Influence of Fatigue Loads on the Laxity of Lumbar Intervertebral Joints

Mechanical Engineerin

THE EFFECT OF SIMULATED MICROGRAVITY ON ANTI-CMV AND ANTI-LEUKEMIA IMMUNE RESPONSES IN VITRO AND IN VIVO.

As the National Aeronautics and Space Administration (NASA), plans to return to the moon and send humans to Mars, crew health and safety concerns need to be examined with a focus on longer duration exploratory class missions. One of the transient changes that has been observed during the ISS missions is dysregulation of immune system. Latent viral reactivation and diminished cellular mediated immunity along with a TH2-cytokine shift being the consistently observed effects of spaceflight on immune system. However, most of the changes observed in spaceflight are a composite effect of stress, microgravity, radiation, circadian disruption, altered nutrition, and sleep disturbances, all of which have an immune-altering effect. Discerning precise effects of the various components of spaceflight become crucial to devise appropriate countermeasures. Ground-based simulated microgravity systems can be used to understand the specific effects of microgravity on human immune system. Rotary cell culture system (RCCS) is a NASA validated ground-based model to simulate microgravity. Using this quiescent low-shear stress environment, human immune cells can be exposed to simulated microgravity (SMG) for brief periods by randomizing the gravity vector and facilitating continuous free fall. In short-term spaceflight, latent viral reactivation along with lowered viral-specific T cell function has been recorded. Healthy functioning of viral-specific T cells is a prerequisite to controlling viral infections. So, it becomes imperative to examine if microgravity plays a role in this reduction of viral-specific T-cell function. There is also a paucity of data on effect of spaceflight and simulated microgravity on γδ-T cells. These crucial effector lymphocytes are considered a connecting link between innate and adaptive immune system. Their function in spaceflight, especially Vγ9Vδ2 T cell subset, becomes important to control hematological malignancies in early stages. Therefore, this dissertation examined the effect of simulated microgravity on expansion potential and function of viral specific T-cells and γδ-T cells. Exposure to SMG impaired in vitro expansion of CMV-specific T-cells (RM ANOVA, F(1.571, 6.283)= 8.367, p=0.0198). 10-million PBMCs at day1 of the expansion in SMG-exposed condition yielded 17.63±3.75 million (MEAN±SEM, N=5) CMV-specific T-cells at the end of the expansion. In comparison, STATIC-1G control cells expanded to 33.8±7.57 million, while 1G-rotational control exposed cells expanded to 28.32±7.21 million cells. However, exposure to SMG did not affect in vitro function of CMV-specific T-cells (RM ANOVA, F(1.357,12.21)=0.7434, p=0.4457). Hundred thousand cytotoxic (CD8+) CMV-specific T-cells exposed to SMG at the end of expansion killed 804.6±166.3 (MEAN±SEM, N=10) autologous PHA blasts pulsed with CMV peptides. In comparison, 1G-control and 1G-rotational control killed 909.2±160.6 and 669.7±125 PHA blasts respectively. Exposure to SMG also impaired in vitro expansion of γδ-T cells (Wilcoxon signed ranks test, p=0.039). 10 million PBMCs at day 1 of the expansion in SMG-exposed condition yielded 102.3±23.07 million (MEAN±SEM, N=9) γδ-T cells at the end of the expansion. In comparison, 1G-control PBMCs yielded 113.7±23.91 million γδ-T cells. Γδ-T cells that were exposed to SMG and later expanded in 1G showed downregulation of inhibitory receptor CD158b (paired t-test, p=0.03) and killed more U266 target cells (paired t-test, p=0.04) compared to γδ-T cells that were expanded in 1G. Exposure to SMG upregulated activating receptor NKG2D on γδ-T cells that were expanded in 1G (Wilcoxon matched-pairs signed rank test, p=0.0078), without concomitant increase in function. Exposure