bouts of exercise and exercise training appear to modulate complement system proteins. Future research is needed to assess the clinical implications of these changes, for example on the efficacy of monoclonal antibody therapies dependent on CDC.

Reframing How Physical Activity Reduces The Incidence of Clinically-Diagnosed Cancers: Appraising Exercise-Induced Immuno-Modulation As An Integral Mechanism.

Undertaking a high volume of physical activity is associated with reduced risk of a broad range of clinically diagnosed cancers. These findings, which imply that physical activity induces physiological changes that avert or suppress neoplastic activity, are supported by preclinical intervention studies in rodents demonstrating that structured regular exercise commonly represses tumour growth. In Part 1 of this review, we summarise epidemiology and preclinical evidence linking physical activity or regular structured exercise with reduced cancer risk or tumour growth. Despite abundant evidence that physical activity commonly exerts anti-cancer effects, the mechanism(s)-of-action responsible for these beneficial outcomes is undefined and remains subject to ongoing speculation. In Part 2, we outline why altered immune regulation from physical activity - specifically to T cells - is likely an integral mechanism. We do this by first explaining how physical activity appears to modulate the cancer immunoediting process. In doing so, we highlight that augmented elimination of immunogenic cancer cells predominantly leads to the containment of cancers in a 'precancerous' or 'covert' equilibrium state, thus reducing the incidence of clinically diagnosed cancers among physically active individuals. In seeking to understand how physical activity might augment T cell function to avert cancer outgrowth, in Part 3 we appraise how physical activity affects the determinants of a successful T cell response against immunogenic cancer cells. Using the cancer immunogram as a basis for this evaluation, we assess the effects of physical activity on: (i) general T cell status in blood, (ii) T cell infiltration to tissues, (iii) presence of immune checkpoints associated with T cell exhaustion and anergy, (iv) presence of inflammatory inhibitors of T cells and (v) presence of metabolic inhibitors of T cells. The extent to which physical activity alters these determinants to reduce the risk of clinically diagnosed cancers - and whether physical activity changes these determinants in an interconnected or unrelated manner - is unresolved. Accordingly, we analyse how physical activity might alter each determinant, and we show how these changes may interconnect to explain how physical activity alters T cell regulation to prevent cancer outgrowth.

'You're kind of left to your own devices': a qualitative focus group study of patients with breast, prostate or blood cancer at a hospital in the South West of England, exploring their engagement with exercise and physical activity during cancer treatment and in the months following standard care.

OBJECTIVES: The aim of this study was to explore the experiences of patients with breast, prostate or blood cancer, regarding their (1) engagement with exercise and physical activity during treatment and in the months following standard care, and (2) the meanings attached to these lifestyle behaviours. DESIGN: A qualitative study using focus groups. The groups were audio recorded, transcribed and analysed using Framework analysis. SETTING: A hospital-based cancer treatment centre in the South-West of England. PARTICIPANTS: Eighteen people who had either completed treatment or were currently on maintenance therapy for breast, prostate or blood cancer (non-Hodgkin lymphoma or Hodgkin lymphoma). RESULTS: Participants reported treatment limiting their ability to engage in exercise and physical activity. However, participants were aware of the physiological, emotional and social benefits of exercise and expressed a desire to maintain a physically active lifestyle before, during and after treatment. They noted a lack of concrete guidance and appropriate exercise classes for people with cancer and felt poorly informed about the type, intensity, duration and frequency of exercise they should be undertaking. As such, participants reported making decisions on their own, relying on their intuition and listening to their bodies to gauge whether they were doing enough exercise (or not). CONCLUSIONS: Participants were aware of the benefits of a physically active lifestyle during and following cancer treatment, but were not familiar with exercise and physical activity guidelines for people living with and beyond cancer. There is a need for healthcare professionals, academics and policy makers to determine how exercise and physical activity can be supported in clinical settings in realistic and meaningful ways accommodating individual patient circumstances.

The Impact of Long-term Physical Inactivity on Adipose Tissue Immunometabolism.

CONTEXT: Adipose tissue and physical inactivity both influence metabolic health and systemic inflammation, but how adipose tissue responds to chronic physical inactivity is unknown. OBJECTIVE: This work aimed to characterize the impact of chronic physical inactivity on adipose tissue in healthy, young males. METHODS: We collected subcutaneous adipose tissue from 20 healthy, young men before and after 60 days of complete bed rest with energy intake reduced to maintain energy balance and fat mass. We used RNA sequencing, flow cytometry, ex vivo tissue culture, and targeted protein analyses to examine adipose tissue phenotype. RESULTS: Our results indicate that the adipose tissue transcriptome, stromal cellular compartment, and insulin signaling protein abundance are largely unaffected by bed rest when fat mass is kept stable. However, there was an increase in the circulating concentration of several adipokines, including plasma leptin, which was associated with inactivity-induced increases in plasma insulin and absent from adipose tissue cultured ex vivo under standardized culture conditions. CONCLUSION: Physical inactivity-induced disturbances to adipokine concentrations such as leptin, without changes to fat mass, could have profound metabolic implications outside a clinical facility when energy intake is not tightly controlled.

Divergent immunometabolic changes in adipose tissue and skeletal muscle with ageing in healthy humans.