to SMG did not impair γδ-T cells’ ability to kill tumor target cells (K562: paired t-test, t(8) =0.5032, p=0.628; U266: paired t-test, t(8) =0.1479, p=0.886). Another limitation of spaceflight data is lack of in vivo functional data. Although we have observed decreased in vitro function of various immune cells in several flight and ground-based simulation studies, how this translates to a more physiologically relevant in vivo model remains to be explored. Ergo, this dissertation also improved upon the current in vitro data by examining how exposure to SMG using a RCCS, affects in vivo anti-leukemia activity of human effector lymphocytes. A humanized NSG-tg(hu-IL15) mice model, which is used in a pre-clinical setting for hematopoietic stem cell transplantation studies, was used to examine the in vivo effect of exposure to SMG on PBMCs. Furthermore, we examined the efficacy of Zoledronic acid+IL2 (ZOL+IL2) therapy as a possible spaceflight countermeasure to revive the in vivo anti-leukemia activity of SMG exposed PBMCs. ZOL+IL2 is a clinical therapeutic strategy to accelerate favorable immune reconstitution. This therapy also improves NK cell and γδ-T cell activity in vivo, after a hematopoietic stem cell transplantation. Therefore, we expected administration of ZOL+IL2 to abrogate the effect of exposure to SMG on human PBMCs, by stimulating NK cells and γδ-T cells. Exposure to SMG impaired anti-leukemia activity of human immune cells in vivo. Tumor growth control was compared between mice that were injected with PBMCs exposed to SMG (TUMOR+SMG PBMCs) or 1G-control (TUMOR+1G PBMCs) to evaluate the effect of SMG on anti-leukemia activity of human immune cells in vivo. Mice injected with tumor cells only was used as a reference for unrestrained tumor growth. Bioluminescent intensity (BLI) score was used as a measure of tumor burden. A mixed effects model was used to analyze BLI scores with ‘condition’ (TUMOR control, TUMOR+SMG PBMCs, TUMOR+1G PBMCs) and ‘time’ as main effects and an interaction term ‘condition*time’ in the model. This revealed that tumor grew differentially over time in different conditions. A pairwise comparison revealed that 1G-exposed PBMCs controlled tumor growth better than SMG-exposed PBMCs (pTUMOR+1G PBMCs). Exposure to SMG did not alter engraftment, survival or graft-versus-host-disease (GVHD) dynamics. ZOL+IL2 therapy improved anti-leukemia activity of human immune cells in vivo. Mice that received SMG PBMCs and given ZOL+IL2 therapy controlled their tumor better compared to mice that received SMG PBMCs without ZOL+IL2 therapy (Mixed effects model, p=0.0004). There were no differences in tumor control between mice that received SMG PBMCs along with ZOL+IL2 therapy and mice that received 1G-exposed PBMCs. This showed that ZOL+IL2 therapy abrogated the loss of in vivo function after exposure to SMG. ZOL+IL2 therapy did not alter survival and GVHD dynamics. There were non-significant increases in NK cell and γδ-T cell engraftment throughout the experiment in blood of the animals, showing that ZOL+IL2 therapy improved anti-leukemia effector immune cell engraftment, which helped in tumor control. In summary, these experiments advance our understanding of the effect of simulated microgravity on immune cells. Exposure to SMG detrimentally affects immune cell function and expansion potential both in vitro and in vivo. These negative effects could impair crew health and performance during an exploratory class missions. These experiments also highlight the importance of microgravity as a contributor to the immune dysregulation observed in spaceflight. Future studies should explore the sustenance of this detrimental effect in confluence with other perturbations of spaceflight. Future investigations should also include relevant immunotherapeutic countermeasures to improve crew health and performance during long-duration exploration class missions

Lean and obese Zucker rats exhibit different patterns of p70s6 kinase regulation in the tibialis anterior muscle in response to high-force muscle contraction

Increased phosphorylation of the 70-kDa ribosomal S6 kinase (p70S6k) signaling is strongly correlated with the degree of muscle adaptation following exercise. Herein we compare the phosphorylation of p70S6k, Akt, and mammalian target of rapamycin (mTOR) in the tibialis anterior (TA) muscles of lean and obese Zucker rats following a bout of eccentric exercise. Exercise increased p70S6k (Thr389) phosphorylation immediately after (33.3 ± 7.2%) and during [1 h (24.0 ± 14.9%) and 3 h (24.6 ± 11.3%)] recovery in the lean TA and at 3 h (33.5 ± 8.0%) in the obese TA Zucker rats. mTOR (Ser2448) phosphorylation was elevated in the lean TA immediately after exercise (96.5 ± 40.3%) but remained unaltered in the obese TA. Exercise increased Akt (Thr308) and Akt (Ser473) phosphorylation in the lean but not the obese TA. These results suggest that insulin resistance is associated with alterations in the ability of muscle to activate p70S6k signaling following an acute bout of exercise. Muscle Nerve 39: 503–511 2009. Type 2 (non–insulin-dependent) diabetes mellitus (DM) is an emerging epidemic in Western cultures, and it is believed to afflict 150 million people worldwide.11 Insulin resistance is frequently accompanied by a variety of metabolic and cardiovascular abnormalities, including hypertension, glucose intolerance, type 2 diabetes, dyslipidemia, atherosclerosis, and central obesity. A number of studies that employ strength training regimens have been shown to improve glycemic control, increase skeletal muscle size and strength, and positively change body composition. The data suggest that anaerobic exercise may be an effective strategy for the treatment of insulin resistance and type 2 diabetes.6, 7, 49, 53 Recent reports have suggested that differences exist between normal and insulin-resistant muscle in their adaptation to an exercise regimen.5, 8, 20, 24, 48, 50 However, the direct effects of exercise on the phenotype of insulin-resistant muscle have not been widely studied. It is thought that the beneficial effects of exercise on muscle are mediated through activation of the various signaling cascades involved in regulating changes in gene expression, glucose uptake, and protein synthesis.2Whether insulin resistance alters exercise-induced signal transduction processes in muscle is unknown, but the differences, if present, may help to explain why exercise-induced skeletal muscle adaptations can differ between normal and insulin-resistant populations. It is well established that increased muscle loading increases the rates of muscle protein synthesis.27 This increase in protein synthesis, at least in part, is thought to be regulated by the phosphorylation of the p70 ribosomal protein S6 kinase (p70S6k),26 whose activation has been proposed to promote increased translation of messages that have a polypyrimide motif just downstream of the 5′ cap.45 It is believed that p70S6k activity is regulated by the mammalian target of rapamycin (mTOR), which functions as a growth factor and nutrient-sensing signaling molecule in mammalian cells.40 How mTOR activity is modulated is not clear; however, recent evidence suggests that mTOR is controlled by Akt or protein kinase B (PKB), which is activated in response to phospholipid products of the phosphatidylinositol 3-kinase (PI3K) reaction. It is well documented that binding of insulin to the membrane receptor stimulates a cascade of phosphorylation events resulting in activation of PI3K. It is likely that PKB/Akt directly increases mTOR activity by phosphorylating mTOR at Ser2448, and it has been hypothesized that this event is a critical point of control in the regulation of protein synthesis.4 It has been postulated that p70S6k signaling may be particularly important in mediating muscle adaptation given that the phosphorylation of this molecule following an exercise bout has been found to be strongly associated with the increase in muscle weight after 6 weeks of chronic stimulation. The purpose of the present study was to determine whether insulin resistance alters p70S6k signaling after an acute episode of contractile activity. To investigate this possibility, muscle signaling was examined in 12-week-old lean and obese Zucker rats, as it is widely accepted that the insulin resistance exhibited by these animals closely models the development of type 2 diabetes seen in humans.3, 18, 25, 39 We hypothesized that insulin resistance would be associated with differences in how muscle contraction regulates the phosphorylation of the Akt/TOR/p70S6k signaling cascade. To test this hypothesis, the contraction-mediated activation of Akt, mTOR, and p70S6k was assessed either immediately after or 1 or 3 h after a single bout of sciatic nerve stimulation. Taken together, our data suggest that insulin resistance alters contraction-induced p70S6k phosphorylation in skeletal muscle. These findings are consistent with the possibility that insulin resistance alters the way skeletal muscle “senses and responds” to contractile stimuli

Exercise-induced β2-adrenergic receptor activation enhances the anti-leukemic activity of expanded γδ T-Cells via DNAM-1 upregulation and PVR/Nectin-2 recognition