KEY POINTS: Ageing is associated with increased systemic inflammation and metabolic dysfunction that contributes to the development of age-associated diseases. The role of adipose tissue in immunometabolic alterations that take place with ageing is unknown in humans. We show, in healthy, active and lean older adults, that adipose tissue, but not skeletal muscle, displays considerable pro-inflammatory transcriptomic, cellular and secretory changes, as well as a reduction in insulin signalling proteins compared to younger adults. These findings indicate that adipose tissue undergoes substantial immunometabolic alterations with ageing, and that these changes are tissue-specific and more profound than those observed in skeletal muscle or in the circulation. These results identify adipose tissue as an important tissue in the biological ageing process in humans, which may exhibit signs of immunometabolic dysfunction prior to systemic manifestation. ABSTRACT: Ageing and obesity are both characterized by inflammation and a deterioration in metabolic health. It is now clear that adipose tissue plays a major role in inflammation and metabolic control in obesity, although little is known about the role of adipose tissue in human ageing. To understand how ageing impacts adipose tissue, we characterized subcutaneous adipose tissue and skeletal muscle samples from twelve younger (27 ± 4 years [Young]) and twelve older (66 ± 5 years [Old]) active/non-obese males. We performed a wide-range of whole-body and tissue measures, including RNA-sequencing and multicolour flow cytometry. We also measured a range of inflammatory and metabolic proteins in the circulation and their release by adipose tissue, ex vivo. Both adipose tissue and muscle had ∼2-fold more immune cells per gram of tissue with ageing. In adipose tissue, this immune cell infiltration was driven by increased memory/effector T-cells, whereas, in muscle, the accumulation was driven by memory/effector T-cells and macrophages. Transcriptomic analysis revealed that, with ageing, adipose tissue, but not muscle, was enriched for inflammatory transcripts/pathways related to acquired and innate immunity. Ageing also increased the adipose tissue pro-inflammatory secretory profile. Insulin signalling protein content was reduced in adipose tissue, but not muscle. Our findings indicate that adipose tissue undergoes substantial immunometabolic changes with ageing in humans, and that these changes are tissue-specific and more profound than those observed in the circulation and skeletal muscle.

The influence of a supervised group exercise intervention combined with active lifestyle recommendations on breast cancer survivors' health, physical functioning, and quality of life indices: study protocol for a randomized and controlled trial.

BACKGROUND: Most cancer patients, under active treatment or not, are sedentary, despite increasing scientific and clinical understanding of the benefits of exercise and physical activity, such as improving quality of life, limiting disease symptoms, decreasing cancer recurrence, and increasing overall survival. Studies have shown that both supervised exercise and unsupervised physical activity programs have low adherence and limited long-term benefits among cancer survivors. Therefore, interventions focused on increasing physical activity levels have clinical and psychological relevance. The present study will examine the feasibility and efficacy of an intervention that combines supervised group exercise with active lifestyle recommendations, analyzing its clinical, psychological, physiological, functional, and immunological effects in breast cancer survivors. METHODS: Women aged 35-75 years who have completed chemotherapy, radiotherapy, and surgery for breast cancer will be recruited from the Cancer Institute of the State of Sao Paulo (ICESP) and take part in a 16-week, parallel-group, randomized, and controlled trial. They will receive a booklet with recommendations for achieving a physically active lifestyle by increasing overall daily movement and undertaking at least 150 min/week of structured exercise. Then, they will be randomized into two groups: the supervised group will take part in two canoeing group exercise sessions every week, and the unsupervised group will increase their overall physical activity level by any means, such as active commuting, daily activities, or home-based exercise. Primary outcome includes aerobic capacity. Secondary outcomes are physical activity, physical functioning, self-reported quality of life, fatigue, presence of lymphedema, body composition, immune function, adherence to physical activity guidelines, and perceptions of self-image. DISCUSSION: Results should contribute to advance knowledge on the impact of a supervised group exercise intervention to improve aspects related to health, physical functioning, and quality of life in female breast cancer survivors. TRIAL REGISTRATION: Brazilian Registry of Clinical Trials Number: RBR-3fw9xf. Retrospectively Registered on 27 December 2018. Items from the World Health Organization Trial Registration Data Set can be accessed on http://www.ensaiosclinicos.gov.br/rg/RBR-3fw9xf/ .

Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors.

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.

One night of sleep fragmentation does not affect exercise-induced leukocyte trafficking or mitogen-stimulated leukocyte oxidative burst in healthy men.

PURPOSE: This study examined whether one night of sleep fragmentation alters circulating leukocyte counts and mitogen-stimulated oxidative burst by leukocytes at rest and in response to an acute bout of vigorous exercise. METHODS: In a randomised crossover design, nine healthy men (mean ± SD: age 22 ± 2 years; BMI 24.9 ± 1.9 kg/m2) were exposed to one night of fragmented or uninterrupted sleep before cycling for 45 min at 71% ± 4% V̇O2peak. Finger-tip blood samples were collected at rest, immediately post-exercise and one-hour post-exercise. Total leukocytes, lymphocytes, monocytes and neutrophils were counted. Leukocyte oxidative burst was assessed in whole blood by measuring Reactive Oxygen Species (ROS) production with luminol-amplified chemiluminescence after stimulation with phorbol 12-myristate 13-acetate (PMA). RESULTS: Exercise elicited the expected trafficking pattern of leukocytes, lymphocytes, monocytes and neutrophils. Compared to rest, PMA-stimulated ROS production was increased one-hour post-exercise (+73% ± 65%; p = 0.019; data combined for fragmented and uninterrupted sleep). There were no statistically significant effects of fragmented sleep on leukocyte, lymphocyte, monocyte, and neutrophil counts or on ROS production at rest, immediately post-exercise or one-hour post-exercise (p > 0.05). However, with fragmented sleep, there was a +10% greater lymphocytosis immediately post-exercise (fragmented +40% ± 37%; uninterrupted +30% ± 35%; p = 0.51) and a -19% smaller neutrophilia by one-hour post-exercise (fragmented +103% ± 88%; uninterrupted +122% ± 131%; p = 0.72). CONCLUSION: Fragmented sleep did not substantially alter the magnitude or pattern of exercise-induced leukocyte trafficking or mitogen-stimulated oxidative burst by leukocytes.