Exercise mobilizes cytotoxic lymphocytes to blood which may allow superior cell products to be manufactured for cancer therapy. Gamma-Delta (γδ) T-cells have shown promise for treating solid tumors, but there is a need to increase their potency against hematologic malignancies. Here, we show that human γδ T-cells mobilized to blood in response to just 20-minutes of graded exercise have surface phenotypes and transcriptomic profiles associated with cytotoxicity, adhesion, migration and cytokine signaling. Following 14-days ex vivo expansion with zoledronic acid and interleukin (IL)-2, exercise mobilized γδ T-cells had surface phenotypes and transcriptomic profiles associated with enhanced effector functions, and demonstrated superior cytotoxic activity against multiple hematologic tumors in vitro, and in vivo in leukemia bearing xenogeneic mice. Infusing humans with the β1+β2-agonist isoproterenol and administering β1 or β1+β2 antagonists prior to exercise revealed these effects to be β2-adrenergic receptor (AR) dependent. Antibody blocking of DNAM-1 on expanded γδ T-cells, as well as the DNAM-1 ligands PVR and Nectin-2 on leukemic targets, abolished the enhanced anti-leukemic effects of exercise. These findings provide a mechanistic link between exercise, β2-AR activation, and the manufacture of superior γδ T-cell products for adoptive cell therapy against hematological malignancies

Human lymphocytes mobilized with exercise have an anti-tumor transcriptomic profile and exert enhanced graft-versus-leukemia effects in xenogeneic mice

BackgroundEvery bout of exercise mobilizes and redistributes large numbers of effector lymphocytes with a cytotoxic and tissue migration phenotype. The frequent redistribution of these cells is purported to increase immune surveillance and play a mechanistic role in reducing cancer risk and slowing tumor progression in physically active cancer survivors. Our aim was to provide the first detailed single cell transcriptomic analysis of exercise-mobilized lymphocytes and test their effectiveness as a donor lymphocyte infusion (DLI) in xenogeneic mice engrafted with human leukemia.MethodsPeripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers at rest and at the end of an acute bout of cycling exercise. Flow cytometry and single-cell RNA sequencing was performed to identify phenotypic and transcriptomic differences between resting and exercise-mobilized cells using a targeted gene expression panel curated for human immunology. PBMCs were injected into the tail vein of xenogeneic NSG-IL-15 mice and subsequently challenged with a luciferase tagged chronic myelogenous leukemia cell line (K562). Tumor growth (bioluminescence) and xenogeneic graft-versus-host disease (GvHD) were monitored bi-weekly for 40-days.ResultsExercise preferentially mobilized NK-cell, CD8+ T-cell and monocyte subtypes with a differentiated and effector phenotype, without significantly mobilizing CD4+ regulatory T-cells. Mobilized effector lymphocytes, particularly effector-memory CD8+ T-cells and NK-cells, displayed differentially expressed genes and enriched gene sets associated with anti-tumor activity, including cytotoxicity, migration/chemotaxis, antigen binding, cytokine responsiveness and alloreactivity (e.g. graft-versus-host/leukemia). Mice receiving exercise-mobilized PBMCs had lower tumor burden and higher overall survival (4.14E+08 photons/s and 47%, respectively) at day 40 compared to mice receiving resting PBMCs (12.1E+08 photons/s and 22%, respectively) from the same donors (p<0.05). Human immune cell engraftment was similar for resting and exercise-mobilized DLI. However, when compared to non-tumor bearing mice, K562 increased the expansion of NK-cell and CD3+/CD4-/CD8- T-cells in mice receiving exercise-mobilized but not resting lymphocytes, 1-2 weeks after DLI. No differences in GvHD or GvHD-free survival was observed between groups either with or without K562 challenge.ConclusionExercise in humans mobilizes effector lymphocytes with an anti-tumor transcriptomic profile and their use as DLI extends survival and enhances the graft-versus-leukemia (GvL) effect without exacerbating GvHD in human leukemia bearing xenogeneic mice. Exercise may serve as an effective and economical adjuvant to increase the GvL effects of allogeneic cell therapies without intensifying GvHD

Pre-injury Variables and Risk of Sport Concussion

A retrospective study explored the predictive ability of pre-injury cognitive functioning (CF), history of concussion (HOC), and sex in a sample (n=708, 41.2% female) of University of Toronto varsity athletes. A series of logistic regressions were conducted. HOC was a significant predictor in both sexes. For every previous concussion, the risk of sustaining another sport concussion increased by 1.5 (CI: 1.1, 2.1 [females]; 1.2, 1.9 [males]). Males with HOC had three times the odds of sustaining another concussion than those without a HOC (CI: 1.7, 5.6). For females, these odds were two times higher (CI: 1.1, 4.0). CF (as measured by the Automated Neuropsychological Assessment Metrics), and sex were not meaningful predictors of risk of concussion. The results support previous findings on HOC and sex as predictors of sport concussion, provide novel findings on the predictive ability of CF, and may be useful in informing concussion prevention and management practices.M.Sc

Assessment of symptoms and complications in treatment of naive newly diagnosed type 2 diabetes mellitus and their correlation with glycemic parameters

Introduction: Diabetes mellitus (DM) is a metabolic disorder that occurs in the body because of decreased insulin activity and/or insulin secretion. Pathological changes such as nephropathy, retinopathy, and cardiovascular complications inevitably occur in the body with the progression of the disease. DM is mainly categorized into 2 sub-types, type I DM and type II DM. While type I DM is generally treated through insulin replacement therapy, type II DM is treated with oral hypoglycaemics.&nbsp

IL-2 and Zoledronic Acid Therapy Restores the In Vivo Anti-Leukemic Activity of Human Lymphocytes Pre-Exposed to Simulated Microgravity

BACKGROUND: We have previously shown that the anti-tumor activity of human lymphocytes is diminished in vitro after 12-hours pre-exposure to simulated microgravity (SMG). Here we used an immunocompromised mouse model to determine if this loss of function would extend in vivo, and to also test the efficacy of IL-2 and zoledronic acid (ZOL) therapy as a potential countermeasure against SMG-induced immune dysfunction. We adoptively transferred human lymphocytes that were exposed to either SMG or 1G-control into NSG-Tg (Hu-IL15) mice 1-week after they were injected with a luciferase-tagged human chronic myeloid leukemia (K562) cell line. Tumor growth was monitored 2x weekly with bioluminescence imaging (BLI) for up to 6-weeks. RESULTS: Mice that received lymphocytes exposed to SMG showed greater tumor burden compared to those receiving lymphocytes exposed to 1G (week 6 BLI: 1.8e10 ± 8.07e9 versus 2.22e8 ± 1.39e8 photons/second; p < 0.0001). Peak BLI was also higher in the SMG group compared to 1G-control (2.34e10 ± 1.23e10 versus 3.75e8 ± 1.56e8 photons/second; p = 0.0062). Exposure to SMG did not affect the ability of human lymphocytes to engraft or evoke xeno-graft-versus-host disease in the mice. Additionally, we injected the mice with IL-2 and zoledronic acid (ZOL) to expand and activate the anti-tumor activity of NK cells and γ δ-T cells, respectively. This treatment was found to revive the loss of anti-leukemic function observed in vivo when lymphocytes were pre-exposed to SMG. CONCLUSIONS: Microgravity plays a contributory role in loss of tumor control in vivo. Immuno-stimulating agents like ZOL+IL-2 may offer an important countermeasure for immune dysregulation during prolonged spaceflight. © 2022 The Author(s). Published by IMR Press.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Human lymphocytes mobilized with exercise have an anti-tumor transcriptomic profile and exert enhanced graft-versus-leukemia effects in xenogeneic mice

Background: Every bout of exercise mobilizes and redistributes large numbers of effector lymphocytes with a cytotoxic and tissue migration phenotype. The frequent redistribution of these cells is purported to increase immune surveillance and play a mechanistic role in reducing cancer risk and slowing tumor progression in physically active cancer survivors. Our aim was to provide the first detailed single cell transcriptomic analysis of exercise-mobilized lymphocytes and test their effectiveness as a donor lymphocyte infusion (DLI) in xenogeneic mice engrafted with human leukemia. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers at rest and at the end of an acute bout of cycling exercise. Flow cytometry and single-cell RNA sequencing was performed to identify phenotypic and transcriptomic differences between resting and exercise-mobilized cells using a targeted gene expression panel curated for human immunology. PBMCs were injected into the tail vein of xenogeneic NSG-IL-15 mice and subsequently challenged with a luciferase tagged chronic myelogenous leukemia cell line (K562). Tumor growth (bioluminescence) and xenogeneic graft-versus-host disease (GvHD) were monitored bi-weekly for 40-days. Results: Exercise preferentially mobilized NK-cell, CD8+ T-cell and monocyte subtypes with a differentiated and effector phenotype, without significantly mobilizing CD4+ regulatory T-cells. Mobilized effector lymphocytes, particularly effector-memory CD8+ T-cells and NK-cells, displayed differentially expressed genes and enriched gene sets associated with anti-tumor activity, including cytotoxicity, migration/chemotaxis, antigen binding, cytokine responsiveness and alloreactivity (e.g. graft-versus-host/leukemia). Mice receiving exercise-mobilized PBMCs had lower tumor burden and higher overall survival (4.14E+08 photons/s and 47%, respectively) at day 40 compared to mice receiving resting PBMCs (12.1E+08 photons/s and 22%, respectively) from the same donors (p<0.05). Human immune cell engraftment was similar for resting and exercise-mobilized DLI. However, when compared to non-tumor bearing mice, K562 increased the expansion of NK-cell and CD3+/CD4-/CD8- T-cells in mice receiving exercise-mobilized but not resting lymphocytes, 1-2 weeks after DLI. No differences in GvHD or GvHD-free survival was observed between groups either with or without K562 challenge. Conclusion: Exercise in humans mobilizes effector lymphocytes with an anti-tumor transcriptomic profile and their use as DLI extends survival and enhances the graft-versus-leukemia (GvL) effect without exacerbating GvHD in human leukemia bearing xenogeneic mice. Exercise may serve as an effective and economical adjuvant to increase the GvL effects of allogeneic cell therapies without intensifying GvHD. Copyright © 2023 Batatinha, Diak, Niemiro, Baker, Smith, Zúñiga, Mylabathula, Seckeler, Lau, LaVoy, Gustafson, Katsanis and Simpson.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The effects of normoxic endurance exercise on erythropoietin (EPO) production and the impact of selective β1 and non-selective β1 + β2 adrenergic receptor blockade

Purpose: Habitual endurance exercise results in increased erythropoiesis, which is primarily controlled by erythropoietin (EPO), yet studies demonstrating upregulation of EPO via a single bout of endurance exercise have been equivocal. This study compares the acute EPO response to 30 minutes of high versus 90 minutes of moderate intensity endurance exercise and whether that response can be upregulated via selective adrenergic receptor blockade. Methods: Using a counterbalanced, cross-over design, fifteen individuals (age 28±8) completed two bouts of running (30-minute, high intensity vs 90-minute, moderate intensity) matched for overall training stress. A separate cohort of fourteen individuals (age 31±6) completed three bouts of 30-minute high intensity cycling after ingesting the beta antagonist bisoprolol, nadolol or placebo. Venous blood was collected before, during and after exercise and EPO response assessed. Results: No detectable EPO response was observed during or after high intensity running, however in the moderate intensity trial EPO was significantly elevated at both during-exercise timepoints (+6.8% ± 2.3% at 15 minutes and +8.7% ± 2.2% at 60 minutes). No significant change in EPO was observed post-cycling or between βAR drug trials. Conclusion: Neither training mode (running or cycling), nor beta-blockade significantly influenced the EPO response to 30 minutes of high-intensity exercise, however 90 minutes of moderate-intensity running elevated EPO during exercise, returning to baseline immediately post exercise. This exercise dependent and transient elevation may explain equivocal data regarding the effect of exercise on serum EPO and allow prescription of exercise to maximize EPO response for both performance and clinical applications